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Sample records for arx transcriptional targets

  1. Identification of Arx targets unveils new candidates for controlling cortical interneuron migration and differentiation

    Directory of Open Access Journals (Sweden)

    Gaelle M Friocourt

    2011-12-01

    Full Text Available Mutations in the homeobox transcription factor ARX have been found to be responsible for a wide spectrum of disorders extending from phenotypes with severe neuronal migration defects, such as lissencephaly, to mild forms of intellectual disabilities without apparent brain abnormalities, but with associated features of dystonia and epilepsy. Arx expression is mainly restricted to populations of GABA-containing neurons. Studies of the effects of ARX loss of function, either in humans or mutant mice, revealed varying defects, suggesting multiple roles of this gene in brain patterning, neuronal proliferation and migration, cell maturation and differentiation, as well as axonal outgrowth and connectivity. However, to date, little is known about how Arx functions as a transcription factor or which genes it binds and regulates. Recently, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified approximately 1000 gene promoters bound by Arx in transfected neuroblastoma N2a cells and mouse embryonic brain. To narrow the analysis of Arx targets to those most likely to control cortical interneuron migration and/or differentiation, we compare here our data to previously published studies searching for genes enriched or down-regulated in cortical interneurons between E13.5 and E15.5. We thus identified 14 Arx-target genes enriched (Cxcr7, Meis1, Ppap2a, Slc12a5, Ets2, Phlda1, Zif268, Igf1, Lmo3, Sema6, Lgi1, Alk, Tgfb3, Napb and 5 genes specifically down-regulated (Hmgn3, Lmo1, Ebf3, Rasgef1b and Slit2 in cortical migrating neurons. In this review, we present these genes and discuss how their possible regulation by Arx may lead to the dysfunction of GABAergic neurons, resulting in mental retardation and epilepsy.

  2. MUTATIONS IN THE ARX GENE: CLINICAL, ELECTROENCEPHALOGRAPHIC AND NEUROIMAGING FEATURES IN 3 PATIENTS

    Directory of Open Access Journals (Sweden)

    I. V. Ivanova

    2017-01-01

    Full Text Available The Aristaless-related homeobox (ARX gene is a member of the paired-type homeodomain transcription factor family with critical roles in embryonic development, particularly in the developing brain. Mutations in ARX gene demonstrate striking intra- and interfamilial pleiotropy together with genetic heterogeneity and lead to a broad spectrum of diseases. They give rise to 4 key phenotypic features: a different types of brain malformation, abnormal genitalia, epilepsy and intellectual disability. Authors present 3 clinical cases: a girl with duplication on the short arm of X-chromosome (Xp11.22-p22.33, which include genes ARX and CDKL5; a girl and a boy with a missense mutation in ARX gene that have not been previously described (chrX:25031522C>A, causes the substitution of an amino acid in the 197 protein position (p.Gly197Val, NM_139058.2. All patients suffer from severe epilepsy, that is refractory to antiepileptic drugs, and all of them have different degrees of psychomotor delay. The patients with missense mutation also have movement disorders: stereotypic movements in the girl and choreo athetosis and dystonia in the boy. Electroencephalographic abnormalities have been identified in all patients, and there were not significant abnormalities on magnetic resonance imaging in all cases. The described cases broaden the clinical spectrum of mutations in ARX gene.

  3. Microarray-Based Identification of Transcription Factor Target Genes

    NARCIS (Netherlands)

    Gorte, M.; Horstman, A.; Page, R.B.; Heidstra, R.; Stromberg, A.; Boutilier, K.A.

    2011-01-01

    Microarray analysis is widely used to identify transcriptional changes associated with genetic perturbation or signaling events. Here we describe its application in the identification of plant transcription factor target genes with emphasis on the design of suitable DNA constructs for controlling TF

  4. Comparison of ARX and ARMAX models for thermoelectric refrigerator

    International Nuclear Information System (INIS)

    Saifizi, M; Yaacob, Sazali; Mohamad, M S; Ab Muin, M Z

    2013-01-01

    System identification is a procedure to characterize the dynamic behavior of a system, a system component based on measured data. This paper presents a study of the modeling and parameter identification of a refrigerator process by using the mathematical black-box modeling technique- ARX and ARMAX structure. A general ARX and ARMAX structure has been constructed for usual thermoelectric refrigerator systems and the Recursive Least Square (RLS) process is used for model parameters identification. The capabilities of the model constructed compared with that of ARX and ARMAX model. Several compositions of ARX and ARMAX orders and forgetting factor are analyzed for thermoelectric air-conditioning model to examine the optimal adjustments that improve their efficiency of PID controller

  5. Role of ARX Gene in Infantile Spasms and Dystonia

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2007-08-01

    Full Text Available The role of ARX gene in a syndrome of infantile spasms with generalized dystonia was investigated in 6 boys from 4 families at the University of Florence, Italy, and other centers in Italy, Japan, and USA.

  6. Targeted genome regulation via synthetic programmable transcriptional regulators

    KAUST Repository

    Piatek, Agnieszka Anna

    2016-04-19

    Regulation of gene transcription controls cellular functions and coordinates responses to developmental, physiological and environmental cues. Precise and efficient molecular tools are needed to characterize the functions of single and multiple genes in linear and interacting pathways in a native context. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like proteins (TALE) are amenable to bioengineering to bind DNA target sequences of interest. As a result, ZF and TALE proteins were used to develop synthetic programmable transcription factors. However, these systems are limited by the requirement to re-engineer proteins for each new target sequence. The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9) genome editing tool was recently repurposed for targeted transcriptional regulation by inactivation of the nuclease activity of Cas9. Due to the facile engineering, simplicity, precision and amenability to library construction, the CRISPR/Cas9 system is poised to revolutionize the functional genomics field across diverse eukaryotic species. In this review, we discuss the development of synthetic customizable transcriptional regulators and provide insights into their current and potential applications, with special emphasis on plant systems, in characterization of gene functions, elucidation of molecular mechanisms and their biotechnological applications. © 2016 Informa UK Limited, trading as Taylor & Francis Group

  7. Targeted genome regulation via synthetic programmable transcriptional regulators

    KAUST Repository

    Piatek, Agnieszka Anna; Mahfouz, Magdy M.

    2016-01-01

    genes in linear and interacting pathways in a native context. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like proteins (TALE) are amenable to bioengineering to bind DNA target sequences of interest. As a result, ZF

  8. 78 FR 63559 - Order Of Suspension of Trading; In the Matter of ARX Gold Corp.

    Science.gov (United States)

    2013-10-24

    ... of ARX Gold Corp. October 22, 2013. It appears to the Securities and Exchange Commission (``Commission'') that there is a lack of current and accurate information concerning the securities of ARX Gold Corp. (``ARX Gold''), quoted under the ticker symbol DUCP, because of questions regarding the...

  9. Signatures of DNA target selectivity by ETS transcription factors.

    Science.gov (United States)

    Poon, Gregory M K; Kim, Hye Mi

    2017-05-27

    The ETS family of transcription factors is a functionally heterogeneous group of gene regulators that share a structurally conserved, eponymous DNA-binding domain. DNA target specificity derives from combinatorial interactions with other proteins as well as intrinsic heterogeneity among ETS domains. Emerging evidence suggests molecular hydration as a fundamental feature that defines the intrinsic heterogeneity in DNA target selection and susceptibility to epigenetic DNA modification. This perspective invokes novel hypotheses in the regulation of ETS proteins in physiologic osmotic stress, their pioneering potential in heterochromatin, and the effects of passive and pharmacologic DNA demethylation on ETS regulation.

  10. Transcription profile of Escherichia coli: genomic SELEX search for regulatory targets of transcription factors.

    Science.gov (United States)

    Ishihama, Akira; Shimada, Tomohiro; Yamazaki, Yukiko

    2016-03-18

    Bacterial genomes are transcribed by DNA-dependent RNA polymerase (RNAP), which achieves gene selectivity through interaction with sigma factors that recognize promoters, and transcription factors (TFs) that control the activity and specificity of RNAP holoenzyme. To understand the molecular mechanisms of transcriptional regulation, the identification of regulatory targets is needed for all these factors. We then performed genomic SELEX screenings of targets under the control of each sigma factor and each TF. Here we describe the assembly of 156 SELEX patterns of a total of 116 TFs performed in the presence and absence of effector ligands. The results reveal several novel concepts: (i) each TF regulates more targets than hitherto recognized; (ii) each promoter is regulated by more TFs than hitherto recognized; and (iii) the binding sites of some TFs are located within operons and even inside open reading frames. The binding sites of a set of global regulators, including cAMP receptor protein, LeuO and Lrp, overlap with those of the silencer H-NS, suggesting that certain global regulators play an anti-silencing role. To facilitate sharing of these accumulated SELEX datasets with the research community, we compiled a database, 'Transcription Profile of Escherichia coli' (www.shigen.nig.ac.jp/ecoli/tec/). © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Targeted deficiency of the transcriptional activator Hnf1alpha alters subnuclear positioning of its genomic targets.

    Directory of Open Access Journals (Sweden)

    Reini F Luco

    2008-05-01

    Full Text Available DNA binding transcriptional activators play a central role in gene-selective regulation. In part, this is mediated by targeting local covalent modifications of histone tails. Transcriptional regulation has also been associated with the positioning of genes within the nucleus. We have now examined the role of a transcriptional activator in regulating the positioning of target genes. This was carried out with primary beta-cells and hepatocytes freshly isolated from mice lacking Hnf1alpha, an activator encoded by the most frequently mutated gene in human monogenic diabetes (MODY3. We show that in Hnf1a-/- cells inactive endogenous Hnf1alpha-target genes exhibit increased trimethylated histone H3-Lys27 and reduced methylated H3-Lys4. Inactive Hnf1alpha-targets in Hnf1a-/- cells are also preferentially located in peripheral subnuclear domains enriched in trimethylated H3-Lys27, whereas active targets in wild-type cells are positioned in more central domains enriched in methylated H3-Lys4 and RNA polymerase II. We demonstrate that this differential positioning involves the decondensation of target chromatin, and show that it is spatially restricted rather than a reflection of non-specific changes in the nuclear organization of Hnf1a-deficient cells. This study, therefore, provides genetic evidence that a single transcriptional activator can influence the subnuclear location of its endogenous genomic targets in primary cells, and links activator-dependent changes in local chromatin structure to the spatial organization of the genome. We have also revealed a defect in subnuclear gene positioning in a model of a human transcription factor disease.

