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Sample records for changed transcription factor-binding

  1. Binding site turnover produces pervasive quantitative changes in transcription factor binding between closely related Drosophila species.

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    Robert K Bradley

    2010-03-01

    Full Text Available Changes in gene expression play an important role in evolution, yet the molecular mechanisms underlying regulatory evolution are poorly understood. Here we compare genome-wide binding of the six transcription factors that initiate segmentation along the anterior-posterior axis in embryos of two closely related species: Drosophila melanogaster and Drosophila yakuba. Where we observe binding by a factor in one species, we almost always observe binding by that factor to the orthologous sequence in the other species. Levels of binding, however, vary considerably. The magnitude and direction of the interspecies differences in binding levels of all six factors are strongly correlated, suggesting a role for chromatin or other factor-independent forces in mediating the divergence of transcription factor binding. Nonetheless, factor-specific quantitative variation in binding is common, and we show that it is driven to a large extent by the gain and loss of cognate recognition sequences for the given factor. We find only a weak correlation between binding variation and regulatory function. These data provide the first genome-wide picture of how modest levels of sequence divergence between highly morphologically similar species affect a system of coordinately acting transcription factors during animal development, and highlight the dominant role of quantitative variation in transcription factor binding over short evolutionary distances.

  2. Comparison of Transcription Factor Binding Site Models

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

    Modeling of transcription factor binding sites (TFBSs) and TFBS prediction on genomic sequences are important steps to elucidate transcription regulatory mechanism. Dependency of transcription regulation on a great number of factors such as chemical specificity, molecular structure, genomic and epigenetic characteristics, long distance interaction, makes this a challenging problem. Different experimental procedures generate evidence that DNA-binding domains of transcription factors show considerable DNA sequence specificity. Probabilistic modeling of TFBSs has been moderately successful in identifying patterns from a family of sequences. In this study, we compare performances of different probabilistic models and try to estimate their efficacy over experimental TFBSs data. We build a pipeline to calculate sensitivity and specificity from aligned TFBS sequences for several probabilistic models, such as Markov chains, hidden Markov models, Bayesian networks. Our work, containing relevant statistics and evaluation for the models, can help researchers to choose the most appropriate model for the problem at hand.

  3. Adaptive evolution of transcription factor binding sites

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    Berg Johannes

    2004-10-01

    Full Text Available Abstract Background The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection. Results We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background mutation rate, the selection coefficient, and the effective population size. Conclusions The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed.

  4. Statistics for Transcription Factor Binding Sites

    OpenAIRE

    2008-01-01

    Transcription factors (TFs) play a key role in gene regulation. They interact with specific binding sites or motifs on the DNA sequence and regulate expression of genes downstream of these binding sites. In silico prediction of potential binding of a TF to a binding site is an important task in computational biology. From a statistical point of view, the DNA sequence is a long text consisting of four different letters ('A','C','G', and 'T'). The binding of a TF to the sequence corresponds to ...

  5. DNA methylation changes are a late event in acute promyelocytic leukemia and coincide with loss of transcription factor binding

    DEFF Research Database (Denmark)

    Schoofs, Till; Rohde, Christian; Hebestreit, Katja;

    2013-01-01

    The origin of aberrant DNA methylation in cancer remains largely unknown. In the present study, we elucidated the DNA methylome in primary acute promyelocytic leukemia (APL) and the role of promyelocytic leukemia-retinoic acid receptor α (PML-RARα) in establishing these patterns. Cells from APL...... patients showed increased genome-wide DNA methylation with higher variability than healthy CD34(+) cells, promyelocytes, and remission BM cells. A core set of differentially methylated regions in APL was identified. Age at diagnosis, Sanz score, and Flt3-mutation status characterized methylation subtypes......-trans retinoic acid also did not result in immediate DNA methylation changes. The results of the present study suggest that aberrant DNA methylation is associated with leukemia phenotype but is not required for PML-RARα-mediated initiation of leukemogenesis....

  6. Identifying differential transcription factor binding in ChIP-seq.

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    Wu, Dai-Ying; Bittencourt, Danielle; Stallcup, Michael R; Siegmund, Kimberly D

    2015-01-01

    ChIP seq is a widely used assay to measure genome-wide protein binding. The decrease in costs associated with sequencing has led to a rise in the number of studies that investigate protein binding across treatment conditions or cell lines. In addition to the identification of binding sites, new studies evaluate the variation in protein binding between conditions. A number of approaches to study differential transcription factor binding have recently been developed. Several of these methods build upon established methods from RNA-seq to quantify differences in read counts. We compare how these new approaches perform on different data sets from the ENCODE project to illustrate the impact of data processing pipelines under different study designs. The performance of normalization methods for differential ChIP-seq depends strongly on the variation in total amount of protein bound between conditions, with total read count outperforming effective library size, or variants thereof, when a large variation in binding was studied. Use of input subtraction to correct for non-specific binding showed a relatively modest impact on the number of differential peaks found and the fold change accuracy to biological validation, however a larger impact might be expected for samples with more extreme copy number variations between them. Still, it did identify a small subset of novel differential regions while excluding some differential peaks in regions with high background signal. These results highlight proper scaling for between-sample data normalization as critical for differential transcription factor binding analysis and suggest bioinformaticians need to know about the variation in level of total protein binding between conditions to select the best analysis method. At the same time, validation using fold-change estimates from qRT-PCR suggests there is still room for further method improvement.

  7. Structural Fingerprints of Transcription Factor Binding Site Regions

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    Peter Willett

    2009-03-01

    Full Text Available Fourier transforms are a powerful tool in the prediction of DNA sequence properties, such as the presence/absence of codons. We have previously compiled a database of the structural properties of all 32,896 unique DNA octamers. In this work we apply Fourier techniques to the analysis of the structural properties of human chromosomes 21 and 22 and also to three sets of transcription factor binding sites within these chromosomes. We find that, for a given structural property, the structural property power spectra of chromosomes 21 and 22 are strikingly similar. We find common peaks in their power spectra for both Sp1 and p53 transcription factor binding sites. We use the power spectra as a structural fingerprint and perform similarity searching in order to find transcription factor binding site regions. This approach provides a new strategy for searching the genome data for information. Although it is difficult to understand the relationship between specific functional properties and the set of structural parameters in our database, our structural fingerprints nevertheless provide a useful tool for searching for function information in sequence data. The power spectrum fingerprints provide a simple, fast method for comparing a set of functional sequences, in this case transcription factor binding site regions, with the sequences of whole chromosomes. On its own, the power spectrum fingerprint does not find all transcription factor binding sites in a chromosome, but the results presented here show that in combination with other approaches, this technique will improve the chances of identifying functional sequences hidden in genomic data.

  8. Evolutionary computation for discovery of composite transcription factor binding sites

    OpenAIRE

    Fogel, Gary B.; Porto, V. William; Varga, Gabor; Dow, Ernst R.; Craven, Andrew M.; Powers, David M.; Harlow, Harry B.; Su, Eric W.; Onyia, Jude E.; Su, Chen

    2008-01-01

    Previous research demonstrated the use of evolutionary computation for the discovery of transcription factor binding sites (TFBS) in promoter regions upstream of coexpressed genes. However, it remained unclear whether or not composite TFBS elements, commonly found in higher organisms where two or more TFBSs form functional complexes, could also be identified by using this approach. Here, we present an important refinement of our previous algorithm and test the identification of composite elem...

  9. Leveraging cross-species transcription factor binding site patterns

    DEFF Research Database (Denmark)

    Claussnitzer, Melina; Dankel, Simon N; Klocke, Bernward;

    2014-01-01

    diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele......Genome-wide association studies have revealed numerous risk loci associated with diverse diseases. However, identification of disease-causing variants within association loci remains a major challenge. Divergence in gene expression due to cis-regulatory variants in noncoding regions is central...... to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2...

  10. A systems biology approach to transcription factor binding site prediction.

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    Xiang Zhou

    Full Text Available BACKGROUND: The elucidation of mammalian transcriptional regulatory networks holds great promise for both basic and translational research and remains one the greatest challenges to systems biology. Recent reverse engineering methods deduce regulatory interactions from large-scale mRNA expression profiles and cross-species conserved regulatory regions in DNA. Technical challenges faced by these methods include distinguishing between direct and indirect interactions, associating transcription regulators with predicted transcription factor binding sites (TFBSs, identifying non-linearly conserved binding sites across species, and providing realistic accuracy estimates. METHODOLOGY/PRINCIPAL FINDINGS: We address these challenges by closely integrating proven methods for regulatory network reverse engineering from mRNA expression data, linearly and non-linearly conserved regulatory region discovery, and TFBS evaluation and discovery. Using an extensive test set of high-likelihood interactions, which we collected in order to provide realistic prediction-accuracy estimates, we show that a careful integration of these methods leads to significant improvements in prediction accuracy. To verify our methods, we biochemically validated TFBS predictions made for both transcription factors (TFs and co-factors; we validated binding site predictions made using a known E2F1 DNA-binding motif on E2F1 predicted promoter targets, known E2F1 and JUND motifs on JUND predicted promoter targets, and a de novo discovered motif for BCL6 on BCL6 predicted promoter targets. Finally, to demonstrate accuracy of prediction using an external dataset, we showed that sites matching predicted motifs for ZNF263 are significantly enriched in recent ZNF263 ChIP-seq data. CONCLUSIONS/SIGNIFICANCE: Using an integrative framework, we were able to address technical challenges faced by state of the art network reverse engineering methods, leading to significant improvement in direct

  11. Evolutionary computation for discovery of composite transcription factor binding sites

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    Fogel, Gary B.; Porto, V. William; Varga, Gabor; Dow, Ernst R.; Craven, Andrew M.; Powers, David M.; Harlow, Harry B.; Su, Eric W.; Onyia, Jude E.; Su, Chen

    2008-01-01

    Previous research demonstrated the use of evolutionary computation for the discovery of transcription factor binding sites (TFBS) in promoter regions upstream of coexpressed genes. However, it remained unclear whether or not composite TFBS elements, commonly found in higher organisms where two or more TFBSs form functional complexes, could also be identified by using this approach. Here, we present an important refinement of our previous algorithm and test the identification of composite elements using NFAT/AP-1 as an example. We demonstrate that by using appropriate existing parameters such as window size, novel-scoring methods such as central bonusing and methods of self-adaptation to automatically adjust the variation operators during the evolutionary search, TFBSs of different sizes and complexity can be identified as top solutions. Some of these solutions have known experimental relationships with NFAT/AP-1. We also indicate that even after properly tuning the model parameters, the choice of the appropriate window size has a significant effect on algorithm performance. We believe that this improved algorithm will greatly augment TFBS discovery. PMID:18927103

  12. Prediction of nucleosome positioning based on transcription factor binding sites.

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    Xianfu Yi

    Full Text Available BACKGROUND: The DNA of all eukaryotic organisms is packaged into nucleosomes, the basic repeating units of chromatin. The nucleosome consists of a histone octamer around which a DNA core is wrapped and the linker histone H1, which is associated with linker DNA. By altering the accessibility of DNA sequences, the nucleosome has profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is of great importance for the study of genomic control mechanisms. Transcription factors (TFs have been suggested to play a role in nucleosome positioning in vivo. PRINCIPAL FINDINGS: Here, the minimum redundancy maximum relevance (mRMR feature selection algorithm, the nearest neighbor algorithm (NNA, and the incremental feature selection (IFS method were used to identify the most important TFs that either favor or inhibit nucleosome positioning by analyzing the numbers of transcription factor binding sites (TFBSs in 53,021 nucleosomal DNA sequences and 50,299 linker DNA sequences. A total of nine important families of TFs were extracted from 35 families, and the overall prediction accuracy was 87.4% as evaluated by the jackknife cross-validation test. CONCLUSIONS: Our results are consistent with the notion that TFs are more likely to bind linker DNA sequences than the sequences in the nucleosomes. In addition, our results imply that there may be some TFs that are important for nucleosome positioning but that play an insignificant role in discriminating nucleosome-forming DNA sequences from nucleosome-inhibiting DNA sequences. The hypothesis that TFs play a role in nucleosome positioning is, thus, confirmed by the results of this study.

  13. Incorporating evolution of transcription factor binding sites into annotated alignments

    Indian Academy of Sciences (India)

    Abha S Bais; Steffen Grossmann; Martin Vingron

    2007-08-01

    Identifying transcription factor binding sites (TFBSs) is essential to elucidate putative regulatory mechanisms. A common strategy is to combine cross-species conservation with single sequence TFBS annotation to yield ``conserved TFBSs”. Most current methods in this field adopt a multi-step approach that segregates the two aspects. Again, it is widely accepted that the evolutionary dynamics of binding sites differ from those of the surrounding sequence. Hence, it is desirable to have an approach that explicitly takes this factor into account. Although a plethora of approaches have been proposed for the prediction of conserved TFBSs, very few explicitly model TFBS evolutionary properties, while additionally being multi-step. Recently, we introduced a novel approach to simultaneously align and annotate conserved TFBSs in a pair of sequences. Building upon the standard Smith-Waterman algorithm for local alignments, SimAnn introduces additional states for profiles to output extended alignments or annotated alignments. That is, alignments with parts annotated as gaplessly aligned TFBSs (pair-profile hits) are generated. Moreover, the pair-profile related parameters are derived in a sound statistical framework. In this article, we extend this approach to explicitly incorporate evolution of binding sites in the SimAnn framework. We demonstrate the extension in the theoretical derivations through two position-specific evolutionary models, previously used for modelling TFBS evolution. In a simulated setting, we provide a proof of concept that the approach works given the underlying assumptions, as compared to the original work. Finally, using a real dataset of experimentally verified binding sites in human-mouse sequence pairs, we compare the new approach (eSimAnn) to an existing multi-step tool that also considers TFBS evolution. Although it is widely accepted that binding sites evolve differently from the surrounding sequences, most comparative TFBS identification

  14. Transcription factor binding site positioning in yeast: proximal promoter motifs characterize TATA-less promoters.

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    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of 6 'proximal promoter motifs' (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters.

  15. Transcription factor binding site positioning in yeast: proximal promoter motifs characterize TATA-less promoters.

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    Ionas Erb

    Full Text Available The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of 6 'proximal promoter motifs' (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1 occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters.

  16. Effects of cytosine methylation on transcription factor binding sites

    KAUST Repository

    Medvedeva, Yulia A

    2014-03-26

    Background: DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important.Results: We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines " traffic lights" We observed a strong selection against CpG " traffic lights" within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions.Conclusions: Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription. 2013 Medvedeva et al.; licensee BioMed Central Ltd.

  17. Imputation for transcription factor binding predictions based on deep learning

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    Qin, Qian

    2017-01-01

    Understanding the cell-specific binding patterns of transcription factors (TFs) is fundamental to studying gene regulatory networks in biological systems, for which ChIP-seq not only provides valuable data but is also considered as the gold standard. Despite tremendous efforts from the scientific community to conduct TF ChIP-seq experiments, the available data represent only a limited percentage of ChIP-seq experiments, considering all possible combinations of TFs and cell lines. In this study, we demonstrate a method for accurately predicting cell-specific TF binding for TF-cell line combinations based on only a small fraction (4%) of the combinations using available ChIP-seq data. The proposed model, termed TFImpute, is based on a deep neural network with a multi-task learning setting to borrow information across transcription factors and cell lines. Compared with existing methods, TFImpute achieves comparable accuracy on TF-cell line combinations with ChIP-seq data; moreover, TFImpute achieves better accuracy on TF-cell line combinations without ChIP-seq data. This approach can predict cell line specific enhancer activities in K562 and HepG2 cell lines, as measured by massively parallel reporter assays, and predicts the impact of SNPs on TF binding. PMID:28234893

  18. Probabilistic inference of transcription factor binding from multiple data sources.

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    Harri Lähdesmäki

    Full Text Available An important problem in molecular biology is to build a complete understanding of transcriptional regulatory processes in the cell. We have developed a flexible, probabilistic framework to predict TF binding from multiple data sources that differs from the standard hypothesis testing (scanning methods in several ways. Our probabilistic modeling framework estimates the probability of binding and, thus, naturally reflects our degree of belief in binding. Probabilistic modeling also allows for easy and systematic integration of our binding predictions into other probabilistic modeling methods, such as expression-based gene network inference. The method answers the question of whether the whole analyzed promoter has a binding site, but can also be extended to estimate the binding probability at each nucleotide position. Further, we introduce an extension to model combinatorial regulation by several TFs. Most importantly, the proposed methods can make principled probabilistic inference from multiple evidence sources, such as, multiple statistical models (motifs of the TFs, evolutionary conservation, regulatory potential, CpG islands, nucleosome positioning, DNase hypersensitive sites, ChIP-chip binding segments and other (prior sequence-based biological knowledge. We developed both a likelihood and a Bayesian method, where the latter is implemented with a Markov chain Monte Carlo algorithm. Results on a carefully constructed test set from the mouse genome demonstrate that principled data fusion can significantly improve the performance of TF binding prediction methods. We also applied the probabilistic modeling framework to all promoters in the mouse genome and the results indicate a sparse connectivity between transcriptional regulators and their target promoters. To facilitate analysis of other sequences and additional data, we have developed an on-line web tool, ProbTF, which implements our probabilistic TF binding prediction method using multiple

  19. BaalChIP: Bayesian analysis of allele-specific transcription factor binding in cancer genomes.

    Science.gov (United States)

    de Santiago, Ines; Liu, Wei; Yuan, Ke; O'Reilly, Martin; Chilamakuri, Chandra Sekhar Reddy; Ponder, Bruce A J; Meyer, Kerstin B; Markowetz, Florian

    2017-02-24

    Allele-specific measurements of transcription factor binding from ChIP-seq data are key to dissecting the allelic effects of non-coding variants and their contribution to phenotypic diversity. However, most methods of detecting an allelic imbalance assume diploid genomes. This assumption severely limits their applicability to cancer samples with frequent DNA copy-number changes. Here we present a Bayesian statistical approach called BaalChIP to correct for the effect of background allele frequency on the observed ChIP-seq read counts. BaalChIP allows the joint analysis of multiple ChIP-seq samples across a single variant and outperforms competing approaches in simulations. Using 548 ENCODE ChIP-seq and six targeted FAIRE-seq samples, we show that BaalChIP effectively corrects allele-specific analysis for copy-number variation and increases the power to detect putative cis-acting regulatory variants in cancer genomes.

  20. DNA-MATRIX: a tool for constructing transcription factor binding sites Weight matrix

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    Chandra Prakash Singh,

    2009-12-01

    Full Text Available Despite considerable effort to date, DNA transcription factor binding sites prediction in whole genome remains a challenge for the researchers. Currently the genome wide transcription factor binding sites prediction tools required either direct pattern sequence or weight matrix. Although there are known transcription factor binding sites pattern databases and tools for genome level prediction but no tool for weight matrix construction. Considering this, we developed a DNA-MATRIX tool for searching putative transcription factor binding sites in genomic sequences. DNA-MATRIX uses the simple heuristic approach for weight matrix construction, which can be transformed into different formats as per the requirement of researcher’s for further genome wide prediction and therefore provides the possibility to identify the conserved known DNA binding sites in the coregulated genes and also to search for a great variety of different regulatory binding patterns. The user may construct and save specific weight or frequency matrices in different formats derived through user selected set of known motif sequences.

  1. Transcription factor binding sites are highly enriched within microRNA precursor sequences

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    Piriyapongsa Jittima

    2011-12-01

    Full Text Available Abstract Background Transcription factors are thought to regulate the transcription of microRNA genes in a manner similar to that of protein-coding genes; that is, by binding to conventional transcription factor binding site DNA sequences located in or near promoter regions that lie upstream of the microRNA genes. However, in the course of analyzing the genomics of human microRNA genes, we noticed that annotated transcription factor binding sites commonly lie within 70- to 110-nt long microRNA small hairpin precursor sequences. Results We report that about 45% of all human small hairpin microRNA (pre-miR sequences contain at least one predicted transcription factor binding site motif that is conserved across human, mouse and rat, and this rises to over 75% if one excludes primate-specific pre-miRs. The association is robust and has extremely strong statistical significance; it affects both intergenic and intronic pre-miRs and both isolated and clustered microRNA genes. We also confirmed and extended this finding using a separate analysis that examined all human pre-miR sequences regardless of conservation across species. Conclusions The transcription factor binding sites localized within small hairpin microRNA precursor sequences may possibly regulate their transcription. Transcription factors may also possibly bind directly to nascent primary microRNA gene transcripts or small hairpin microRNA precursors and regulate their processing. Reviewers This article was reviewed by Guillaume Bourque (nominated by Jerzy Jurka, Dmitri Pervouchine (nominated by Mikhail Gelfand, and Yuriy Gusev.

  2. rVISTA for Comparative Sequence-Based Discovery of Functional Transcription Factor Binding Sites

    Energy Technology Data Exchange (ETDEWEB)

    Loots, Gabriela G.; Ovcharenko, Ivan; Pachter, Lior; Dubchak, Inna; Rubin, Edward M.

    2002-03-08

    Identifying transcriptional regulatory elements represents a significant challenge in annotating the genomes of higher vertebrates. We have developed a computational tool, rVISTA, for high-throughput discovery of cis-regulatory elements that combines transcription factor binding site prediction and the analysis of inter-species sequence conservation. Here, we illustrate the ability of rVISTA to identify true transcription factor binding sites through the analysis of AP-1 and NFAT binding sites in the 1 Mb well-annotated cytokine gene cluster1 (Hs5q31; Mm11). The exploitation of orthologous human-mouse data set resulted in the elimination of 95 percent of the 38,000 binding sites predicted upon analysis of the human sequence alone, while it identified 87 percent of the experimentally verified binding sites in this region.

  3. PhyloScan: identification of transcription factor binding sites using cross-species evidence

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    Newberg Lee A

    2007-01-01

    Full Text Available Abstract Background When transcription factor binding sites are known for a particular transcription factor, it is possible to construct a motif model that can be used to scan sequences for additional sites. However, few statistically significant sites are revealed when a transcription factor binding site motif model is used to scan a genome-scale database. Methods We have developed a scanning algorithm, PhyloScan, which combines evidence from matching sites found in orthologous data from several related species with evidence from multiple sites within an intergenic region, to better detect regulons. The orthologous sequence data may be multiply aligned, unaligned, or a combination of aligned and unaligned. In aligned data, PhyloScan statistically accounts for the phylogenetic dependence of the species contributing data to the alignment and, in unaligned data, the evidence for sites is combined assuming phylogenetic independence of the species. The statistical significance of the gene predictions is calculated directly, without employing training sets. Results In a test of our methodology on synthetic data modeled on seven Enterobacteriales, four Vibrionales, and three Pasteurellales species, PhyloScan produces better sensitivity and specificity than MONKEY, an advanced scanning approach that also searches a genome for transcription factor binding sites using phylogenetic information. The application of the algorithm to real sequence data from seven Enterobacteriales species identifies novel Crp and PurR transcription factor binding sites, thus providing several new potential sites for these transcription factors. These sites enable targeted experimental validation and thus further delineation of the Crp and PurR regulons in E. coli. Conclusion Better sensitivity and specificity can be achieved through a combination of (1 using mixed alignable and non-alignable sequence data and (2 combining evidence from multiple sites within an intergenic

  4. Asap: a framework for over-representation statistics for transcription factor binding sites

    DEFF Research Database (Denmark)

    Marstrand, Troels T; Frellsen, Jes; Moltke, Ida

    2008-01-01

    BACKGROUND: In studies of gene regulation the efficient computational detection of over-represented transcription factor binding sites is an increasingly important aspect. Several published methods can be used for testing whether a set of hypothesised co-regulated genes share a common regulatory...... promoter sequences. Controlling all aspects of our input data we are able to identify the optimal statistics across multiple threshold values and for sequence sets containing different distributions of transcription factor binding sites. CONCLUSIONS: We show that our implementation is significantly faster...... than more naïve scanning algorithms when searching with many weight matrices in large sequence sets. When comparing the various statistics, we show that those based on binomial over-representation and Fisher's exact test performs almost equally good and better than the others. An online server...

  5. Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively

    CERN Document Server

    Clifford, Jacob

    2015-01-01

    Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through Position Weight Matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain a...

  6. Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively.

    Science.gov (United States)

    Clifford, Jacob; Adami, Christoph

    2015-09-02

    Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through position weight matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

  7. Using TESS to predict transcription factor binding sites in DNA sequence.

    Science.gov (United States)

    Schug, Jonathan

    2008-03-01

    This unit describes how to use the Transcription Element Search System (TESS). This Web site predicts transcription factor binding sites (TFBS) in DNA sequence using two different kinds of models of sites, strings and positional weight matrices. The binding of transcription factors to DNA is a major part of the control of gene expression. Transcription factors exhibit sequence-specific binding; they form stronger bonds to some DNA sequences than to others. Identification of a good binding site in the promoter for a gene suggests the possibility that the corresponding factor may play a role in the regulation of that gene. However, the sequences transcription factors recognize are typically short and allow for some amount of mismatch. Because of this, binding sites for a factor can typically be found at random every few hundred to a thousand base pairs. TESS has features to help sort through and evaluate the significance of predicted sites.

  8. Predicting Polymerase Ⅱ Core Promoters by Cooperating Transcription Factor Binding Sites in Eukaryotic Genes

    Institute of Scientific and Technical Information of China (English)

    Xiao-Tu MA; Min-Ping QIAN; Hai-Xu TANG

    2004-01-01

    Several discriminate functions for predicting core promoters that based on the potential cooperation between transcription factor binding sites (TFBSs) are discussed. It is demonstrated that the promoter predicting accuracy is improved when the cooperation among TFBSs is taken into consideration.The core promoter region of a newly discovered gene CKLFSF1 is predicted to locate more than 1.5 kb far away from the 5′ end of the transcript and in the last intron of its upstream gene, which is experimentally confirmed later. The core promoters of 3402 human RefSeq sequences, obtained by extending the mRNAs in human genome sequences, are predicted by our algorithm, and there are about 60% of the predicted core promoters locating within the ± 500 bp region relative to the annotated transcription start site.

  9. Number of active transcription factor binding sites is essential for the Hes7 oscillator

    Directory of Open Access Journals (Sweden)

    de Angelis Martin

    2006-02-01

    Full Text Available Abstract Background It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region. Results We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site. Conclusion Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which

  10. ECRbase: Database of Evolutionary Conserved Regions, Promoters, and Transcription Factor Binding Sites in Vertebrate Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Loots, G; Ovcharenko, I

    2006-08-08

    Evolutionary conservation of DNA sequences provides a tool for the identification of functional elements in genomes. We have created a database of evolutionary conserved regions (ECRs) in vertebrate genomes entitled ECRbase that is constructed from a collection of pairwise vertebrate genome alignments produced by the ECR Browser database. ECRbase features a database of syntenic blocks that recapitulate the evolution of rearrangements in vertebrates and a collection of promoters in all vertebrate genomes presented in the database. The database also contains a collection of annotated transcription factor binding sites (TFBS) in all ECRs and promoter elements. ECRbase currently includes human, rhesus macaque, dog, opossum, rat, mouse, chicken, frog, zebrafish, and two pufferfish genomes. It is freely accessible at http://ECRbase.dcode.org.

  11. JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles

    DEFF Research Database (Denmark)

    Mathelier, Anthony; Fornes, Oriol; Arenillas, David J;

    2016-01-01

    JASPAR (http://jaspar.genereg.net) is an open-access database storing curated, non-redundant transcription factor (TF) binding profiles representing transcription factor binding preferences as position frequency matrices for multiple species in six taxonomic groups. For this 2016 release, we...

  12. GTRD: a database of transcription factor binding sites identified by ChIP-seq experiments

    Science.gov (United States)

    Yevshin, Ivan; Sharipov, Ruslan; Valeev, Tagir; Kel, Alexander; Kolpakov, Fedor

    2017-01-01

    GTRD—Gene Transcription Regulation Database (http://gtrd.biouml.org)—is a database of transcription factor binding sites (TFBSs) identified by ChIP-seq experiments for human and mouse. Raw ChIP-seq data were obtained from ENCODE and SRA and uniformly processed: (i) reads were aligned using Bowtie2; (ii) ChIP-seq peaks were called using peak callers MACS, SISSRs, GEM and PICS; (iii) peaks for the same factor and peak callers, but different experiment conditions (cell line, treatment, etc.), were merged into clusters; (iv) such clusters for different peak callers were merged into metaclusters that were considered as non-redundant sets of TFBSs. In addition to information on location in genome, the sets contain structured information about cell lines and experimental conditions extracted from descriptions of corresponding ChIP-seq experiments. A web interface to access GTRD was developed using the BioUML platform. It provides: (i) browsing and displaying information; (ii) advanced search possibilities, e.g. search of TFBSs near the specified gene or search of all genes potentially regulated by a specified transcription factor; (iii) integrated genome browser that provides visualization of the GTRD data: read alignments, peaks, clusters, metaclusters and information about gene structures from the Ensembl database and binding sites predicted using position weight matrices from the HOCOMOCO database. PMID:27924024

  13. Comparative Analysis of Regulatory Motif Discovery Tools for Transcription Factor Binding Sites

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In the post-genomic era, identification of specific regulatory motifs or transcription factor binding sites (TFBSs) in non-coding DNA sequences, which is essential to elucidate transcriptional regulatory networks, has emerged as an obstacle that frustrates many researchers. Consequently, numerous motif discovery tools and correlated databases have been applied to solving this problem. However, these existing methods, based on different computational algorithms, show diverse motif prediction efficiency in non-coding DNA sequences. Therefore, understanding the similarities and differences of computational algorithms and enriching the motif discovery literatures are important for users to choose the most appropriate one among the online available tools. Moreover, there still lacks credible criterion to assess motif discovery tools and instructions for researchers to choose the best according to their own projects. Thus integration of the related resources might be a good approach to improve accuracy of the application. Recent studies integrate regulatory motif discovery tools with experimental methods to offer a complementary approach for researchers, and also provide a much-needed model for current researches on transcriptional regulatory networks. Here we present a comparative analysis of regulatory motif discovery tools for TFBSs.

  14. Model-based Comparative Prediction of Transcription-Factor Binding Motifs in Anabolic Responses in Bone

    Institute of Scientific and Technical Information of China (English)

    Andy; B.; Chen; Kazunori; Hamamura; Guohua; Wang; Weirong; Xing; Subburaman; Mohan; Hiroki; Yokota; Yunlong; Liu

    2007-01-01

    Understanding the regulatory mechanism that controls the alteration of global gene expression patterns continues to be a challenging task in computational biology. We previously developed an ant algorithm, a biologically-inspired computational technique for microarray data, and predicted putative transcription-factor binding motifs (TFBMs) through mimicking interactive behaviors of natural ants. Here we extended the algorithm into a set of web-based software, Ant Modeler, and applied it to investigate the transcriptional mechanism underlying bone formation. Mechanical loading and administration of bone morphogenic proteins (BMPs) are two known treatments to strengthen bone. We addressed a question: Is there any TFBM that stimulates both "anabolic responses of mechanical loading" and "BMP-mediated osteogenic signaling"? Although there is no significant overlap among genes in the two responses, a comparative model-based analysis suggests that the two independent osteogenic processes employ common TFBMs, such as a stress responsive element and a motif for peroxisome proliferator-activated recep- tor (PPAR). The post-modeling in vitro analysis using mouse osteoblast cells sup- ported involvements of the predicted TFBMs such as PPAR, Ikaros 3, and LMO2 in response to mechanical loading. Taken together, the results would be useful to derive a set of testable hypotheses and examine the role of specific regulators in complex transcriptional control of bone formation.

  15. rVISTA 2.0: Evolutionary Analysis of Transcription Factor Binding Sites

    Energy Technology Data Exchange (ETDEWEB)

    Loots, G G; Ovcharenko, I

    2004-01-28

    Identifying and characterizing the patterns of DNA cis-regulatory modules represents a challenge that has the potential to reveal the regulatory language the genome uses to dictate transcriptional dynamics. Several studies have demonstrated that regulatory modules are under positive selection and therefore are often conserved between related species. Using this evolutionary principle we have created a comparative tool, rVISTA, for analyzing the regulatory potential of noncoding sequences. The rVISTA tool combines transcription factor binding site (TFBS) predictions, sequence comparisons and cluster analysis to identify noncoding DNA regions that are highly conserved and present in a specific configuration within an alignment. Here we present the newly developed version 2.0 of the rVISTA tool that can process alignments generated by both zPicture and PipMaker alignment programs or use pre-computed pairwise alignments of seven vertebrate genomes available from the ECR Browser. The rVISTA web server is closely interconnected with the TRANSFAC database, allowing users to either search for matrices present in the TRANSFAC library collection or search for user-defined consensus sequences. rVISTA tool is publicly available at http://rvista.dcode.org/.

  16. A structural-based strategy for recognition of transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Beisi Xu

    Full Text Available Scanning through genomes for potential transcription factor binding sites (TFBSs is becoming increasingly important in this post-genomic era. The position weight matrix (PWM is the standard representation of TFBSs utilized when scanning through sequences for potential binding sites. However, many transcription factor (TF motifs are short and highly degenerate, and methods utilizing PWMs to scan for sites are plagued by false positives. Furthermore, many important TFs do not have well-characterized PWMs, making identification of potential binding sites even more difficult. One approach to the identification of sites for these TFs has been to use the 3D structure of the TF to predict the DNA structure around the TF and then to generate a PWM from the predicted 3D complex structure. However, this approach is dependent on the similarity of the predicted structure to the native structure. We introduce here a novel approach to identify TFBSs utilizing structure information that can be applied to TFs without characterized PWMs, as long as a 3D complex structure (TF/DNA exists. This approach utilizes an energy function that is uniquely trained on each structure. Our approach leads to increased prediction accuracy and robustness compared with those using a more general energy function. The software is freely available upon request.

  17. Bidirectional Transcription Arises from Two Distinct Hubs of Transcription Factor Binding and Active Chromatin.

    Science.gov (United States)

    Scruggs, Benjamin S; Gilchrist, Daniel A; Nechaev, Sergei; Muse, Ginger W; Burkholder, Adam; Fargo, David C; Adelman, Karen

    2015-06-18

    Anti-sense transcription originating upstream of mammalian protein-coding genes is a well-documented phenomenon, but remarkably little is known about the regulation or function of anti-sense promoters and the non-coding RNAs they generate. Here we define at nucleotide resolution the divergent transcription start sites (TSSs) near mouse mRNA genes. We find that coupled sense and anti-sense TSSs precisely define the boundaries of a nucleosome-depleted region (NDR) that is highly enriched in transcription factor (TF) motifs. Notably, as the distance between sense and anti-sense TSSs increases, so does the size of the NDR, the level of signal-dependent TF binding, and gene activation. We further discover a group of anti-sense TSSs in macrophages with an enhancer-like chromatin signature. Interestingly, this signature identifies divergent promoters that are activated during immune challenge. We propose that anti-sense promoters serve as platforms for TF binding and establishment of active chromatin to further regulate or enhance sense-strand mRNA expression.

  18. A deeper look into transcription regulatory code by preferred pair distance templates for transcription factor binding sites

    KAUST Repository

    Kulakovskiy, Ivan V.

    2011-08-18

    Motivation: Modern experimental methods provide substantial information on protein-DNA recognition. Studying arrangements of transcription factor binding sites (TFBSs) of interacting transcription factors (TFs) advances understanding of the transcription regulatory code. Results: We constructed binding motifs for TFs forming a complex with HIF-1α at the erythropoietin 3\\'-enhancer. Corresponding TFBSs were predicted in the segments around transcription start sites (TSSs) of all human genes. Using the genome-wide set of regulatory regions, we observed several strongly preferred distances between hypoxia-responsive element (HRE) and binding sites of a particular cofactor protein. The set of preferred distances was called as a preferred pair distance template (PPDT). PPDT dramatically depended on the TF and orientation of its binding sites relative to HRE. PPDT evaluated from the genome-wide set of regulatory sequences was used to detect significant PPDT-consistent binding site pairs in regulatory regions of hypoxia-responsive genes. We believe PPDT can help to reveal the layout of eukaryotic regulatory segments. © The Author 2011. Published by Oxford University Press. All rights reserved.

  19. Arabidopsis sigma factor binding proteins are activators of the WRKY33 transcription factor in plant defense.

    Science.gov (United States)

    Lai, Zhibing; Li, Ying; Wang, Fei; Cheng, Yuan; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2011-10-01

    Necrotrophic pathogens are important plant pathogens that cause many devastating plant diseases. Despite their impact, our understanding of the plant defense response to necrotrophic pathogens is limited. The WRKY33 transcription factor is important for plant resistance to necrotrophic pathogens; therefore, elucidation of its functions will enhance our understanding of plant immunity to necrotrophic pathogens. Here, we report the identification of two WRKY33-interacting proteins, nuclear-encoded SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2, which also interact with plastid-encoded plastid RNA polymerase SIGMA FACTOR1. Both SIB1 and SIB2 contain an N-terminal chloroplast targeting signal and a putative nuclear localization signal, suggesting that they are dual targeted. Bimolecular fluorescence complementation indicates that WRKY33 interacts with SIBs in the nucleus of plant cells. Both SIB1 and SIB2 contain a short VQ motif that is important for interaction with WRKY33. The two VQ motif-containing proteins recognize the C-terminal WRKY domain and stimulate the DNA binding activity of WRKY33. Like WRKY33, both SIB1 and SIB2 are rapidly and strongly induced by the necrotrophic pathogen Botrytis cinerea. Resistance to B. cinerea is compromised in the sib1 and sib2 mutants but enhanced in SIB1-overexpressing transgenic plants. These results suggest that dual-targeted SIB1 and SIB2 function as activators of WRKY33 in plant defense against necrotrophic pathogens.

  20. Predicting transcription factor binding sites using local over-representation and comparative genomics

    Directory of Open Access Journals (Sweden)

    Touzet Hélène

    2006-08-01

    Full Text Available Abstract Background Identifying cis-regulatory elements is crucial to understanding gene expression, which highlights the importance of the computational detection of overrepresented transcription factor binding sites (TFBSs in coexpressed or coregulated genes. However, this is a challenging problem, especially when considering higher eukaryotic organisms. Results We have developed a method, named TFM-Explorer, that searches for locally overrepresented TFBSs in a set of coregulated genes, which are modeled by profiles provided by a database of position weight matrices. The novelty of the method is that it takes advantage of spatial conservation in the sequence and supports multiple species. The efficiency of the underlying algorithm and its robustness to noise allow weak regulatory signals to be detected in large heterogeneous data sets. Conclusion TFM-Explorer provides an efficient way to predict TFBS overrepresentation in related sequences. Promising results were obtained in a variety of examples in human, mouse, and rat genomes. The software is publicly available at http://bioinfo.lifl.fr/TFM-Explorer.

  1. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  2. A general pairwise interaction model provides an accurate description of in vivo transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Marc Santolini

    Full Text Available The identification of transcription factor binding sites (TFBSs on genomic DNA is of crucial importance for understanding and predicting regulatory elements in gene networks. TFBS motifs are commonly described by Position Weight Matrices (PWMs, in which each DNA base pair contributes independently to the transcription factor (TF binding. However, this description ignores correlations between nucleotides at different positions, and is generally inaccurate: analysing fly and mouse in vivo ChIPseq data, we show that in most cases the PWM model fails to reproduce the observed statistics of TFBSs. To overcome this issue, we introduce the pairwise interaction model (PIM, a generalization of the PWM model. The model is based on the principle of maximum entropy and explicitly describes pairwise correlations between nucleotides at different positions, while being otherwise as unconstrained as possible. It is mathematically equivalent to considering a TF-DNA binding energy that depends additively on each nucleotide identity at all positions in the TFBS, like the PWM model, but also additively on pairs of nucleotides. We find that the PIM significantly improves over the PWM model, and even provides an optimal description of TFBS statistics within statistical noise. The PIM generalizes previous approaches to interdependent positions: it accounts for co-variation of two or more base pairs, and predicts secondary motifs, while outperforming multiple-motif models consisting of mixtures of PWMs. We analyse the structure of pairwise interactions between nucleotides, and find that they are sparse and dominantly located between consecutive base pairs in the flanking region of TFBS. Nonetheless, interactions between pairs of non-consecutive nucleotides are found to play a significant role in the obtained accurate description of TFBS statistics. The PIM is computationally tractable, and provides a general framework that should be useful for describing and predicting

  3. Identifying functional transcription factor binding sites in yeast by considering their positional preference in the promoters.

    Directory of Open Access Journals (Sweden)

    Fu-Jou Lai

    Full Text Available Transcription factor binding site (TFBS identification plays an important role in deciphering gene regulatory codes. With comprehensive knowledge of TFBSs, one can understand molecular mechanisms of gene regulation. In the recent decades, various computational approaches have been proposed to predict TFBSs in the genome. The TFBS dataset of a TF generated by each algorithm is a ranked list of predicted TFBSs of that TF, where top ranked TFBSs are statistically significant ones. However, whether these statistically significant TFBSs are functional (i.e. biologically relevant is still unknown. Here we develop a post-processor, called the functional propensity calculator (FPC, to assign a functional propensity to each TFBS in the existing computationally predicted TFBS datasets. It is known that functional TFBSs reveal strong positional preference towards the transcriptional start site (TSS. This motivates us to take TFBS position relative to the TSS as the key idea in building our FPC. Based on our calculated functional propensities, the TFBSs of a TF in the original TFBS dataset could be reordered, where top ranked TFBSs are now the ones with high functional propensities. To validate the biological significance of our results, we perform three published statistical tests to assess the enrichment of Gene Ontology (GO terms, the enrichment of physical protein-protein interactions, and the tendency of being co-expressed. The top ranked TFBSs in our reordered TFBS dataset outperform the top ranked TFBSs in the original TFBS dataset, justifying the effectiveness of our post-processor in extracting functional TFBSs from the original TFBS dataset. More importantly, assigning functional propensities to putative TFBSs enables biologists to easily identify which TFBSs in the promoter of interest are likely to be biologically relevant and are good candidates to do further detailed experimental investigation. The FPC is implemented as a web tool at http://santiago.ee.ncku.edu.tw/FPC/.

  4. FISim: A new similarity measure between transcription factor binding sites based on the fuzzy integral

    Directory of Open Access Journals (Sweden)

    Cano Carlos

    2009-07-01

    Full Text Available Abstract Background Regulatory motifs describe sets of related transcription factor binding sites (TFBSs and can be represented as position frequency matrices (PFMs. De novo identification of TFBSs is a crucial problem in computational biology which includes the issue of comparing putative motifs with one another and with motifs that are already known. The relative importance of each nucleotide within a given position in the PFMs should be considered in order to compute PFM similarities. Furthermore, biological data are inherently noisy and imprecise. Fuzzy set theory is particularly suitable for modeling imprecise data, whereas fuzzy integrals are highly appropriate for representing the interaction among different information sources. Results We propose FISim, a new similarity measure between PFMs, based on the fuzzy integral of the distance of the nucleotides with respect to the information content of the positions. Unlike existing methods, FISim is designed to consider the higher contribution of better conserved positions to the binding affinity. FISim provides excellent results when dealing with sets of randomly generated motifs, and outperforms the remaining methods when handling real datasets of related motifs. Furthermore, we propose a new cluster methodology based on kernel theory together with FISim to obtain groups of related motifs potentially bound by the same TFs, providing more robust results than existing approaches. Conclusion FISim corrects a design flaw of the most popular methods, whose measures favour similarity of low information content positions. We use our measure to successfully identify motifs that describe binding sites for the same TF and to solve real-life problems. In this study the reliability of fuzzy technology for motif comparison tasks is proven.

  5. Effect of positional dependence and alignment strategy on modeling transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Quader Saad

    2012-07-01

    Full Text Available Abstract Background Many consensus-based and Position Weight Matrix-based methods for recognizing transcription factor binding sites (TFBS are not well suited to the variability in the lengths of binding sites. Besides, many methods discard known binding sites while building the model. Moreover, the impact of Information Content (IC and the positional dependence of nucleotides within an aligned set of TFBS has not been well researched for modeling variable-length binding sites. In this paper, we propose ML-Consensus (Mixed-Length Consensus: a consensus model for variable-length TFBS which does not exclude any reported binding sites. Methods We consider Pairwise Score (PS as a measure of positional dependence of nucleotides within an alignment of TFBS. We investigate how the prediction accuracy of ML-Consensus is affected by the incorporation of IC and PS with a particular binding site alignment strategy. We perform cross-validations for datasets of six species from the TRANSFAC public database, and analyze the results using ROC curves and the Wilcoxon matched-pair signed-ranks test. Results We observe that the incorporation of IC and PS in ML-Consensus results in statistically significant improvement in the prediction accuracy of the model. Moreover, the existence of a core region among the known binding sites (of any length is witnessed by the pairwise coexistence of nucleotides within the core length. Conclusions These observations suggest the possibility of an efficient multiple sequence alignment algorithm for aligning TFBS, accommodating known binding sites of any length, for optimal (or near-optimal TFBS prediction. However, designing such an algorithm is a matter of further investigation.

  6. MONKEY: Identifying conserved transcription-factor binding sitesin multiple alignments using a binding site-specific evolutionarymodel

    Energy Technology Data Exchange (ETDEWEB)

    Moses, Alan M.; Chiang, Derek Y.; Pollard, Daniel A.; Iyer, VenkyN.; Eisen, Michael B.

    2004-10-28

    We introduce a method (MONKEY) to identify conserved transcription-factor binding sites in multispecies alignments. MONKEY employs probabilistic models of factor specificity and binding site evolution, on which basis we compute the likelihood that putative sites are conserved and assign statistical significance to each hit. Using genomes from the genus Saccharomyces, we illustrate how the significance of real sites increases with evolutionary distance and explore the relationship between conservation and function.

  7. CORE_TF: a user-friendly interface to identify evolutionary conserved transcription factor binding sites in sets of co-regulated genes

    Science.gov (United States)

    Hestand, Matthew S; van Galen, Michiel; Villerius, Michel P; van Ommen, Gert-Jan B; den Dunnen, Johan T; 't Hoen, Peter AC

    2008-01-01

    Background The identification of transcription factor binding sites is difficult since they are only a small number of nucleotides in size, resulting in large numbers of false positives and false negatives in current approaches. Computational methods to reduce false positives are to look for over-representation of transcription factor binding sites in a set of similarly regulated promoters or to look for conservation in orthologous promoter alignments. Results We have developed a novel tool, "CORE_TF" (Conserved and Over-REpresented Transcription Factor binding sites) that identifies common transcription factor binding sites in promoters of co-regulated genes. To improve upon existing binding site predictions, the tool searches for position weight matrices from the TRANSFACR database that are over-represented in an experimental set compared to a random set of promoters and identifies cross-species conservation of the predicted transcription factor binding sites. The algorithm has been evaluated with expression and chromatin-immunoprecipitation on microarray data. We also implement and demonstrate the importance of matching the random set of promoters to the experimental promoters by GC content, which is a unique feature of our tool. Conclusion The program CORE_TF is accessible in a user friendly web interface at . It provides a table of over-represented transcription factor binding sites in the users input genes' promoters and a graphical view of evolutionary conserved transcription factor binding sites. In our test data sets it successfully predicts target transcription factors and their binding sites. PMID:19036135

  8. Gains and Losses of Transcription Factor Binding Sites in Saccharomyces cerevisiae and Saccharomyces paradoxus.

    Science.gov (United States)

    Schaefke, Bernhard; Wang, Tzi-Yuan; Wang, Chuen-Yi; Li, Wen-Hsiung

    2015-07-27

    Gene expression evolution occurs through changes in cis- or trans-regulatory elements or both. Interactions between transcription factors (TFs) and their binding sites (TFBSs) constitute one of the most important points where these two regulatory components intersect. In this study, we investigated the evolution of TFBSs in the promoter regions of different Saccharomyces strains and species. We divided the promoter of a gene into the proximal region and the distal region, which are defined, respectively, as the 200-bp region upstream of the transcription starting site and as the 200-bp region upstream of the proximal region. We found that the predicted TFBSs in the proximal promoter regions tend to be evolutionarily more conserved than those in the distal promoter regions. Additionally, Saccharomyces cerevisiae strains used in the fermentation of alcoholic drinks have experienced more TFBS losses than gains compared with strains from other environments (wild strains, laboratory strains, and clinical strains). We also showed that differences in TFBSs correlate with the cis component of gene expression evolution between species (comparing S. cerevisiae and its sister species Saccharomyces paradoxus) and within species (comparing two closely related S. cerevisiae strains).

  9. Hybrid incompatibility despite pleiotropic constraint in a sequence-based bioenergetic model of transcription factor binding.

    Science.gov (United States)

    Tulchinsky, Alexander Y; Johnson, Norman A; Porter, Adam H

    2014-12-01

    Hybrid incompatibility can result from gene misregulation produced by divergence in trans-acting regulatory factors and their cis-regulatory targets. However, change in trans-acting factors may be constrained by pleiotropy, which would in turn limit the evolution of incompatibility. We employed a mechanistically explicit bioenergetic model of gene expression wherein parameter combinations (number of transcription factor molecules, energetic properties of binding to the regulatory site, and genomic background size) determine the shape of the genotype-phenotype (G-P) map, and interacting allelic variants of mutable cis and trans sites determine the phenotype along that map. Misregulation occurs when the phenotype differs from its optimal value. We simulated a pleiotropic regulatory pathway involving a positively selected and a conserved trait regulated by a shared transcription factor (TF), with two populations evolving in parallel. Pleiotropic constraints shifted evolution in the positively selected trait to its cis-regulatory locus. We nevertheless found that the TF genotypes often evolved, accompanied by compensatory evolution in the conserved trait, and both traits contributed to hybrid misregulation. Compensatory evolution resulted in "developmental system drift," whereby the regulatory basis of the conserved phenotype changed although the phenotype itself did not. Pleiotropic constraints became stronger and in some cases prohibitive when the bioenergetic properties of the molecular interaction produced a G-P map that was too steep. Likewise, compensatory evolution slowed and hybrid misregulation was not evident when the G-P map was too shallow. A broad pleiotropic "sweet spot" nevertheless existed where evolutionary constraints were moderate to weak, permitting substantial hybrid misregulation in both traits. None of these pleiotropic constraints manifested when the TF contained nonrecombining domains independently regulating the respective traits.

  10. Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs.

    Science.gov (United States)

    Ohmura, Sakie; Mizuno, Seiya; Oishi, Hisashi; Ku, Chia-Jui; Hermann, Mary; Hosoya, Tomonori; Takahashi, Satoru; Engel, James Douglas

    2016-03-01

    The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell-specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte-specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte-specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell-regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell-specific transcriptional activity.

  11. msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding.

    Science.gov (United States)

    Raj, Anil; Shim, Heejung; Gilad, Yoav; Pritchard, Jonathan K; Stephens, Matthew

    2015-01-01

    Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede.

  12. msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding.

    Directory of Open Access Journals (Sweden)

    Anil Raj

    Full Text Available Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede.

  13. SNP2TFBS – a database of regulatory SNPs affecting predicted transcription factor binding site affinity

    Science.gov (United States)

    Kumar, Sunil; Ambrosini, Giovanna; Bucher, Philipp

    2017-01-01

    SNP2TFBS is a computational resource intended to support researchers investigating the molecular mechanisms underlying regulatory variation in the human genome. The database essentially consists of a collection of text files providing specific annotations for human single nucleotide polymorphisms (SNPs), namely whether they are predicted to abolish, create or change the affinity of one or several transcription factor (TF) binding sites. A SNP's effect on TF binding is estimated based on a position weight matrix (PWM) model for the binding specificity of the corresponding factor. These data files are regenerated at regular intervals by an automatic procedure that takes as input a reference genome, a comprehensive SNP catalogue and a collection of PWMs. SNP2TFBS is also accessible over a web interface, enabling users to view the information provided for an individual SNP, to extract SNPs based on various search criteria, to annotate uploaded sets of SNPs or to display statistics about the frequencies of binding sites affected by selected SNPs. Homepage: http://ccg.vital-it.ch/snp2tfbs/. PMID:27899579

  14. SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics.

    Science.gov (United States)

    Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

    2016-09-15

    Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression.

  15. CORE_TF: a user-friendly interface to identify evolutionary conserved transcription factor binding sites in sets of co-regulated genes

    Directory of Open Access Journals (Sweden)

    den Dunnen Johan T

    2008-11-01

    Full Text Available Abstract Background The identification of transcription factor binding sites is difficult since they are only a small number of nucleotides in size, resulting in large numbers of false positives and false negatives in current approaches. Computational methods to reduce false positives are to look for over-representation of transcription factor binding sites in a set of similarly regulated promoters or to look for conservation in orthologous promoter alignments. Results We have developed a novel tool, "CORE_TF" (Conserved and Over-REpresented Transcription Factor binding sites that identifies common transcription factor binding sites in promoters of co-regulated genes. To improve upon existing binding site predictions, the tool searches for position weight matrices from the TRANSFACR database that are over-represented in an experimental set compared to a random set of promoters and identifies cross-species conservation of the predicted transcription factor binding sites. The algorithm has been evaluated with expression and chromatin-immunoprecipitation on microarray data. We also implement and demonstrate the importance of matching the random set of promoters to the experimental promoters by GC content, which is a unique feature of our tool. Conclusion The program CORE_TF is accessible in a user friendly web interface at http://www.LGTC.nl/CORE_TF. It provides a table of over-represented transcription factor binding sites in the users input genes' promoters and a graphical view of evolutionary conserved transcription factor binding sites. In our test data sets it successfully predicts target transcription factors and their binding sites.

  16. Promoter-proximal transcription factor binding is transcriptionally active when coupled with nucleosome repositioning in immediate vicinity

    Science.gov (United States)

    Yadav, Vinod Kumar; Thakur, Ram Krishna; Eckloff, Bruce; Baral, Aradhita; Singh, Ankita; Halder, Rashi; Kumar, Akinchan; Alam, Mohammad Parwez; Kundu, Tapas K.; Pandita, Raj; Pandita, Tej K.; Wieben, Eric D.; Chowdhury, Shantanu

    2014-01-01

    Previous studies have analyzed patterns of transcription, transcription factor (TF) binding or mapped nucleosome occupancy across the genome. These suggest that the three aspects are genetically connected but the cause and effect relationships are still unknown. For example, physiologic TF binding studies involve many TFs, consequently, it is difficult to assign nucleosome reorganization to the binding site occupancy of any particular TF. Therefore, several aspects remain unclear: does TF binding influence nucleosome (re)organizations locally or impact the chromatin landscape at a more global level; are all or only a fraction of TF binding a result of reorganization in nucleosome occupancy and do all TF binding and associated changes in nucleosome occupancy result in altered gene expression? With these in mind, following characterization of two states (before and after induction of a single TF of choice) we determined: (i) genomic binding sites of the TF, (ii) promoter nucleosome occupancy and (iii) transcriptome profiles. Results demonstrated that promoter-proximal TF binding influenced expression of the target gene when it was coupled to nucleosome repositioning at or close to its binding site in most cases. In contrast, only in few cases change in target gene expression was found when TF binding occurred without local nucleosome reorganization. PMID:25081206

  17. JASPAR 2010: the greatly expanded open-access database of transcription factor binding profiles

    DEFF Research Database (Denmark)

    Portales-Casamar, Elodie; Thongjuea, Supat; Kwon, Andrew T

    2009-01-01

    JASPAR (http://jaspar.genereg.net) is the leading open-access database of matrix profiles describing the DNA-binding patterns of transcription factors (TFs) and other proteins interacting with DNA in a sequence-specific manner. Its fourth major release is the largest expansion of the core database...

  18. HOCOMOCO: expansion and enhancement of the collection of transcription factor binding sites models

    KAUST Repository

    Kulakovskiy, Ivan V.

    2015-11-19

    Models of transcription factor (TF) binding sites provide a basis for a wide spectrum of studies in regulatory genomics, from reconstruction of regulatory networks to functional annotation of transcripts and sequence variants. While TFs may recognize different sequence patterns in different conditions, it is pragmatic to have a single generic model for each particular TF as a baseline for practical applications. Here we present the expanded and enhanced version of HOCOMOCO (http://hocomoco.autosome.ru and http://www.cbrc.kaust.edu.sa/hocomoco10), the collection of models of DNA patterns, recognized by transcription factors. HOCOMOCO now provides position weight matrix (PWM) models for binding sites of 601 human TFs and, in addition, PWMs for 396 mouse TFs. Furthermore, we introduce the largest up to date collection of dinucleotide PWM models for 86 (52) human (mouse) TFs. The update is based on the analysis of massive ChIP-Seq and HT-SELEX datasets, with the validation of the resulting models on in vivo data. To facilitate a practical application, all HOCOMOCO models are linked to gene and protein databases (Entrez Gene, HGNC, UniProt) and accompanied by precomputed score thresholds. Finally, we provide command-line tools for PWM and diPWM threshold estimation and motif finding in nucleotide sequences.

  19. Selective influence of Sox2 on POU transcription factor binding in embryonic and neural stem cells.

    Science.gov (United States)

    Mistri, Tapan Kumar; Devasia, Arun George; Chu, Lee Thean; Ng, Wei Ping; Halbritter, Florian; Colby, Douglas; Martynoga, Ben; Tomlinson, Simon R; Chambers, Ian; Robson, Paul; Wohland, Thorsten

    2015-09-01

    Embryonic stem cell (ESC) identity is orchestrated by co-operativity between the transcription factors (TFs) Sox2 and the class V POU-TF Oct4 at composite Sox/Oct motifs. Neural stem cells (NSCs) lack Oct4 but express Sox2 and class III POU-TFs Oct6, Brn1 and Brn2. This raises the question of how Sox2 interacts with POU-TFs to transcriptionally specify ESCs versus NSCs. Here, we show that Oct4 alone binds the Sox/Oct motif and the octamer-containing palindromic MORE equally well. Sox2 binding selectively increases the affinity of Oct4 for the Sox/Oct motif. In contrast, Oct6 binds preferentially to MORE and is unaffected by Sox2. ChIP-Seq in NSCs shows the MORE to be the most enriched motif for class III POU-TFs, including MORE subtypes, and that the Sox/Oct motif is not enriched. These results suggest that in NSCs, co-operativity between Sox2 and class III POU-TFs may not occur and that POU-TF-driven transcription uses predominantly the MORE cis architecture. Thus, distinct interactions between Sox2 and POU-TF subclasses distinguish pluripotent ESCs from multipotent NSCs, providing molecular insight into how Oct4 alone can convert NSCs to pluripotency.

  20. Transcription Factors Bind Thousands of Active and InactiveRegions in the Drosophila Blastoderm

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiao-Yong; MacArthur, Stewart; Bourgon, Richard; Nix, David; Pollard, Daniel A.; Iyer, Venky N.; Hechmer, Aaron; Simirenko, Lisa; Stapleton, Mark; Luengo Hendriks, Cris L.; Chu, Hou Cheng; Ogawa, Nobuo; Inwood, William; Sementchenko, Victor; Beaton, Amy; Weiszmann, Richard; Celniker, Susan E.; Knowles, David W.; Gingeras, Tom; Speed, Terence P.; Eisen, Michael B.; Biggin, Mark D.

    2008-01-10

    Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. Here, we use whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior-posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched in bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over forty well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs) transcribed in the blastoderm, as well as all major zygotically transcribed dorsal-ventral patterning genes, whose expression we show to be quantitatively modulated by anterior-posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly-bound regions are not involved in early

  1. Transcription factors bind thousands of active and inactive regions in the Drosophila blastoderm.

    Directory of Open Access Journals (Sweden)

    Xiao-yong Li

    2008-02-01

    Full Text Available Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. We used whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior-posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched in bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over 40 well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs transcribed in the blastoderm, as well as all major zygotically transcribed dorsal-ventral patterning genes, whose expression we show to be quantitatively modulated by anterior-posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly bound regions are not involved in

  2. Computational identification of transcription factor binding sites by functional analysis of sets of genes sharing overrep-resented upstream motifs

    Directory of Open Access Journals (Sweden)

    Silengo Lorenzo

    2004-05-01

    Full Text Available Abstract Background Transcriptional regulation is a key mechanism in the functioning of the cell, and is mostly effected through transcription factors binding to specific recognition motifs located upstream of the coding region of the regulated gene. The computational identification of such motifs is made easier by the fact that they often appear several times in the upstream region of the regulated genes, so that the number of occurrences of relevant motifs is often significantly larger than expected by pure chance. Results To exploit this fact, we construct sets of genes characterized by the statistical overrepresentation of a certain motif in their upstream regions. Then we study the functional characterization of these sets by analyzing their annotation to Gene Ontology terms. For the sets showing a statistically significant specific functional characterization, we conjecture that the upstream motif characterizing the set is a binding site for a transcription factor involved in the regulation of the genes in the set. Conclusions The method we propose is able to identify many known binding sites in S. cerevisiae and new candidate targets of regulation by known transcritpion factors. Its application to less well studied organisms is likely to be valuable in the exploration of their regulatory interaction network.

  3. Statistical Mechanics of Transcription-Factor Binding Site Discovery Using Hidden Markov Models.

    Science.gov (United States)

    Mehta, Pankaj; Schwab, David J; Sengupta, Anirvan M

    2011-04-01

    Hidden Markov Models (HMMs) are a commonly used tool for inference of transcription factor (TF) binding sites from DNA sequence data. We exploit the mathematical equivalence between HMMs for TF binding and the "inverse" statistical mechanics of hard rods in a one-dimensional disordered potential to investigate learning in HMMs. We derive analytic expressions for the Fisher information, a commonly employed measure of confidence in learned parameters, in the biologically relevant limit where the density of binding sites is low. We then use techniques from statistical mechanics to derive a scaling principle relating the specificity (binding energy) of a TF to the minimum amount of training data necessary to learn it.

  4. Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction

    Science.gov (United States)

    Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K.; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G.; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H.

    2017-01-01

    The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively. PMID:27899623

  5. HOCOMOCO: A comprehensive collection of human transcription factor binding sites models

    KAUST Repository

    Kulakovskiy, Ivan V.

    2012-11-21

    Transcription factor (TF) binding site (TFBS) models are crucial for computational reconstruction of transcription regulatory networks. In existing repositories, a TF often has several models (also called binding profiles or motifs), obtained from different experimental data. Having a single TFBS model for a TF is more pragmatic for practical applications. We show that integration of TFBS data from various types of experiments into a single model typically results in the improved model quality probably due to partial correction of source specific technique bias. We present the Homo sapiens comprehensive model collection (HOCOMOCO, http://autosome.ru/HOCOMOCO/, http://cbrc.kaust.edu.sa/ hocomoco/) containing carefully hand-curated TFBS models constructed by integration of binding sequences obtained by both low- and high-throughput methods. To construct position weight matrices to represent these TFBS models, we used ChIPMunk software in four computational modes, including newly developed periodic positional prior mode associated with DNA helix pitch. We selected only one TFBS model per TF, unless there was a clear experimental evidence for two rather distinct TFBS models. We assigned a quality rating to each model. HOCOMOCO contains 426 systematically curated TFBS models for 401 human TFs, where 172 models are based on more than one data source. The Author(s) 2012.

  6. Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction.

    Science.gov (United States)

    Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H

    2017-01-09

    The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively.

  7. Targeted protein footprinting: where different transcription factors bind to RNA polymerase.

    Science.gov (United States)

    Traviglia, S L; Datwyler, S A; Yan, D; Ishihama, A; Meares, C F

    1999-11-30

    Gene transcription is regulated through the interactions of RNA polymerase (RNAP) with transcription factors, such as the bacterial sigma proteins. We have devised a new strategy that relies on targeted protein footprinting to make an extensive survey of proximity to the protein surface. This involves attaching cutting reagents randomly to lysine residues on the surface of a protein such as sigma. The lysine-labeled sigma protein is then used to cleave the polypeptide backbones of the RNAP proteins at exposed residues adjacent to the sigma binding site. We used targeted protein footprinting to compare the areas near which sigma(70), sigma(54), sigma(38), sigma(E), NusA, GreA, and omega bind to the protein subunits of Escherichia coli RNAP. The sigma proteins and NusA cut sites in similar regions of the two large RNAP subunits, beta and beta', outlining a common surface. GreA cuts a larger set of sites, whereas omega shows no overlap with the others, cutting only the beta' subunit at a unique location.

  8. Using DNA duplex stability information for transcription factor binding site discovery.

    Science.gov (United States)

    Gordân, Raluca; Hartemink, Alexander J

    2008-01-01

    Transcription factor (TF) binding site discovery is an important step in understanding transcriptional regulation. Many computational tools have already been developed, but their success in detecting TF motifs is still limited. We believe one of the main reasons for the low accuracy of current methods is that they do not take into account the structural aspects of TF-DNA interaction. We have previously shown that knowledge about the structural class of the TF and information about nucleosome occupancy can be used to improve motif discovery. Here, we demonstrate the benefits of using information about the DNA double-helical stability for motif discovery. We notice that, in general, the energy needed to destabilize the DNA double helix is higher at TF binding sites than at random DNA sites. We use this information to derive informative positional priors that we incorporate into a motif finding algorithm. When applied to yeast ChIP-chip data, the new informative priors improve the performance of the motif finder significantly when compared to priors that do not use the energetic stability information.

  9. HOCOMOCO: a comprehensive collection of human transcription factor binding sites models

    Science.gov (United States)

    Kulakovskiy, Ivan V.; Medvedeva, Yulia A.; Schaefer, Ulf; Kasianov, Artem S.; Vorontsov, Ilya E.; Bajic, Vladimir B.; Makeev, Vsevolod J.

    2013-01-01

    Transcription factor (TF) binding site (TFBS) models are crucial for computational reconstruction of transcription regulatory networks. In existing repositories, a TF often has several models (also called binding profiles or motifs), obtained from different experimental data. Having a single TFBS model for a TF is more pragmatic for practical applications. We show that integration of TFBS data from various types of experiments into a single model typically results in the improved model quality probably due to partial correction of source specific technique bias. We present the Homo sapiens comprehensive model collection (HOCOMOCO, http://autosome.ru/HOCOMOCO/, http://cbrc.kaust.edu.sa/hocomoco/) containing carefully hand-curated TFBS models constructed by integration of binding sequences obtained by both low- and high-throughput methods. To construct position weight matrices to represent these TFBS models, we used ChIPMunk software in four computational modes, including newly developed periodic positional prior mode associated with DNA helix pitch. We selected only one TFBS model per TF, unless there was a clear experimental evidence for two rather distinct TFBS models. We assigned a quality rating to each model. HOCOMOCO contains 426 systematically curated TFBS models for 401 human TFs, where 172 models are based on more than one data source. PMID:23175603

  10. Hybrid incompatibility arises in a sequence-based bioenergetic model of transcription factor binding.

    Science.gov (United States)

    Tulchinsky, Alexander Y; Johnson, Norman A; Watt, Ward B; Porter, Adam H

    2014-11-01

    Postzygotic isolation between incipient species results from the accumulation of incompatibilities that arise as a consequence of genetic divergence. When phenotypes are determined by regulatory interactions, hybrid incompatibility can evolve even as a consequence of parallel adaptation in parental populations because interacting genes can produce the same phenotype through incompatible allelic combinations. We explore the evolutionary conditions that promote and constrain hybrid incompatibility in regulatory networks using a bioenergetic model (combining thermodynamics and kinetics) of transcriptional regulation, considering the bioenergetic basis of molecular interactions between transcription factors (TFs) and their binding sites. The bioenergetic parameters consider the free energy of formation of the bond between the TF and its binding site and the availability of TFs in the intracellular environment. Together these determine fractional occupancy of the TF on the promoter site, the degree of subsequent gene expression and in diploids, and the degree of dominance among allelic interactions. This results in a sigmoid genotype-phenotype map and fitness landscape, with the details of the shape determining the degree of bioenergetic evolutionary constraint on hybrid incompatibility. Using individual-based simulations, we subjected two allopatric populations to parallel directional or stabilizing selection. Misregulation of hybrid gene expression occurred under either type of selection, although it evolved faster under directional selection. Under directional selection, the extent of hybrid incompatibility increased with the slope of the genotype-phenotype map near the derived parental expression level. Under stabilizing selection, hybrid incompatibility arose from compensatory mutations and was greater when the bioenergetic properties of the interaction caused the space of nearly neutral genotypes around the stable expression level to be wide. F2's showed higher

  11. Nonlinearity arising from noncooperative transcription factor binding enhances negative feedback and promotes genetic oscillations

    CERN Document Server

    Lengyel, Iván M; Oates, Andrew C; Morelli, Luis G

    2015-01-01

    We study the effects of multiple binding sites in the promoter of a genetic oscillator. We evaluate the regulatory function of a promoter with multiple binding sites in the absence of cooperative binding, and consider different hypotheses for how the number of bound repressors affects transcription rate. Effective Hill exponents of the resulting regulatory functions reveal an increase in the nonlinearity of the feedback with the number of binding sites. We identify optimal configurations that maximize the nonlinearity of the feedback. We use a generic model of a biochemical oscillator to show that this increased nonlinearity is reflected in enhanced oscillations, with larger amplitudes over wider oscillatory ranges. Although the study is motivated by genetic oscillations in the zebrafish segmentation clock, our findings may reveal a general principle for gene regulation.

  12. Cell-type specificity of ChIP-predicted transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Håndstad Tony

    2012-08-01

    Full Text Available Abstract Background Context-dependent transcription factor (TF binding is one reason for differences in gene expression patterns between different cellular states. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identifies genome-wide TF binding sites for one particular context—the cells used in the experiment. But can such ChIP-seq data predict TF binding in other cellular contexts and is it possible to distinguish context-dependent from ubiquitous TF binding? Results We compared ChIP-seq data on TF binding for multiple TFs in two different cell types and found that on average only a third of ChIP-seq peak regions are common to both cell types. Expectedly, common peaks occur more frequently in certain genomic contexts, such as CpG-rich promoters, whereas chromatin differences characterize cell-type specific TF binding. We also find, however, that genotype differences between the cell types can explain differences in binding. Moreover, ChIP-seq signal intensity and peak clustering are the strongest predictors of common peaks. Compared with strong peaks located in regions containing peaks for multiple transcription factors, weak and isolated peaks are less common between the cell types and are less associated with data that indicate regulatory activity. Conclusions Together, the results suggest that experimental noise is prevalent among weak peaks, whereas strong and clustered peaks represent high-confidence binding events that often occur in other cellular contexts. Nevertheless, 30-40% of the strongest and most clustered peaks show context-dependent regulation. We show that by combining signal intensity with additional data—ranging from context independent information such as binding site conservation and position weight matrix scores to context dependent chromatin structure—we can predict whether a ChIP-seq peak is likely to be present in other cellular contexts.

  13. Spatial distribution of predicted transcription factor binding sites in Drosophila ChIP peaks.

    Science.gov (United States)

    Pettie, Kade P; Dresch, Jacqueline M; Drewell, Robert A

    2016-08-01

    In the development of the Drosophila embryo, gene expression is directed by the sequence-specific interactions of a large network of protein transcription factors (TFs) and DNA cis-regulatory binding sites. Once the identity of the typically 8-10bp binding sites for any given TF has been determined by one of several experimental procedures, the sequences can be represented in a position weight matrix (PWM) and used to predict the location of additional TF binding sites elsewhere in the genome. Often, alignments of large (>200bp) genomic fragments that have been experimentally determined to bind the TF of interest in Chromatin Immunoprecipitation (ChIP) studies are trimmed under the assumption that the majority of the binding sites are located near the center of all the aligned fragments. In this study, ChIP/chip datasets are analyzed using the corresponding PWMs for the well-studied TFs; CAUDAL, HUNCHBACK, KNIRPS and KRUPPEL, to determine the distribution of predicted binding sites. All four TFs are critical regulators of gene expression along the anterio-posterior axis in early Drosophila development. For all four TFs, the ChIP peaks contain multiple binding sites that are broadly distributed across the genomic region represented by the peak, regardless of the prediction stringency criteria used. This result suggests that ChIP peak trimming may exclude functional binding sites from subsequent analyses.

  14. Systematic dissection of genomic features determining transcription factor binding and enhancer function

    Science.gov (United States)

    Grossman, Sharon R.; Zhang, Xiaolan; Wang, Li; Engreitz, Jesse; Melnikov, Alexandre; Rogov, Peter; Tewhey, Ryan; Isakova, Alina; Deplancke, Bart; Bernstein, Bradley E.; Mikkelsen, Tarjei S.; Lander, Eric S.

    2017-01-01

    Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function—including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation. PMID:28137873

  15. Probabilistic Inference on Multiple Normalized Signal Profiles from Next Generation Sequencing: Transcription Factor Binding Sites

    KAUST Repository

    Wong, Ka-Chun

    2015-04-20

    With the prevalence of chromatin immunoprecipitation (ChIP) with sequencing (ChIP-Seq) technology, massive ChIP-Seq data has been accumulated. The ChIP-Seq technology measures the genome-wide occupancy of DNA-binding proteins in vivo. It is well-known that different DNA-binding protein occupancies may result in a gene being regulated in different conditions (e.g. different cell types). To fully understand a gene\\'s function, it is essential to develop probabilistic models on multiple ChIP-Seq profiles for deciphering the gene transcription causalities. In this work, we propose and describe two probabilistic models. Assuming the conditional independence of different DNA-binding proteins\\' occupancies, the first method (SignalRanker) is developed as an intuitive method for ChIP-Seq genome-wide signal profile inference. Unfortunately, such an assumption may not always hold in some gene regulation cases. Thus, we propose and describe another method (FullSignalRanker) which does not make the conditional independence assumption. The proposed methods are compared with other existing methods on ENCODE ChIP-Seq datasets, demonstrating its regression and classification ability. The results suggest that FullSignalRanker is the best-performing method for recovering the signal ranks on the promoter and enhancer regions. In addition, FullSignalRanker is also the best-performing method for peak sequence classification. We envision that SignalRanker and FullSignalRanker will become important in the era of next generation sequencing. FullSignalRanker program is available on the following website: http://www.cs.toronto.edu/∼wkc/FullSignalRanker/ © 2015 IEEE.

  16. The Role of Genome Accessibility in Transcription Factor Binding in Bacteria.

    Directory of Open Access Journals (Sweden)

    Antonio L C Gomes

    2016-04-01

    Full Text Available ChIP-seq enables genome-scale identification of regulatory regions that govern gene expression. However, the biological insights generated from ChIP-seq analysis have been limited to predictions of binding sites and cooperative interactions. Furthermore, ChIP-seq data often poorly correlate with in vitro measurements or predicted motifs, highlighting that binding affinity alone is insufficient to explain transcription factor (TF-binding in vivo. One possibility is that binding sites are not equally accessible across the genome. A more comprehensive biophysical representation of TF-binding is required to improve our ability to understand, predict, and alter gene expression. Here, we show that genome accessibility is a key parameter that impacts TF-binding in bacteria. We developed a thermodynamic model that parameterizes ChIP-seq coverage in terms of genome accessibility and binding affinity. The role of genome accessibility is validated using a large-scale ChIP-seq dataset of the M. tuberculosis regulatory network. We find that accounting for genome accessibility led to a model that explains 63% of the ChIP-seq profile variance, while a model based in motif score alone explains only 35% of the variance. Moreover, our framework enables de novo ChIP-seq peak prediction and is useful for inferring TF-binding peaks in new experimental conditions by reducing the need for additional experiments. We observe that the genome is more accessible in intergenic regions, and that increased accessibility is positively correlated with gene expression and anti-correlated with distance to the origin of replication. Our biophysically motivated model provides a more comprehensive description of TF-binding in vivo from first principles towards a better representation of gene regulation in silico, with promising applications in systems biology.

  17. Different papillomaviruses have different repertoires of transcription factor binding sites: convergence and divergence in the upstream regulatory region

    Directory of Open Access Journals (Sweden)

    Alonso Ángel

    2006-03-01

    Full Text Available Abstract Background Papillomaviruses (PVs infect stratified squamous epithelia in warm-blooded vertebrates and have undergone a complex evolutionary process. The control of the expression of the early ORFs in PVs depends on the binding of cellular and viral transcription factors to the upstream regulatory region (URR of the virus. It is believed that there is a core of transcription factor binding sites (TFBS common to all PVs, with additional individual differences, although most of the available information focuses only on a handful of viruses. Results We have studied the URR of sixty-one PVs, covering twenty different hosts. We have predicted the TFBS present in the URR and analysed these results by principal component analysis and genetic algorithms. The number and nature of TFBS in the URR might be much broader than thus far described, and different PVs have different repertoires of TFBS. Conclusion There are common fingerprints in the URR in PVs that infect primates, although the ancestors of these viruses diverged a long time ago. Additionally, there are obvious differences between the URR of alpha and beta PVs, despite these PVs infect similar histological cell types in the same host, i.e. human. A thorough analysis of the TFBS in the URR might provide crucial information about the differential biology of cancer-associated PVs.

  18. Pipeline for Efficient Mapping of Transcription Factor Binding Sites and Comparison of Their Models

    KAUST Repository

    Ba alawi, Wail

    2011-06-01

    The control of genes in every living organism is based on activities of transcription factor (TF) proteins. These TFs interact with DNA by binding to the TF binding sites (TFBSs) and in that way create conditions for the genes to activate. Of the approximately 1500 TFs in human, TFBSs are experimentally derived only for less than 300 TFs and only in generally limited portions of the genome. To be able to associate TF to genes they control we need to know if TFs will have a potential to interact with the control region of the gene. For this we need to have models of TFBS families. The existing models are not sufficiently accurate or they are too complex for use by ordinary biologists. To remove some of the deficiencies of these models, in this study we developed a pipeline through which we achieved the following: 1. Through a comparison analysis of the performance we identified the best models with optimized thresholds among the four different types of models of TFBS families. 2. Using the best models we mapped TFBSs to the human genome in an efficient way. The study shows that a new scoring function used with TFBS models based on the position weight matrix of dinucleotides with remote dependency results in better accuracy than the other three types of the TFBS models. The speed of mapping has been improved by developing a parallelized code and shows a significant speed up of 4x when going from 1 CPU to 8 CPUs. To verify if the predicted TFBSs are more accurate than what can be expected with the conventional models, we identified the most frequent pairs of TFBSs (for TFs E4F1 and ATF6) that appeared close to each other (within the distance of 200 nucleotides) over the human genome. We show unexpectedly that the genes that are most close to the multiple pairs of E4F1/ATF6 binding sites have a co-expression of over 90%. This indirectly supports our hypothesis that the TFBS models we use are more accurate and also suggests that the E4F1/ATF6 pair is exerting the

  19. Stratifying melanoma and breast cancer TCGA datasets on the basis of the CNV of transcription factor binding sites common to proliferation- and apoptosis-effector genes.

    Science.gov (United States)

    Mauro, James A; Yavorski, John M; Blanck, George

    2017-02-28

    Transcription factors that activate both proliferation- and apoptosis-effector genes, along with a number of related observations, have led to a proposal for a feed forward mechanism of activating the two gene classes, whereby a certain concentration of a transcription factor activates the proliferation-effector genes and a higher concentration of the transcription factor activates the apoptosis-effector genes. We reasoned that this paradigm of regulation could lead to, in the cancer setting, a selection for relatively reduced copy numbers of apoptosis-effector gene, transcription factor binding sites (TFBS). Thus, the aim of this investigation was to examine the DNA sequencing read depths of TFBS for a set of proliferation- and apoptosis-effector genes, normalized to the read depths found in matching blood samples, as provided by the cancer genome atlas (TCGA); and thereby document copy number differences among these TFBS. We determined that the melanoma and breast cancer, TCGA datasets could be divided into three categories: (i) no detectable copy number variation for the proliferation- and apoptosis-effector, shared TFBS; (ii) a relative increase in the copy number of proliferation-effector gene TFBS, compared with the copy number of the apoptosis-effector gene TFBS; and (iii) a relative decrease in the number of proliferation-effector gene TFBS. Thus, we conclude that changes in the relative copies of the shared TFBS, for proliferation- and apoptosis-effector genes, have the potential of impacting tumor cell proliferative and apoptotic capacities.

  20. The thumb subdomain of yeast mitochondrial RNA polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding.

    Science.gov (United States)

    Velazquez, Gilberto; Sousa, Rui; Brieba, Luis G

    2015-01-01

    Single subunit RNA polymerases have evolved 2 mechanisms to synthesize long transcripts without falling off a DNA template: binding of nascent RNA and interactions with an RNA:DNA hybrid. Mitochondrial RNA polymerases share a common ancestor with T-odd bacteriophage single subunit RNA polymerases. Herein we characterized the role of the thumb subdomain of the yeast mtRNA polymerase gene (RPO41) in complex stability, processivity, and fidelity. We found that deletion and point mutants of the thumb subdomain of yeast mtRNA polymerase increase the synthesis of abortive transcripts and the probability that the polymerase will disengage from the template during the formation of the late initial transcription and elongation complexes. Mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric template and, unexpectedly, thumb subdomain deletions decrease the binding affinity for mitochondrial transcription factor (Mtf1). The latter suggests that the thumb subdomain is part of an extended binding surface area involved in binding Mtf1.

  1. Does positive selection drive transcription factor binding site turnover? A test with Drosophila cis-regulatory modules.

    Directory of Open Access Journals (Sweden)

    Bin Z He

    2011-04-01

    Full Text Available Transcription factor binding site(s (TFBS gain and loss (i.e., turnover is a well-documented feature of cis-regulatory module (CRM evolution, yet little attention has been paid to the evolutionary force(s driving this turnover process. The predominant view, motivated by its widespread occurrence, emphasizes the importance of compensatory mutation and genetic drift. Positive selection, in contrast, although it has been invoked in specific instances of adaptive gene expression evolution, has not been considered as a general alternative to neutral compensatory evolution. In this study we evaluate the two hypotheses by analyzing patterns of single nucleotide polymorphism in the TFBS of well-characterized CRM in two closely related Drosophila species, Drosophila melanogaster and Drosophila simulans. An important feature of the analysis is classification of TFBS mutations according to the direction of their predicted effect on binding affinity, which allows gains and losses to be evaluated independently along the two phylogenetic lineages. The observed patterns of polymorphism and divergence are not compatible with neutral evolution for either class of mutations. Instead, multiple lines of evidence are consistent with contributions of positive selection to TFBS gain and loss as well as purifying selection in its maintenance. In discussion, we propose a model to reconcile the finding of selection driving TFBS turnover with constrained CRM function over long evolutionary time.

  2. MicroRNA genes preferentially expressed in dendritic cells contain sites for conserved transcription factor binding motifs in their promoters

    Directory of Open Access Journals (Sweden)

    Huynen Martijn A

    2011-06-01

    Full Text Available Abstract Background MicroRNAs (miRNAs play a fundamental role in the regulation of gene expression by translational repression or target mRNA degradation. Regulatory elements in miRNA promoters are less well studied, but may reveal a link between their expression and a specific cell type. Results To explore this link in myeloid cells, miRNA expression profiles were generated from monocytes and dendritic cells (DCs. Differences in miRNA expression among monocytes, DCs and their stimulated progeny were observed. Furthermore, putative promoter regions of miRNAs that are significantly up-regulated in DCs were screened for Transcription Factor Binding Sites (TFBSs based on TFBS motif matching score, the degree to which those TFBSs are over-represented in the promoters of the up-regulated miRNAs, and the extent of conservation of the TFBSs in mammals. Conclusions Analysis of evolutionarily conserved TFBSs in DC promoters revealed preferential clustering of sites within 500 bp upstream of the precursor miRNAs and that many mRNAs of cognate TFs of the conserved TFBSs were indeed expressed in the DCs. Taken together, our data provide evidence that selected miRNAs expressed in DCs have evolutionarily conserved TFBSs relevant to DC biology in their promoters.

  3. Wide-scale analysis of human functional transcription factor binding reveals a strong bias towards the transcription start site.

    Directory of Open Access Journals (Sweden)

    Yuval Tabach

    Full Text Available BACKGROUND: Transcription factors (TF regulate expression by binding to specific DNA sequences. A binding event is functional when it affects gene expression. Functionality of a binding site is reflected in conservation of the binding sequence during evolution and in over represented binding in gene groups with coherent biological functions. Functionality is governed by several parameters such as the TF-DNA binding strength, distance of the binding site from the transcription start site (TSS, DNA packing, and more. Understanding how these parameters control functionality of different TFs in different biological contexts is a must for identifying functional TF binding sites and for understanding regulation of transcription. METHODOLOGY/PRINCIPAL FINDINGS: We introduce a novel method to screen the promoters of a set of genes with shared biological function (obtained from the functional Gene Ontology (GO classification against a precompiled library of motifs, and find those motifs which are statistically over-represented in the gene set. More than 8,000 human (and 23,000 mouse genes, were assigned to one of 134 GO sets. Their promoters were searched (from 200 bp downstream to 1,000 bp upstream the TSS for 414 known DNA motifs. We optimized the sequence similarity score threshold, independently for every location window, taking into account nucleotide heterogeneity along the promoters of the target genes. The method, combined with binding sequence and location conservation between human and mouse, identifies with high probability functional binding sites for groups of functionally-related genes. We found many location-sensitive functional binding events and showed that they clustered close to the TSS. Our method and findings were tested experimentally. CONCLUSIONS/SIGNIFICANCE: We identified reliably functional TF binding sites. This is an essential step towards constructing regulatory networks. The promoter region proximal to the TSS is of central

  4. Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli.

    Directory of Open Access Journals (Sweden)

    Alfredo Mendoza-Vargas

    Full Text Available Despite almost 40 years of molecular genetics research in Escherichia coli a major fraction of its Transcription Start Sites (TSSs are still unknown, limiting therefore our understanding of the regulatory circuits that control gene expression in this model organism. RegulonDB (http://regulondb.ccg.unam.mx/ is aimed at integrating the genetic regulatory network of E. coli K12 as an entirely bioinformatic project up till now. In this work, we extended its aims by generating experimental data at a genome scale on TSSs, promoters and regulatory regions. We implemented a modified 5' RACE protocol and an unbiased High Throughput Pyrosequencing Strategy (HTPS that allowed us to map more than 1700 TSSs with high precision. From this collection, about 230 corresponded to previously reported TSSs, which helped us to benchmark both our methodologies and the accuracy of the previous mapping experiments. The other ca 1500 TSSs mapped belong to about 1000 different genes, many of them with no assigned function. We identified promoter sequences and type of sigma factors that control the expression of about 80% of these genes. As expected, the housekeeping sigma(70 was the most common type of promoter, followed by sigma(38. The majority of the putative TSSs were located between 20 to 40 nucleotides from the translational start site. Putative regulatory binding sites for transcription factors were detected upstream of many TSSs. For a few transcripts, riboswitches and small RNAs were found. Several genes also had additional TSSs within the coding region. Unexpectedly, the HTPS experiments revealed extensive antisense transcription, probably for regulatory functions. The new information in RegulonDB, now with more than 2400 experimentally determined TSSs, strengthens the accuracy of promoter prediction, operon structure, and regulatory networks and provides valuable new information that will facilitate the understanding from a global perspective the complex and

  5. Bioinformatics Identification of Modules of Transcription Factor Binding Sites in Alzheimer's Disease-Related Genes by In Silico Promoter Analysis and Microarrays

    Directory of Open Access Journals (Sweden)

    Regina Augustin

    2011-01-01

    Full Text Available The molecular mechanisms and genetic risk factors underlying Alzheimer's disease (AD pathogenesis are only partly understood. To identify new factors, which may contribute to AD, different approaches are taken including proteomics, genetics, and functional genomics. Here, we used a bioinformatics approach and found that distinct AD-related genes share modules of transcription factor binding sites, suggesting a transcriptional coregulation. To detect additional coregulated genes, which may potentially contribute to AD, we established a new bioinformatics workflow with known multivariate methods like support vector machines, biclustering, and predicted transcription factor binding site modules by using in silico analysis and over 400 expression arrays from human and mouse. Two significant modules are composed of three transcription factor families: CTCF, SP1F, and EGRF/ZBPF, which are conserved between human and mouse APP promoter sequences. The specific combination of in silico promoter and multivariate analysis can identify regulation mechanisms of genes involved in multifactorial diseases.

  6. Distinct patterns of epigenetic marks and transcription factor binding sites across promoters of sense-intronic long noncoding RNAs

    Indian Academy of Sciences (India)

    Saurav Ghosh; Satish Sati; Shantanu Sengupta; Vinod Scaria

    2015-03-01

    Long noncoding RNAs (lncRNAs) are a new class of noncoding RNAs that have been extensively studied in the recent past as a regulator of gene expression, including modulation of epigenetic regulation. The lncRNAs class encompasses a number of subclasses, classified based on their genomic loci and relation to protein-coding genes. Functional differences between subclasses have been increasingly studied in the recent years, though the regulation of expression and biogenesis of lncRNAs have been poorly studied. The availability of genome-scale datasets of epigenetic marks has motivated us to understand the patterns and processes of epigenetic regulation of lncRNAs. Here we analysed the occurrence of expressive and repressive histone marks at the transcription start site (TSS) of lncRNAs and their subclasses, and compared these profiles with that of the protein-coding regions. We observe distinct differences in the density of histone marks across the TSS of a few lncRNA subclasses. The sense-intronic lncRNA subclass showed a paucity for mapped histone marks across the TSS which were significantly different than all the lncRNAs and protein-coding genes in most cases. Similar pattern was also observed for the density of transcription factor binding sites (TFBS). These observations were generally consistent across cell and tissue types. The differences in density across the promoter were significantly associated with the expression level of the genes, but the differences between the densities across long noncoding and protein-coding gene promoters were consistent irrespective of the expression levels. Apart from suggesting general differences in epigenetic regulatory marks across long noncoding RNA promoters, our analysis suggests a possible alternative mechanism of regulation and/or biogenesis of sense-intronic lncRNAs.

  7. Data in support of FSH induction of IRS-2 in human granulosa cells: Mapping the transcription factor binding sites in human IRS-2 promoter

    OpenAIRE

    Surleen Kaur; Anjali, G.; Priya Bhardwaj; Jyoti Taneja; Rita Singh

    2015-01-01

    Insulin receptor substrate-2 (IRS-2) plays critical role in the regulation of various metabolic processes by insulin and IGF-1. The defects in its expression and/or function are linked to diseases like polycystic ovary syndrome (PCOS), insulin resistance and cancer. To predict the transcription factors (TFs) responsible for the regulation of human IRS-2 gene expression, the transcription factor binding sites (TFBS) and the corresponding TFs were investigated by analysis of IRS-2 promoter sequ...

  8. Methylene Blue Attenuates iNOS Induction Through Suppression of Transcriptional Factor Binding Amid iNOS mRNA Transcription.

    Science.gov (United States)

    Huang, Chao; Tong, Lijuan; Lu, Xu; Wang, Jia; Yao, Wenjuan; Jiang, Bo; Zhang, Wei

    2015-08-01

    Inducible nitric oxide synthase (iNOS) critically contributes to the development of endotoxin-mediated inflammation. It can be induced by cytokines or endotoxins via distinct signaling pathways. Lipopolysaccharide (LPS) triggers iNOS expression through activation of the inhibitor of κB-α (IκB-α)-nuclear factor κB (NF-κB) cascade, whereas interferon-γ (IFN-γ) acts primarily through Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1). Methylene blue (MB), an agent used clinically to treat numerous ailments, has been shown to reduce NO accumulation through suppression of iNOS activity. But it remains unclear whether MB affects iNOS induction. This knowledge gap is addressed in the present study using cultured cells and endotoxemic mice. With mouse macrophages, MB treatment prevented the LPS- and/or IFN-γ-stimulated iNOS protein expression. Real-time PCR experiments showed that iNOS mRNA transcription was robustly blocked by MB treatment. The inhibitory effect of MB on iNOS expression was confirmed in vivo in endotoxemic mice. Further analysis showed that MB had no significant effect on IκB-α degradation and NF-κB or STAT1 phosphorylation in LPS/IFN-γ-stimulated cells. The nuclear transport of active NF-κB or STAT1 was also not affected by MB treatment. But MB treatment markedly reduced the binding of NF-κB and STAT1 to their DNA elements. Chromatin immunoprecipitation assays confirmed that MB reduced NF-κB and STAT1 bindings to iNOS promoter inside the cell. These studies show that MB attenuates transcriptional factor binding amid iNOS mRNA transcription, providing further insight into the molecular mechanism of MB in disease therapy.

  9. A novel HMM-based method for detecting enriched transcription factor binding sites reveals RUNX3 as a potential target in pancreatic cancer biology.

    Directory of Open Access Journals (Sweden)

    Liron Levkovitz

    Full Text Available BACKGROUND: Pancreatic adenocarcinoma (PAC is one of the most intractable malignancies. In order to search for potential new therapeutic targets, we relied on computational methods aimed at identifying transcription factor binding sites (TFBSs over-represented in the promoter regions of genes differentially expressed in PAC. Though many computational methods have been implemented to accomplish this, none has gained overall acceptance or produced proven novel targets in PAC. To this end we have developed DEMON, a novel method for motif detection. METHODOLOGY: DEMON relies on a hidden Markov model to score the appearance of sequence motifs, taking into account all potential sites in a promoter of potentially varying binding affinities. We demonstrate DEMON's accuracy on simulated and real data sets. Applying DEMON to PAC-related data sets identifies the RUNX family as highly enriched in PAC-related genes. Using a novel experimental paradigm to distinguish between normal and PAC cells, we find that RUNX3 mRNA (but not RUNX1 or RUNX2 mRNAs exhibits time-dependent increases in normal but not in PAC cells. These increases are accompanied by changes in mRNA levels of putative RUNX gene targets. CONCLUSIONS: The integrated application of DEMON and a novel differentiation system led to the identification of a single family member, RUNX3, which together with four of its putative targets showed a robust response to a differentiation stimulus in healthy cells, whereas this regulatory mechanism was absent in PAC cells, emphasizing RUNX3 as a promising target for further studies.

  10. Nutritional status and growth hormone regulate insulin-like growth factor binding protein (igfbp) transcripts in Mozambique tilapia.

    Science.gov (United States)

    Breves, Jason P; Tipsmark, Christian K; Stough, Beth A; Seale, Andre P; Flack, Brenda R; Moorman, Benjamin P; Lerner, Darren T; Grau, E Gordon

    2014-10-01

    Growth in teleosts is controlled in large part by the activities of the growth hormone (Gh)/insulin-like growth factor (Igf) system. In this study, we initially identified igf-binding protein (bp)1b, -2b, -4, -5a and -6b transcripts in a tilapia EST library. In Mozambique tilapia (Oreochromis mossambicus), tissue expression profiling of igfbps revealed that igfbp1b and -2b had the highest levels of expression in liver while igfbp4, -5a and -6b were expressed at comparable levels in most other tissues. We compared changes in hepatic igfbp1b, -2b and -5a expression during catabolic conditions (28days of fasting) along with key components of the Gh/Igf system, including plasma Gh and Igf1 and hepatic gh receptor (ghr2), igf1 and igf2 expression. In parallel with elevated plasma Gh and decreased Igf1 levels, we found that hepatic igfbp1b increased substantially in fasted animals. We then tested whether systemic Gh could direct the expression of igfbps in liver. A single intraperitoneal injection of ovine Gh into hypophysectomized tilapia specifically stimulated liver igfbp2b expression along with plasma Igf1 and hepatic ghr2 levels. Our collective data suggest that hepatic endocrine signaling during fasting may involve post-translational regulation of plasma Igf1 via a shift towards the expression of igfbp1b. Thus, Igfbp1b may operate as a molecular switch to restrict Igf1 signaling in tilapia; furthermore, we provide new details regarding isoform-specific regulation of igfbp expression by Gh.

  11. Transcription factors binding to the cis-elements of GADD45 gene in ML-1 cells after low-dose irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Daino, Kazuhiro [Chiba Univ., Faculty of Science, Chiba (Japan); Ichimura, Sachiko; Nenoi, Mitsuru [National Inst. of Radiological Sciences, Chiba (Japan)

    2003-07-01

    Several stress-responsive genes, including p53-target genes, are induced by low-dose of ionizing radiation ranging from 0.02 to 0.5 Gy. We show here that 0.5 Gy of X-rays increase the transcription rate of the GADD45 gene, with maximum induction at 0.5 to 1 h after irradiation, much earlier than the maximum accumulation of stabilized p53 protein. It could be suggested that some transcription factors cooperate with p53 in regulating the GADD45 gene at an early time after low-dose irradiation. This idea is supported by the studies that showed GKLF and Spl are required for p53-dependent transcriptional activation of the p21{sup WAFI/Cipl} and BAX genes, respectively. To examine the possible involvement of cooperating transcription factors in regulation of the GADD45 gene after low-dose radiation, we attempted a comprehensive EMSA, in which 136 species of double-stranded DNA probes were used to identify X-ray-inducible factor-bindings to the upstream and the third intron regions of the gene after exposure to 0.5 Gy of X-rays in human myeloblastic leukemia ML-1 cells. Several X-ray-inducible DNA-protein complexes were observed. The factors related to forkhead transcription factors, POU domain transcription factors and kruppel-like factors were putatively identified by the competition assay. It is possible that these factors cooperate with p53 to mediate the transcriptional regulation of the GADD45 genes after low-dose irradiation. (author)

  12. Genome-wide profiling of transcription factor binding and epigenetic marks in adipocytes by ChIP-seq

    DEFF Research Database (Denmark)

    Nielsen, Ronni; Mandrup, Susanne

    2014-01-01

    The recent advances in high-throughput sequencing combined with various other technologies have allowed detailed and genome-wide insight into the transcriptional networks that control adipogenesis. Chromatin immunoprecipitation (ChIP) combined with high-throughput sequencing (ChIP-seq) is one...

  13. Genome-scale study of the importance of binding site context for transcription factor binding and gene regulation

    Directory of Open Access Journals (Sweden)

    Ronne Hans

    2008-11-01

    Full Text Available Abstract Background The rate of mRNA transcription is controlled by transcription factors that bind to specific DNA motifs in promoter regions upstream of protein coding genes. Recent results indicate that not only the presence of a motif but also motif context (for example the orientation of a motif or its location relative to the coding sequence is important for gene regulation. Results In this study we present ContextFinder, a tool that is specifically aimed at identifying cases where motif context is likely to affect gene regulation. We used ContextFinder to examine the role of motif context in S. cerevisiae both for DNA binding by transcription factors and for effects on gene expression. For DNA binding we found significant patterns of motif location bias, whereas motif orientations did not seem to matter. Motif context appears to affect gene expression even more than it affects DNA binding, as biases in both motif location and orientation were more frequent in promoters of co-expressed genes. We validated our results against data on nucleosome positioning, and found a negative correlation between preferred motif locations and nucleosome occupancy. Conclusion We conclude that the requirement for stable binding of transcription factors to DNA and their subsequent function in gene regulation can impose constraints on motif context.

  14. CONREAL : conserved regulatory elements anchored alignment algorithm for identification of transcription factor binding sites by phylogenetic footprinting

    NARCIS (Netherlands)

    Berezikov, Eugene; Guryev, Victor; Plasterk, Ronald H A; Cuppen, Edwin

    2004-01-01

    Prediction of transcription-factor target sites in promoters remains difficult due to the short length and degeneracy of the target sequences. Although the use of orthologous sequences and phylogenetic footprinting approaches may help in the recognition of conserved and potentially functional sequen

  15. JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update

    DEFF Research Database (Denmark)

    Bryne, J.C.; Valen, E.; Tang, M.H.E.

    2008-01-01

    JASPAR is a popular open-access database for matrix models describing DNA-binding preferences for transcription factors and other DNA patterns. With its third major release, JASPAR has been expanded and equipped with additional functions aimed at both casual and power users. The heart of the JASPAR...

  16. DNase I-hypersensitive sites and transcription factor-binding motifs within the mouse E beta meiotic recombination hot spot.

    Science.gov (United States)

    Shenkar, R; Shen, M H; Arnheim, N

    1991-04-01

    The second intron of the E beta gene in the mouse major histocompatibility complex is the site of a meiotic recombination hot spot. We detected two DNase I-hypersensitive sites in this intron in meiotic cells isolated from mouse testes. One site appears to be constitutive and is found in other tissues regardless of whether or not they express the E beta gene. Near this hypersensitive site are potential binding motifs for H2TF1/KBF1, NF kappa B, and octamer transcription factors. Gel retardation studies with mouse lymphoma cell nuclear extracts confirmed that each of these motifs is capable of binding protein. The binding of transcription factors may contribute to the enhancement of recombination potential by altering chromatin structure and increasing the accessibility of the DNA to the recombination machinery.

  17. Role of ChIP-seq in the discovery of transcription factor binding sites, differential gene regulation mechanism, epigenetic marks and beyond.

    Science.gov (United States)

    Mundade, Rasika; Ozer, Hatice Gulcin; Wei, Han; Prabhu, Lakshmi; Lu, Tao

    2014-01-01

    Many biologically significant processes, such as cell differentiation and cell cycle progression, gene transcription and DNA replication, chromosome stability and epigenetic silencing etc. depend on the crucial interactions between cellular proteins and DNA. Chromatin immunoprecipitation (ChIP) is an important experimental technique for studying interactions between specific proteins and DNA in the cell and determining their localization on a specific genomic locus. In recent years, the combination of ChIP with second generation DNA-sequencing technology (ChIP-seq) allows precise genomic functional assay. This review addresses the important applications of ChIP-seq with an emphasis on its role in genome-wide mapping of transcription factor binding sites, the revelation of underlying molecular mechanisms of differential gene regulation that are governed by specific transcription factors, and the identification of epigenetic marks. Furthermore, we also describe the ChIP-seq data analysis workflow and a perspective for the exciting potential advancement of ChIP-seq technology in the future.

  18. In Silico Analysis of Gene Expression Network Components Underlying Pigmentation Phenotypes in the Python Identified Evolutionarily Conserved Clusters of Transcription Factor Binding Sites

    Science.gov (United States)

    2016-01-01

    Color variation provides the opportunity to investigate the genetic basis of evolution and selection. Reptiles are less studied than mammals. Comparative genomics approaches allow for knowledge gained in one species to be leveraged for use in another species. We describe a comparative vertebrate analysis of conserved regulatory modules in pythons aimed at assessing bioinformatics evidence that transcription factors important in mammalian pigmentation phenotypes may also be important in python pigmentation phenotypes. We identified 23 python orthologs of mammalian genes associated with variation in coat color phenotypes for which we assessed the extent of pairwise protein sequence identity between pythons and mouse, dog, horse, cow, chicken, anole lizard, and garter snake. We next identified a set of melanocyte/pigment associated transcription factors (CREB, FOXD3, LEF-1, MITF, POU3F2, and USF-1) that exhibit relatively conserved sequence similarity within their DNA binding regions across species based on orthologous alignments across multiple species. Finally, we identified 27 evolutionarily conserved clusters of transcription factor binding sites within ~200-nucleotide intervals of the 1500-nucleotide upstream regions of AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR from Python bivittatus. Our results provide insight into pigment phenotypes in pythons. PMID:27698666

  19. In Silico Analysis of Gene Expression Network Components Underlying Pigmentation Phenotypes in the Python Identified Evolutionarily Conserved Clusters of Transcription Factor Binding Sites

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    Kristopher J. L. Irizarry

    2016-01-01

    Full Text Available Color variation provides the opportunity to investigate the genetic basis of evolution and selection. Reptiles are less studied than mammals. Comparative genomics approaches allow for knowledge gained in one species to be leveraged for use in another species. We describe a comparative vertebrate analysis of conserved regulatory modules in pythons aimed at assessing bioinformatics evidence that transcription factors important in mammalian pigmentation phenotypes may also be important in python pigmentation phenotypes. We identified 23 python orthologs of mammalian genes associated with variation in coat color phenotypes for which we assessed the extent of pairwise protein sequence identity between pythons and mouse, dog, horse, cow, chicken, anole lizard, and garter snake. We next identified a set of melanocyte/pigment associated transcription factors (CREB, FOXD3, LEF-1, MITF, POU3F2, and USF-1 that exhibit relatively conserved sequence similarity within their DNA binding regions across species based on orthologous alignments across multiple species. Finally, we identified 27 evolutionarily conserved clusters of transcription factor binding sites within ~200-nucleotide intervals of the 1500-nucleotide upstream regions of AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR from Python bivittatus. Our results provide insight into pigment phenotypes in pythons.

  20. Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.

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    Yaron Orenstein

    Full Text Available The new technology of protein binding microarrays (PBMs allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.

  1. An integrated pipeline for the genome-wide analysis of transcription factor binding sites from ChIP-Seq.

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    Mercier, Eloi; Droit, Arnaud; Li, Leping; Robertson, Gordon; Zhang, Xuekui; Gottardo, Raphael

    2011-02-16

    ChIP-Seq has become the standard method for genome-wide profiling DNA association of transcription factors. To simplify analyzing and interpreting ChIP-Seq data, which typically involves using multiple applications, we describe an integrated, open source, R-based analysis pipeline. The pipeline addresses data input, peak detection, sequence and motif analysis, visualization, and data export, and can readily be extended via other R and Bioconductor packages. Using a standard multicore computer, it can be used with datasets consisting of tens of thousands of enriched regions. We demonstrate its effectiveness on published human ChIP-Seq datasets for FOXA1, ER, CTCF and STAT1, where it detected co-occurring motifs that were consistent with the literature but not detected by other methods. Our pipeline provides the first complete set of Bioconductor tools for sequence and motif analysis of ChIP-Seq and ChIP-chip data.

  2. A DNA-Centric Protein Interaction Map of Ultraconserved Elements Reveals Contribution of Transcription Factor Binding Hubs to Conservation

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    Tar Viturawong

    2013-10-01

    Full Text Available Ultraconserved elements (UCEs have been the subject of great interest because of their extreme sequence identity and their seemingly cryptic and largely uncharacterized functions. Although in vivo studies of UCE sequences have demonstrated regulatory activity, protein interactors at UCEs have not been systematically identified. Here, we combined high-throughput affinity purification, high-resolution mass spectrometry, and SILAC quantification to map intrinsic protein interactions for 193 UCE sequences. The interactome contains over 400 proteins, including transcription factors with known developmental roles. We demonstrate based on our data that UCEs consist of strongly conserved overlapping binding sites. We also generated a fine-resolution interactome of a UCE, confirming the hub-like nature of the element. The intrinsic interactions mapped here are reflected in open chromatin, as indicated by comparison with existing ChIP data. Our study argues for a strong contribution of protein-DNA interactions to UCE conservation and provides a basis for further functional characterization of UCEs.

  3. Intronic rs2147363 variant in ATP7B transcription factor-binding site associated with Alzheimer's disease.

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    Bucossi, Serena; Polimanti, Renato; Ventriglia, Mariacarla; Mariani, Stefania; Siotto, Mariacristina; Ursini, Francesca; Trotta, Laura; Scrascia, Federica; Callea, Antonio; Vernieri, Fabrizio; Squitti, Rosanna

    2013-01-01

    Copper homeostasis abnormalities have been shown to be associated with Alzheimer's disease (AD), possibly by accelerating amyloid-β toxicity and plaque formation. The ATP7B gene plays a key role in controlling body copper balance. Our previous studies showed an association between ATP7B variants and AD risk. Among these variants, an intronic single nucleotide polymorphism, rs2147363, was associated with AD risk. In order to understand this intronic association, we screened a population of 286 AD patients and 283 healthy controls, and verified the presence of other functional coding variants in linkage disequilibrium (LD). Then we searched for a regulatory function region close to rs2147363. An LD analysis revealed the presence of an LD between rs2147363 and a Wilson's disease-causing variant, rs7334118. However, this mutation did not explain the observed genetic association. Conversely, in silico analyses of rs2147363 functionality highlighted that this variant is located in a binding site of a transcription factor, and is, consequently, associated with regulatory function. These data suggest that the genetic variation in cis-regulatory elements located in non-coding regions can have a role in determining ATP7B functionality and account for some of the AD missing hereditability.

  4. In silico analysis of transcription factor binding sites in promoters of germin-like protein genes in rice

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    Ilyas Muhammad

    2016-01-01

    Full Text Available Germins (GERs and germin-like proteins (GLPs play important roles in responses to various stresses; however, their function is still not fully understood. Significant insight into their function can be obtained by analyzing their promoters. In the present study, the 5' upstream promoters (1000 bp of 43 Asian rice (Oryza sativa var. Japonica GLP genes were retrieved from the Plant Ensemble, based on the Rice Annotation Project database (RAP-DB. Phylogenetic analysis via MEGA6 showed a narrow genetic background (0.2% with a Tajima neutrality value (π of 0.69. Overall, 4234 transcription factor (TF binding sites (TFBSs were found on chromosomes 1, 2, 3, 4, 5, 8, 9, 11 and 12 via “MatInspector” from 90 different TF families using a total of 444 families. Common TFs and DiAlign analyses showed that arabidopsis homeobox protein (AHBP, MYB-like proteins (MYBL and vertebrate TATA-box-binding protein (VTBP were the most abundant, common and evolutionarily conserved elements in the upstream region from 0 to -800. Finding their mutual interaction via Farmworker analysis uncovered three new cisregulatory modules (VTBP_VTBP, MYBS_MYBS, and AHBP_VTBP, which appear to be decisive for OsGLP regulation. In silico functional analysis via ModelInspector revealed 77 cis-regulatory modules, each comprised of two elements, among which DOFF_OPAQ_03 and GTBX_MYCL_01 were the most frequent and mostly found on chromosome 8 and 12, indicating that the combinatorial interaction of these elements has a fundamental role in various biological processes. The study revealed the importance of these elements in regulating OsGLP expression that will help in predicting the role of these genes in various stresses, and can have application in biotechnology.

  5. Assessment of clusters of transcription factor binding sites in relationship to human promoter, CpG islands and gene expression

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    Sakaki Yoshiyuki

    2004-02-01

    Full Text Available Abstract Background Gene expression is regulated mainly by transcription factors (TFs that interact with regulatory cis-elements on DNA sequences. To identify functional regulatory elements, computer searching can predict TF binding sites (TFBS using position weight matrices (PWMs that represent positional base frequencies of collected experimentally determined TFBS. A disadvantage of this approach is the large output of results for genomic DNA. One strategy to identify genuine TFBS is to utilize local concentrations of predicted TFBS. It is unclear whether there is a general tendency for TFBS to cluster at promoter regions, although this is the case for certain TFBS. Also unclear is the identification of TFs that have TFBS concentrated in promoters and to what level this occurs. This study hopes to answer some of these questions. Results We developed the cluster score measure to evaluate the correlation between predicted TFBS clusters and promoter sequences for each PWM. Non-promoter sequences were used as a control. Using the cluster score, we identified a PWM group called PWM-PCP, in which TFBS clusters positively correlate with promoters, and another PWM group called PWM-NCP, in which TFBS clusters negatively correlate with promoters. The PWM-PCP group comprises 47% of the 199 vertebrate PWMs, while the PWM-NCP group occupied 11 percent. After reducing the effect of CpG islands (CGI against the clusters using partial correlation coefficients among three properties (promoter, CGI and predicted TFBS cluster, we identified two PWM groups including those strongly correlated with CGI and those not correlated with CGI. Conclusion Not all PWMs predict TFBS correlated with human promoter sequences. Two main PWM groups were identified: (1 those that show TFBS clustered in promoters associated with CGI, and (2 those that show TFBS clustered in promoters independent of CGI. Assessment of PWM matches will allow more positive interpretation of TFBS in

  6. Data in support of FSH induction of IRS-2 in human granulosa cells: Mapping the transcription factor binding sites in human IRS-2 promoter

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    Surleen Kaur

    2016-03-01

    Full Text Available Insulin receptor substrate-2 (IRS-2 plays critical role in the regulation of various metabolic processes by insulin and IGF-1. The defects in its expression and/or function are linked to diseases like polycystic ovary syndrome (PCOS, insulin resistance and cancer. To predict the transcription factors (TFs responsible for the regulation of human IRS-2 gene expression, the transcription factor binding sites (TFBS and the corresponding TFs were investigated by analysis of IRS-2 promoter sequence using MatInspector Genomatix software (Cartharius et al., 2005 [1]. The ibid data is part of author׳s publication (Anjali et al., 2015 [2] that explains Follicle stimulating hormone (FSH mediated IRS-2 promoter activation in human granulosa cells and its importance in the pathophysiology of PCOS. Further analysis was carried out for binary interactions of TF regulatory genes in IRS-2 network using Cytoscape software tool and R-code. In this manuscript, we describe the methodology used for the identification of TFBSs in human IRS-2 promoter region and provide details on experimental procedures, analysis method, validation of data and also the raw files. The purpose of this article is to provide the data on all TFBSs in the promoter region of human IRS-2 gene as it has the potential for prediction of the regulation of IRS-2 gene in normal or diseased cells from patients with metabolic disorders and cancer.

  7. Integration of Known Transcription Factor Binding Site Information and Gene Expression Data to Advance from Co-Expression to Co-Regulation

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The common approach to find co-regulated genes is to cluster genes based on gene expression. However, due to the limited information present in any dataset, genes in the same cluster might be co-expressed but not necessarily co-regulated. In this paper, we propose to integrate known transcription factor binding site informa tion and gene expression data into a single clustering scheme. This scheme will find clusters of co-regulated genes that are not only expressed similarly under the measured conditions, but also share a regulatory structure that may explain their common regulation. We demonstrate the utility of this approach on a microarray dataset of yeast grown under different nutrient and oxygen limitations. Our in tegrated clustering method not only unravels many regulatory modules that are consistent with current biological knowledge, but also provides a more profound understanding of the underlying process. The added value of our approach, compared with the clustering solely based on gene expression, is its ability to uncover clusters of genes that are involved in more specific biological processes and are evidently regulated by a set of transcription factors.

  8. Computational identification of developmental enhancers:conservation and function of transcription factor binding-site clustersin drosophila melanogaster and drosophila psedoobscura

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    Berman, Benjamin P.; Pfeiffer, Barret D.; Laverty, Todd R.; Salzberg, Steven L.; Rubin, Gerald M.; Eisen, Michael B.; Celniker, SusanE.

    2004-08-06

    The identification of sequences that control transcription in metazoans is a major goal of genome analysis. In a previous study, we demonstrated that searching for clusters of predicted transcription factor binding sites could discover active regulatory sequences, and identified 37 regions of the Drosophila melanogaster genome with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. Here, we report the results of in vivo functional analysis of 27 remaining clusters. We generated transgenic flies carrying each cluster attached to a basal promoter and reporter gene, and assayed embryos for reporter gene expression. Six clusters are enhancers of adjacent genes: giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three drive expression in patterns unrelated to those of neighboring genes; the remaining 18 do not appear to have enhancer activity. We used the Drosophila pseudoobscura genome to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. We incorporated conservation of binding-site clustering into a new genome-wide enhancer screen, and predict several hundred new regulatory sequences, including 85 adjacent to genes with embryonic patterns. Measuring conservation of sequence features closely linked to function--such as binding-site clustering--makes better use of comparative sequence data than commonly used methods that examine only sequence identity.

  9. A Transcription Factor-Binding Domain of the Coactivator CBP Is Essential for Long-Term Memory and the Expression of Specific Target Genes

    Science.gov (United States)

    Oliveira, Ana M. M.; Brindle, Paul K.; Abel, Ted; Wood, Marcelo A.; Attner, Michelle A.

    2006-01-01

    Transcriptional activation is a key process required for long-term memory formation. Recently, the transcriptional coactivator CREB-binding protein (CBP) was shown to be critical for hippocampus-dependent long-term memory and hippocampal synaptic plasticity. As a coactivator with intrinsic histone acetyltransferase activity, CBP interacts with…

  10. Point mutations within 663-666 bp of intron 6 of the human TDO2 gene, associated with a number of psychiatric disorders, damage the YY-1 transcription factor binding site.

    Science.gov (United States)

    Vasiliev, G V; Merkulov, V M; Kobzev, V F; Merkulova, T I; Ponomarenko, M P; Kolchanov, N A

    1999-11-26

    Single base mutations G-->A at position 663 and G-->T at position 666 of intron 6 of the human tryptophan oxygenase gene (TDO2) are associated with a variety of psychiatric disorders [Comings, D.E. et al. (1996) Pharmacogenetics 6, 307-318]. Binding of rat liver nuclear extract proteins to synthetic double-strand oligonucleotides corresponding to three allelic states of the region between 651 bp and 680 bp of human TDO2 intron 6 has been studied by gel shift assay. It has been demonstrated that to each allelic state of the region there corresponds a specific set of proteins that interacts with it. With the aid of computer analysis and using specific anti-YY-1 antibodies it has been shown that both mutations damage the YY-1 transcription factor binding site.

  11. ChIP on chip and ChIP-Seq assays: genome-wide analysis of transcription factor binding and histone modifications.

    Science.gov (United States)

    Pillai, Smitha; Chellappan, Srikumar P

    2015-01-01

    Deregulation of transcriptional activity of many genes has been causatively linked to human diseases including cancer. Altered patterns of gene expression in normal and cancer cells are the result of inappropriate expression of transcription factors and chromatin modifying proteins. Chromatin immunoprecipitation assay is a well-established tool for investigating the interactions between regulatory proteins and DNA at distinct stages of gene activation. ChIP coupled with DNA microarrays, known as ChIP on chip, or sequencing of DNA associated with the factors (ChIP-Seq) allow us to determine the entire spectrum of in vivo DNA binding sites for a given protein. This has been of immense value because ChIP on chip assays and ChIP-Seq experiments can provide a snapshot of the transcriptional regulatory mechanisms on a genome-wide scale. This chapter outlines the general strategies used to carry out ChIP-chip assays to study the differential recruitment of regulatory molecules based on the studies conducted in our lab as well as other published protocols; these can be easily modified to a ChIP-Seq analysis.

  12. Oct-2 transcription factor binding activity and expression up-regulation in rat cerebral ischaemia is associated with a diminution of neuronal damage in vitro.

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    Camós, Susanna; Gubern, Carme; Sobrado, Mónica; Rodríguez, Rocío; Romera, Víctor G; Moro, María Ángeles; Lizasoain, Ignacio; Serena, Joaquín; Mallolas, Judith; Castellanos, Mar

    2014-06-01

    Brain plasticity provides a mechanism to compensate for lesions produced as a result of stroke. The present study aims to identify new transcription factors (TFs) following focal cerebral ischaemia in rat as potential therapeutic targets. A transient focal cerebral ischaemia model was used for TF-binding activity and TF-TF interaction profile analysis. A permanent focal cerebral ischaemia model was used for the transcript gene analysis and for the protein study. The identification of TF variants, mRNA analysis, and protein study was performed using conventional polymerase chain reaction (PCR), qPCR, and Western blot and immunofluorescence, respectively. Rat cortical neurons were transfected with small interfering RNA against the TF in order to study its role. The TF-binding analysis revealed a differential binding activity of the octamer family in ischaemic brain in comparison with the control brain samples both in acute and late phases. In this study, we focused on Oct-2 TF. Five of the six putative Oct-2 transcript variants are expressed in both control and ischaemic rat brain, showing a significant increase in the late phase of ischaemia. Oct-2 protein showed neuronal localisation both in control and ischaemic rat brain cortical slices. Functional studies revealed that Oct-2 interacts with TFs involved in important brain processes (neuronal and vascular development) and basic cellular functions and that Oct-2 knockdown promotes neuronal injury. The present study shows that Oct-2 expression and binding activity increase in the late phase of cerebral ischaemia and finds Oct-2 to be involved in reducing ischaemic-mediated neuronal injury.

  13. Global MYCN transcription factor binding analysis in neuroblastoma reveals association with distinct E-box motifs and regions of DNA hypermethylation.

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    Derek M Murphy

    Full Text Available BACKGROUND: Neuroblastoma, a cancer derived from precursor cells of the sympathetic nervous system, is a major cause of childhood cancer related deaths. The single most important prognostic indicator of poor clinical outcome in this disease is genomic amplification of MYCN, a member of a family of oncogenic transcription factors. METHODOLOGY: We applied MYCN chromatin immunoprecipitation to microarrays (ChIP-chip using MYCN amplified/non-amplified cell lines as well as a conditional knockdown cell line to determine the distribution of MYCN binding sites within all annotated promoter regions. CONCLUSION: Assessment of E-box usage within consistently positive MYCN binding sites revealed a predominance for the CATGTG motif (p<0.0016, with significant enrichment of additional motifs CATTTG, CATCTG, CAACTG in the MYCN amplified state. For cell lines over-expressing MYCN, gene ontology analysis revealed enrichment for the binding of MYCN at promoter regions of numerous molecular functional groups including DNA helicases and mRNA transcriptional regulation. In order to evaluate MYCN binding with respect to other genomic features, we determined the methylation status of all annotated CpG islands and promoter sequences using methylated DNA immunoprecipitation (MeDIP. The integration of MYCN ChIP-chip and MeDIP data revealed a highly significant positive correlation between MYCN binding and DNA hypermethylation. This association was also detected in regions of hemizygous loss, indicating that the observed association occurs on the same homologue. In summary, these findings suggest that MYCN binding occurs more commonly at CATGTG as opposed to the classic CACGTG E-box motif, and that disease associated over expression of MYCN leads to aberrant binding to additional weaker affinity E-box motifs in neuroblastoma. The co-localization of MYCN binding and DNA hypermethylation further supports the dual role of MYCN, namely that of a classical transcription

  14. Global MYCN transcription factor binding analysis in neuroblastoma reveals association with distinct E-box motifs and regions of DNA hypermethylation.

    LENUS (Irish Health Repository)

    Murphy, Derek M

    2009-01-01

    BACKGROUND: Neuroblastoma, a cancer derived from precursor cells of the sympathetic nervous system, is a major cause of childhood cancer related deaths. The single most important prognostic indicator of poor clinical outcome in this disease is genomic amplification of MYCN, a member of a family of oncogenic transcription factors. METHODOLOGY: We applied MYCN chromatin immunoprecipitation to microarrays (ChIP-chip) using MYCN amplified\\/non-amplified cell lines as well as a conditional knockdown cell line to determine the distribution of MYCN binding sites within all annotated promoter regions. CONCLUSION: Assessment of E-box usage within consistently positive MYCN binding sites revealed a predominance for the CATGTG motif (p<0.0016), with significant enrichment of additional motifs CATTTG, CATCTG, CAACTG in the MYCN amplified state. For cell lines over-expressing MYCN, gene ontology analysis revealed enrichment for the binding of MYCN at promoter regions of numerous molecular functional groups including DNA helicases and mRNA transcriptional regulation. In order to evaluate MYCN binding with respect to other genomic features, we determined the methylation status of all annotated CpG islands and promoter sequences using methylated DNA immunoprecipitation (MeDIP). The integration of MYCN ChIP-chip and MeDIP data revealed a highly significant positive correlation between MYCN binding and DNA hypermethylation. This association was also detected in regions of hemizygous loss, indicating that the observed association occurs on the same homologue. In summary, these findings suggest that MYCN binding occurs more commonly at CATGTG as opposed to the classic CACGTG E-box motif, and that disease associated over expression of MYCN leads to aberrant binding to additional weaker affinity E-box motifs in neuroblastoma. The co-localization of MYCN binding and DNA hypermethylation further supports the dual role of MYCN, namely that of a classical transcription factor affecting the

  15. Defining the plasticity of transcription factor binding sites by Deconstructing DNA consensus sequences: the PhoP-binding sites among gamma/enterobacteria.

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    Oscar Harari

    Full Text Available Transcriptional regulators recognize specific DNA sequences. Because these sequences are embedded in the background of genomic DNA, it is hard to identify the key cis-regulatory elements that determine disparate patterns of gene expression. The detection of the intra- and inter-species differences among these sequences is crucial for understanding the molecular basis of both differential gene expression and evolution. Here, we address this problem by investigating the target promoters controlled by the DNA-binding PhoP protein, which governs virulence and Mg(2+ homeostasis in several bacterial species. PhoP is particularly interesting; it is highly conserved in different gamma/enterobacteria, regulating not only ancestral genes but also governing the expression of dozens of horizontally acquired genes that differ from species to species. Our approach consists of decomposing the DNA binding site sequences for a given regulator into families of motifs (i.e., termed submotifs using a machine learning method inspired by the "Divide & Conquer" strategy. By partitioning a motif into sub-patterns, computational advantages for classification were produced, resulting in the discovery of new members of a regulon, and alleviating the problem of distinguishing functional sites in chromatin immunoprecipitation and DNA microarray genome-wide analysis. Moreover, we found that certain partitions were useful in revealing biological properties of binding site sequences, including modular gains and losses of PhoP binding sites through evolutionary turnover events, as well as conservation in distant species. The high conservation of PhoP submotifs within gamma/enterobacteria, as well as the regulatory protein that recognizes them, suggests that the major cause of divergence between related species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous target

  16. Chromatin immunoprecipitation and multiplex sequencing (ChIP-Seq) to identify global transcription factor binding sites in the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Brdlik, Cathleen M; Niu, Wei; Snyder, Michael

    2014-01-01

    The global identification of transcription factor (TF) binding sites is a critical step in the elucidation of the functional elements of the genome. Several methods have been developed that map TF binding in human cells, yeast, and other model organisms. These methods make use of chromatin immunoprecipitation, or ChIP, and take advantage of the fact that formaldehyde fixation of living cells can be used to cross-link DNA sequences to the TFs that bind them in vivo. In ChIP, the cross-linked TF-DNA complexes are sheared by sonication, size fractionated, and incubated with antibody specific to the TF of interest to generate a library of TF-bound DNA sequences. ChIP-chip was the first technology developed to globally identify TF-bound DNA sequences and involves subsequent hybridization of the ChIP DNA to oligonucleotide microarrays. However, ChIP-chip proved to be costly, labor-intensive, and limited by the fixed number of probes available on the microarray chip. ChIP-Seq combines ChIP with massively parallel high-throughput sequencing (see Explanatory Chapter: Next Generation Sequencing) and has demonstrated vast improvement over ChIP-chip with respect to time and cost, signal-to-noise ratio, and resolution. In particular, multiplex sequencing can be used to achieve a higher throughput in ChIP-Seq analyses involving organisms with genomes of lower complexity than that of human (Lefrançois et al., 2009) and thereby reduce the cost and amount of time needed for each result. The multiplex ChIP-Seq method described in this section has been developed for Caenorhabditis elegans, but is easily adaptable for other organisms.

  17. Multi-species comparative analysis of the equine ACE gene identifies a highly conserved potential transcription factor binding site in intron 16.

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    Natasha A Hamilton

    Full Text Available Angiotensin converting enzyme (ACE is essential for control of blood pressure. The human ACE gene contains an intronic Alu indel (I/D polymorphism that has been associated with variation in serum enzyme levels, although the functional mechanism has not been identified. The polymorphism has also been associated with cardiovascular disease, type II diabetes, renal disease and elite athleticism. We have characterized the ACE gene in horses of breeds selected for differing physical abilities. The equine gene has a similar structure to that of all known mammalian ACE genes. Nine common single nucleotide polymorphisms (SNPs discovered in pooled DNA were found to be inherited in nine haplotypes. Three of these SNPs were located in intron 16, homologous to that containing the Alu polymorphism in the human. A highly conserved 18 bp sequence, also within that intron, was identified as being a potential binding site for the transcription factors Oct-1, HFH-1 and HNF-3β, and lies within a larger area of higher than normal homology. This putative regulatory element may contribute to regulation of the documented inter-individual variation in human circulating enzyme levels, for which a functional mechanism is yet to be defined. Two equine SNPs occurred within the conserved area in intron 16, although neither of them disrupted the putative binding site. We propose a possible regulatory mechanism of the ACE gene in mammalian species which was previously unknown. This advance will allow further analysis leading to a better understanding of the mechanisms underpinning the associations seen between the human Alu polymorphism and enzyme levels, cardiovascular disease states and elite athleticism.

  18. Changes of insulin-like growth factor-Ⅱ and insulin-like growth factor binding protein-3 in cerebrospinal fluid of children with tuberculous meningitis

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    BACKGROUND: Recent studies have found that insulin-like growth factors (IGFs) and insulin-like growth factor binding protein-3 (IGFBP-3) have stronger neurotrophic and neuroprotective effects. But whether their levels in cerebrospinal fluid could be used as an auxiliary indicator in differentially diagnosing tuberculous meningitis and viral encephalitis is not yet clear.OBJECTIVE: To explore the changes of insulin-like growth factor-Ⅱ (IGF-Ⅱ ) and IGFBP-3 in cerebrospinal fluid (CSF) of children with tuberculous meningitis and the significance of the changes.DESIGN: A non-randomized concurrent controlled study.SETTING: Department of Pediatric Internal Medicine, the First Affiliated Hospital of Xinxiang Medical College.PARTICIPANTS: Thirty children with tuberculous meningitis (14 males and 16 females) were selected from the Department of Pediatric Internal Medicine, the First Affiliated Hospital of Xinxiang Medical College from January 2005 to December 2006. Tuberculous meningitis was diagnosed according to their clinical manifestations, the history of close contact with tuberculosis, typical cerebrospinal fluid changes of tuberculous meningitis, positive tuberculosis antibody and effective antituberculosis treatment. There were 30 children (13 males and 17 females) with viral encephalitis, and viral encephalitis was diagnosed according to epidemiological history, clinical manifestations, conventional and biochemical changes of cerebrospinal fluid, and negative bacteriology judgment. Meanwhile, 30 children (13 males and 17 females) without infectious and central nervous system disease were selected as the control group. Informed consent was obtained from the parents of all the enrolled children.METHODS: ① The lumbar puncture operation was implemented immediately to obtain cerebrospinal fluid (3 mL). The contents of IGF-Ⅱ and IGFBP-3 were detected with immunoradiometric assay. The concentrations of glucose and protein in cerebrospinal fluid were determined

  19. Skp2B overexpression alters a prohibitin-p53 axis and the transcription of PAPP-A, the protease of insulin-like growth factor binding protein 4.

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    Harish Chander

    Full Text Available BACKGROUND: We previously reported that the degradation of prohibitin by the SCF(Skp2B ubiquitin ligase results in a defect in the activity of p53. We also reported that MMTV-Skp2B transgenic mice develop mammary gland tumors that are characterized by an increased proteolytic cleavage of the insulin-like growth factor binding protein 4 (IGFBP-4, an inhibitor of IGF signaling. However, whether a link exists between a defect in p53 activity and proteolysis of IGFBP-4 was not established. METHODS AND RESULTS: We analyzed the levels of pregnancy-associated plasma protein A (PAPP-A, the protease of IGFBP-4, in MMTV-Skp2B transgenic mice and found that PAPP-A levels are elevated. Further, we found a p53 binding site in intron 1 of the PAPP-A gene and that both wild type and mutant p53 bind to this site. However, binding of wild type p53 results in the transcriptional repression of PAPP-A, while binding of mutant p53 results in the transcriptional activation of PAPP-A. Since MMTV-Skp2B mice express wild type p53 and yet show elevated levels of PAPP-A, at first, these observations appeared contradictory. However, further analysis revealed that the defect in p53 activity in Skp2B overexpressing cells does not only abolish the activity of wild type of p53 but actually mimics that of mutant p53. Our results suggest that in absence of prohibitin, the half-life of p53 is increased and like mutant p53, the conformation of p53 is denatured. CONCLUSIONS: These observations revealed a novel function of prohibitin as a chaperone of p53. Further, they suggest that binding of denatured p53 in intron 1 causes an enhancer effect and increases the transcription of PAPP-A. Therefore, these findings indicate that the defect in p53 function and the increased proteolysis of IGFBP-4, we had observed, represent two components of the same pathway, which contributes to the oncogenic function of Skp2B.

  20. Heritable change caused by transient transcription errors.

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    Alasdair J E Gordon

    2013-06-01

    Full Text Available Transmission of cellular identity relies on the faithful transfer of information from the mother to the daughter cell. This process includes accurate replication of the DNA, but also the correct propagation of regulatory programs responsible for cellular identity. Errors in DNA replication (mutations and protein conformation (prions can trigger stable phenotypic changes and cause human disease, yet the ability of transient transcriptional errors to produce heritable phenotypic change ('epimutations' remains an open question. Here, we demonstrate that transcriptional errors made specifically in the mRNA encoding a transcription factor can promote heritable phenotypic change by reprogramming a transcriptional network, without altering DNA. We have harnessed the classical bistable switch in the lac operon, a memory-module, to capture the consequences of transient transcription errors in living Escherichia coli cells. We engineered an error-prone transcription sequence (A9 run in the gene encoding the lac repressor and show that this 'slippery' sequence directly increases epigenetic switching, not mutation in the cell population. Therefore, one altered transcript within a multi-generational series of many error-free transcripts can cause long-term phenotypic consequences. Thus, like DNA mutations, transcriptional epimutations can instigate heritable changes that increase phenotypic diversity, which drives both evolution and disease.

  1. Auxin-dependent compositional change in Mediator in ARF7- and ARF19-mediated transcription.

    Science.gov (United States)

    Ito, Jun; Fukaki, Hidehiro; Onoda, Makoto; Li, Lin; Li, Chuanyou; Tasaka, Masao; Furutani, Masahiko

    2016-06-07

    Mediator is a multiprotein complex that integrates the signals from transcription factors binding to the promoter and transmits them to achieve gene transcription. The subunits of Mediator complex reside in four modules: the head, middle, tail, and dissociable CDK8 kinase module (CKM). The head, middle, and tail modules form the core Mediator complex, and the association of CKM can modify the function of Mediator in transcription. Here, we show genetic and biochemical evidence that CKM-associated Mediator transmits auxin-dependent transcriptional repression in lateral root (LR) formation. The AUXIN/INDOLE 3-ACETIC ACID 14 (Aux/IAA14) transcriptional repressor inhibits the transcriptional activity of its binding partners AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 by making a complex with the CKM-associated Mediator. In addition, TOPLESS (TPL), a transcriptional corepressor, forms a bridge between IAA14 and the CKM component MED13 through the physical interaction. ChIP assays show that auxin induces the dissociation of MED13 but not the tail module component MED25 from the ARF7 binding region upstream of its target gene. These findings indicate that auxin-induced degradation of IAA14 changes the module composition of Mediator interacting with ARF7 and ARF19 in the upstream region of their target genes involved in LR formation. We suggest that this regulation leads to a quick switch of signal transmission from ARFs to target gene expression in response to auxin.

  2. Transcriptional control of the F0F1-ATP synthase operon of Corynebacterium glutamicum: SigmaH factor binds to its promoter and regulates its expression at different pH values.

    Science.gov (United States)

    Barriuso-Iglesias, Mónica; Barreiro, Carlos; Sola-Landa, Alberto; Martín, Juan F

    2013-03-01

    Corynebacterium glutamicum used in the amino acid fermentation industries is an alkaliphilic microorganism. Its F(0)F(1)-ATPase operon (atpBEFHAGDC) is expressed optimally at pH 9.0 forming a polycistronic (7.5 kb) and a monocistronic (1.2 kb) transcripts both starting upstream of the atpB gene. Expression of this operon is controlled by the SigmaH factor. The sigmaH gene (sigH) was cloned and shown to be co-transcribed with a small gene, cg0877, encoding a putative anti-sigma factor. A mutant deleted in the sigH gene expressed the atpBEFHAGDC operon optimally at pH 7.0 at difference of the wild-type strain (optimal expression at pH 9.0). These results suggested that the SigmaH factor is involved in pH control of expression of the F(0) F(1) ATPase operon. The SigmaH protein was expressed in Escherichia coli fused to the GST (glutathione-S-transferase) and purified to homogeneity by affinity chromatography on a GSTrap HP column. The fused protein was identified by immunodetection with anti-GST antibodies. DNA-binding studies by electrophoretic mobility shift assays showed that the SigH protein binds to a region of the atpB promoter containing the sigmaH recognition sequence (-35)TTGGAT…18nt…GTTA(-10). SigmaH plays an important role in the cascade of control of pH stress in Corynebacterium.

  3. Position specific variation in the rate of evolution intranscription factor binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Moses, Alan M.; Chiang, Derek Y.; Kellis, Manolis; Lander, EricS.; Eisen, Michael B.

    2003-08-28

    The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Here we analyze the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikataeto study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artifacts of computational motif finding algorithms. As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative

  4. Transcript changes in Vibrio cholerae in response to salt stress.

    Science.gov (United States)

    Fu, Xiuping; Liang, Weili; Du, Pengcheng; Yan, Meiying; Kan, Biao

    2014-01-01

    Vibrio cholerae, which is a serious human intestinal pathogen, often resides and thrives in estuaries but requires major self-regulation to overcome intestinal hyperosmotic stress or high salt stress in water and food. In the present study, we selected multiple O1 and O139 group V. cholerae strains that were isolated from different regions and during different years to study their salt tolerance. Based on the mechanisms that other bacteria use to respond to high salt stress, we selected salt stress-response related genes to study the mechanisms which V. cholerae responds to high salt stress. V. cholerae strains showed salt-resistance characteristics that varied in salt concentrations from 4% to 6%. However, group O1 and group O139 showed no significant difference in the degree of salt tolerance. The primary responses of bacteria to salt stress, including Na(+) exclusion, K(+) uptake and glutamate biosynthesis, were observed in V. cholerae strains. In addition, some sigma factors were up-regulated in V. cholerae strains, suggesting that V. cholerae may recruit common sigma factors to achieve an active salt stress response. However, some changes in gene transcript levels in response to salt stress in V. cholerae were strain-specific. In particular, hierarchical clustering of differentially expressed genes indicated that transcript levels of these genes were correlated with the degree of salt tolerance. Therefore, elevated transcript levels of some genes, including sigma factors and genes involved in peptidoglycan biosynthesis, may be due to the salt tolerance of strains. In addition, high salt-tolerant strains may recruit common as well as additional sigma factors to activate the salt stress response.

  5. Transcriptional Control During Hematopoietic Development : Transcription factor binding and chromatin conformation dynamics

    NARCIS (Netherlands)

    A. van den Heuvel (Anita)

    2015-01-01

    markdownabstractA cell’s identity is primarily determined by the proteins it produces and therefore by the genes it expresses. During development, correct cell fate specification and determination therefore requires a strictly controlled upregulation or downregulation of lineage-specific gene expres

  6. Preaxial polydactyly/triphalangeal thumb is associated with changed transcription factor-binding affinity in a family with a novel point mutation in the long-range cis-regulatory element ZRS

    DEFF Research Database (Denmark)

    Farooq, Muhammad; Troelsen, Jesper T; Boyd, Mette

    2010-01-01

    A cis-regulatory sequence also known as zone of polarizing activity (ZPA) regulatory sequence (ZRS) located in intron 5 of LMBR1 is essential for expression of sonic hedgehog (SHH) in the developing posterior limb bud mesenchyme. Even though many point mutations causing preaxial duplication defects...

  7. Butyrate-induced transcriptional changes in human colonic mucosa.

    Directory of Open Access Journals (Sweden)

    Steven A L W Vanhoutvin

    Full Text Available BACKGROUND: Fermentation of dietary fiber in the colon results in the production of short chain fatty acids (mainly propionate, butyrate and acetate. Butyrate modulates a wide range of processes, but its mechanism of action is mostly unknown. This study aimed to determine the effects of butyrate on the transcriptional regulation of human colonic mucosa in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Five hundred genes were found to be differentially expressed after a two week daily butyrate administration with enemas. Pathway analysis showed that the butyrate intervention mainly resulted in an increased transcriptional regulation of the pathways representing fatty acid oxidation, electron transport chain and oxidative stress. In addition, several genes associated with epithelial integrity and apoptosis, were found to be differentially expressed after the butyrate intervention. CONCLUSIONS/SIGNIFICANCE: Colonic administration of butyrate in concentrations that can be achieved by consumption of a high-fiber diet enhances the maintenance of colonic homeostasis in healthy subjects, by regulating fatty acid metabolism, electron transport and oxidative stress pathways on the transcriptional level and provide for the first time, detailed molecular insight in the transcriptional response of gut mucosa to butyrate.

  8. Controlling for gene expression changes in transcription factor protein networks.

    Science.gov (United States)

    Banks, Charles A S; Lee, Zachary T; Boanca, Gina; Lakshminarasimhan, Mahadevan; Groppe, Brad D; Wen, Zhihui; Hattem, Gaye L; Seidel, Chris W; Florens, Laurence; Washburn, Michael P

    2014-06-01

    The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein-protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFκB1, NFκB2, Rel, RelB, IκBα, IκBβ, and IκBε). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFκB family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions.