  12. The Drosophila Helicase MLE Targets Hairpin Structures in Genomic Transcripts.

    Directory of Open Access Journals (Sweden)

    Simona Cugusi

    2016-01-01

    Full Text Available RNA hairpins are a common type of secondary structures that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi and microRNA (miRNA pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE was shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. Here we report that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, we show that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. We use two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway.

  13. Targeting HOX and PBX transcription factors in ovarian cancer

    International Nuclear Information System (INIS)

    Morgan, Richard; Plowright, Lynn; Harrington, Kevin J; Michael, Agnieszka; Pandha, Hardev S

    2010-01-01

    Ovarian cancer still has a relatively poor prognosis due to the frequent occurrence of drug resistance, making the identification of new therapeutic targets an important goal. We have studied the role of HOX genes in the survival and proliferation of ovarian cancer cells. These are a family of homeodomain-containing transcription factors that determine cell and tissue identity in the early embryo, and have an anti-apoptotic role in a number of malignancies including lung and renal cancer. We used QPCR to determine HOX gene expression in normal ovary and in the ovarian cancer cell lines SK-OV3 and OV-90. We used a short peptide, HXR9, to disrupt the formation of HOX/PBX dimers and alter transcriptional regulation by HOX proteins. In this study we show that the ovarian cancer derived line SK-OV3, but not OV-90, exhibits highly dysregulated expression of members of the HOX gene family. Disrupting the interaction between HOX proteins and their co-factor PBX induces apoptosis in SK-OV3 cells and retards tumour growth in vivo. HOX/PBX binding is a potential target in ovarian cancer

  14. Adaptive MPC based on MIMO ARX-Laguerre model.

    Science.gov (United States)

    Ben Abdelwahed, Imen; Mbarek, Abdelkader; Bouzrara, Kais

    2017-03-01

    This paper proposes a method for synthesizing an adaptive predictive controller using a reduced complexity model. This latter is given by the projection of the ARX model on Laguerre bases. The resulting model is entitled MIMO ARX-Laguerre and it is characterized by an easy recursive representation. The adaptive predictive control law is computed based on multi-step-ahead finite-element predictors, identified directly from experimental input/output data. The model is tuned in each iteration by an online identification algorithms of both model parameters and Laguerre poles. The proposed approach avoids time consuming numerical optimization algorithms associated with most common linear predictive control strategies, which makes it suitable for real-time implementation. The method is used to synthesize and test in numerical simulations adaptive predictive controllers for the CSTR process benchmark. Copyright © 2016 ISA. Published by Elsevier Ltd. All rights reserved.

  15. Ar-Xe Laser: The Path to a Robust, All-Electric Shipboard Directed Energy Weapon

    National Research Council Canada - National Science Library

    Apruzese, J. P; Sethian, J. D; Giuliani, J. L; Wolford, M. F

    2008-01-01

    .... The electron beam pumped Ar-Xe laser has been investigated in an ONR-sponsored 6.1 program at NRL. The results of this program are summarized in this Memorandum Report, and indicate that the Ar-Xe laser has strong potential to meet these requirements...

  16. Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

    Science.gov (United States)

    Osato, Naoki

    2018-01-19

    Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional

  17. Vibrio elicits targeted transcriptional responses from copepod hosts.

    Science.gov (United States)

    Almada, Amalia A; Tarrant, Ann M

    2016-06-01

    Copepods are abundant crustaceans that harbor diverse bacterial communities, yet the nature of their interactions with microbiota are poorly understood. Here, we report that Vibrio elicits targeted transcriptional responses in the estuarine copepod Eurytemora affinis We pre-treated E. affinis with an antibiotic cocktail and exposed them to either a zooplankton specialist (Vibrio sp. F10 9ZB36) or a free-living species (Vibrio ordalii 12B09) for 24 h. We then identified via RNA-Seq a total of 78 genes that were differentially expressed following Vibrio exposure, including homologs of C-type lectins, chitin-binding proteins and saposins. The response differed between the two Vibrio treatments, with the greatest changes elicited upon inoculation with V. sp. F10 We suggest that these differentially regulated genes play important roles in cuticle integrity, the innate immune response, and general stress response, and that their expression may enable E. affinis to recognize and regulate symbiotic vibrios. We further report that V. sp. F10 culturability is specifically altered upon colonization of E. affinis These findings suggest that rather than acting as passive environmental vectors, copepods discriminately interact with vibrios, which may ultimately impact the abundance and activity of copepod-associated bacteria. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Transcription Factors in Heart: Promising Therapeutic Targets in Cardiac Hypertrophy

    OpenAIRE

    Kohli, Shrey; Ahuja, Suchit; Rani, Vibha

    2011-01-01

    Regulation of gene expression is central to cell growth, differentiation and diseases. Context specific and signal dependent regulation of gene expression is achieved to a large part by transcription factors. Cardiac transcription factors regulate heart development and are also involved in stress regulation of the adult heart, which may lead to cardiac hypertrophy. Hypertrophy of cardiac myocytes is an outcome of the imbalance between prohypertrophic factors and anti-hypertrophic factors. Thi...

  19. A mechanistic overview of herbal medicine and botanical compounds to target transcriptional factors in Breast cancer.

    Science.gov (United States)

    Zhao, Yingke; Liu, Yue

    2018-04-01

    The abnormalities of transcription factors, such as NF-κB, STAT, estrogen receptor, play a critical role in the initiation and progression of breast cancer. Due to the limitation of current treatment, transcription factors could be promising therapeutic targets, which have received close attention. In this review, we introduced herbal medicines, as well as botanical compounds that had been verified with anti-tumor properties via regulating transcription factors. Herbs, compounds, as well as formulae reported with various transcriptional targets, were summarized thoroughly, to provide implication for the future research on basic experiment and clinical application. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Targeting Transcription Elongation Machinery for Breast Cancer Therapy

    Science.gov (United States)

    2017-05-01

    ABSTRACT: This project focuses on the important but under-studied role of the P-TEFb- dependent transcription elongation machinery in human breast...molecule CDK9 inhibitors can be used to halt breast cancer metastasis. 8 experimental groups to test various drug dosage and frequency regimes will...tumor cells, which are said to be ’ addicted ’ to this protein. Consistently, pharmacological inhibition of Hsp90 has demonstrated great promise in

  1. Genomewide identification of pheromone-targeted transcription in fission yeast

    Directory of Open Access Journals (Sweden)

    Wright Anthony

    2006-11-01

    Full Text Available Abstract Background Fission yeast cells undergo sexual differentiation in response to nitrogen starvation. In this process haploid M and P cells first mate to form diploid zygotes, which then enter meiosis and sporulate. Prior to mating, M and P cells communicate with diffusible mating pheromones that activate a signal transduction pathway in the opposite cell type. The pheromone signalling orchestrates mating and is also required for entry into meiosis. Results Here we use DNA microarrays to identify genes that are induced by M-factor in P cells and by P-factor in M-cells. The use of a cyr1 genetic background allowed us to study pheromone signalling independently of nitrogen starvation. We identified a total of 163 genes that were consistently induced more than two-fold by pheromone stimulation. Gene disruption experiments demonstrated the involvement of newly discovered pheromone-induced genes in the differentiation process. We have mapped Gene Ontology (GO categories specifically associated with pheromone induction. A direct comparison of the M- and P-factor induced expression pattern allowed us to identify cell-type specific transcripts, including three new M-specific genes and one new P-specific gene. Conclusion We found that the pheromone response was very similar in M and P cells. Surprisingly, pheromone control extended to genes fulfilling their function well beyond the point of entry into meiosis, including numerous genes required for meiotic recombination. Our results suggest that the Ste11 transcription factor is responsible for the majority of pheromone-induced transcription. Finally, most cell-type specific genes now appear to be identified in fission yeast.

  2. Apaf-1 is a transcriptional target for E2F and p53

    DEFF Research Database (Denmark)

    Moroni, M C; Hickman, E S; Lazzerini Denchi, E

    2001-01-01

    between the deregulation of the pRB pathway and apoptosis. Furthermore, because the pRB pathway is functionally inactivated in most cancers, the identification of Apaf-1 as a transcriptional target for E2F might explain the increased sensitivity of tumour cells to chemotherapy. We also show that......, independently of the pRB pathway, Apaf-1 is a direct transcriptional target of p53, suggesting that p53 might sensitize cells to apoptosis by increasing Apaf-1 levels....