  9. Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Ludmila R.P. Ferreira

    2008-03-01

    Full Text Available The differentiation of proliferating epimastigote forms of Trypanosoma cruzi , the protozoan parasite that causes Chagas’ disease, into the infective and non-proliferating metacyclic forms can be reproduced in the laboratory by incubating the cells in a chemically-defined medium that mimics the urine of the insect vector. Epimastigotes have a spherical nucleus, a flagellum protruding from the middle of the protozoan cell, and a disk-shaped kinetoplast - an organelle that corresponds to the mitochondrial DNA. Metacyclic trypomastigotes have an elongated shape with the flagellum protruding from the posterior portion of the cell and associated with a spherical kinetoplast. Here we describe the morphological events of this transformation and characterize a novel intermediate stage by three-dimensional reconstruction of electron microscope serial sections. This new intermediate stage is characterized by a kinetoplast compressing an already elongated nucleus, indicating that metacyclogenesis involves active movements of the flagellar structure relative to the cell body. As transcription occurs more intensely in proliferating epimastigotes than in metacyclics, we also examined the presence of RNA polymerase II and measured transcriptional activity during the differentiation process. Both the presence of the enzyme and transcriptional activity remain unchanged during all steps of metacyclogenesis. RNA polymerase II levels and transcriptional activity only decrease after metacyclics are formed. We suggest that transcription is required during the epimastigote-to-metacyclic trypomastigote differentiation process, until the kinetoplast and flagellum reach the posterior position of the parasites in the infective form.A diferenciação de formas epimastigotas (proliferativas do Trypanosoma cruzi, parasita protozoário causador da doença de Chagas, em formas metacíclicas tripomastigotas (infectivas e não proliferativas, pode ser reproduzida em laborat

  10. Differentiation driven changes in the dynamic organization of Basal transcription initiation.

    Directory of Open Access Journals (Sweden)

    Giuseppina Giglia-Mari

    2009-10-01

    Full Text Available Studies based on cell-free systems and on in vitro-cultured living cells support the concept that many cellular processes, such as transcription initiation, are highly dynamic: individual proteins stochastically bind to their substrates and disassemble after reaction completion. This dynamic nature allows quick adaptation of transcription to changing conditions. However, it is unknown to what extent this dynamic transcription organization holds for postmitotic cells embedded in mammalian tissue. To allow analysis of transcription initiation dynamics directly into living mammalian tissues, we created a knock-in mouse model expressing fluorescently tagged TFIIH. Surprisingly and in contrast to what has been observed in cultured and proliferating cells, postmitotic murine cells embedded in their tissue exhibit a strong and long-lasting transcription-dependent immobilization of TFIIH. This immobilization is both differentiation driven and development dependent. Furthermore, although very statically bound, TFIIH can be remobilized to respond to new transcriptional needs. This divergent spatiotemporal transcriptional organization in different cells of the soma revisits the generally accepted highly dynamic concept of the kinetic framework of transcription and shows how basic processes, such as transcription, can be organized in a fundamentally different fashion in intact organisms as previously deduced from in vitro studies.

  11. Heat shock response in yeast involves changes in both transcription rates and mRNA stabilities.

    Directory of Open Access Journals (Sweden)

    Laia Castells-Roca

    Full Text Available We have analyzed the heat stress response in the yeast Saccharomyces cerevisiae by determining mRNA levels and transcription rates for the whole transcriptome after a shift from 25 °C to 37 °C. Using an established mathematical algorithm, theoretical mRNA decay rates have also been calculated from the experimental data. We have verified the mathematical predictions for selected genes by determining their mRNA decay rates at different times during heat stress response using the regulatable tetO promoter. This study indicates that the yeast response to heat shock is not only due to changes in transcription rates, but also to changes in the mRNA stabilities. mRNA stability is affected in 62% of the yeast genes and it is particularly important in shaping the mRNA profile of the genes belonging to the environmental stress response. In most cases, changes in transcription rates and mRNA stabilities are homodirectional for both parameters, although some interesting cases of antagonist behavior are found. The statistical analysis of gene targets and sequence motifs within the clusters of genes with similar behaviors shows that both transcriptional and post-transcriptional regulons apparently contribute to the general heat stress response by means of transcriptional factors and RNA binding proteins.

  12. 拟南芥bZIP1转录因子通过与ABRE元件结合调节ABA信号传导%Arabidopisis bZIP1 Transcription Factor Binding to the ABRE Cis-Element Regulates Abscisic Acid Signal Transduction

    Institute of Scientific and Technical Information of China (English)

    孙晓丽; 李勇; 才华; 柏锡; 纪巍; 季佐军; 朱延明

    2011-01-01

    Abscisic acid (ABA) is a phytohormone and mediates the response and adaptation of higher plants to various environmental stresses during vegetative growth.The basic leucine zipper (bZIP) transcription factors are also important regulators of plant development and abiotic resistance, acting through either ABA-dependent or ABA-independent pathways.In this study, we investigated and characterized the involvement of the AtbZIP1 gene in plant responsiveness to ABA.As confirmed by PCR and RT-PCR, AtbZIP1 has been silenced in mutant Arabidopsis ko-1 (SALK_059343) and ko-2 (SALK_069489C).The AtbZIP1 knockout plants demonstrated reduced sensitivity to ABA both at the seed germination and seedling stage with improvements in rates of germination, leaf opening/greening, and primary root length.In order to investigate whether the regulation of AtbZIP1-mediated ABA responsiveness depended on the ABA-responsive elements (ABRE), we expressed the AtbZIP1 HIS6 fusion protein in E.coli and found that the AtbZIP1 HIS6 specifically bound to the ABRE cis-elements.Semi-quantitive RT PCR showed that AtbZIP1 disruption altered expressions of some ABA responsive genes, such as NCED3, RD22, KIN1, and RD29A.Our results indicated that AtbZIP1 regulates abscisic acid signal transduction by binding to the ABREs and altered the expressions of the ABA responsive genes.%ABA作为一种重要的植物激素和生长凋节剂,介导了高等植物在营养生长阶段对各种外界环境的响应和适应.bZIP类转录因子可以通过ABA依赖途径和ABA非依赖途径调节植物的生长发育和对非生物胁迫的耐性.本研究通过AtbZIP1 T-DNA插入突变的拟南芥植株ko-1(SALK_059343)和ko-2(SALK_069489C)在ABA处理后的表型实验,验证了AtbZIP1参与ABA依赖的信号传导通路.采用"三引物法",分别在DNA水平和RNA水平通过PCR和RT-PCR验证了AtbZIP1基因在拟南芥突变体中的沉默效果.定量分析数据表明,在种子萌发阶段,经过0.6μmolL-1

  13. Transcriptional changes are involved in phenotype switching in Streptococcus equi subspecies equi.

    Science.gov (United States)

    Steward, Karen F; Robinson, Carl; Waller, Andrew S

    2016-04-01

    Phenotypic heterogeneity within a population of bacteria, through genetic or transcriptional variation, enables survival and persistence in challenging and changing environments. We report here that a recent clinical isolate of S. equi, strain 1691 (Se1691), yielded a mixture of reduced capsule and mucoid colonies on primary isolation when grown on colistin-oxolinic acid blood agar (COBA) streptococcal selective plates. Passaging colonies of Se1691, with a reduced capsule phenotype maintained this mixed phenotype. In contrast, passaging mucoid colonies fixed the mucoid phenotype, suggesting adaptive genetic or transcriptional changes in response to growth on artificial media. However, despite obvious phenotypic and transcriptional differences, there were no apparent differences in the genome sequences of Se1691 recovered from colonies with a mucoid or reduced capsule phenotype. We identified 105 differentially transcribed genes in the transcriptomes of reduced capsule and mucoid colonies. The reduced capsule phenotype was associated with a significant reduction in transcription of the has locus (SEQ_0269 Q = 0.0015, SEQ_0270 Q = 0.0015, SEQ_0271 Q = 0.0285) and the amount of hyaluronic acid on the surface of S. equi recovered from non-mucoid colonies (P = 0.017). Significant differences in the transcription of 21 surface and secreted proteins were also observed. Our data show that changes in the bacterial transcriptome are linked to the mixed colony phenotype of Se1691.

  14. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  15. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites

    DEFF Research Database (Denmark)

    Tornøe, Jens; Kusk, P.; Johansen, T.E.;

    2002-01-01

    The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human beta-actin and ubiquitin C...

  16. Isolation of transcription factors binding auxin response elements using a yeast one-hybrid system

    Institute of Scientific and Technical Information of China (English)

    齐眉; 黄美娟; 陈凡

    2002-01-01

    Plant hormones play an important role during higher plant embryogenesis. Auxin is central to the development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses. Auxin response element (AuxRE), present in the promoters of many auxin-induced genes, can confer auxin responsiveness. Using carrot somatic embryo under specific developmental phase, a cDNA expression library was constructed. Several plasmids were recombined containing the tetramer of AuxRE as a bait. After screening by a yeast one-hy- brid system, one positive clone was confirmed and characterized. Electrophoretic mobility shift assay showed that AxRF1 protein expressed in yeast cell could bind AuxRE in vitro. It suggests that AxRF1 participates in regulation of the expression of auxin responsive gene during carrot somatic embryogenesis.

  17. Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities.

    Science.gov (United States)

    Glick, Yair; Orenstein, Yaron; Chen, Dana; Avrahami, Dorit; Zor, Tsaffrir; Shamir, Ron; Gerber, Doron

    2016-04-07

    Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo In vitro methodologies provide valuable complementary information on protein-DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein-DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein-DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein-DNA binding.

  18. BROMATE-INDUCED TRANSCRIPTIONAL CHANGES IN LONG-EVANS RAT KIDNEYS

    Science.gov (United States)

    Bromate-Induced Transcriptional Changes in Long-Evans Rat Kidneys.Ozone disinfection of surface waters containing bromide ion (Br-) results in the oxidation of bromide to bromate, which can be found in finished drinking water as a by-product. Potassium bromate (KBrO3)...

  19. Yeast mitochondrial RNAP conformational changes are regulated by interactions with the mitochondrial transcription factor.

    Science.gov (United States)

    Drakulic, Srdja; Wang, Liping; Cuéllar, Jorge; Guo, Qing; Velázquez, Gilberto; Martín-Benito, Jaime; Sousa, Rui; Valpuesta, José M

    2014-01-01

    Mitochondrial RNA polymerases (MtRNAPs) are members of the single-subunit RNAP family, the most well-characterized member being the RNAP from T7 bacteriophage. MtRNAPs are, however, functionally distinct in that they depend on one or more transcription factors to recognize and open the promoter and initiate transcription, while the phage RNAPs are capable of performing these tasks alone. Since the transcriptional mechanisms that are conserved in phage and mitochondrial RNAPs have been so effectively characterized in the phage enzymes, outstanding structure-mechanism questions concern those aspects that are distinct in the MtRNAPs, particularly the role of the mitochondrial transcription factor(s). To address these questions we have used both negative staining and cryo-EM to generate three-dimensional reconstructions of yeast MtRNAP initiation complexes with and without the mitochondrial transcription factor (MTF1), and of the elongation complex. Together with biochemical experiments, these data indicate that MTF1 uses multiple mechanisms to drive promoter opening, and that its interactions with the MtRNAP regulate the conformational changes undergone by the latter enzyme as it traverses the template strand.

  20. Sequential changes in chromatin structure during transcriptional activation in the beta globin LCR and its target gene.

    Science.gov (United States)

    Kim, Kihoon; Kim, AeRi

    2010-09-01

    Chromatin structure is modulated during transcriptional activation. The changes include the association of transcriptional activators, formation of hypersensitive sites and covalent modifications of histones. To understand the order of the various changes accompanying transcriptional activation, we analyzed the mouse beta globin gene, which is transcriptionally inducible in erythroid MEL cells over a time course of HMBA treatment. Transcription of the globin genes requires the locus control region (LCR) consisting of several hypersensitive sites (HSs). Erythroid specific transcriptional activators such as NF-E2, GATA-1, TAL1 and EKLF were associated with the LCR in the uninduced state before transcriptional activation. The HSs of the LCR were formed in this state as revealed by high sensitivity to DNase I and MNase attack. However the binding of transcriptional activators and the depletion of histones were observed in the promoter of the beta globin gene only after transcriptional activation. In addition, various covalent histone modifications were sequentially detected in lysine residues of histone H3 during the activation. Acetylation of K9, K36 and K27 was notable in both LCR HSs and gene after induction but before transcriptional initiation. Inactive histone marks such as K9me2, K36me2 and K27me2 were removed coincident with transcriptional initiation in the gene region. Taken together, these results indicate that LCR has a substantially active structure in the uninduced state while transcriptional activation serially adds active marks, including histone modifications, and removes inactive marks in the target gene of the LCR.

  1. The Inflammatory Transcription Factors NFκB, STAT1 and STAT3 Drive Age-Associated Transcriptional Changes in the Human Kidney.

    Science.gov (United States)

    O'Brown, Zach K; Van Nostrand, Eric L; Higgins, John P; Kim, Stuart K

    2015-12-01

    Human kidney function declines with age, accompanied by stereotyped changes in gene expression and histopathology, but the mechanisms underlying these changes are largely unknown. To identify potential regulators of kidney aging, we compared age-associated transcriptional changes in the human kidney with genome-wide maps of transcription factor occupancy from ChIP-seq datasets in human cells. The strongest candidates were the inflammation-associated transcription factors NFκB, STAT1 and STAT3, the activities of which increase with age in epithelial compartments of the renal cortex. Stimulation of renal tubular epithelial cells with the inflammatory cytokines IL-6 (a STAT3 activator), IFNγ (a STAT1 activator), or TNFα (an NFκB activator) recapitulated age-associated gene expression changes. We show that common DNA variants in RELA and NFKB1, the two genes encoding subunits of the NFκB transcription factor, associate with kidney function and chronic kidney disease in gene association studies, providing the first evidence that genetic variation in NFκB contributes to renal aging phenotypes. Our results suggest that NFκB, STAT1 and STAT3 underlie transcriptional changes and chronic inflammation in the aging human kidney.

  2. Transcriptional infidelity promotes heritable phenotypic change in a bistable gene network.

    Directory of Open Access Journals (Sweden)

    Alasdair J E Gordon

    2009-02-01

    Full Text Available Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS. Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour.

  3. Identification of chemicals that mimic transcriptional changes associated with autism, brain aging and neurodegeneration.

    Science.gov (United States)

    Pearson, Brandon L; Simon, Jeremy M; McCoy, Eric S; Salazar, Gabriela; Fragola, Giulia; Zylka, Mark J

    2016-03-31

    Environmental factors, including pesticides, have been linked to autism and neurodegeneration risk using retrospective epidemiological studies. Here we sought to prospectively identify chemicals that share transcriptomic signatures with neurological disorders, by exposing mouse cortical neuron-enriched cultures to hundreds of chemicals commonly found in the environment and on food. We find that rotenone, a pesticide associated with Parkinson's disease risk, and certain fungicides, including pyraclostrobin, trifloxystrobin, famoxadone and fenamidone, produce transcriptional changes in vitro that are similar to those seen in brain samples from humans with autism, advanced age and neurodegeneration (Alzheimer's disease and Huntington's disease). These chemicals stimulate free radical production and disrupt microtubules in neurons, effects that can be reduced by pretreating with a microtubule stabilizer, an antioxidant, or with sulforaphane. Our study provides an approach to prospectively identify environmental chemicals that transcriptionally mimic autism and other brain disorders.

  4. Insulin-Like Growth Factor Binding Protein-4 as a Marker of Chronic Lupus Nephritis.

    Directory of Open Access Journals (Sweden)

    Tianfu Wu

    Full Text Available Kidney biopsy remains the mainstay of Lupus Nephritis (LN diagnosis and prognostication. The objective of this study is to identify non-invasive biomarkers that closely parallel renal pathology in LN. Previous reports have demonstrated that serum Insulin-like growth factor binding protein 4 (IGFBP-4 was increased in diabetic nephropathy in both animal models and patients. We proceeded to assess if IGFBP4 could be associated with LN. We performed ELISA using the serum of 86 patients with LN. Normal healthy adults (N = 23 and patients with other glomerular diseases (N = 20 served as controls. Compared to the healthy controls or other glomerular disease controls, serum IGFBP-4 levels were significantly higher in the patients with LN. Serum IGFBP-4 did not correlate well with systemic lupus erythematosus disease activity index (SLEDAI, renal SLEDAI or proteinuria, but it did correlate with estimated glomerular filtration rate (R = 0.609, P < 0.0001. Interestingly, in 18 patients with proliferative LN whose blood samples were obtained at the time of renal biopsy, serum IGFBP-4 levels correlated strongly with the chronicity index of renal pathology (R = 0.713, P < 0.001. IGFBP-4 emerges a potential marker of lupus nephritis, reflective of renal pathology chronicity changes.

  5. Aspergillus flavus infection induces transcriptional and physical changes in developing maize kernels

    Directory of Open Access Journals (Sweden)

    Andrea L Dolezal

    2014-07-01

    Full Text Available Maize kernels are susceptible to infection by the opportunistic pathogen Aspergillus flavus. Infection results in reduction of grain quality and contamination of kernels with the highly carcinogenic mycotoxin, aflatoxin. To understanding host response to infection by the fungus, transcription of approximately 9,000 maize genes were monitored during the host-pathogen interaction with a custom designed Affymetrix GeneChip® DNA array. More than 1,000 maize genes were found differentially expressed at a fold change of 2 or greater. This included the up regulation of defense related genes and signaling pathways. Transcriptional changes also were observed in primary metabolism genes. Starch biosynthetic genes were down regulated during infection, while genes encoding maize hydrolytic enzymes, presumably involved in the degradation of host reserves, were up regulated. These data indicate that infection of the maize kernel by A. flavus induced metabolic changes in the kernel, including the production of a defense response, as well as a disruption in kernel development.

  6. Expression of insulin-like growth factor binding protein-1 and -2 genes through the perinatal period in the rat.

    Science.gov (United States)

    Babajko, S; Hardouin, S; Segovia, B; Groyer, A; Binoux, M

    1993-06-01

    Insulin-like growth factor binding proteins (IGFBPs) are essential mediators of the bioavailability and biological effects of the IGFs. Liver expression of the rat (r) IGFBP-1 and rIGFBP-2 genes has been characterized between day 16 in utero (16 diu) and 16 days postnatally (+16 dpn). Run-on experiments showed transcriptional activity of the rIGFBP-1 and rIGFBP-2 genes at birth (B) to be 25 and 5 times that at 16 diu, respectively. After B, transcriptional activity of the rIGFBP-1 gene remained high (140% B at +6 dpn), but that of the rIGFBP-2 gene dropped to 70% B by +6 dpn. Northern blot analysis done simultaneously showed rIGFBP-1 messenger RNA (mRNA) levels to increase approximately 50-fold between 16 diu and B, whereas rIGFBP-2 mRNA increased only 5- to 10-fold. rIGFBP-1 mRNA levels decreased after birth, reaching about 20% B by +6 dpn; rIGFBP-2 mRNA, however, remained stable (about 80% B) at least up to +6 dpn. Parallel Western ligand blot and immunoblot analyses of serum rIGFBPs revealed rIGFBP-1 and rIGFBP-2 concentrations to be increased 3- and 2-fold, respectively between 20 diu and B. Maximal expression of rIGFBP-1 was at +1 dpn (220% B), and of rIGFBP-2, at B. Both rIGFBPs then decreased, reaching about 5% B at adulthood. All these data indicate that increased transcriptional activity of the rIGFBP-1 and rIGFBP-2 genes at birth would determine the increased synthesis in the liver and circulating levels of these proteins. In addition, it would seem that post-transcriptional events (reduced half-life of the rIGFBP-1 messenger after birth, translation efficiency of the rIGFBP-2 messenger) modulate transcriptional regulation.

  7. RNA-Seq Reveals OTA-Related Gene Transcriptional Changes in Aspergillus carbonarius

    Science.gov (United States)

    Gerin, Donato; De Miccolis Angelini, Rita M.; Pollastro, Stefania; Faretra, Francesco

    2016-01-01

    Ochratoxin A (OTA) is a mycotoxin harmful for animals and humans. Aspergillus carbonarius is the main responsible for OTA contamination of grapes and derived products. Gene transcriptional profiling of 4 A. carbonarius strains was carried out by RNA-Seq analysis to study transcriptome changes associated with OTA production. By comparing OTA inducing (OTAI) vs. non-inducing (OTAN) cultural conditions, a total of 3,705 differentially expressed genes (DEGs) (fold change > |2| and FDR ≤ 0.05) were identified. Several genes involved in primary metabolic processes, with particular regard to carbohydrate and amino acid metabolisms, secondary metabolic processes, transport, response to stress and sporulation were up-regulated by OTAI conditions at all the analysed sampling times (4, 6 and 8 DAI) or starting from 6 DAI. Highly up-regulated DEGs encoding enzymes involved in biosynthesis of secondary metabolites, oxidoreductases, transporters and transcription factors were examined for their potential involvement in OTA biosynthesis and related metabolic pathways. Differential expression of genes encoding polyketide synthases (pks), non-ribosomal peptide synthetases (nrps) and chloroperoxidase (cpo) was validated by RT-qPCR. Among clusters of co-regulated genes involved in SM biosynthesis, one putative OTA-gene cluster, including both pks and nrps genes, was detected in the A. carbonarius genome. PMID:26765536

  8. Soybean roots grown under heat stress show global changes in their transcriptional and proteomic profiles

    Directory of Open Access Journals (Sweden)

    Oswaldo eValdes-Lopez

    2016-04-01

    Full Text Available Heat stress is likely to be a key factor in the negative impact of climate change on crop production. Heat stress significantly influences the functions of roots, which provide support, water and nutrients to other plant organs. Likewise, roots play an important role in the establishment of symbiotic associations with different microorganisms. Despite the physiological relevance of roots, few studies have examined their response to heat stress. In this study, we performed genome-wide transcriptomic and proteomic analyses on isolated root hairs, which are a single, epidermal cell type, and compared their response to stripped roots. On average, we identified 1,849 and 3,091 genes differentially regulated in root hairs and stripped roots, respectively, in response to heat stress. Our gene regulatory module analysis identified ten key modules that might control the majority of the transcriptional response to heat stress. We also conducted proteomic analysis on membrane fractions isolated from root hairs and compared these responses to stripped roots. These experiments identified a variety of proteins whose expression changed within 3 hours of application of heat stress. Most of these proteins were predicted to play a significant role in thermo-tolerance, as well as in chromatin remodeling and post-transcriptional regulation. The data presented represent an in-depth analysis of the heat stress response of a single cell type in soybean.

  9. Soybean Roots Grown under Heat Stress Show Global Changes in Their Transcriptional and Proteomic Profiles

    Energy Technology Data Exchange (ETDEWEB)

    Valdes-Lopez, Oswaldo; Batek Rios, Josef M.; Gomez-Hernandez, Nicolas; Nguyen, Cuong T.; Isidra-Arellano, Mariel C.; Zhang, Ning; Joshi, Trupti; Xu, Dong; Hixson, Kim K.; Weitz, Karl K.; Aldrich, Joshua T.; Pasa-Tolic, Ljiljana; Stacey, Gary

    2016-04-25

    Heat stress is likely to be a key factor in the negative impact of climate change on crop production. Roots provide support, water and nutrients to other plant organs. Likewise, roots play an important role in the establishment of symbiotic associations with different microorganisms. Despite the physiological relevance of roots, few studies have examined the response of these plant organs to heat stress. In this study, we performed genome-wide transcriptomic and proteomic analyses on isolated root hairs, which are a single, epidermal cell type, and compared their response to whole roots. We identified 2,013 genes differentially regulated in root hairs in response to heat stress. Our gene regulatory module analysis identified ten, key modules that controlled the majority of the transcriptional response to heat stress. We also conducted proteomic analysis on membrane fractions isolated from roots and root hairs. These experiments identified a variety of proteins whose expression changed within 3 hours of application of heat stress. Most of these proteins were predicted to play a role in thermotolerance, as well as in chromatin remodeling and post-transcriptional regulation. The data presented represent an in-depth analysis of the heat stress response of a single cell type in soybean.

  10. RNA-Seq Reveals OTA-Related Gene Transcriptional Changes in Aspergillus carbonarius.

    Science.gov (United States)

    Gerin, Donato; De Miccolis Angelini, Rita M; Pollastro, Stefania; Faretra, Francesco

    2016-01-01

    Ochratoxin A (OTA) is a mycotoxin harmful for animals and humans. Aspergillus carbonarius is the main responsible for OTA contamination of grapes and derived products. Gene transcriptional profiling of 4 A. carbonarius strains was carried out by RNA-Seq analysis to study transcriptome changes associated with OTA production. By comparing OTA inducing (OTAI) vs. non-inducing (OTAN) cultural conditions, a total of 3,705 differentially expressed genes (DEGs) (fold change > |2| and FDR ≤ 0.05) were identified. Several genes involved in primary metabolic processes, with particular regard to carbohydrate and amino acid metabolisms, secondary metabolic processes, transport, response to stress and sporulation were up-regulated by OTAI conditions at all the analysed sampling times (4, 6 and 8 DAI) or starting from 6 DAI. Highly up-regulated DEGs encoding enzymes involved in biosynthesis of secondary metabolites, oxidoreductases, transporters and transcription factors were examined for their potential involvement in OTA biosynthesis and related metabolic pathways. Differential expression of genes encoding polyketide synthases (pks), non-ribosomal peptide synthetases (nrps) and chloroperoxidase (cpo) was validated by RT-qPCR. Among clusters of co-regulated genes involved in SM biosynthesis, one putative OTA-gene cluster, including both pks and nrps genes, was detected in the A. carbonarius genome.

  11. RNA-Seq Reveals OTA-Related Gene Transcriptional Changes in Aspergillus carbonarius.

    Directory of Open Access Journals (Sweden)

    Donato Gerin

    Full Text Available Ochratoxin A (OTA is a mycotoxin harmful for animals and humans. Aspergillus carbonarius is the main responsible for OTA contamination of grapes and derived products. Gene transcriptional profiling of 4 A. carbonarius strains was carried out by RNA-Seq analysis to study transcriptome changes associated with OTA production. By comparing OTA inducing (OTAI vs. non-inducing (OTAN cultural conditions, a total of 3,705 differentially expressed genes (DEGs (fold change > |2| and FDR ≤ 0.05 were identified. Several genes involved in primary metabolic processes, with particular regard to carbohydrate and amino acid metabolisms, secondary metabolic processes, transport, response to stress and sporulation were up-regulated by OTAI conditions at all the analysed sampling times (4, 6 and 8 DAI or starting from 6 DAI. Highly up-regulated DEGs encoding enzymes involved in biosynthesis of secondary metabolites, oxidoreductases, transporters and transcription factors were examined for their potential involvement in OTA biosynthesis and related metabolic pathways. Differential expression of genes encoding polyketide synthases (pks, non-ribosomal peptide synthetases (nrps and chloroperoxidase (cpo was validated by RT-qPCR. Among clusters of co-regulated genes involved in SM biosynthesis, one putative OTA-gene cluster, including both pks and nrps genes, was detected in the A. carbonarius genome.

  12. Systematic repression of transcription factors reveals limited patterns of gene expression changes in ES cells

    Science.gov (United States)

    Nishiyama, Akira; Sharov, Alexei A.; Piao, Yulan; Amano, Misa; Amano, Tomokazu; Hoang, Hien G.; Binder, Bernard Y.; Tapnio, Richard; Bassey, Uwem; Malinou, Justin N.; Correa-Cerro, Lina S.; Yu, Hong; Xin, Li; Meyers, Emily; Zalzman, Michal; Nakatake, Yuhki; Stagg, Carole; Sharova, Lioudmila; Qian, Yong; Dudekula, Dawood; Sheer, Sarah; Cadet, Jean S.; Hirata, Tetsuya; Yang, Hsih-Te; Goldberg, Ilya; Evans, Michele K.; Longo, Dan L.; Schlessinger, David; Ko, Minoru S. H.

    2013-01-01

    Networks of transcription factors (TFs) are thought to determine and maintain the identity of cells. Here we systematically repressed each of 100 TFs with shRNA and carried out global gene expression profiling in mouse embryonic stem (ES) cells. Unexpectedly, only the repression of a handful of TFs significantly affected transcriptomes, which changed in two directions/trajectories: one trajectory by the repression of either Pou5f1 or Sox2; the other trajectory by the repression of either Esrrb, Sall4, Nanog, or Tcfap4. The data suggest that the trajectories of gene expression change are already preconfigured by the gene regulatory network and roughly correspond to extraembryonic and embryonic fates of cell differentiation, respectively. These data also indicate the robustness of the pluripotency gene network, as the transient repression of most TFs did not alter the transcriptomes. PMID:23462645

  13. RNA-SEQ reveals transcriptional level changes of poplar roots in different forms of nitrogen treatments

    Directory of Open Access Journals (Sweden)

    Chunpu eQu

    2016-02-01

    Full Text Available Poplar has emerged as a model plant for understanding molecular mechanisms of tree growth, development and response to environment. Long-term application of different forms of nitrogen (such as NO3--N and NH4+-N may cause morphological changes of poplar roots; however, the molecular level changes are still not well known. In this study, we analyzed the expression profiling of poplar roots treated by three forms of nitrogen: S1 (NH4+, S2 (NH4NO3 and S3 (NO3- by using RNA-SEQ technique. We found 463 genes significantly differentially expressed in roots by different N treatments, of which a total of 116 genes were found to differentially express between S1 and S2, 173 genes between S2 and S3, and 327 genes between S1 and S3. A cluster analysis shows significant difference in many transcription factor families and functional genes family under different N forms. Through an analysis of Mapman metabolic pathway, we found that the significantly differentially expressed genes are associated with fermentation, glycolysis and tricarboxylic acid cycle (TCA, secondary metabolism, hormone metabolism, and transport processing. Interestingly, we did not find significantly differentially expressed genes in N metabolism pathway, mitochondrial electron transport / ATP synthesis and mineral nutrition. We also found abundant candidate genes (20 transcription factors and 30 functional genes regulating morphology changes of poplar roots under the three N forms. The results obtained are beneficial to a better understanding of the potential molecular and cellular mechanisms regulating root morphology changes under different N treatments.

  14. Transcriptional changes in canine distemper virus-induced demyelinating leukoencephalitis favor a biphasic mode of demyelination.

    Science.gov (United States)

    Ulrich, Reiner; Puff, Christina; Wewetzer, Konstantin; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang

    2014-01-01

    Canine distemper virus (CDV)-induced demyelinating leukoencephalitis in dogs (Canis familiaris) is suggested to represent a naturally occurring translational model for subacute sclerosing panencephalitis and multiple sclerosis in humans. The aim of this study was a hypothesis-free microarray analysis of the transcriptional changes within cerebellar specimens of five cases of acute, six cases of subacute demyelinating, and three cases of chronic demyelinating and inflammatory CDV leukoencephalitis as compared to twelve non-infected control dogs. Frozen cerebellar specimens were used for analysis of histopathological changes including demyelination, transcriptional changes employing microarrays, and presence of CDV nucleoprotein RNA and protein using microarrays, RT-qPCR and immunohistochemistry. Microarray analysis revealed 780 differentially expressed probe sets. The dominating change was an up-regulation of genes related to the innate and the humoral immune response, and less distinct the cytotoxic T-cell-mediated immune response in all subtypes of CDV leukoencephalitis as compared to controls. Multiple myelin genes including myelin basic protein and proteolipid protein displayed a selective down-regulation in subacute CDV leukoencephalitis, suggestive of an oligodendrocyte dystrophy. In contrast, a marked up-regulation of multiple immunoglobulin-like expressed sequence tags and the delta polypeptide of the CD3 antigen was observed in chronic CDV leukoencephalitis, in agreement with the hypothesis of an immune-mediated demyelination in the late inflammatory phase of the disease. Analysis of pathways intimately linked to demyelination as determined by morphometry employing correlation-based Gene Set Enrichment Analysis highlighted the pathomechanistic importance of up-regulated genes comprised by the gene ontology terms "viral replication" and "humoral immune response" as well as down-regulated genes functionally related to "metabolite and energy generation".

  15. Transcriptional changes in canine distemper virus-induced demyelinating leukoencephalitis favor a biphasic mode of demyelination.

    Directory of Open Access Journals (Sweden)

    Reiner Ulrich

    Full Text Available Canine distemper virus (CDV-induced demyelinating leukoencephalitis in dogs (Canis familiaris is suggested to represent a naturally occurring translational model for subacute sclerosing panencephalitis and multiple sclerosis in humans. The aim of this study was a hypothesis-free microarray analysis of the transcriptional changes within cerebellar specimens of five cases of acute, six cases of subacute demyelinating, and three cases of chronic demyelinating and inflammatory CDV leukoencephalitis as compared to twelve non-infected control dogs. Frozen cerebellar specimens were used for analysis of histopathological changes including demyelination, transcriptional changes employing microarrays, and presence of CDV nucleoprotein RNA and protein using microarrays, RT-qPCR and immunohistochemistry. Microarray analysis revealed 780 differentially expressed probe sets. The dominating change was an up-regulation of genes related to the innate and the humoral immune response, and less distinct the cytotoxic T-cell-mediated immune response in all subtypes of CDV leukoencephalitis as compared to controls. Multiple myelin genes including myelin basic protein and proteolipid protein displayed a selective down-regulation in subacute CDV leukoencephalitis, suggestive of an oligodendrocyte dystrophy. In contrast, a marked up-regulation of multiple immunoglobulin-like expressed sequence tags and the delta polypeptide of the CD3 antigen was observed in chronic CDV leukoencephalitis, in agreement with the hypothesis of an immune-mediated demyelination in the late inflammatory phase of the disease. Analysis of pathways intimately linked to demyelination as determined by morphometry employing correlation-based Gene Set Enrichment Analysis highlighted the pathomechanistic importance of up-regulated genes comprised by the gene ontology terms "viral replication" and "humoral immune response" as well as down-regulated genes functionally related to "metabolite and energy

  16. Genome-wide transcriptional changes induced by phagocytosis or growth on bacteria in Dictyostelium

    Directory of Open Access Journals (Sweden)

    Peracino Barbara

    2008-06-01

    Full Text Available Abstract Background Phagocytosis plays a major role in the defense of higher organisms against microbial infection and provides also the basis for antigen processing in the immune response. Cells of the model organism Dictyostelium are professional phagocytes that exploit phagocytosis of bacteria as the preferred way to ingest food, besides killing pathogens. We have investigated Dictyostelium differential gene expression during phagocytosis of non-pathogenic bacteria, using DNA microarrays, in order to identify molecular functions and novel genes involved in phagocytosis. Results The gene expression profiles of cells incubated for a brief time with bacteria were compared with cells either incubated in axenic medium or growing on bacteria. Transcriptional changes during exponential growth in axenic medium or on bacteria were also compared. We recognized 443 and 59 genes that are differentially regulated by phagocytosis or by the different growth conditions (growth on bacteria vs. axenic medium, respectively, and 102 genes regulated by both processes. Roughly one third of the genes are up-regulated compared to macropinocytosis and axenic growth. Functional annotation of differentially regulated genes with different tools revealed that phagocytosis induces profound changes in carbohydrate, aminoacid and lipid metabolism, and in cytoskeletal components. Genes regulating translation and mitochondrial biogenesis are mostly up-regulated. Genes involved in sterol biosynthesis are selectively up-regulated, suggesting a shift in membrane lipid composition linked to phagocytosis. Very few changes were detected in genes required for vesicle fission/fusion, indicating that the intracellular traffic machinery is mostly in common between phagocytosis and macropinocytosis. A few putative receptors, including GPCR family 3 proteins, scaffolding and adhesion proteins, components of signal transduction and transcription factors have been identified, which could

  17. Luteolin modulates 6-hydroxydopamine-induced transcriptional changes of stress response pathways in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Ling-Wei Hu

    Full Text Available The neurotoxin 6-hydroxydopamine (6-OHDA, which causes transcriptional changes associated with oxidative and proteotoxic stress, has been widely used to generate an experimental model of Parkinson's disease. The food-derived compound luteolin has multi-target actions including antioxidant, anti-inflammatory and neurotrophic activities. The aim of this study is to investigate how luteolin affects 6-OHDA-mediated stress response pathways. The results showed that when PC12 cells were pre-treated with luteolin (20 µM 30 min prior to 6-OHDA (100 µM exposure, 6-OHDA-induced ROS overproduction, cytotoxicity, caspase-3 activation, and mRNA expression of BIM, TRB3 and GADD34 were significantly attenuated. Moreover, 6-OHDA-mediated cell cycle arrest and transcription of p53 target genes, p21, GADD45α and PUMA, were reduced by luteolin. Luteolin also significantly down-regulated 6-OHDA-mediated unfolded protein response (UPR, leading to decreases in phospho-eIF2α, ATF4, GRP78 and CHOP. In addition, luteolin attenuated 6-OHDA-induced Nrf2-mediated HO-1 and GCLC. Taken together, these results suggest that diminishing intracellular ROS formation and down-regulation of p53, UPR and Nrf2-ARE pathways may be involved in the neuroprotective effect of luteolin.

  18. In silico and wet lab approaches to study transcriptional regulation

    NARCIS (Netherlands)

    Hestand, Matthew Scott

    2010-01-01

    Gene expression is a complicated process with multiple types of regulation, including binding of proteins termed transcription factors. This thesis looks at transcription factors and transcription factor binding site discovery through computational predictions and wet lab work to better elucidate th

  19. Water deficit-induced changes in transcription factor expression in maize seedlings

    Science.gov (United States)

    Plants tolerate water deficits by regulating gene networks controlling cellular and physiological traits to modify growth and development. Transcription factor (TFs) directed regulation of transcription within these gene networks is key to eliciting appropriate responses. In this study, reverse tran...

  20. Insulin-like growth factor binding protein-5 influences pancreatic cancer cell growth

    Institute of Scientific and Technical Information of China (English)

    Sarah K Johnson; Randy S Haun

    2009-01-01

    AIM: To investigate the functional significance of insulin-like growth factor binding protein-5 (IGFBP-5) overexpression in pancreatic cancer (PaC).METHODS: The effects of IGFBP-5 on cell growth were assessed by stable transfection of BxPC-3 and PANC-1 cell lines and measuring cell number and DNA synthesis. Alterations in the cell cycle were assessed by flow cytometry and immunoblot analyses.Changes in cell survival and signal transduction were evaluated after mitogen activated protein kinase and phosphatidylinositol 3-kinase (PI3K) inhibitor treatment.RESULTS: After serum depr ivat ion, IGFBP-5 expression increased both cell number and DNA synthesis in BxPC-3 cells, but reduced cell number in PANC-1 cells. Consistent with this observation, cell cycle analysis of IGFBP-5-expressing cells revealed accelerated cell cycle progression in BxPC-3 and G2/M arrest of PANC-1 cells. Signal transduction analysis revealed that Akt activation was increased in BxPC-3, but reduced in PANC-1 cells that express IGFBP-5. Inhibition of PI3K with LY294002 suppressed extracellular signal-regulated kinase-1 and -2 (ERK1/2) activation in BxPC-3, but enhanced ERK1/2 activation in PANC-1 cells that express IGFBP-5. When MEK1/2 was blocked, Akt activation remained elevated in IGFBP-5 expressing PaC cells; however, inhibition of PI3K or MEK1/2 abrogated IGFBP-5-mediated cell survival.CONCLUSION: These results indicate that IGFBP-5 expression affects the cell cycle and survival signal pathways and thus it may be an important mediator of PaC cell growth.

  1. High fat diet-induced changes of mouse hepatic transcription and enhancer activity can be reversed by subsequent weight loss

    DEFF Research Database (Denmark)

    Siersbæk, Majken; Varticovski, Lyuba; Yang, Shutong;

    2017-01-01

    Abstract Epigenetic factors have been suggested to play an important role in metabolic memory by trapping and maintaining initial metabolic changes within the transcriptional regulatory machinery. In this study we fed mice a high fat diet (HFD) for seven weeks followed by additional five weeks...... of chow, to identify HFD-mediated changes to the hepatic transcriptional program that may persist after weight loss. Mice fed a HFD displayed increased fasting insulin levels, hepatosteatosis and major changes in hepatic gene transcription associated with modulation of H3K27Ac at enhancers...... for efficient treatment of early obesity-associated changes to hepatic complications by simple weight loss intervention without persistent reprograming of the liver transcriptome....

  2. Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation.

    Science.gov (United States)

    Fraser, James; Ferrai, Carmelo; Chiariello, Andrea M; Schueler, Markus; Rito, Tiago; Laudanno, Giovanni; Barbieri, Mariano; Moore, Benjamin L; Kraemer, Dorothee C A; Aitken, Stuart; Xie, Sheila Q; Morris, Kelly J; Itoh, Masayoshi; Kawaji, Hideya; Jaeger, Ines; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R R; Semple, Colin A; Dostie, Josée; Pombo, Ana; Nicodemi, Mario

    2015-12-23

    Mammalian chromosomes fold into arrays of megabase-sized topologically associating domains (TADs), which are arranged into compartments spanning multiple megabases of genomic DNA. TADs have internal substructures that are often cell type specific, but their higher-order organization remains elusive. Here, we investigate TAD higher-order interactions with Hi-C through neuronal differentiation and show that they form a hierarchy of domains-within-domains (metaTADs) extending across genomic scales up to the range of entire chromosomes. We find that TAD interactions are well captured by tree-like, hierarchical structures irrespective of cell type. metaTAD tree structures correlate with genetic, epigenomic and expression features, and structural tree rearrangements during differentiation are linked to transcriptional state changes. Using polymer modelling, we demonstrate that hierarchical folding promotes efficient chromatin packaging without the loss of contact specificity, highlighting a role far beyond the simple need for packing efficiency.

  3. DksA involvement in transcription fidelity buffers stochastic epigenetic change.

    Science.gov (United States)

    Satory, Dominik; Gordon, Alasdair J E; Wang, Mengyu; Halliday, Jennifer A; Golding, Ido; Herman, Christophe

    2015-12-01

    DksA is an auxiliary transcription factor that interacts with RNA polymerase and influences gene expression. Depending on the promoter, DksA can be a positive or negative regulator of transcription initiation. Moreover, DksA has a substantial effect on transcription elongation where it prevents the collision of transcription and replication machineries, plays a key role in maintaining transcription elongation when translation and transcription are uncoupled and has been shown to be involved in transcription fidelity. Here, we assessed the role of DksA in transcription fidelity by monitoring stochastic epigenetic switching in the lac operon (with and without an error-prone transcription slippage sequence), partial phenotypic suppression of a lacZ nonsense allele, as well as monitoring the number of lacI mRNA transcripts produced in the presence and absence of DksA via an operon fusion and single molecule fluorescent in situ hybridization studies. We present data showing that DksA acts to maintain transcription fidelity in vivo and the role of DksA seems to be distinct from that of the GreA and GreB transcription fidelity factors.

  4. High fat diet-induced changes of mouse hepatic transcription and enhancer activity can be reversed by subsequent weight loss.

    Science.gov (United States)

    Siersbæk, Majken; Varticovski, Lyuba; Yang, Shutong; Baek, Songjoon; Nielsen, Ronni; Mandrup, Susanne; Hager, Gordon L; Chung, Jay H; Grøntved, Lars

    2017-01-10

    Epigenetic factors have been suggested to play an important role in metabolic memory by trapping and maintaining initial metabolic changes within the transcriptional regulatory machinery. In this study we fed mice a high fat diet (HFD) for seven weeks followed by additional five weeks of chow, to identify HFD-mediated changes to the hepatic transcriptional program that may persist after weight loss. Mice fed a HFD displayed increased fasting insulin levels, hepatosteatosis and major changes in hepatic gene transcription associated with modulation of H3K27Ac at enhancers, but no significant changes in chromatin accessibility, indicating that HFD-regulated gene transcription is primarily controlled by modulating the activity of pre-established enhancers. After return to the same body weight as chow fed control mice, the fasting insulin, glucose, and hepatic triglyceride levels were fully restored to normal levels. Moreover, HFD-regulated H3K27Ac and mRNA levels returned to similar levels as control mice. These data demonstrates that the transcription regulatory landscape in the liver induced by HFD is highly dynamic and can be reversed by weight loss. This provides hope for efficient treatment of early obesity-associated changes to hepatic complications by simple weight loss intervention without persistent reprograming of the liver transcriptome.

  5. Gene Transcriptional and Metabolic Profile Changes in Mimetic Aging Mice Induced by D-Galactose.

    Directory of Open Access Journals (Sweden)

    Yue-Yue Zhou

    Full Text Available D-galactose injection has been shown to induce many changes in mice that represent accelerated aging. This mouse model has been widely used for pharmacological studies of anti-aging agents. The underlying mechanism of D-galactose induced aging remains unclear, however, it appears to relate to glucose and 1ipid metabolic disorders. Currently, there has yet to be a study that focuses on investigating gene expression changes in D-galactose aging mice. In this study, integrated analysis of gas chromatography/mass spectrometry-based metabonomics and gene expression profiles was used to investigate the changes in transcriptional and metabolic profiles in mimetic aging mice injected with D-galactose. Our findings demonstrated that 48 mRNAs were differentially expressed between control and D-galactose mice, and 51 potential biomarkers were identified at the metabolic level. The effects of D-galactose on aging could be attributed to glucose and 1ipid metabolic disorders, oxidative damage, accumulation of advanced glycation end products (AGEs, reduction in abnormal substance elimination, cell apoptosis, and insulin resistance.

  6. High fat diet-induced changes of mouse hepatic transcription and enhancer activity can be reversed by subsequent weight loss

    DEFF Research Database (Denmark)

    Siersbæk, Majken; Varticovski, Lyuba; Yang, Shutong

    2017-01-01

    of chow, to identify HFD-mediated changes to the hepatic transcriptional program that may persist after weight loss. Mice fed a HFD displayed increased fasting insulin levels, hepatosteatosis and major changes in hepatic gene transcription associated with modulation of H3K27Ac at enhancers...... fully restored to normal levels. Moreover, HFD-regulated H3K27Ac and mRNA levels returned to similar levels as control mice. These data demonstrates that the transcription regulatory landscape in the liver induced by HFD is highly dynamic and can be reversed by weight loss. This provides hope...... for efficient treatment of early obesity-associated changes to hepatic complications by simple weight loss intervention without persistent reprograming of the liver transcriptome....

  7. Transcriptional changes of cytokines in rooster testis and epididymis during sexual maturation stages and Salmonella infection.

    Science.gov (United States)

    Anastasiadou, M; Michailidis, G

    2016-08-01

    Infection of rooster testis and epididymis by pathogens can lead to impaired fertility, resulting in economic losses in the poultry industry. Antimicrobial protection of rooster reproductive organs is, therefore, an important aspect of reproductive physiology. Salmonellosis is one of the most important zoonotic diseases, caused by Salmonella bacteria including Salmonella Enteritidis (SE) and is usually the result of infection of the reproductive organs. Thus, knowledge of the endogenous innate immune mechanisms of the rooster testis and epididymis is an emerging aspect of reproductive physiology. Cytokines are key factors for stimulating the immune response and inflammation in chickens to Salmonella infection. In the present study the expression profile of 11 pro-inflammatory cytokine genes in the rooster testis and epididymis in vivo and transcriptional changes in these organs during sexual maturation and SE infection were investigated. Gene expression analysis data revealed that in both testis and epididymis nine cytokines namely the IL-1β, IL-6, IL-8, IL-10, IL-12, IL-15, IL-16, IL-17 and IL-18 genes were expressed, while no mRNA transcripts were detected in both organs for IL-2 and IL-4. Furthermore, the expression of various cytokine genes during sexual maturation appeared to be developmentally regulated, while SE infection resulted in a significant up-regulation of IL-1β, -6, -12 and -18 genes in the testis and an increase in the mRNA relative abundance of IL-1β, -6, -12, -16 and -18 in the epididymis of SE-infected sexually mature 28-week-old roosters. These results suggest a cytokine-mediated immune response mechanism against Salmonella infection in the rooster reproductive tract.

  8. Erythroid-specific transcriptional changes in PBMCs from pulmonary hypertension patients.

    Directory of Open Access Journals (Sweden)

    Chris Cheadle

    Full Text Available BACKGROUND: Gene expression profiling of peripheral blood mononuclear cells (PBMCs is a powerful tool for the identification of surrogate markers involved in disease processes. The hypothesis tested in this study was that chronic exposure of PBMCs to a hypertensive environment in remodeled pulmonary vessels would be reflected by specific transcriptional changes in these cells. METHODOLOGY/PRINCIPAL FINDINGS: The transcript profiles of PBMCs from 30 idiopathic pulmonary arterial hypertension patients (IPAH, 19 patients with systemic sclerosis without pulmonary hypertension (SSc, 42 scleroderma-associated pulmonary arterial hypertensio patients (SSc-PAH, and 8 patients with SSc complicated by interstitial lung disease and pulmonary hypertension (SSc-PH-ILD were compared to the gene expression profiles of PBMCs from 41 healthy individuals. Multiple gene expression signatures were identified which could distinguish various disease groups from controls. One of these signatures, specific for erythrocyte maturation, is enriched specifically in patients with PH. This association was validated in multiple published datasets. The erythropoiesis signature was strongly correlated with hemodynamic measures of increasing disease severity in IPAH patients. No significant correlation of the same type was noted for SSc-PAH patients, this despite a clear signature enrichment within this group overall. These findings suggest an association of the erythropoiesis signature in PBMCs from patients with PH with a variable presentation among different subtypes of disease. CONCLUSIONS/SIGNIFICANCE: In PH, the expansion of immature red blood cell precursors may constitute a response to the increasingly hypoxic conditions prevalent in this syndrome. A correlation of this erythrocyte signature with more severe hypertension cases may provide an important biomarker of disease progression.

  9. Differential expression of serum glycodelin and insulin-like growth factor binding protein 1 in early pregnancy.

    Science.gov (United States)

    Douglas, Nataki C; Thornton, Melvin H; Nurudeen, Sahadat K; Bucur, Maria; Lobo, Rogerio A; Sauer, Mark V

    2013-11-01

    This prospective study evaluated whether serum glycodelin and insulin-like growth factor binding protein 1 (IGFBP-1) predict the likelihood of embryo implantation in recipients undergoing donor egg in vitro fertilization. We measured glycodelin and IGFBP-1 at 6 points from lining check to lutenizing hormone (LH) + 31. β-Human chorionic gonadotropin levels were first measured at LH + 17. The recipients were divided into those without embryo implantation (group 1, n = 6) and those with successful implantation (group 2, n = 30). Although this is a negative study in that neither glycodelin nor IGFBP-1 alone reflected endometrial (EM) receptivity, the glycodelin/IGFBP-1 ratio on the day of blastocyst transfer was higher in recipients who achieved pregnancy (P = .05). At LH + 17, glycodelin was higher (P = .04), and IGFBP-1 was lower (P = .004) in recipients who achieved pregnancy when compared to those who did not. These observations are likely due to EM changes induced by successful embryo implantation.

  10. Stable Binding of the Conserved Transcription Factor Grainy Head to its Target Genes Throughout Drosophila melanogaster Development.

    Science.gov (United States)

    Nevil, Markus; Bondra, Eliana R; Schulz, Katharine N; Kaplan, Tommy; Harrison, Melissa M

    2017-02-01

    It has been suggested that transcription factor binding is temporally dynamic, and that changes in binding determine transcriptional output. Nonetheless, this model is based on relatively few examples in which transcription factor binding has been assayed at multiple developmental stages. The essential transcription factor Grainy head (Grh) is conserved from fungi to humans, and controls epithelial development and barrier formation in numerous tissues. Drosophila melanogaster, which possess a single grainy head (grh) gene, provide an excellent system to study this conserved factor. To determine whether temporally distinct binding events allow Grh to control cell fate specification in different tissue types, we used a combination of ChIP-seq and RNA-seq to elucidate the gene regulatory network controlled by Grh during four stages of embryonic development (spanning stages 5-17) and in larval tissue. Contrary to expectations, we discovered that Grh remains bound to at least 1146 genomic loci over days of development. In contrast to this stable DNA occupancy, the subset of genes whose expression is regulated by Grh varies. Grh transitions from functioning primarily as a transcriptional repressor early in development to functioning predominantly as an activator later. Our data reveal that Grh binds to target genes well before the Grh-dependent transcriptional program commences, suggesting it sets the stage for subsequent recruitment of additional factors that execute stage-specific Grh functions.

  11. Evaluating Transcription Factor Activity Changes by Scoring Unexplained Target Genes in Expression Data

    Science.gov (United States)

    Berchtold, Evi; Csaba, Gergely; Zimmer, Ralf

    2016-01-01

    Several methods predict activity changes of transcription factors (TFs) from a given regulatory network and measured expression data. But available gene regulatory networks are incomplete and contain many condition-dependent regulations that are not relevant for the specific expression measurement. It is not known which combination of active TFs is needed to cause a change in the expression of a target gene. A method to systematically evaluate the inferred activity changes is missing. We present such an evaluation strategy that indicates for how many target genes the observed expression changes can be explained by a given set of active TFs. To overcome the problem that the exact combination of active TFs needed to activate a gene is typically not known, we assume a gene to be explained if there exists any combination for which the predicted active TFs can possibly explain the observed change of the gene. We introduce the i-score (inconsistency score), which quantifies how many genes could not be explained by the set of activity changes of TFs. We observe that, even for these minimal requirements, published methods yield many unexplained target genes, i.e. large i-scores. This holds for all methods and all expression datasets we evaluated. We provide new optimization methods to calculate the best possible (minimal) i-score given the network and measured expression data. The evaluation of this optimized i-score on a large data compendium yields many unexplained target genes for almost every case. This indicates that currently available regulatory networks are still far from being complete. Both the presented Act-SAT and Act-A* methods produce optimal sets of TF activity changes, which can be used to investigate the difficult interplay of expression and network data. A web server and a command line tool to calculate our i-score and to find the active TFs associated with the minimal i-score is available from https://services.bio.ifi.lmu.de/i-score. PMID:27723775

  12. Identification of identical transcript changes in liver and whole blood during acetaminophen toxicity

    Directory of Open Access Journals (Sweden)

    Liwen eZhang

    2012-09-01

    Full Text Available Abstract The ability to identify mechanisms underlying drug-induced liver injury (DILI in man has been hampered by the difficulty in obtaining liver tissue from patients. It has recently been proposed that whole blood toxicogenomics may provide a noninvasive means for mechanistic studies of human DILI. However, it remains unclear to what extent changes in whole blood transcriptome mirror those in liver mechanistically linked to hepatotoxicity. To address this question, we applied the program Extracting Patterns and Identifying co-expressed Genes (EPIG to publically available toxicogenomic data obtained from rats treated with both toxic and subtoxic doses of acetaminophen (APAP. In a training set of animals, we identified genes (760 at 6 h and 185 at 24 h post dose with similar patterns of expression in blood and liver during APAP induced hepatotoxicity. The pathways represented in the coordinately regulated genes largely involved mitochondrial and immune functions. The identified expression signatures were then evaluated in a separate set of animals for discernment of APAP exposure level or APAP induced hepatotoxicity. At 6 h, the gene sets from liver and blood had equally sufficient classification of APAP exposure levels. At 24 h when toxicity was evident, the gene sets did not perform well in evaluating APAP exposure doses, but provided accurate classification of dose-independent liver injury that was evaluated by serum ALT elevation in the blood. Only thirty eight genes were common to both the 6 and 24h gene sets, but these genes had the same capability as the parent gene sets to discern the exposure level and degree of liver injury. Some of the parallel transcript changes reflect pathways that are relevant to APAP hepatotoxicity, including mitochondria and immune functions. However, the extent to which these changes reflect similar mechanisms of action in both tissues remains to be determined.

  13. Transcriptional changes in Crassostrea gigas oyster spat following a parental exposure to the herbicide diuron

    Energy Technology Data Exchange (ETDEWEB)

    Rondon, R. [Ifremer, IHPE UMR 5244, Univ. Perpignan Via Domitia, CNRS, Univ. Montpellier, F-34095 Montpellier (France); Univ. Perpignan Via Domitia, IHPE UMR 5244, CNRS, IFREMER, Univ. Montpellier, F-66860 Perpignan (France); Akcha, F. [Ifremer, Department of Biogeochemistry and Ecotoxicology, Laboratory of Ecotoxicology, Rue de l’ile d’Yeu, BP 21105, 44311 Nantes Cedex 03 (France); Alonso, P. [CNRS, IHPE UMR 5244, Univ. Perpignan Via Domitia, IFREMER, Univ. Montpellier, F-34095 Montpellier (France); Menard, D.; Rouxel, J. [Ifremer, Department of Biogeochemistry and Ecotoxicology, Laboratory of Ecotoxicology, Rue de l’ile d’Yeu, BP 21105, 44311 Nantes Cedex 03 (France); Montagnani, C., E-mail: cmontagn@ifremer.fr [Ifremer, IHPE UMR 5244, Univ. Perpignan Via Domitia, CNRS, Univ. Montpellier, F-34095 Montpellier (France); Mitta, G.; Cosseau, C.; Grunau, C. [Univ. Perpignan Via Domitia, IHPE UMR 5244, CNRS, IFREMER, Univ. Montpellier, F-66860 Perpignan (France)

    2016-06-15

    Highlights: • Remodeling of the transcriptome of the offspring of oysters exposed to diuron (838 gene which expression is modulated). • Functions related to cytoskeleton organization, translation, ATP synthesis were activated. • Functions linked to transcription and protein degradation were altered. • Up-regulation of genes involved in energy production, protein synthesis and mitosis. • Catch-up growth phenomenon could allow the spats to compensate for slower growth. - Abstract: The Pacific oyster Crassostrea gigas is the main oyster species produced in the world, and a key coastal economic resource in France. High mortalities affect Pacific oysters since 2008 in France and Europe. Their origins have been attributed to a combination of biotic and abiotic factors, underlining the importance of environment quality. The impact of water pollution has been pointed out and one of the pollutants, the genotoxic herbicide diuron, occurs at high concentrations all along the French coasts. Previous work has revealed that a parental exposure to diuron had a strong impact on hatching rates and offspring development even if spats were not exposed to diuron themselves. In this study, we explored for the first time the transcriptional changes occurring in oyster spats (non exposed) originating from genitors exposed to an environmentally relevant concentration of diuron during gametogenesis using the RNAseq methodology. We identified a transcriptomic remodeling revealing an effect of the herbicide. Different molecular pathways involved in energy production, translation and cell proliferation are particularly disturbed. This analysis revealed modulated candidate genes putatively involved in response to oxidative stress and mitochondrial damage in offspring of genitors exposed to diuron. Complementary measures of the activity of enzymes involved in these latter processes corroborate the results obtained at the transcriptomic level. In addition, our results suggested an

  14. Behavioural and transcriptional changes in the amphipod Echinogammarus marinus exposed to two antidepressants, fluoxetine and sertraline.

    Science.gov (United States)

    Bossus, Maryline C; Guler, Yasmin Z; Short, Stephen J; Morrison, Edward R; Ford, Alex T

    2014-06-01

    In the past decade, there have been increasing concerns over the effects of pharmaceutical compounds in the aquatic environment, however very little is known about the effects of antidepressants such as the selective serotonin re-uptake inhibitors (SSRIs). Many biological functions within invertebrates are under the control of serotonin, such as reproduction, metabolism, moulting and behaviour. The effects of serotonin and fluoxetine have recently been shown to alter the behaviour of the marine amphipod, Echinogammarus marinus (Leach, 1815). The purpose of this study was to observe behavioural and transcriptional modifications in this crustacean exposed to the two most prescribed SSRIs (fluoxetine and sertraline) and to develop biomarkers of neurological endocrine disruption. The animals were exposed to both drugs at environmentally relevant concentrations from 0.001 to 1μg/L during short-term (1h and 1day) and medium-term (8 days) experiments. The movement of the amphipods was tracked using the behavioural analysis software during 12min alternating dark/light conditions. The behavioural analysis revealed a significant effect on velocity which was observed after 1h exposure to sertraline at 0.01μg/L and after 1 day exposure to fluoxetine as low as 0.001μg/L. The most predominant effect of drugs on velocity was recorded after 1 day exposure for the 0.1 and 0.01μg/L concentrations of fluoxetine and sertraline, respectively. Subsequently, the expression (in this article gene expression is taken to represent only transcription, although it is acknowledged that gene expression can also be regulated at translation, mRNA and protein stability levels) of several E. marinus neurological genes, potentially involved in the serotonin metabolic pathway or behaviour regulation, were analysed in animals exposed to various SSRIs concentrations using RT-qPCR. The expression of a tryptophan hydroxylase (Ph), a neurocan core protein (Neuc), a Rhodopsin (Rhod1) and an Arrestin

  15. Environmental cues induce changes of steviol glycosides contents and transcription of corresponding biosynthetic genes in Stevia rebaudiana.

    Science.gov (United States)

    Yang, Yongheng; Huang, Suzhen; Han, Yulin; Yuan, Haiyan; Gu, Chunsun; Wang, Zhongwei

    2015-01-01

    Plant growth and secondary metabolism are commonly regulated by external cues such as light, temperature and water availability. In this study, the influences of low and high temperatures, dehydration, photoperiods, and different growing stages on the changes of steviol glycosides (SGs) contents and transcription levels of fifteen genes involved in SGs biosynthesis of Stevia rebaudiana Bertoni were examined using HPLC and RT-PCR. The observations showed that the transcript levels of all the fifteen genes were maximum under 25 °C treatment, and the transcription of SrDXS, SrDXR, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI, SrGGDPS, SrCPPS1, SrUGT85C2 and SrUGT76G1 were restrained both in low temperature (15 °C) and high temperature (35 °C). Most genes in SGs biosynthesis pathway exhibited down-regulation in dehydration. To elucidate the effect of photoperiods, the plants were treated by different simulated photoperiods (8 L/16 D, 1 0L/14 D, 14 L/10 D and 16 L/8 D), but no significant transcription changes were observed. In the study of growing stages, there were evident changes of SGs contents, and the transcript levels of all the fifteen genes were minimal in fast growing period, and exhibited evident increase both in flower-bud appearing stage and flowering stage. The obtained results strongly suggest that the effect of environmental cues on steviol glycosides contents and transcription of corresponding biosynthetic genes in S. rebaudiana is significant. It is worth to study deeply.

  16. Transcriptional changes during neuronal death and replacement in the olfactory epithelium.

    Science.gov (United States)

    Shetty, Ranjit S; Bose, Soma C; Nickell, Melissa D; McIntyre, Jeremy C; Hardin, Debra H; Harris, Andrew M; McClintock, Timothy S

    2005-12-01

    The olfactory epithelium has the unusual ability to replace its neurons.We forced replacement of mouse olfactory sensory neurons by bulbectomy. Microarray, bioinformatics, and in situ hybridization techniques detected a rapid shift in favor of pro-apoptotic proteins, a progressive immune response by macrophages and dendritic cells, and identified or predicted 439 mRNAs enriched in olfactory sensory neurons, including gene silencing factors and sperm flagellar proteins. Transcripts encoding cell cycle regulators, axonogenesis proteins, and transcription factors and signaling proteins that promote proliferation and differentiation were increased at 5-7 days after bulbectomy and were expressed by basal progenitor cells or immature neurons. The transcription factors included Nhlhl, Hes6, Lmycl, c-Myc, Mxd4, Idl,Nmycl, Cited2, c-Myb, Mybll, Tead2, Dpl, Gata2, Lmol, and Soxll. The data reveal significant similarities with embryonic neurogenesis and make several mechanistic predictions, including the roles of the transcription factors in the olfactory sensory neuron lineage.

  17. Transcriptomic Changes Due to Cytoplasmic TDP-43 Expression Reveal Dysregulation of Histone Transcripts and Nuclear Chromatin.

    Directory of Open Access Journals (Sweden)

    Alexandre Amlie-Wolf

    Full Text Available TAR DNA-binding protein 43 (TDP-43 is normally a nuclear RNA-binding protein that exhibits a range of functions including regulation of alternative splicing, RNA trafficking, and RNA stability. However, in amyotrophic lateral sclerosis (ALS and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP, TDP-43 is abnormally phosphorylated, ubiquitinated, and cleaved, and is mislocalized to the cytoplasm where it forms distinctive aggregates. We previously developed a mouse model expressing human TDP-43 with a mutation in its nuclear localization signal (ΔNLS-hTDP-43 so that the protein preferentially localizes to the cytoplasm. These mice did not exhibit a significant number of cytoplasmic aggregates, but did display dramatic changes in gene expression as measured by microarray, suggesting that cytoplasmic TDP-43 may be associated with a toxic gain-of-function. Here, we analyze new RNA-sequencing data from the ΔNLS-hTDP-43 mouse model, together with published RNA-sequencing data obtained previously from TDP-43 antisense oligonucleotide (ASO knockdown mice to investigate further the dysregulation of gene expression in the ΔNLS model. This analysis reveals that the transcriptomic effects of the overexpression of the ΔNLS-hTDP-43 transgene are likely due to a gain of cytoplasmic function. Moreover, cytoplasmic TDP-43 expression alters transcripts that regulate chromatin assembly, the nucleolus, lysosomal function, and histone 3' untranslated region (UTR processing. These transcriptomic alterations correlate with observed histologic abnormalities in heterochromatin structure and nuclear size in transgenic mouse and human brains.

  18. Computational Analysis of the Transcriptional Regulation of the Actin Family

    Institute of Scientific and Technical Information of China (English)

    郑家顺; 吴加金; 孙之荣

    2002-01-01

    Transcriptional regulation is a very important regulatory step in the regulation of gene expression. Transcription factors (TFs) play an important role in controlling the temporal special specificity of gene expression. The regulation area of actin genes was analyzed statistically to predict the transcription factor binding sites in the regulatory area. A group of transcription factors located in most of the sequences is believed to play an important role in co-regulating the expression of actin genes.

  19. DELLA-induced early transcriptional changes during etiolated development in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Javier Gallego-Bartolomé

    Full Text Available The hormones gibberellins (GAs control a wide variety of processes in plants, including stress and developmental responses. This task largely relies on the activity of the DELLA proteins, nuclear-localized transcriptional regulators that do not seem to have DNA binding capacity. The identification of early target genes of DELLA action is key not only to understand how GAs regulate physiological responses, but also to get clues about the molecular mechanisms by which DELLAs regulate gene expression. Here, we have investigated the global, early transcriptional response triggered by the Arabidopsis DELLA protein GAI during skotomorphogenesis, a developmental program tightly regulated by GAs. Our results show that the induction of GAI activity has an almost immediate effect on gene expression. Although this transcriptional regulation is largely mediated by the PIFs and HY5 transcription factors based on target meta-analysis, additional evidence points to other transcription factors that would be directly involved in DELLA regulation of gene expression. First, we have identified cis elements recognized by Dofs and type-B ARRs among the sequences enriched in the promoters of GAI targets; and second, an enrichment in additional cis elements appeared when this analysis was extended to a dataset of early targets of the DELLA protein RGA: CArG boxes, bound by MADS-box proteins, and the E-box CACATG that links the activity of DELLAs to circadian transcriptional regulation. Finally, Gene Ontology analysis highlights the impact of DELLA regulation upon the homeostasis of the GA, auxin, and ethylene pathways, as well as upon pre-existing transcriptional networks.

  20. Genome Binding and Gene Regulation by Stem Cell Transcription Factors

    NARCIS (Netherlands)

    J.H. Brandsma (Johan)

    2016-01-01

    markdownabstractNearly all cells of an individual organism contain the same genome. However, each cell type transcribes a different set of genes due to the presence of different sets of cell type-specific transcription factors. Such transcription factors bind to regulatory regions such as promoters

  1. Transcription Factors in Xylem Development. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Sederoff, Ronald; Whetten, Ross; O' Malley, David; Campbell, Malcolm

    1999-07-01

    Answers to the following questions are answered in this report. do the two pine Byb proteins previously identified as candidate transcription factors bind to DNA and activate transcription? In what cell types are tehse Myb proteins expressed? Are these proteins localized to the nucleus? Do other proteins in pine xylem interact with these Myb proteins? Does altered expression of these genes have an impact on xylogenesis, specifically the expression of monolignol biosynthetic genes?

  2. Evidence for a common evolutionary rate in metazoan transcriptional networks.

    Science.gov (United States)

    Carvunis, Anne-Ruxandra; Wang, Tina; Skola, Dylan; Yu, Alice; Chen, Jonathan; Kreisberg, Jason F; Ideker, Trey

    2015-12-18

    Genome sequences diverge more rapidly in mammals than in other animal lineages, such as birds or insects. However, the effect of this rapid divergence on transcriptional evolution remains unclear. Recent reports have indicated a faster divergence of transcription factor binding in mammals than in insects, but others found the reverse for mRNA expression. Here, we show that these conflicting interpretations resulted from differing methodologies. We performed an integrated analysis of transcriptional network evolution by examining mRNA expression, transcription factor binding and cis-regulatory motifs across >25 animal species, including mammals, birds and insects. Strikingly, we found that transcriptional networks evolve at a common rate across the three animal lineages. Furthermore, differences in rates of genome divergence were greatly reduced when restricting comparisons to chromatin-accessible sequences. The evolution of transcription is thus decoupled from the global rate of genome sequence evolution, suggesting that a small fraction of the genome regulates transcription.

  3. SIGffRid: A tool to search for sigma factor binding sites in bacterial genomes using comparative approach and biologically driven statistics

    Directory of Open Access Journals (Sweden)

    Kucherov Gregory

    2008-01-01

    Full Text Available Abstract Background Many programs have been developed to identify transcription factor binding sites. However, most of them are not able to infer two-word motifs with variable spacer lengths. This case is encountered for RNA polymerase Sigma (σ Factor Binding Sites (SFBSs usually composed of two boxes, called -35 and -10 in reference to the transcription initiation point. Our goal is to design an algorithm detecting SFBS by using combinational and statistical constraints deduced from biological observations. Results We describe a new approach to identify SFBSs by comparing two related bacterial genomes. The method, named SIGffRid (SIGma Factor binding sites Finder using R'MES to select Input Data, performs a simultaneous analysis of pairs of promoter regions of orthologous genes. SIGffRid uses a prior identification of over-represented patterns in whole genomes as selection criteria for potential -35 and -10 boxes. These patterns are then grouped using pairs of short seeds (of which one is possibly gapped, allowing a variable-length spacer between them. Next, the motifs are extended guided by statistical considerations, a feature that ensures a selection of motifs with statistically relevant properties. We applied our method to the pair of related bacterial genomes of Streptomyces coelicolor and Streptomyces avermitilis. Cross-check with the well-defined SFBSs of the SigR regulon in S. coelicolor is detailed, validating the algorithm. SFBSs for HrdB and BldN were also found; and the results suggested some new targets for these σ factors. In addition, consensus motifs for BldD and new SFBSs binding sites were defined, overlapping previously proposed consensuses. Relevant tests were carried out also on bacteria with moderate GC content (i.e. Escherichia coli/Salmonella typhimurium and Bacillus subtilis/Bacillus licheniformis pairs. Motifs of house-keeping σ factors were found as well as other SFBSs such as that of SigW in Bacillus strains

  4. Nitrate induction triggers different transcriptional changes in a high and a low nitrogen use efficiency maize inbred line

    Institute of Scientific and Technical Information of China (English)

    Anita Zamboni; Stefania Astolfi; Sabrina Zuchi; Youry Pii; Katia Guardini; Paola Tononi; Zeno Varanini

    2014-01-01

    In higher plants, NO3? can induce its own uptake and the magnitude of this induction is positively related to the external anion concentration. This phenomenon has been characterized in both herbaceous and woody plants. Here, different adaptation strategies of roots from two maize (Zea mays L., ZmAGOs) inbred lines differing in nitrogen use efficiency (NUE) and exhibiting different timing of induction were discussed by investigating NO3?‐induced changes in their transcriptome. Lo5 line (high NUE) showing the maximum rate of NO3? uptake 4 h after the provision of 200 mmol/L NO3? treatment modulated a higher number of transcripts relative to T250 (low NUE) that peaked after 12 h. The two inbred lines share only 368 transcripts that are modulated by the treatment with NO3? and behaved differently when transcripts involved in anion uptake and assimilation were analyzed. T250 line responded to the NO3? induction modulating this group of genes as reported for several plant species. On the contrary, the Lo5 line did not exhibit during the induction changes in this set of genes. Obtained data suggest the importance of exploring the physiological and molecular variations among different maize genotypes in response to environmental clues like NO3? provision, in order to understand mechanisms underlying NUE.

  5. Nitrate induction triggers different transcriptional changes in a high and a low nitrogen use efficiency maize inbred line.

    Science.gov (United States)

    Zamboni, Anita; Astolfi, Stefania; Zuchi, Sabrina; Pii, Youry; Guardini, Katia; Tononi, Paola; Varanini, Zeno

    2014-11-01

    In higher plants, NO3(-) can induce its own uptake and the magnitude of this induction is positively related to the external anion concentration. This phenomenon has been characterized in both herbaceous and woody plants. Here, different adaptation strategies of roots from two maize (Zea mays L., ZmAGOs) inbred lines differing in nitrogen use efficiency (NUE) and exhibiting different timing of induction were discussed by investigating NO3(-) -induced changes in their transcriptome. Lo5 line (high NUE) showing the maximum rate of NO3(-) uptake 4 h after the provision of 200 μmol/L NO3(-) treatment modulated a higher number of transcripts relative to T250 (low NUE) that peaked after 12 h. The two inbred lines share only 368 transcripts that are modulated by the treatment with NO3(-) and behaved differently when transcripts involved in anion uptake and assimilation were analyzed. T250 line responded to the NO3(-) induction modulating this group of genes as reported for several plant species. On the contrary, the Lo5 line did not exhibit during the induction changes in this set of genes. Obtained data suggest the importance of exploring the physiological and molecular variations among different maize genotypes in response to environmental clues like NO3(-) provision, in order to understand mechanisms underlying NUE.

  6. Growth factor stimulation of cardiomyocytes induces changes in the transcriptional contents of secreted exosomes

    Directory of Open Access Journals (Sweden)

    Gunnar Ronquist

    2013-05-01

    Full Text Available Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or repress responses in targets cells. We recently reported that cultured cardiomyocytes are able to release exosomes and that they, in turn, are involved in facilitating events in target cells by alteration of gene expression. We investigated whether external stimuli of the cardiomyocyte might influence the transcriptional content of the released exosomes.Exosomes were isolated from media collected from cultured cardiomyocytes (HL-1 with or without growth factor treatment (TGF-β2 and PDGF-BB, with a series of differential centrifugations, including preparative ultracentrifugation and separation with a sucrose gradient. The exosomes were characterized with dynamic light scattering (DLS, electron microscopy (EM and Western blot and analyzed with Illumina whole genome microarray gene expression.The exosomes were rounded in shape and had an average size of 50–90 nm in diameter with no difference between treatment groups. Analysis of the mRNA content in repeated experiments conclusively revealed 505 transcripts in the control group, 562 in the TGF-β2-treated group and 300 in the PDGF-BB-treated group. Common transcripts (217 were found in all 3 groups.We show that the mode of stimulation of parental cells affects the characteristics of exosomes released. Hence, there is a difference in mRNA content between exosomes derived from cultured cardiomyocytes stimulated, or not stimulated, with growth factors. We also conclude that all exosomes contain a basic package consisting of ribosomal transcripts and mRNAs coding for proteins with functions within the energy supply system. To access the supplementary material to this article, please see Supplementary files under Article Tools online.

  7. Changes in nucleosome position at transcriptional start sites of specific genes in Zea mays mediator of paramutation1 mutants.

    Science.gov (United States)

    Labonne, Jonathan D J; Dorweiler, Jane E; McGinnis, Karen M

    2013-04-01

    Nucleosomes facilitate compaction of DNA within the confines of the eukaryotic nucleus. This packaging of DNA and histone proteins must accommodate cellular processes, such as transcription and DNA replication. The repositioning of nucleosomes to facilitate cellular processes is likely regulated by several factors. In Zea mays, Mediator of paramutation1 (MOP1) has been demonstrated to be an epigenetic regulator of gene expression. Based on sequence orthology and mutant phenotypes, MOP1 is likely to function in an RNA-dependent pathway to mediate changes to chromatin. High-resolution microarrays were used to assay the distribution of nucleosomes across the transcription start sites (TSSs) of ~400 maize genes in wild type and mutant mop1-1 tissues. Analysis of nucleosome distribution in leaf, immature tassel and ear shoot tissues resulted in the identification of three genes showing consistent differences in nucleosome positioning and occupancy between wild type and mutant mop1-1. These specific changes in nucleosome distribution were located upstream as well as downstream of the TSS. No direct relationship between the specific changes in nucleosome distribution and transcription were observed through quantitative expression analysis in these tissues. In silico prediction suggests that nucleosome positioning is not dictated by intrinsic DNA sequence signals in the TSSs of two of the identified genes, suggesting a role for chromatin remodeling proteins in MOP1-mediated pathways. These results also indicate that MOP1 contributions to nucleosome position may be either separate from changes in gene expression, or cooperative with development and other levels of regulation in coordinating gene expression.

  8. Insulinlike growth factor-binding protein proteolysis an emerging paradigm in insulinlike growth factor physiology.

    Science.gov (United States)

    Fowlkes, J L

    1997-10-01

    In biologic fluids, insulinlike growth factors (IGF-I and IGF-II) are bound to high-affinity insulinlike growth factor binding proteins (IGFBPs) of which seven have now been identified (IGFBPs 1-7). In a variety of biologic fluids, several IGFBPs undergo proteolytic degradation. Such degradation can lead to increased IGF bioavailability at the cell surface, facilitating receptor interactions. Herein, recent data identifying several IGFBP-degrading proteinases and their effects on IGF bioactivity is reviewed, and how IGFBP proteolysis is regulated by IGFs and IGFBPs, as well as how IGFBP cleavage analysis provides insights into the structure and function of IGFBPs, is explored. (Trends Endocrinol Metab 1997;8:299-306). (c) 1997, Elsevier Science Inc.

  9. Insulin-like growth factor binding protein 3 in inflammatory bowel disease

    DEFF Research Database (Denmark)

    Kirman, Irena; Whelan, Richard Larry; Jain, Suvinit;

    2005-01-01

    Epithelial cell growth regulation has been reported to be altered in inflammatory bowel disease (IBD) patients. The cell growth regulatory factor, insulin-like growth factor binding protein 3 (IGFBP-3), may be partly responsible for this phenomenon. So far, IGFBP-3 levels have been assessed...... as values of total protein, which is a sum of bioactive intact 43- to 45-kDa protein and its inactive proteolytic cleavage fragments. We aimed to assess the levels of intact IGFBP-3 and its cleaving protease MMP-9 in IBD. Patients with IBD and controls were included. Total plasma IGFBP-3 concentration...... and MMP-9 levels were determined in ELISA. The concentration of intact IGFBP-3 was significantly decreased in patients with moderate to severe IBD activity compared to those in remission or controls. Of note, a dramatic depletion of intact IGFBP-3 was found in 7.4% of patients with IBD. Zymography...

  10. Transcription of hexose transporters of Saccharomyces cerevisiae is affected by change in oxygen provision

    Directory of Open Access Journals (Sweden)

    Ruohonen Laura

    2008-03-01

    Full Text Available Abstract Background The gene family of hexose transporters in Saccharomyces cerevisiae consists of 20 members; 18 genes encoding transporters (HXT1-HXT17, GAL2 and two genes encoding sensors (SNF3, RGT2. The effect of oxygen provision on the expression of these genes was studied in glucose-limited chemostat cultivations (D = 0.10 h-1, pH 5, 30°C. Transcript levels were measured from cells grown in five steady state oxygen levels (0, 0.5, 1, 2.8 and 20.9% O2, and from cells under conditions in which oxygen was introduced to anaerobic cultures or removed from cultures receiving oxygen. Results The expression pattern of the HXT gene family was distinct in cells grown under aerobic, hypoxic and anaerobic conditions. The transcription of HXT2, HXT4 and HXT5 was low when the oxygen concentration in the cultures was low, both under steady state and non-steady state conditions, whereas the expression of HXT6, HXT13 and HXT15/16 was higher in hypoxic than in fully aerobic or anaerobic conditions. None of the HXT genes showed higher transcript levels in strictly anaerobic conditions. Expression of HXT9, HXT14 and GAL2 was not detected under the culture conditions studied. Conclusion When oxygen becomes limiting in a glucose-limited chemostat cultivation, the glucose uptake rate per cell increases. However, the expression of none of the hexose transporter encoding genes was increased in anaerobic conditions. It thus seems that the decrease in the moderately low affinity uptake and consequently the relative increase of high affinity uptake may itself allow the higher specific glucose consumption rate to occur in anaerobic compared to aerobic conditions.

  11. Imatinib treatment causes substantial transcriptional changes in adult Schistosoma mansoni in vitro exhibiting pleiotropic effects.

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    Christin Buro

    2014-06-01

    Full Text Available BACKGROUND: Schistosome parasites cause schistosomiasis, one of the most important infectious diseases worldwide. For decades Praziquantel (PZQ is the only drug widely used for controlling schistosomiasis. The absence of a vaccine and fear of PZQ resistance have motivated the search for alternatives. Studies on protein kinases (PKs demonstrated their importance for diverse physiological processes in schistosomes. Among others two Abl tyrosine kinases, SmAbl1 and SmAbl2, were identified in Schistosoma mansoni and shown to be transcribed in the gonads and the gastrodermis. SmAbl1 activity was blocked by Imatinib, a known Abl-TK inhibitor used in human cancer therapy (Gleevec/Glivec. Imatinib exhibited dramatic effects on the morphology and physiology of adult schistosomes in vitro causing the death of the parasites. METHODOLOGY/PRINCIPAL FINDINGS: Here we show modeling data supporting the targeting of SmAbl1/2 by Imatinib. A biochemical assay confirmed that SmAbl2 activity is also inhibited by Imatinib. Microarray analyses and qRT-PCR experiments were done to unravel transcriptional processes influenced by Imatinib in adult schistosomes in vitro demonstrating a wide influence on worm physiology. Surface-, muscle-, gut and gonad-associated processes were affected as evidenced by the differential transcription of e.g. the gynecophoral canal protein gene GCP, paramyosin, titin, hemoglobinase, and cathepsins. Furthermore, transcript levels of VAL-7 and egg formation-associated genes such as tyrosinase 1, p14, and fs800-like were affected as well as those of signaling genes including a ribosomal protein S6 kinase and a glutamate receptor. Finally, a comparative in silico analysis of the obtained microarray data sets and previous data analyzing the effect of a TGFβR1 inhibitor on transcription provided first evidence for an association of TGFβ and Abl kinase signaling. Among others GCP and egg formation-associated genes were identified as common

  12. Environmental stress-mediated changes in transcriptional and translational regulation of protein synthesis in crop plants. Final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-12-31

    The research described in this final report focused on the influence of stress agents on protein synthesis in crop plants (primarily soybean). Investigations into the `heat shock` (HS) stress mediated changes in transcriptional and translocational regulation of protein synthesis coupled with studies on anaerobic water deficit and other stress mediated alterations in protein synthesis in plants provided the basis of the research. Understanding of the HS gene expression and function(s) of the HSPs may clarify regulatory mechanisms operative in development. Since the reproductive systems of plants if often very temperature sensitive, it may be that the system could be manipulated to provide greater thermotolerance.

  13. Mapping temperature-induced conformational changes in the Escherichia coli heat shock transcription factor sigma 32 by amide hydrogen exchange

    DEFF Research Database (Denmark)

    Rist, Wolfgang; Jørgensen, Thomas J D; Roepstorff, Peter;

    2003-01-01

    gene transcription. To investigate possible heat-induced conformational changes in sigma 32 we performed amide hydrogen (H/D) exchange experiments under optimal growth and heat shock conditions combined with mass spectrometry. We found a rapid exchange of around 220 of the 294 amide hydrogens at 37...... degrees C, indicating that sigma 32 adopts a highly flexible structure. At 42 degrees C we observed a slow correlated exchange of 30 additional amide hydrogens and localized it to a helix-loop-helix motif within domain sigma 2 that is responsible for the recognition of the -10 region in heat shock...

  14. DEVELOPMENT OF A NOVEL METHOD FOR ANALYSIS OF TRANSCRIPTIONAL CHANGES IN TRANSITIONAL EPITHELIUM FROM URINARY BLADDERS OF RATS EXPOSED TO DRINKING WATER DISINFECTION BY-PRODUCTS

    Science.gov (United States)

    Development of a Novel Method for Analysis of Transcriptional Changes in Transitional Epithelium from Urinary Bladders of Rats Exposed to Drinking Water Disinfection By- products.Epidemiologic studies in human populations that drink chemically disinfected drinking wa...

  15. Mitosis gives a brief window of opportunity for a change in gene transcription.

    Directory of Open Access Journals (Sweden)

    Richard P Halley-Stott

    2014-07-01

    Full Text Available Cell differentiation is remarkably stable but can be reversed by somatic cell nuclear transfer, cell fusion, and iPS. Nuclear transfer to amphibian oocytes provides a special opportunity to test transcriptional reprogramming without cell division. We show here that, after nuclear transfer to amphibian oocytes, mitotic chromatin is reprogrammed up to 100 times faster than interphase nuclei. We find that, as cells traverse mitosis, their genes pass through a temporary phase of unusually high responsiveness to oocyte reprogramming factors (mitotic advantage. Mitotic advantage is not explained by nuclear penetration, DNA modifications, histone acetylation, phosphorylation, methylation, nor by salt soluble chromosomal proteins. Our results suggest that histone H2A deubiquitination may account, at least in part, for the acquisition of mitotic advantage. They support the general principle that a temporary access of cytoplasmic factors to genes during mitosis may facilitate somatic cell nuclear reprogramming and the acquisition of new cell fates in normal development.

  16. Changes in Dietary Fat Content Rapidly Alters the Mouse Plasma Coagulation Profile without Affecting Relative Transcript Levels of Coagulation Factors.

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    Audrey C A Cleuren

    Full Text Available Obesity is associated with a hypercoagulable state and increased risk for thrombotic cardiovascular events.Establish the onset and reversibility of the hypercoagulable state during the development and regression of nutritionally-induced obesity in mice, and its relation to transcriptional changes and clearance rates of coagulation factors as well as its relation to changes in metabolic and inflammatory parameters.Male C57BL/6J mice were fed a low fat (10% kcal as fat; LFD or high fat diet (45% kcal as fat; HFD for 2, 4, 8 or 16 weeks. To study the effects of weight loss, mice were fed the HFD for 16 weeks and switched to the LFD for 1, 2 or 4 weeks. For each time point analyses of plasma and hepatic mRNA levels of coagulation factors were performed after overnight fasting, as well as measurements of circulating metabolic and inflammatory parameters. Furthermore, in vivo clearance rates of human factor (F VII, FVIII and FIX proteins were determined after 2 weeks of HFD-feeding.HFD feeding gradually increased the body and liver weight, which was accompanied by a significant increase in plasma glucose levels from 8 weeks onwards, while insulin levels were affected after 16 weeks. Besides a transient rise in cytokine levels at 2 weeks after starting the HFD, no significant effect on inflammation markers was present. Increased plasma levels of fibrinogen, FII, FVII, FVIII, FIX, FXI and FXII were observed in mice on a HFD for 2 weeks, which in general persisted throughout the 16 weeks of HFD-feeding. Interestingly, with the exception of FXI the effects on plasma coagulation levels were not paralleled by changes in relative transcript levels in the liver, nor by decreased clearance rates. Switching from HFD to LFD reversed the HFD-induced procoagulant shift in plasma, again not coinciding with transcriptional modulation.Changes in dietary fat content rapidly alter the mouse plasma coagulation profile, thereby preceding plasma metabolic changes, which

  17. Understanding Transcription Factor Regulation by Integrating Gene Expression and DNase I Hypersensitive Sites

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    Guohua Wang

    2015-01-01

    Full Text Available Transcription factors are proteins that bind to DNA sequences to regulate gene transcription. The transcription factor binding sites are short DNA sequences (5–20 bp long specifically bound by one or more transcription factors. The identification of transcription factor binding sites and prediction of their function continue to be challenging problems in computational biology. In this study, by integrating the DNase I hypersensitive sites with known position weight matrices in the TRANSFAC database, the transcription factor binding sites in gene regulatory region are identified. Based on the global gene expression patterns in cervical cancer HeLaS3 cell and HelaS3-ifnα4h cell (interferon treatment on HeLaS3 cell for 4 hours, we present a model-based computational approach to predict a set of transcription factors that potentially cause such differential gene expression. Significantly, 6 out 10 predicted functional factors, including IRF, IRF-2, IRF-9, IRF-1 and IRF-3, ICSBP, belong to interferon regulatory factor family and upregulate the gene expression levels responding to the interferon treatment. Another factor, ISGF-3, is also a transcriptional activator induced by interferon alpha. Using the different transcription factor binding sites selected criteria, the prediction result of our model is consistent. Our model demonstrated the potential to computationally identify the functional transcription factors in gene regulation.

  18. Insulin-like growth factor binding protein 1 and human embryonic development during 6-10 gestational weeks

    Institute of Scientific and Technical Information of China (English)

    方群; 王艳霞; 周祎

    2004-01-01

    Background Insulin-like growth factor binding protein-1 (IGFBP-1), which is a carrier of Insulin-like growth factors (IGFs) regulates the fetal development by working as an active factor controlling the combination of IGFs with their receptors. This study was designed to investigate the relationship between IGFBP-1 and human embryonic development during weeks 6 -10 of gestation.Methods A total of 44 pregnant women with singleton pregnancy were divided into two groups: one with abnormal embryo development (n = 32) and the other with normal embryo development (n = 12).Enzyme-linked immunosorbent assay (ELISA) was employed to detect IGFBP-1 levels in maternal serum and decidual tissue. The expression of IGFBP-1 mRNA in deciduas was examined by reverse transcription polymerase chain reaction (RT-PCR) technique.Results The level of IGFBP-1 protein in maternal serum was significantly higher in the abnormal group [ (125.36 ± 47.93) μg/ml] than in the normal group [(70.72 ± 21.21) μg/ml ]. Both of IGFBP-1 and IGFBP-1 mRNA in deciduas were higher in abnormal group [ (1.60 ± 1.39) μg/ml and 1.66 ± 1.64, respectively ] than in the normal group [ (0.35 ± 0.23) μg/mi and 0.40 ± 0.20,respectively]. The level of IGFBP-1 in maternal serum was positively correlated with IGFBP-1 mRNA (r=0. 90, P<0.05) and IGFBP-1 protein (r=0.92, P<0.05) in decidual tissue.Conclusions During weeks 6 -10 of gestation, abnormal embryonic development is correlated with elevated IGFBP-1. The level of IGFBP-1 in maternal serum is related to the concentrations of IGFBP1 mRNA and IGFBP-1 in decidual tissue. The IGFBP-1 level in maternal serum may be used as a predictive marker to evaluate embryonic development.

  19. Insulin-like growth factor binding protein-3 in preterm infants with retinopathy of prematurity

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    Manizheh Mostafa Gharehbaghi

    2012-01-01

    Full Text Available Background: Retinopathy of prematurity (ROP is the main cause of visual impairment in preterm newborn infants. Objective: This study was conducted to determine whether insulin-like growth factor binding protein -3 (IGFBP-3 is associated with proliferative ROP and has a role in pathogenesis of the disease in premature infants. Materials and Methods: A total of 71 preterm infants born at or before 32 weeks of gestation participated in this study. Studied patients consisted of 41 neonates without vaso-proliferative findings of ROP as the control group and 30 preterm infants with evidence of severe ROP in follow up eye examination as the case group. Blood samples obtained from these infants 6-8 weeks after birth and blood levels of IGFBP-3 were measured using enzyme-linked immunosorbent assay (ELISA. Results: The mean gestation age and birth weight of the studied patients were 28.2±1.6 weeks and 1120.7±197 gram in the case group and 28.4±1.6 weeks and 1189.4±454 gram in the control group (P=0.25 and P=0.44 respectively. The infants in the case group had significantly lower Apgar score at first and 5 min after birth. Insulin-like growth factor binding protein -3 (IGFBP-3 was significantly lower in the patients with proliferative ROP than the patients without ROP [592.5±472.9 vs. 995.5±422.2 ng/ml (P=0.009]. Using a cut-off point 770.45 ng/ml for the plasma IGFBP-3, we obtained a sensitivity of 65.9% and a specificity of 66.7% in the preterm infants with vasoproliferative ROP. Conclusion: Our data demonstrated that the blood levels IGFBP-3 was significantly lower in the patients with ROP and it is suspected that IGFBP-3 deficiency in the premature infants may have a pathogenetic role in proliferative ROP.

  20. Skeletal muscle transcriptional coactivator PGC-1α mediates mitochondrial, but not metabolic, changes during calorie restriction.

    Science.gov (United States)

    Finley, Lydia W S; Lee, Jaewon; Souza, Amanda; Desquiret-Dumas, Valérie; Bullock, Kevin; Rowe, Glenn C; Procaccio, Vincent; Clish, Clary B; Arany, Zoltan; Haigis, Marcia C

    2012-02-21

    Calorie restriction (CR) is a dietary intervention that extends lifespan and healthspan in a variety of organisms. CR improves mitochondrial energy production, fuel oxidation, and reactive oxygen species (ROS) scavenging in skeletal muscle and other tissues, and these processes are thought to be critical to the benefits of CR. PGC-1α is a transcriptional coactivator that regulates mitochondrial function and is induced by CR. Consequently, many of the mitochondrial and metabolic benefits of CR are attributed to increased PGC-1α activity. To test this model, we examined the metabolic and mitochondrial response to CR in mice lacking skeletal muscle PGC-1α (MKO). Surprisingly, MKO mice demonstrated a normal improvement in glucose homeostasis in response to CR, indicating that skeletal muscle PGC-1α is dispensable for the whole-body benefits of CR. In contrast, gene expression profiling and electron microscopy (EM) demonstrated that PGC-1α is required for the full CR-induced increases in mitochondrial gene expression and mitochondrial density in skeletal muscle. These results demonstrate that PGC-1α is a major regulator of the mitochondrial response to CR in skeletal muscle, but surprisingly show that neither PGC-1α nor mitochondrial biogenesis in skeletal muscle are required for the whole-body metabolic benefits of CR.

  1. Fibronectin Growth Factor-Binding Domains Are Required for Fibroblast Survival

    Science.gov (United States)

    Lin, Fubao; Ren, Xiang-Dong; Pan, Zhi; Macri, Lauren; Zong, Wei-Xing; Tonnesen, Marcia G.; Rafailovich, Miriam; Bar-Sagi, Dafna; Clark, Richard A.F.

    2011-01-01

    Fibronectin (FN) is required for embryogenesis, morphogenesis, and wound repair, and its Arg–Gly–Asp-containing central cell-binding domain (CCBD) is essential for mesenchymal cell survival and growth. Here, we demonstrate that FN contains three growth factor-binding domains (FN-GFBDs) that bind platelet-derived growth factor-BB (PDGF-BB), a potent fibroblast survival and mitogenic factor. These sites bind PDGF-BB with dissociation constants of 10–100 nm. FN-null cells cultured on recombinant CCBD (FNIII8–11) without a FN-GFBD demonstrated minimal metabolism and underwent autophagy at 24 hours, followed by apoptosis at 72 hours, even in the presence of PDGF-BB. In contrast, FN-null cells plated on FNIII8–11 contiguous with FN-GFBD survived without, and proliferated with, PDGF-BB. FN-null cell survival on FNIII8–11 and noncontiguous arrays of FN-GFBDs required these domains to be adsorbed on the same surface, suggesting the existence of a mesenchymal cell-extracellular matrix synapse. Thus, fibroblast survival required GF stimulation in the presence of a FN-GFBD, as well as adhesion to FN through the CCBD. The findings that fibroblast survival is dependent on FN-GFBD underscore the critical importance of pericellular matrix for cell survival and have significant implications for cutaneous wound healing and regeneration. PMID:20811396

  2. Insulin-like growth factor binding protein 2 promotes ovarian cancer cell invasion

    Directory of Open Access Journals (Sweden)

    Liu Jinsong

    2005-02-01

    Full Text Available Abstract Background Insulin-like growth factor binding protein 2 (IGFBP2 is overexpressed in ovarian malignant tissues and in the serum and cystic fluid of ovarian cancer patients, suggesting an important role of IGFBP2 in the biology of ovarian cancer. The purpose of this study was to assess the role of increased IGFBP2 in ovarian cancer cells. Results Using western blotting and tissue microarray analyses, we showed that IGFBP2 was frequently overexpressed in ovarian carcinomas compared with normal ovarian tissues. Furthermore, IGFBP2 was significantly overexpressed in invasive serous ovarian carcinomas compared with borderline serous ovarian tumors. To test whether increased IGFBP2 contributes to the highly invasive nature of ovarian cancer cells, we generated IGFBP2-overexpressing cells from an SKOV3 ovarian cancer cell line, which has a very low level of endogenous IGFBP2. A Matrigel invasion assay showed that these IGFBP2-overexpressing cells were more invasive than the control cells. We then designed small interference RNA (siRNA molecules that attenuated IGFBP2 expression in PA-1 ovarian cancer cells, which have a high level of endogenous IGFBP2. The Matrigel invasion assay showed that the attenuation of IGFBP2 expression indeed decreased the invasiveness of PA-1 cells. Conclusions We therefore showed that IGFBP2 enhances the invasion capacity of ovarian cancer cells. Blockage of IGFBP2 may thus constitute a viable strategy for targeted cancer therapy.

  3. Detection and properties of A-factor-binding protein from Streptomyces griseus

    Energy Technology Data Exchange (ETDEWEB)

    Miyake, K.; Horinouchi, S.; Yoshida, M.; Chiba, N.; Mori, K.; Nogawa, N.; Morikawa, N.; Beppu, T. (Univ. of Tokyo (Japan))

    1989-08-01

    The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel filtration. Scatchard analysis suggested that A-factor bound the protein in the molar ratio of 1:1 with a binding constant, Kd, of 0.7 nM. The number of the binding protein was roughly estimated to be 37 per genome. The inducing material virginiae butanolide C (VB-C), which has a structure very similar to that of A-factor and is essential for virginiamycin production in Streptomyces virginiae, did not inhibit binding. In addition, no protein capable of specifically binding {sup 3}H-labeled VB-C was found in S. griseus. Together with the observation that VB-C had almost no biological activity on the restoration of streptomycin production or sporulation in an A-factor-deficient mutant of S. griseus, these results indicated that the binding protein had a strict ligand specificity. Examination for an A-factor-binding protein in Streptomyces coelicolor A3(2) and Streptomyces lividans showed the absence of any specifically binding protein.

  4. Characterization of the insulin-like growth factor binding protein family in Xenopus tropicalis.

    Science.gov (United States)

    Haramoto, Yoshikazu; Oshima, Tomomi; Takahashi, Shuji; Ito, Yuzuru

    2014-01-01

    The insulin-like growth factor binding protein (Igfbp) family consists of six members designated Igfbp1-6. Igfbps are involved in many vital biological functions. They physically interact with IGFs (IGF1 and IGF2) and act as carriers, thereby protecting IGFs from proteolytic degradation. Thus, they function as modulators of IGF activity. Furthermore, Igfbps have been reported to have IGF-independent activities. They interact with other proteins, including cell surface proteins, extra-cellular matrix proteins, and potentially intracellular molecules. In Xenopus tropicalis (X. tropicalis), only four igfbp genes (igfbp1, igfbp2, igfbp4, and igfbp5) have been identified, and their expression is not well characterized. We report that X. tropicalis genome lacks the igfbp3 and igfbp6 genes based on synteny analyses. We also examined the spatio-temporal expression patterns of igfbp genes in early X. tropicalis development. Expression analyses indicated that they are differentially expressed during early development. Each igfbp gene showed a characteristic spatial expression pattern. Except for igfbp5, they demonstrated overlapping expression in the pronephros. The Xenopus pronephros is composed of four domains (i.e., the proximal tubule, intermediate tubule, distal tubule, and connecting tubule). Our results showed that at least two igfbp genes are co-expressed in all pronephric domains, suggesting that redundant functions of igfbp genes are required in early pronephric kidney development.

  5. Downy mildew resistance induced by Trichoderma harzianum T39 in susceptible grapevines partially mimics transcriptional changes of resistant genotypes

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    Perazzolli Michele

    2012-11-01

    Full Text Available Abstract Background Downy mildew, caused by Plasmopara viticola, is one of the most severe diseases of grapevine and is commonly controlled by fungicide treatments. The beneficial microorganism Trichoderma harzianum T39 (T39 can induce resistance to downy mildew, although the molecular events associated with this process have not yet been elucidated in grapevine. A next generation RNA sequencing (RNA-Seq approach was used to study global transcriptional changes associated with resistance induced by T39 in Vitis vinifera Pinot Noir leaves. The long-term aim was to develop strategies to optimize the use of this agent for downy mildew control. Results More than 14.8 million paired-end reads were obtained for each biological replicate of T39-treated and control leaf samples collected before and 24 h after P. viticola inoculation. RNA-Seq analysis resulted in the identification of 7,024 differentially expressed genes, highlighting the complex transcriptional reprogramming of grapevine leaves during resistance induction and in response to pathogen inoculation. Our data show that T39 has a dual effect: it directly modulates genes related to the microbial recognition machinery, and it enhances the expression of defence-related processes after pathogen inoculation. Whereas several genes were commonly affected by P. viticola in control and T39-treated plants, opposing modulation of genes related to responses to stress and protein metabolism was found. T39-induced resistance partially inhibited some disease-related processes and specifically activated defence responses after P. viticola inoculation, causing a significant reduction of downy mildew symptoms. Conclusions The global transcriptional analysis revealed that defence processes known to be implicated in the reaction of resistant genotypes to downy mildew were partially activated by T39-induced resistance in susceptible grapevines. Genes identified in this work are an important source of markers

  6. Sodium Stress in the Halophyte Thellungiella halophila and Transcriptional Changes in a thsos1-RNA Interference Line

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The plasma membrane Na+/H+-antiporter salt overly sensitive1 (SOS1) from the halophytic Arabidopsis-relative Thellunglella halophila (ThSOS1) shows conserved sequence and domain structure with the orthologous genes from Arabidopsis thaliana and other plants. When expression of ThSOS1 was reduced by RNA interference (RNAi), pronounced characteristics of salt-sensitivity were observed. We were interested in monitoring altered transcriptional responses between Thellungiella wild type and thsos1-4, a representative RNAI line with particular emphasis on root responses to salt stress at 350 mmol/L NaCl, a concentration that is only moderately stressful for mature wild type plants. Transcript profiling revealed several functional categories of genes that were differently affected in wild-type and RNAi plants. Down-regulation of SOS1 resulted In different gene expression even In the absence of stress. The pattern of gene induction in the RNAi plant under salt stress was similar to that of glycophytic Arabidopsis rather than that of wild type Thellungiella. The RNAi plants failed to down-regulate functions that are normally reduced in wild type Thellungiella upon stress and did not up-regulate functions that characterize the Thellungiella salt stress response. Metabolite changes observed in wild type Thellungiella after salt stress were less pronounced or absent in RNAi plants. Transcript and metabolite behavior suggested SOS1 functions including but also extending its established function as a sodium transporter. The down-regulation of ThSOS1 converted the halophyte Thellungiella into a salt-sensitive plant.

  7. Vitamin C modulates cadmium-induced hepatic antioxidants' gene transcripts and toxicopathic changes in Nile tilapia, Oreochromis niloticus.

    Science.gov (United States)

    El-Sayed, Yasser S; El-Gazzar, Ahmed M; El-Nahas, Abeer F; Ashry, Khaled M

    2016-01-01

    Cadmium (Cd) is one of the naturally occurring heavy metals having adverse effects, while vitamin C (L-ascorbic acid) is an essential micronutrient for fish, which can attenuate tissue damage owing to its chain-breaking antioxidant and free radical scavenger properties. The adult Nile tilapia fish were exposed to Cd at 5 mg/l with and without vitamin C (500 mg/kg diet) for 45 days in addition to negative and positive controls fed with the basal diet and basal diet supplemented with vitamin C, respectively. Hepatic relative mRNA expression of genes involved in antioxidant function, metallothionein (MT), glutathione S-transferase (GST-α1), and glutathione peroxidase (GPx1), was assessed using real-time reverse transcription polymerase chain reaction (RT-PCR). Hepatic architecture was also histopathologically examined. Tilapia exposed to Cd exhibited upregulated antioxidants' gene transcript levels, GST-⍺1, GPx1, and MT by 6.10-, 4.60-, and 4.29-fold, respectively. Histopathologically, Cd caused severe hepatic changes of multifocal hepatocellular and pancreatic acinar necrosis, and lytic hepatocytes infiltrated with eosinophilic granular cells. Co-treatment of Cd-exposed fish with vitamin C overexpressed antioxidant enzyme-related genes, GST-⍺1 (16.26-fold) and GPx1 (18.68-fold), and maintained the expression of MT gene close to control (1.07-fold), averting the toxicopathic lesions induced by Cd. These results suggested that vitamin C has the potential to protect Nile tilapia from Cd hepatotoxicity via sustaining hepatic antioxidants' genes transcripts and normal histoarchitecture.

  8. Hippocampal transcriptional and neurogenic changes evoked by combination yohimbine and imipramine treatment.

    Science.gov (United States)

    Husain, Basma Fatima Anwar; Nanavaty, Ishira N; Marathe, Swananda V; Rajendran, Rajeev; Vaidya, Vidita A

    2015-08-03

    Adjunct α2-adrenoceptor antagonism is a potential strategy to accelerate the behavioral effects of antidepressants. Co-administration of the α2-adrenoceptor antagonist yohimbine hastens the behavioral and neurogenic effects of the antidepressant imipramine. We examined the transcriptional targets of short duration (7days), combination treatment of yohimbine and imipramine (Y+I) within the adult rat hippocampus. Using microarray and qPCR analysis we observed functional enrichment of genes involved in intracellular signaling cascades, plasma membrane, cellular metal ion homeostasis, multicellular stress responses and neuropeptide signaling pathways in the Y+I transcriptome. We noted reduced expression of the α2A-adrenoceptor (Adra2a), serotonin 5HT2C receptor (Htr2c) and the somatostatin receptor 1 (Sstr1), which modulate antidepressant action. Further, we noted a regulation of signaling pathway genes like inositol monophosphatase 2 (Impa2), iodothyronine deiodinase 3 (Dio3), regulator of G-protein signaling 4 (Rgs4), alkaline ceramidase 2 (Acer2), doublecortin-like kinase 2 (Dclk2), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (Nfkbia) and serum/glucocorticoid-regulated kinase 1 (Sgk1), several of which are implicated in the pathophysiology of mood disorders. Comparative analysis revealed an overlap in the hippocampal regulation of Acer2, Nfkbia, Sgk1 and Impa2 between Y+I treatment, the fast-acting electroconvulsive seizure (ECS) paradigm, and the slow-onset chronic (21days) imipramine treatment. Further, Y+I treatment enhanced the quiescent neural progenitor pool in the hippocampal neurogenic niche similar to ECS, and distinct from chronic imipramine treatment. Taken together, our results provide insight into the molecular and cellular targets of short duration Y+I treatment, and identify potential leads for the development of rapid-action antidepressants.

  9. Zea mays Taxilin protein negatively regulates opaque-2 transcriptional activity by causing a change in its sub-cellular distribution.

    Directory of Open Access Journals (Sweden)

    Nan Zhang

    Full Text Available Zea mays (maize Opaque-2 (ZmO2 protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2 as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as in vivo assay system, we found that ZmTaxilin could change the sub-cellular distribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellular distribution of ZmO2 may have important functional consequences for ZmO2 activity.

  10. Global changes in transcription orchestrate metabolic differentiation during symbiotic nitrogen fixation in Lotus japonicus

    DEFF Research Database (Denmark)

    Colebatch, Gillian; Desbrosses, Guilhem; Ott, Thomas;

    2004-01-01

    Research on legume nodule metabolism has contributed greatly to our knowledge of primary carbon and nitrogen metabolism in plants in general, and in symbiotic nitrogen fixation in particular. However, most previous studies focused on one or a few genes/enzymes involved in selected metabolic...... coupled to mass spectrometry revealed a distinct metabolic phenotype for nodules that reflected the global changes in metabolism inferred from transcriptome analysis. Udgivelsesdato: 2004-Aug...

  11. Root inoculation with Pseudomonas putida KT2440 induces transcriptional and metabolic changes and systemic resistance in maize plants

    Directory of Open Access Journals (Sweden)

    Chantal ePlanchamp

    2015-01-01

    Full Text Available Pseudomonas putida KT2440 (KT2440 rhizobacteria colonize a wide range of plants. They have been extensively studied for their capacity to adhere to maize seeds, to tolerate toxic secondary metabolites produced by maize roots and to be attracted by maize roots. However, the response of maize plants to KT2440 colonization has not been investigated yet. Maize roots were inoculated with KT2440 and the local (roots and systemic (leaves early plant responses were investigated. The colonization behavior of KT2440 following application to maize seedlings was investigated and transcriptional analysis of stress- and defense-related genes as well as metabolite profiling of local and systemic maize tissues of KT2440-inoculated were performed. The local and systemic responses differed and more pronounced changes were observed in roots compared to leaves. Early in the interaction roots responded via jasmonic acid- and abscisic acid-dependent signaling. Interestingly, during later steps, the salicylic acid pathway was suppressed. Metabolite profiling revealed the importance of plant phospholipids in KT2440-maize interactions. An additional important maize secondary metabolite, a form of benzoxazinone, was also found to be differently abundant in roots three days after KT2440 inoculation. However, the transcriptional and metabolic changes observed in bacterized plants early during the interaction were minor and became even less pronounced with time, indicating an accommodation state of the plant to the presence of KT2440. Since the maize plants reacted to the presence of KT2440 in the rhizosphere, we also investigated the ability of these bacteria to trigger induced systemic resistance (ISR against the maize anthracnose fungus Colletotrichum graminicola. The observed resistance was expressed as strongly reduced leaf necrosis and fungal development in infected bacterized plants compared to non-bacterized controls, showing the potential of KT2440 to act as

  12. Root inoculation with Pseudomonas putida KT2440 induces transcriptional and metabolic changes and systemic resistance in maize plants.

    Science.gov (United States)

    Planchamp, Chantal; Glauser, Gaetan; Mauch-Mani, Brigitte

    2014-01-01

    Pseudomonas putida KT2440 (KT2440) rhizobacteria colonize a wide range of plants. They have been extensively studied for their capacity to adhere to maize seeds, to tolerate toxic secondary metabolites produced by maize roots and to be attracted by maize roots. However, the response of maize plants to KT2440 colonization has not been investigated yet. Maize roots were inoculated with KT2440 and the local (roots) and systemic (leaves) early plant responses were investigated. The colonization behavior of KT2440 following application to maize seedlings was investigated and transcriptional analysis of stress- and defense-related genes as well as metabolite profiling of local and systemic maize tissues of KT2440-inoculated were performed. The local and systemic responses differed and more pronounced changes were observed in roots compared to leaves. Early in the interaction roots responded via jasmonic acid- and abscisic acid-dependent signaling. Interestingly, during later steps, the salicylic acid pathway was suppressed. Metabolite profiling revealed the importance of plant phospholipids in KT2440-maize interactions. An additional important maize secondary metabolite, a form of benzoxazinone, was also found to be differently abundant in roots 3 days after KT2440 inoculation. However, the transcriptional and metabolic changes observed in bacterized plants early during the interaction were minor and became even less pronounced with time, indicating an accommodation state of the plant to the presence of KT2440. Since the maize plants reacted to the presence of KT2440 in the rhizosphere, we also investigated the ability of these bacteria to trigger induced systemic resistance (ISR) against the maize anthracnose fungus Colletotrichum graminicola. The observed resistance was expressed as strongly reduced leaf necrosis and fungal growth in infected bacterized plants compared to non-bacterized controls, showing the potential of KT2440 to act as resistance inducers.

  13. Atmospheric-Pressure Cold Plasma Induces Transcriptional Changes in Ex Vivo Human Corneas.

    Directory of Open Access Journals (Sweden)

    Umberto Rosani

    Full Text Available Atmospheric pressure cold plasma (APCP might be considered a novel tool for tissue disinfection in medicine since the active chemical species produced by low plasma doses, generated by ionizing helium gas in air, induces reactive oxygen species (ROS that kill microorganisms without substantially affecting human cells.In this study, we evaluated morphological and functional changes in human corneas exposed for 2 minutes (min to APCP and tested if the antioxidant n-acetyl l-cysteine (NAC was able to inhibit or prevent damage and cell death.Immunohistochemistry and western blotting analyses of corneal tissues collected at 6 hours (h post-APCP treatment demonstrated no morphological tissue changes, but a transient increased expression of OGG1 glycosylase that returned to control levels in 24 h. Transcriptome sequencing and quantitative real time PCR performed on different corneas revealed in the treated corneas many differentially expressed genes: namely, 256 and 304 genes showing expression changes greater than ± 2 folds in the absence and presence of NAC, respectively. At 6 h post-treatment, the most over-expressed gene categories suggested an active or enhanced cell functioning, with only a minority of genes specifically concerning oxidative DNA damage and repair showing slight over-expression values (<2 folds. Moreover, time-related expression analysis of eight genes up-regulated in the APCP-treated corneas overall demonstrated the return to control expression levels after 24 h.These findings of transient oxidative stress accompanied by wide-range transcriptome adjustments support the further development of APCP as an ocular disinfectant.

  14. Fluctuation sensitivity of a transcriptional signaling cascade

    Science.gov (United States)

    Pilkiewicz, Kevin R.; Mayo, Michael L.

    2016-09-01

    The internal biochemical state of a cell is regulated by a vast transcriptional network that kinetically correlates the concentrations of numerous proteins. Fluctuations in protein concentration that encode crucial information about this changing state must compete with fluctuations caused by the noisy cellular environment in order to successfully transmit information across the network. Oftentimes, one protein must regulate another through a sequence of intermediaries, and conventional wisdom, derived from the data processing inequality of information theory, leads us to expect that longer sequences should lose more information to noise. Using the metric of mutual information to characterize the fluctuation sensitivity of transcriptional signaling cascades, we find, counter to this expectation, that longer chains of regulatory interactions can instead lead to enhanced informational efficiency. We derive an analytic expression for the mutual information from a generalized chemical kinetics model that we reduce to simple, mass-action kinetics by linearizing for small fluctuations about the basal biological steady state, and we find that at long times this expression depends only on a simple ratio of protein production to destruction rates and the length of the cascade. We place bounds on the values of these parameters by requiring that the mutual information be at least one bit—otherwise, any received signal would be indistinguishable from noise—and we find not only that nature has devised a way to circumvent the data processing inequality, but that it must be circumvented to attain this one-bit threshold. We demonstrate how this result places informational and biochemical efficiency at odds with one another by correlating high transcription factor binding affinities with low informational output, and we conclude with an analysis of the validity of our assumptions and propose how they might be tested experimentally.

  15. Time since onset of disease and individual clinical markers associate with transcriptional changes in uncomplicated dengue.

    Directory of Open Access Journals (Sweden)

    Cornelia A M van de Weg

    2015-03-01

    Full Text Available BACKGROUND: Dengue virus (DENV infection causes viral haemorrhagic fever that is characterized by extensive activation of the immune system. The aim of this study is to investigate the kinetics of the transcriptome signature changes during the course of disease and the association of genes in these signatures with clinical parameters. METHODOLOGY/PRINCIPLE FINDINGS: Sequential whole blood samples from DENV infected patients in Jakarta were profiled using affymetrix microarrays, which were analysed using principal component analysis, limma, gene set analysis, and weighted gene co-expression network analysis. We show that time since onset of disease, but not diagnosis, has a large impact on the blood transcriptome of patients with non-severe dengue. Clinical diagnosis (according to the WHO classification does not associate with differential gene expression. Network analysis however, indicated that the clinical markers platelet count, fibrinogen, albumin, IV fluid distributed per day and liver enzymes SGOT and SGPT strongly correlate with gene modules that are enriched for genes involved in the immune response. Overall, we see a shift in the transcriptome from immunity and inflammation to repair and recovery during the course of a DENV infection. CONCLUSIONS/SIGNIFICANCE: Time since onset of disease associates with the shift in transcriptome signatures from immunity and inflammation to cell cycle and repair mechanisms in patients with non-severe dengue. The strong association of time with blood transcriptome changes hampers both the discovery as well as the potential application of biomarkers in dengue. However, we identified gene expression modules that associate with key clinical parameters of dengue that reflect the systemic activity of disease during the course of infection. The expression level of these gene modules may support earlier detection of disease progression as well as clinical management of dengue.

  16. Seasonal changes in CRF-I and urotensin I transcript levels in masu salmon: correlation with cortisol secretion during spawning.

    Science.gov (United States)

    Westring, Christian G; Ando, Hironori; Kitahashi, Takashi; Bhandari, Ramji Kumar; Ueda, Hiroshi; Urano, Akihisa; Dores, Robert M; Sher, Anna A; Danielson, Phillip B

    2008-01-01

    Pacific salmon employ a semelparous reproductive strategy where sexual maturation is followed by rapid senescence and death. Cortisol overproduction has been implicated as the central physiologic event responsible for the post-spawning demise of these fish. Cortisol homeostasis is regulated through the action of hormones of the hypothalamus-pituitary-interrenal (HPI) axis. These include corticotropin-releasing factor (CRF) and urotensin-I (UI). In the present study, masu salmon (Oncorhynchus masou) were assayed for changes in the levels CRF-I and UI mRNA transcripts by quantitative real-time PCR (qRT-PCR). These results were compared to plasma cortisol levels in juvenile, adult, and spawning masu salmon to identify specific regulatory factors that appear to be functionally associated with changes in cortisol levels. Intramuscular implantation of GnRH analog (GnRHa) capsules was also used to determine whether GnRH influences stress hormone levels. In both male and female masu salmon, spawning fish experienced a 5- to 7-fold increase in plasma cortisol levels relative to juvenile non-spawning salmon. Changes in CRF-I mRNA levels were characterized by 1-2 distinctive short-term surges in adult masu salmon. Conversely, seasonal changes in UI mRNA levels displayed broad and sustained increases during the pre-spawning and spawning periods. The increases in UI mRNA levels were positively correlated (R(2)=0.21 male and 0.26 female, pcortisol in the pre-spawning and spawning periods. Despite the importance of GnRH in sexual maturation and reproduction, the administration of GnRHa to test animals failed to produce broad changes in CRF-I, UI or plasma cortisol levels. These findings suggest a more direct role for UI than for CRF-I in the regulation of cortisol levels in spawning Pacific salmon.

  17. AtRTD2: A Reference Transcript Dataset for accurate quantification of alternative splicing and expression changes in Arabidopsis thaliana RNA-seq data

    KAUST Repository

    Zhang, Runxuan

    2016-05-06

    Background Alternative splicing is the major post-transcriptional mechanism by which gene expression is regulated and affects a wide range of processes and responses in most eukaryotic organisms. RNA-sequencing (RNA-seq) can generate genome-wide quantification of individual transcript isoforms to identify changes in expression and alternative splicing. RNA-seq is an essential modern tool but its ability to accurately quantify transcript isoforms depends on the diversity, completeness and quality of the transcript information. Results We have developed a new Reference Transcript Dataset for Arabidopsis (AtRTD2) for RNA-seq analysis containing over 82k non-redundant transcripts, whereby 74,194 transcripts originate from 27,667 protein-coding genes. A total of 13,524 protein-coding genes have at least one alternatively spliced transcript in AtRTD2 such that about 60% of the 22,453 protein-coding, intron-containing genes in Arabidopsis undergo alternative splicing. More than 600 putative U12 introns were identified in more than 2,000 transcripts. AtRTD2 was generated from transcript assemblies of ca. 8.5 billion pairs of reads from 285 RNA-seq data sets obtained from 129 RNA-seq libraries and merged along with the previous version, AtRTD, and Araport11 transcript assemblies. AtRTD2 increases the diversity of transcripts and through application of stringent filters represents the most extensive and accurate transcript collection for Arabidopsis to date. We have demonstrated a generally good correlation of alternative splicing ratios from RNA-seq data analysed by Salmon and experimental data from high resolution RT-PCR. However, we have observed inaccurate quantification of transcript isoforms for genes with multiple transcripts which have variation in the lengths of their UTRs. This variation is not effectively corrected in RNA-seq analysis programmes and will therefore impact RNA-seq analyses generally. To address this, we have tested different genome

  18. Changes in transcript related to osmosis and intracellular ion homeostasis in Paulownia tomentosa under salt stress

    Directory of Open Access Journals (Sweden)

    Guoqiang eFan

    2016-03-01

    Full Text Available Paulownia tomentosa is an important economic and greening tree species that is cultivated widely, including salt environment. Our previous studies indicated its autotetraploid induced by colchicine showed better stress tolerance, but the underlying molecular mechanism related to ploidy and salt stress is still unclear. To investigate this issue, physiological measurements and transcriptome profiling of diploid and autotetraploid plants untreated and treated with NaCl were performed. Through the comparisons among four accessions, for one thing, we found different physiological changes between diploid and autotetraploid P. tomentosa; for another, and we detected many differentially expressed unigenes involved in salt stress response. These differentially expressed unigenes were assigned to several metabolic pathways, including plant hormone signal transduction, RNA transporter, protein processing in endoplasmic reticulum and plant-pathogen interaction, which constructed the complex regulatory network to maintain osmotic and intracellular ion homeostasis. Quantitative real-time polymerase chain reaction was used to confirm the expression patterns of 20 unigenes. The results establish the foundation for the genetic basis of salt tolerance in P. tomentosa, which in turn accelerates Paulownia breeding and expands available arable land.

  19. Transcriptional changes in sensory ganglia associated with primary afferent axon collateral sprouting in spared dermatome model

    Directory of Open Access Journals (Sweden)

    Benjamin J. Harrison

    2015-12-01

    Full Text Available Primary afferent collateral sprouting is a process whereby non-injured primary afferent neurons respond to some stimulus and extend new branches from existing axons. Neurons of both the central and peripheral nervous systems undergo this process, which contributes to both adaptive and maladaptive plasticity (e.g., [1–9]. In the model used here (the “spared dermatome” model, the intact sensory neurons respond to the denervation of adjacent areas of skin by sprouting new axon branches into that adjacent denervated territory. Investigations of gene expression changes associated with collateral sprouting can provide a better understanding of the molecular mechanisms controlling this process. Consequently, it can be used to develop treatments to promote functional recovery for spinal cord injury and other similar conditions. This report includes raw gene expression data files from microarray experiments in order to study the gene regulation in spared sensory ganglia in the initiation (7 days and maintenance (14 days phases of the spared dermatome model relative to intact (“naïve” sensory ganglia. Data has been deposited into GEO (GSE72551.

  20. Changes in Transcript Related to Osmosis and Intracellular Ion Homeostasis in Paulownia tomentosa under Salt Stress.

    Science.gov (United States)

    Fan, Guoqiang; Wang, Limin; Deng, Minjie; Zhao, Zhenli; Dong, Yanpeng; Zhang, Xiaoshen; Li, Yongsheng

    2016-01-01

    Paulownia tomentosa is an important economic and greening tree species that is cultivated widely, including salt environment. Our previous studies indicated its autotetraploid induced by colchicine showed better stress tolerance, but the underlying molecular mechanism related to ploidy and salt stress is still unclear. To investigate this issue, physiological measurements and transcriptome profiling of diploid and autotetraploid plants untreated and treated with NaCl were performed. Through the comparisons among four accessions, for one thing, we found different physiological changes between diploid and autotetraploid P. tomentosa; for another, and we detected many differentially expressed unigenes involved in salt stress response. These differentially expressed unigenes were assigned to several metabolic pathways, including "plant hormone signal transduction," "RNA transporter," "protein processing in endoplasmic reticulum," and "plant-pathogen interaction," which constructed the complex regulatory network to maintain osmotic and intracellular ion homeostasis. Quantitative real-time polymerase chain reaction was used to confirm the expression patterns of 20 unigenes. The results establish the foundation for the genetic basis of salt tolerance in P. tomentosa, which in turn accelerates Paulownia breeding and expands available arable land.

  1. Methylmercury-induced changes in gene transcription associated with neuroendocrine disruption in largemouth bass (Micropterus salmoides)

    Science.gov (United States)

    Richter, Catherine A.; Martyniuk, Christopher J.; Annis, Mandy L.; Brumbaugh, William G.; Chasar, Lia C.; Denslow, Nancy D.; Tillitt, Donald E.

    2014-01-01

    Methyl-mercury (MeHg) is a potent neuroendocrine disruptor that impairs reproductive processes in fish. The objectives of this study were to (1) characterize transcriptomic changes induced by MeHg exposure in the female largemouth bass (LMB) hypothalamus under controlled laboratory conditions, (2) investigate the health and reproductive impacts of MeHg exposure on male and female largemouth bass (LMB) in the natural environment, and (3) identify MeHg-associated gene expression patterns in whole brain of female LMB from MeHg-contaminated habitats. The laboratory experiment was a single injection of 2.5 μg MeHg/g body weight for 96 h exposure. The field survey compared river systems in Florida, USA with comparably lower concentrations of MeHg (Wekiva, Santa Fe, and St. Johns Rivers) in fish and one river system with LMB that contained elevated concentrations of MeHg (St. Marys River). Microarray analysis was used to quantify transcriptomic responses to MeHg exposure. Although fish at the high-MeHg site did not show overt health or reproductive impairment, there were MeHg-responsive genes and pathways identified in the laboratory study that were also altered in fish from the high-MeHg site relative to fish at the low-MeHg sites. Gene network analysis suggested that MeHg regulated the expression targets of neuropeptide receptor and steroid signaling, as well as structural components of the cell. Disease-associated gene networks related to MeHg exposure, based upon expression data, included cerebellum ataxia, movement disorders, and hypercalcemia. Gene responses in the CNS are consistent with the documented neurotoxicological and neuroendocrine disrupting effects of MeHg in vertebrates.

  2. Transcription Factor Substitution during the Evolution of Fungal Ribosome Regulation

    OpenAIRE

    Hogues, Hervé; Lavoie, Hugo; Sellam, Adnane; Mangos, Maria; Roemer, Terry; Purisima, Enrico; Nantel, André; Whiteway, Malcolm

    2008-01-01

    Coordinated ribosomal protein (RP) gene expression is crucial for cellular viability, but the transcriptional network controlling this regulon has only been well characterized in the yeast Saccharomyces cerevisiae. We have used whole-genome transcriptional and location profiling to establish that, in Candida albicans, the RP regulon is controlled by the Myb domain protein Tbf1 working in conjunction with Cbf1. These two factors bind both the promoters of RP genes and the rDNA locus; Tbf1 acti...

  3. A discrete role for FNR in the transcriptional response to moderate changes in oxygen by Haemophilus influenzae Rd KW20.

    Science.gov (United States)

    Jiang, Donald; Tikhomirova, Alexandra; Bent, Stephen J; Kidd, Stephen P

    2016-01-01

    The survival by pathogenic bacteria within the specific conditions of an anatomical niche is critical for their persistence. These conditions include the combination of toxic chemicals, such as reactive oxygen (ROS) and reactive nitrogen species (RNS), with factors relevant to cell growth, such as oxygen. Haemophilus influenzae senses oxygen levels largely through the redox state of the intracellular fumarate-nitrate global regulator (FNR). H. influenzae certainly encounters oxygen levels that fluctuate, but in reality, these would rarely reach a state that results in FNR being fully reduced or oxidized. We were therefore interested in the response of H. influenzae to ROS and RNS at moderately high or low oxygen levels and the corresponding role of FNR. At these levels of oxygen, even though the growth rate of an H. influenzae fnr mutant was similar to wild type, its ROS and RNS tolerance was significantly different. Additionally, the subtle changes in oxygen did alter the whole cell transcriptional profile and this was different between the wild type and fnr mutant strains. It was the changed whole cell profile that impacted on ROS/RNS defence, but surprisingly, the FNR-regulated, anaerobic nitrite reductase (NrfA) continued to be expressed and had a role in this phenotype.

  4. Capsaicin-induced transcriptional changes in hypothalamus and alterations in gut microbial count in high fat diet fed mice.

    Science.gov (United States)

    Baboota, Ritesh K; Murtaza, Nida; Jagtap, Sneha; Singh, Dhirendra P; Karmase, Aniket; Kaur, Jaspreet; Bhutani, Kamlesh K; Boparai, Ravneet K; Premkumar, Louis S; Kondepudi, Kanthi Kiran; Bishnoi, Mahendra

    2014-09-01

    Obesity is a global health problem and recently it has been seen as a growing concern for developing countries. Several bioactive dietary molecules have been associated with amelioration of obesity and associated complications and capsaicin is one among them. The present work is an attempt to understand and provide evidence for the novel mechanisms of anti-obesity activity of capsaicin in high fat diet (HFD)-fed mice. Swiss albino mice divided in three groups (n=8-10) i.e. control, HFD fed and capsaicin (2mg/kg, po)+HFD fed were administered respective treatment for 3months. After measuring phenotypic and serum related biochemical changes, effect of capsaicin on HFD-induced transcriptional changes in hypothalamus, white adipose tissue (WAT) (visceral and subcutaneous), brown adipose tissue (BAT) and gut microbial alterations was studied and quantified. Our results suggest that, in addition to its well-known effects, oral administration of capsaicin (a) modulates hypothalamic satiety associated genotype, (b) alters gut microbial composition, (c) induces "browning" genotype (BAT associated genes) in subcutaneous WAT and (d) increases expression of thermogenesis and mitochondrial biogenesis genes in BAT. The present study provides evidence for novel and interesting mechanisms to explain the anti-obesity effect of capsaicin.

  5. Circulating insulin-like growth factor-binding protein 3 as prognostic biomarker in liver cirrhosis

    Institute of Scientific and Technical Information of China (English)

    Carina; Gabriela; Correa; Bruno; da; Silveira; Colombo; Marcelo; Fernando; Ronsoni; Pedro; Eduardo; Soares; e; Silva; Leonardo; Fayad; Telma; Erotides; Silva; Letícia; Muraro; Wildner; Maria; Luiza; Bazzo; Esther; Buzaglo; Dantas-Correa; Janaína; Luz; Narciso-Schiavon; Leonardo; de; Lucca; Schiavon

    2016-01-01

    AIM: To investigate the prognostic significance of insulin-like growth factor-binding protein 3(IGFBP-3) in patients with cirrhosis.METHODS: Prospective study that included two cohorts: outpatients with stable cirrhosis(n = 138) and patients hospitalized for acute decompensation(n = 189). Development of complications, mortality or liver transplantation was assessed by periodical phone calls and during outpatient visits. The cohort of stable cirrhosis also underwent clinical and laboratory evaluation yearly(2013 and 2014) in predefined study visits. In patients with stable cirrhosis, IGFBP-3 levels were measured at baseline(2012) and at second re-evaluation(2014). In hospitalized subjects, IGFBP-3 levels were measured in serum samples collected in the first and in the third day after admission and stored at-80 ℃. IGFBP-3 levels were measured by immunochemiluminescence.RESULTS: IGFBP-3 levels were lower in hospitalized patients as compared to outpatients(0.94 mcg/mL vs 1.69 mcg/m L, P < 0.001) and increased after liver transplantation(3.81 mcg/m L vs 1.33 mcg/mL, P = 0.008). During the follow-up of the stable cohort, 17 patients died and 11 received liver transplantation. Bivariate analysis showed that death or transplant was associated with lower IGFBP-3 levels(1.44 mcg/mL vs 1.74 mcg/m L, P = 0.027). The Kaplan-Meier transplant-free survival probability was 88.6% in patients with IGFBP-3 ≥ 1.67 mcg/mL and 72.1% for those with IGFBP3 < 1.67 mcg/mL(P = 0.015). In the hospitalized cohort, 30-d mortality was 24.3% and was independently associated with creatinine, INR, SpO2/FiO2 ratio and IGFBP-3 levels in the logistic regression. The 90-d transplant-free survival probability was 80.4% in patients with IGFBP-3 ≥ 0.86 mcg/mL and 56.1% for those with IGFBP3 < 0.86 mcg/mL(P < 0.001). CONCLUSION: Lower IGFBP-3 levels were associated with worse outcomes in patients with cirrhosis, and might represent a promising prognostic

  6. PPARγ induces growth inhibition and apoptosis through upregulation of insulin-like growth factor-binding protein-3 in gastric cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, S.Y. [Department of Pediatrics, Chonbuk National University Hospital, Jeonju (Korea, Republic of); Biomedical Research Institute, School of Medicine, Chonbuk National University Hospital, Jeonju (Korea, Republic of); Kim, M.S.; Lee, M.K. [Department of Pediatrics, Chonbuk National University Hospital, Jeonju (Korea, Republic of); Kim, J.S.; Yi, H.K. [Department of Biochemistry, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Nam, S.Y. [Department of Alternative Therapy, Jeonju University, Jeonju (Korea, Republic of); Lee, D.Y.; Hwang, P.H. [Department of Pediatrics, Chonbuk National University Hospital, Jeonju (Korea, Republic of); Biomedical Research Institute, School of Medicine, Chonbuk National University Hospital, Jeonju (Korea, Republic of)

    2015-01-13

    Peroxisome proliferator activator receptor-gamma (PPARγ) is a ligand-activated transcriptional factor involved in the carcinogenesis of various cancers. Insulin-like growth factor-binding protein-3 (IGFBP-3) is a tumor suppressor gene that has anti-apoptotic activity. The purpose of this study was to investigate the anticancer mechanism of PPARγ with respect to IGFBP-3. PPARγ was overexpressed in SNU-668 gastric cancer cells using an adenovirus gene transfer system. The cells in which PPARγ was overexpressed exhibited growth inhibition, induction of apoptosis, and a significant increase in IGFBP-3 expression. We investigated the underlying molecular mechanisms of PPARγ in SNU-668 cells using an IGFBP-3 promoter/luciferase reporter system. Luciferase activity was increased up to 15-fold in PPARγ transfected cells, suggesting that PPARγ may directly interact with IGFBP-3 promoter to induce its expression. Deletion analysis of the IGFBP-3 promoter showed that luciferase activity was markedly reduced in cells without putative p53-binding sites (-Δ1755, -Δ1795). This suggests that the critical PPARγ-response region is located within the p53-binding region of the IGFBP-3 promoter. We further demonstrated an increase in PPARγ-induced luciferase activity even in cells treated with siRNA to silence p53 expression. Taken together, these data suggest that PPARγ exhibits its anticancer effect by increasing IGFBP-3 expression, and that IGFBP-3 is a significant tumor suppressor.

  7. Transcript profiles uncover temporal and stress-induced changes of metabolic pathways in germinating sugar beet seeds

    Directory of Open Access Journals (Sweden)

    Windhövel Andrea

    2008-12-01

    Full Text Available Abstract Background With a cultivation area of 1.75 Mio ha and sugar yield of 16.7 Mio tons in 2006, sugar beet is a crop of great economic importance in Europe. The productivity of sugar beet is determined significantly by seed vigour and field emergence potential; however, little is known about the molecular mechanisms underlying these traits. Both traits exhibit large variations within sugar beet germplasm that have been difficult to ascribe to either environmental or genetic causes. Among potential targets for trait improvement, an enhancement of stress tolerance is considered because of the high negative influence of environmental stresses on trait parameters. Extending our knowledge of genetic and molecular determinants of sugar beet germination, stress response and adaptation mechanisms would facilitate the detection of new targets for breeding crop with an enhanced field emergence potential. Results To gain insight into the sugar beet germination we initiated an analysis of gene expression in a well emerging sugar beet hybrid showing high germination potential under various environmental conditions. A total of 2,784 ESTs representing 2,251 'unigenes' was generated from dry mature and germinating seeds. Analysis of the temporal expression of these genes during germination under non-stress conditions uncovered drastic transcriptional changes accompanying a shift from quiescent to metabolically active stages of the plant life cycle. Assay of germination under stressful conditions revealed 157 genes showing significantly different expression patterns in response to stress. As deduced from transcriptome data, stress adaptation mechanisms included an alteration in reserve mobilization pathways, an accumulation of the osmoprotectant glycine betaine, late embryogenesis abundant proteins and detoxification enzymes. The observed transcriptional changes are supposed to be regulated by ABA-dependent signal transduction pathway. Conclusion This study

  8. A New Microsphere-Based Immunoassay for Measuring the Activity of Transcription Factors

    Directory of Open Access Journals (Sweden)

    Tsai Chueh-Jen

    2010-01-01

    Full Text Available Abstract There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α and nuclear factor-kappa B (NF-κB were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors.

  9. An MSC2 Promoter-lacZ Fusion Gene Reveals Zinc-Responsive Changes in Sites of Transcription Initiation That Occur across the Yeast Genome

    Science.gov (United States)

    Wu, Yi-Hsuan; Taggart, Janet; Song, Pamela Xiyao; MacDiarmid, Colin; Eide, David J.

    2016-01-01

    The Msc2 and Zrg17 proteins of Saccharomyces cerevisiae form a complex to transport zinc into the endoplasmic reticulum. ZRG17 is transcriptionally induced in zinc-limited cells by the Zap1 transcription factor. In this report, we show that MSC2 mRNA also increases (~1.5 fold) in zinc-limited cells. The MSC2 gene has two in-frame ATG codons at its 5’ end, ATG1 and ATG2; ATG2 is the predicted initiation codon. When the MSC2 promoter was fused at ATG2 to the lacZ gene, we found that unlike the chromosomal gene this reporter showed a 4-fold decrease in lacZ mRNA in zinc-limited cells. Surprisingly, β-galactosidase activity generated by this fusion gene increased ~7 fold during zinc deficiency suggesting the influence of post-transcriptional factors. Transcription of MSC2ATG2-lacZ was found to start upstream of ATG1 in zinc-replete cells. In zinc-limited cells, transcription initiation shifted to sites just upstream of ATG2. From the results of mutational and polysome profile analyses, we propose the following explanation for these effects. In zinc-replete cells, MSC2ATG2-lacZ mRNA with long 5’ UTRs fold into secondary structures that inhibit translation. In zinc-limited cells, transcripts with shorter unstructured 5’ UTRs are generated that are more efficiently translated. Surprisingly, chromosomal MSC2 did not show start site shifts in response to zinc status and only shorter 5’ UTRs were observed. However, the shifts that occur in the MSC2ATG2-lacZ construct led us to identify significant transcription start site changes affecting the expression of ~3% of all genes. Therefore, zinc status can profoundly alter transcription initiation across the yeast genome. PMID:27657924

  10. Analysis of Cryptococcus neoformans sexual development reveals rewiring of the pheromone-response network by a change in transcription factor identity.

    Science.gov (United States)

    Kruzel, Emilia K; Giles, Steven S; Hull, Christina M

    2012-06-01

    The fundamental mechanisms that control eukaryotic development include extensive regulation at the level of transcription. Gene regulatory networks, composed of transcription factors, their binding sites in DNA, and their target genes, are responsible for executing transcriptional programs. While divergence of these control networks drives species-specific gene expression that contributes to biological diversity, little is known about the mechanisms by which these networks evolve. To investigate how network evolution has occurred in fungi, we used a combination of microarray expression profiling, cis-element identification, and transcription-factor characterization during sexual development of the human fungal pathogen Cryptococcus neoformans. We first defined the major gene expression changes that occur over time throughout sexual development. Through subsequent bioinformatic and molecular genetic analyses, we identified and functionally characterized the C. neoformans pheromone-response element (PRE). We then discovered that transcriptional activation via the PRE requires direct binding of the high-mobility transcription factor Mat2, which we conclude functions as the elusive C. neoformans pheromone-response factor. This function of Mat2 distinguishes the mechanism of regulation through the PRE of C. neoformans from all other fungal systems studied to date and reveals species-specific adaptations of a fungal transcription factor that defies predictions on the basis of sequence alone. Overall, our findings reveal that pheromone-response network rewiring has occurred at the level of transcription factor identity, despite the strong conservation of upstream and downstream components, and serve as a model for how selection pressures act differently on signaling vs. gene regulatory components during eukaryotic evolution.

  11. Differential roles of epigenetic changes and Foxp3 expression in regulatory T cell-specific transcriptional regulation

    NARCIS (Netherlands)

    Morikawa, Hiromasa; Ohkura, Naganari; Vandenbon, Alexis; Itoh, Masayoshi; Nagao-Sato, Sayaka; Kawaji, Hideya; Lassmann, Timo; Carninci, Piero; Hayashizaki, Yoshihide; Forrest, Alistair R R; Standley, Daron M; Date, Hiroshi; Sakaguchi, Shimon; Clevers, Hans

    2014-01-01

    Naturally occurring regulatory T (Treg) cells, which specifically express the transcription factor forkhead box P3 (Foxp3), are engaged in the maintenance of immunological self-tolerance and homeostasis. By transcriptional start site cluster analysis, we assessed here how genome-wide patterns of DNA

  12. Transcriptional changes in the brains of cattle orally infected with the bovine spongiform encephalopathy agent precede detection of infectivity.

    Science.gov (United States)

    Tang, Yue; Xiang, Wei; Hawkins, Steve A C; Kretzschmar, Hans A; Windl, Otto

    2009-09-01

    Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. BSE can be transmitted experimentally between cattle through the oral route, and in this study, brain tissue samples from animals at different time points postinoculation were analyzed for changes in gene expression. The aims of this study were to identify differentially regulated genes during the progression of BSE using microarray-based gene expression profiling and to understand the effect of prion pathogenesis on gene expression. A total of 114 genes were found to be differentially regulated over the time course of the infection, and many of these 114 genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response, and transcription. This study also revealed a broad correlation between gene expression profiles and the progression of BSE in cattle. At 21 months postinoculation, the largest number of differentially regulated genes was detected, suggesting that there are many pathogenic processes in the animal brain even prior to the detection of infectivity in the central nervous systems of these orally infected cattle. Moreover, evidence is presented to suggest that it is possible to predict the infectious status of animals using the expression profiles from this study.