  3. Identifying MicroRNAs and Transcript Targets in Jatropha Seeds

    Science.gov (United States)

    Galli, Vanessa; Guzman, Frank; de Oliveira, Luiz F. V.; Loss-Morais, Guilherme; Körbes, Ana P.; Silva, Sérgio D. A.; Margis-Pinheiro, Márcia M. A. N.; Margis, Rogério

    2014-01-01

    MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs) as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs. PMID:24551031

  4. Targeting cancer stem cells: emerging role of Nanog transcription factor

    Directory of Open Access Journals (Sweden)

    Wang ML

    2013-09-01

    Full Text Available Mong-Lien Wang,1 Shih-Hwa Chiou,2,3 Cheng-Wen Wu1,4–61Institute of Biochemistry and Molecular Biology, 2Institute of Pharmacology, National Yang Ming University, Taipei, Taiwan; 3Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan; 4Institute of Microbiology and Immunology, 5Institute of Clinical Medicine, National Yang Ming University, Taipei, Taiwan; 6Institute of Biomedical Science, Academia Sinica, Taipei, TaiwanAbstract: The involvement of stemness factors in cancer initiation and progression has drawn much attention recently, especially after the finding that introducing four stemness factors in somatic cells is able to reprogram the cells back to an embryonic stem cell-like state. Following accumulating data revealing abnormal elevated expression levels of key stemness factors, like Nanog, Oct4, and Sox2, in several types of cancer stem cells; the importance and therapeutic potential of targeting these stemness regulators in cancers has turned to research focus. Nanog determines cell fate in both embryonic and cancer stem cells; activating Nanog at an inappropriate time would result in cancer stem cells rather than normal pluripotent stem cells or differentiated somatic cells. Upregulated Nanog is correlated with poor survival outcome of patients with various types of cancer. The discoveries of downstream regulatory pathways directly or indirectly mediated by Nanog indicate that Nanog regulates several aspects of cancer development such as tumor cell proliferation, self-renewal, motility, epithelial-mesenchymal transition, immune evasion, and drug-resistance, which are all defined features for cancer stem cells. The current review paper illustrates the central role of Nanog in the regulatory networks of cancer malignant development and stemness acquirement, as well as in the communication between cancer cells and the surrounding stroma. Though a more defined model is needed to test the

  5. CDKL5 and ARX mutations in males with early-onset epilepsy.

    Science.gov (United States)

    Mirzaa, Ghayda M; Paciorkowski, Alex R; Marsh, Eric D; Berry-Kravis, Elizabeth M; Medne, Livija; Alkhateeb, Asem; Grix, Art; Wirrell, Elaine C; Powell, Berkley R; Nickels, Katherine C; Burton, Barbara; Paras, Andrea; Kim, Katherine; Chung, Wendy; Dobyns, William B; Das, Soma

    2013-05-01

    Mutations in CDKL5 and ARX are known causes of early-onset epilepsy and severe developmental delay in males and females. Although numerous males with ARX mutations associated with various phenotypes have been reported in the literature, the majority of CDKL5 mutations have been identified in females with a phenotype characterized by early-onset epilepsy, severe global developmental delay, absent speech, and stereotypic hand movements. To date, only 10 males with CDKL5 mutations have been reported. Our retrospective study reports on the clinical, neuroimaging, and molecular findings of 18 males with early-onset epilepsy caused by either CDKL5 or ARX mutations. These 18 patients include eight new males with CDKL5 mutations and 10 with ARX mutations identified through sequence analysis of 266 and 346 males, respectively, at our molecular diagnostic laboratory. Our large dataset therefore expands on the number of reported males with CDKL5 mutations and highlights that aberrations of CDKL5 and ARX combined are an important consideration in the genetic forms of early-onset epilepsy in boys. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. A PCA3 gene-based transcriptional amplification system targeting primary prostate cancer

    OpenAIRE

    Neveu, Bertrand; Jain, Pallavi; T?tu, Bernard; Wu, Lily; Fradet, Yves; Pouliot, Fr?d?ric

    2015-01-01

    Targeting specifically primary prostate cancer (PCa) cells for immune therapy, gene therapy or molecular imaging is of high importance. The PCA3 long non-coding RNA is a unique PCa biomarker and oncogene that has been widely studied. This gene has been mainly exploited as an accurate diagnostic urine biomarker for PCa detection. In this study, the PCA3 promoter was introduced into a new transcriptional amplification system named the 3-Step Transcriptional Amplification System (PCA3-3STA) and ...

  7. FATS is a transcriptional target of p53 and associated with antitumor activity

    OpenAIRE

    Zhang Xifeng; Zhang Qian; Zhang Jun; Qiu Li; Yan Shuang-shuang; Feng Juling; Sun Yan; Huang Xingxu; Lu Karen H; Li Zheng

    2010-01-01

    Abstract Frequent mutations of p53 in human cancers exemplify its crucial role as a tumor suppressor transcription factor, and p21, a transcriptional target of p53, plays a central role in surveillance of cell-cycle checkpoints. Our previous study has shown that FATS stabilize p21 to preserve genome integrity. In this study we identified a novel transcript variant of FATS (GenBank: GQ499374) through screening a cDNA library from mouse testis, which uncovered the promoter region of mouse FATS....

  8. Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems.

    Directory of Open Access Journals (Sweden)

    Julia K Bialek

    Full Text Available CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs, act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5' long terminal repeat (LTR, for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.

  9. Targeting Transcriptional Addictions in Small Cell Lung Cancer with a Covalent CDK7 Inhibitor

    DEFF Research Database (Denmark)

    Christensen, Camilla L; Kwiatkowski, Nicholas; Abraham, Brian J

    2014-01-01

    Small cell lung cancer (SCLC) is an aggressive disease with high mortality, and the identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library, we observe that SCLC is sensitive...... to transcription-targeting drugs, in particular to THZ1, a recently identified covalent inhibitor of cyclin-dependent kinase 7. We find that expression of super-enhancer-associated transcription factor genes, including MYC family proto-oncogenes and neuroendocrine lineage-specific factors, is highly vulnerability...

  10. NGF-mediated transcriptional targets of p53 in PC12 neuronal differentiation

    Directory of Open Access Journals (Sweden)

    Labhart Paul

    2007-05-01

    Full Text Available Abstract Background p53 is recognized as a critical regulator of the cell cycle and apoptosis. Mounting evidence also suggests a role for p53 in differentiation of cells including neuronal precursors. We studied the transcriptional role of p53 during nerve growth factor-induced differentiation of the PC12 line into neuron-like cells. We hypothesized that p53 contributed to PC12 differentiation through the regulation of gene targets distinct from its known transcriptional targets for apoptosis or DNA repair. Results Using a genome-wide chromatin immunoprecipitation cloning technique, we identified and validated 14 novel p53-regulated genes following NGF treatment. The data show p53 protein was transcriptionally activated and contributed to NGF-mediated neurite outgrowth during differentiation of PC12 cells. Furthermore, we describe stimulus-specific regulation of a subset of these target genes by p53. The most salient differentiation-relevant target genes included wnt7b involved in dendritic extension and the tfcp2l4/grhl3 grainyhead homolog implicated in ectodermal development. Additional targets included brk, sdk2, sesn3, txnl2, dusp5, pon3, lect1, pkcbpb15 and other genes. Conclusion Within the PC12 neuronal context, putative p53-occupied genomic loci spanned the entire Rattus norvegicus genome upon NGF treatment. We conclude that receptor-mediated p53 transcriptional activity is involved in PC12 differentiation and may suggest a contributory role for p53 in neuronal development.

  11. Transcript profiling of Elf5+/- mammary glands during pregnancy identifies novel targets of Elf5.

    Directory of Open Access Journals (Sweden)

    Renee L Rogers

    Full Text Available BACKGROUND: Elf5, an epithelial specific Ets transcription factor, plays a crucial role in the pregnancy-associated development of the mouse mammary gland. Elf5(-/- embryos do not survive, however the Elf5(+/- mammary gland displays a severe pregnancy-associated developmental defect. While it is known that Elf5 is crucial for correct mammary development and lactation, the molecular mechanisms employed by Elf5 to exert its effects on the mammary gland are largely unknown. PRINCIPAL FINDINGS: Transcript profiling was used to investigate the transcriptional changes that occur as a result of Elf5 haploinsufficiency in the Elf5(+/- mouse model. We show that the development of the mouse Elf5(+/- mammary gland is delayed at a transcriptional and morphological level, due to the delayed increase in Elf5 protein in these glands. We also identify a number of potential Elf5 target genes, including Mucin 4, whose expression, is directly regulated by the binding of Elf5 to an Ets binding site within its promoter. CONCLUSION: We identify novel transcriptional targets of Elf5 and show that Muc4 is a direct target of Elf5, further elucidating the mechanisms through which Elf5 regulates proliferation and differentiation in the mammary gland.

  12. Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein

    KAUST Repository

    Mahfouz, Magdy M.

    2011-12-14

    Transcriptional activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria when they infect plants. TALEs contain a modular DNA binding domain that can be easily engineered to bind any sequence of interest, and have been used to provide user-selected DNA-binding modules to generate chimeric nucleases and transcriptional activators in mammalian cells and plants. Here we report the use of TALEs to generate chimeric sequence-specific transcriptional repressors. The dHax3 TALE was used as a scaffold to provide a DNA-binding module fused to the EAR-repression domain (SRDX) to generate a chimeric repressor that targets the RD29A promoter. The dHax3. SRDX protein efficiently repressed the transcription of the RD29A

  13. Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein

    KAUST Repository

    Mahfouz, Magdy M.; Li, Lixin; Piatek, Marek J.; Fang, Xiaoyun; Mansour, Hicham; Bangarusamy, Dhinoth K.; Zhu, Jian-Kang

    2011-01-01

    Transcriptional activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria when they infect plants. TALEs contain a modular DNA binding domain that can be easily engineered to bind any sequence of interest, and have been used to provide user-selected DNA-binding modules to generate chimeric nucleases and transcriptional activators in mammalian cells and plants. Here we report the use of TALEs to generate chimeric sequence-specific transcriptional repressors. The dHax3 TALE was used as a scaffold to provide a DNA-binding module fused to the EAR-repression domain (SRDX) to generate a chimeric repressor that targets the RD29A promoter. The dHax3. SRDX protein efficiently repressed the transcription of the RD29A

  14. Histone Deacetylase Rpd3 Regulates Olfactory Projection Neuron Dendrite Targeting via the Transcription Factor Prospero

    Science.gov (United States)

    Tea, Joy S.; Chihara, Takahiro; Luo, Liqun

    2010-01-01

    Compared to the mechanisms of axon guidance, relatively little is known about the transcriptional control of dendrite guidance. The Drosophila olfactory system with its stereotyped organization provides an excellent model to study the transcriptional control of dendrite wiring specificity. Each projection neuron (PN) targets its dendrites to a specific glomerulus in the antennal lobe and its axon stereotypically to higher brain centers. Using a forward genetic screen, we identified a mutation in Rpd3 that disrupts PN targeting specificity. Rpd3 encodes a class I histone deacetylase (HDAC) homologous to mammalian HDAC1 and HDAC2. Rpd3−/− PN dendrites that normally target to a dorsolateral glomerulus mistarget to medial glomeruli in the antennal lobe, and axons exhibit a severe overbranching phenotype. These phenotypes can be rescued by postmitotic expression of Rpd3 but not HDAC3, the only other class I HDAC in Drosophila. Furthermore, disruption of the atypical homeodomain transcription factor Prospero (Pros) yields similar phenotypes, which can be rescued by Pros expression in postmitotic neurons. Strikingly, overexpression of Pros can suppress Rpd3−/− phenotypes. Our study suggests a specific function for the general chromatin remodeling factor Rpd3 in regulating dendrite targeting in neurons, largely through the postmitotic action of the Pros transcription factor. PMID:20660276

  15. Computational design and application of endogenous promoters for transcriptionally targeted gene therapy for rheumatoid arthritis.

    NARCIS (Netherlands)

    Geurts, J.; Joosten, L.A.B.; Takahashi, N.; Arntz, O.J.; Gluck, A.; Bennink, M.B.; Berg, W.B. van den; Loo, F.A.J. van de

    2009-01-01

    The promoter regions of genes that are differentially regulated in the synovial membrane during the course of rheumatoid arthritis (RA) represent attractive candidates for application in transcriptionally targeted gene therapy. In this study, we applied an unbiased computational approach to define

  16. saRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription.

    Science.gov (United States)

    Portnoy, Victoria; Lin, Szu Hua Sharon; Li, Kathy H; Burlingame, Alma; Hu, Zheng-Hui; Li, Hao; Li, Long-Cheng

    2016-03-01

    Small activating RNAs (saRNAs) targeting specific promoter regions are able to stimulate gene expression at the transcriptional level, a phenomenon known as RNA activation (RNAa). It is known that RNAa depends on Ago2 and is associated with epigenetic changes at the target promoters. However, the precise molecular mechanism of RNAa remains elusive. Using human CDKN1A (p21) as a model gene, we characterized the molecular nature of RNAa. We show that saRNAs guide Ago2 to and associate with target promoters. saRNA-loaded Ago2 facilitates the assembly of an RNA-induced transcriptional activation (RITA) complex, which, in addition to saRNA-Ago2 complex, includes RHA and CTR9, the latter being a component of the PAF1 complex. RITA interacts with RNA polymerase II to stimulate transcription initiation and productive elongation, accompanied by monoubiquitination of histone 2B. Our results establish the existence of a cellular RNA-guided genome-targeting and transcriptional activation mechanism and provide important new mechanistic insights into the RNAa process.

  17. A Novel PCR Assay for Listeria welshimeri Targeting Transcriptional Regulator Gene lwe1801

    Science.gov (United States)

    Transcriptional regulator genes encode a group of specialized molecules that play essential roles in microbial responses to changing external conditions. These genes have been shown to possess species or group specificity and are useful as detection targets for diagnostic application. The present st...

  18. A single gene target of an ETS-family transcription factor determines neuronal CO2-chemosensitivity

    DEFF Research Database (Denmark)

    Brandt, Julia P; Aziz-Zaman, Sonya; Juozaityte, Vaida

    2012-01-01

    . We report here a mechanism that endows C. elegans neurons with the ability to detect CO(2). The ETS-5 transcription factor is necessary for the specification of CO(2)-sensing BAG neurons. Expression of a single ETS-5 target gene, gcy-9, which encodes a receptor-type guanylate cyclase, is sufficient...

  19. Genome-wide identification of the regulatory targets of a transcription factor using biochemical characterization and computational genomic analysis

    Directory of Open Access Journals (Sweden)

    Jolly Emmitt R

    2005-11-01

    Full Text Available Abstract Background A major challenge in computational genomics is the development of methodologies that allow accurate genome-wide prediction of the regulatory targets of a transcription factor. We present a method for target identification that combines experimental characterization of binding requirements with computational genomic analysis. Results Our method identified potential target genes of the transcription factor Ndt80, a key transcriptional regulator involved in yeast sporulation, using the combined information of binding affinity, positional distribution, and conservation of the binding sites across multiple species. We have also developed a mathematical approach to compute the false positive rate and the total number of targets in the genome based on the multiple selection criteria. Conclusion We have shown that combining biochemical characterization and computational genomic analysis leads to accurate identification of the genome-wide targets of a transcription factor. The method can be extended to other transcription factors and can complement other genomic approaches to transcriptional regulation.

  20. Target-Derived Neurotrophins Coordinate Transcription and Transport of Bclw to Prevent Axonal Degeneration

    Science.gov (United States)

    Cosker, Katharina E.; Pazyra-Murphy, Maria F.; Fenstermacher, Sara J.

    2013-01-01

    Establishment of neuronal circuitry depends on both formation and refinement of neural connections. During this process, target-derived neurotrophins regulate both transcription and translation to enable selective axon survival or elimination. However, it is not known whether retrograde signaling pathways that control transcription are coordinated with neurotrophin-regulated actions that transpire in the axon. Here we report that target-derived neurotrophins coordinate transcription of the antiapoptotic gene bclw with transport of bclw mRNA to the axon, and thereby prevent axonal degeneration in rat and mouse sensory neurons. We show that neurotrophin stimulation of nerve terminals elicits new bclw transcripts that are immediately transported to the axons and translated into protein. Bclw interacts with Bax and suppresses the caspase6 apoptotic cascade that fosters axonal degeneration. The scope of bclw regulation at the levels of transcription, transport, and translation provides a mechanism whereby sustained neurotrophin stimulation can be integrated over time, so that axonal survival is restricted to neurons connected within a stable circuit. PMID:23516285

  1. Transcription factor HBP1 is a direct anti-cancer target of transcription factor FOXO1 in invasive oral cancer.

    Science.gov (United States)

    Chan, Chien-Yi; Huang, Shih-Yi; Sheu, Jim Jinn-Chyuan; Roth, Mendel M; Chou, I-Tai; Lien, Chia-Hsien; Lee, Ming-Fen; Huang, Chun-Yin

    2017-02-28

    Either FOXO1 or HBP1 transcription factor is a downstream effector of the PI3K/Akt pathway and associated with tumorigenesis. However, the relationship between FOXO1 and HBP1 in oral cancer remains unclear. Analysis of 30 oral tumor specimens revealed that mean mRNA levels of both FOXO1 and HBP1 in non-invasive and invasive oral tumors were found to be significantly lower than that of the control tissues, and the status of low FOXO1 and HBP1 (oral tumors. To investigate if HBP1 is a direct transcription target of FOXO1, we searched potential FOXO1 binding sites in the HBP1 promoter using the MAPPER Search Engine, and two putative FOXO1 binding sites located in the HBP1 promoter -132 to -125 bp and -343 to -336 bp were predicted. These binding sites were then confirmed by both reporter gene assays and the in cellulo ChIP assay. In addition, Akt activity manipulated by PI3K inhibitor LY294002 or Akt mutants was shown to negatively affect FOXO1-mediated HBP1 promoter activation and gene expression. Last, the biological significance of the FOXO1-HBP1 axis in oral cancer malignancy was evaluated in cell growth, colony formation, and invasiveness. The results indicated that HBP1 knockdown potently promoted malignant phenotypes of oral cancer and the suppressive effect of FOXO1 on cell growth, colony formation, and invasion was alleviated upon HBP1 knockdown in invasive oral cancer cells. Taken together, our data provide evidence for HBP1 as a direct downstream target of FOXO1 in oral cancer malignancy.

  2. Compressive system identification of LTI and LTV ARX models: The limited data set case

    NARCIS (Netherlands)

    Sanandaij, B. M.; Vincent, T. L.; Wakin, M. B.; Toth, R.; Poolla, K.

    2011-01-01

    In this paper, we consider identifying Auto Regressive with eXternal input (ARX) models for both Linear Time-Invariant (LTI) and Linear Time-Variant (LTV) systems. We aim at doing the identification from the smallest possible number of observations. This is inspired by the field of Compressive

  3. A MISO-ARX-Based Method for Single-Trial Evoked Potential Extraction

    Directory of Open Access Journals (Sweden)

    Nannan Yu

    2017-01-01

    Full Text Available In this paper, we propose a novel method for solving the single-trial evoked potential (EP estimation problem. In this method, the single-trial EP is considered as a complex containing many components, which may originate from different functional brain sites; these components can be distinguished according to their respective latencies and amplitudes and are extracted simultaneously by multiple-input single-output autoregressive modeling with exogenous input (MISO-ARX. The extraction process is performed in three stages: first, we use a reference EP as a template and decompose it into a set of components, which serve as subtemplates for the remaining steps. Then, a dictionary is constructed with these subtemplates, and EPs are preliminarily extracted by sparse coding in order to roughly estimate the latency of each component. Finally, the single-trial measurement is parametrically modeled by MISO-ARX while characterizing spontaneous electroencephalographic activity as an autoregression model driven by white noise and with each component of the EP modeled by autoregressive-moving-average filtering of the subtemplates. Once optimized, all components of the EP can be extracted. Compared with ARX, our method has greater tracking capabilities of specific components of the EP complex as each component is modeled individually in MISO-ARX. We provide exhaustive experimental results to show the effectiveness and feasibility of our method.