  13. Transcriptional and phenotypic changes in aorta and aortic valve with aging and MnSOD deficiency in mice.

    Science.gov (United States)

    Roos, Carolyn M; Hagler, Michael; Zhang, Bin; Oehler, Elise A; Arghami, Arman; Miller, Jordan D

    2013-11-15

    The purpose of this study was to characterize changes in antioxidant and age-related gene expression in aorta and aortic valve with aging, and test the hypothesis that increased mitochondrial oxidative stress accelerates age-related endothelial and aortic valve dysfunction. Wild-type (MnSOD(+/+)) and manganese SOD heterozygous haploinsufficient (MnSOD(+/-)) mice were studied at 3 and 18 mo of age. In aorta from wild-type mice, antioxidant expression was preserved, although there were age-associated increases in Nox2 expression. Haploinsufficiency of MnSOD did not alter antioxidant expression in aorta, but increased expression of Nox2. When compared with that of aorta, age-associated reductions in antioxidant expression were larger in aortic valves from wild-type and MnSOD haploinsufficient mice, although Nox2 expression was unchanged. Similarly, sirtuin expression was relatively well-preserved in aorta from both genotypes, whereas expression of SIRT1, SIRT2, SIRT3, SIRT4, and SIRT6 were significantly reduced in the aortic valve. Expression of p16(ink4a), a marker of cellular senescence, was profoundly increased in both aorta and aortic valve from MnSOD(+/+) and MnSOD(+/-) mice. Functionally, we observed comparable age-associated reductions in endothelial function in aorta from both MnSOD(+/+) and MnSOD(+/-) mice. Interestingly, inhibition of NAD(P)H oxidase with apocynin or gp91ds-tat improved endothelial function in MnSOD(+/+) mice but significantly impaired endothelial function in MnSOD(+/-) mice at both ages. Aortic valve function was not impaired by aging or MnSOD haploinsufficiency. Changes in antioxidant and sirtuin gene expression with aging differ dramatically between aorta and aortic valve. Furthermore, although MnSOD does not result in overt cardiovascular dysfunction with aging, compensatory transcriptional responses to MnSOD deficiency appear to be tissue specific.

  14. Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts.

    Science.gov (United States)

    Oppert, Brenda; Martynov, Alexander G; Elpidina, Elena N

    2012-09-01

    The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the Bacillus thuringiensis (Bt) Cry3Aa toxin. As digestive peptidases are a determining factor in Cry toxicity and resistance, we evaluated the expression of peptidase transcripts in the midgut of T. molitor larvae fed either a control or Cry3Aa protoxin diet for 24 h (RNA-Seq), or in larvae exposed to the protoxin for 6, 12, or 24 h (microarrays). Cysteine peptidase transcripts (9) were similar to cathepsins B, L, and K, and their expression did not vary more than 2.5-fold in control and Cry3Aa-treated larvae. Serine peptidase transcripts (48) included trypsin, chymotrypsin and chymotrypsin-like, elastase 1-like, and unclassified serine peptidases, as well as homologs lacking functional amino acids. Highly expressed trypsin and chymotrypsin transcripts were severely repressed, and most serine peptidase transcripts were expressed 2- to 15-fold lower in Cry3Aa-treated larvae. Many serine peptidase and homolog transcripts were found only in control larvae. However, expression of a few serine peptidase transcripts was increased or found only in Cry3Aa-treated larvae. Therefore, Bt intoxication significantly impacted the expression of serine peptidases, potentially important in protoxin processing, while the insect maintained the production of critical digestive cysteine peptidases.

  15. Transcriptional changes in the nuc-2A mutant strain of Neurospora crassa cultivated under conditions of phosphate shortage.

    Science.gov (United States)

    Gras, Diana E; Silveira, Henrique C S; Peres, Nalu T A; Sanches, Pablo R; Martinez-Rossi, Nilce M; Rossi, Antonio

    2009-01-01

    The molecular mechanism that controls the response to phosphate shortage in Neurospora crassa involves four regulatory genes -nuc-2, preg, pgov, and nuc-1. Phosphate shortage is sensed by the nuc-2 gene, the product of which inhibits the functioning of the PREG-PGOV complex. This allows the translocation of the transcriptional factor NUC-1 into the nucleus, which activates the transcription of phosphate-repressible phosphatases. The nuc-2A mutant strain of N. crassa carries a loss-of-function mutation in the nuc-2 gene, which encodes an ankyrin-like repeat protein. In this study, we identified transcripts that are downregulated in the nuc-2A mutant strain. Functional grouping of these expressed sequence tags allowed the identification of genes that play essential roles in different cellular processes such as transport, transcriptional regulation, signal transduction, metabolism, protein synthesis, protein fate, and development. These results reveal novel aspects of the phosphorus-sensing network in N. crassa.

  16. Previous history of chronic stress changes the transcriptional response to glucocorticoid challenge in the dentate gyrus region of the male rat hippocampus.

    Science.gov (United States)

    Datson, Nicole A; van den Oever, Jessica M E; Korobko, Oksana B; Magarinos, Ana Maria; de Kloet, E Ronald; McEwen, Bruce S

    2013-09-01

    Chronic stress is a risk factor for several neuropsychiatric diseases, such as depression and psychosis. In response to stress glucocorticoids (GCs) are secreted that bind to mineralocorticoid and glucocorticoid receptors, ligand-activated transcription factors that regulate the transcription of gene networks in the brain necessary for coping with stress, recovery, and adaptation. Chronic stress particularly affects the dentate gyrus (DG) subregion of the hippocampus, causing several functional and morphological changes with consequences for learning and memory, which are likely adaptive but at the same time make DG neurons more vulnerable to subsequent challenges. The aim of this study was to investigate the transcriptional response of DG neurons to a GC challenge in male rats previously exposed to chronic restraint stress (CRS). An intriguing finding of the current study was that having a history of CRS had profound consequences for the subsequent response to acute GC challenge, differentially affecting the expression of several hundreds of genes in the DG compared with challenged nonstressed control animals. This enduring effect of previous stress exposure suggests that epigenetic processes may be involved. In line with this, CRS indeed affected the expression of several genes involved in chromatin structure and epigenetic processes, including Asf1, Ash1l, Hist1h3f, and Tp63. The data presented here indicate that CRS alters the transcriptional response to a subsequent GC injection. We propose that this altered transcriptional potential forms part of the molecular mechanism underlying the enhanced vulnerability for stress-related disorders like depression caused by chronic stress.

  17. Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells

    DEFF Research Database (Denmark)

    Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani;

    2015-01-01

    Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge...... such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure...

  18. Interaction with diurnal and circadian regulation results in dynamic metabolic and transcriptional changes during cold acclimation in Arabidopsis.

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    Carmen Espinoza

    Full Text Available In plants, there is a large overlap between cold and circadian regulated genes and in Arabidopsis, we have shown that cold (4°C affects the expression of clock oscillator genes. However, a broader insight into the significance of diurnal and/or circadian regulation of cold responses, particularly for metabolic pathways, and their physiological relevance is lacking. Here, we performed an integrated analysis of transcripts and primary metabolites using microarrays and gas chromatography-mass spectrometry. As expected, expression of diurnally regulated genes was massively affected during cold acclimation. Our data indicate that disruption of clock function at the transcriptional level extends to metabolic regulation. About 80% of metabolites that showed diurnal cycles maintained these during cold treatment. In particular, maltose content showed a massive night-specific increase in the cold. However, under free-running conditions, maltose was the only metabolite that maintained any oscillations in the cold. Furthermore, although starch accumulates during cold acclimation we show it is still degraded at night, indicating significance beyond the previously demonstrated role of maltose and starch breakdown in the initial phase of cold acclimation. Levels of some conventional cold induced metabolites, such as γ-aminobutyric acid, galactinol, raffinose and putrescine, exhibited diurnal and circadian oscillations and transcripts encoding their biosynthetic enzymes often also cycled and preceded their cold-induction, in agreement with transcriptional regulation. However, the accumulation of other cold-responsive metabolites, for instance homoserine, methionine and maltose, did not have consistent transcriptional regulation, implying that metabolic reconfiguration involves complex transcriptional and post-transcriptional mechanisms. These data demonstrate the importance of understanding cold acclimation in the correct day-night context, and are further

  19. DNA damage and transcriptional changes in the gills of mytilus galloprovincialis exposed to nanomolar doses of combined metal salts (Cd, Cu, Hg.

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    Laura Varotto

    Full Text Available Aiming at an integrated and mechanistic view of the early biological effects of selected metals in the marine sentinel organism Mytilus galloprovincialis, we exposed mussels for 48 hours to 50, 100 and 200 nM solutions of equimolar Cd, Cu and Hg salts and measured cytological and molecular biomarkers in parallel. Focusing on the mussel gills, first target of toxic water contaminants and actively proliferating tissue, we detected significant dose-related increases of cells with micronuclei and other nuclear abnormalities in the treated mussels, with differences in the bioconcentration of the three metals determined in the mussel flesh by atomic absorption spectrometry. Gene expression profiles, determined in the same individual gills in parallel, revealed some transcriptional changes at the 50 nM dose, and substantial increases of differentially expressed genes at the 100 and 200 nM doses, with roughly similar amounts of up- and down-regulated genes. The functional annotation of gill transcripts with consistent expression trends and significantly altered at least in one dose point disclosed the complexity of the induced cell response. The most evident transcriptional changes concerned protein synthesis and turnover, ion homeostasis, cell cycle regulation and apoptosis, and intracellular trafficking (transcript sequences denoting heat shock proteins, metal binding thioneins, sequestosome 1 and proteasome subunits, and GADD45 exemplify up-regulated genes while transcript sequences denoting actin, tubulins and the apoptosis inhibitor 1 exemplify down-regulated genes. Overall, nanomolar doses of co-occurring free metal ions have induced significant structural and functional changes in the mussel gills: the intensity of response to the stimulus measured in laboratory supports the additional validation of molecular markers of metal exposure to be used in Mussel Watch programs.

  20. The dynamic changes of DNA methylation and histone modifications of salt responsive transcription factor genes in soybean.

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    Yuguang Song

    Full Text Available Epigenetic modification contributes to the regulation of gene expression and plant development under salinity stress. Here we describe the identification of 49 soybean transcription factors by microarray analysis as being inducible by salinity stress. A semi-quantitative RT-PCR-based expression assay confirmed the salinity stress inducibility of 45 of these 49 transcription factors, and showed that ten of them were up-regulated when seedlings were exposed to the demethylation agent 5-aza-2-deoxycytidine. Salinity stress was shown to affect the methylation status of four of these ten transcription factors (one MYB, one b-ZIP and two AP2/DREB family members using a combination of bisulfite sequencing and DNA methylation-sensitive DNA gel blot analysis. ChIP analysis indicated that the activation of three of the four DNA methylated transcription factors was correlated with an increased level of histone H3K4 trimethylation and H3K9 acetylation, and/or a reduced level of H3K9 demethylation in various parts of the promoter or coding regions. Our results suggest a critical role for some transcription factors' activation/repression by DNA methylation and/or histone modifications in soybean tolerance to salinity stress.

  1. Genome-wide prediction of transcriptional regulatory elements of human promoters using gene expression and promoter analysis data

    Directory of Open Access Journals (Sweden)

    Kim Seon-Young

    2006-07-01

    Full Text Available Abstract Background A complete understanding of the regulatory mechanisms of gene expression is the next important issue of genomics. Many bioinformaticians have developed methods and algorithms for predicting transcriptional regulatory mechanisms from sequence, gene expression, and binding data. However, most of these studies involved the use of yeast which has much simpler regulatory networks than human and has many genome wide binding data and gene expression data under diverse conditions. Studies of genome wide transcriptional networks of human genomes currently lag behind those of yeast. Results We report herein a new method that combines gene expression data analysis with promoter analysis to infer transcriptional regulatory elements of human genes. The Z scores from the application of gene set analysis with gene sets of transcription factor binding sites (TFBSs were successfully used to represent the activity of TFBSs in a given microarray data set. A significant correlation between the Z scores of gene sets of TFBSs and individual genes across multiple conditions permitted successful identification of many known human transcriptional regulatory elements of genes as well as the prediction of numerous putative TFBSs of many genes which will constitute a good starting point for further experiments. Using Z scores of gene sets of TFBSs produced better predictions than the use of mRNA levels of a transcription factor itself, suggesting that the Z scores of gene sets of TFBSs better represent diverse mechanisms for changing the activity of transcription factors in the cell. In addition, cis-regulatory modules, combinations of co-acting TFBSs, were readily identified by our analysis. Conclusion By a strategic combination of gene set level analysis of gene expression data sets and promoter analysis, we were able to identify and predict many transcriptional regulatory elements of human genes. We conclude that this approach will aid in decoding

  2. Second Intron of Mouse Nestin Gene Directs its Expression in Pluripotent Embryonic Carcinoma Cells through POU Factor Binding Site

    Institute of Scientific and Technical Information of China (English)

    Zhi-Gang JIN; Li LIU; Hua ZHONG; Ke-Jing ZHANG; Yong-Feng CHEN; Wei BIAN; Le-Ping CHENG; Nai-He JING

    2006-01-01

    Nestin, an intermediate filament protein, is expressed in the neural stem cells of the developing central nervous system. This tissue-specific expression is driven by the neural stem cell-specific enhancer in the second intron of the nestin gene. In this study, we showed that the mouse nestin gene was expressed in pluripotent embryonic carcinoma (EC) P19 and F9 cells, not in the differentiated cell types. This cell typespecific expression was conferred by the enhancer in the second intron. Mutation of the conserved POU factor-binding site in the enhancer abolished the reporter gene expression in EC cells. Oct4, a Class V POU factor, was found to be coexpressed with nestin in EC cells. Electrophoretic mobility-shift assays and supershift assays showed that a unique protein-DNA complex was formed specifically with nuclear extracts of EC cells, and Oct4 protein was included. Together, these results suggest the functional relevance between the conserved POU factor-binding site and the expression of the nestin gene in pluripotent EC cells.

  3. Insulin-like growth factor binding protein-5 modulates muscle differentiation through an insulin-like growth factor-dependent mechanism.

    Science.gov (United States)

    James, P L; Stewart, C E; Rotwein, P

    1996-05-01

    The insulin-like growth factor binding proteins (IGFBPs) are a family of six secreted proteins which bind to and modulate the actions of insulin-like growth factors-I and -II (IGF-I and -II). IGFBP-5 is more conserved than other IGFBPs characterized to date, and is expressed in adult rodent muscle and in the developing myotome. We have shown previously that C2 myoblasts secrete IGFBP-5 as their sole IGFBP. Here we use these cells to study the function of IGFBP-5 during myogenesis, a process stimulated by IGFs. We stably transfected C2 cells with IGFBP-5 cDNAs under control of a constitutively active promoter. Compared with vector-transfected control cells, C2 myoblasts expressing the IGFBP-5 transgene in the sense orientation exhibit increased IGFBP-5 levels in the extracellular matrix during proliferation, and subsequently fail to differentiate normally, as assessed by both morphological and biochemical criteria. Compared to controls, IGFBP-5 sense myoblasts show enhanced survival in low serum medium, remaining viable for at least four weeks in culture. By contrast, myoblasts expressing the IGFBP-5 antisense transcript differentiate prematurely and more extensively than control cells. The inhibition of myogenic differentiation by high level expression of IGFBP-5 could be overcome by exogenous IGFs, with des (1-3) IGF-I, an analogue with decreased affinity for IGFBP-5 but normal affinity for the IGF-I receptor, showing the highest potency. These results are consistent with a model in which IGFBP-5 blocks IGF-stimulated myogenesis, and indicate that sequestration of IGFs in the extracellular matrix could be a possible mechanism of action. Our observations also suggest that IGFBP-5 normally inhibits muscle differentiation, and imply a role for IGFBP-5 in regulating IGF action during myogenic development in vivo.

  4. Notch2 controls prolactin and insulin-like growth factor binding protein-1 expression in decidualizing human stromal cells of early pregnancy.

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    Gerlinde R Otti

    Full Text Available Decidualization, the transformation of the human uterine mucosa into the endometrium of pregnancy, is critical for successful implantation and embryonic development. However, key regulatory factors controlling differentiation of uterine stromal cells into hormone-secreting decidual cells have not been fully elucidated. Hence, we herein investigated the role of the Notch signaling pathway in human decidual stromal cells (HDSC isolated from early pregnancy samples. Immunofluorescence of first trimester decidual tissues revealed expression of Notch2 receptor and its putative, membrane-anchored interaction partners Jagged1, Delta-like (DLL 1 and DLL4 in stromal cells whereas other Notch receptors and ligands were absent from these cells. During in vitro differentiation with estrogen/progesterone (E2P4 and/or cyclic adenosine monophosphate (cAMP HDSC constitutively expressed Notch2 and weakly downregulated Jagged1 mRNA and protein, measured by quantitative PCR (qPCR and Western blotting, respectively. However, increased levels of DLL1 and DLL4 were observed in the decidualizing cultures. Transfection of a Notch luciferase reporter and qPCR of the Notch target gene hairy and enhancer of split 1 (HES1 revealed an induction of canonical Notch activity during in vitro differentiation. In contrast, treatment of HDSC with a chemical Notch/γ-secretase inhibitor decreased cAMP/E2P4-stimulated Notch luciferase activity, HES1 transcript levels and mRNA expression of the decidual marker genes prolactin (PRL and insulin-like growth factor binding protein 1 (IGFBP1. Similarly, siRNA-mediated gene silencing or antibody-mediated blocking of Notch2 diminished HES1, PRL and IGFBP1 mRNA levels as well as secreted PRL protein. In summary, the data suggest that canonical, Notch2-dependent signaling plays a role in human decidualization.

  5. Evolution of the insulin-like growth factor binding protein (IGFBP) family.

    Science.gov (United States)

    Daza, Daniel Ocampo; Sundström, Görel; Bergqvist, Christina A; Duan, Cunming; Larhammar, Dan

    2011-06-01

    The evolution of the IGF binding protein (IGFBP) gene family has been difficult to resolve. Both chromosomal and serial duplications have been suggested as mechanisms for the expansion of this gene family. We have identified and annotated IGFBP sequences from a wide selection of vertebrate species as well as Branchiostoma floridae and Ciona intestinalis. By combining detailed sequence analysis with sequence-based phylogenies and chromosome information, we arrive at the following scenario: the ancestral chordate IGFBP gene underwent a local gene duplication, resulting in a gene pair adjacent to a HOX cluster. Subsequently, the gene family expanded in the two basal vertebrate tetraploidization (2R) resulting in the six IGFBP types that are presently found in placental mammals. The teleost fish ancestor underwent a third tetraploidization (3R) that further expanded the IGFBP repertoire. The five sequenced teleost fish genomes retain 9-11 of IGFBP genes. This scenario is supported by the phylogenies of three adjacent gene families in the HOX gene regions, namely the epidermal growth factor receptors (EGFR) and the Ikaros and distal-less (DLX) transcription factors. Our sequence comparisons show that several important structural components in the IGFBPs are ancestral vertebrate features that have been maintained in all orthologs, for instance the integrin interaction motif Arg-Gly-Asp in IGFBP-2. In contrast, the Arg-Gly-Asp motif in IGFBP-1 has arisen independently in mammals. The large degree of retention of IGFBP genes after the ancient expansion of the gene family strongly suggests that each gene evolved distinct and important functions early in vertebrate evolution.

  6. After-ripening induced transcriptional changes of hormonal genes in wheat seeds: the cases of brassinosteroids, ethylene, cytokinin and salicylic acid.

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    Vijaya R Chitnis

    Full Text Available Maintenance and release of seed dormancy is regulated by plant hormones; their levels and seed sensitivity being the critical factors. This study reports transcriptional regulation of brassinosteroids (BR, ethylene (ET, cytokinin (CK and salicylic acid (SA related wheat genes by after-ripening, a period of dry storage that decays dormancy. Changes in the expression of hormonal genes due to seed after-ripening did not occur in the anhydrobiotic state but rather in the hydrated state. After-ripening induced dormancy decay appears to be associated with imbibition mediated increase in the synthesis and signalling of BR, via transcriptional activation of de-etiolated2, dwarf4 and brassinosteroid signaling kinase, and repression of brassinosteroid insensitive 2. Our analysis is also suggestive of the significance of increased ET production, as reflected by enhanced transcription of 1-aminocyclopropane-1-carboxylic acid oxidase in after-ripened seeds, and tight regulation of seed response to ET in regulating dormancy decay. Differential transcriptions of lonely guy, zeatin O-glucosyltransferases and cytokinin oxidases, and pseudo-response regulator between dormant and after-ripened seeds implicate CK in the regulation of seed dormancy in wheat. Our analysis also reflects the association of dormancy decay in wheat with seed SA level and NPR independent SA signaling that appear to be regulated transcriptionally by phenylalanine ammonia lyase, and whirly and suppressor of npr1 inducible1 genes, respectively. Co-expression clustering of the hormonal genes implies the significance of synergistic and antagonistic interaction between the different plant hormones in regulating wheat seed dormancy. These results contribute to further our understanding of the molecular features controlling seed dormancy in wheat.

  7. Transcriptional Regulation of Telomerase Reverse Transcriptase (TERT) by MYC

    Science.gov (United States)

    Khattar, Ekta; Tergaonkar, Vinay

    2017-01-01

    Telomerase elongates telomeres and is crucial for maintaining genomic stability. While stem cells and cancer cells display high telomerase activity, normal somatic cells lack telomerase activity primarily due to transcriptional repression of telomerase reverse transcriptase (TERT), the catalytic component of telomerase. Transcription factor binding, chromatin status as well as epigenetic modifications at the TERT promoter regulates TERT transcription. Myc is an important transcriptional regulator of TERT that directly controls its expression by promoter binding and associating with other transcription factors. In this review, we discuss the current understanding of the molecular mechanisms behind regulation of TERT transcription by Myc. We also discuss future perspectives in investigating the regulation of Myc at TERT promoter during cancer development.

  8. Three transcription factors and the way immune cells affected by different plasma change in opposite ways in the development of the syndrome of pre-eclampsia

    Institute of Scientific and Technical Information of China (English)

    Liang Zhou; Zhu Jing; Wang Yunfei; Wang You; Zhang Yu; Lin Jianhua; Di Wen

    2014-01-01

    Background How the transcriptional factors regulated the innate and adaptive immune system in pregnancy and preeclampsia are less understood.Nevertheless,what the plasma work in the development of this disease was not sure.The present study was design to evaluate what the transcriptional factors change in innate and adaptive immune system and what the plasma do in this filed.Methods Peripheral blood mononuclear cells (PBMC) from non-pregnant women (n=18),women with clinically normal pregnancies (n=23) and women with pre-eclampsia (n=20) were separated from peripheral blood to isolate monocytes and T cells.The purity of monocytes and T cells were analysed by flow cytometry.Monocytes and T cells were stimulated in either lipopolysaccharides (LPS) or phorbol-myristate-acetate (PMA),respectively.Transcription Factor Arrays were used to screen the transcription factors of interest in comparing of different groups.PBMC were isolated from another 8 nonpregnant samples were co-incubated with different groups of plasma.Polymerase chain reaction (PCR) was performed using whole cell extractions of the samples.Results Nuclear factor of activated T-cells-1 (NFAT-1),signal transducers and activators of transcription-1 (STAT-1) and activator protein-1 (AP-1) are up-regulated in monocytes in pregnancy and more so in pre-eclampsia.On the the contrary,NFAT-1,STAT-1 and AP-1 are down-regulated in T cells in pregnancy and more so in pre-eclampsia.A reduction was observed in interferon (IFN)-y,interleukin (IL)-12 and IL-4 expression in T cells incubated with pre-eclamptic plasma.An elevation was observed in tumor necrosis factor (TNF)-α,IL-1 and IL-12 expression in monocytes incubated with preeclamptic plasma.Conclusions Innate immunity is over activated and adaptive immunity is over suppressed in the development of preeclampsia.NFAT-1,STAT-1 and AP-1 might be the central transcription factors in the pathogenesis of pre-eclampsia.They induced some changes in plasma and "educate" the

  9. Non-transcriptional regulatory processes shape transcriptional network dynamics

    OpenAIRE

    Ray, J. Christian J; Tabor, Jeffrey J.; Igoshin, Oleg A.

    2011-01-01

    Information about the extra- or intracellular environment is often captured as biochemical signals propagating through regulatory networks. These signals eventually drive phenotypic changes, typically by altering gene expression programs in the cell. Reconstruction of transcriptional regulatory networks has given a compelling picture of bacterial physiology, but transcriptional network maps alone often fail to describe phenotypes. In many cases, the dynamical performance of transcriptional re...

  10. Inactivation of fibroblast growth factor binding protein 3 causes anxiety-related behaviors.

    Science.gov (United States)

    Yamanaka, Yasunari; Kitano, Ayumi; Takao, Keizo; Prasansuklab, Anchalee; Mushiroda, Taisei; Yamazaki, Keiko; Kumada, Tomohiro; Shibata, Minoru; Takaoka, Yuki; Awaya, Tomonari; Kato, Takeo; Abe, Takaya; Iwata, Nakao; Miyakawa, Tsuyoshi; Nakamura, Yusuke; Nakahata, Tatsutoshi; Heike, Toshio

    2011-01-01

    The neurobiological mechanisms of emotional modulation and the molecular pathophysiology of anxiety disorders are largely unknown. The fibroblast growth factor (FGF) family has been implicated in the regulation of many physiological and pathological processes, which include the control of emotional behaviors. The present study examined mice with a targeted deletion of the fgf-bp3 gene, which encodes a novel FGF-binding protein, in animal models relevant to anxiety. To define the behavioral consequences of FGF-BP3 deficiency, we evaluated fgf-bp3-deficient mice using anxiety-related behavioral paradigms that provide a conflict between the desire to explore an unknown area or objects and the aversion to a brightly lit open space. The fgf-bp3-deficient mice exhibited alterations in time spent in the central area of the open-field arena, were less active in the lit areas of a light/dark transition test, and had a prolonged latency to feed during a novelty-induced hypophagia test. These changes were associated with alterations in light-induced orbitofrontal cortex (OFC) activation in an extracellular signal-regulated kinase (ERK) pathway-dependent manner. These results demonstrate that FGF-BP3 is a potent mediator of anxiety-related behaviors in mice and suggest that distinct pathways regulate emotional behaviors. Therefore, FGF-BP3 plays a critical role in the regulation of emotional states and in the development of anxiety disorders and should be investigated as a therapeutic target for anxiety disease in humans.

  11. Molecular determinants of epidermal growth factor binding: a molecular dynamics study.

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    Jeffrey M Sanders

    Full Text Available The epidermal growth factor receptor (EGFR is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of

  12. Extensive neuroadaptive changes in cortical gene-transcript expressions of the glutamate system in response to repeated intermittent MDMA administration in adolescent rats

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    Malki Rana

    2008-04-01

    Full Text Available Abstract Background Many studies have focused on the implication of the serotonin and dopamine systems in neuroadaptive responses to the recreational drug 3,4-methylenedioxy-metamphetamine (MDMA. Less attention has been given to the major excitatory neurotransmitter glutamate known to be implicated in schizophrenia and drug addiction. The aim of the present study was to investigate the effect of repeated intermittent MDMA administration upon gene-transcript expression of the glutamate transporters (EAAT1, EAAT2-1, EAAT2-2, the glutamate receptor subunits of AMPA (GluR1, GluR2, GluR3, the glutamate receptor subunits of NMDA (NR1, NR2A and NR2B, as well as metabotropic glutamate receptors (mGluR1, mGluR2, mGluR3, mGluR5 in six different brain regions. Adolescent male Sprague Dawley rats received MDMA at the doses of 3 × 1 and 3 × 5 mg/kg/day, or 3× vehicle 3 hours apart, every 7th day for 4 weeks. The gene-transcript levels were assessed using real-time PCR validated with a range of housekeeping genes. Results The findings showed pronounced enhancements in gene-transcript expression of GluR2, mGluR1, mGluR5, NR1, NR2A, NR2B, EAAT1, and EAAT2-2 in the cortex at bregma +1.6. In the caudate putamen, mRNA levels of GluR3, NR2A, and NR2B receptor subunits were significantly increased. In contrast, the gene-transcript expression of GluR1 was reduced in the hippocampus. In the hypothalamus, there was a significant increase of GluR1, GluR3, mGluR1, and mGluR3 gene-transcript expressions. Conclusion Repeated intermittent MDMA administration induces neuroadaptive changes in gene-transcript expressions of glutamatergic NMDA and AMPA receptor subunits, metabotropic receptors and transporters in regions of the brain regulating reward-related associative learning, cognition, and memory and neuro-endocrine functions.

  13. DNA damage and transcriptional changes induced by tributyltin (TBT) after short in vivo exposures of Chironomus riparius (Diptera) larvae.

    Science.gov (United States)

    Morales, Mónica; Martínez-Paz, Pedro; Ozáez, Irene; Martínez-Guitarte, José Luis; Morcillo, Gloria

    2013-08-01

    Tributyltin (TBT) is a widespread environmental contaminant in aquatic systems whose adverse effects in development and reproduction are related to its well-known endocrine-disrupting activity. In this work, the early molecular effects of TBT in Chironomus riparius (Diptera) were evaluated by analyzing its DNA damaging potential and the transcriptional response of different endocrine-related genes. Twenty-four-hour in vivo exposures of the aquatic larvae, at environmentally relevant doses of TBT, revealed genotoxic activity as shown by significant increases in DNA strand breaks quantified with the comet assay. TBT was also able to induce significant increases in transcripts from the ecdysone receptor gene (EcR), the ultraspiracle gene (usp) (insect ortholog of the retinoid X receptor), the estrogen-related receptor (ERR) gene and the E74 early ecdysone-inducible gene, as measured by real-time RT-PCR. In contrast, the expression of the vitellogenin (vg) gene remained unaltered, while the hsp70 gene appeared to be down-regulated. The ability of TBT to up-regulate hormonal target genes provides the first evidence, at genomic level, of its endocrine disruptive effects and also suggests a mechanism of action that mimics ecdysteroid hormones in insects. These data reveal for the first time the early genomic effects of TBT on an insect genome.

  14. A novel computational approach for the prediction of networked transcription factors of aryl hydrocarbon-receptor-regulated genes.

    Science.gov (United States)

    Kel, Alexander; Reymann, Susanne; Matys, Volker; Nettesheim, Paul; Wingender, Edgar; Borlak, Jürgen

    2004-12-01

    A novel computational method based on a genetic algorithm was developed to study composite structure of promoters of coexpressed genes. Our method enabled an identification of combinations of multiple transcription factor binding sites regulating the concerted expression of genes. In this article, we study genes whose expression is regulated by a ligand-activated transcription factor, aryl hydrocarbon receptor (AhR), that mediates responses to a variety of toxins. AhR-mediated change in expression of AhR target genes was measured by oligonucleotide microarrays and by reverse transcription-polymerase chain reaction in human and rat hepatocytes. Promoters and long-distance regulatory regions (>10 kb) of AhR-responsive genes were analyzed by the genetic algorithm and a variety of other computational methods. Rules were established on the local oligonucleotide context in the flanks of the AhR binding sites, on the occurrence of clusters of AhR recognition elements, and on the presence in the promoters of specific combinations of multiple binding sites for the transcription factors cooperating in the AhR regulatory network. Our rules were applied to search for yet unknown Ah-receptor target genes. Experimental evidence is presented to demonstrate high fidelity of this novel in silico approach.

  15. A model for aryl hydrocarbon receptor-activated gene expression shows potency and efficacy changes and predicts squelching due to competition for transcription co-activators.

    Directory of Open Access Journals (Sweden)

    Ted W Simon

    Full Text Available A stochastic model of nuclear receptor-mediated transcription was developed based on activation of the aryl hydrocarbon receptor (AHR by 2,3,7,8-tetrachlorodibenzodioxin (TCDD and subsequent binding the activated AHR to xenobiotic response elements (XREs on DNA. The model was based on effects observed in cells lines commonly used as in vitro experimental systems. Following ligand binding, the AHR moves into the cell nucleus and forms a heterodimer with the aryl hydrocarbon nuclear translocator (ARNT. In the model, a requirement for binding to DNA is that a generic coregulatory protein is subsequently bound to the AHR-ARNT dimer. Varying the amount of coregulator available within the nucleus altered both the potency and efficacy of TCDD for inducing for transcription of CYP1A1 mRNA, a commonly used marker for activation of the AHR. Lowering the amount of available cofactor slightly increased the EC50 for the transcriptional response without changing the efficacy or maximal response. Further reduction in the amount of cofactor reduced the efficacy and produced non-monotonic dose-response curves (NMDRCs at higher ligand concentrations. The shapes of these NMDRCs were reminiscent of the phenomenon of squelching. Resource limitations for transcriptional machinery are becoming apparent in eukaryotic cells. Within single cells, nuclear receptor-mediated gene expression appears to be a stochastic process; however, intercellular communication and other aspects of tissue coordination may represent a compensatory process to maintain an organism's ability to respond on a phenotypic level to various stimuli within an inconstant environment.

  16. The TERT promoter SNP rs2853669 decreases E2F1 transcription factor binding and increases mortality and recurrence risks in liver cancer.

    Science.gov (United States)

    Ko, Eunkyong; Seo, Hyun-Wook; Jung, Eun Sun; Kim, Baek-hui; Jung, Guhung

    2016-01-05

    A common single-nucleotide polymorphism in the telomerase reverse transcriptase (TERT) promoter, rs2853669 influences patient survival rates and the risk of developing cancer. Recently, several lines of evidence suggest that the rs2853669 suppresses TERT promoter mutation-mediated TERT expression levels and cancer mortality as well as recurrence rates. However, no reports are available on the impact of rs2853669 on TERT expression in hepatocellular carcinoma (HCC) and its association with patient survival. Here, we found that HCC-related overall and recurrence-free survival rates were not associated with TERT promoter mutation individually, but rs2853669 and the TERT promoter mutation in combination were associated with poor survival rates. TERT mRNA expression and telomere fluorescence levels were greater in patients with HCC who had both the combination. The combination caused TERT promoter methylation through regulating the binding of DNA methyltransferase 1 and histone deacetylase 1 to the TERT promoter in HCC cell lines. The TERT expression level was significantly higher in HCC tumor with a methylated promoter than in that with an unmethylated promoter. In conclusion, we demonstrate a substantial role for the rs2853669 in HCC with TERT promoter mutation, which suggests that the combination of the rs2853669 and the mutation indicate poor prognoses in liver cancer.

  17. The transcription factor ATF7 mediates lipopolysaccharide-induced epigenetic changes in macrophages involved in innate immunological memory.

    Science.gov (United States)

    Yoshida, Keisuke; Maekawa, Toshio; Zhu, Yujuan; Renard-Guillet, Claire; Chatton, Bruno; Inoue, Kentaro; Uchiyama, Takeru; Ishibashi, Ken-ichi; Yamada, Takuji; Ohno, Naohito; Shirahige, Katsuhiko; Okada-Hatakeyama, Mariko; Ishii, Shunsuke

    2015-10-01

    Immunological memory is thought to be mediated exclusively by lymphocytes. However, enhanced innate immune responses caused by a previous infection increase protection against reinfection, which suggests the presence of innate immunological memory. Here we identified an important role for the stress-response transcription factor ATF7 in innate immunological memory. ATF7 suppressed a group of genes encoding factors involved in innate immunity in macrophages by recruiting the histone H3K9 dimethyltransferase G9a. Treatment with lipopolysaccharide, which mimics bacterial infection, induced phosphorylation of ATF7 via the kinase p38, which led to the release of ATF7 from chromatin and a decrease in repressive histone H3K9me2 marks. A partially disrupted chromatin structure and increased basal expression of target genes were maintained for long periods, which enhanced resistance to pathogens. ATF7 might therefore be important in controlling memory in cells of the innate immune system.

  18. Changes in transcription during recovery from heat injury in Salmonella typhimurium and effects of BCAA on recovery.

    Science.gov (United States)

    Hsu-Ming, Wen; Naito, Kimitaka; Kinoshita, Yoshimasa; Kobayashi, Hiroshi; Honjoh, Ken-ichi; Tashiro, Kousuke; Miyamoto, Takahisa

    2012-06-01

    Mechanisms of recovery from heat injury in Salmonella typhimurium were elucidated. Recovery of the heat-injured S. typhimurium cells in TSB resulted in full recovery after 3 h of incubation at 37°C. The DNA microarray analysis of 30- and 60-min recovering cells resulted in an increase in transcription of 89 and 141 genes, respectively. Among them, 15 genes, with known function, seemed to be somewhat involved in recovery. They encoded proteins involved in branched-chain amino acid (BCAA) transport (livJ, livH), cell envelope integrity (ddg), heat-shock response (cpxP, rrmJ), phage shock protein (pspA), ribosome modulation factor (rmf), virulence (sseB) transcriptional regulation (rpoE, rpoH, rseA, rseB, rseC) and ArcB signal transduction (sixA) and cytoplasmic membrane protein (fxsA). Among them, the effects of BCAA supplementation on recovery from heat injury were studied to confirm the importance of the BCAA transport liv genes during recovery. It was found that supplementation of TSB with 0.1% BCAA resulted in an enhanced recovery of injured cells in comparison to those recovered in TSB without BCAA. Supplementation of BCAA at 0.1% resulted in a cell count increase 4.4-fold greater than that of the control after 1 h incubation. It seems that BCAA promoted the recovery by promoting protein synthesis either directly through their use in translation or indirectly through stimulation of protein synthesis by activation of the Lrp protein.

  19. Changes in the Expression of Transcription Factors Involved in Modulating the Expression of EPO-R in Activated Human CD4-Positive Lymphocytes.

    Science.gov (United States)

    Lisowska, Katarzyna A; Frackowiak, Joanna E; Mikosik, Anna; Witkowski, Jacek M

    2013-01-01

    We have recently described the presence of the erythropoietin receptor (EPO-R) on CD4(+) lymphocytes and demonstrated that its expression increases during their activation, reaching a level reported to be typical for erythroid progenitors. This observation suggests that EPO-R expression is modulated during lymphocyte activation, which may be important for the cells' function. Here we investigated whether the expression of GATA1, GATA3 and Sp1 transcription factors is correlated with the expression of EPO-R in human CD4(+) lymphocytes stimulated with monoclonal anti-CD3 antibody. The expression of GATA1, GATA3 and Sp1 transcription factors in CD4(+) cells was estimated before and after stimulation with anti-CD3 antibody by Western Blot and flow cytometry. The expression of EPO-R was measured using real-time PCR and flow cytometry. There was no change in the expression of GATA1 and GATA3 in CD4(+) lymphocytes after stimulation with anti-CD3 antibody. However, stimulation resulted in the significantly increased expression of the Sp1 factor. CD4(+) lymphocytes stimulated with anti-CD3 antibody exhibited an increase in both the expression level of EPOR gene and the number of EPO-R molecules on the cells' surface, the latter being significantly correlated with the increased expression of Sp1. Sp1 is noted to be the single transcription factor among the ones studied whose level changes as a result of CD4(+) lymphocyte stimulation. It seems that Sp1 may significantly affect the number of EPO-R molecules present on the surface of activated CD4(+) lymphocytes.

  20. Genome-wide transcriptional changes and defence-related chemical profiling of rice in response to infestation by the rice striped stem borer Chilo suppressalis.

    Science.gov (United States)

    Zhou, Guoxin; Wang, Xia; Yan, Feng; Wang, Xia; Li, Ran; Cheng, Jiaan; Lou, Yonggen

    2011-09-01

    How rice defends itself against pathogen infection is well documented, but little is known about how it defends itself against herbivore attack. We measured changes in the transcriptome and chemical profile of rice when the plant is infested by the striped stem borer (SSB) Chilo suppressalis. Infestation by SSBs resulted in changes in the expression levels of 4545 rice genes; this number accounts for about 8% of the genome and is made up of 18 functional groups with broad functions. The largest group comprised genes involved in metabolism, followed by cellular transport, transcription and cellular signaling. Infestation by SSBs modulated many genes responsible for the biosynthesis of plant hormones and plant signaling. Jasmonic acid (JA), salicylic acid (SA) and ethylene were the major hormones that shaped the SSB-induced defence responses of rice. Many secondary signal transduction components, such as those involved in Ca²⁺ signaling and G-protein signaling, receptor and non-receptor protein kinases, and transcription factors were involved in the SSB-induced responses of rice. Photosynthesis and ATP synthesis from photophosphorylation were restricted by SSB feeding. In addition, SSB infestation induced the accumulation of defence compounds, including trypsin proteinase inhibitors (TrypPIs) and volatile organic compounds. These results demonstrate that SSB-induced defences required rice to reconfigure a wide variety of its metabolic, physiological and biochemical processes.

  1. DIAGNOSTIC VALUE OF SERUM INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 IN CHILDREN WITH OR WITHOUT GROWTH HORMONE DEFICIENCY

    Institute of Scientific and Technical Information of China (English)

    覃舒文; 史轶蘩; 邓洁英

    2002-01-01

    Objective. To study the value of serum insulin-like growth factor binding protein-3 (IGFBP-3) levels in differential diagnosis of growth hormone deficiency (GHD). Methods. To measure serum IGFBP-3 levels by RIA in normal children and adolescents, GHD children and short-stature children without GHD. Results. Serum level of IGFBP-3 in 129 children with untreated GHD and with no pubertal development was 1.6± 0.9 mg/L, which was less than that in normal group of the same age, but overlapped with the normal children in Tanner stage I. After six -month treatment with recombinant human growth hormone (rhGH), serum level of IGFBP-3 in 59 GHD significantly increased from 1.3± 0.7 mg/L to 2.7± 0.9 mg/L, accompanied by an increase of body heights, growth velocities and serum level of IGF-1. Serum level of IGFBP-3 in 55 short-stature children without GHD was 3.3± 2.2 mg/L, which was not significantly different from that in normal group. Conclusion. Serum IGFBP-3 level can reflect the status of GH secretion in children with GHD and is a useful marker for differential diagnosis of GHD.

  2. DIAGNOSTIC VALUE OF SERUM INSULIN—LIKE GROWTH FACTOR BINDING PROTEIN—3 IN CHILDREN WITH OR WITHOUT GROWTH HORMONE DEFICIENCY

    Institute of Scientific and Technical Information of China (English)

    覃舒文; 史轶蘩; 等

    2002-01-01

    Objective:To study the value of serum insulin-like growth factor binding protein-3 (IGFBP-3) levels in differential diagnosis of growth hormone deficiency(GHD).Methods:To measure serum IGFBP-3 levels by RIA in normal children and adolescents,GHD children and short-stature children without (GHD).Methods:To measure serum IGFBP-3 levels by RIA in normal children and adolescents,GHD children and short-stature children without GHD.Results.Serum level of IGFBP-3 in 129 children with untreated GHD and with no pubertal development was 1.6±0.9mg/L,which was less than that in normal group of the same age,but overlapped with the normal children in Tanner stage I.After six-month treatment with recombinant human growth hormone(rhGH),serum level of IGFBP-3 in 59 GHD significantly increased from 1.3±0.7mg/L to 2.7±0.9mg/L,accompanied by an increase of body heights,growth velocities and serum level of IGF-1.Serum level of IGFBP-3 in 55 shortstature children without GHD was 3.3±2.2mg/L,which was not significantly different from that in normal group.Conclusion:Serum IGFBP-3 level can reflect the status of GH secretion in children with GHD and is a useful marker for differential diagnosis of GHD.

  3. Expression of insulin-like growth factor binding protein-2 in gastric carcinoma and its relationship with cell proliferation

    Institute of Scientific and Technical Information of China (English)

    Liang-Hui Shi; Xiao-Qun Zhu; Guo-Hai Zhao; Ya-Bin Xia; Yi-Sheng Zhang

    2006-01-01

    AIM: To investigate the expression of insulin-like growth factor binding protein-2 (IGFBP-2) in gastric carcinoma and its clinical significance and to explore its relationship with cell proliferation.METHODS: Expressions of IGFBP-2 and Ki-67 in 118cases of gastric carcinoma and 40 cases of normal gastric mucosa were detected by EnVision immunohistochemical technique.RESULTS: Expression of IGFBP-2 in gastric carcinoma was higher than that in normal gastric mucosa (P 0.05). Fxpression of IGFBP-2 in aclvancecl gastric carcinoma was higher than that in early gastric carcinoma (P < 0.05). Expression of IGFBP-2 in gastric carcinoma with lymph node metastasis was higher than that without lymph node metastasis (P < 0.01).IGFBP-2 expression was a positively related to the clinical stage of gastric carcinoma (P < 0.01). There was a positive correlation between IGFBP-2 and Ki-67 (P < 0.05).CONCLUSION: IGFBP-2 may be involved in carcinogenesis and progression of gastric carcinoma by promoting cell proliferation.

  4. Quantitative Proteomics Identifies Serum Response Factor Binding Protein 1 as a Host Factor for Hepatitis C Virus Entry

    Directory of Open Access Journals (Sweden)

    Gisa Gerold

    2015-08-01

    Full Text Available Hepatitis C virus (HCV enters human hepatocytes through a multistep mechanism involving, among other host proteins, the virus receptor CD81. How CD81 governs HCV entry is poorly characterized, and CD81 protein interactions after virus binding remain elusive. We have developed a quantitative proteomics protocol to identify HCV-triggered CD81 interactions and found 26 dynamic binding partners. At least six of these proteins promote HCV infection, as indicated by RNAi. We further characterized serum response factor binding protein 1 (SRFBP1, which is recruited to CD81 during HCV uptake and supports HCV infection in hepatoma cells and primary human hepatocytes. SRFBP1 facilitates host cell penetration by all seven HCV genotypes, but not of vesicular stomatitis virus and human coronavirus. Thus, SRFBP1 is an HCV-specific, pan-genotypic host entry factor. These results demonstrate the use of quantitative proteomics to elucidate pathogen entry and underscore the importance of host protein-protein interactions during HCV invasion.

  5. Functional roles and clinical values of insulin-like growth factor-binding protein-5 in different types of cancers

    Institute of Scientific and Technical Information of China (English)

    G(o)k(c)e Güllü; Sevgi Karabulut; Mustafa Akkiprik

    2012-01-01

    Insulin-like growth factor-binding proteins (IGFBPs) are critical regulators of the mitogenic activity of insulin-like growth factors (IGFs).IGFBP5,one of these IGFBPs,has special structural features,including a nuclear transport domain,heparin-binding motif,and IGF/extracellular matrix/acid-labile subunit-binding sites.Furthermore,IGFBP5 has several functional effects on carcinogenesis and even normal cell processes,such as cell growth,death,motility,and tissue remodeling.These biological effects are sometimes related with IGF (IGF-dependent effects) and sometimes not (IGF-independent effects).The functional role of IGFBP5 is most likely determined in a cell-type and tissue-type specific manner but also depends on cell context,especially in terms of the diversity of interacting proteins and the potential for nuclear localization.Clinical findings show that IGFBP5 has the potential to be a useful clinical biomarker for predicting response to therapy and clinical outcome of cancer patients.In this review,we summarize the functional diversity and clinical importance of IGFBP5 in different types of cancers.

  6. Advance on Insulin-like Growth Factor Binding Protein 2 in Lung Cancer and Other Solid Tumors

    Institute of Scientific and Technical Information of China (English)

    Wu Weiqin; Lu Kaihua

    2013-01-01

    Increasing evidence has revealed that IGF signalling plays a key role in cellular proliferation, survival, differentiation and senescence. Dysregulation of this signalling pathway is related to the development and progression of many human diseases, including cancer, diabetes and atherosclerosis. Insulin-like growth factor binding protein-2 (IGFBP-2) is reported to be a modulator of the action of insulin-like growth factors (IGFs), whereas IGF-independent effects of IGFBP-2 on cellular proliferation, apoptosis, and mobility have been revealed not only during the embryonic state but also in the pathological state of cancer. IGFBP-2 is involved in the genesis and progress of various malignancies including lung cancer. Recent ifndings show in many pre-clinical trials that IGFBP-2 may contribute to the transformation and progression of lung cancer. These studies suggest that IGFBP-2 may be a potential therapeutic target for lung cancer. In this review, we provide an overview on IGFBP-2, review corresponding studies investigating the role of IGFBP-2 as a cancer target in multiple tumors and discuss its possible mechanism in lung cancer.

  7. Genome-Wide Transcriptional Regulation Mediated by Biochemically Distinct SWI/SNF Complexes.

    Directory of Open Access Journals (Sweden)

    Jesse R Raab

    2015-12-01

    Full Text Available Multiple positions within the SWI/SNF chromatin remodeling complex can be filled by mutually exclusive subunits. Inclusion or exclusion of these proteins defines many unique forms of SWI/SNF and has profound functional consequences. Often this complex is studied as a single entity within a particular cell type and we understand little about the functional relationship between these biochemically distinct forms of the remodeling complex. Here we examine the functional relationships among three complex-specific ARID (AT-Rich Interacting Domain subunits using genome-wide chromatin immunoprecipitation, transcriptome analysis, and transcription factor binding maps. We find widespread overlap in transcriptional regulation and the genomic binding of distinct SWI/SNF complexes. ARID1B and ARID2 participate in wide-spread cooperation to repress hundreds of genes. Additionally, we find numerous examples of competition between ARID1A and another ARID, and validate that gene expression changes following loss of one ARID are dependent on the function of an alternative ARID. These distinct regulatory modalities are correlated with differential occupancy by transcription factors. Together, these data suggest that distinct SWI/SNF complexes dictate gene-specific transcription through functional interactions between the different forms of the SWI/SNF complex and associated co-factors. Most genes regulated by SWI/SNF are controlled by multiple biochemically distinct forms of the complex, and the overall expression of a gene is the product of the interaction between these different SWI/SNF complexes. The three mutually exclusive ARID family members are among the most frequently mutated chromatin regulators in cancer, and understanding the functional interactions and their role in transcriptional regulation provides an important foundation to understand their role in cancer.

  8. Genome-Wide Transcriptional Regulation Mediated by Biochemically Distinct SWI/SNF Complexes

    Science.gov (United States)

    Raab, Jesse R.; Resnick, Samuel; Magnuson, Terry

    2015-01-01

    Multiple positions within the SWI/SNF chromatin remodeling complex can be filled by mutually exclusive subunits. Inclusion or exclusion of these proteins defines many unique forms of SWI/SNF and has profound functional consequences. Often this complex is studied as a single entity within a particular cell type and we understand little about the functional relationship between these biochemically distinct forms of the remodeling complex. Here we examine the functional relationships among three complex-specific ARID (AT-Rich Interacting Domain) subunits using genome-wide chromatin immunoprecipitation, transcriptome analysis, and transcription factor binding maps. We find widespread overlap in transcriptional regulation and the genomic binding of distinct SWI/SNF complexes. ARID1B and ARID2 participate in wide-spread cooperation to repress hundreds of genes. Additionally, we find numerous examples of competition between ARID1A and another ARID, and validate that gene expression changes following loss of one ARID are dependent on the function of an alternative ARID. These distinct regulatory modalities are correlated with differential occupancy by transcription factors. Together, these data suggest that distinct SWI/SNF complexes dictate gene-specific transcription through functional interactions between the different forms of the SWI/SNF complex and associated co-factors. Most genes regulated by SWI/SNF are controlled by multiple biochemically distinct forms of the complex, and the overall expression of a gene is the product of the interaction between these different SWI/SNF complexes. The three mutually exclusive ARID family members are among the most frequently mutated chromatin regulators in cancer, and understanding the functional interactions and their role in transcriptional regulation provides an important foundation to understand their role in cancer. PMID:26716708

  9. Peri/nuclear localization of intact insulin-like growth factor binding protein-2 and a distinct carboxyl-terminal IGFBP-2 fragment in vivo

    NARCIS (Netherlands)

    Hoeflich, A; Reisinger, R; Schuett, BS; Elmlinger, MW; Russo, VC; Vargas, GA; Jehle, PM; Lahm, H; Renner-Muller, [No Value; Wolf, E

    2004-01-01

    Insulin-like growth factor binding protein-2 (IGFBP-2) as one of the most important IGFBPs has never been assessed in the intracellular compartment in vivo. Since there is evidence for novel intracellular functions of distinct IGFBPs, we investigated the presence of IGFBP-2 inside the cell. In peri/

  10. Insulin-like growth factor binding proteins: regulation in chronic active plaques in multiple sclerosis and functional analysis of glial cells

    NARCIS (Netherlands)

    Chesik, D.; De Keyser, J.; Glazenburg, L.; Wilczak, N.

    2006-01-01

    Studies in experimental allergic encephalomyelitis, an animal model of multiple sclerosis (MS), suggest that astrocyte-secreted insulin-like growth factor binding protein-2 (IGFBP-2) helps target IGF-1 to IGF-1 receptor-expressing oligodendrocytes and promote remyelination. We examined the presence

  11. Insulin-like growth factor binding proteins : regulation in chronic active plaques in multiple sclerosis and functional analysis of glial cells

    NARCIS (Netherlands)

    Chesik, Daniel; De Keyser, Jacques; Glazenburg, Lisa; Wilczak, Nadine

    2006-01-01

    Studies in experimental allergic encephalomyelitis, an animal model of multiple sclerosis (MS), suggest that astrocyte-secreted insulin-like growth factor binding protein-2 (IGFBP-2) helps target IGF-1 to IGF-1 receptor-expressing oligodendrocytes and promote remyelination. We examined the presence

  12. No DNA damage response and negligible genome-wide transcriptional changes in human embryonic stem cells exposed to terahertz radiation.

    Science.gov (United States)

    Bogomazova, A N; Vassina, E M; Goryachkovskaya, T N; Popik, V M; Sokolov, A S; Kolchanov, N A; Lagarkova, M A; Kiselev, S L; Peltek, S E

    2015-01-13

    Terahertz (THz) radiation was proposed recently for use in various applications, including medical imaging and security scanners. However, there are concerns regarding the possible biological effects of non-ionising electromagnetic radiation in the THz range on cells. Human embryonic stem cells (hESCs) are extremely sensitive to environmental stimuli, and we therefore utilised this cell model to investigate the non-thermal effects of THz irradiation. We studied DNA damage and transcriptome responses in hESCs exposed to narrow-band THz radiation (2.3 THz) under strict temperature control. The transcription of approximately 1% of genes was subtly increased following THz irradiation. Functional annotation enrichment analysis of differentially expressed genes revealed 15 functional classes, which were mostly related to mitochondria. Terahertz irradiation did not induce the formation of γH2AX foci or structural chromosomal aberrations in hESCs. We did not observe any effect on the mitotic index or morphology of the hESCs following THz exposure.

  13. Early and delayed long-term transcriptional changes and short-term transient responses during cold acclimation in olive leaves

    Science.gov (United States)

    Leyva-Pérez, María de la O; Valverde-Corredor, Antonio; Valderrama, Raquel; Jiménez-Ruiz, Jaime; Muñoz-Merida, Antonio; Trelles, Oswaldo; Barroso, Juan Bautista; Mercado-Blanco, Jesús; Luque, Francisco

    2015-01-01

    Low temperature severely affects plant growth and development. To overcome this constraint, several plant species from regions having a cool season have evolved an adaptive response, called cold acclimation. We have studied this response in olive tree (Olea europaea L.) cv. Picual. Biochemical stress markers and cold-stress symptoms were detected after the first 24 h as sagging leaves. After 5 days, the plants were found to have completely recovered. Control and cold-stressed plants were sequenced by Illumina HiSeq 1000 paired-end technique. We also assembled a new olive transcriptome comprising 157,799 unigenes and found 6,309 unigenes differentially expressed in response to cold. Three types of response that led to cold acclimation were found: short-term transient response, early long-term response, and late long-term response. These subsets of unigenes were related to different biological processes. Early responses involved many cold-stress-responsive genes coding for, among many other things, C-repeat binding factor transcription factors, fatty acid desaturases, wax synthesis, and oligosaccharide metabolism. After long-term exposure to cold, a large proportion of gene down-regulation was found, including photosynthesis and plant growth genes. Up-regulated genes after long-term cold exposure were related to organelle fusion, nucleus organization, and DNA integration, including retrotransposons. PMID:25324298

  14. Loss of BAF (mSWI/SNF Complexes Causes Global Transcriptional and Chromatin State Changes in Forebrain Development

    Directory of Open Access Journals (Sweden)

    Ramanathan Narayanan

    2015-12-01

    Full Text Available BAF (Brg/Brm-associated factors complexes play important roles in development and are linked to chromatin plasticity at selected genomic loci. Nevertheless, a full understanding of their role in development and chromatin remodeling has been hindered by the absence of mutants completely lacking BAF complexes. Here, we report that the loss of BAF155/BAF170 in double-conditional knockout (dcKO mice eliminates all known BAF subunits, resulting in an overall reduction in active chromatin marks (H3K9Ac, a global increase in repressive marks (H3K27me2/3, and downregulation of gene expression. We demonstrate that BAF complexes interact with H3K27 demethylases (JMJD3 and UTX and potentiate their activity. Importantly, BAF complexes are indispensable for forebrain development, including proliferation, differentiation, and cell survival of neural progenitor cells. Our findings reveal a molecular mechanism mediated by BAF complexes that controls the global transcriptional program and chromatin state in development.

  15. Identification and characterization of transcription networks in environmentally significant species

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, Charles E.; McCue, Lee Ann

    2005-11-30

    Understanding the regulation of gene expression, transcription regulation in particular, is one of the grand challenges of molecular biology. Transcription regulation is arguably the most important foundation of cellular function, since it exerts the most fundamental control of the abundance of virtually all of a cell's functional macromolecules. Nevertheless, this process, perhaps because of its difficulty, has been the subject of only a limited number of genomic level analyses. We have undertaken bioinformatics projects to address this issue by developing (1) a cross-species comparison method (i.e. phylogenetic footprinting) for the identification of transcription factor binding sites, (2) a Bayesian clustering method to identify regulons, (3) an improved scanning algorithm that uses a position weight matrix and several related species sequence data to locate transcription factor binding sites, and (4) a method to predict cognate binding sites for transcription factors of unknown specificity. These bioinformatics methods were developed using the model proteobacterium Escherichia coli, with further applications to the genomes of environmentally significant microbes (Rhodopseudomonas palustris, Shewanella oneidensis) in later years of the grant.

  16. Non-transcriptional regulatory processes shape transcriptional network dynamics.

    Science.gov (United States)

    Ray, J Christian J; Tabor, Jeffrey J; Igoshin, Oleg A

    2011-10-11

    Information about the extra- or intracellular environment is often captured as biochemical signals that propagate through regulatory networks. These signals eventually drive phenotypic changes, typically by altering gene expression programmes in the cell. Reconstruction of transcriptional regulatory networks has given a compelling picture of bacterial physiology, but transcriptional network maps alone often fail to describe phenotypes. Cellular response dynamics are ultimately determined by interactions between transcriptional and non-transcriptional networks, with dramatic implications for physiology and evolution. Here, we provide an overview of non-transcriptional interactions that can affect the performance of natural and synthetic bacterial regulatory networks.

  17. Effect of sample size and P-value filtering techniques on the detection of transcriptional changes induced in rat neuroblastoma (NG108 cells by mefloquine

    Directory of Open Access Journals (Sweden)

    Dow Geoffrey S

    2003-02-01

    Full Text Available Abstract Background There is no known biochemical basis for the adverse neurological events attributed to mefloquine. Identification of genes modulated by toxic agents using microarrays may provide sufficient information to generate hypotheses regarding their mode of action. However, this utility may be compromised if sample sizes are too low or the filtering methods used to identify differentially expressed genes are inappropriate. Methods The transcriptional changes induced in rat neuroblastoma cells by a physiological dose of mefloquine (10 micro-molar were investigated using Affymetrix arrays. A large sample size was used (total of 16 arrays. Genes were ranked by P-value (t-test. RT-PCR was used to confirm (or reject the expression changes of several of the genes with the lowest P-values. Different P-value filtering methods were compared in terms of their ability to detect these differentially expressed genes. A retrospective power analysis was then performed to determine whether the use of lower sample sizes might also have detected those genes with altered transcription. Results Based on RT-PCR, mefloquine upregulated cJun, IkappaB and GADD153. Reverse Holm-Bonferroni P-value filtering was superior to other methods in terms of maximizing detection of differentially expressed genes but not those with unaltered expression. Reduction of total microarray sample size ( Conclusions Adequate sample sizes and appropriate selection of P-value filtering methods are essential for the reliable detection of differentially expressed genes. The changes in gene expression induced by mefloquine suggest that the ER might be a neuronal target of the drug.

  18. Transcriptional and Bioinformatic Analysis Provide a Relationship between Host Response Changes to Marek’s Disease Viruses Infection and an Integrated Long Terminal Repeat

    Directory of Open Access Journals (Sweden)

    Ning eCui

    2016-04-01

    Full Text Available GX0101, Marek’s disease virus (MDV strain with a long terminal repeat (LTR insert of reticuloendotheliosis virus (REV, was isolated from CVI988/Rispens vaccinated birds showing tumors. We have constructed a LTR deleted strain GX0101∆LTR in our previous study. To compare the host responses to GX0101 and GX0101∆LTR, chicken embryo fibroblasts (CEF cells were infected with two MDV strains and a gene-chip containing chicken genome was employed to examine gene transcription changes in host cells in the present study. Of the 42 368 chicken transcripts on the chip, there were 2199 genes that differentially expressed in CEF infected with GX0101 compared to GX0101∆LTR significantly. Differentially expressed genes were distributed to 25 possible gene networks according to their intermolecular connections and were annotated to 56 pathways. The insertion of REV LTR showed the greatest influence on cancer formation and metastasis, followed with immune changes, atherosclerosis and nervous system disorders in MDV-infected CEF cells. Based on these bio functions, GX0101 infection was predicated with a greater growth and survival inhibition but lower oncogenicity in chickens than GX0101∆LTR, at least in the acute phase of infection. In summary, the insertion of REV LTR altered the expression of host genes in response to MDV infection, possibly resulting in novel phenotypic properties in chickens. Our study has provided the evidence of retroviral insertional changes of host responses to herpesvirus infection for the first time, which will promote to elucidation of the possible relationship between the LTR insertion and the observed phenotypes.

  19. Insulin-like growth factor binding protein-3 affects osteogenic efficacy on dental implants in rat mandible

    Energy Technology Data Exchange (ETDEWEB)

    Bhattarai, Govinda; Lee, Young-Hee [Department of Oral Biochemistry, Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Lee, Min-Ho [Department of Dental Materials, Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Park, Il-Song [Division of Advanced Materials Engineering, Research Center for Advanced Materials, Development and Institute of Biodegradable Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Yi, Ho-Keun, E-mail: yihokn@chonbuk.ac.kr [Department of Oral Biochemistry, Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of)

    2015-10-01

    Insulin like growth factor binding protein-3 (IGFBP-3) in bone cells and its utilization in dental implants have not been well studied. The aim of this study was to determine the osteogenic efficacy of chitosan gold nanoparticles (Ch-GNPs) conjugated with IGFBP-3 coated titanium (Ti) implants. Ch-GNPs were conjugated with IGFBP-3 plasmid DNA through a coacervation process. Conjugation was cast over Ti surfaces, and cells were seeded on coated surfaces. For in vitro analysis the expression of different proteins was analyzed by immunoblotting. For in vivo analysis, Ch-GNP/IGFBP-3 coated implants were installed in rat mandibles. Four weeks post-implantation, mandibles were examined by microcomputed tomography (μCT), immunohistochemistry, hematoxylin & eosin and tartrate resistance acid phosphatase staining. In vitro overexpressed Ch-GNP/IGFBP-3 coated Ti surfaces was associated with activation of extracellular signal related kinase (ERK), inhibition of the stress activated protein c-Jun N-terminal kinase (JNK) and enhanced bone morphogenetic protein (BMP)-2 and 7 compared to control. Further, in vivo, Ch-GNP/IGFBP-3 coated implants were associated with inhibition of implant induced osteoclastogenesis molecules, receptor activator of nuclear factor kappa-B ligand (RANKL) and enhanced expression of osteogenic molecules including BMP2/7 and osteopontin (OPN). The μCT analysis demonstrated that IGFBP-3 increased the volume of newly formed bone surrounding the implants compared to control (n = 5; p < 0.05). These results support the view that IGFBP-3 overexpression diminishes osteoclastogenesis and enhances osteogenesis of Ti implants, and can serve as a potent molecule for the development of good implantation. - Highlights: • Chitosan gold nanoparticles were conjugated with IGFBP-3 and coated onto surface of the titanium implants for gene delivery to bone. • Implants were inserted in rat mandible for 4 weeks. • Parameters studied: histopathology and radiology.

  20. Characterization of an Oct1 orthologue in the channel catfish, Ictalurus punctatus: A negative regulator of immunoglobulin gene transcription?

    NARCIS (Netherlands)

    Lennard, M.L.; Hikima, J.I.; Ross, D.A.; Kruiswijk, C.P.; Wilson, M.R.; Miller, N.W.; Warr, G.W.

    2007-01-01

    Background - The enhancer (E¿3') of the immunoglobulin heavy chain locus (IGH) of the channel catfish (Ictalurus punctatus) has been well characterized. The functional core region consists of two variant Oct transcription factor binding octamer motifs and one E-protein binding ¿E5 site. An orthologu

  1. Dietary flavanols modulate the transcription of genes associated with cardiovascular pathology without changes in their DNA methylation state.

    Directory of Open Access Journals (Sweden)

    Dragan Milenkovic

    Full Text Available BACKGROUND: In a recent intervention study, the daily supplementation with 200 mg monomeric and oligomeric flavanols (MOF from grape seeds for 8 weeks revealed a vascular health benefit in male smokers. The objective of the present study was to determine the impact of MOF consumption on the gene expression profile of leukocytes and to assess changes in DNA methylation. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression profiles were determined using whole genome microarrays (Agilent and DNA methylation was assessed using HumanMethylation450 BeadChips (Illumina. MOF significantly modulated the expression of 864 genes. The majority of the affected genes are involved in chemotaxis, cell adhesion, cell infiltration or cytoskeleton organisation, suggesting lower immune cell adhesion to endothelial cells. This was corroborated by in vitro experiments showing that MOF exposure of monocytes attenuates their adhesion to TNF-α-stimulated endothelial cells. Nuclear factor kappa B (NF-κB reporter gene assays confirmed that MOF decrease the activity of NF-κB. Strong inter-individual variability in the leukocytes' DNA methylation was observed. As a consequence, on group level, changes due to MOF supplementation could not be found. CONCLUSION: Our study revealed that an 8 week daily supplementation with 200 mg MOF modulates the expression of genes associated with cardiovascular disease pathways without major changes of their DNA methylation state. However, strong inter-individual variation in leukocyte DNA methylation may obscure the subtle epigenetic response to dietary flavanols. Despite the lack of significant changes in DNA methylation, the modulation of gene expression appears to contribute to the observed vascular health effect of MOF in humans.

  2. Insulin resistance induced by physical inactivity is associated with multiple transcriptional changes in skeletal muscle in young men

    DEFF Research Database (Denmark)

    Alibegovic, A C; Sonne, M P; Højbjerre, L

    2010-01-01

    Physical inactivity is a risk factor for insulin resistance. We examined the effect of 9 days of bed rest on basal and insulin-stimulated expression of genes potentially involved in insulin action by applying hypothesis-generating microarray in parallel with candidate gene real-time PCR approaches...... contribute to the development of insulin resistance induced by bed rest. Lack of complete normalization of changes after 4 wk of retraining underscores the importance of maintaining a minimum of daily physical activity....

  3. Trinucleotide Repeat Expansion in the Transcription Factor 4 (TCF4) Gene Leads to Widespread mRNA Splicing Changes in Fuchs' Endothelial Corneal Dystrophy

    Science.gov (United States)

    Wieben, Eric D.; Aleff, Ross A.; Tang, Xiaojia; Butz, Malinda L.; Kalari, Krishna R.; Highsmith, Edward W.; Jen, Jin; Vasmatzis, George; Patel, Sanjay V.; Maguire, Leo J.; Baratz, Keith H.; Fautsch, Michael P.

    2017-01-01

    Purpose To identify RNA missplicing events in human corneal endothelial tissue isolated from Fuchs' endothelial corneal dystrophy (FECD). Methods Total RNA was isolated and sequenced from corneal endothelial tissue obtained during keratoplasty from 12 patients with FECD and 4 patients undergoing keratoplasty or enucleation for other indications. The length of the trinucleotide repeat (TNR) CTG in the transcription factor 4 (TCF4) gene was determined using leukocyte-derived DNA analyzed by a combination of Southern blotting and Genescan analysis. Commercial statistical software was used to quantify expression of alternatively spliced genes. Validation of selected alternative splicing events was performed by using RT-PCR. Gene sets identified were analyzed for overrepresentation using Web-based analysis system. Results Corneal endothelial tissue from FECD patients containing a CTG TNR expansion sequence in the TCF4 gene revealed widespread changes in mRNA splicing, including a novel splicing event involving FGFR2. Differential splicing of NUMA1, PPFIBP1, MBNL1, and MBNL2 transcripts were identified in all FECD samples containing a TNR expansion. The differentially spliced genes were enriched for products that localize to the cell cortex and bind cytoskeletal and cell adhesion proteins. Conclusions Corneal endothelium from FECD patients harbors a unique signature of mis-splicing events due to CTG TNR expansion in the TCF4 gene, consistent with the hypothesis that RNA toxicity contributes to the pathogenesis of FECD. Changes to the endothelial barrier function, a known event in the development of FECD, was identified as a key biological process influenced by the missplicing events. PMID:28118661

  4. Prenatal exposure to dietary fat induces changes in the transcriptional factors, TEF and YAP, which may stimulate differentiation of peptide neurons in rat hypothalamus.