  4. CDKL5 and ARX mutations are not responsible for early onset severe myoclonic epilepsy in infancy.

    Science.gov (United States)

    Nabbout, Rima; Depienne, Christel; Chipaux, Mathilde; Girard, Benoit; Souville, Isabelle; Trouillard, Oriane; Dulac, Olivier; Chelly, Jamel; Afenjar, Alexandra; Héron, Delphine; Leguern, Eric; Beldjord, Cherif; Bienvenu, Thierry; Bahi-Buisson, Nadia

    2009-11-01

    Severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome (DS) is a distinctive epilepsy syndrome often associated with de novo mutations in the SCN1A gene. However, 25-30% patients with SMEI/DS are negative for SCN1A mutation screening, suggesting that other molecular mechanisms may account for these disorders. Given the overlapping and heterogeneous clinical features of CDKL5- and ARX-related epilepsies and SMEI/DS, we postulated that CDKL5 mutations in females and ARX mutations gene in males may be associated with early onset seizures forms of SMEI/DS. Twenty-eight patients with early onset SMEI/DS before 6 months negative for SCN1A mutational screening were selected and screened for mutations in the ARX gene in males (n=14) or the CDKL5 gene in females (n=14). No mutations in either gene were found except one intronic variation of uncertain pathogenicity in the CDKL5 gene. All patients started seizures at mean age of 3.48 months. Thirteen patients had familial history of epilepsy or febrile seizures. Patients evolved toward refractory epilepsy with generalized tonic clonic seizures (18/28) and myoclonia (23/28) and severe neurological impairment with autistic features (13/28), ataxia (14/28) and spasticity (5/28). No patient ever exhibited infantile spasms, dystonia, or Rett-like features. Our results illustrate that mutation screening of ARX and CDKL5 is not effective in patients selected on the basis of clinical signs associated to early onset SMEI/DS. In addition, they might reflect that other phenotypic features associated with CDKL5 mutations (Rett-like features, infantile spasm) or ARX mutations (dystonia, spasticity) are more distinctive. 2009 Elsevier B.V. All rights reserved.

  5. Targeting Endothelial Adhesion Molecule Transcription for Treatment of Inflammatory Disease: A Proof-of-Concept Study

    Directory of Open Access Journals (Sweden)

    Liam M. Ashander

    2016-01-01

    Full Text Available Targeting the endothelial adhesion molecules that control leukocyte trafficking into a tissue has been explored as a biological therapy for inflammatory diseases. However, these molecules also participate in leukocyte migration for immune surveillance, and inhibiting the physiological level of an adhesion molecule might promote infection or malignancy. We explored the concept of targeting endothelial adhesion molecule transcription during inflammation in a human system. Intercellular adhesion molecule 1 (ICAM-1 mediates leukocyte migration across the retinal endothelium in noninfectious posterior uveitis. We observed an increase in the transcription factor, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB1, in parallel with ICAM-1, in human retinal endothelial cells treated with tumor necrosis factor-alpha (TNF-α, and identified putative binding sites for NF-κB1 within the ICAM-1 regulatory region. We targeted induced NF-κB1 expression in endothelial cells with small interfering (siRNA. Knockdown of NF-κB1 significantly decreased cell surface expression of ICAM-1 protein induced by TNF-α but did not reduce constitutive ICAM-1 expression. Consistently, NF-κB1 knockdown significantly reduced leukocyte binding to cell monolayers in the presence of TNF-α but did not impact baseline binding. Findings of this proof-of-concept study indicate that induced transcription of endothelial adhesion molecules might be targeted therapeutically for inflammatory disease in humans.

  6. Targeting GLI by GANT61 involves mechanisms dependent on inhibition of both transcription and DNA licensing.

    Science.gov (United States)

    Zhang, Ruowen; Wu, Jiahui; Ferrandon, Sylvain; Glowacki, Katie J; Houghton, Janet A

    2016-12-06

    The GLI genes are transcription factors and in cancers are oncogenes, aberrantly and constitutively activated. GANT61, a specific GLI inhibitor, has induced extensive cytotoxicity in human models of colon cancer. The FOXM1 promoter was determined to be a transcriptional target of GLI1. In HT29 cells, inhibition of GLI1 binding at the GLI consensus sequence by GANT61 led to inhibited binding of Pol II, the pause-release factors DSIF, NELF and p-TEFb. The formation of R-loops (RNA:DNA hybrids, ssDNA), were reduced by GANT61 at the FOXM1 promoter. Pretreatment of HT29 cells with α-amanitin reduced GANT61-induced γH2AX foci. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication to be linked. By co-immunoprecipitation and confocal microscopy, GLI1 co-localized with the DNA licensing factors ORC4, CDT1, and MCM2. Significant co-localization of GLI1 and ORC4 was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. CDT1 was found to be a transcription target of GLI1. Overexpression of CDT1 in HT29 and SW480 cells reduced GANT61-induced cell death, gH2AX foci, and cleavage of caspase-3. Data demonstrate involvement of transcription and of DNA replication licensing factors by non-transcriptional and transcriptional mechanisms in the GLI-dependent mechanism of action of GANT61.

  7. Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice

    Science.gov (United States)

    Overman, Jeroen; Fontaine, Frank; Moustaqil, Mehdi; Mittal, Deepak; Sierecki, Emma; Sacilotto, Natalia; Zuegg, Johannes; Robertson, Avril AB; Holmes, Kelly; Salim, Angela A; Mamidyala, Sreeman; Butler, Mark S; Robinson, Ashley S; Lesieur, Emmanuelle; Johnston, Wayne; Alexandrov, Kirill; Black, Brian L; Hogan, Benjamin M; De Val, Sarah; Capon, Robert J; Carroll, Jason S; Bailey, Timothy L; Koopman, Peter; Jauch, Ralf; Smyth, Mark J; Cooper, Matthew A; Gambin, Yann; Francois, Mathias

    2017-01-01

    Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics. DOI: http://dx.doi.org/10.7554/eLife.21221.001 PMID:28137359

  8. UBE2C Is a Transcriptional Target of the Cell Cycle Regulator FOXM1

    Directory of Open Access Journals (Sweden)

    Pedro Nicolau-Neto

    2018-03-01

    Full Text Available FOXM1 (forkhead box protein M1 is a transcription factor that participates in all stages of tumor development, mainly through the control of cell cycle and proliferation, regulating the expression of genes involved in G1/S and G2/M transition and M phase progression. The ubiquitin conjugating enzyme E2 (UBE2C is a member of the anaphase promoting complex/cyclosome, promoting the degradation of several target proteins along cell cycle progression, during metaphase/anaphase transition. FOXM1 and UBE2C have been found overexpressed in a wide range of different solid tumors. Therefore, the aim of this study was to investigate whether UBE2C is a transcriptional target of FOXM1, using esophageal squamous cell carcinoma (ESCC as a model, in addition to several cancer-deposited data. Our results show that FOXM1 and UBE2C expression present a positive correlation in normal tissues and in 25 distinct tumor types, including ESCC, where these genes are overexpressed. Moreover, FOXM1 binds to UBE2C promoter region in ESCC cell line and transcriptionally activates it, leading to UBE2C upregulation. In conclusion, this study provides evidences that FOXM1 transcriptionally regulates UBE2C expression in ESCC and their deregulation may be a general phenomenon in human neoplasias.

  9. Novel Hematopoietic Target Genes in the NRF2-Mediated Transcriptional Pathway

    Directory of Open Access Journals (Sweden)

    Michelle R. Campbell

    2013-01-01

    Full Text Available Nuclear factor- (erythroid-derived 2 like 2 (NFE2L2, NRF2 is a key transcriptional activator of the antioxidant response pathway and is closely related to erythroid transcription factor NFE2. Under oxidative stress, NRF2 heterodimerizes with small Maf proteins and binds cis-acting enhancer sequences found near oxidative stress response genes. Using the dietary isothiocyanate sulforaphane (SFN to activate NRF2, chromatin immunoprecipitation sequencing (ChIP-seq identified several hundred novel NRF2-mediated targets beyond its role in oxidative stress. Activated NRF2 bound the antioxidant response element (ARE in promoters of several known and novel target genes involved in iron homeostasis and heme metabolism, including known targets FTL and FTH1, as well as novel binding in the globin locus control region. Five novel NRF2 target genes were chosen for followup: AMBP, ABCB6, FECH, HRG-1 (SLC48A1, and TBXAS1. SFN-induced gene expression in erythroid K562 and lymphoid cells were compared for each target gene. NRF2 silencing showed reduced expression in lymphoid, lung, and hepatic cells. Furthermore, stable knockdown of NRF2 negative regulator KEAP1 in K562 cells resulted in increased NQO1, AMBP, and TBXAS1 expression. NFE2 binding sites in K562 cells revealed similar binding profiles as lymphoid NRF2 sites in all potential NRF2 candidates supporting a role for NRF2 in heme metabolism and erythropoiesis.

  10. Insulators target active genes to transcription factories and polycomb-repressed genes to polycomb bodies.

    Directory of Open Access Journals (Sweden)

    Hua-Bing Li

    2013-04-01

    Full Text Available Polycomb bodies are foci of Polycomb proteins in which different Polycomb target genes are thought to co-localize in the nucleus, looping out from their chromosomal context. We have shown previously that insulators, not Polycomb response elements (PREs, mediate associations among Polycomb Group (PcG targets to form Polycomb bodies. Here we use live imaging and 3C interactions to show that transgenes containing PREs and endogenous PcG-regulated genes are targeted by insulator proteins to different nuclear structures depending on their state of activity. When two genes are repressed, they co-localize in Polycomb bodies. When both are active, they are targeted to transcription factories in a fashion dependent on Trithorax and enhancer specificity as well as the insulator protein CTCF. In the absence of CTCF, assembly of Polycomb bodies is essentially reduced to those representing genomic clusters of Polycomb target genes. The critical role of Trithorax suggests that stable association with a specialized transcription factory underlies the cellular memory of the active state.

  11. NPPB and ACAN, two novel SHOX2 transcription targets implicated in skeletal development.

    Directory of Open Access Journals (Sweden)

    Miriam Aza-Carmona

    Full Text Available SHOX and SHOX2 transcription factors are highly homologous, with even identical homeodomains. Genetic alterations in SHOX result in two skeletal dysplasias; Léri-Weill dyschondrosteosis (LWD and Langer mesomelic dysplasia (LMD, while no human genetic disease has been linked to date with SHOX2. SHOX2 is, though, involved in skeletal development, as shown by different knockout mice models. Due to the high homology between SHOX and SHOX2, and their functional redundancy during heart development, we postulated that SHOX2 might have the same transcriptional targets and cofactors as SHOX in limb development. We selected two SHOX transcription targets regulated by different mechanisms: 1 the natriuretic peptide precursor B gene (NPPB involved in the endochondral ossification signalling and directly activated by SHOX; and 2 Aggrecan (ACAN, a major component of cartilage extracellular matrix, regulated by the cooperation of SHOX with the SOX trio (SOX5, SOX6 and SOX9 via the protein interaction between SOX5/SOX6 and SHOX. Using the luciferase assay we have demonstrated that SHOX2, like SHOX, regulates NPPB directly whilst activates ACAN via its cooperation with the SOX trio. Subsequently, we have identified and characterized the protein domains implicated in the SHOX2 dimerization and also its protein interaction with SOX5/SOX6 and SHOX using the yeast-two hybrid and co-immunoprecipitation assays. Immunohistochemistry of human fetal growth plates from different time points demonstrated that SHOX2 is coexpressed with SHOX and the members of the SOX trio. Despite these findings, no mutation was identified in SHOX2 in a cohort of 83 LWD patients with no known molecular defect, suggesting that SHOX2 alterations do not cause LWD. In conclusion, our work has identified the first cofactors and two new transcription targets of SHOX2 in limb development, and we hypothesize a time- and tissue-specific functional redundancy between SHOX and SHOX2.

  12. NPPB and ACAN, two novel SHOX2 transcription targets implicated in skeletal development.

    Science.gov (United States)

    Aza-Carmona, Miriam; Barca-Tierno, Veronica; Hisado-Oliva, Alfonso; Belinchón, Alberta; Gorbenko-del Blanco, Darya; Rodriguez, Jose Ignacio; Benito-Sanz, Sara; Campos-Barros, Angel; Heath, Karen E

    2014-01-01

    SHOX and SHOX2 transcription factors are highly homologous, with even identical homeodomains. Genetic alterations in SHOX result in two skeletal dysplasias; Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LMD), while no human genetic disease has been linked to date with SHOX2. SHOX2 is, though, involved in skeletal development, as shown by different knockout mice models. Due to the high homology between SHOX and SHOX2, and their functional redundancy during heart development, we postulated that SHOX2 might have the same transcriptional targets and cofactors as SHOX in limb development. We selected two SHOX transcription targets regulated by different mechanisms: 1) the natriuretic peptide precursor B gene (NPPB) involved in the endochondral ossification signalling and directly activated by SHOX; and 2) Aggrecan (ACAN), a major component of cartilage extracellular matrix, regulated by the cooperation of SHOX with the SOX trio (SOX5, SOX6 and SOX9) via the protein interaction between SOX5/SOX6 and SHOX. Using the luciferase assay we have demonstrated that SHOX2, like SHOX, regulates NPPB directly whilst activates ACAN via its cooperation with the SOX trio. Subsequently, we have identified and characterized the protein domains implicated in the SHOX2 dimerization and also its protein interaction with SOX5/SOX6 and SHOX using the yeast-two hybrid and co-immunoprecipitation assays. Immunohistochemistry of human fetal growth plates from different time points demonstrated that SHOX2 is coexpressed with SHOX and the members of the SOX trio. Despite these findings, no mutation was identified in SHOX2 in a cohort of 83 LWD patients with no known molecular defect, suggesting that SHOX2 alterations do not cause LWD. In conclusion, our work has identified the first cofactors and two new transcription targets of SHOX2 in limb development, and we hypothesize a time- and tissue-specific functional redundancy between SHOX and SHOX2.

  13. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

    Science.gov (United States)

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-05-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

  14. The simian immunodeficiency virus targets central cell cycle functions through transcriptional repression in vivo.

    Directory of Open Access Journals (Sweden)

    Carl-Magnus Hogerkorp

    Full Text Available A massive and selective loss of CD4+ memory T cells occurs during the acute phase of immunodeficiency virus infections. The mechanism of this depletion is poorly understood but constitutes a key event with implications for progression. We assessed gene expression of purified T cells in Rhesus Macaques during acute SIVmac239 infection in order to define mechanisms of pathogenesis. We observe a general transcriptional program of over 1,600 interferon-stimulated genes induced in all T cells by the infection. Furthermore, we identify 113 transcriptional changes that are specific to virally infected cells. A striking downregulation of several key cell cycle regulator genes was observed and shared promotor-region E2F binding sites in downregulated genes suggested a targeted transcriptional control of an E2F regulated cell cycle program. In addition, the upregulation of the gene for the fundamental regulator of RNA polymerase II, TAF7, demonstrates that viral interference with the cell cycle and transcriptional regulation programs may be critical components during the establishment of a pathogenic infection in vivo.

  15. FATS is a transcriptional target of p53 and associated with antitumor activity

    Directory of Open Access Journals (Sweden)

    Zhang Xifeng

    2010-09-01

    Full Text Available Abstract Frequent mutations of p53 in human cancers exemplify its crucial role as a tumor suppressor transcription factor, and p21, a transcriptional target of p53, plays a central role in surveillance of cell-cycle checkpoints. Our previous study has shown that FATS stabilize p21 to preserve genome integrity. In this study we identified a novel transcript variant of FATS (GenBank: GQ499374 through screening a cDNA library from mouse testis, which uncovered the promoter region of mouse FATS. Mouse FATS was highly expressed in testis. The p53-responsive elements existed in proximal region of both mouse and human FATS promoters. Functional study indicated that the transcription of FATS gene was activated by p53, whereas such effect was abolished by site-directed mutagenesis in the p53-RE of FATS promoter. Furthermore, the expression of FATS increased upon DNA damage in a p53-dependent manner. FATS expression was silent or downregulated in human cancers, and overexpression of FATS suppressed tumorigenicity in vivo independently of p53. Our results reveal FATS as a p53-regulated gene to monitor genomic stability.

  16. DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes.

    Science.gov (United States)

    Guha, Mithu; Saare, Mario; Maslovskaja, Julia; Kisand, Kai; Liiv, Ingrid; Haljasorg, Uku; Tasa, Tõnis; Metspalu, Andres; Milani, Lili; Peterson, Pärt

    2017-04-21

    The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Analysis of miRNAs targeting transcription factors in Persicaria minor induced by Fusarium oxysporum

    Science.gov (United States)

    Samad, Abdul Fatah A.; Ali, Nazaruddin Muhammad; Ismail, Ismanizan; Murad, Abdul Munir Abdul

    2016-11-01

    A recent discovery showed small non-coding RNA known as microRNA has a crucial role in plant development and plant survival in extreme condition. In the past few years, researchers have managed to identify the various families of transcription factors that play a crucial role in regulating plant development and plant responses to stresses. This study focuses on the expression pattern of miRNA targeted transcription factor under biotic stress in a plant rich with secondary metabolite, Persicaria minor. A pathogenic fungus, Fusarium oxysporum was used in the biotic stress treatment since the previous study revealed this fungus could trigger plant defense system. Two small RNA libraries were constructed which consist of control and treated samples. In order to identify the potential target, psRobot target prediction software was used for each miRNA that shows significant change due to the infection. The result showed miR156b/c, miR172a, miR319, miR858, and miR894 were found to be targeting a wide range of transcription factors that involve in plant development and plant response towards stresses. The expression of miR156b/c and miR172 were up-regulated while the expression of miR319, miR858, and miR894 was found to be down-regulated. These results may provide a certain level of networking between those two regulatory molecules in plant genetic system under biotic stress.

  18. Tuning SISO offset-free Model Predictive Control based on ARX models

    DEFF Research Database (Denmark)

    Huusom, Jakob Kjøbsted; Poulsen, Niels Kjølstad; Jørgensen, Sten Bay

    2012-01-01

    , the proposed controller is simple to tune as it has only one free tuning parameter. These two features are advantageous in predictive process control as they simplify industrial commissioning of MPC. Disturbance rejection and offset-free control is important in industrial process control. To achieve offset......In this paper, we present a tuning methodology for a simple offset-free SISO Model Predictive Controller (MPC) based on autoregressive models with exogenous inputs (ARX models). ARX models simplify system identification as they can be identified from data using convex optimization. Furthermore......-free control in face of unknown disturbances or model-plant mismatch, integrators must be introduced in either the estimator or the regulator. Traditionally, offset-free control is achieved using Brownian disturbance models in the estimator. In this paper we achieve offset-free control by extending the noise...