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    Kinning Poon

    Full Text Available Gestational exposure to a high-fat diet (HFD stimulates the differentiation of orexigenic peptide-expressing neurons in the hypothalamus of offspring. To examine possible mechanisms that mediate this phenomenon, this study investigated the transcriptional factor, transcription enhancer factor-1 (TEF, and co-activator, Yes-associated protein (YAP, which when inactivated stimulate neuronal differentiation. In rat embryos and postnatal offspring prenatally exposed to a HFD compared to chow, changes in hypothalamic TEF and YAP and their relationship to the orexigenic peptide, enkephalin (ENK, were measured. The HFD offspring at postnatal day 15 (P15 exhibited in the hypothalamic paraventricular nucleus a significant reduction in YAP mRNA and protein, and increased levels of inactive and total TEF protein, with no change in mRNA. Similarly, HFD-exposed embryos at embryonic day 19 (E19 showed in whole hypothalamus significantly decreased levels of YAP mRNA and protein and TEF mRNA, and increased levels of inactive TEF protein, suggesting that HFD inactivates TEF and YAP. This was accompanied by increased density and fluorescence intensity of ENK neurons. A close relationship between TEF and ENK was suggested by the finding that TEF co-localizes with this peptide in hypothalamic neurons and HFD reduced the density of TEF/ENK co-labeled neurons, even while the number and fluorescence intensity of single-labeled TEF neurons were increased. Increased YAP inactivity by HFD was further evidenced by a decrease in number and fluorescence intensity of YAP-containing neurons, although the density of YAP/ENK co-labeled neurons was unaltered. Genetic knockdown of TEF or YAP stimulated ENK expression in hypothalamic neurons, supporting a close relationship between these transcription factors and neuropeptide. These findings suggest that prenatal HFD exposure inactivates both hypothalamic TEF and YAP, by either decreasing their levels or increasing their inactive

  5. Variation in branchial expression among insulin-like growth-factor binding proteins (igfbps) during Atlantic salmon smoltification and seawater exposure

    Science.gov (United States)

    Breves, Jason P.; Fujimoto, Chelsea K.; Phipps-Costin, Silas K.; Einarsdottir, Ingibjörg E.; Björnsson, Björn Thrandur; McCormick, Stephen

    2017-01-01

    BackgroundIn preparation for migration from freshwater to marine habitats, Atlantic salmon (Salmo salar L.) undergo smoltification, a transformation that includes the acquisition of hyposmoregulatory capacity. The growth hormone (Gh)/insulin-like growth-factor (Igf) axis promotes the development of branchial ionoregulatory functions that underlie ion secretion. Igfs interact with a suite of Igf binding proteins (Igfbps) that modulate hormone activity. In Atlantic salmon smolts, igfbp4,−5a,−5b1,−5b2,−6b1 and−6b2 transcripts are highly expressed in gill. We measured mRNA levels of branchial and hepatic igfbps during smoltification (March, April, and May), desmoltification (July) and following seawater (SW) exposure in March and May. We also characterized parallel changes in a broad suite of osmoregulatory (branchial Na+/K+-ATPase (Nka) activity, Na+ /K + /2Cl − cotransporter 1 (nkcc1) and cystic fibrosis transmembrane regulator 1 (cftr1) transcription) and endocrine (plasma Gh and Igf1) parameters.ResultsIndicative of smoltification, we observed increased branchial Nka activity, nkcc1 and cftr1 transcription in May. Branchial igfbp6b1 and -6b2 expression increased coincidentally with smoltification. Following a SW challenge in March, igfbp6b1 showed increased expression while igfbp6b2 exhibited diminished expression. igfbp5a,−5b1 and−5b2 mRNA levels did not change during smolting, but each had lower levels following a SW exposure in March.ConclusionsSalmonids express an especially large suite of igfbps. Our data suggest that dynamic expression of particular igfbps accompanies smoltification and SW challenges; thus, transcriptional control of igfbps may provide a mechanism for the local modulation of Igf activity in salmon gill.

  6. Transcriptional rewiring of the sex determining dmrt1 gene duplicate by transposable elements.

    Directory of Open Access Journals (Sweden)

    Amaury Herpin

    2010-02-01

    Full Text Available Control and coordination of eukaryotic gene expression rely on transcriptional and posttranscriptional regulatory networks. Evolutionary innovations and adaptations often require rapid changes of such networks. It has long been hypothesized that transposable elements (TE might contribute to the rewiring of regulatory interactions. More recently it emerged that TEs might bring in ready-to-use transcription factor binding sites to create alterations to the promoters by which they were captured. A process where the gene regulatory architecture is of remarkable plasticity is sex determination. While the more downstream components of the sex determination cascades are evolutionary conserved, the master regulators can switch between groups of organisms even on the interspecies level or between populations. In the medaka fish (Oryzias latipes a duplicated copy of dmrt1, designated dmrt1bY or DMY, on the Y chromosome was shown to be the master regulator of male development, similar to Sry in mammals. We found that the dmrt1bY gene has acquired a new feedback downregulation of its expression. Additionally, the autosomal dmrt1a gene is also able to regulate transcription of its duplicated paralog by binding to a unique target Dmrt1 site nested within the dmrt1bY proximal promoter region. We could trace back this novel regulatory element to a highly conserved sequence within a new type of TE that inserted into the upstream region of dmrt1bY shortly after the duplication event. Our data provide functional evidence for a role of TEs in transcriptional network rewiring for sub- and/or neo-functionalization of duplicated genes. In the particular case of dmrt1bY, this contributed to create new hierarchies of sex-determining genes.

  7. Insulin-like growth factor-binding protein 2-driven glioma progression is prevented by blocking a clinically significant integrin, integrin-linked kinase, and NF-κB network

    Science.gov (United States)

    Holmes, Kristen M.; Annala, Matti; Chua, Corrine Y. X.; Dunlap, Sarah M.; Liu, Yuexin; Hugen, Niek; Moore, Lynette M.; Cogdell, David; Hu, Limei; Nykter, Matti; Hess, Kenneth; Fuller, Gregory N.; Zhang, Wei

    2012-01-01

    Insulin-like growth factor-binding protein 2 (IGFBP2) is increasingly recognized as a glioma oncogene, emerging as a target for therapeutic intervention. In this study, we used an integrative approach to characterizing the IGFBP2 network, combining transcriptional profiling of human glioma with validation in glial cells and the replication-competent ASLV long terminal repeat with a splice acceptor/tv-a glioma mouse system. We demonstrated that IGFBP2 expression is closely linked to genes in the integrin and integrin-linked kinase (ILK) pathways and that these genes are associated with prognosis. We further showed that IGFBP2 activates integrin β1 and downstream invasion pathways, requires ILK to induce cell motility, and activates NF-κB. Most significantly, the IGFBP2/integrin/ILK/NF-κB network functions as a physiologically active signaling pathway in vivo by driving glioma progression; interfering with any point in the pathway markedly inhibits progression. The results of this study reveal a signaling pathway that is both targetable and highly relevant to improving the survival of glioma patients. PMID:22345562

  8. Genomic organization of the Neurospora crassa gsn gene: possible involvement of the STRE and HSE elements in the modulation of transcription during heat shock.

    Science.gov (United States)

    Freitas, F Zanolli; Bertolini, M C

    2004-12-01

    Glycogen synthase, an enzyme involved in glycogen biosynthesis, is regulated by phosphorylation and by the allosteric ligand glucose-6-phosphate (G6P). In addition, enzyme levels can be regulated by changes in gene expression. We recently cloned a cDNA for glycogen synthase ( gsn) from Neurospora crassa, and showed that gsn transcription decreased when cells were exposed to heat shock (shifted from 30 degrees C to 45 degrees C). In order to understand the mechanisms that control gsn expression, we isolated the gene, including its 5' and 3' flanking regions, from the genome of N. crassa. An ORF of approximately 2.4 kb was identified, which is interrupted by four small introns (II-V). Intron I (482 bp) is located in the 5'UTR region. Three putative Transcription Initiation Sites (TISs) were mapped, one of which lies downstream of a canonical TATA-box sequence (5'-TGTATAAA-3'). Analysis of the 5'-flanking region revealed the presence of putative transcription factor-binding sites, including Heat Shock Elements (HSEs) and STress Responsive Elements (STREs). The possible involvement of these motifs in the negative regulation of gsn transcription was investigated using Electrophoretic Mobility Shift Assays (EMSA) with nuclear extracts of N. crassa mycelium obtained before and after heat shock, and DNA fragments encompassing HSE and STRE elements from the 5'-flanking region. While elements within the promoter region are involved in transcription under heat shock, elements in the 5'UTR intron may participate in transcription during vegetative growth. The results thus suggest that N. crassa possesses trans -acting elements that interact with the 5'-flanking region to regulate gsn transcription during heat shock and vegetative growth.

  9. Simulated herbivory in chickpea causes rapid changes in defense pathways and hormonal transcription networks of JA/ethylene/GA/auxin within minutes of wounding.

    Science.gov (United States)

    Pandey, Saurabh Prakash; Srivastava, Shruti; Goel, Ridhi; Lakhwani, Deepika; Singh, Priya; Asif, Mehar Hasan; Sane, Aniruddha P

    2017-03-16

    Chickpea (C. arietinum L.) is an important pulse crop in Asian and African countries that suffers significant yield losses due to attacks by insects like H. armigera. To obtain insights into early responses of chickpea to insect attack, a transcriptomic analysis of chickpea leaves just 20 minutes after simulated herbivory was performed, using oral secretions of H. armigera coupled with mechanical wounding. Expression profiles revealed differential regulation of 8.4% of the total leaf transcriptome with 1334 genes up-regulated and 501 down-regulated upon wounding at log2-fold change (|FC| ≤ -1 and ≥1) and FDR value ≤ 0.05. In silico analysis showed the activation of defenses through up-regulation of genes of the phenylpropanoid pathway, pathogenesis, oxidases and CYTP450 besides differential regulation of kinases, phosphatases and transcription factors of the WRKY, MYB, ERFs, bZIP families. A substantial change in the regulation of hormonal networks was observed with up-regulation of JA and ethylene pathways and suppression of growth associated hormone pathways like GA and auxin within 20 minutes of wounding. Secondary qPCR comparison of selected genes showed that oral secretions often increased differential expression relative to mechanical damage alone. The studies provide new insights into early wound responses in chickpea.

  10. Isl1 is a direct transcriptional target of Forkhead transcription factors in second heart field-derived mesoderm

    Science.gov (United States)

    Kang, Jione; Nathan, Elisha; Xu, Shan-Mei; Tzahor, Eldad; Black, Brian L.

    2009-01-01

    The cells of the second heart field (SHF) contribute to the outflow tract and right ventricle, as well as to parts of the left ventricle and atria. Isl1, a member of the LIM-homeodomain transcription factor family, is expressed early in this cardiac progenitor population and functions near the top of a transcriptional pathway essential for heart development. Isl1 is required for the survival and migration of SHF-derived cells into the early developing heart at the inflow and outflow poles. Despite this important role for Isl1 in early heart formation, the transcriptional regulation of Isl1 has remained largely undefined. Therefore, to identify transcription factors that regulate Isl1 expression in vivo, we screened the conserved noncoding sequences from the mouse Isl1 locus for enhancer activity in transgenic mouse embryos. Here, we report the identification of an enhancer from the mouse Isl1 gene that is sufficient to direct expression to the SHF and its derivatives. The Isl1 SHF enhancer contains three consensus Forkhead transcription factor binding sites that are efficiently and specifically bound by Forkhead transcription factors. Importantly, the activity of the enhancer is dependent on these three Forkhead binding sites in transgenic mouse embryos. Thus, these studies demonstrate that Isl1 is a direct transcriptional target of Forkhead transcription factors in the SHF and establish a transcriptional pathway upstream of Isl1 in the SHF. PMID:19580802

  11. Changes in the transcriptional profile of transporters in the intestine along the anterior-posterior and crypt-villus axes

    Directory of Open Access Journals (Sweden)

    Delorenzi Mauro

    2005-05-01

    Full Text Available Abstract Background The purpose of this work was to characterize the expression of drug and nutrient carriers along the anterior-posterior and crypt-villus axes of the intestinal epithelium and to study the validity of utilizing whole gut tissue rather than purified epithelial cells to examine regional variations in gene expression. Results We have characterized the mRNA expression profiles of 76 % of all currently known transporters along the anterior-posterior axis of the gut. This is the first study to describe the expression profiles of the majority of all known transporters in the intestine. The expression profiles of transporters, as defined according to the Gene Ontology consortium, were measured in whole tissue of the murine duodenum, jejunum, ileum and colon using high-density microarrays. For nine transporters (Abca1, Abcc1, Abcc3, Abcg8, Slc10a2, Slc28a2, Slc2a1, Slc34a2 and Slc5a8, the mRNA profiles were further measured by RT-PCR in laser micro-dissected crypt and villus epithelial cells corresponding to the aforementioned intestinal regions. With respect to differentially regulated transporters, the colon had a distinct expression profile from small intestinal segments. The majority (59 % for p cutoff ≤ 0.05 of transporter mRNA levels were constant across the intestinal sections studied. For the transporter subclass "carrier activity", which contains the majority of known carriers for biologically active compounds, a significant change (p ≤ 0.05 along the anterior-posterior axis was observed. Conclusion All nine transporters examined in laser-dissected material demonstrated good replication of the region-specific profiles revealed by microarray. Furthermore, we suggest that the distribution characteristics of Slc5a8 along the intestinal tract render it a suitable candidate carrier for monocarboxylate drugs in the posterior portion of the intestine. Our findings also predict that there is a significant difference in the

  12. TRANSFAC: transcriptional regulation, from patterns to profiles.

    Science.gov (United States)

    Matys, V; Fricke, E; Geffers, R; Gössling, E; Haubrock, M; Hehl, R; Hornischer, K; Karas, D; Kel, A E; Kel-Margoulis, O V; Kloos, D-U; Land, S; Lewicki-Potapov, B; Michael, H; Münch, R; Reuter, I; Rotert, S; Saxel, H; Scheer, M; Thiele, S; Wingender, E

    2003-01-01

    The TRANSFAC database on eukaryotic transcriptional regulation, comprising data on transcription factors, their target genes and regulatory binding sites, has been extended and further developed, both in number of entries and in the scope and structure of the collected data. Structured fields for expression patterns have been introduced for transcription factors from human and mouse, using the CYTOMER database on anatomical structures and developmental stages. The functionality of Match, a tool for matrix-based search of transcription factor binding sites, has been enhanced. For instance, the program now comes along with a number of tissue-(or state-)specific profiles and new profiles can be created and modified with Match Profiler. The GENE table was extended and gained in importance, containing amongst others links to LocusLink, RefSeq and OMIM now. Further, (direct) links between factor and target gene on one hand and between gene and encoded factor on the other hand were introduced. The TRANSFAC public release is available at http://www.gene-regulation.com. For yeast an additional release including the latest data was made available separately as TRANSFAC Saccharomyces Module (TSM) at http://transfac.gbf.de. For CYTOMER free download versions are available at http://www.biobase.de:8080/index.html.

  13. ATF3, an HTLV-1 bZip factor binding protein, promotes proliferation of adult T-cell leukemia cells

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    Ohshima Koichi

    2011-03-01

    Full Text Available Abstract Background Adult T-cell leukemia (ATL is an aggressive malignancy of CD4+ T-cells caused by human T-cell leukemia virus type 1 (HTLV-1. The HTLV-1 bZIP factor (HBZ gene, which is encoded by the minus strand of the viral genome, is expressed as an antisense transcript in all ATL cases. By using yeast two-hybrid screening, we identified activating transcription factor 3 (ATF3 as an HBZ-interacting protein. ATF3 has been reported to be expressed in ATL cells, but its biological significance is not known. Results Immunoprecipitation analysis confirmed that ATF3 interacts with HBZ. Expression of ATF3 was upregulated in ATL cell lines and fresh ATL cases. Reporter assay revealed that ATF3 could interfere with the HTLV-1 Tax's transactivation of the 5' proviral long terminal repeat (LTR, doing so by affecting the ATF/CRE site, as well as HBZ. Suppressing ATF3 expression inhibited proliferation and strongly reduced the viability of ATL cells. As mechanisms of growth-promoting activity of ATF3, comparative expression profiling of ATF3 knockdown cells identified candidate genes that are critical for the cell cycle and cell death, including cell division cycle 2 (CDC2 and cyclin E2. ATF3 also enhanced p53 transcriptional activity, but this activity was suppressed by HBZ. Conclusions Thus, ATF3 expression has positive and negative effects on the proliferation and survival of ATL cells. HBZ impedes its negative effects, leaving ATF3 to promote proliferation of ATL cells via mechanisms including upregulation of CDC2 and cyclin E2. Both HBZ and ATF3 suppress Tax expression, which enables infected cells to escape the host immune system.

  14. Time series analysis of benzo[a]pyrene-induced transcriptome changes suggests that a network of transcription factors regulates the effects on functional gene sets

    NARCIS (Netherlands)

    Delft, J.H.M. van; Mathijs, K.; Staal, Y.C.M.; Herwijnen, M.H.M. van; Brauers, K.J.J.; Boorsma, A.; Kleinjans, J.C.S.

    2010-01-01

    Chemical carcinogens may cause a multitude of effects inside cells, thereby affecting transcript levels of genes by direct activation of transcription factors (TF) or indirectly through the formation of DNA damage. As the temporal profiles of these responses may be profoundly different, examining ti

  15. Transcriptional profiling of intrinsic PNS factors in the postnatal mouse.

    Science.gov (United States)

    Smith, Robin P; Lerch-Haner, Jessica K; Pardinas, Jose R; Buchser, William J; Bixby, John L; Lemmon, Vance P

    2011-01-01

    Neurons in the peripheral nervous system (PNS) display a higher capacity to regenerate after injury than those in the central nervous system, suggesting cell specific transcriptional modules underlying axon growth and inhibition. We report a systems biology based search for PNS specific transcription factors (TFs). Messenger RNAs enriched in dorsal root ganglion (DRG) neurons compared to cerebellar granule neurons (CGNs) were identified using subtractive hybridization and DNA microarray approaches. Network and transcription factor binding site enrichment analyses were used to further identify TFs that may be differentially active. Combining these techniques, we identified 32 TFs likely to be enriched and/or active in the PNS. Twenty-five of these TFs were then tested for an ability to promote CNS neurite outgrowth in an overexpression screen. Real-time PCR and immunohistochemical studies confirmed that one representative TF, STAT3, is intrinsic to PNS neurons, and that constitutively active STAT3 is sufficient to promote CGN neurite outgrowth.

  16. Physical training and weight loss in dogs lead to transcriptional changes in genes involved in the glucose-transport pathway in muscle and adipose tissues.

    Science.gov (United States)

    Herrera Uribe, Juber; Vitger, Anne D; Ritz, Christian; Fredholm, Merete; Bjørnvad, Charlotte R; Cirera, Susanna

    2016-02-01

    Obesity is a worldwide problem in humans and domestic animals. Interventions, including a combination of dietary management and exercise, have proven to be effective for inducing weight loss in humans. In companion animals, the role of exercise in the management of obesity has received relatively little attention. The aim of the present study was to investigate changes in the transcriptome of key energy metabolism genes in muscle and adipose tissues in response to diet-induced weight loss alone, or combined with exercise in dogs. Overweight pet dogs were enrolled on a weight loss programme, based on calorie restriction and physical training (FD group, n = 5) or calorie restriction alone (DO group, n = 7). mRNA expression of 12 genes and six microRNAs were investigated using quantitative real-time PCR (qPCR). In the FD group, FOXO1 and RAC1 were expressed at lower levels in adipose tissue, whereas ESRRA and AKT2 were more highly expressed in muscle, when compared with the DO group. Comparing expression before and after the intervention, in the DO group, nine genes and three microRNAs showed significant altered expression in adipose tissue (PPARG, ADIPOQ and FOXO1; P ESRRA, AKT2, PGC1a and mir-23; P < 0.001) in muscle. Thus, calorie restriction causes regulation of several metabolic genes in both tissues. The mild exercise, incorporated into this study design, was sufficient to elicit transcriptional changes in adipose and muscle tissues, suggesting a positive effect on glucose metabolism. The study findings support inclusion of exercise in management of canine obesity.

  17. Genome-wide analysis of brain and gonad transcripts reveals changes of key sex reversal-related genes expression and signaling pathways in three stages of Monopterus albus

    Science.gov (United States)

    Hu, Qing; Guo, Wei; Li, Dapeng

    2017-01-01

    Background The natural sex reversal severely affects the sex ratio and thus decreases the productivity of the rice field eel (Monopterus albus). How to understand and manipulate this process is one of the major issues for the rice field eel stocking. So far the genomics and transcriptomics data available for this species are still scarce. Here we provide a comprehensive study of transcriptomes of brain and gonad tissue in three sex stages (female, intersex and male) from the rice field eel to investigate changes in transcriptional level during the sex reversal process. Results Approximately 195 thousand unigenes were generated and over 44.4 thousand were functionally annotated. Comparative study between stages provided multiple differentially expressed genes in brain and gonad tissue. Overall 4668 genes were found to be of unequal abundance between gonad tissues, far more than that of the brain tissues (59 genes). These genes were enriched in several different signaling pathways. A number of 231 genes were found with different levels in gonad in each stage, with several reproduction-related genes included. A total of 19 candidate genes that could be most related to sex reversal were screened out, part of these genes’ expression patterns were validated by RT-qPCR. The expression of spef2, maats1, spag6 and dmc1 were abundant in testis, but was barely detected in females, while the 17β-hsd12, zpsbp3, gal3 and foxn5 were only expressed in ovary. Conclusion This study investigated the complexity of brain and gonad transcriptomes in three sex stages of the rice field eel. Integrated analysis of different gene expression and changes in signaling pathways, such as PI3K-Akt pathway, provided crucial data for further study of sex transformation mechanisms. PMID:28319194

  18. Transcriptional changes in response to X chromosome dosage in the mouse: implications for X inactivation and the molecular basis of Turner Syndrome

    Directory of Open Access Journals (Sweden)

    Sargent Carole A

    2010-02-01

    Full Text Available Abstract Background X monosomic mice (39,XO have a remarkably mild phenotype when compared to women with Turner syndrome (45,XO. The generally accepted hypothesis to explain this discrepancy is that the number of genes on the mouse X chromosome which escape X inactivation, and thus are expressed at higher levels in females, is very small. However this hypothesis has never been tested and only a small number of genes have been assayed for their X-inactivation status in the mouse. We performed a global expression analysis in four somatic tissues (brain, liver, kidney and muscle of adult 40,XX and 39,XO mice using the Illumina Mouse WG-6 v1_1 Expression BeadChip and an extensive validation by quantitative real time PCR, in order to identify which genes are expressed from both X chromosomes. Results We identified several genes on the X chromosome which are overexpressed in XX females, including those previously reported as escaping X inactivation, as well as new candidates. However, the results obtained by microarray and qPCR were not fully concordant, illustrating the difficulty in ascertaining modest fold changes, such as those expected for genes escaping X inactivation. Remarkably, considerable variation was observed between tissues, suggesting that inactivation patterns may be tissue-dependent. Our analysis also exposed several autosomal genes involved in mitochondrial metabolism and in protein translation which are differentially expressed between XX and XO mice, revealing secondary transcriptional changes to the alteration in X chromosome dosage. Conclusions Our results support the prediction that the mouse inactive X chromosome is largely silent, while providing a list of the genes potentially escaping X inactivation in rodents. Although the lower expression of X-linked genes in XO mice may not be relevant in the particular tissues/systems which are affected in human X chromosome monosomy, genes deregulated in XO mice are good candidates for

  19. Identification of a GCC transcription factor responding to fruit colour change events in citrus through the transcriptomic analyses of two mutants

    Directory of Open Access Journals (Sweden)

    Cercós Manuel

    2010-12-01

    Full Text Available Abstract Background External ripening in Citrus fruits is morphologically characterized by a colour shift from green to orange due to the degradation of chlorophylls and the accumulation of carotenoid pigments. Although numerous genes coding for enzymes involved in such biochemical pathways have been identified, the molecular control of this process has been scarcely studied. In this work we used the Citrus clementina mutants 39B3 and 39E7, showing delayed colour break, to isolate genes potentially related to the regulation of peel ripening and its physiological or biochemical effects. Results Pigment analyses revealed different profiles of carotenoid and chlorophyll modification in 39B3 and 39E7 mutants. Flavedo from 39B3 fruits showed an overall delay in carotenoid accumulation and chlorophyll degradation, while the flavedo of 39E7 was devoid of the apocarotenoid β-citraurin among other carotenoid alterations. A Citrus microarray containing about 20,000 cDNA fragments was used to identify genes that were differentially expressed during colour change in the flavedo of 39B3 and 39E7 mutants respect to the parental variety. The results highlighted 73 and 90 genes that were respectively up- and down-regulated in both mutants. CcGCC1 gene, coding for a GCC type transcriptional factor, was found to be down-regulated. CcGCC1 expression was strongly induced at the onset of colour change in the flavedo of parental clementine fruit. Moreover, treatment of fruits with gibberellins, a retardant of external ripening, delayed both colour break and CcGCC1 overexpression. Conclusions In this work, the citrus fruit ripening mutants 39B3 and 39E7 have been characterized at the phenotypic, biochemical and transcriptomic level. A defective synthesis of the apocarotenoid β-citraurin has been proposed to cause the yellowish colour of fully ripe 39E7 flavedo. The analyses of the mutant transcriptomes revealed that colour change during peel ripening was strongly

  20. Bioinformatic detection of E47, E2F1 and SREBP1 transcription factors as potential regulators of genes associated to acquisition of endometrial receptivity

    Science.gov (United States)

    2011-01-01

    Background The endometrium is a dynamic tissue whose changes are driven by the ovarian steroidal hormones. Its main function is to provide an adequate substrate for embryo implantation. Using microarray technology, several reports have provided the gene expression patterns of human endometrial tissue during the window of implantation. However it is required that biological connections be made across these genomic datasets to take full advantage of them. The objective of this work was to perform a research synthesis of available gene expression profiles related to acquisition of endometrial receptivity for embryo implantation, in order to gain insights into its molecular basis and regulation. Methods Gene expression datasets were intersected to determine a consensus endometrial receptivity transcript list (CERTL). For this cluster of genes we determined their functional annotations using available web-based databases. In addition, promoter sequences were analyzed to identify putative transcription factor binding sites using bioinformatics tools and determined over-represented features. Results We found 40 up- and 21 down-regulated transcripts in the CERTL. Those more consistently increased were C4BPA, SPP1, APOD, CD55, CFD, CLDN4, DKK1, ID4, IL15 and MAP3K5 whereas the more consistently decreased were OLFM1, CCNB1, CRABP2, EDN3, FGFR1, MSX1 and MSX2. Functional annotation of CERTL showed it was enriched with transcripts related to the immune response, complement activation and cell cycle regulation. Promoter sequence analysis of genes revealed that DNA binding sites for E47, E2F1 and SREBP1 transcription factors were the most consistently over-represented and in both up- and down-regulated genes during the window of implantation. Conclusions Our research synthesis allowed organizing and mining high throughput data to explore endometrial receptivity and focus future research efforts on specific genes and pathways. The discovery of possible new transcription factors

  1. Total and free insulin-like growth factor I, insulin-like growth factor binding protein 3 and acid-labile subunit reflect clinical activity in acromegaly

    DEFF Research Database (Denmark)

    Sneppen, S B; Lange, Merete Wolder; Pedersen, L M;

    2001-01-01

    The aim was to evaluate, markers of disease activity in acromegaly in relation to perceived disease activity. Thirty-seven consecutively treated, acromegalic patients, classified by clinical symptoms as inactive (n=16), slightly active (n=10) and active (n=11), entered the study. When evaluating......-like growth factor binding protein-3 (IGFBP-3) with PV(pos) of 0.69 and 0.71 and PV(neg) of 0.91 and 0.92 respectively. We conclude that free IGF-I is more closely related than total IGF-I to perceived disease activity and is as such useful when evaluating previously treated acromegaly for disease activity...

  2. Human transcriptional coactivator with PDZ-binding motif (TAZ) is downregulated during decidualization.

    Science.gov (United States)

    Strakova, Zuzana; Reed, Jennifer; Ihnatovych, Ivanna

    2010-06-01

    Transcriptional coactivator with PDZ-binding motif (TAZ) is known to bind to a variety of transcription factors to control cell differentiation and organ development. However, its role in uterine physiology has not yet been described. To study its regulation during the unique process of differentiation of fibroblasts into decidual cells (decidualization), we utilized the human uterine fibroblast (HuF) in vitro cell model. Immunocytochemistry data demonstrated that the majority of the TAZ protein is localized in the nucleus. Treatment of HuF cells with the embryonic stimulus cytokine interleukin 1 beta in the presence of steroid hormones (estradiol-17 beta and medroxyprogesterone acetate) for 13 days did not cause any apparent TAZ mRNA changes but resulted in a significant TAZ protein decline (approximately 62%) in total cell lysates. Analysis of cytosolic and nuclear extracts revealed that the decline of total TAZ was caused primarily by a drop of TAZ protein levels in the nucleus. TAZ was localized on the peroxisome proliferator-activated receptor response element site (located at position -1200 bp relative to the transcription start site) of the genomic region of decidualization marker insulin-like growth factor-binding protein 1 (IGFBP1) in HuF cells as detected by chromatin immunoprecipitation. TAZ is also present in human endometrium tissue as confirmed by immunohistochemistry. During the secretory phase of the menstrual cycle, specific TAZ staining particularly diminishes in the stroma, suggesting its participation during the decidualization process, as well as implantation. During early baboon pregnancy, TAZ protein expression remains minimal in the endometrium close to the implantation site. In summary, the presented evidence shows for the first time to date TAZ protein in the human uterine tract, its downregulation during in vitro decidualization, and its localization on the IGFBP1 promoter region, all of which indicate its presence in the uterine

  3. Sequence changes in predicted promoter elements of STK11/LKB1 are unlikely to contribute to Peutz-Jeghers syndrome

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    Morrison Patrick J

    2005-03-01

    Full Text Available Abstract Background Germline mutations or large-scale deletions in the coding region and splice sites of STK11/LKB1 do not account for all cases of Peutz-Jeghers syndrome (PJS. It is conceivable that, on the basis of data from other diseases, inherited variation in promoter elements of STK11/LKB1 may cause PJS. Results Phylogenetic foot printing and transcription factor binding site prediction of sequence 5' to the coding sequence of STK11/LKB1 was performed to identify non-coding sequences of DNA indicative of regulatory elements. A series of 33 PJS cases in whom no mutation in STK11/LKB1 could be identified were screened for sequence changes in the putative promoter defined by nucleotides -1090 to -1472. Two novel sequence changes were identified, but were found to be present in healthy individuals. Conclusion These findings indicate that promoter sequence changes are unlikely to contribute to PJS.

  4. Effect of desipramine and citalopram treatment on forced swimming test-induced changes in cocaine- and amphetamine-regulated transcript (CART) immunoreactivity in mice.

    Science.gov (United States)

    Chung, Sung; Kim, Hee Jeong; Kim, Hyun Ju; Choi, Sun Hye; Kim, Jin Wook; Kim, Jeong Min; Shin, Kyung Ho

    2014-05-01

    Recent study demonstrates antidepressant-like effect of cocaine- and amphetamine-regulated transcript (CART) in the forced swimming test (FST), but less is known about whether antidepressant treatments alter levels of CART immunoreactivity (CART-IR) in the FST. To explore this possibility, we assessed the treatment effects of desipramine and citalopram, which inhibit the reuptake of norepinephrine and serotonin into the presynaptic terminals, respectively, on changes in levels of CART-IR before and after the test swim in mouse brain. Levels of CART-IR in the nucleus accumbens shell (AcbSh), dorsal bed nucleus of the stria terminalis (dBNST), and hypothalamic paraventricular nucleus (PVN) were significantly increased before the test swim by desipramine and citalopram treatments. This increase in CART-IR in the AcbSh, dBNST, and PVN before the test swim remained elevated by desipramine treatment after the test swim, but this increase in these brain areas returned to near control levels after test swim by citalopram treatment. Citalopram, but not desipramine, treatment increased levels of CART-IR in the central nucleus of the amygdala (CeA) and the locus ceruleus (LC) before the test swim, and this increase was returned to control levels after the test swim in the CeA, but not in the LC. These results suggest common and distinct regulation of CART by desipramine and citalopram treatments in the FST and raise the possibility that CART in the AcbSh, dBNST, and CeA may be involved in antidepressant-like effect in the FST.

  5. Supra-optimal expression of the cold-regulated OsMyb4 transcription factor in transgenic rice changes the complexity of transcriptional network with major effects on stress tolerance and panicle development.

    Science.gov (United States)

    Park, Myoung-Ryoul; Yun, Kil-Young; Mohanty, Bijayalaxmi; Herath, Venura; Xu, Fuyu; Wijaya, Edward; Bajic, Vladimir B; Yun, Song-Joong; De Los Reyes, Benildo G

    2010-12-01

    The R2R3-type OsMyb4 transcription factor of rice has been shown to play a role in the regulation of osmotic adjustment in heterologous overexpression studies. However, the exact composition and organization of its underlying transcriptional network has not been established to be a robust tool for stress tolerance enhancement by regulon engineering. OsMyb4 network was dissected based on commonalities between the global chilling stress transcriptome and the transcriptome configured by OsMyb4 overexpression. OsMyb4 controls a hierarchical network comprised of several regulatory sub-clusters associated with cellular defense and rescue, metabolism and development. It regulates target genes either directly or indirectly through intermediary MYB, ERF, bZIP, NAC, ARF and CCAAT-HAP transcription factors. Regulatory sub-clusters have different combinations of MYB-like, GCC-box-like, ERD1-box-like, ABRE-like, G-box-like, as1/ocs/TGA-like, AuxRE-like, gibberellic acid response element (GARE)-like and JAre-like cis-elements. Cold-dependent network activity enhanced cellular antioxidant capacity through radical scavenging mechanisms and increased activities of phenylpropanoid and isoprenoid metabolic processes involving various abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), ethylene and reactive oxygen species (ROS) responsive genes. OsMyb4 network is independent of drought response element binding protein/C-repeat binding factor (DREB/CBF) and its sub-regulons operate with possible co-regulators including nuclear factor-Y. Because of its upstream position in the network hierarchy, OsMyb4 functions quantitatively and pleiotrophically. Supra-optimal expression causes misexpression of alternative targets with costly trade-offs to panicle development.

  6. Supra-optimal expression of the cold-regulated OsMyb4 transcription factor in transgenic rice changes the complexity of transcriptional network with major effects on stress tolerance and panicle development

    KAUST Repository

    Park, Myoungryoul

    2010-09-28

    The R2R3-type OsMyb4 transcription factor of rice has been shown to play a role in the regulation of osmotic adjustment in heterologous overexpression studies. However, the exact composition and organization of its underlying transcriptional network has not been established to be a robust tool for stress tolerance enhancement by regulon engineering. OsMyb4 network was dissected based on commonalities between the global chilling stress transcriptome and the transcriptome configured by OsMyb4 overexpression. OsMyb4 controls a hierarchical network comprised of several regulatory sub-clusters associated with cellular defense and rescue, metabolism and development. It regulates target genes either directly or indirectly through intermediary MYB, ERF, bZIP, NAC, ARF and CCAAT-HAP transcription factors. Regulatory sub-clusters have different combinations of MYB-like, GCC-box-like, ERD1-box-like, ABRE-like, G-box-like, as1/ocs/TGA-like, AuxRE-like, gibberellic acid response element (GARE)-like and JAre-like cis-elements. Cold-dependent network activity enhanced cellular antioxidant capacity through radical scavenging mechanisms and increased activities of phenylpropanoid and isoprenoid metabolic processes involving various abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), ethylene and reactive oxygen species (ROS) responsive genes. OsMyb4 network is independent of drought response element binding protein/C-repeat binding factor (DREB/CBF) and its sub-regulons operate with possible co-regulators including nuclear factor-Y. Because of its upstream position in the network hierarchy, OsMyb4 functions quantitatively and pleiotrophically. Supra-optimal expression causes misexpression of alternative targets with costly trade-offs to panicle development. © 2010 Blackwell Publishing Ltd.

  7. The transcriptional response in human umbilical vein endothelial cells exposed to insulin: a dynamic gene expression approach.

    Directory of Open Access Journals (Sweden)

    Barbara Di Camillo

    Full Text Available BACKGROUND: In diabetes chronic hyperinsulinemia contributes to the instability of the atherosclerotic plaque and stimulates cellular proliferation through the activation of the MAP kinases, which in turn regulate cellular proliferation. However, it is not known whether insulin itself could increase the transcription of specific genes for cellular proliferation in the endothelium. Hence, the characterization of transcriptional modifications in endothelium is an important step for a better understanding of the mechanism of insulin action and the relationship between endothelial cell dysfunction and insulin resistance. METHODOLOGY AND PRINCIPAL FINDINGS: The transcriptional response of endothelial cells in the 440 minutes following insulin stimulation was monitored using microarrays and compared to a control condition. About 1700 genes were selected as differentially expressed based on their treated minus control profile, thus allowing the detection of even small but systematic changes in gene expression. Genes were clustered in 7 groups according to their time expression profile and classified into 15 functional categories that can support the biological effects of insulin, based on Gene Ontology enrichment analysis. In terms of endothelial function, the most prominent processes affected were NADH dehydrogenase activity, N-terminal myristoylation domain binding, nitric-oxide synthase regulator activity and growth factor binding. Pathway-based enrichment analysis revealed "Electron Transport Chain" significantly enriched. Results were validated on genes belonging to "Electron Transport Chain" pathway, using quantitative RT-PCR. CONCLUSIONS: As far as we know, this is the first systematic study in the literature monitoring transcriptional response to insulin in endothelial cells, in a time series microarray experiment. Since chronic hyperinsulinemia contributes to the instability of the atherosclerotic plaque and stimulates cellular proliferation

  8. Changes in abundance of an abscisic acid-responsive, early cysteine-labeled metallothionein transcript during pollen embryogenesis in bread wheat (Triticum aestivum).

    Science.gov (United States)

    Reynolds, T L; Crawford, R L

    1996-12-01

    A clone for an embryoid-abundant, early cysteine-labeled metallothionein (EcMt) gene has been isolated from a wheat pollen embryoid cDNA library. The transcript of this gene was only expressed in embryogenic microspores, pollen embryoids, and developing zygotic embryos of wheat. Accumulation of the EcMt mRNA showed a direct and positive correlation with an increase of the plant hormone, abscisic acid (ABA) in developing pollen embryoids. Treating cultures with an inhibitor of ABA biosynthesis, fluridone, suppressed not only ABA accumulation but also the appearance of the EcMt gene transcript and the ability of microspores to form embryoids. These results suggest that the EcMt gene may act as a molecular marker for pollen embryogenesis because ABA biosynthesis is accompanied by the increased expression of the EcMt transcript that coincides with the differentiation of pollen embryoids in wheat anther cultures.

  9. Genome-wide RNA polymerase II profiles and RNA accumulation reveal kinetics of transcription and associated epigenetic changes during diurnal cycles.

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    Gwendal Le Martelot

    Full Text Available Interactions of cell-autonomous circadian oscillators with diurnal cycles govern the temporal compartmentalization of cell physiology in mammals. To understand the transcriptional and epigenetic basis of diurnal rhythms in mouse liver genome-wide, we generated temporal DNA occupancy profiles by RNA polymerase II (Pol II as well as profiles of the histone modifications H3K4me3 and H3K36me3. We used these data to quantify the relationships of phases and amplitudes between different marks. We found that rhythmic Pol II recruitment at promoters rather than rhythmic transition from paused to productive elongation underlies diurnal gene transcription, a conclusion further supported by modeling. Moreover, Pol II occupancy preceded mRNA accumulation by 3 hours, consistent with mRNA half-lives. Both methylation marks showed that the epigenetic landscape is highly dynamic and globally remodeled during the 24-hour cycle. While promoters of transcribed genes had tri-methylated H3K4 even at their trough activity times, tri-methylation levels reached their peak, on average, 1 hour after Pol II. Meanwhile, rhythms in tri-methylation of H3K36 lagged transcription by 3 hours. Finally, modeling profiles of Pol II occupancy and mRNA accumulation identified three classes of genes: one showing rhythmicity both in transcriptional and mRNA accumulation, a second class with rhythmic transcription but flat mRNA levels, and a third with constant transcription but rhythmic mRNAs. The latter class emphasizes widespread temporally gated posttranscriptional regulation in the mouse liver.

  10. A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity

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    Granier Thierry

    2011-08-01

    Full Text Available Abstract Background Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR. In grapevine (Vitis vinifera L., several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood. Results In this work, we describe the effects of a single amino acid substitution (R69L located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5bL, the native protein being referred as VvMYB5bR was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5bL exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5bR and VvMYB5bL genes in tobacco. Flowers from 35S::VvMYB5bL transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5bR and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation. Conclusions Taken together, our findings indicate that VvMYB5bL is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s. In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment

  11. ETS transcription factors in hematopoietic stem cell development.

    Science.gov (United States)

    Ciau-Uitz, Aldo; Wang, Lu; Patient, Roger; Liu, Feng

    2013-12-01

    Hematopoietic stem cells (HSCs) are essential for the maintenance of the hematopoietic system. However, these cells cannot be maintained or created in vitro, and very little is known about their generation during embryogenesis. Many transcription factors and signaling pathways play essential roles at various stages of HSC development. Members of the ETS ('E twenty-six') family of transcription factors are recognized as key regulators within the gene regulatory networks governing hematopoiesis, including the ontogeny of HSCs. Remarkably, although all ETS transcription factors bind the same DNA consensus sequence and overlapping tissue expression is observed, individual ETS transcription factors play unique roles in the development of HSCs. Also, these transcription factors are recurrently used throughout development and their functions are context-dependent, increasing the challenge of studying their mechanism of action. Critically, ETS factors also play roles under pathological conditions, such as leukemia and, therefore, deciphering their mechanism of action will not only enhance our knowledge of normal hematopoiesis, but also inform protocols for their creation in vitro from pluripotent stem cells and the design of new therapeutic approaches for the treatment of malignant blood cell diseases. In this review, we summarize the key findings on the roles of ETS transcription factors in HSC development and discuss novel mechanisms by which they could control hematopoiesis.

  12. Functionality of intergenic transcription: an evolutionary comparison.

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    Philipp Khaitovich

    2006-10-01

    Full Text Available Although a large proportion of human transcription occurs outside the boundaries of known genes, the functional significance of this transcription remains unknown. We have compared the expression patterns of known genes as well as intergenic transcripts within the ENCODE regions between humans and chimpanzees in brain, heart, testis, and lymphoblastoid cell lines. We find that intergenic transcripts show patterns of tissue-specific conservation of their expression, which are comparable to exonic transcripts of known genes. This suggests that intergenic transcripts are subject to functional constraints that restrict their rate of evolutionary change as well as putative positive selection to an extent comparable to that of classical protein-coding genes. In brain and testis, we find that part of this intergenic transcription is caused by widespread use of alternative promoters. Further, we find that about half of the expression differences between humans and chimpanzees are due to intergenic transcripts.

  13. Functionality of Intergenic Transcription: An Evolutionary Comparison

    Science.gov (United States)

    Visagie, Johann; Giger, Thomas; Joerchel, Sabrina; Petzold, Ekkehard; Green, Richard E; Lachmann, Michael; Pääbo, Svante

    2006-01-01

    Although a large proportion of human transcription occurs outside the boundaries of known genes, the functional significance of this transcription remains unknown. We have compared the expression patterns of known genes as well as intergenic transcripts within the ENCODE regions between humans and chimpanzees in brain, heart, testis, and lymphoblastoid cell lines. We find that intergenic transcripts show patterns of tissue-specific conservation of their expression, which are comparable to exonic transcripts of known genes. This suggests that intergenic transcripts are subject to functional constraints that restrict their rate of evolutionary change as well as putative positive selection to an extent comparable to that of classical protein-coding genes. In brain and testis, we find that part of this intergenic transcription is caused by widespread use of alternative promoters. Further, we find that about half of the expression differences between humans and chimpanzees are due to intergenic transcripts. PMID:17040132

  14. A Trematode Parasite Derived Growth Factor Binds and Exerts Influences on Host Immune Functions via Host Cytokine Receptor Complexes

    Science.gov (United States)

    Sulaiman, Azad A.; Zolnierczyk, Katarzyna; Japa, Ornampai; Owen, Jonathan P.; Maddison, Ben C.; Hodgkinson, Jane E.; Gough, Kevin C.

    2016-01-01

    The trematode Fasciola hepatica is responsible for chronic zoonotic infection globally. Despite causing a potent T-helper 2 response, it is believed that potent immunomodulation is responsible for rendering this host reactive non-protective host response thereby allowing the parasite to remain long-lived. We have previously identified a growth factor, FhTLM, belonging to the TGF superfamily can have developmental effects on the parasite. Herein we demonstrate that FhTLM can exert influence over host immune functions in a host receptor specific fashion. FhTLM can bind to receptor members of the Transforming Growth Factor (TGF) superfamily, with a greater affinity for TGF-β RII. Upon ligation FhTLM initiates the Smad2/3 pathway resulting in phenotypic changes in both fibroblasts and macrophages. The formation of fibroblast CFUs is reduced when cells are cultured with FhTLM, as a result of TGF-β RI kinase activity. In parallel the wound closure response of fibroblasts is also delayed in the presence of FhTLM. When stimulated with FhTLM blood monocyte derived macrophages adopt an alternative or regulatory phenotype. They express high levels interleukin (IL)-10 and arginase-1 while displaying low levels of IL-12 and nitric oxide. Moreover they also undergo significant upregulation of the inhibitory receptor PD-L1 and the mannose receptor. Use of RNAi demonstrates that this effect is dependent on TGF-β RII and mRNA knock-down leads to a loss of IL-10 and PD-L1. Finally, we demonstrate that FhTLM aids newly excysted juveniles (NEJs) in their evasion of antibody-dependent cell cytotoxicity (ADCC) by reducing the NO response of macrophages—again dependent on TGF-β RI kinase. FhTLM displays restricted expression to the F. hepatica gut resident NEJ stages. The altered fibroblast responses would suggest a role for dampened tissue repair responses in facilitating parasite migration. Furthermore, the adoption of a regulatory macrophage phenotype would allow for a reduced

  15. A novel Rieske-type protein derived from an apoptosis-inducing factor-like (AIFL) transcript with a retained intron 4 induces change in mitochondrial morphology and growth arrest

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Yasuhiko, E-mail: 97318@ib.k.u-tokyo.ac.jp [Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561 (Japan); Furuyama, Isao; Oda, Shoji [Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561 (Japan); Mitani, Hiroshi, E-mail: mitani@k.u-tokyo.ac.jp [Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561 (Japan)

    2011-04-01

    Highlights: {yields} A novel major transcript, AIFL-I4, is found. {yields} Nuclear localization of AIFL-I4 induces mitochondrial morphology change and suppression of cell proliferation. {yields} AIFL-I4 mutant with a lesion in [2Fe-2S] cluster binding site does not induce these phenotypes. {yields} [2Fe-2S] cluster binding site is essential for these phenotypes. -- Abstract: Apoptosis-inducing factor-like (AIFL) protein contains a Rieske domain and pyridine nucleotide-disulfide oxidoreductase (Pyr{sub r}edox) domain that shows 35% homology to that of apoptosis-inducing factor (AIF) protein. We identified a novel major transcript of the medaka (Oryzias latipes) AIFL gene that retained intron 4 (AIFL-I4) in embryos and tissues from adult fish. The product of this transcript, AIFL-I4 protein, lacked the Pyr{sub r}edox domain because of a nonsense codon in intron 4. Both AIFL-I4 and full-length AIFL (fAIFL) transcripts were highly expressed in the brain and late embryos, and relative fAIFL and AIFL-I4 expression levels differed among tissues. Transient expression of AIFL-I4 and fAIFL tagged with GFP showed that AIFL-I4 localized in the nucleus, while fAIFL localized throughout the cytoplasm. We also found that overexpression of AIFL-I4 induced a change in mitochondrial morphology and suppression of cell proliferation. AIFL-I4 mutant with a lesion in [2Fe-2S] cluster binding site of the Rieske domain did not induce these phenotypes. This report is the first to demonstrate nuclear localization of a Rieske-type protein translated from the AIFL gene. Our data suggested that the [2Fe-2S] cluster binding site was essential for the nuclear localization and involved in mitochondrial morphology and suppression of cell proliferation.

  16. Genome-Wide Association between Transcription Factor Expression and Chromatin Accessibility Reveals Regulators of Chromatin Accessibility

    Science.gov (United States)

    Rueedi, Rico

    2017-01-01

    To better understand genome regulation, it is important to uncover the role of transcription factors in the process of chromatin structure establishment and maintenance. Here we present a data-driven approach to systematically characterise transcription factors that are relevant for this process. Our method uses a linear mixed modelling approach to combine datasets of transcription factor binding motif enrichments in open chromatin and gene expression across the same set of cell lines. Applying this approach to the ENCODE dataset, we confirm already known and imply numerous novel transcription factors that play a role in the establishment or maintenance of open chromatin. In particular, our approach rediscovers many factors that have been annotated as pioneer factors. PMID:28118358

  17. Transcriptional and post-transcriptional regulation of SPAST, the gene most frequently mutated in hereditary spastic paraplegia.

    Directory of Open Access Journals (Sweden)

    Brian J Henson

    Full Text Available Hereditary spastic paraplegias (HSPs comprise a group of neurodegenerative disorders that are characterized by progressive spasticity of the lower extremities, due to axonal degeneration in the corticospinal motor tracts. HSPs are genetically heterogeneous and show autosomal dominant inheritance in ∼70-80% of cases, with additional cases being recessive or X-linked. The most common type of HSP is SPG4 with mutations in the SPAST gene, encoding spastin, which occurs in 40% of dominantly inherited cases and in ∼10% of sporadic cases. Both loss-of-function and dominant-negative mutation mechanisms have been described for SPG4, suggesting that precise or stoichiometric levels of spastin are necessary for biological function. Therefore, we hypothesized that regulatory mechanisms controlling expression of SPAST are important determinants of spastin biology, and if altered, could contribute to the development and progression of the disease. To examine the transcriptional and post-transcriptional regulation of SPAST, we used molecular phylogenetic methods to identify conserved sequences for putative transcription factor binding sites and miRNA targeting motifs in the SPAST promoter and 3'-UTR, respectively. By a variety of molecular methods, we demonstrate that SPAST transcription is positively regulated by NRF1 and SOX11. Furthermore, we show that miR-96 and miR-182 negatively regulate SPAST by effects on mRNA stability and protein level. These transcriptional and miRNA regulatory mechanisms provide new functional targets for mutation screening and therapeutic targeting in HSP.

  18. Physical training and weight loss in dogs lead to transcriptional changes in genes involved in the glucose-transport pathway in muscle and adipose tissues

    DEFF Research Database (Denmark)

    Herrera Uribe, Juber; Vitger, Anne Désiré; Ritz, Christian

    2016-01-01

    on calorie restriction and physical training (FD group, n = 5) or calorie restriction alone (DO group, n = 7). mRNA expression of 12 genes and six microRNAs were investigated using quantitative real-time PCR (qPCR). In the FD group, FOXO1 and RAC1 were expressed at lower levels in adipose tissue, whereas...... and two microRNAs were significantly downregulated (NRF2, RAC1, ESRRA, AKT2, PGC1a and mir-23; P calorie restriction causes regulation of several metabolic genes in both tissues. The mild exercise, incorporated into this study design, was sufficient to elicit transcriptional...

  19. Transcriptional changes in steroidogenesis by perfluoroalkyl acids (PFOA and PFOS) regulate the synthesis of sex hormones in H295R cells.

    Science.gov (United States)

    Kang, Jae Soon; Choi, Jin-Soo; Park, June-Woo

    2016-07-01

    Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are two of the most widely used perfluoroalkyl acids (PFAAs). Because of their strong persistence, they have become widely distributed throughout the environment and human bodies. PFOA and PFOS are suspected to disrupt the endocrine system based upon many in vivo studies, but the underlying mechanisms are currently unclear. In this study, we investigated the endocrine-related effects of PFOA and PFOS using in vitro estrogen receptor (ER) and androgen receptor (AR) transactivation assays and steroidogenesis assay. The results showed that PFOA and PFOS exhibited weak antagonistic ER transactivation but did not exhibit agonistic ER or AR transactivation. In the steroidogenesis assay, PFOA and PFOS induced 17β-estradiol (E2) level and reduced testosterone level, which would be caused by the induction of aromatase activity. The qPCR analysis of genes involved in steroidogenesis indicates that PFOA and PFOS associate with sex hormone synthesis by the transcriptional induction of two genes, cyp19 and 3β-hsd2. Moreover, the transcriptional induction of cyp11b2 by PFOS suggests that this chemical may underlie the disruption of several physiological functions related to aldosterone. The results of the current study suggest that PFOA and PFOS are potential endocrine disrupting chemicals (EDCs) and provide information for further studies on the molecular events that initiate the adverse endocrine effects.

  20. The crystal structures of apo and cAMP-bound GlxR from Corynebacterium glutamicum reveal structural and dynamic changes upon cAMP binding in CRP/FNR family transcription factors.

    Directory of Open Access Journals (Sweden)

    Philip D Townsend

    Full Text Available The cyclic AMP-dependent transcriptional regulator GlxR from Corynebacterium glutamicum is a member of the super-family of CRP/FNR (cyclic AMP receptor protein/fumarate and nitrate reduction regulator transcriptional regulators that play central roles in bacterial metabolic regulatory networks. In C. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the Corynebacteriales including Mycobacterium tuberculosis, the GlxR homodimer controls the transcription of a large number of genes involved in carbon metabolism. GlxR therefore represents a key target for understanding the regulation and coordination of C. glutamicum metabolism. Here we investigate cylic AMP and DNA binding of GlxR from C. glutamicum and describe the crystal structures of apo GlxR determined at a resolution of 2.5 Å, and two crystal forms of holo GlxR at resolutions of 2.38 and 1.82 Å, respectively. The detailed structural analysis and comparison of GlxR with CRP reveals that the protein undergoes a distinctive conformational change upon cyclic AMP binding leading to a dimer structure more compatible to DNA-binding. As the two binding sites in the GlxR homodimer are structurally identical dynamic changes upon binding of the first ligand are responsible for the allosteric behavior. The results presented here show how dynamic and structural changes in GlxR lead to optimization of orientation and distance of its two DNA-binding helices for optimal DNA recognition.

  1. Using cell fate attractors to uncover transcriptional regulation of HL60 neutrophil differentiation

    Directory of Open Access Journals (Sweden)

    Kauffman Stuart A

    2009-02-01

    Full Text Available Abstract Background The process of cellular differentiation is governed by complex dynamical biomolecular networks consisting of a multitude of genes and their products acting in concert to determine a particular cell fate. Thus, a systems level view is necessary for understanding how a cell coordinates this process and for developing effective therapeutic strategies to treat diseases, such as cancer, in which differentiation plays a significant role. Theoretical considerations and recent experimental evidence support the view that cell fates are high dimensional attractor states of the underlying molecular networks. The temporal behavior of the network states progressing toward different cell fate attractors has the potential to elucidate the underlying molecular mechanisms governing differentiation. Results Using the HL60 multipotent promyelocytic leukemia cell line, we performed experiments that ultimately led to two different cell fate attractors by two treatments of varying dosage and duration of the differentiation agent all-trans-retinoic acid (ATRA. The dosage and duration combinations of the two treatments were chosen by means of flow cytometric measurements of CD11b, a well-known early differentiation marker, such that they generated two intermediate populations that were poised at the apparently same stage of differentiation. However, the population of one treatment proceeded toward the terminally differentiated neutrophil attractor while that of the other treatment reverted back toward the undifferentiated promyelocytic attractor. We monitored the gene expression changes in the two populations after their respective treatments over a period of five days and identified a set of genes that diverged in their expression, a subset of which promotes neutrophil differentiation while the other represses cell cycle progression. By employing promoter based transcription factor binding site analysis, we found enrichment in the set of divergent

  2. Boosting transcription by transcription: enhancer-associated transcripts.

    Science.gov (United States)

    Darrow, Emily M; Chadwick, Brian P

    2013-12-01

    Enhancers are traditionally viewed as DNA sequences located some distance from a promoter that act in cis and in an orientation-independent fashion to increase utilization of specific promoters and thereby regulate gene expression. Much progress has been made over the last decade toward understanding how these distant elements interact with target promoters, but how transcription is enhanced remains an object of active inquiry. Recent reports convey the prevalence and diversity of enhancer transcription and transcripts and support both as key factors with mechanistically distinct, but not mutually exclusive roles in enhancer function. Decoupling the causes and effects of transcription on the local chromatin landscape and understanding the role of enhancer transcripts in the context of long-range interactions are challenges that require additional attention. In this review, we focus on the possible functions of enhancer transcription by highlighting several recent enhancer RNA papers and, within the context of other enhancer studies, speculate on the role of enhancer transcription in regulating differential gene expression.

  3. Effects of tibolone and its metabolites on prolactin and insulin-like growth factor binding protein-1 expression in human endometrial stromal cells.

    Science.gov (United States)

    Guzel, Elif; Buchwalder, Lynn; Basar, Murat; Kayisli, Umit; Ocak, Nehir; Bozkurt, Idil; Lockwood, Charles J; Schatz, Frederick

    2015-05-01

    The effects of the postmenopausal replacement steroid tibolone and its 3α-, 3β-OH and Δ-4 tibolone metabolites were evaluated on progesterone receptor-mediated classic decidualization markers insulin-like growth factor binding protein-1 (IGFBP-1) and prolactin expression in human endometrial stromal cells (HESCs). Supernatants of conditioned medium or erxtracted RNA from experimental cell incubations of confluent HESCs were subjected to ELISAs, Western blot analysis and RT/PCR, and results were statisically assesed. Over 21 days, specific ELISAs observed linear increases in secreted IGFBP-1 and prolactin levels elicited by tibolone and its metabolites. Cultured HESCs were refractory to E2 and dexamethasone, whereas tibolone and each metabolite exceeded medroxyprogesterone acetate in significantly elevating IGFBP-1 and prolactin output. Anti-progestins eliminated IGFBP-1 and prolactin induction by tibolone and its metabolites. Immunoblotting and RT/PCR confirmed ELISA results. These observations of IGFBP-1 and prolactin expression: (a) indicate the relevance of cultured HESCs in evaluating the chronic effects of tibolone administration to women; (b) are consistent with PR-mediated endometrial atrophy and protection against endometrial bleeding despite the persistence of circulating ER-binding, but not PR-binding metabolites following tibolone administration to women.

  4. Serum insulin-like growth factors I and II, insulin-like growth factor binding protein-3 and risk of breast cancer in the Japan Collaborative Cohort study.

    Science.gov (United States)

    Sakauchi, Fumio; Nojima, Masanori; Mori, Mitsuru; Wakai, Kenji; Suzuki, Sadao; Tamakoshi, Akiko; Ito, Yoshinori; Watanabe, Yoshiyuki; Inaba, Yutaka; Tajima, Kazuo; Nakachi, Kei

    2009-12-01

    The Japan Collaborative Cohort Study for Evaluation of Cancer Risk (JACC Study) was planned in the late 1980s as a large-scale cohort study of persons in various areas of Japan. In the present study, we conducted a nested case-control study and examined associations of breast cancer risk with serum levels of insulin-like growth factors I and II (IGF-I, IGF-II), as well as insulin-like growth factor binding protein-3 (IGFBP-3), among women who participated in the JACC Study and donated blood at the baseline. Sixty-three women who died or suffered from breast cancer were examined. Two or three controls were selected to match each case for age at recruitment and the study area. Controls were alive and not diagnosed as having breast cancer at the diagnosis date of the cases. Associations between the serum IGF-I, IGF-II, IGFBP-3 and breast cancer risk were evaluated using a conditional logistic regression model. In premenopausal Japanese women, IGF-I showed a marginal negative dose-dependent association with the breast cancer risk (trend P= 0.08), but any link disappeared on taking into account IGFBP-3 (trend P= 0.47), which was likely to be inversely associated with the risk. In postmenopausal women, IGFBP-3 showed a marginal dose-dependent association with the risk (trend P= 0.06). Further studies are needed to confirm these findings.

  5. Elevation of insulin-like growth factor binding protein-2 level in Pallister-Killian syndrome: implications for the postnatal growth retardation phenotype.

    Science.gov (United States)

    Izumi, Kosuke; Kellogg, Emily; Fujiki, Katsunori; Kaur, Maninder; Tilton, Richard K; Noon, Sarah; Wilkens, Alisha; Shirahige, Katsuhiko; Krantz, Ian D

    2015-06-01

    Pallister-Killian syndrome (PKS) is a multi-system developmental disorder caused by tetrasomy 12p that exhibits tissue-limited mosaicism. Probands with PKS often demonstrate a unique growth profile consisting of macrosomia at birth with deceleration of growth postnatally. We have previously demonstrated that cultured skin fibroblasts from PKS probands have significantly elevated expression of insulin-like growth factor binding protein-2 (IGFBP2). To further evaluate the role of IGFBP2 in PKS, the amount of IGFBP2 secreted from cultured skin fibroblast cell lines and serum IGFBP2 levels were measured in probands with PKS. Approximately 60% of PKS fibroblast cell lines secreted higher levels of IGFBP2 compared to control fibroblasts, although the remaining 40% of PKS samples produced comparable level of IGFBP2 to that of control fibroblasts. Serum IGFBP2 levels were also measured in PKS probands and were elevated in 40% of PKS probands. PKS probands with elevated IGFBP2 manifested with severe postnatal growth retardation. IGFBPs are the family of related proteins that bind IGFs with high affinity and are typically thought to attenuate IGF action. We suggest that elevated IGFBP2 levels might play a role in the growth retardation phenotype of PKS.

  6. Detection of placental alpha microglobulin-1 versus insulin-like growth factor-binding protein-1 in amniotic fluid at term: a comparative study.

    Science.gov (United States)

    Pollet-Villard, Marie; Cartier, Régine; Gaucherand, Pascal; Doret, Muriel

    2011-06-01

    We compared two biochemical tests of premature rupture of membranes (PROM) in vitro: Actim PROM (Medix Biochemica, Kauniainen, Finland), which detects insulin-like growth factor binding protein-1, and AmniSure (AmniSure International LLC, Cambridge, MA), which detects placental alpha microglobulin-1. Samples of amniotic fluid were collected during caesarean section in 41 patients. A dilution series was prepared and both tests were performed twice at each dilution. Sensitivity, detection limit, response time, and reproducibility of both tests were compared. Both tests' sensitivity was 100% at dilution 1:10 and 1:20. AmniSure sensitivity was higher at dilution 1:40 and 1:80 ( P AmniSure had a lower detection limit than Actim PROM. AmniSure response times were shorter and reproducibility was higher than Actim PROM ( P AmniSure had a lower detection limit of amniotic fluid than Actim PROM, with a shorter response time, a higher sensitivity, and a better reproducibility.

  7. Changes in Gene Transcription Induced by Hydrogen Peroxide Treatment of Verotoxin-Producing Escherichia coli O157:H7 and Non-O157 Serotypes on Romaine Lettuce

    Science.gov (United States)

    Mei, Gui-Ying; Tang, Joshua; Bach, Susan; Kostrzynska, Magdalena

    2017-01-01

    Disease outbreaks of verotoxin-producing Escherichia coli (VTEC) O157:H7 and non-O157 serotypes associated with leafy green vegetables are becoming a growing concern. A better understanding of the behavior of VTEC, particularly non-O157 serotypes, on lettuce under stress conditions is necessary for designing more effective control strategies. Hydrogen peroxide (H2O2) can be used as a sanitizer to reduce the microbial load in leafy green vegetables, particularly in fresh produce destined for the organic market. In this study, we tested the hypothesis that H2O2 treatment of contaminated lettuce affects in the same manner transcription of stress-associated and virulence genes in VTEC strains representing O157 and non-O157 serotypes. Six VTEC isolates representing serotypes O26:H11, O103:H2, O104:H4, O111:NM, O145:NM, and O157:H7 were included in this study. The results indicate that 50 mM H2O2 caused a population reduction of 2.4–2.8 log10 (compared to non-treated control samples) in all six VTEC strains present on romaine lettuce. Following the treatment, the transcription of genes related to oxidative stress (oxyR and sodA), general stress (uspA and rpoS), starvation (phoA), acid stress (gadA, gadB, and gadW), and virulence (stx1A, stx2A, and fliC) were dramatically downregulated in all six VTEC serotypes (P ≤ 0.05) compared to not treated control samples. Therefore, VTEC O157:H7 and non-O157 serotypes on lettuce showed similar survival rates and gene transcription profiles in response to 50 mM H2O2 treatment. Thus, the results derived from this study provide a basic understanding of the influence of H2O2 treatment on the survival and virulence of VTEC O157:H7 and non-O157 serotypes on lettuce.

  8. Prediction of the outcome of growth hormone provocative testing in short children by measurement of serum levels of insulin-like growth factor I and insulin-like growth factor binding protein 3

    DEFF Research Database (Denmark)

    Juul, A; Skakkebaek, N E

    1997-01-01

    Serum levels of insulin-like growth factor I (IGF-I) and insulin-like growth factor binding protein 3 (IGFBP-3) reflect the secretion of endogenous growth hormone (GH) in healthy children and exhibit little diurnal variation, which makes them potential candidates for screening of GH deficiency (G...

  9. Transcriptional networks inferred from molecular signatures of breast cancer.

    Science.gov (United States)

    Tongbai, Ron; Idelman, Gila; Nordgard, Silje H; Cui, Wenwu; Jacobs, Jonathan L; Haggerty, Cynthia M; Chanock, Stephen J; Børresen-Dale, Anne-Lise; Livingston, Gary; Shaunessy, Patrick; Chiang, Chih-Hung; Kristensen, Vessela N; Bilke, Sven; Gardner, Kevin

    2008-02-01

    Global genomic approaches in cancer research have provided new and innovative strategies for the identification of signatures that differentiate various types of human cancers. Computational analysis of the promoter composition of the genes within these signatures may provide a powerful method for deducing the regulatory transcriptional networks that mediate their collective function. In this study we have systematically analyzed the promoter composition of gene classes derived from previously established genetic signatures that recently have been shown to reliably and reproducibly distinguish five molecular subtypes of breast cancer associated with distinct clinical outcomes. Inferences made from the trends of transcription factor binding site enrichment in the promoters of these gene groups led to the identification of regulatory pathways that implicate discrete transcriptional networks associated with specific molecular subtypes of breast cancer. One of these inferred pathways predicted a role for nuclear factor-kappaB in a novel feed-forward, self-amplifying, autoregulatory module regulated by the ERBB family of growth factor receptors. The existence of this pathway was verified in vivo by chromatin immunoprecipitation and shown to be deregulated in breast cancer cells overexpressing ERBB2. This analysis indicates that approaches of this type can provide unique insights into the differential regulatory molecular programs associated with breast cancer and will aid in identifying specific transcriptional networks and pathways as potential targets for tumor subtype-specific therapeutic intervention.

  10. TRANSFAC and its module TRANSCompel: transcriptional gene regulation in eukaryotes.

    Science.gov (United States)

    Matys, V; Kel-Margoulis, O V; Fricke, E; Liebich, I; Land, S; Barre-Dirrie, A; Reuter, I; Chekmenev, D; Krull, M; Hornischer, K; Voss, N; Stegmaier, P; Lewicki-Potapov, B; Saxel, H; Kel, A E; Wingender, E

    2006-01-01

    The TRANSFAC database on transcription factors, their binding sites, nucleotide distribution matrices and regulated genes as well as the complementing database TRANSCompel on composite elements have been further enhanced on various levels. A new web interface with different search options and integrated versions of Match and Patch provides increased functionality for TRANSFAC. The list of databases which are linked to the common GENE table of TRANSFAC and TRANSCompel has been extended by: Ensembl, UniGene, EntrezGene, HumanPSD and TRANSPRO. Standard gene names from HGNC, MGI and RGD, are included for human, mouse and rat genes, respectively. With the help of InterProScan, Pfam, SMART and PROSITE domains are assigned automatically to the protein sequences of the transcription factors. TRANSCompel contains now, in addition to the COMPEL table, a separate table for detailed information on the experimental EVIDENCE on which the composite elements are based. Finally, for TRANSFAC, in respect of data growth, in particular the gain of Drosophila transcription factor binding sites (by courtesy of the Drosophila DNase I footprint database) and of Arabidopsis factors (by courtesy of DATF, Database of Arabidopsis Transcription Factors) has to be stressed. The here described public releases, TRANSFAC 7.0 and TRANSCompel 7.0, are accessible under http://www.gene-regulation.com/pub/databases.html.

  11. Implementing arithmetic and other analytic operations by transcriptional regulation.

    Directory of Open Access Journals (Sweden)

    Sean M Cory

    2008-04-01

    Full Text Available The transcriptional regulatory machinery of a gene can be viewed as a computational device, with transcription factor concentrations as inputs and expression level as the output. This view begs the question: what kinds of computations are possible? We show that different parameterizations of a simple chemical kinetic model of transcriptional regulation are able to approximate all four standard arithmetic operations: addition, subtraction, multiplication, and division, as well as various equality and inequality operations. This contrasts with other studies that emphasize logical or digital notions of computation in biological networks. We analyze the accuracy and precision of these approximations, showing that they depend on different sets of parameters, and are thus independently tunable. We demonstrate that networks of these "arithmetic" genes can be combined to accomplish yet more complicated computations by designing and simulating a network that detects statistically significant elevations in a time-varying signal. We also consider the much more general problem of approximating analytic functions, showing that this can be achieved by allowing multiple transcription factor binding sites on the promoter. These observations are important for the interpretation of naturally occurring networks and imply new possibilities for the design of synthetic networks.

  12. Insulin-like growth factor-binding protein-2 promotes prostate cancer cell growth via IGF-dependent or -independent mechanisms and reduces the efficacy of docetaxel

    Science.gov (United States)

    Uzoh, C C; Holly, J M P; Biernacka, K M; Persad, R A; Bahl, A; Gillatt, D; Perks, C M

    2011-01-01

    Background: The development of androgen independence, chemo-, and radioresistance are critical markers of prostate cancer progression and the predominant reasons for its high mortality. Understanding the resistance to therapy could aid the development of more effective treatments. Aim: The aim of this study is to investigate the effects of insulin-like growth factor-binding protein-2 (IGFBP-2) on prostate cancer cell proliferation and its effects on the response to docetaxel. Methods: DU145 and PC3 cells were treated with IGFBP-2, insulin-like growth factor I (IGF-I) alone or in combination with blockade of the IGF-I receptor or integrin receptors. Cells were also treated with IGFBP-2 short interfering ribonucleic acid with or without a PTEN (phosphatase and tensin homologue deleted on chromosome 10) inhibitor or docetaxel. Tritiated thymidine incorporation was used to measure cell proliferation and Trypan blue cell counting for cell death. Levels of IGFBP-2 mRNA were measured using RT–PCR. Abundance and phosphorylation of proteins were assessed using western immunoblotting. Results: The IGFBP-2 promoted cell growth in both cell lines but with PC3 cells this was in an IGF-dependent manner, whereas with DU145 cells the effect was independent of IGF receptor activation. This IGF-independent effect of IGFBP-2 was mediated by interaction with β-1-containing integrins and a consequent increase in PTEN phosphorylation. We also determined that silencing IGFBP-2 in both cell lines increased the sensitivity of the cells to docetaxel. Conclusion: The IGFBP-2 has a key role in the growth of prostate cancer cells, and silencing IGFBP-2 expression reduced the resistance of these cells to docetaxel. Targeting IGFBP-2 may increase the efficacy of docetaxel. PMID:21487405

  13. Amblyomma americanum tick saliva insulin-like growth factor binding protein-related protein 1 binds insulin but not insulin-like growth factors.

    Science.gov (United States)

    Radulović, Ž M; Porter, L M; Kim, T K; Bakshi, M; Mulenga, A

    2015-10-01

    Silencing Amblyomma americanum insulin-like growth factor binding protein-related protein 1 (AamIGFBP-rP1) mRNA prevented ticks from feeding to repletion. In this study, we used recombinant (r)AamIGFBP-rP1 in a series of assays to obtain further insight into the role(s) of this protein in tick feeding regulation. Our results suggest that AamIGFBP-1 is an antigenic protein that is apparently exclusively expressed in salivary glands. We found that both males and females secrete AamIGFBP-rP1 into the host during feeding and confirmed that female ticks secrete this protein from within 24-48 h after attachment. Our data suggest that native AamIGFBP-rP1 is a functional insulin binding protein in that both yeast- and insect cell-expressed rAamIGFBP-rP1 bound insulin, but not insulin-like growth factors. When subjected to anti-blood clotting and platelet aggregation assays, rAamIGFBP-rP1 did not have any effect. Unlike human IGFBP-rP1, which is controlled by trypsinization, rAamIGFBP-rP1 is resistant to digestion, suggesting that the tick protein may not be under mammalian host control at the tick feeding site. The majority of tick-borne pathogens are transmitted 48 h after the tick has attached. Thus, the demonstrated antigenicity and secretion into the host within 24-48 h of the tick starting to feed makes AamIGFBP-rP1 an attractive target for antitick vaccine development.

  14. The joint effects of arsenic and risk diplotypes of insulin-like growth factor binding protein-3 in renal cell carcinoma.

    Science.gov (United States)

    Huang, Chao-Yuan; Huang, Ya-Li; Pu, Yeong-Shiau; Shiue, Horng-Sheng; Chen, Wei-Jen; Chen, Shih-Shan; Lin, Ying-Chin; Su, Chien-Tien; Hsueh, Yu-Mei

    2016-07-01

    The association between renal cell carcinoma (RCC) and diabetes mellitus (DM), alcohol consumption, insulin-like growth factor binding protein-3 (IGFBP-3) gene, and arsenic exposure, has been the subject of independent studies. However, few studies have examined the combined effect of these factors on RCC risk. The aim of this study was to examine the association between these risk factors and the odds ratio (OR) of RCC. A hospital-based case-control study was conducted in 398 RCC patients and 756 age- and gender-matched non-cancer controls. Genomic DNA was used to examine the genotype of IRS-1 (Gly972Arg), PI3-K (Met362Ile), IGFBP-3 (A[-202]C), and IGFBP-3 (C[-1590]A) by PCR-RFLP. Profiles of urinary arsenic were measured by high performance liquid chromatography linked with hydride generator and atomic absorption spectrometry. Participants who had never consumed alcohol and who had high total levels of urinary arsenic and DM had a high OR of RCC. IGFBP-3 (A[-202]C) and IGFBP-3 (C[-1590]A) were in linkage disequilibrium. Participants carrying high-risk IGFBP-3 diplotypes A-C/C-C, A-A/A-C, and C-A/C-A had a significantly higher odds ratio (OR) and 95% confidence interval (2.80, 1.91-4.12) of RCC compared to those carrying other IGFBP-3 diplotypes. This is the first study to show that borderline significant interaction of high total levels of urinary arsenic and IGFBP-3 high-risk diplotypes significantly enhanced the OR of RCC. Our data also provide evidence that subjects with more risk factors (e.g., high total levels of urinary arsenic, never consumed alcohol, IGFBP-3 high-risk diplotypes) may experience a higher OR of RCC.

  15. Urinary bladder cancer risk in relation to a single nucleotide polymorphism (rs2854744) in the insulin-like growth factor-binding protein-3 (IGFBP3) gene.

    Science.gov (United States)

    Selinski, Silvia; Lehmann, Marie-Louise; Blaszkewicz, Meinolf; Ovsiannikov, Daniel; Moormann, Oliver; Guballa, Christoph; Kress, Alexander; Truss, Michael C; Gerullis, Holger; Otto, Thomas; Barski, Dimitri; Niegisch, Günter; Albers, Peter; Frees, Sebastian; Brenner, Walburgis; Thüroff, Joachim W; Angeli-Greaves, Miriam; Seidel, Thilo; Roth, Gerhard; Volkert, Frank; Ebbinghaus, Rainer; Prager, Hans-Martin; Lukas, Cordula; Bolt, Hermann M; Falkenstein, Michael; Zimmermann, Anna; Klein, Torsten; Reckwitz, Thomas; Roemer, Hermann C; Hartel, Mark; Weistenhöfer, Wobbeke; Schöps, Wolfgang; Rizvi, S Adibul Hassan; Aslam, Muhammad; Bánfi, Gergely; Romics, Imre; Ickstadt, Katja; Hengstler, Jan G; Golka, Klaus

    2012-02-01

    Currently, twelve validated genetic variants have been identified that are associated with urinary bladder cancer (UBC) risk. However, those validated variants explain only 5-10% of the overall inherited risk. In addition, there are more than 100 published polymorphisms still awaiting validation or disproval. A particularly promising of the latter unconfirmed polymorphisms is rs2854744 that recently has been published to be associated with UBC risk. The [A] allele of rs2854744 has been reported to be associated with a higher promoter activity of the insulin-like growth factor-binding protein-3 (IGFBP3) gene, which may lead to increased IGFBP-3 plasma levels and cancer risk. Therefore, we investigated the association of rs2854744 with UBC in the IfADo case-control series consisting of 1,450 cases and 1,725 controls from Germany, Hungary, Venezuela and Pakistan. No significant association of rs2854744 with UBC risk was obtained (all study groups combined: unadjusted P = 0.4446; adjusted for age, gender and smoking habits P = 0.6510), besides a small effect of the [A] allele in the Pakistani study group opposed to the original findings (unadjusted P = 0.0508, odds ratio (OR) = 1.43 for the multiplicative model) that diminished after adjustment for age, gender and smoking habits (P = 0.7871; OR = 0.93). Associations of rs2854744 with occupational exposure to urinary bladder carcinogens and smoking habits were also not present. A meta-analysis of all available case-control series including the original discovery study resulted in an OR of 1.00 (P = 0.9562). In conclusion, we could not confirm the recently published hypothesis that rs2854744 in the IGFBP3 gene is associated with UBC risk.

  16. Prognostic usefulness of insulin-like growth factor-binding protein 7 in heart failure with reduced ejection fraction: a novel biomarker of myocardial diastolic function?

    Science.gov (United States)

    Gandhi, Parul U; Gaggin, Hanna K; Sheftel, Alex D; Belcher, Arianna M; Weiner, Rory B; Baggish, Aaron L; Motiwala, Shweta R; Liu, Peter P; Januzzi, James L

    2014-11-15

    Insulin-like growth factor-binding protein 7 (IGFBP7) is a biomarker that has recently been associated with heart failure and cardiac hypertrophy. The aim of this study was to examine IGFBP7 relative to echocardiographic abnormalities reflecting diastolic dysfunction. One hundred twenty-four patients with ambulatory heart failure with reduced ejection fraction and baseline detailed 2-dimensional echocardiograms were followed for a mean of 10 months. IGFBP7 was measured serially at each office visit; 108 patients underwent follow-up echocardiography. Echocardiographic parameters of diastolic function were compared at baseline and over time. IGFBP7 concentrations were not linked to left ventricular size or systolic function. In contrast, those with elevated baseline IGFBP7 concentrations were more likely to have abnormalities of parameters describing diastolic function, such as higher left atrial volume index, transmitral E/A ratio, E/E' ratio, and right ventricular systolic pressure. IGFBP7 was correlated with left atrial volume index (ρ = 0.237, p = 0.008), transmitral E/A ratio (ρ = 0.304, p = 0.001), E/E' ratio (ρ = 0.257, p = 0.005), and right ventricular systolic pressure (ρ = 0.316, p = 0.001). Furthermore, each was found to be independently predictive of IGFBP7 in adjusted analysis. In subjects with baseline and final echocardiograms, more time spent with elevated IGFBP7 concentrations in serial measurement was associated with worsening diastolic function and increasing left atrial volume index or right ventricular systolic pressure. IGFBP7 concentrations were predictive of an increased risk for cardiovascular events independent of echocardiographic measures of diastolic function (p = 0.006). In conclusion, IGFBP7 is a novel prognostic biomarker for heart failure with reduced ejection fraction and shows significant links to the presence and severity of echocardiographic parameters of abnormal diastolic function.

  17. Identification and localization of insulin-like growth factor-binding protein (IGFBP) messenger RNAs in human hair follicle dermal papilla.

    Science.gov (United States)

    Batch, J A; Mercuri, F A; Werther, G A

    1996-03-01

    The role of the insulin-like growth factors (IGFs) in hair follicle biology has recently been recognized, although their actions, sites of production, and modulation by the insulin-like growth factor-binding proteins (IGFBPs) have not to date been defined. IGF-I is essential for normal hair growth and development, and may be important in regulation of the hair growth cycle. In many culture systems, IGF-I actions are modulated by the IGFBPs. Thus, if IGFBPs are produced in the human hair follicle, they may play a role in targeting IGF-I to its receptor or may modulate IGF-I action by interaction with matrix proteins. We have used in situ hybridization to localize messenger RNA for the six IGFBPs in anagen hair follicles. Anti-sense and sense RNA probes for the IGFBPs (IGFBP-1 to -6) were produced, and 5-micrometer sections of adult facial skin were probed. Messenger RNA for IGFBP-3, -4, and -5 were identified, with predominantly IGFBP-3 and -5 mRNA found in the dermal papilla, and to a lesser extent IGFBP-4 mRNA. IGFBP-4 mRNA was also found at the dermal papilla/epithelial matrix border. Messenger RNAs for both IGFBP-4 and -5 were also demonstrated in the dermal sheath surrounding the hair follicle. Messenger RNAs for IGFBP-1, -2, and -6 were not identified. These studies demonstrate specific localization of IGFBP mRNAs in hair follicles, suggesting that they each play specific roles in the local modulation of IGF action during the hair growth cycle.

  18. Two grass carp (Ctenopharyngodon idella) insulin-like growth factor-binding protein 5 genes exhibit different yet conserved functions in development and growth.

    Science.gov (United States)

    Zheng, Guo-Dong; Zhou, Chun-Xue; Lin, Si-Tong; Chen, Jie; Jiang, Xia-Yun; Zou, Shu-Ming

    2017-02-01

    Insulin-like growth factor binding-protein 5 (igfbp5), the most conserved member of the IGFBP family in vertebrates, plays a critical role in controlling cell survival, growth, differentiation, and apoptosis. Here, we characterized the expression patterns of igfbp5a and igfbp5b in grass carp (Ctenopharyngodon idella), which are retained in many fish species, likely from the teleost-specific whole-genome duplication. Both igfbp5a and igfbp5b encode 268- and 263-aa peptides, respectively, which share a sequence identity of 71%. Their mRNAs are not detected in zygotes. At 14hpf, grass carp igfbp5b mRNA was detected in the somites, while igfbp5a mRNA has some possible signal around the eye and head region. At 24hpf, both igfbp5a and igfbp5b mRNA appear to be limited to the presomitic mesoderm. At 36hpf, igfbp5a mRNA was only detected in the midbrain, while igfbp5b mRNA was detected in both the midbrain and notochord. Overall, both mRNAs were expressed in most adult tissues. igfbp5a and igfbp5b were significantly upregulated in the muscle and liver after injection of 10μg per kilogram body weight of zebrafish growth hormone (zGH), while their hepatic expression was downregulated by 50μg zGH. During fasting, both igfbp5a and igfbp5b mRNAs were significantly downregulated in the muscle but upregulated in the liver. Collectively, the results suggest that the two igfbp5 genes play important but different roles in the regulation of growth and development in grass carp.

  19. Presence of Insulin-Like Growth Factor Binding Proteins Correlates With Tumor-Promoting Effects of Matrix Metalloproteinase 9 in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Jae-Hyun Park

    2015-05-01

    Full Text Available The stroma of breast cancer can promote the disease’s progression, but whether its composition and functions are shared among different subtypes is poorly explored. We compared stromal components of a luminal [mouse mammary tumor virus (MMTV–Neu] and a triple-negative/basal-like [C3(1–Simian virus 40 large T antigen (Tag] genetically engineered breast cancer mouse model. The types of cytokines and their expression levels were very different in the two models, as was the extent of innate immune cell infiltration; however, both models showed infiltration of innate immune cells that expressed matrix metalloproteinase 9 (MMP9, an extracellular protease linked to the progression of many types of cancer. By intercrossing with Mmp9 null mice, we found that the absence of MMP9 delayed tumor onset in the C3(1-Tag model but had no effect on tumor onset in the MMTV-Neu model. We discovered that protein levels of insulin-like growth factor binding protein-1 (IGFBP-1, an MMP9 substrate, were increased in C3(1-Tag;Mmp9−/− compared to C3(1-Tag;Mmp9+/+ tumors. In contrast, IGFBP-1 protein expression was low in MMTV-Neu tumors regardless of Mmp9 status. IGFBP-1 binds and antagonizes IGFs, preventing them from activating their receptors to promote cell proliferation and survival. Tumors from C3(1-Tag;Mmp9−/− mice had reduced IGF-1 receptor phosphorylation, consistent with slower tumor onset. Finally, gene expression analysis of human breast tumors showed that high expression of IGFBP mRNA was strongly correlated with good prognosis but not when MMP9 mRNA was also highly expressed. In conclusion, MMP9 has different effects on breast cancer progression depending on whether IGFBPs are expressed.

  20. Regulation of the MEF2 Family of Transcription Factors by p38†

    OpenAIRE

    Zhao, Ming; New, Liguo; Kravchenko, Vladimir V.; KATO, Yutaka; Gram, Hermann; Di Padova, Franco; Olson, Eric N.; Ulevitch, Richard J.; Han, Jiahuai

    1999-01-01

    Members of the MEF2 family of transcription factors bind as homo- and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate...

  1. Adult glucocorticoid exposure leads to transcriptional and DNA methylation changes in nuclear steroid receptors in the hippocampus and kidney of mouse male offspring.

    Science.gov (United States)

    Petropoulos, Sophie; Matthews, Stephen G; Szyf, Moshe

    2014-02-01

    Synthetic glucocorticoids (sGCs) are commonly prescribed for the management of inflammatory and endocrine disorders. However, nothing is known regarding the effects of sGC on adult germline methylome and whether these effects can be transmitted to the next generation. We hypothesized that administration of sGC to adult male mice alters DNA methylation in mature sperm and modifies the transcription and methylation of steroid receptors in male F1 offspring. Adult C57BL/6 males (n = 10/group) were injected on five consecutive days with 1 mg/kg sGC (i.e., dexamethasone) or vehicle and euthanized 35 or 60 days after initial treatment or bred with control females (60 days postinitial treatment; n = 5/group). A significant increase in global non-CpG methylation was observed in F0 sperm 60 days following sGC treatment. In the hippocampus and kidney of Postnatal Day 50 (PND50) and PND240 male offspring derived from fathers exposed to sGC, significant differences in mineralocorticoid receptor (Nr3c2; Mr), estrogen alpha receptor (Nr3a1; Ers1), and glucocorticoid receptor (Nr3c1; Gr) expression were observed. Furthermore, significant demethylation in regulatory regions of Mr, Gr, and Esr1 was observed in the PND50 kidney derived from fathers exposed to sGC. This is the first demonstration that paternal pharmacological exposure to sGC can alter the expression and DNA methylation of nuclear steroid receptors in brain and somatic tissues of offspring. These findings provide proof of principle that adult male exposure to sGC can affect DNA methylation and gene expression in offspring, indicating the possibility that adult experiences that evoke increases in endogenous glucocorticoid (i.e., stress) might have similar effects.

  2. Aestivation induces changes in transcription and translation of coagulation factor II and fibrinogen gamma chain in the liver of the African lungfish Protopterus annectens.

    Science.gov (United States)

    Hiong, Kum C; Tan, Xiang R; Boo, Mel V; Wong, Wai P; Chew, Shit F; Ip, Yuen K

    2015-12-01

    This study aimed to sequence and characterize two pro-coagulant genes, coagulation factor II (f2) and fibrinogen gamma chain (fgg), from the liver of the African lungfish Protopterus annectens, and to determine their hepatic mRNA expression levels during three phases of aestivation. The protein abundance of F2 and Fgg in the liver and plasma was determined by immunoblotting. The results indicated that F2 and Fgg of P. annectens were phylogenetically closer to those of amphibians than those of teleosts. Three days of aestivation resulted in an up-regulation in the hepatic fgg mRNA expression level, while 6 days of aestivation led to a significant increase (3-fold) in the protein abundance of Fgg in the plasma. Hence, there could be an increase in the blood-clotting ability in P. annectens during the induction phase of aestivation. By contrast, the blood-clotting ability in P. annectens might be reduced in response to decreased blood flow and increased possibility of thrombosis during the maintenance phase of aestivation, as 6 months of aestivation led to significant decreases in mRNA expression levels of f2 and fgg in the liver. There could also be a decrease in the export of F2 and Fgg from the liver to the plasma so as to avert thrombosis. Three to 6 days after arousal from 6 months of aestivation, the protein abundance of F2 and Fgg recovered partially in the plasma of P. annectens; a complete recovery of the transcription and translation of f2/F2 in the liver might occur only after refeeding.

  3. Integration of retroviral vectors induces minor changes in the transcriptional activity of T cells from ADA-SCID patients treated with gene therapy.

    Science.gov (United States)

    Cassani, Barbara; Montini, Eugenio; Maruggi, Giulietta; Ambrosi, Alessandro; Mirolo, Massimiliano; Selleri, Silvia; Biral, Erika; Frugnoli, Ilaria; Hernandez-Trujillo, Vivian; Di Serio, Clelia; Roncarolo, Maria Grazia; Naldini, Luigi; Mavilio, Fulvio; Aiuti, Alessandro

    2009-10-22

    Gene transfer into hematopoietic stem cells by gamma-retroviral vectors (RVs) is an effective treatment for inherited blood disorders, although potentially limited by the risk of insertional mutagenesis. We evaluated the genomic impact of RV integration in T lymphocytes from adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) patients 10 to 30 months after infusion of autologous, genetically corrected CD34(+) cells. Expression profiling on ex vivo T-cell bulk population revealed no difference with respect to healthy controls. To assess the effect of vector integration on gene expression at the single-cell level, primary T-cell clones were isolated from 2 patients. T-cell clones harbored either 1 (89.8%) or 2 (10.2%) vector copies per cell and displayed partial to full correction of ADA expression, purine metabolism, and T-cell receptor-driven functions. Analysis of RV integration sites indicated a high diversity in T-cell origin, consistently with the polyclonal T-cell receptor-Vbeta repertoire. Quantitative transcript analysis of 120 genes within a 200-kb window around RV integration sites showed modest (2.8- to 5.2-fold) dysregulation of 5.8% genes in 18.6% of the T-cell clones compared with controls. Nonetheless, affected clones maintained a stable phenotype and normal in vitro functions. These results confirm that RV-mediated gene transfer for ADA-SCID is safe, and provide crucial information for the development of future gene therapy protocols. The trials described herein have been registered at http://www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

  4. Cocaine- and amphetamine-regulated transcript peptide (CART) in the brain of zebra finch, Taeniopygia guttata: Organization, interaction with neuropeptide Y, and response to changes in energy status.

    Science.gov (United States)

    Singh, Omprakash; Kumar, Santosh; Singh, Uday; Kumar, Vinod; Lechan, Ronald M; Singru, Praful S

    2016-10-15

    Cocaine- and amphetamine-regulated transcript (CART) has emerged as a potent anorectic agent. CART is widely distributed in the brain of mammals, amphibians, and teleosts, but the relevant information in avian brain is not available. In birds, CART inhibits food intake, whereas neuropeptide Y (NPY), a well-known orexigenic peptide, stimulates it. How these neuropeptides interact in the brain to regulate energy balance is not known. We studied the distribution of CART-immunoreactivity in the brain of zebra finch, Taeniopygia guttata, its interaction with NPY, and their response to dynamic energy states. CART-immunoreactive fibers were found in the subpallium, hypothalamus, midbrain, and brainstem. Conspicuous CART-immunoreactive cells were observed in the bed nucleus of the stria terminalis, hypothalamic paraventricular, supraoptic, dorsomedial, infundibular (IN), lateral hypothalamic, Edinger-Westphal, and parabrachial nuclei. Hypothalamic sections of fed, fasted, and refed animals were immunostained with cFos, NPY, and CART antisera. Fasting dramatically increased cFos- and NPY-immunoreactivity in the IN, followed by rapid reduction by 2 hours and restoration to normal fed levels 6-10 hours after refeeding. CART-immunoreactive fibers in IN showed a significant reduction during fasting and upregulation with refeeding. Within the IN, double immunofluorescence revealed that 94 ± 2.1% of NPY-immunoreactive neurons were contacted by CART-immunoreactive fibers and 96 ± 2.8% NPY-immunoreactive neurons expressed cFos during fasting. Compared to controls, superfused hypothalamic slices of fasted birds treated with CART-peptide showed a significant reduction (P CART in the brain of T. guttata may perform several functions, and has a particularly important role in the hypothalamic regulation of energy homeostasis. J. Comp. Neurol. 524:3014-3041, 2016. © 2016 Wiley Periodicals, Inc.

  5. Evolution of transcriptional regulation in closely related bacteria

    Directory of Open Access Journals (Sweden)

    Tsoy Olga V

    2012-10-01

    Full Text Available Abstract Background The exponential growth of the number of fully sequenced genomes at varying taxonomic closeness allows one to characterize transcriptional regulation using comparative-genomics analysis instead of time-consuming experimental methods. A transcriptional regulatory unit consists of a transcription factor, its binding site and a regulated gene. These units constitute a graph which contains so-called “network motifs”, subgraphs of a given structure. Here we consider genomes of closely related Enterobacteriales and estimate the fraction of conserved network motifs and sites as well as positions under selection in various types of non-coding regions. Results Using a newly developed technique, we found that the highest fraction of positions under selection, approximately 50%, was observed in synvergon spacers (between consecutive genes from the same strand, followed by ~45% in divergon spacers (common 5’-regions, and ~10% in convergon spacers (common 3’-regions. The fraction of selected positions in functional regions was higher, 60% in transcription factor-binding sites and ~45% in terminators and promoters. Small, but significant differences were observed between Escherichia coli and Salmonella enterica. This fraction is similar to the one observed in eukaryotes. The conservation of binding sites demonstrated some differences between types of regulatory units. In E. coli, strains the interactions of the type “local transcriptional factor gene” turned out to be more conserved in feed-forward loops (FFLs compared to non-motif interactions. The coherent FFLs tend to be less conserved than the incoherent FFLs. A natural explanation is that the former imply functional redundancy. Conclusions A naïve hypothesis that FFL would be highly conserved turned out to be not entirely true: its conservation depends on its status in the transcriptional network and also from its usage. The fraction of positions under selection in

  6. Dynamic changes in binding of immunoglobulin heavy chain 3' regulatory region to protein factors during class switching.

    Science.gov (United States)

    Chatterjee, Sanjukta; Ju, Zhongliang; Hassan, Rabih; Volpi, Sabrina A; Emelyanov, Alexander V; Birshtein, Barbara K

    2011-08-19

    The 3' regulatory region (3' RR) of the Igh locus works at long distances on variable region (V(H)) and switch region (I) region promoters to initiate germ line (non-coding) transcription (GT) and promote class switch recombination (CSR). The 3' RR contains multiple elements, including enhancers (hs3a, hs1.2, hs3b, and hs4) and a proposed insulator region containing CTCF (CCCTC-binding factor) binding sites, i.e. hs5/6/7 and the downstream region ("38"). Notably, deletion of each individual enhancer (hs3a-hs4) has no significant phenotypic consequence, suggesting that the 3' RR has considerable structural flexibility in its function. To better understand how the 3' RR functions, we identified transcription factor binding sites and used chromatin immunoprecipitation (ChIP) assays to monitor their occupancy in splenic B cells that initiate GT and undergo CSR (LPS±IL4), are deficient in GT and CSR (p50(-/-)), or do not undergo CSR despite efficient GT (anti-IgM+IL4). Like 3' RR enhancers, hs5-7 and the 38 region were observed to contain multiple Pax5 binding sites (in addition to multiple CTCF sites). We found that the Pax5 binding profile to the 3' RR dynamically changed during CSR independent of the specific isotype to which switching was induced, and binding focused on hs1.2, hs4, and hs7. CTCF-associated and CTCF-independent cohesin interactions were also identified. Our observations are consistent with a scaffold model in which a platform of active protein complexes capable of facilitating GT and CSR can be formed by varying constellations of 3' RR elements.

  7. Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts

    Science.gov (United States)

    McCarthy, T. L.; Casinghino, S.; Mittanck, D. W.; Ji, C. H.; Centrella, M.; Rotwein, P.

    1996-01-01

    Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

  8. Predicting Virulence of Aeromonas Isolates Based-on Changes in Transcription of c-jun and c-fos in Human Tissue Culture Cells

    Science.gov (United States)

    Aims: To assess virulence of Aeromonas isolates based on the change in regulation of c-jun and c-fos in the human intestinal tissue culture cell line Caco-2. Methods and Results: Aeromonas cells were added to Caco-2 cells at approximately a one to one ratio. After 1, 2 and 3 ...

  9. Urinary Tissue Inhibitor of Metalloproteinase-2 (TIMP-2 • Insulin-Like Growth Factor-Binding Protein 7 (IGFBP7 Predicts Adverse Outcome in Pediatric Acute Kidney Injury.

    Directory of Open Access Journals (Sweden)

    Jens H Westhoff

    Full Text Available The G1 cell cycle inhibitors tissue inhibitor of metalloproteinase-2 (TIMP-2 and insulin-like growth factor-binding protein 7 (IGFBP7 have been identified as promising biomarkers for the prediction of adverse outcomes including renal replacement therapy (RRT and mortality in critically ill adult patients who develop acute kidney injury (AKI. However, the prognostic value of urinary TIMP-2 and IGFBP7 in neonatal and pediatric AKI for adverse outcome has not been investigated yet.The product of the urinary concentration of TIMP-2 and IGFBP7 ([TIMP-2]•[IGFBP7] was assessed by a commercially available immunoassay (NephroCheck™ in a prospective cohort study in 133 subjects aged 0-18 years including 46 patients with established AKI according to pRIFLE criteria, 27 patients without AKI (non-AKI group I and 60 apparently healthy neonates and children (non-AKI group II. AKI etiologies were: dehydration/hypovolemia (n = 7, hemodynamic instability (n = 7, perinatal asphyxia (n = 9, septic shock (n = 7, typical hemolytic-uremic syndrome (HUS; n = 5, interstitial nephritis (n = 5, vasculitis (n = 4, nephrotoxic injury (n = 1 and renal vein thrombosis (n = 1.When AKI patients were classified into pRIFLE criteria, 6/46 (13% patients fulfilled the criteria for the category "Risk", 13/46 (28% for "Injury", 26/46 (57% for "Failure" and 1/46 (2% for "Loss". Patients in the "Failure" stage had a median 3.7-fold higher urinary [TIMP-2]•[IGFBP7] compared to non-AKI subjects (P<0.001. When analyzed for AKI etiology, highest [TIMP-2]•[IGFBP7] values were found in patients with septic shock (P<0.001 vs. non-AKI I+II. Receiver operating characteristic (ROC curve analyses in the AKI group revealed good performance of [TIMP-2]•[IGFBP7] in predicting 30-day (area under the curve (AUC 0.79; 95% CI, 0.61-0.97 and 3-month mortality (AUC 0.84; 95% CI, 0.67-0.99 and moderate performance in predicting RRT (AUC 0.67; 95% CI, 0.50-0.84.This study shows that urinary [TIMP

  10. Insulin-like growth factor binding protein-3 is required for the regulation of rat oval cell proliferation and differentiation in the 2AAF/PHX model

    Directory of Open Access Journals (Sweden)

    Nicole C Steiger-Luther

    2010-02-01

    Full Text Available Nicole C Steiger-Luther1, Houda Darwiche1, Seh-Hoon Oh1, Jennifer M Williams1, Bryon E Petersen1,21Department of Pathology, Immunology and Laboratory Medicine, 2Program in Stem Cell Biology and Regenerative Medicine, College of Medicine, University of Florida, Gainesville, FL, USAAbstract: Oval cell-mediated liver regeneration is a highly complex process that involves the coordination of several signaling factors, chemokines and cytokines to allow for proper maintenance of the liver architecture. When hepatocyte proliferation is inhibited, an hepatic stem cell population, often referred to as “oval cells”, is activated to aid in liver regeneration. The function of insulin-like growth factor binding protein-3 (IGFBP-3 during this process of oval cell activation is of particular interest because it is produced in liver and has been shown to induce migration and differentiation of other stem cell populations both in vitro and in vivo. Additionally, IGFBP-3 production has been linked to the transforming growth factor-β (TGF-β superfamily, a pathway known to be induced during oval cell proliferation. In this study, we set out to determine whether IGFBP-3 plays a role in oval cell proliferation, migration and differentiation during this specific type of regeneration. Through activation of the oval cell-mediated liver regeneration in a rat model, we found that IGFBP-3 is elevated in the liver and serum of animals during peak days of oval cell activation and proliferation. Furthermore, in vitro assays found that WB-344 cells, a liver stem cell line similar to oval cells, were induced to migrate in the presence of IGFBP-3. When expression of IGFBP-3 was knocked down during oval cell activation in vivo, we found that oval cell proliferation was increased and observed the appearance of numerous atypical ductular structures, which were OV-6 and Ki67-positive. Finally, quantitative realtime PCR analysis of liver tissue from IGFBP-3 small interfering

  11. Cervical length and phosphorilated insulin like growth factor binding protein-1 as the predictors of spontaneus preterm delivery in symptomatic women

    Directory of Open Access Journals (Sweden)

    Hadži-Lega Marija

    2014-07-01

    Full Text Available Objective: To assess the combined use of cervical length and cervical phosphorylated insulin-like growth factor binding protein-1 (phIGFBP-1 in the prediction of preterm delivery in symptomatic women in next 14 days. Methods: Cervical length was prospectively measured in 58 consecutive singleton pregnancies with intact membranes and regular contractions at 24-36 weeks, and phIGFBP-1 was assessed. Demographic data was evaluated(history of previous preterm delivery, history of spontaneous abortion, parity, BMI, maternal age, Orthodox or Muslims. Results: Values of all variables were evaluated (demographic data, cervical length and values ofphIGFBP-1 alone and in combination with cervical length of ≤ 15 mm and more than 15 mm. In women with cervical length less than 15 mm/ /phIGFPB-1 was positive in 30 patients(22 of them delivered in 14 days. In women with cervical length less than 15 mm/phIGFBP-1was positive in 9 of delivered pregnant women in 14 days. In women with cervical length less than 25 mm/phIGFBP-1was positive in 26 patients (2 of them delivered in 14 days. In patients with cervical length more than 25 mm/ph IGFBP-1 was positive in 4 patients (2 of them delivered in 14 days. With logistic regression we confirmed that with OR 0.117 and CI 95% (0.046-0.295 and p < 0.01 odds for preterm birth among patients with negative phIGFBP-1 is 0.117 lower than the odds for preterm birth among patients with positive test results. With same test for p = 0.009 (p < 0.01 we confirmed with OR and CI 95% (0.06 to 0.671 that cervical length less than 25 mm is good predictor of preterm delivery with symptomatic patients. Probability for delivery in the following 14 days with patients with positive phIGFBP-1 and cervical length ≤ 15 mm is 0.88 or probability for not delivering in those patients is 0.12. In 88% patients with positive phIGFBP-1 and cervical length ≤ 15 mm will deliver in the following 14 days. Conclusions: In symptomatic women phIGFBP-1

  12. Expression pattern and transcriptional regulatory mechanism of noxa gene in grass carp (Ctenopharyngodon idella).

    Science.gov (United States)

    Pei, Yongyan; Lu, Xiaonan; He, Libo; Wang, Hao; Zhang, Aidi; Li, Yongming; Huang, Rong; Liao, Lanjie; Zhu, Zuoyan; Wang, Yaping

    2015-12-01

    Noxa, a pro-apoptotic protein, plays an important role in cell apoptosis. The researches about noxa gene were concentrated in mammalians, whereas the role and transcriptional regulatory mechanism of noxa in fish were still unclear. In this study, the expression pattern and transcriptional regulatory mechanism of noxa gene in grass carp were analyzed. Noxa was constitutively expressed in all the examined tissues but the relative expression level differed. After exposure to grass carp reovirus (GCRV), mRNA expression level of noxa was down-regulated at the early phase whereas up-regulated at the late phase of infection. Luciferase assays showed that the promoter region -867 ∼ +107 of noxa had high activity and the region -678 ∼ -603 was important in the response to GCRV infection. By deleting the predicted transcription factor binding sites, transcription factors FOXO1 and CEBPβ were found important for noxa in response to GCRV infection. Moreover, the noxa promoter was biotin-labeled and incubated with nuclear extracts from GCRV infected cells. Mass spectrometry analysis showed that transcription factors FOXO1 and CEBPβ were also enriched in the combined proteins. Therefore, the results suggested that transcription factors FOXO1 and CEBPβ may play an important role in the regulation of noxa. Our study would provide new insight into the transcriptional regulatory mechanism of noxa in teleost fish.

  13. How much is enough? Modulation of dose-response curve for steroid receptor-regulated gene expression by changing concentrations of transcription factor.

    Science.gov (United States)

    Simons, S Stoney

    2006-01-01

    The position of the dose-response curve for steroid-regulated gene expression determines how much variation in response will accompany the normal physiological changes in circulating steroid. Over the last several years, it has become clear that the concentration of steroid hormone required for half-maximal induction or repression by a given receptor-steroid complex, which is normally called the EC50, is not constant for all responsive genes. Thus, the position of the dose-response curve can change so that a single concentration of steroid produces very different percentages of maximal activity. This, in turn, allows for the differential expression of genes by a common steroid hormone concentration during development, differentiation, and homeostasis. Here we review the variety of factors that influence the EC50 and position of the dose-response curve for steroid hormone receptors, discuss what is known about the mechanisms, and highlight promising areas for future research.

  14. Transcriptional responses in Lactococcus lactis subsp. cremoris to the changes in oxygen and redox potential during milk acidification

    DEFF Research Database (Denmark)

    Larsen, Nadja; Werner, Birgit Brøsted; Jespersen, Lene

    2016-01-01

    genes were detected during aerobic reduction phase. Upregulated genes were implicated in lactose utilization, glycogen biosynthesis, amino sugar metabolism, oxidation-reduction, pyrimidine biosynthesis and DNA integration processes. Genes of purine nucleotide biosynthesis and genes encoding amino acid...... responses to oxygen depletion and the changes of redox potential with the fermentation kinetics and clarification of molecular factors specifically expressed in milk which might be essential for bacterial performance and the final quality of cheeses....

  15. ATF6 as a Transcription Activator of the Endoplasmic Reticulum Stress Element: Thapsigargin Stress-Induced Changes and Synergistic Interactions with NF-Y and YY1

    OpenAIRE

    Li, Mingqing; Baumeister, Peter; Roy, Binayak; Phan, Trevor; Foti, Dolly; Luo, Shengzhan; Lee, Amy S.

    2000-01-01

    ATF6, a member of the leucine zipper protein family, can constitutively induce the promoter of glucose-regulated protein (grp) genes through activation of the endoplasmic reticulum (ER) stress element (ERSE). To understand the mechanism of grp78 induction by ATF6 in cells subjected to ER calcium depletion stress mediated by thapsigargin (Tg) treatment, we discovered that ATF6 itself undergoes Tg stress-induced changes. In nonstressed cells, ATF6, which contains a putative short transmembrane ...

  16. In silico detection of sequence variations modifying transcriptional regulation.

    Directory of Open Access Journals (Sweden)

    Malin C Andersen

    2008-01-01

    Full Text Available Identification of functional genetic variation associated with increased susceptibility to complex diseases can elucidate genes and underlying biochemical mechanisms linked to disease onset and progression. For genes linked to genetic diseases, most identified causal mutations alter an encoded protein sequence. Technological advances for measuring RNA abundance suggest that a significant number of undiscovered causal mutations may alter the regulation of gene transcription. However, it remains a challenge to separate causal genetic variations from linked neutral variations. Here we present an in silico driven approach to identify possible genetic variation in regulatory sequences. The approach combines phylogenetic footprinting and transcription factor binding site prediction to identify variation in candidate cis-regulatory elements. The bioinformatics approach has been tested on a set of SNPs that are reported to have a regulatory function, as well as background SNPs. In the absence of additional information about an analyzed gene, the poor specificity of binding site prediction is prohibitive to its application. However, when additional data is available that can give guidance on which transcription factor is involved in the regulation of the gene, the in silico binding site prediction improves the selection of candidate regulatory polymorphisms for further analyses. The bioinformatics software generated for the analysis has been implemented as a Web-based application system entitled RAVEN (regulatory analysis of variation in enhancers. The RAVEN system is available at http://www.cisreg.ca for all researchers interested in the detection and characterization of regulatory sequence variation.

  17. SigmoID: a user-friendly tool for improving bacterial genome annotation through analysis of transcription control signals.

    Science.gov (United States)

    Nikolaichik, Yevgeny; Damienikan, Aliaksandr U

    2016-01-01

    The majority of bacterial genome annotations are currently automated and based on a 'gene by gene' approach. Regulatory signals and operon structures are rarely taken into account which often results in incomplete and even incorrect gene function assignments. Here we present SigmoID, a cross-platform (OS X, Linux and Windows) open-source application aiming at simplifying the identification of transcription regulatory sites (promoters, transcription factor binding sites and terminators) in bacterial genomes and providing assistance in correcting annotations in accordance with regulatory information. SigmoID combines a user-friendly graphical interface to well known command line tools with a genome browser for visualising regulatory elements in genomic context. Integrated access to online databases with regulatory information (RegPrecise and RegulonDB) and web-based search engines speeds up genome analysis and simplifies correction of genome annotation. We demonstrate some features of SigmoID by constructing a series of regulatory protein binding site profiles for two groups of bacteria: Soft Rot Enterobacteriaceae (Pectobacterium and Dickeya spp.) and Pseudomonas spp. Furthermore, we inferred over 900 transcription factor binding sites and alternative sigma factor promoters in the annotated genome of Pectobacterium atrosepticum. These regulatory signals control putative transcription units covering about 40% of the P. atrosepticum chromosome. Reviewing the annotation in cases where it didn't fit with regulatory information allowed us to correct product and gene names for over 300 loci.

  18. SigmoID: a user-friendly tool for improving bacterial genome annotation through analysis of transcription control signals

    Directory of Open Access Journals (Sweden)

    Yevgeny Nikolaichik

    2016-05-01

    Full Text Available The majority of bacterial genome annotations are currently automated and based on a ‘gene by gene’ approach. Regulatory signals and operon structures are rarely taken into account which often results in incomplete and even incorrect gene function assignments. Here we present SigmoID, a cross-platform (OS X, Linux and Windows open-source application aiming at simplifying the identification of transcription regulatory sites (promoters, transcription factor binding sites and terminators in bacterial genomes and providing assistance in correcting annotations in accordance with regulatory information. SigmoID combines a user-friendly graphical interface to well known command line tools with a genome browser for visualising regulatory elements in genomic context. Integrated access to online databases with regulatory information (RegPrecise and RegulonDB and web-based search engines speeds up genome analysis and simplifies correction of genome annotation. We demonstrate some features of SigmoID by constructing a series of regulatory protein binding site profiles for two groups of bacteria: Soft Rot Enterobacteriaceae (Pectobacterium and Dickeya spp. and Pseudomonas spp. Furthermore, we inferred over 900 transcription factor binding sites and alternative sigma factor promoters in the annotated genome of Pectobacterium atrosepticum. These regulatory signals control putative transcription units covering about 40% of the P. atrosepticum chromosome. Reviewing the annotation in cases where it didn’t fit with regulatory information allowed us to correct product and gene names for over 300 loci.

  19. The WRKY57 Transcription Factor Affects the Expression of Jasmonate ZIM-Domain Genes Transcriptionally to Compromise Botrytis cinerea Resistance.

    Science.gov (United States)

    Jiang, Yanjuan; Yu, Diqiu

    2016-08-01

    Although necrotrophic pathogens cause many devastating plant diseases, our understanding of the plant defense response to them is limited. Here, we found that loss of function of WRKY57 enhanced the resistance of Arabidopsis (Arabidopsis thaliana) against Botrytis cinerea infection. Further investigation suggested that the negative regulation of WRKY57 against B cinerea depends on the jasmonic acid (JA) signaling pathway. Chromatin immunoprecipitation experiments revealed that WRKY57 directly binds to the promoters of JASMONATE ZIM-DOMAIN1 (JAZ1) and JAZ5, encoding two important repressors of the JA signaling pathway, and activates their transcription. In vivo and in vitro experiments demonstrated that WRKY57 interacts with nuclear-encoded SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2. Further experiments display that the same domain, the VQ motif, of SIB1 and SIB2 interact with WRKY33 and WRKY57. Moreover, transient transcriptional activity assays confirmed that WRKY57 and WRKY33 competitively regulate JAZ1 and JAZ5, SIB1 and SIB2 further enhance these competitions of WRKY57 to WRKY33. Therefore, coordinated regulation of Arabidopsis against B cinerea by transcription activators and repressors would benefit plants by allowing fine regulation of defense.

  20. Mapping Yeast Transcriptional Networks

    OpenAIRE

    Hughes, Timothy R; de Boer, Carl G.

    2013-01-01

    The term “transcriptional network” refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face....

  1. Transcriptional changes in epigenetic modifiers associated with gene silencing in the intestine of the sea cucumber,Apostichopus japonicus (Selenka), during aestivation

    Institute of Scientific and Technical Information of China (English)

    WANG Tianming; YANG Hongsheng; ZHAO Huan; CHEN Muyan; WANG Bing

    2011-01-01

    The sea cucumber,Apostichopusjaponicus,undergoes aestivation to improve survival during periods of high-temperature.During aestivation,the metabolic rate is depressed to reduce the consumption of reserved energy.We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber.We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers.The expression of three genes involved in DNA methylation and chromatin remodeling (DNA (cytosine-5)-methyltransferase 1,Methyl-CpG-binding domain protein 2),and Chromodomain-helicase-DNA-binding protein 5) was significantly higher during aestivation (Days 20 and 40).Similarly,we observed an increase in the expression of genes involved in histone acetylation (Histone deacetylase 3) and Histone-binding protein RBBP4) during the early (Days 5 and 10) and late phases (Days 20 and 40) of aestivation.There was no change in the expression of KAT2B,a histone acetyltransferase.However,the expression of histone methylation associated modifiers (Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5) was significantly higher after 5 d in the aestivating group.The results suggest that the expression of epigenetic modifiers involved in DNA methylation,chromatin remodeling,histone acetylation,and histone methylation is upregulated during aestivation.We hypothesize that these changes regulate global gene silencing during aestivation in A.japonicus.

  2. Transcriptional Changes in nAChRs, Interactive Proteins and P450s in Locusta migratoria manilensis (Orthoptera: Acrididae) CNS in Response to High and Low Oral Doses of Imidacloprid.

    Science.gov (United States)

    Wang, Xin; Sun, Huahua; Zhang, Yixi; Liu, Chuanjun; Liu, Zewen

    2015-01-01

    The insect central nervous system (CNS) is the target for many insecticides, and changes in transcript levels could be expected after insecticide applications. In this study, differentially expressed genes in the locust (Locusta migratoria manilensis) CNS in response to imidacloprid treatments at low dose (LD, 10% mortality) and high dose (HD, 80% mortality) were identified. Two nicotine acetylcholine receptor (nAChR) subunits genes and 18 interacting protein genes were regulated at LD, and only one nAChR subunit gene and 11 interacting proteins were regulated at HD. Among the 110 annotated P450 unigenes, 43 unigenes were regulated at LD and 34 unigenes were regulated at HD. Most of the differentially expressed P450 unigenes were mapped to CYP4, in which most unigenes were upregulated at LD, but downregulated at HD. Totally, the numbers and regulation levels of the regulated genes were more at LD than that at HD. Seventeen unigenes were selected to test their expression changes following insecticide treatments by qRT-PCR, in which the changes in more than half of the selected genes were verified. The results revealed the variation in the response of locusts to different insecticide pressure, such as different doses.

  3. Demethylation of oligogalacturonides by FaPE1 in the fruits of the wild strawberry Fragaria vesca triggers metabolic and transcriptional changes associated with defence and development of the fruit.

    Science.gov (United States)

    Osorio, Sonia; Bombarely, Aureliano; Giavalisco, Patrick; Usadel, Björn; Stephens, Camilla; Aragüez, Irene; Medina-Escobar, Nieves; Botella, Miguel A; Fernie, Alisdair R; Valpuesta, Victoriano

    2011-05-01

    Ectopic expression of the strawberry (Fragaria×ananassa) gene FaPE1 encoding pectin methyl esterase produced in the wild species Fragaria vesca partially demethylated oligogalacturonides (OGAs), which conferred partial resistance of ripe fruits to the fungus Botrytis cinerea. Analyses of metabolic and transcriptional changes in the receptacle of the transgenic fruits revealed channelling of metabolites to aspartate and aromatic amino acids as well as phenolics, flavanones, and sesquiterpenoids, which was in parallel with the increased expression of some genes related to plant defence. The results illustrate the changes associated with resistance to B. cinerea in the transgenic F. vesca. These changes were accompanied by a significant decrease in the auxin content of the receptacle of the ripe fruits of transgenic F. vesca, and enhanced expression of some auxin-repressed genes. The role of these OGAs in fruit development was revealed by the larger size of the ripe fruits in transgenic F. vesca. When taken together these results show that in cultivated F. ananassa FaPE1 participates in the de-esterification of pectins and the generation of partially demethylated OGAs, which might reinforce the plant defence system and play an active role in fruit development.

  4. Massively Systematic Transcript End Readout (MASTER): Transcription Start Site Selection, Transcriptional Slippage, and Transcript Yields

    Science.gov (United States)

    Vvedenskaya, Irina O.; Zhang, Yuanchao; Goldman, Seth R.; Valenti, Anna; Visone, Valeria; Taylor, Deanne M.; Ebright, Richard H.; Nickels, Bryce E.

    2015-01-01

    SUMMARY We report the development of a next-generation sequencing-based technology that entails construction of a DNA library comprising up to at least 47 (~16,000) bar-coded sequences, production of RNA transcripts, and analysis of transcript ends and transcript yields ("massively systematic transcript end readout," MASTER). Using MASTER, we define full inventories of transcription start sites ("TSSomes") of Escherichia coli RNA polymerase for initiation at a consensus core promoter in vitro and in vivo, we define the TSS-region DNA-sequence determinants for TSS selection, reiterative initiation ("slippage synthesis"), and transcript yield, and we define effects of DNA topology and NTP concentration. The results reveal that slippage synthesis occurs from the majority of TSS-region DNA sequences and that TSS-region DNA sequences have profound, up to 100-fold, effects on transcript yield. The results further reveal that TSSomes depend on DNA topology, consistent with the proposal that TSS selection involves transcription-bubble expansion ("scrunching") and transcription-bubble contraction ("anti-scrunching"). PMID:26626484

  5. Massively Systematic Transcript End Readout, "MASTER": Transcription Start Site Selection, Transcriptional Slippage, and Transcript Yields.

    Science.gov (United States)

    Vvedenskaya, Irina O; Zhang, Yuanchao; Goldman, Seth R; Valenti, Anna; Visone, Valeria; Taylor, Deanne M; Ebright, Richard H; Nickels, Bryce E

    2015-12-17

    We report the development of a next-generation sequencing-based technology that entails construction of a DNA library comprising up to at least 4(7) (∼ 16,000) barcoded sequences, production of RNA transcripts, and analysis of transcript ends and transcript yields (massively systematic transcript end readout, "MASTER"). Using MASTER, we define full inventories of transcription start sites ("TSSomes") of Escherichia coli RNA polymerase for initiation at a consensus core promoter in vitro and in vivo; we define the TSS-region DNA sequence determinants for TSS selection, reiterative initiation ("slippage synthesis"), and transcript yield; and we define effects of DNA topology and NTP concentration. The results reveal that slippage synthesis occurs from the majority of TSS-region DNA sequences and that TSS-region DNA sequences have profound, up to 100-fold, effects on transcript yield. The results further reveal that TSSomes depend on DNA topology, consistent with the proposal that TSS selection involves transcription-bubble expansion ("scrunching") and transcription-bubble contraction ("anti-scrunching").

  6. A Nonnatural Transcriptional Coactivator

    Science.gov (United States)

    Nyanguile, Origene; Uesugi, Motonari; Austin, David J.; Verdine, Gregory L.

    1997-12-01

    In eukaryotes, sequence-specific DNA-binding proteins activate gene expression by recruiting the transcriptional apparatus and chromatin remodeling proteins to the promoter through protein-protein contacts. In many instances, the connection between DNA-binding proteins and the transcriptional apparatus is established through the intermediacy of adapter proteins known as coactivators. Here we describe synthetic molecules with low molecular weight that act as transcriptional coactivators. We demonstrate that a completely nonnatural activation domain in one such molecule is capable of stimulating transcription in vitro and in vivo. The present strategy provides a means of gaining external control over gene activation through intervention using small molecules.

  7. Combinatorial Regulation in Yeast Transcription Networks

    Science.gov (United States)

    Li, Hao

    2006-03-01

    Yeast has evolved a complex network to regulate its transcriptional program in response to changes in environment. It is quite common that in response to an external stimulus, several transcription factors will be activated and they work in combinations to control different subsets of genes in the genome. We are interested in how the promoters of genes are designed to integrate signals from multiple transcription factors and what are the functional and evolutionary constraints. To answer how, we have developed a number of computational algorithms to systematically map the binding sites and target genes of transcription factors using sequence and gene expression data. To analyze the functional constraints, we have employed mechanistic models to study the dynamic behavior of genes regulated by multiple factors. We have also developed methods to trace the evolution of transcriptional networks via comparative analysis of multiple species.

  8. The Transcription Factor Encyclopedia

    DEFF Research Database (Denmark)

    Yusuf, Dimas; Butland, Stefanie L; Swanson, Magdalena I

    2012-01-01

    ABSTRACT: Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130...

  9. The transcriptional landscape

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2011-01-01

    The application of new and less biased methods to study the transcriptional output from genomes, such as tiling arrays and deep sequencing, has revealed that most of the genome is transcribed and that there is substantial overlap of transcripts derived from the two strands of DNA. In protein codi...

  10. The Global Regulatory Architecture of Transcription during the Caulobacter Cell Cycle

    Science.gov (United States)

    Zhou, Bo; Schrader, Jared M.; Kalogeraki, Virginia S.; Abeliuk, Eduardo; Dinh, Cong B.; Pham, James Q.; Cui, Zhongying Z.; Dill, David L.; McAdams, Harley H.; Shapiro, Lucy

    2015-01-01

    Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5′ RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle. PMID:25569173

  11. The global regulatory architecture of transcription during the Caulobacter cell cycle.

    Directory of Open Access Journals (Sweden)

    Bo Zhou

    2015-01-01

    Full Text Available Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs and a DNA methyltransferase. Using a modified global 5' RACE protocol, we globally mapped transcription start sites (TSSs at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle.

  12. Modeling transcriptional networks regulating secondary growth and wood formation in forest trees.

    Science.gov (United States)

    Liu, Lijun; Filkov, Vladimir; Groover, Andrew

    2014-06-01

    The complex interactions among the genes that underlie a biological process can be modeled and presented as a transcriptional network, in which genes (nodes) and their interactions (edges) are shown in a graphical form similar to a wiring diagram. A large number of genes have been identified that are expressed during the radial woody growth of tree stems (secondary growth), but a comprehensive understanding of how these genes interact to influence woody growth is currently lacking. Modeling transcriptional networks has recently been made tractable by next-generation sequencing-based technologies that can comprehensively catalog gene expression and transcription factor-binding genome-wide, but has not yet been extensively applied to undomesticated tree species or woody growth. Here we discuss basic features of transcriptional networks, approaches for modeling biological networks, and examples of biological network models developed for forest trees to date. We discuss how transcriptional network research is being developed in the model forest tree genus, Populus, and how this research area can be further developed and applied. Transcriptional network models for forest tree secondary growth and wood formation could ultimately provide new predictive models to accelerate hypothesis-driven research and develop new breeding applications.

  13. NFAT5 regulates transcription of the mouse telomerase reverse transcriptase gene

    Energy Technology Data Exchange (ETDEWEB)

    Fujiki, Tsukasa; Udono, Miyako; Kotake, Yojiro; Yamashita, Makiko; Shirahata, Sanetaka; Katakura, Yoshinori, E-mail: katakura.yoshinori.528@m.kyushu-u.ac.jp

    2010-12-10

    We aimed to clarify the transcription-regulation mechanisms of the mouse telomerase reverse transcriptase gene (mTERT). First, we searched for the promoter region required for transcriptional activation of mTERT and identified an enhancer cis-element (named mTERT-EE) located between - 200 and - 179 bp of the mouse TERT gene (mTERT). EMSA results suggested that nuclear factor of activated T cells (NFAT) member proteins bind to mTERT-EE. We then identified NFAT5 as the factor binding to mTERT-EE and found that it activates the transcription of the mTERT core promoter. The results that siRNA directed against NFAT5 significantly reduced mTERT expression and mTERT core promoter activity and that the expressions of NFAT5 and mTERT were well correlated in various mouse tissues except liver suggest that NFAT5 dominantly and directly regulates mTERT expression. To clarify their functionality further, we investigated the effect of hypertonic stress, a known stimulus affecting the expression and transcriptional activity of NFAT5, on mTERT expression. The result indicated that hypertonic stress activates mTERT transcription via the activation and recruitment of NFAT5 to the mTERT promoter. These results provide useful information about the transcription-regulation mechanisms of mTERT.

  14. Control of transcription by cell size.

    Directory of Open Access Journals (Sweden)

    Chia-Yung Wu

    Full Text Available Cell size increases significantly with increasing ploidy. Differences in cell size and ploidy are associated with alterations in gene expression, although no direct connection has been made between cell size and transcription. Here we show that ploidy-associated changes in gene expression reflect transcriptional adjustment to a larger cell size, implicating cellular geometry as a key parameter in gene regulation. Using RNA-seq, we identified genes whose expression was altered in a tetraploid as compared with the isogenic haploid. A significant fraction of these genes encode cell surface proteins, suggesting an effect of the enlarged cell size on the differential regulation of these genes. To test this hypothesis, we examined expression of these genes in haploid mutants that also produce enlarged size. Surprisingly, many genes differentially regulated in the tetraploid are identically regulated in the enlarged haploids, and the magnitude of change in gene expression correlates with the degree of size enlargement. These results indicate a causal relationship between cell size and transcription, with a size-sensing mechanism that alters transcription in response to size. The genes responding to cell size are enriched for those regulated by two mitogen-activated protein kinase pathways, and components in those pathways were found to mediate size-dependent gene regulation. Transcriptional adjustment to enlarged cell size could underlie other cellular changes associated with polyploidy. The causal relationship between cell size and transcription suggests that cell size homeostasis serves a regulatory role in transcriptome maintenance.

  15. Using both strands: The fundamental nature of antisense transcription.

    Science.gov (United States)

    Murray, Struan C; Mellor, Jane

    2016-01-01

    Non-coding transcription across the antisense strands of genes is an abundant, pervasive process in eukaryotes from yeast to humans, however its biological function remains elusive. Here, we provide commentary on a recent study of ours, which demonstrates a genome-wide role for antisense transcription: establishing a unique, dynamic chromatin architecture over genes. Antisense transcription increases the level of nucleosome occupancy and histone acetylation at the promoter and body of genes, without necessarily modulating the level of protein-coding sense transcription. It is also associated with high levels of histone turnover. By allowing genes to sample a wider range of chromatin configurations, antisense transcription could serve to make genes more sensitive to changing signals, priming them for responses to developmental programs or stressful cellular environments. Given the abundance of antisense transcription and the breadth of these chromatin changes, we propose that antisense transcription represents a fundamental, canonical feature of eukaryotic genes.

  16. The Chromatin Remodelling Enzymes SNF2H and SNF2L Position Nucleosomes adjacent to CTCF and Other Transcription Factors.

    Directory of Open Access Journals (Sweden)

    Nicola Wiechens

    2016-03-01

    Full Text Available Within the genomes of metazoans, nucleosomes are highly organised adjacent to the binding sites for a subset of transcription factors. Here we have sought to investigate which chromatin remodelling enzymes are responsible for this. We find that the ATP-dependent chromatin remodelling enzyme SNF2H plays a major role organising arrays of nucleosomes adjacent to the binding sites for the architectural transcription factor CTCF sites and acts to promote CTCF binding. At many other factor binding sites SNF2H and the related enzyme SNF2L contribute to nucleosome organisation. The action of SNF2H at CTCF sites is functionally important as depletion of CTCF or SNF2H affects transcription of a common group of genes. This suggests that chromatin remodelling ATPase's most closely related to the Drosophila ISWI protein contribute to the function of many human gene regulatory elements.

  17. The Chromatin Remodelling Enzymes SNF2H and SNF2L Position Nucleosomes adjacent to CTCF and Other Transcription Factors.

    Science.gov (United States)

    Wiechens, Nicola; Singh, Vijender; Gkikopoulos, Triantaffyllos; Schofield, Pieta; Rocha, Sonia; Owen-Hughes, Tom

    2016-03-01

    Within the genomes of metazoans, nucleosomes are highly organised adjacent to the binding sites for a subset of transcription factors. Here we have sought to investigate which chromatin remodelling enzymes are responsible for this. We find that the ATP-dependent chromatin remodelling enzyme SNF2H plays a major role organising arrays of nucleosomes adjacent to the binding sites for the architectural transcription factor CTCF sites and acts to promote CTCF binding. At many other factor binding sites SNF2H and the related enzyme SNF2L contribute to nucleosome organisation. The action of SNF2H at CTCF sites is functionally important as depletion of CTCF or SNF2H affects transcription of a common group of genes. This suggests that chromatin remodelling ATPase's most closely related to the Drosophila ISWI protein contribute to the function of many human gene regulatory elements.

  18. DNA supercoiling during transcription

    Science.gov (United States)

    Ma, Jie; Wang, Michelle D.

    2017-01-01

    The twin-supercoiled-domain model describes how transcription can drive DNA supercoiling, and how DNA supercoiling, in turn plays an important role in regulating gene transcription. In vivo and in vitro experiments have disclosed many details of the complex interactions in this relationship, and recently new insights have been gained with the help of genome-wide DNA supercoiling mapping techniques and single molecule methods. This review summarizes the general mechanisms of the interplay between DNA supercoiling and transcription, considers the biological implications, and focuses on recent important discoveries and technical advances in this field. We highlight the significant impact of DNA supercoiling in transcription, but also more broadly in all processes operating on DNA.

  19. Mutations and binding sites of human transcription factors

    KAUST Repository

    Kamanu, Frederick Kinyua

    2012-06-01

    Mutations in any genome may lead to phenotype characteristics that determine ability of an individual to cope with adaptation to environmental challenges. In studies of human biology, among the most interesting ones are phenotype characteristics that determine responses to drug treatments, response to infections, or predisposition to specific inherited diseases. Most of the research in this field has been focused on the studies of mutation effects on the final gene products, peptides, and their alterations. Considerably less attention was given to the mutations that may affect regulatory mechanism(s) of gene expression, although these may also affect the phenotype characteristics. In this study we make a pilot analysis of mutations observed in the regulatory regions of 24,667 human RefSeq genes. Our study reveals that out of eight studied mutation types, insertions are the only one that in a statistically significant manner alters predicted transcription factor binding sites (TFBSs). We also find that 25 families of TFBSs have been altered by mutations in a statistically significant manner in the promoter regions we considered. Moreover, we find that the related transcription factors are, for example, prominent in processes related to intracellular signaling; cell fate; morphogenesis of organs and epithelium; development of urogenital system, epithelium, and tube; neuron fate commitment. Our study highlights the significance of studying mutations within the genes regulatory regions and opens way for further detailed investigations on this topic, particularly on the downstream affected pathways. 2012 Kamanu, Medvedeva, Schaefer, Jankovic, Archer and Bajic.

  20. Nuclear Actin in Development and Transcriptional Reprogramming.

    Science.gov (United States)

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

    Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin's roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation.

  1. The regulation of transcription in memory consolidation.

    Science.gov (United States)

    Alberini, Cristina M; Kandel, Eric R

    2014-12-04

    De novo transcription of DNA is a fundamental requirement for the formation of long-term memory. It is required during both consolidation and reconsolidation, the posttraining and postreactivation phases that change the state of the memory from a fragile into a stable and long-lasting form. Transcription generates both mRNAs that are translated into proteins, which are necessary for the growth of new synaptic connections, as well as noncoding RNA transcripts that have regulatory or effector roles in gene expression. The result is a cascade of events that ultimately leads to structural changes in the neurons that mediate long-term memory storage. The de novo transcription, critical for synaptic plasticity and memory formation, is orchestrated by chromatin and epigenetic modifications. The complexity of transcription regulation, its temporal progression, and the effectors produced all contribute to the flexibility and persistence of long-term memory formation. In this article, we provide an overview of the mechanisms contributing to this transcriptional regulation underlying long-term memory formation.

  2. The synergetic effects of two CCAAT boxes in Aspergillus niger glaA gene promoter on activation of PglaA transcription

    Institute of Scientific and Technical Information of China (English)

    ZHU; Xingguo; WANG; H.; M.; QIU; Runxiang; LIU; Li; DONG; Zh

    2004-01-01

    EMSA and footprinting analyses have revealed that the 489-414 bp and the 390-345 bp (designated DC and PC respectively) upstream of the Aspergillus niger T21 glaA gene were bound by one protein factor in the A. niger T21 whole cell extract. Both DC and PC contained CCAAT pentanucleotides. The functions of DC and PC in regulation of expression of glucoamylase (GLA) were studied. CCAAT pentanucleotides were replaced with CGTAA and the mutated DNA fragments DCm and PCm lost the binding activities of protein factors in vitro. In vivo when either DC or PC was mutated or the relative orientations between them were changed on the PglaA, the transcriptional activity of PglaA decreased to a basal level. Introduction of multi-copies of DC into the original site at the PglaA in A. niger T21 decreased the expression of endogenous GLA expression and the exogenous reporter E. coli uidA gene introduced under the PglaA promoter, while having no effect on the uidA gene under the control of PgpdA. EMSA revealed that the levels of the specific DNA-binding protein factors in the transformants maintained the same meaning that introduction of multi-copies of DC caused the titration effect. AnghapC gene was cloned from A. niger T21 cDNA and introduced into the DC multi-copied strains. The expression of AnghapC improved the expression of the endogenous GLA and the exogenous gene controlled by PglaA. These results showed that both the CCAAT pentanucleotides were necessary for DC and PC binding to the protein factors, and the simultaneous binding of DC and PC to the protein was necessary for promoting the transcriptional activity of PglaA. AngHapC was the specific positive trans-acting protein factor binding to DC.

  3. Distinguishing the Transcription Regulation Patterns in Promoters of Human Genes with Different Function or Evolutionary Age

    KAUST Repository

    Alam, Tanvir

    2012-07-01

    Distinguishing transcription regulatory patterns of different gene groups is a common problem in various bioinformatics studies. In this work we developed a methodology to deal with such a problem based on machine learning techniques. We applied our method to two biologically important problems related to detecting a difference in transcription regulation of: a/ protein-coding and long non-coding RNAs (lncRNAs) in human, as well as b/ a difference between primate-specific and non-primate-specific long non-coding RNAs. Our method is capable to classify RNAs using various regulatory features of genes that transcribe into these RNAs, such as nucleotide frequencies, transcription factor binding sites, de novo sequence motifs, CpG islands, repetitive elements, histone modification marks, and others. Ten-fold cross-validation tests suggest that our model can distinguish protein-coding and non-coding RNAs with accuracy above 80%. Twenty-fold cross-validation tests suggest that our model can distinguish primate-specific from non-primate-specific promoters of lncRNAs with accuracy above 80%. Consequently, we can hypothesize that transcription of the groups of genes mentioned above are regulated by different mechanisms. Feature selection techniques allowed us to reduce the number of features significantly while keeping the accuracy around 80%. Consequently, we can conclude that selected features play significant role in transcription regulation of coding and non-coding genes, as well as primate-specific and non-primate-specific lncRNA genes.

  4. Transcriptional and post-transcriptional regulation of a NAC1 transcription factor in Medicago truncatula roots.

    Science.gov (United States)

    D'haeseleer, Katrien; Den Herder, Griet; Laffont, Carole; Plet, Julie; Mortier, Virginie; Lelandais-Brière, Christine; De Bodt, Stefanie; De Keyser, Annick; Crespi, Martin; Holsters, Marcelle; Frugier, Florian; Goormachtig, Sofie

    2011-08-01

    • Legume roots develop two types of lateral organs, lateral roots and nodules. Nodules develop as a result of a symbiotic interaction with rhizobia and provide a niche for the bacteria to fix atmospheric nitrogen for the plant. • The Arabidopsis NAC1 transcription factor is involved in lateral root formation, and is regulated post-transcriptionally by miRNA164 and by SINAT5-dependent ubiquitination. We analyzed in Medicago truncatula the role of the closest NAC1 homolog in lateral root formation and in nodulation. • MtNAC1 shows a different expression pattern in response to auxin than its Arabidopsis homolog and no changes in lateral root number or nodulation were observed in plants affected in MtNAC1 expression. In addition, no interaction was found with SINA E3 ligases, suggesting that post-translational regulation of MtNAC1 does not occur in M. truncatula. Similar to what was found in Arabidopsis, a conserved miR164 target site was retrieved in MtNAC1, which reduced protein accumulation of a GFP-miR164 sensor. Furthermore, miR164 and MtNAC1 show an overlapping expression pattern in symbiotic nodules, and overexpression of this miRNA led to a reduction in nodule number. • This work suggests that regulatory pathways controlling a conserved transcription factor are complex and divergent between M. truncatula and Arabidopsis.

  5. Mecasermin rinfabate: insulin-like growth factor-I/insulin-like growth factor binding protein-3, mecaserimin rinfibate, rhIGF-I/rhIGFBP-3.

    Science.gov (United States)

    2005-01-01

    Insmed is developing mecasermin rinfabate, a recombinant complex of insulin-like growth factor-I (rhIGF-I) and binding protein-3 (rhIGFBP-3) [insulin-like growth factor-I/insulin-like growth factor binding protein-3, rhIGF-I/rhIGFBP-3, SomatoKine], for a number of metabolic and endocrine indications. In the human body, IGF-I circulates in the blood bound to a binding protein-3 (IGFBP-3), which regulates the delivery of IGF-I to target tissues, and particular proteases clip them apart in response to stresses and release IGF-I as needed. IGF-I, a naturally occurring hormone, is necessary for normal growth and metabolism. For the treatment of IGF-I deficiency, it is desirable to administer IGF-I bound to IGFBP-3 to maintain the normal equilibrium of these proteins in the blood. Mecasermin rinfabate (rhIGF-I/rhIGFBP-3) mimics the effects of the natural protein complex in the bloodstream and would augment the natural supply of these linked compounds. The most advanced indication in development of mecasermin rinfabate is the treatment of severe growth disorders due to growth hormone insensitivity syndrome (GHIS), also called Laron syndrome. GHIS is a genetic condition in which patients do not produce adequate quantities of IGF because of a failure to respond to the growth hormone signal. This results in a slower growth rate and short stature. Mecasermin rinfabate also has potential as replacement therapy for IGF-I, which may become depleted in indications such as major surgery, organ damage/failure, traumatic injury, cachexia and severe burn trauma. It also has potential for the treatment of osteoporosis. Mecasermin rinfabate was developed by Celtrix using its proprietary recombinant protein production technology. Subsequently, Celtrix was acquired by Insmed Pharmaceuticals on 1 June 2000. Insmed and Avecia of the UK have signed an agreement for manufacturing mecasermin rinfabate and its components, rhIGF-1 and rhIGFBP-3. CGMP clinical production of mecasermin rinfabate

  6. Oleanolic Acid Induces Differentiation of Neural Stem Cells to Neurons: An Involvement of Transcription Factor Nkx-2.5

    Directory of Open Access Journals (Sweden)

    You Ning

    2015-01-01

    Full Text Available Neural stem cells (NSCs harbor the potential to differentiate into neurons, astrocytes, and oligodendrocytes under normal conditions and/or in response to tissue damage. NSCs open a new way of treatment of the injured central nervous system and neurodegenerative disorders. Thus far, few drugs have been developed for controlling NSC functions. Here, the effect as well as mechanism of oleanolic acid (OA, a pentacyclic triterpenoid, on NSC function was investigated. We found OA significantly inhibited neurosphere formation in a dose-dependent manner and achieved a maximum effect at 10 nM. OA also reduced 5-ethynyl-2′-deoxyuridine (EdU incorporation into NSCs, which was indicative of inhibited NSC proliferation. Western blotting analysis revealed the protein levels of neuron-specific marker tubulin-βIII (TuJ1 and Mash1 were increased whilst the astrocyte-specific marker glial fibrillary acidic protein (GFAP decreased. Immunofluorescence analysis showed OA significantly elevated the percentage of TuJ1-positive cells and reduced GFAP-positive cells. Using DNA microarray analysis, 183 genes were differentially regulated by OA. Through transcription factor binding site analyses of the upstream regulatory sequences of these genes, 87 genes were predicted to share a common motif for Nkx-2.5 binding. Finally, small interfering RNA (siRNA methodology was used to silence Nkx-2.5 expression and found silence of Nkx-2.5 alone did not change the expression of TuJ-1 and the percentage of TuJ-1-positive cells. But in combination of OA treatment and silence of Nkx-2.5, most effects of OA on NSCs were abolished. These results indicated that OA is an effective inducer for NSCs differentiation into neurons at least partially by Nkx-2.5-dependent mechanism.

  7. Genome-wide integration on transcription factors, histone acetylation and gene expression reveals genes co-regulated by histone modification patterns.

    Directory of Open Access Journals (Sweden)

    Yayoi Natsume-Kitatani

    Full Text Available N-terminal tails of H2A, H2B, H3 and H4 histone families are subjected to posttranslational modifications that take part in transcriptional regulation mechanisms, such as transcription factor binding and gene expression. Regulation mechanisms under control of histone modification are important but remain largely unclear, despite of emerging datasets for comprehensive analysis of histone modification. In this paper, we focus on what we call genetic harmonious units (GHUs, which are co-occurring patterns among transcription factor binding, gene expression and histone modification. We present the first genome-wide approach that captures GHUs by combining ChIP-chip with microarray datasets from Saccharomyces cerevisiae. Our approach employs noise-robust soft clustering to select patterns which share the same preferences in transcription factor-binding, histone modification and gene expression, which are all currently implied to be closely correlated. The detected patterns are a well-studied acetylation of lysine 16 of H4 in glucose depletion as well as co-acetylation of five lysine residues of H3 with H4 Lys12 and H2A Lys7 responsible for ribosome biogenesis. Furthermore, our method further suggested the recognition of acetylated H4 Lys16 being crucial to histone acetyltransferase ESA1, whose essential role is still under controversy, from a microarray dataset on ESA1 and its bypass suppressor mutants. These results demonstrate that our approach allows us to provide clearer principles behind gene regulation mechanisms under histone modifications and detect GHUs further by applying to other microarray and ChIP-chip datasets. The source code of our method, which was implemented in MATLAB (http://www.mathworks.com/, is available from the supporting page for this paper: http://www.bic.kyoto-u.ac.jp/pathway/natsume/hm_detector.htm.