  19. Tuning of methods for offset free MPC based on ARX model representations

    DEFF Research Database (Denmark)

    Huusom, Jakob Kjøbsted; Poulsen, Niels Kjølstad; Jørgensen, Sten Bay

    2010-01-01

    In this paper we investigate model predictive control (MPC) based on ARX models. ARX models can be identified from data using convex optimization technologies and is linear in the system parameters. Compared to other model parameterizations this feature is an advantage in embedded applications...... for robust and automatic system identification. Standard MPC is not able to reject a sustained, unmeasured, non zero mean disturbance and will therefore not provide offset free tracking. Offset free tracking can be guaranteed for this type of disturbances if Δ variables are used or if the state space...... is extended with a disturbance model state. The relation between the base case and the two extended methods are illustrated which provides good understanding and a platform for discussing tuning for good closed loop performance....

  20. A Tuning Procedure for ARX-based MPC of Multivariate Processes

    DEFF Research Database (Denmark)

    Olesen, Daniel; Huusom, Jakob Kjøbsted; Jørgensen, John Bagterp

    2013-01-01

    We present an optimization based tuning procedure with certain robustness properties for an offset free Model Predictive Controller (MPC). The MPC is designed for multivariate processes that can be represented by an ARX model. The stochastic model of the ARX model identified from input-output data...... is modified with an ARMA model designed as part of the MPC-design procedure to ensure offset-free control. The MPC is designed and implemented based on a state space model in innovation form. Expressions for the closed-loop dynamics of the unconstrained system is used to derive the sensitivity function...... to a constraint on the maximum of the sensitivity function. The latter constraint provides a robustness measure that is essential for the procedure. The method is demonstrated for two simulated examples: A Wood-Berry distillation column example and a cement mill example....

  1. Identification of direct regulatory targets of the transcription factor Sox10 based on function and conservation

    Directory of Open Access Journals (Sweden)

    Lee Sanghyuk

    2008-09-01

    Full Text Available Abstract Background Sox10, a member of the Sry-related HMG-Box gene family, is a critical transcription factor for several important cell lineages, most notably the neural crest stem cells and the derivative peripheral glial cells and melanocytes. Thus far, only a handful of direct target genes are known for this transcription factor limiting our understanding of the biological network it governs. Results We describe identification of multiple direct regulatory target genes of Sox10 through a procedure based on function and conservation. By combining RNA interference technique and DNA microarray technology, we have identified a set of genes that show significant down-regulation upon introduction of Sox10 specific siRNA into Schwannoma cells. Subsequent comparative genomics analyses led to potential binding sites for Sox10 protein conserved across several mammalian species within the genomic region proximal to these genes. Multiple sites belonging to 4 different genes (proteolipid protein, Sox10, extracellular superoxide dismutase, and pleiotrophin were shown to directly interact with Sox10 by chromatin immunoprecipitation assay. We further confirmed the direct regulation through the identified cis-element for one of the genes, extracellular superoxide dismutase, using electrophoretic mobility shift assay and reporter assay. Conclusion In sum, the process of combining differential expression profiling and comparative genomics successfully led to further defining the role of Sox10, a critical transcription factor for the development of peripheral glia. Our strategy utilizing relatively accessible techniques and tools should be applicable to studying the function of other transcription factors.

  2. Kaiso Directs the Transcriptional Corepressor MTG16 to the Kaiso Binding Site in Target Promoters

    Science.gov (United States)

    Barrett, Caitlyn W.; Smith, J. Joshua; Lu, Lauren C.; Markham, Nicholas; Stengel, Kristy R.; Short, Sarah P.; Zhang, Baolin; Hunt, Aubrey A.; Fingleton, Barbara M.; Carnahan, Robert H.; Engel, Michael E.; Chen, Xi; Beauchamp, R. Daniel; Wilson, Keith T.; Hiebert, Scott W.; Reynolds, Albert B.; Williams, Christopher S.

    2012-01-01

    Myeloid translocation genes (MTGs) are transcriptional corepressors originally identified in acute myelogenous leukemia that have recently been linked to epithelial malignancy with non-synonymous mutations identified in both MTG8 and MTG16 in colon, breast, and lung carcinoma in addition to functioning as negative regulators of WNT and Notch signaling. A yeast two-hybrid approach was used to discover novel MTG binding partners. This screen identified the Zinc fingers, C2H2 and BTB domain containing (ZBTB) family members ZBTB4 and ZBTB38 as MTG16 interacting proteins. ZBTB4 is downregulated in breast cancer and modulates p53 responses. Because ZBTB33 (Kaiso), like MTG16, modulates Wnt signaling at the level of TCF4, and its deletion suppresses intestinal tumorigenesis in the ApcMin mouse, we determined that Kaiso also interacted with MTG16 to modulate transcription. The zinc finger domains of Kaiso as well as ZBTB4 and ZBTB38 bound MTG16 and the association with Kaiso was confirmed using co-immunoprecipitation. MTG family members were required to efficiently repress both a heterologous reporter construct containing Kaiso binding sites (4×KBS) and the known Kaiso target, Matrix metalloproteinase-7 (MMP-7/Matrilysin). Moreover, chromatin immunoprecipitation studies placed MTG16 in a complex occupying the Kaiso binding site on the MMP-7 promoter. The presence of MTG16 in this complex, and its contributions to transcriptional repression both required Kaiso binding to its binding site on DNA, establishing MTG16-Kaiso binding as functionally relevant in Kaiso-dependent transcriptional repression. Examination of a large multi-stage CRC expression array dataset revealed patterns of Kaiso, MTG16, and MMP-7 expression supporting the hypothesis that loss of either Kaiso or MTG16 can de-regulate a target promoter such as that of MMP-7. These findings provide new insights into the mechanisms of transcriptional control by ZBTB family members and broaden the scope of co

  3. An adaptive ARX model to estimate the RUL of aluminum plates based on its crack growth

    Science.gov (United States)

    Barraza-Barraza, Diana; Tercero-Gómez, Víctor G.; Beruvides, Mario G.; Limón-Robles, Jorge

    2017-01-01

    A wide variety of Condition-Based Maintenance (CBM) techniques deal with the problem of predicting the time for an asset fault. Most statistical approaches rely on historical failure data that might not be available in several practical situations. To address this issue, practitioners might require the use of self-starting approaches that consider only the available knowledge about the current degradation process and the asset operating context to update the prognostic model. Some authors use Autoregressive (AR) models for this purpose that are adequate when the asset operating context is constant, however, if it is variable, the accuracy of the models can be affected. In this paper, three autoregressive models with exogenous variables (ARX) were constructed, and their capability to estimate the remaining useful life (RUL) of a process was evaluated following the case of the aluminum crack growth problem. An existing stochastic model of aluminum crack growth was implemented and used to assess RUL estimation performance of the proposed ARX models through extensive Monte Carlo simulations. Point and interval estimations were made based only on individual history, behavior, operating conditions and failure thresholds. Both analytic and bootstrapping techniques were used in the estimation process. Finally, by including recursive parameter estimation and a forgetting factor, the ARX methodology adapts to changing operating conditions and maintain the focus on the current degradation level of an asset.

  4. Nonlinear System Identification Using Quasi-ARX RBFN Models with a Parameter-Classified Scheme

    Directory of Open Access Journals (Sweden)

    Lan Wang

    2017-01-01

    Full Text Available Quasi-linear autoregressive with exogenous inputs (Quasi-ARX models have received considerable attention for their usefulness in nonlinear system identification and control. In this paper, identification methods of quasi-ARX type models are reviewed and categorized in three main groups, and a two-step learning approach is proposed as an extension of the parameter-classified methods to identify the quasi-ARX radial basis function network (RBFN model. Firstly, a clustering method is utilized to provide statistical properties of the dataset for determining the parameters nonlinear to the model, which are interpreted meaningfully in the sense of interpolation parameters of a local linear model. Secondly, support vector regression is used to estimate the parameters linear to the model; meanwhile, an explicit kernel mapping is given in terms of the nonlinear parameter identification procedure, in which the model is transformed from the nonlinear-in-nature to the linear-in-parameter. Numerical and real cases are carried out finally to demonstrate the effectiveness and generalization ability of the proposed method.

  5. APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death

    DEFF Research Database (Denmark)

    Fortin, A; Cregan, S P; MacLaurin, J G

    2001-01-01

    p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulati...

  6. Transcription Factor STAT3 as a Novel Molecular Target for Cancer Prevention

    International Nuclear Information System (INIS)

    Xiong, Ailian; Yang, Zhengduo; Shen, Yicheng; Zhou, Jia; Shen, Qiang

    2014-01-01

    Signal Transducers and Activators of Transcription (STATs) are a family of transcription factors that regulate cell proliferation, differentiation, apoptosis, immune and inflammatory responses, and angiogenesis. Cumulative evidence has established that STAT3 has a critical role in the development of multiple cancer types. Because it is constitutively activated during disease progression and metastasis in a variety of cancers, STAT3 has promise as a drug target for cancer therapeutics. Recently, STAT3 was found to have an important role in maintaining cancer stem cells in vitro and in mouse tumor models, suggesting STAT3 is integrally involved in tumor initiation, progression and maintenance. STAT3 has been traditionally considered as nontargetable or undruggable, and the lag in developing effective STAT3 inhibitors contributes to the current lack of FDA-approved STAT3 inhibitors. Recent advances in cancer biology and drug discovery efforts have shed light on targeting STAT3 globally and/or specifically for cancer therapy. In this review, we summarize current literature and discuss the potential importance of STAT3 as a novel target for cancer prevention and of STAT3 inhibitors as effective chemopreventive agents

  7. Preclinical evaluation of transcriptional targeting strategy for human hepatocellular carcinoma in an orthotopic xenograft mouse model.