  8. The transcription factor encyclopedia.

    Science.gov (United States)

    Yusuf, Dimas; Butland, Stefanie L; Swanson, Magdalena I; Bolotin, Eugene; Ticoll, Amy; Cheung, Warren A; Zhang, Xiao Yu Cindy; Dickman, Christopher T D; Fulton, Debra L; Lim, Jonathan S; Schnabl, Jake M; Ramos, Oscar H P; Vasseur-Cognet, Mireille; de Leeuw, Charles N; Simpson, Elizabeth M; Ryffel, Gerhart U; Lam, Eric W-F; Kist, Ralf; Wilson, Miranda S C; Marco-Ferreres, Raquel; Brosens, Jan J; Beccari, Leonardo L; Bovolenta, Paola; Benayoun, Bérénice A; Monteiro, Lara J; Schwenen, Helma D C; Grontved, Lars; Wederell, Elizabeth; Mandrup, Susanne; Veitia, Reiner A; Chakravarthy, Harini; Hoodless, Pamela A; Mancarelli, M Michela; Torbett, Bruce E; Banham, Alison H; Reddy, Sekhar P; Cullum, Rebecca L; Liedtke, Michaela; Tschan, Mario P; Vaz, Michelle; Rizzino, Angie; Zannini, Mariastella; Frietze, Seth; Farnham, Peggy J; Eijkelenboom, Astrid; Brown, Philip J; Laperrière, David; Leprince, Dominique; de Cristofaro, Tiziana; Prince, Kelly L; Putker, Marrit; del Peso, Luis; Camenisch, Gieri; Wenger, Roland H; Mikula, Michal; Rozendaal, Marieke; Mader, Sylvie; Ostrowski, Jerzy; Rhodes, Simon J; Van Rechem, Capucine; Boulay, Gaylor; Olechnowicz, Sam W Z; Breslin, Mary B; Lan, Michael S; Nanan, Kyster K; Wegner, Michael; Hou, Juan; Mullen, Rachel D; Colvin, Stephanie C; Noy, Peter John; Webb, Carol F; Witek, Matthew E; Ferrell, Scott; Daniel, Juliet M; Park, Jason; Waldman, Scott A; Peet, Daniel J; Taggart, Michael; Jayaraman, Padma-Sheela; Karrich, Julien J; Blom, Bianca; Vesuna, Farhad; O'Geen, Henriette; Sun, Yunfu; Gronostajski, Richard M; Woodcroft, Mark W; Hough, Margaret R; Chen, Edwin; Europe-Finner, G Nicholas; Karolczak-Bayatti, Magdalena; Bailey, Jarrod; Hankinson, Oliver; Raman, Venu; LeBrun, David P; Biswal, Shyam; Harvey, Christopher J; DeBruyne, Jason P; Hogenesch, John B; Hevner, Robert F; Héligon, Christophe; Luo, Xin M; Blank, Marissa Cathleen; Millen, Kathleen Joyce; Sharlin, David S; Forrest, Douglas; Dahlman-Wright, Karin; Zhao, Chunyan; Mishima, Yuriko; Sinha, Satrajit; Chakrabarti, Rumela; Portales-Casamar, Elodie; Sladek, Frances M; Bradley, Philip H; Wasserman, Wyeth W

    2012-01-01

    Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.

  9. Evolution of transcriptional networks in yeast: alternative teams of transcriptional factors for different species

    OpenAIRE

    Adriana Muñoz; Daniella Santos Muñoz; Aleksey Zimin; Yorke, James A.

    2016-01-01

    Background The diversity in eukaryotic life reflects a diversity in regulatory pathways. Nocedal and Johnson argue that the rewiring of gene regulatory networks is a major force for the diversity of life, that changes in regulation can create new species. Results We have created a method (based on our new “ping-pong algorithm) for detecting more complicated rewirings, where several transcription factors can substitute for one or more transcription factors in the regulation of a family of co-r...

  10. Transcriptional regulation of human thromboxane synthase gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Lee, K.D.; Baek, S.J.; Fleischer, T [Univ. of Maryland Medical School, Baltimore, MD (United States)] [and others

    1994-09-01

    The human thromboxane synthase (TS) gene encodes a microsomal enzyme catalyzing the conversion of prostaglandin endoperoxide into thromboxane A{sub 2}(TxA{sub 2}), a potent inducer of vasoconstriction and platelet aggregation. A deficiency in platelet TS activity results in bleeding disorders, but the underlying molecular mechanism remains to be elucidated. Increased TxA{sub 2} has been associated with many pathophysiological conditions such as cardiovascular disease, pulmonary hypertension, pre-eclampsia, and thrombosis in sickle cell patients. Since the formation of TxA{sub 2} is dependent upon TS, the regulation of TS gene expression may presumably play a crucial role in vivo. Abrogation of the regulatory mechanism in TS gene expression might contribute, in part, to the above clinical manifestations. To gain insight into TS gene regulation, a 1.7 kb promoter of the human TS gene was cloned and sequenced. RNase protection assay and 5{prime} RACE protocols were used to map the transcription initiation site to nucleotide A, 30 bp downstream from a canonical TATA box. Several transcription factor binding sites, including AP-1, PU.1, and PEA3, were identified within this sequence. Transient expression studies in HL-60 cells transfected with constructs containing various lengths (0.2 to 5.5 kb) of the TS promoter/luciferase fusion gene indicated the presence of multiple repressor elements within the 5.5 kb TS promoter. However, a lineage-specific up-regulation of TS gene expression was observed in HL-60 cells induced by TPA to differentiate along the macrophage lineage. The increase in TS transcription was not detectable until 36 hr after addition of the inducer. These results suggest that expression of the human TS gene may be regulated by a mechanism involving repression and derepression of the TS promoter.

  11. Cutting the chain of command: specific inhibitors of transcription.

    Science.gov (United States)

    Holt, J T

    1991-01-01

    Cell growth and differentiation are regulated (at least in part) by changes in gene transcription. The cloning and characterization of transcription factors has revealed that these factors coordinately regulate the transcription of specific genetic programs; for example, a number of phorbol ester-induced genes are activated by binding of the transcription factors Fos and Jun to specific DNA sequences. Clearly, inhibition of either the production or function of specific transcription factors would alter complete genetic programs, changing the expression of a great number of genes (analogous to cutting the chain of military command and affecting an entire brigade or division). Our laboratory and others have employed genetic methods to specifically inhibit transcription by two distinct methods: (1) antisense inhibition of the production of transcription factors; and (2) introduction of target DNA sequences to "soak up"or quench transcription factors. In this report, we present data showing that serum-stimulated induction of the c-fos gene may be reduced more than 90% by introduction of target DNA sequences containing the serum response element (SRE); identical amounts of mutant SRE sequences have no effect on gene induction. These studies demonstrate that specific inhibitors of transcription can have significant effects on cellular gene expression. The challenge is to modulate transcriptional programs without deleterious effects on normal cells.

  12. Biophysical models of transcription in cells

    Science.gov (United States)

    Choubey, Sandeep

    Cells constantly face environmental challenges and deal with them by changing their gene expression patterns. They make decisions regarding which genes to express and which genes not to express based on intra-cellular and environmental cues. These decisions are often made by regulating the process of transcription. While the identities of the different molecules that take part in regulating transcription have been determined for a number of different genes, their dynamics inside the cell are still poorly understood. One key feature of these regulatory dynamics is that the numbers of the bio-molecules involved is typically small, resulting in large temporal fluctuations in transcriptional outputs (mRNA and protein). In this thesis I show that measurements of the cell-to-cell variability of the distribution of transcribing RNA polymerases along a gene provide a previously unexplored method for deciphering the mechanism of its transcription in vivo. First, I propose a simple kinetic model of transcription initiation and elongation from which I calculate transcribing RNA polymerase copy-number fluctuations. I test my theory against published data obtained for yeast genes and propose a novel mechanism of transcription. Rather than transcription being initiated through a single rate-limiting step, as was previously proposed, my single-cell analysis reveals the presence of at least two rate limiting steps. Second, I compute the distribution of inter-polymerase distance distribution along a gene and propose a method for analyzing inter-polymerase distance distributions acquired in experiments. By applying this method to images of polymerases transcribing ribosomal genes in E.coli I show that one model of regulation of these genes is consistent with inter-polymerase distance data while a number of other models are not. The analytical framework described in this thesis can be used to extract quantitative information about the dynamics of transcription from single

  13. 基因启动子甲基化对转录因子结合的抑制作用分析方法%Method to Analyze Gene Promoter Methylation Inhibition Effect on Binding of Transcription Factors

    Institute of Scientific and Technical Information of China (English)

    冯伟兴; 王科俊; 贺波; 李霞

    2011-01-01

    DNA methylation is identified as an elaborate epigenetic element to regulate binding of transcription factor to gene promoter region. With latest highthroughput technology, it is convenient to accurately test methylation level in experiment, which opens a door to investigate how methylation affects transcription factor. A general model is presented to sense methylation effect on transcription factor in a specific cell. In the model, an inverse sigmoid function is adopted to depict effect of DNA methylation to binding ability of transcription factors with two parameters as center C and steepness S. For each transcription factor, the parameters of model can be fixed by analysis of relativity between transcription factor binding scores in promoter regions and gene expression levels. Here three relativity values should be computed while different formula is used to calculate transcription factor binding score. Relativity value A is obtained when transcription factor binding scores are calculated without considering methylation effect. Relativity value B is analyzed from transcription factor binding scores considering methylation effect with the proposed model. On the contrary, normal sigmoid function is used to depict effect of DNA methylation and relativity value C is just calculated with transcription factor binding scores considering methylation effect using such model. For a transcription factor, if relativity value B is found obviously larger than relativity value A and relativity value C is always less than relativity value A, the transcription factor can be figured out to be apparently affected by DNA methylation and the model with optimal fixed parameters can be used to depict the methylation effect. In neuroblastoma cell, with the proposed model, 10 transcriptional factors were found to be apparently affected by methylation of promoter regions which proves the effectiveness of the model.Based on the proposed model, TF binding status in genome promoter region

  14. Gene-Silencing-Induced Changes in Carbohydrate Conformation in Relation to Bioenergy Value and Carbohydrate Subfractions in Modeled Plant (Medicago sativa) with Down-Regulation of HB12 and TT8 Transcription Factors.

    Science.gov (United States)

    Li, Xinxin; Hannoufa, Abdelali; Zhang, Yonggen; Yu, Peiqiang

    2016-05-13

    Gene silencing with RNA interference (RNAi) technology may be capable of modifying internal structure at a molecular level. This structural modification could affect biofunctions in terms of biodegradation, biochemical metabolism, and bioactive compound availability. The objectives of this study were to (1) Detect gene silencing-induced changes in carbohydrate molecular structure in an alfalfa forage (Medicago sativa spp. sativa: alfalfa) with down-regulation of genes that encode transcription factors TT8 and HB12; (2) Determine gene silencing-induced changes in nutrient bioutilization and bioavailability in the alfalfa forage (Medicago sativa); and (3) Quantify the correlation between gene silencing-induced molecular structure changes and the nutrient bioutilization and bioavailability in animals of ruminants. The experimental treatments included: T1 = Non-transgenic and no-gene silenced alfalfa forage (code "NT"); T2 = HB12-RNAi forage with HB12 gene down regulation (code "HB12"); T3 = TT8-RNAi forage with TT8 gene down regulation (code "TT8"). The HB12 and TT8 gene silencing-induced molecular structure changes were determined by non-invasive and non-destructive advanced molecular spectroscopy in a middle infrared radiation region that focused on structural, non-structural and total carbohydrate compounds. The nutrient bioutilization and bioavailability of the modified forage were determined using NRC-2001 system in terms of total digestive nutrient (TDN), truly digestible fiber (tdNDF), non-fiber carbohydrate (tdNDF), fatty acid (tdFA), crude protein (tdCP) and bioenergy profiles (digestible energy, metabolizable energy, net energy) for ruminants. The carbohydrate subfractions were evaluated using the updated CNCPS 6.0 system. The results showed that gene silencing significantly affected tdNFC (42.3 (NT) vs. 38.7 (HB12) vs. 37.4% Dry Matter (TT8); p = 0.016) and tdCP (20.8 (NT) vs. 19.4 (HB12) vs. 22.3% DM (TT8); p = 0.009). The gene-silencing also affected

  15. Rhythm quantization for transcription

    NARCIS (Netherlands)

    Cemgil, A.T.; Desain, P.W.M.; Kappen, H.J.

    1999-01-01

    Automatic Music Transcription is the extraction of an acceptable notation from performed music. One important task in this problem is rhythm quantization which refers to categorization of note durations. Although quantization of a pure mechanical performance is rather straightforward, the task becom

  16. Mapping yeast transcriptional networks.

    Science.gov (United States)

    Hughes, Timothy R; de Boer, Carl G

    2013-09-01

    The term "transcriptional network" refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face. For most yeast TFs, data have been collected on their sequence preferences, in vivo promoter occupancy, and gene expression profiles in deletion mutants. These systematic studies have led to the identification of new regulators of numerous cellular functions and shed light on the overall organization of yeast gene regulation. However, many yeast TFs appear to be inactive under standard laboratory growth conditions, and many of the available data were collected using techniques that have since been improved. Perhaps as a consequence, comprehensive and accurate mapping among TF sequence preferences, promoter binding, and gene expression remains an open challenge. We propose that the time is ripe for renewed systematic efforts toward a complete mapping of yeast transcriptional regulatory mechanisms.

  17. Mitochondrial transcription: How does it end

    Energy Technology Data Exchange (ETDEWEB)

    J Byrnes; M Garcia-Diaz

    2011-12-31

    The structure of the mitochondrial transcription termination factor (MTERF1) provides novel insight into the mechanism of binding, recognition of the termination sequence and the conformational changes involved in mediating termination. Besides its functional implications, this structure provides a framework to understand the consequences of numerous diseases associated with mitochondrial DNA mutations.

  18. Mitochondrial transcription: how does it end?

    Science.gov (United States)

    Byrnes, James; Garcia-Diaz, Miguel

    2011-01-01

    The structure of the mitochondrial transcription termination factor (MTERF1) provides novel insight into the mechanism of binding, recognition of the termination sequence and the conformational changes involved in mediating termination. Besides its functional implications, this structure provides a framework to understand the consequences of numerous diseases associated with mitochondrial DNA mutations.

  19. Transcription Dynamics in Living Cells.

    Science.gov (United States)

    Lenstra, Tineke L; Rodriguez, Joseph; Chen, Huimin; Larson, Daniel R

    2016-07-01

    The transcription cycle can be roughly divided into three stages: initiation, elongation, and termination. Understanding the molecular events that regulate all these stages requires a dynamic view of the underlying processes. The development of techniques to visualize and quantify transcription in single living cells has been essential in revealing the transcription kinetics. They have revealed that (a) transcription is heterogeneous between cells and (b) transcription can be discontinuous within a cell. In this review, we discuss the progress in our quantitative understanding of transcription dynamics in living cells, focusing on all parts of the transcription cycle. We present the techniques allowing for single-cell transcription measurements, review evidence from different organisms, and discuss how these experiments have broadened our mechanistic understanding of transcription regulation.

  20. Uncovering transcriptional regulation of metabolism by using metabolic network topology

    DEFF Research Database (Denmark)

    Patil, Kiran Raosaheb; Nielsen, Jens

    2005-01-01

    therefore developed an algorithm that is based on hypothesis-driven data analysis to uncover the transcriptional regulatory architecture of metabolic networks. By using information on the metabolic network topology from genome-scale metabolic reconstruction, we show that it is possible to reveal patterns...... in the metabolic network that follow a common transcriptional response. Thus, the algorithm enables identification of so-called reporter metabolites (metabolites around which the most significant transcriptional changes occur) and a set of connected genes with significant and coordinated response to genetic...... changes induced by complex regulatory mechanisms coordinating the activity of different metabolic pathways. It is difficult to map such global transcriptional responses by using traditional methods, because many genes in the metabolic network have relatively small changes at their transcription level. We...

  1. Transcriptional regulation of topology modulators and transcription regulators of Mycobacterium tuberculosis.

    Science.gov (United States)

    Ghosh, Soumitra; Padmanabhan, Bhavna; Godbole, Adwait Anand; Tare, Priyanka; Ahmed, Wareed; Vasu, Kommireddy; China, Arnab; Kumar, Rupesh; Mitra, Anirban; Nagaraja, Valakunja

    2016-07-01

    Mycobacterium tuberculosis (Mtb) is a formidable pathogen which has the ability to survive the hostile environment of the host by evading the host defense system. The re-configuration of its transcriptional and metabolic process allows the pathogen to confront the adverse environment within the host macrophages. The factors that assist the transcription and modulate the DNA topology would have to play a key role in the regulation of global gene expression of the organism. How transcription of these essential housekeeping genes alters in response to growth conditions and environmental stress has not been addressed together in a set of experimental conditions in Mtb. Now, we have mapped the transcription start sites (TSS) and promoters of several genes that play a central role in the regulation of DNA topology and transcription in Mtb. Using in vivo reporter assays, we validated the activity of the identified promoter elements in different growth conditions. The variation in transcript abundance of these essential genes was also analyzed in growth phase-dependent manner. These data provide the first glimpse into the specific adaptive changes in the expression of genes involved in transcription and DNA topology modulation in Mtb.

  2. Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription.

    Science.gov (United States)

    Hoffman, Elizabeth A; McCulley, Andrew; Haarer, Brian; Arnak, Remigiusz; Feng, Wenyi

    2015-03-01

    We have previously demonstrated that in Saccharomyces cerevisiae replication, checkpoint inactivation via a mec1 mutation leads to chromosome breakage at replication forks initiated from virtually all origins after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Here we sought to determine whether all replication forks containing single-stranded DNA gaps have equal probability of producing double-strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-seq, that combines our previously described DSB labeling with next generation sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed a greater distance in MEC1 cells than in mec1 cells during recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of those transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. We further propose that replication inhibitors can induce unscheduled encounters between replication and transcription and give rise to distinct patterns of chromosome fragile sites.

  3. Inference of RNA polymerase II transcription dynamics from chromatin immunoprecipitation time course data.

    Directory of Open Access Journals (Sweden)

    Ciira wa Maina

    2014-05-01

    Full Text Available Gene transcription mediated by RNA polymerase II (pol-II is a key step in gene expression. The dynamics of pol-II moving along the transcribed region influence the rate and timing of gene expression. In this work, we present a probabilistic model of transcription dynamics which is fitted to pol-II occupancy time course data measured using ChIP-Seq. The model can be used to estimate transcription speed and to infer the temporal pol-II activity profile at the gene promoter. Model parameters are estimated using either maximum likelihood estimation or via Bayesian inference using Markov chain Monte Carlo sampling. The Bayesian approach provides confidence intervals for parameter estimates and allows the use of priors that capture domain knowledge, e.g. the expected range of transcription speeds, based on previous experiments. The model describes the movement of pol-II down the gene body and can be used to identify the time of induction for transcriptionally engaged genes. By clustering the inferred promoter activity time profiles, we are able to determine which genes respond quickly to stimuli and group genes that share activity profiles and may therefore be co-regulated. We apply our methodology to biological data obtained using ChIP-seq to measure pol-II occupancy genome-wide when MCF-7 human breast cancer cells are treated with estradiol (E2. The transcription speeds we obtain agree with those obtained previously for smaller numbers of genes with the advantage that our approach can be applied genome-wide. We validate the biological significance of the pol-II promoter activity clusters by investigating cluster-specific transcription factor binding patterns and determining canonical pathway enrichment. We find that rapidly induced genes are enriched for both estrogen receptor alpha (ERα and FOXA1 binding in their proximal promoter regions.

  4. Identification of a novel and unique transcription factor in the intraerythrocytic stage of Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Kanako Komaki-Yasuda

    Full Text Available The mechanisms of stage-specific gene regulation in the malaria parasite Plasmodium falciparum are largely unclear, with only a small number of specific regulatory transcription factors (AP2 family having been identified. In particular, the transcription factors that function in the intraerythrocytic stage remain to be elucidated. Previously, as a model case for stage-specific transcription in the P. falciparum intraerythrocytic stage, we analyzed the transcriptional regulation of pf1-cys-prx, a trophozoite/schizont-specific gene, and suggested that some nuclear factors bind specifically to the cis-element of pf1-cys-prx and enhance transcription. In the present study, we purified nuclear factors from parasite nuclear extract by 5 steps of chromatography, and identified a factor termed PREBP. PREBP is not included in the AP2 family, and is a novel protein with four K-homology (KH domains. The KH domain is known to be found in RNA-binding or single-stranded DNA-binding proteins. PREBP is well conserved in Plasmodium species and partially conserved in phylum Apicomplexa. To evaluate the effects of PREBP overexpression, we used a transient overexpression and luciferase assay combined approach. Overexpression of PREBP markedly enhanced luciferase expression under the control of the pf1-cys-prx cis-element. These results provide the first evidence of a novel transcription factor that activates the gene expression in the malaria parasite intraerythrocytic stage. These findings enhance our understanding of the evolution of specific transcription machinery in Plasmodium and other eukaryotes.

  5. Prediction and testing of novel transcriptional networks regulating embryonic stem cell self-renewal and commitment.

    Science.gov (United States)

    Walker, Emily; Ohishi, Minako; Davey, Ryan E; Zhang, Wen; Cassar, Paul A; Tanaka, Tetsuya S; Der, Sandy D; Morris, Quaid; Hughes, Timothy R; Zandstra, Peter W; Stanford, William L

    2007-06-07

    Stem cell fate is governed by the integration of intrinsic and extrinsic positive and negative signals upon inherent transcriptional networks. To identify novel embryonic stem cell (ESC) regulators and assemble transcriptional networks controlling ESC fate, we performed temporal expression microarray analyses of ESCs after the initiation of commitment and integrated these data with known genome-wide transcription factor binding. Effects of forced under- or overexpression of predicted novel regulators, defined as differentially expressed genes with potential binding sites for known regulators of pluripotency, demonstrated greater than 90% correspondence with predicted function, as assessed by functional and high-content assays of self-renewal. We next assembled 43 theoretical transcriptional networks in ESCs, 82% (23 out of 28 tested) of which were supported by analysis of genome-wide expression in Oct4 knockdown cells. By using this integrative approach, we have formulated novel networks describing gene repression of key developmental regulators in undifferentiated ESCs and successfully predicted the outcomes of genetic manipulation of these networks.

  6. Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factorsin flower development

    NARCIS (Netherlands)

    Pajoro, A.; Madrigal, P.; Muiño, J.M.; Tomas Matus, J.; Jin, J.; Mecchia, M.A.; Debernardi, J.M.; Palatnik, J.F.; Balazadeh, S.; Arif, M.; Ó’Maoiléidigh, D.S.; Wellmer, F.; Krajewski, P.; Riechmann, J.L.; Angenent, G.C.

    2014-01-01

    Background: Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanism

  7. DNA Topoisomerases in Transcription

    DEFF Research Database (Denmark)

    Rødgaard, Morten Terpager

    2015-01-01

    This Ph.D. thesis summarizes the main results of my studies on the interplay between DNA topoisomerases and transcription. The work was performed from 2011 to 2015 at Aarhus University in the Laboratory of Genome Research, and was supervised by associate professor Anni H. Andersen. Most of the ex......This Ph.D. thesis summarizes the main results of my studies on the interplay between DNA topoisomerases and transcription. The work was performed from 2011 to 2015 at Aarhus University in the Laboratory of Genome Research, and was supervised by associate professor Anni H. Andersen. Most...... topoisomerase-DNA cleavage complex. The second study is an investigation of how topoisomerases influence gene regulation by keeping the genome in an optimal topological state....

  8. SNFing HIV transcription

    Directory of Open Access Journals (Sweden)

    Bukrinsky Michael

    2006-08-01

    Full Text Available Abstract The SWI/SNF chromatin remodeling complex is an essential regulator of transcription of cellular genes. HIV-1 infection induces exit of a core component of SWI/SNF, Ini1, into the cytoplasm and its association with the viral pre-integration complex. Several recent papers published in EMBO Journal, Journal of Biological Chemistry, and Retrovirology provide new information regarding possible functions of Ini1 and SWI/SNF in HIV life cycle. It appears that Ini1 has an inhibitory effect on pre-integration steps of HIV replication, but also contributes to stimulation of Tat-mediated transcription. This stimulation involves displacement of the nucleosome positioned at the HIV promoter.

  9. Linking high-resolution metabolic flux phenotypes and transcriptional regulation in yeast modulated by the global regulator Gcn4p

    DEFF Research Database (Denmark)

    Moxley, Joel F.; Jewett, Michael Christopher; Antoniewicz, Maciek R.

    2009-01-01

    Genome sequencing dramatically increased our ability to understand cellular response to perturbation. Integrating system-wide measurements such as gene expression with networks of protein protein interactions and transcription factor binding revealed critical insights into cellular behavior. Howe...... that an integrated approach focusing on metabolic measurements will facilitate construction of more realistic models of cellular regulation for understanding diseases and constructing strains for industrial applications.......RNA and metabolic flux data that combines information from both interaction network models and flux determination models. We started by quantifying 5,764 mRNAs, 54 metabolites, and 83 experimental C-13-based reaction fluxes in continuous cultures of yeast under stress in the absence or presence of global regulator...... of metabolic flux (i.e., use of different reaction pathways) by transcriptional regulation and metabolite interaction density (i.e., level of pairwise metabolite-protein interactions) as a key biosynthetic control determinant. Furthermore, this model predicted flux rewiring in studies of follow...

  10. Tumors from rats given 1,2-dimethylhydrazine plus chlorophyllin or indole-3-carbinol contain transcriptional changes in beta-catenin that are independent of beta-catenin mutation status.

    Science.gov (United States)

    Wang, Rong; Dashwood, W Mohaiza; Bailey, George S; Williams, David E; Dashwood, Roderick H

    2006-10-10

    Tumors induced in the rat by 1,2-dimethylhydrazine (DMH) contain mutations in beta-catenin, but the spectrum of such mutations can be influenced by phytochemicals such as chlorophyllin (CHL) and indole-3-carbinol (I3C). In the present study, we determined the mutation status of beta-catenin in more than 50 DMH-induced colon tumors and small intestine tumors, and compared this with the concomitant expression of beta-catenin mRNA using quantitative real-time RT-PCR analysis. In total, 19/57 (33%) of the tumors harbored mutations in beta-catenin, and 14/19 (74%) of the genetic changes substituted amino acids adjacent to Ser33, a key site for phosphorylation and beta-catenin degradation. These tumors were found to express a 10-fold range of beta-catenin mRNA levels, independent of the beta-catenin mutation status and phytochemical exposure, i.e. CHL or I3C given post-initiation. However, beta-catenin mRNA levels were strongly correlated with mRNA levels of c-myc, c-jun and cyclin D1, which are targets of beta-catenin/Tcf signaling. Tumors with the highest levels of beta-catenin mRNA often had over-expressed beta-catenin protein, and those with lower beta-catenin mRNA typically had low beta-catenin protein expression, but there were exceptions (high beta-catenin mRNA/low beta-catenin protein, or vice versa). We conclude that DMH-induced mutations stabilize beta-catenin protein in tumors, which increase c-myc, c-jun and cyclin D1, but there also can be over-expression of beta-catenin itself at the mRNA level, contributing to high beta-catenin protein levels. Similar findings have been reported in primary human colon cancers and their liver metastases, compared with matched normal-looking tissue. Thus, further studies are warranted on the mechanisms that upregulate beta-catenin at the transcriptional level in human and rodent colon cancers.

  11. A compendium of nucleosome and transcript profiles reveals determinants of chromatin architecture and transcription.

    Directory of Open Access Journals (Sweden)

    Harm van Bakel

    2013-05-01

    Full Text Available Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies.

  12. A compendium of nucleosome and transcript profiles reveals determinants of chromatin architecture and transcription.

    Science.gov (United States)

    van Bakel, Harm; Tsui, Kyle; Gebbia, Marinella; Mnaimneh, Sanie; Hughes, Timothy R; Nislow, Corey

    2013-05-01

    Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies.

  13. Structural analysis of nucleosomal barrier to transcription.

    Science.gov (United States)

    Gaykalova, Daria A; Kulaeva, Olga I; Volokh, Olesya; Shaytan, Alexey K; Hsieh, Fu-Kai; Kirpichnikov, Mikhail P; Sokolova, Olga S; Studitsky, Vasily M

    2015-10-27

    Thousands of human and Drosophila genes are regulated at the level of transcript elongation and nucleosomes are likely targets for this regulation. However, the molecular mechanisms of formation of the nucleosomal barrier to transcribing RNA polymerase II (Pol II) and nucleosome survival during/after transcription remain unknown. Here we show that both DNA-histone interactions and Pol II backtracking contribute to formation of the barrier and that nucleosome survival during transcription likely occurs through allosterically stabilized histone-histone interactions. Structural analysis indicates that after Pol II encounters the barrier, the enzyme backtracks and nucleosomal DNA recoils on the octamer, locking Pol II in the arrested state. DNA is displaced from one of the H2A/H2B dimers that remains associated with the octamer. The data reveal the importance of intranucleosomal DNA-protein and protein-protein interactions during conformational changes in the nucleosome structure on transcription. Mechanisms of nucleosomal barrier formation and nucleosome survival during transcription are proposed.

  14. Transcription factor AP1 binds the functional region of the promoter and regulates gene expression of human PPARdelta in LoVo cell.

    Science.gov (United States)

    Jiang, Xiaogang; Yang, Xudong; Han, Yan; Lu, Shemin

    2013-12-01

    Peroxisome proliferator-activated receptor δ gene (PPARδ) is correlated with carcinogenesis of colorectal cancer, but the regulation of its gene transcription remains unclear. We herein report that AP1 binds the promoter and regulates PPARδ gene expression. With a luciferase reporter system, we identified a functional promoter region of 30 bp of PPARδ gene by deletion and electrophoretic mobility shift assays (EMSA). Using site-directed mutagenesis and decoy analyses, we demonstrated that AP1 bound the functional transcriptional factor binding site in a region extending from -176 to -73 of the PPARδ promoter, which was confirmed using EMSA and supershift assays. Consequently, inhibition of the AP1 binding site led to decreased PPARδ mRNA. Our study demonstrated that AP1 is the transcriptional factor that contributes to PPARδ expression in LoVo cells.

  15. Reliable transfer of transcriptional gene regulatory networks between taxonomically related organisms

    Directory of Open Access Journals (Sweden)

    Tauch Andreas

    2009-01-01

    Full Text Available Abstract Background Transcriptional regulation of gene activity is essential for any living organism. Transcription factors therefore recognize specific binding sites within the DNA to regulate the expression of particular target genes. The genome-scale reconstruction of the emerging regulatory networks is important for biotechnology and human medicine but cost-intensive, time-consuming, and impossible to perform for any species separately. By using bioinformatics methods one can partially transfer networks from well-studied model organisms to closely related species. However, the prediction quality is limited by the low level of evolutionary conservation of the transcription factor binding sites, even within organisms of the same genus. Results Here we present an integrated bioinformatics workflow that assures the reliability of transferred gene regulatory networks. Our approach combines three methods that can be applied on a large-scale: re-assessment of annotated binding sites, subsequent binding site prediction, and homology detection. A gene regulatory interaction is considered to be conserved if (1 the transcription factor, (2 the adjusted binding site, and (3 the target gene are conserved. The power of the approach is demonstrated by transferring gene regulations from the model organism Corynebacterium glutamicum to the human pathogens C. diphtheriae, C. jeikeium, and the biotechnologically relevant C. efficiens. For these three organisms we identified reliable transcriptional regulations for ~40% of the common transcription factors, compared to ~5% for which knowledge was available before. Conclusion Our results suggest that trustworthy genome-scale transfer of gene regulatory networks between organisms is feasible in general but still limited by the level of evolutionary conservation.

  16. Deciphering Transcriptional Regulation

    DEFF Research Database (Denmark)

    Valen, Eivind

    in different cell types. This thesis presents several methods for analysis and description of promoters. We focus particularly the binding sites of TFs and computational methods for locating these. We contribute to the ¿eld by compiling a database of binding preferences for TFs which can be used for site...... published providing an unbiased overview of the transcription start site (TSS) usage in a tissue. We have paired this method with high-throughput sequencing technology to produce a library of unprecedented depth (DeepCAGE) for the mouse hippocampus. We investigated this in detail and focused particularly...

  17. Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis

    Science.gov (United States)

    Zhang, Zhengjian; English, Brian P.; Grimm, Jonathan B.; Kazane, Stephanie A.; Hu, Wenxin; Tsai, Albert; Inouye, Carla; You, Changjiang; Piehler, Jacob; Schultz, Peter G.; Lavis, Luke D.; Revyakin, Andrey; Tjian, Robert

    2016-01-01

    Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A “step-wise” preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB–promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II–TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions. PMID:27798851

  18. PlantPAN 2.0: an update of plant promoter analysis navigator for reconstructing transcriptional regulatory networks in plants.

    Science.gov (United States)

    Chow, Chi-Nga; Zheng, Han-Qin; Wu, Nai-Yun; Chien, Chia-Hung; Huang, Hsien-Da; Lee, Tzong-Yi; Chiang-Hsieh, Yi-Fan; Hou, Ping-Fu; Yang, Tien-Yi; Chang, Wen-Chi

    2016-01-01

    Transcription factors (TFs) are sequence-specific DNA-binding proteins acting as critical regulators of gene expression. The Plant Promoter Analysis Navigator (PlantPAN; http://PlantPAN2.itps.ncku.edu.tw) provides an informative resource for detecting transcription factor binding sites (TFBSs), corresponding TFs, and other important regulatory elements (CpG islands and tandem repeats) in a promoter or a set of plant promoters. Additionally, TFBSs, CpG islands, and tandem repeats in the conserve regions between similar gene promoters are also identified. The current PlantPAN release (version 2.0) contains 16 960 TFs and 1143 TF binding site matrices among 76 plant species. In addition to updating of the annotation information, adding experimentally verified TF matrices, and making improvements in the visualization of transcriptional regulatory networks, several new features and functions are incorporated. These features include: (i) comprehensive curation of TF information (response conditions, target genes, and sequence logos of binding motifs, etc.), (ii) co-expression profiles of TFs and their target genes under various conditions, (iii) protein-protein interactions among TFs and their co-factors, (iv) TF-target networks, and (v) downstream promoter elements. Furthermore, a dynamic transcriptional regulatory network under various conditions is provided in PlantPAN 2.0. The PlantPAN 2.0 is a systematic platform for plant promoter analysis and reconstructing transcriptional regulatory networks.

  19. The influence of hepatitis B virus on the expression of insulin-like growth factor-binding protein 7 and its ;clinical significance%乙型肝炎病毒对胰岛素样生长因子结合蛋白7表达的影响及其临床意义

    Institute of Scientific and Technical Information of China (English)

    宋惠; 刘兴晖; 祝成亮; 钟基大; 李毅

    2013-01-01

    目的:探讨乙型肝炎病毒(HBV)对胰岛素样生长因子结合蛋白7(IGFBP7)表达的影响及其临床意义。方法逆转录-聚合酶链反应(PCR)检测IGFBP7 mRNA的表达水平,酶联免疫吸附试验(ELISA)检测血清IGFBP7含量,分析IGFBP7在慢性乙型肝炎(CHB)、肝纤维化(LC)和肝细胞癌(HCC)患者间血清含量的差异。结果 IGFBP7 mRNA在HepG2.2.15细胞中的表达水平较HepG2高;与健康对照组相比,HBV感染者IGFBP7血清学水平明显升高(P<0.05);IGFBP7在LC和HCC患者低于CHB患者(P<0.05)。结论 HBV能够上调IGFBP7的表达,其血清水平与疾病进程呈负相关。%Objective To investigate the influence of hepatitis B virus(HBV)on the expression of insulin-like growth factor-binding protein 7(IGFBP7)and its clinical significance.Methods The mRNA expression of IGFBP7 was measured by reverse transcription-polymerase chain reaction (PCR),and serum levels of IGFBP7 were measured by enzyme-linked immunosorbent assay(ELISA).The different expressions of IGFBP7 among patients with chronic hepatitis B(CHB),liver cirrhosis(LC)and hepatocellular carcinoma(HCC)were analyzed.Results The expression of IGFBP7 mRNA was higher in HepG2.2.15 cells than in HepG2 cells.Compared with healthy controls,serum IGFBP7 levels were much higher in HBV patients (P<0.05),and the serum levels of IGFBP7 were detected at lower levels in patients with LC and HCC than those with CHB(P<0.05).Conclusions HBV can upregulate the expression of IGFBP7, which is negatively correlated with the disease progression.

  20. Bound transcription factor suppresses photoproduct formation in the NF-kappa B promoter.

    Science.gov (United States)

    Ghosh, R; Paniker, L; Mitchell, D L

    2001-01-01

    The relationship between purified transcription factor p50 binding and ultraviolet light-induced DNA damage formation in the NF-kappa B promoter element was investigated. The effect of bound transcription factor on cyclobutane dimer formation was quantified using Maxam-Gilbert analysis of irradiated substrate digested with T4 phage endonuclease V. Two methods were employed for cleaving (6-4) photoproducts. Sites of (6-4) photoproducts cleaved by piperidine showed a general suppression in the presence of bound p50 protein similar to that observed for cyclobutane dimers. In contrast to piperidine, digestion with ultraviolet damage endonuclease (UVDE) from Saccharomyces pombe subsequent to cyclobutane dimer reversal by photolyase displayed a broader spectrum of damaged sites. Whereas some of these sites were suppressed by bound p50 protein, some remained unaffected and one site showed increased (6-4) photoproduct induction. These data illustrate the advantage of UVDE over piperidine for studying (6-4) photoproducts at the sequence level and suggest that this approach may be useful for footprinting transcription factor binding in other promoters.

  1. Inference of transcriptional networks in Arabidopsis through conserved noncoding sequence analysis.

    Science.gov (United States)

    Van de Velde, Jan; Heyndrickx, Ken S; Vandepoele, Klaas

    2014-07-01

    Transcriptional regulation plays an important role in establishing gene expression profiles during development or in response to (a)biotic stimuli. Transcription factor binding sites (TFBSs) are the functional elements that determine transcriptional activity, and the identification of individual TFBS in genome sequences is a major goal to inferring regulatory networks. We have developed a phylogenetic footprinting approach for the identification of conserved noncoding sequences (CNSs) across 12 dicot plants. Whereas both alignment and non-alignment-based techniques were applied to identify functional motifs in a multispecies context, our method accounts for incomplete motif conservation as well as high sequence divergence between related species. We identified 69,361 footprints associated with 17,895 genes. Through the integration of known TFBS obtained from the literature and experimental studies, we used the CNSs to compile a gene regulatory network in Arabidopsis thaliana containing 40,758 interactions, of which two-thirds act through binding events located in DNase I hypersensitive sites. This network shows significant enrichment toward in vivo targets of known regulators, and its overall quality was confirmed using five different biological validation metrics. Finally, through the integration of detailed expression and function information, we demonstrate how static CNSs can be converted into condition-dependent regulatory networks, offering opportunities for regulatory gene annotation.

  2. The BCG Moreau RD16 deletion inactivates a repressor reshaping transcription of an adjacent gene.

    Science.gov (United States)

    Galvão, Teca Calcagno; Lima, Cristiane Rodrigues; Gomes, Leonardo Henrique Ferreira; Pagani, Talita Duarte; Ferreira, Marcelo Alves; Gonçalves, Antonio S; Correa, Paloma Rezende; Degrave, Wim Maurits; Mendonça-Lima, Leila

    2014-01-01

    The Brazilian anti-tuberculosis vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG) BCG Moreau is unique in having a deletion of 7608 bp (RD16) that results in the truncation of a putative TetR transcriptional regulator, the ortholog of Mycobacterium tuberculosis rv3405c, BCG_M3439c. We investigated the effect of this truncation on the expression of the rv3406 ortholog (BCG_M3440), lying 81 bp downstream in the opposite orientation. RT-PCR and western blot experiments show that rv3406 mRNA and Rv3406 accumulate in BCG Moreau but not in BCG Pasteur (strain that bears an intact rv3405c), suggesting this to be a result of rv3405c truncation. Recombinant Rv3405c forms a complex with the rv3405c-rv3406 intergenic region, which contains a characteristic transcription factor binding site, showing it to have DNA binding activity. Complementation of M. bovis BCG Moreau with an intact copy of rv3405c abolishes Rv3406 accumulation. These results show that Rv3405c is a DNA binding protein that acts as a transcriptional repressor of rv3406.

  3. Capsella rubella TGA4, a bZIP transcription factor, causes delayed flowering in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Li Maofu

    2016-01-01

    Full Text Available Flowering time is usually regulated by many environmental factors and endogenous signals. TGA family members are bZIP transcription factors that bind to the octopine synthase element, which has been closely linked to defense/stress responses. Most TGA factors interact with non-expressor of PR1 (NPR1 and plant defense responses are strengthened by this interaction. TGA1and TGA4factors bind to NPR1 only in salicylic acid (SA-induced leaves, suggesting that TGA4 has another function during plant development. Here, we isolated a bZIP transcription factor gene, TGA4, from Capsella rubella. TGA4transcripts were detected in most tissues, with high expression in leaves, low expression in stems and flowering buds, and undetectable in siliques. CruTGA4was over expressed in Arabidopsis thaliana wild typeCol-0 plants. Flowering time and total leaf number in the transgenic plants showed that overexpression of CruTGA4could delay flowering in A. thaliana. Our findings suggest that TGA4 may act as flowering regulator that controls plant flowering.

  4. Problem-Solving Test: The Mechanism of Transcription Termination by the Rho Factor

    Science.gov (United States)

    Szeberenyi, Jozsef

    2012-01-01

    Transcription termination comes in two forms in "E. coli" cells. Rho-dependent termination requires the binding of a termination protein called Rho factor to the transcriptional machinery at the terminator region, whereas Rho-independent termination is achieved by conformational changes in the transcript itself. This article presents a test…

  5. Nucleocytoplasmic shuttling of transcription factors

    DEFF Research Database (Denmark)

    Cartwright, P; Helin, K

    2000-01-01

    To elicit the transcriptional response following intra- or extracellular stimuli, the signals need to be transmitted to their site of action within the nucleus. The nucleocytoplasmic shuttling of transcription factors is a mechanism mediating this process. The activation and inactivation...... of the transcriptional response is essential for cells to progress through the cell cycle in a normal manner. The involvement of cytoplasmic and nuclear accessory molecules, and the general nuclear membrane transport components, are essential for this process. Although nuclear import and export for different...... transcription factor families are regulated by similar mechanisms, there are several differences that allow for the specific activation of each transcription factor. This review discusses the general import and export pathways found to be common amongst many different transcription factors, and highlights...

  6. Transcriptional Silencing of Retroviral Vectors

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M.; Pedersen, F.S.

    1996-01-01

    . Extinction of long-term vector expression has been observed after implantation of transduced hematopoietic cells as well as fibroblasts, myoblasts and hepatocytes. Here we review the influence of vector structure, integration site and cell type on transcriptional silencing. While down-regulation of proviral...... transcription is known from a number of cellular and animal models, major insight has been gained from studies in the germ line and embryonal cells of the mouse. Key elements for the transfer and expression of retroviral vectors, such as the viral transcriptional enhancer and the binding site for the t......RNA primer for reverse transcription may have a major influence on transcriptional silencing. Alterations of these elements of the vector backbone as well as the use of internal promoter elements from housekeeping genes may contribute to reduce transcriptional silencing. The use of cell culture and animal...

  7. DNA topology and transcription.

    Science.gov (United States)

    Kouzine, Fedor; Levens, David; Baranello, Laura

    2014-01-01

    Chromatin is a complex assembly that compacts DNA inside the nucleus while providing the necessary level of accessibility to regulatory factors conscripted by cellular signaling systems. In this superstructure, DNA is the subject of mechanical forces applied by variety of molecular motors. Rather than being a rigid stick, DNA possesses dynamic structural variability that could be harnessed during critical steps of genome functioning. The strong relationship between DNA structure and key genomic processes necessitates the study of physical constrains acting on the double helix. Here we provide insight into the source, dynamics, and biology of DNA topological domains in the eukaryotic cells and summarize their possible involvement in gene transcription. We emphasize recent studies that might inspire and impact future experiments on the involvement of DNA topology in cellular functions.

  8. Cdk phosphorylation of the Ste11 transcription factor constrains differentiation-specific transcription to G1

    DEFF Research Database (Denmark)

    Kjaerulff, Søren; Andersen, Nicoline Resen; Borup, Mia Trolle;

    2007-01-01

    Eukaryotic cells normally differentiate from G(1); here we investigate the mechanism preventing expression of differentiation-specific genes outside G(1). In fission yeast, induction of the transcription factor Ste11 triggers sexual differentiation. We find that Ste11 is only active in G(1) when...... S phase. When we mutated T82 to aspartic acid, mimicking constant phosphorylation, cells no longer underwent differentiation. Conversely, changing T82 to alanine rendered Ste11-controlled transcription constitutive through the cell cycle, and allowed mating from S phase with increased frequency...

  9. The post-transcriptional operon

    DEFF Research Database (Denmark)

    Tenenbaum, Scott A.; Christiansen, Jan; Nielsen, Henrik

    2011-01-01

    A post-transcriptional operon is a set of monocistronic mRNAs encoding functionally related proteins that are co-regulated by a group of RNA-binding proteins and/or small non-coding RNAs so that protein expression is coordinated at the post-transcriptional level. The post-transcriptional operon...... model (PTO) is used to describe data from an assortment of methods (e.g. RIP-Chip, CLIP-Chip, miRNA profiling, ribosome profiling) that globally address the functionality of mRNA. Several examples of post-transcriptional operons have been documented in the literature and demonstrate the usefulness...

  10. A quantitative validated model reveals two phases of transcriptional regulation for the gap gene giant in Drosophila.

    Science.gov (United States)

    Hoermann, Astrid; Cicin-Sain, Damjan; Jaeger, Johannes

    2016-03-15

    Understanding eukaryotic transcriptional regulation and its role in development and pattern formation is one of the big challenges in biology today. Most attempts at tackling this problem either focus on the molecular details of transcription factor binding, or aim at genome-wide prediction of expression patterns from sequence through bioinformatics and mathematical modelling. Here we bridge the gap between these two complementary approaches by providing an integrative model of cis-regulatory elements governing the expression of the gap gene giant (gt) in the blastoderm embryo of Drosophila melanogaster. We use a reverse-engineering method, where mathematical models are fit to quantitative spatio-temporal reporter gene expression data to infer the regulatory mechanisms underlying gt expression in its anterior and posterior domains. These models are validated through prediction of gene expression in mutant backgrounds. A detailed analysis of our data and models reveals that gt is regulated by domain-specific CREs at early stages, while a late element drives expression in both the anterior and the posterior domains. Initial gt expression depends exclusively on inputs from maternal factors. Later, gap gene cross-repression and gt auto-activation become increasingly important. We show that auto-regulation creates a positive feedback, which mediates the transition from early to late stages of regulation. We confirm the existence and role of gt auto-activation through targeted mutagenesis of Gt transcription factor binding sites. In summary, our analysis provides a comprehensive picture of spatio-temporal gene regulation by different interacting enhancer elements for an important developmental regulator.

  11. Nonparallel changes of growth hormone (GH) and insulin-like growth factor-I, insulin-like growth factor binding protein-3, and GH-binding protein, after craniospinal irradiation and chemotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Nivot, S.; Adan, L.; Souberbielle, J.; Rappaport, R.; Brauner, R.; Benelli, C.; Clot, J.P.; Saucet, C. [Hopital des Enfants-Malades, Paris (France); Zucker, J.M. [Institut Curie, Paris (France)

    1994-03-01

    The authors studied the GH-insulin-like growth factor-I (IGF-I) axis serially over 24-36 months in six patients with medulloblastoma who underwent surgical removal of the tumor followed by craniospinal irradiation therapy for 6 weeks and then chemotherapy for 42 weeks. Eighteen and 24 months after beginning irradiation there was a decline in the peak GH secretory response to acute stimulation with arginine/insulin hypoglycemia. Six months after irradiation and during chemotherapy there was a transient decline in IGF-I, IGF binding protein-3 (IGFBP-3), and GH-BP values (respective mean values of 56.1 {+-} 9.0 ng/mL, 1.1 {+-} 0.2 {mu}g/mL, and 7.6 {+-} 3.3% of radioactivity as compared to time 0 values: 139 {+-} 15 ng/mL, 2.2 {+-} 0.2 {mu}g/mL, and 20.0 {+-} 4.0%, P < 0.001), although provoked GH secretion was normal at this time. The IGF-I, IGFBP-3, and GH-BP returned to pretreatment ranges by 12-36 months after initiation of the study. There was also a decline in body mass index and serum protein values at 6 months after irradiation in ligand and immunoblot analysis there was a decline in IGFBP-3 and an abnormal electrophoretic mobility of IGFBP-2 that were both normalized at 36 months. In one patient they observed a high level of IGFBP-3 proteolysis at this time. This study demonstrates that before the decrease of GH secretion in patients receiving cranial irradiation there is a transient phase of GH insensitivity that may be characteristic of the acute therapeutic phase including the chemotherapy. This partial insensitivity may explain the early growth retardation observed in these patients. 28 refs., 4 figs., 1 tab.

  12. Transcriptional networks in epithelial-mesenchymal transition.

    Directory of Open Access Journals (Sweden)

    Christo Venkov

    Full Text Available Epithelial-mesenchymal transition (EMT changes polarized epithelial cells into migratory phenotypes associated with loss of cell-cell adhesion molecules and cytoskeletal rearrangements. This form of plasticity is seen in mesodermal development, fibroblast formation, and cancer metastasis.Here we identify prominent transcriptional networks active during three time points of this transitional process, as epithelial cells become fibroblasts. DNA microarray in cultured epithelia undergoing EMT, validated in vivo, were used to detect various patterns of gene expression. In particular, the promoter sequences of differentially expressed genes and their transcription factors were analyzed to identify potential binding sites and partners. The four most frequent cis-regulatory elements (CREs in up-regulated genes were SRY, FTS-1, Evi-1, and GC-Box, and RNA inhibition of the four transcription factors, Atf2, Klf10, Sox11, and SP1, most frequently binding these CREs, establish their importance in the initiation and propagation of EMT. Oligonucleotides that block the most frequent CREs restrain EMT at early and intermediate stages through apoptosis of the cells.Our results identify new transcriptional interactions with high frequency CREs that modulate the stability of cellular plasticity, and may serve as targets for modulating these transitional states in fibroblasts.

  13. Mastering Transcription: Multiplexed Analysis of Transcription Start Site Sequences.

    Science.gov (United States)

    Hochschild, Ann

    2015-12-17

    In this issue of Molecular Cell, Vvedenskaya et al. (2015) describe a high-throughput sequencing-based methodology for the massively parallel analysis of transcription from a high-complexity barcoded template library both in vitro and in vivo, providing a powerful new tool for the study of transcription.

  14. Gene Transcription Profile of the Detached Retina (An AOS Thesis)

    Science.gov (United States)

    Zacks, David N.

    2009-01-01

    Purpose: Separation of the neurosensory retina from the retinal pigment epithelium (RPE) yields many morphologic and functional consequences, including death of the photoreceptor cells, Müller cell hypertrophy, and inner retinal rewiring. Many of these changes are due to the separation-induced activation of specific genes. In this work, we define the gene transcription profile within the retina as a function of time after detachment. We also define the early activation of kinases that might be responsible for the detachment-induced changes in gene transcription. Methods: Separation of the retina from the RPE was induced in Brown-Norway rats by the injection of 1% hyaluronic acid into the subretinal space. Retinas were harvested at 1, 7, and 28 days after separation. Gene transcription profiles for each time point were determined using the Affymetrix Rat 230A gene microarray chip. Transcription levels in detached retinas were compared to those of nondetached retinas with the BRB-ArrayTools Version 3.6.0 using a random variance analysis of variance (ANOVA) model. Confirmation of the significant transcriptional changes for a subset of the genes was performed using microfluidic quantitative real-time polymerase chain reaction (qRT-PCR) assays. Kinase activation was explored using Western blot analysis to look for early phosphorylation of any of the 3 main families of mitogen-activated protein kinases (MAPK): the p38 family, the Janus kinase family, and the p42/p44 family. Results: Retinas separated from the RPE showed extensive alterations in their gene transcription profile. Many of these changes were initiated as early as 1 day after separation, with significant increases by 7 days. ANOVA analysis defined 144 genes that had significantly altered transcription levels as a function of time after separation when setting a false discovery rate at ≤0.1. Confirmatory RT-PCR was performed on 51 of these 144 genes. Differential transcription detected on the microarray

  15. Evolution of transcriptional networks in yeast: alternative teams of transcriptional factors for different species

    Directory of Open Access Journals (Sweden)

    Adriana Muñoz

    2016-11-01

    Full Text Available Abstract Background The diversity in eukaryotic life reflects a diversity in regulatory pathways. Nocedal and Johnson argue that the rewiring of gene regulatory networks is a major force for the diversity of life, that changes in regulation can create new species. Results We have created a method (based on our new “ping-pong algorithm for detecting more complicated rewirings, where several transcription factors can substitute for one or more transcription factors in the regulation of a family of co-regulated genes. An example is illustrative. A rewiring has been reported by Hogues et al. that RAP1 in Saccharomyces cerevisiae substitutes for TBF1/CBF1 in Candida albicans for ribosomal RP genes. There one transcription factor substitutes for another on some collection of genes. Such a substitution is referred to as a “rewiring”. We agree with this finding of rewiring as far as it goes but the situation is more complicated. Many transcription factors can regulate a gene and our algorithm finds that in this example a “team” (or collection of three transcription factors including RAP1 substitutes for TBF1 for 19 genes. The switch occurs for a branch of the phylogenetic tree containing 10 species (including Saccharomyces cerevisiae, while the remaining 13 species (Candida albicans are regulated by TBF1. Conclusions To gain insight into more general evolutionary mechanisms, we have created a mathematical algorithm that finds such general switching events and we prove that it converges. Of course any such computational discovery should be validated in the biological tests. For each branch of the phylogenetic tree and each gene module, our algorithm finds a sub-group of co-regulated genes and a team of transcription factors that substitutes for another team of transcription factors. In most cases the signal will be small but in some cases we find a strong signal of switching. We report our findings for 23 Ascomycota fungi species.

  16. Controllability analysis of transcriptional regulatory networks reveals circular control patterns among transcription factors.

    Science.gov (United States)

    Österlund, Tobias; Bordel, Sergio; Nielsen, Jens

    2015-05-01

    Transcriptional regulation is the most committed type of regulation in living cells where transcription factors (TFs) control the expression of their target genes and TF expression is controlled by other TFs forming complex transcriptional regulatory networks that can be highly interconnected. Here we analyze the topology and organization of nine transcriptional regulatory networks for E. coli, yeast, mouse and human, and we evaluate how the structure of these networks influences two of their key properties, namely controllability and stability. We calculate the controllability for each network as a measure of the organization and interconnectivity of the network. We find that the number of driver nodes nD needed to control the whole network is 64% of the TFs in the E. coli transcriptional regulatory network in contrast to only 17% for the yeast network, 4% for the mouse network and 8% for the human network. The high controllability (low number of drivers needed to control the system) in yeast, mouse and human is due to the presence of internal loops in their regulatory networks where the TFs regulate each other in a circular fashion. We refer to these internal loops as circular control motifs (CCM). The E. coli transcriptional regulatory network, which does not have any CCMs, shows a hierarchical structure of the transcriptional regulatory network in contrast to the eukaryal networks. The presence of CCMs also has influence on the stability of these networks, as the presence of cycles can be associated with potential unstable steady-states where even small changes in binding affinities can cause dramatic rearrangements of the state of the network.

  17. Transcriptional and post-transcriptional profile of human chromosome 21.

    Science.gov (United States)

    Nikolaev, Sergey I; Deutsch, Samuel; Genolet, Raphael; Borel, Christelle; Parand, Leila; Ucla, Catherine; Schütz, Frederic; Duriaux Sail, Genevieve; Dupré, Yann; Jaquier-Gubler, Pascale; Araud, Tanguy; Conne, Beatrice; Descombes, Patrick; Vassalli, Jean-Dominique; Curran, Joseph; Antonarakis, Stylianos E

    2009-08-01

    Recent studies have demonstrated extensive transcriptional activity across the human genome, a substantial fraction of which is not associated with any functional annotation. However, very little is known regarding the post-transcriptional processes that operate within the different classes of RNA molecules. To characterize the post-transcriptional properties of expressed sequences from human chromosome 21 (HSA21), we separated RNA molecules from three cell lines (GM06990, HeLa S3, and SK-N-AS) according to their ribosome content by sucrose gradient fractionation. Polyribosomal-associated RNA and total RNA were subsequently hybridized to genomic tiling arrays. We found that approximately 50% of the transcriptional signals were located outside of annotated exons and were considered as TARs (transcriptionally active regions). Although TARs were observed among polysome-associated RNAs, RT-PCR and RACE experiments revealed that approximately 40% were likely to represent nonspecific cross-hybridization artifacts. Bioinformatics discrimination of TARs according to conservation and sequence complexity allowed us to identify a set of high-confidence TARs. This set of TARs was significantly depleted in the polysomes, suggesting that it was not likely to be involved in translation. Analysis of polysome representation of RefSeq exons showed that at least 15% of RefSeq transcripts undergo significant post-transcriptional regulation in at least two of the three cell lines tested. Among the regulated transcripts, enrichment analysis revealed an over-representation of genes involved in Alzheimer's disease (AD), including APP and the BACE1 protease that cleaves APP to produce the pathogenic beta 42 peptide. We demonstrate that the combination of RNA fractionation and tiling arrays is a powerful method to assess the transcriptional and post-transcriptional properties of genomic regions.

  18. Changes in barrier health status of the gill for grass carp (Ctenopharyngodon idella) during valine deficiency: Regulation of tight junction protein transcript, antioxidant status and apoptosis-related gene expression.

    Science.gov (United States)

    Feng, Lin; Luo, Jian-Bo; Jiang, Wei-Dan; Liu, Yang; Wu, Pei; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Zhang, Yong-An; Zhou, Xiao-Qiu

    2015-08-01

    This study investigated the effects of dietary valine on tight junction protein transcription, antioxidant status and apoptosis on grass carp gills (Ctenopharyngodon idella). Fish were fed six different experimental diets containing graded levels of valine (4.3, 8.0, 10.6, 13.1, 16.7, 19.1 g/kg). The results indicated that valine deficiency decreased Claudin b, Claudin 3, Occludin and ZO-1 transcription and increased Claudin 15 expression in the fish gill (P valine deficiency and valine supplementation did not have a significant effect on Claudin c and Claudin 12 expression in grass carp gills (P > 0.05). Valine deficiency also disrupted antioxidant status in the gill by decreasing anti-superoxide radicals and hydroxyl radical capacity, glutathione contents and the activities and mRNA levels of Cu/Zn superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) (P valine deficiency induced DNA fragmentation via the up-regulation of Caspase 3, Caspase 8 and Caspase 9 expressions (P valine deficiency impaired the structural integrity of fish gill by disrupted fish antioxidant defenses and regulating the expression of tight junction protein, cytokines, antioxidant enzymes, NF-κB p65, IκBα, TOR, Nrf2, Keap1 and apoptosis-related genes in the fish gill.

  19. Elucidating MicroRNA Regulatory Networks Using Transcriptional, Post-transcriptional, and Histone Modification Measurements

    Directory of Open Access Journals (Sweden)

    Sara J.C. Gosline

    2016-01-01

    Full Text Available MicroRNAs (miRNAs regulate diverse biological processes by repressing mRNAs, but their modest effects on direct targets, together with their participation in larger regulatory networks, make it challenging to delineate miRNA-mediated effects. Here, we describe an approach to characterizing miRNA-regulatory networks by systematically profiling transcriptional, post-transcriptional and epigenetic activity in a pair of isogenic murine fibroblast cell lines with and without Dicer expression. By RNA sequencing (RNA-seq and CLIP (crosslinking followed by immunoprecipitation sequencing (CLIP-seq, we found that most of the changes induced by global miRNA loss occur at the level of transcription. We then introduced a network modeling approach that integrated these data with epigenetic data to identify specific miRNA-regulated transcription factors that explain the impact of miRNA perturbation on gene expression. In total, we demonstrate that combining multiple genome-wide datasets spanning diverse regulatory modes enables accurate delineation of the downstream miRNA-regulated transcriptional network and establishes a model for studying similar networks in other systems.

  20. Elucidating MicroRNA Regulatory Networks Using Transcriptional, Post-transcriptional, and Histone Modification Measurements.

    Science.gov (United States)

    Gosline, Sara J C; Gurtan, Allan M; JnBaptiste, Courtney K; Bosson, Andrew; Milani, Pamela; Dalin, Simona; Matthews, Bryan J; Yap, Yoon S; Sharp, Phillip A; Fraenkel, Ernest

    2016-01-12

    MicroRNAs (miRNAs) regulate diverse biological processes by repressing mRNAs, but their modest effects on direct targets, together with their participation in larger regulatory networks, make it challenging to delineate miRNA-mediated effects. Here, we describe an approach to characterizing miRNA-regulatory networks by systematically profiling transcriptional, post-transcriptional and epigenetic activity in a pair of isogenic murine fibroblast cell lines with and without Dicer expression. By RNA sequencing (RNA-seq) and CLIP (crosslinking followed by immunoprecipitation) sequencing (CLIP-seq), we found that most of the changes induced by global miRNA loss occur at the level of transcription. We then introduced a network modeling approach that integrated these data with epigenetic data to identify specific miRNA-regulated transcription factors that explain the impact of miRNA perturbation on gene expression. In total, we demonstrate that combining multiple genome-wide datasets spanning diverse regulatory modes enables accurate delineation of the downstream miRNA-regulated transcriptional network and establishes a model for studying similar networks in other systems.

  1. Transcriptional networks controlling adipocyte differentiation

    DEFF Research Database (Denmark)

    Siersbæk, R; Mandrup, Susanne

    2011-01-01

    Adipocyte differentiation is regulated by a complex cascade of signals that drive the transcriptional reprogramming of the fibroblastic precursors. Genome-wide analyses of chromatin accessibility and binding of adipogenic transcription factors make it possible to generate "snapshots" of the trans...

  2. Transcriptional responses and regulations to deficient phosphorus in plants

    Institute of Scientific and Technical Information of China (English)

    Jinxiang BAO; Shuhua ZHANG; Wenjing LU; Chengjin GUO; Juntao GU; Kai XIAO

    2009-01-01

    Significant progress has been made over the past several years in the understanding of phosphorus (Pi)-starvation responses in plants and their regulation. The transcriptional changes that occur in response to Pi starvation are beginning to be revealed, although much is left to understand about their significance. In this paper, the recent progresses on the gene expression changes under deficient-Pi, cis-regulatory elements involved in response to deficient-Pi, the transcriptional control of Pi-starvation responses in eukaryotes, transcription factors involved in response to Pi-starvation, the role of MicroRNA on regulation of phosphate homeostasis, and phosphate sensing and signal transduction in plants have been summarized. The purpose of this review is to provide some basis for further elucidation of the transcriptional responses and regulations, and the networks of Pi sensing and signal transduction under deficient-Pi in plants in the future.

  3. Dataset of transcriptional landscape of B cell early activation

    Directory of Open Access Journals (Sweden)

    Alexander S. Garruss

    2015-09-01

    Full Text Available Signaling via B cell receptors (BCR and Toll-like receptors (TLRs result in activation of B cells with distinct physiological outcomes, but transcriptional regulatory mechanisms that drive activation and distinguish these pathways remain unknown. At early time points after BCR and TLR ligand exposure, 0.5 and 2 h, RNA-seq was performed allowing observations on rapid transcriptional changes. At 2 h, ChIP-seq was performed to allow observations on important regulatory mechanisms potentially driving transcriptional change. The dataset includes RNA-seq, ChIP-seq of control (Input, RNA Pol II, H3K4me3, H3K27me3, and a separate RNA-seq for miRNA expression, which can be found at Gene Expression Omnibus Dataset GSE61608. Here, we provide details on the experimental and analysis methods used to obtain and analyze this dataset and to examine the transcriptional landscape of B cell early activation.

  4. Biophysics and bioinformatics of transcription regulation in bacteria and bacteriophages

    Science.gov (United States)

    Djordjevic, Marko

    2005-11-01

    Due to rapid accumulation of biological data, bioinformatics has become a very important branch of biological research. In this thesis, we develop novel bioinformatic approaches and aid design of biological experiments by using ideas and methods from statistical physics. Identification of transcription factor binding sites within the regulatory segments of genomic DNA is an important step towards understanding of the regulatory circuits that control expression of genes. We propose a novel, biophysics based algorithm, for the supervised detection of transcription factor (TF) binding sites. The method classifies potential binding sites by explicitly estimating the sequence-specific binding energy and the chemical potential of a given TF. In contrast with the widely used information theory based weight matrix method, our approach correctly incorporates saturation in the transcription factor/DNA binding probability. This results in a significant reduction in the number of expected false positives, and in the explicit appearance---and determination---of a binding threshold. The new method was used to identify likely genomic binding sites for the Escherichia coli TFs, and to examine the relationship between TF binding specificity and degree of pleiotropy (number of regulatory targets). We next address how parameters of protein-DNA interactions can be obtained from data on protein binding to random oligos under controlled conditions (SELEX experiment data). We show that 'robust' generation of an appropriate data set is achieved by a suitable modification of the standard SELEX procedure, and propose a novel bioinformatic algorithm for analysis of such data. Finally, we use quantitative data analysis, bioinformatic methods and kinetic modeling to analyze gene expression strategies of bacterial viruses. We study bacteriophage Xp10 that infects rice pathogen Xanthomonas oryzae. Xp10 is an unusual bacteriophage, which has morphology and genome organization that most closely

  5. Transcriptional profiling of epidermal differentiation.

    Science.gov (United States)

    Radoja, Nada; Gazel, Alix; Banno, Tomohiro; Yano, Shoichiro; Blumenberg, Miroslav

    2006-10-03

    In epidermal differentiation basal keratinocytes detach from the basement membrane, stop proliferating, and express a new set of structural proteins and enzymes, which results in an impermeable protein/lipid barrier that protects us. To define the transcriptional changes essential for this process, we purified large quantities of basal and suprabasal cells from human epidermis, using the expression of beta4 integrin as the discriminating factor. The expected expression differences in cytoskeletal, cell cycle, and adhesion genes confirmed the effective separation of the cell populations. Using DNA microarray chips, we comprehensively identify the differences in genes expressed in basal and differentiating layers of the epidermis, including the ECM components produced by the basal cells, the proteases in both the basal and suprabasal cells, and the lipid and steroid metabolism enzymes in suprabasal cells responsible for the permeability barrier. We identified the signaling pathways specific for the two populations and found two previously unknown paracrine and one juxtacrine signaling pathway operating between the basal and suprabasal cells. Furthermore, using specific expression signatures, we identified a new set of late differentiation markers and mapped their chromosomal loci, as well as a new set of melanocyte-specific markers. The data represent a quantum jump in understanding the mechanisms of epidermal differentiation.

  6. Nickel-responsive transcriptional regulators.

    Science.gov (United States)

    Musiani, Francesco; Zambelli, Barbara; Bazzani, Micaela; Mazzei, Luca; Ciurli, Stefano

    2015-09-01

    Nickel is an essential micronutrient for a large number of living organisms, but it is also a toxic metal ion when it accumulates beyond the sustainable level as it may result if and when its cellular trafficking is not properly governed. Therefore, the homeostasis and metabolism of nickel is tightly regulated through metal-specific protein networks that respond to the available Ni(II) concentration. These are directed by specific nickel sensors, able to couple Ni(II) binding to a change in their DNA binding affinity and/or specificity, thus translating the cellular level of Ni(II) into a modification of the expression of the proteins devoted to modulating nickel uptake, efflux and cellular utilization. This review describes the Ni(II)-dependent transcriptional regulators discovered so far, focusing on their structural features, metal coordination modes and metal binding thermodynamics. Understanding these properties is essential to comprehend how these sensors correlate nickel availability to metal coordination and functional responses. A broad and comparative study, described here, reveals some general traits that characterize the binding stoichiometry and Ni(II) affinity of these metallo-sensors.

  7. New PAH gene promoter KLF1 and 3'-region C/EBPalpha motifs influence transcription in vitro.

    Science.gov (United States)

    Klaassen, Kristel; Stankovic, Biljana; Kotur, Nikola; Djordjevic, Maja; Zukic, Branka; Nikcevic, Gordana; Ugrin, Milena; Spasovski, Vesna; Srzentic, Sanja; Pavlovic, Sonja; Stojiljkovic, Maja

    2017-02-01

    Phenylketonuria (PKU) is a metabolic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. Although the PAH genotype remains the main determinant of PKU phenotype severity, genotype-phenotype inconsistencies have been reported. In this study, we focused on unanalysed sequences in non-coding PAH gene regions to assess their possible influence on the PKU phenotype. We transiently transfected HepG2 cells with various chloramphenicol acetyl transferase (CAT) reporter constructs which included PAH gene non-coding regions. Selected non-coding regions were indicated by in silico prediction to contain transcription factor binding sites. Furthermore, electrophoretic mobility shift assay (EMSA) and supershift assays were performed to identify which transcriptional factors were engaged in the interaction. We found novel KLF1 motif in the PAH promoter, which decreases CAT activity by 50 % in comparison to basal transcription in vitro. The cytosine at the c.-170 promoter position creates an additional binding site for the protein complex involving KLF1 transcription factor. Moreover, we assessed for the first time the role of a multivariant variable number tandem repeat (VNTR) region located in the 3'-region of the PAH gene. We found that the VNTR3, VNTR7 and VNTR8 constructs had approximately 60 % of CAT activity. The regulation is mediated by the C/EBPalpha transcription factor, present in protein complex binding to VNTR3. Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the PAH gene which decrease transcription in vitro and, thus, could be considered as PAH expression modifiers. New transcription motifs in non-coding regions will contribute to better understanding of the PKU phenotype complexity and may become important for the optimisation of PKU treatment.

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