    Science.gov (United States)

    Sia, Kian Chuan; Huynh, Hung; Chung, Alexander Yaw Fui; Ooi, London Lucien Peng Jin; Lim, Kiat Hon; Hui, Kam Man; Lam, Paula Yeng Po

    2013-08-01

    Gene regulation of many key cell-cycle players in S-, G(2) phase, and mitosis results from transcriptional repression in their respective promoter regions during the G(0) and G(1) phases of cell cycle. Within these promoter regions are phylogenetically conserved sequences known as the cell-cycle-dependent element (CDE) and cell-cycle genes homology regions (CHR) sites. Thus, we hypothesize that transcriptional regulation of cell-cycle regulation via the CDE/CHR region together with liver-specific apolipoprotein E (apoE)-hAAT promoter could bring about a selective transgene expression in proliferating human hepatocellular carcinoma. We show that the newly generated vector AH-6CC-L2C could mediate hepatocyte-targeted luciferase gene expression in tumor cells and freshly isolated short-term hepatocellular carcinoma cultures from patient biopsy. In contrast, normal murine and human hepatocytes infected with AH-6CC-L2C expressed minimal or low luciferase activities. In the presence of prodrug 5-fluorocytosine (5-FC), AH-6CC-L2C effectively suppressed the growth of orthotopic hepatocellular carcinoma patient-derived xenograft mouse model via the expression of yeast cytosine deaminase (yCD) that converts 5-FC to anticancer metabolite 5-fluoruracil. More importantly, we show that combination treatment of AH-6CC-L2C with an EZH2 inhibitor, DZNep, that targets EpCAM-positive hepatocellular carcinoma, can bring about a greater therapeutic efficacy compared with a single treatment of virus or inhibitor. Our study showed that targeting proliferating human hepatocellular carcinoma cells through the transcriptional control of therapeutic gene could represent a feasible approach against hepatocellular carcinoma.

  8. Transcription Factors Expressed in Lateral Organ Boundaries: Identification of Downstream Targets

    Energy Technology Data Exchange (ETDEWEB)

    Springer, Patricia S

    2010-07-12

    The processes of lateral organ initiation and patterning are central to the generation of mature plant form. Characterization of the molecular mechanisms underlying these processes is essential to our understanding of plant development. Communication between the shoot apical meristem and initiating organ primordia is important both for functioning of the meristem and for proper organ patterning, and very little is known about this process. In particular, the boundary between meristem and leaf is emerging as a critical region that is important for SAM maintenance and regulation of organogenesis. The goal of this project was to characterize three boundary-expressed genes that encode predicted transcription factors. Specifically, we have studied LATERAL ORGAN BOUNDARIES (LOB), LATERAL ORGAN FUSION1 (LOF1), and LATERAL ORGAN FUSION2 (LOF2). LOB encodes the founding member of the LOB-DOMAIN (LBD) plant-specific DNA binding transcription factor family and LOF1 and LOF2 encode paralogous MYB-domain transcription factors. We characterized the genetic relationship between these three genes and other boundary and meristem genes. We also used an ectopic inducible expression system to identify direct targets of LOB.

  9. Targeted reduction of highly abundant transcripts using pseudo-random primers.

    Science.gov (United States)

    Arnaud, Ophélie; Kato, Sachi; Poulain, Stéphane; Plessy, Charles

    2016-04-01

    Transcriptome studies based on quantitative sequencing can estimate levels of gene expression by measuring target RNA abundance in sequencing libraries. Sequencing costs are proportional to the total number of sequenced reads, and in order to cover rare RNAs, considerable quantities of abundant and identical reads are needed. This major limitation can be addressed by depleting a proportion of the most abundant sequences from the library. However, such depletion strategies involve either extra handling of the input RNA sample or use of a large number of reverse transcription primers, termed not-so-random (NSR) primers, which are costly to synthesize. Taking advantage of the high tolerance of reverse transcriptase to mis-prime, we found that it is possible to use as few as 40 pseudo-random (PS) reverse transcription primers to decrease the rate of undesirable abundant sequences within a library without affecting the overall transcriptome diversity. PS primers are simple to design and can be used to deplete several undesirable RNAs simultaneously, thus creating a flexible tool for enriching transcriptome libraries for rare transcript sequences.

  10. Identification of target genes of transcription factor activator protein 2 gamma in breast cancer cells

    International Nuclear Information System (INIS)

    Ailan, He; Shuanglin, Xiang; Xiangwen, Xiao; Daolong, Ren; Lu, Gan; Xiaofeng, Ding; Xi, Qiao; Xingwang, Hu; Rushi, Liu; Jian, Zhang

    2009-01-01

    Activator protein 2 gamma (AP-2γ) is a member of the transcription factor activator protein-2 (AP-2) family, which is developmentally regulated and plays a role in human neoplasia. AP-2γ has been found to be overexpressed in most breast cancers, and have a dual role to inhibit tumor initiation and promote tumor progression afterwards during mammary tumorigensis. To identify the gene targets that mediate its effects, we performed chromatin immunoprecipitation (ChIP) to isolate AP-2γ binding sites on genomic DNA from human breast cancer cell line MDA-MB-453. 20 novel DNA fragments proximal to potential AP-2γ targets were obtained. They are categorized into functional groups of carcinogenesis, metabolism and others. A combination of sequence analysis, reporter gene assays, quantitative real-time PCR, electrophoretic gel mobility shift assays and immunoblot analysis further confirmed the four AP-2γ target genes in carcinogenesis group: ErbB2, CDH2, HPSE and IGSF11. Our results were consistent with the previous reports that ErbB2 was the target gene of AP-2γ. Decreased expression and overexpression of AP-2γ in human breast cancer cells significantly altered the expression of these four genes, indicating that AP-2γ directly regulates them. This suggested that AP-2γ can coordinate the expression of a network of genes, involving in carcinogenesis, especially in breast cancer. They could serve as therapeutic targets against breast cancers in the future

  11. Comprehensive reanalysis of transcription factor knockout expression data in Saccharomyces cerevisiae reveals many new targets.

    Science.gov (United States)

    Reimand, Jüri; Vaquerizas, Juan M; Todd, Annabel E; Vilo, Jaak; Luscombe, Nicholas M

    2010-08-01

    Transcription factor (TF) perturbation experiments give valuable insights into gene regulation. Genome-scale evidence from microarray measurements may be used to identify regulatory interactions between TFs and targets. Recently, Hu and colleagues published a comprehensive study covering 269 TF knockout mutants for the yeast Saccharomyces cerevisiae. However, the information that can be extracted from this valuable dataset is limited by the method employed to process the microarray data. Here, we present a reanalysis of the original data using improved statistical techniques freely available from the BioConductor project. We identify over 100,000 differentially expressed genes-nine times the total reported by Hu et al. We validate the biological significance of these genes by assessing their functions, the occurrence of upstream TF-binding sites, and the prevalence of protein-protein interactions. The reanalysed dataset outperforms the original across all measures, indicating that we have uncovered a vastly expanded list of relevant targets. In summary, this work presents a high-quality reanalysis that maximizes the information contained in the Hu et al. compendium. The dataset is available from ArrayExpress (accession: E-MTAB-109) and it will be invaluable to any scientist interested in the yeast transcriptional regulatory system.

  12. RNA glycosidase and other agents target Tat to inhibit HIV-1 transcription.

    Science.gov (United States)

    Harrich, David; Jin, Hongping

    2018-03-20

    The HIV-1 tat gene encodes a small 86-104 amino acid protein depending on the HIV-1 strain. Tat is essential for HIV-1 replication through interactions with numerous cellular transcription factors. The interaction between Tat and P-TEFb, which is a cellular protein complex composed of cyclin T1 and CDK9, delivers P-TEFb to the newly transcribed viral mRNAs where phosphorylation of RNA polymerase II by CDK9 leads to highly efficient mRNA transcription. It has long been recognized that Tat is a potential anti-HIV-1 target and possibly a viral Achilles' heel. However, specifically targeting Tat without affecting normal host cell functions has been challenging. Means to inactivate Tat have been reported that includes small compounds, transdominant negative Tat proteins, and by plant-derived antivirals. Investigations of these agents have reported encouraging outcomes that inform and may hopefully affect strategies for a functional HIV-1 cure. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  13. Kinome-wide transcriptional profiling of uveal melanoma reveals new vulnerabilities to targeted therapeutics.

    Science.gov (United States)

    Bailey, Fiona P; Clarke, Kim; Kalirai, Helen; Kenyani, Jenna; Shahidipour, Haleh; Falciani, Francesco; Coulson, Judy M; Sacco, Joseph J; Coupland, Sarah E; Eyers, Patrick A

    2018-03-01

    Metastatic uveal melanoma (UM) is invariably fatal, usually within a year of diagnosis. There are currently no effective therapies, and clinical studies employing kinase inhibitors have so far demonstrated limited success. This is despite common activating mutations in GNAQ/11 genes, which trigger signalling pathways that might predispose tumours to a variety of targeted drugs. In this study, we have profiled kinome expression network dynamics in various human ocular melanomas. We uncovered a shared transcriptional profile in human primary UM samples and across a variety of experimental cell-based models. The poor overall response of UM cells to FDA-approved kinase inhibitors contrasted with much higher sensitivity to the bromodomain inhibitor JQ1, a broad transcriptional repressor. Mechanistically, we identified a repressed FOXM1-dependent kinase subnetwork in JQ1-exposed cells that contained multiple cell cycle-regulated protein kinases. Consistently, we demonstrated vulnerability of UM cells to inhibitors of mitotic protein kinases within this network, including the investigational PLK1 inhibitor BI6727. We conclude that analysis of kinome-wide signalling network dynamics has the potential to reveal actionable drug targets and inhibitors of potential therapeutic benefit for UM patients. © 2017 The Authors. Pigment Cell & Melanoma Research Published by John Wiley & Sons.

  14. Hourly cooling load forecasting using time-indexed ARX models with two-stage weighted least squares regression

    International Nuclear Information System (INIS)

    Guo, Yin; Nazarian, Ehsan; Ko, Jeonghan; Rajurkar, Kamlakar

    2014-01-01

    Highlights: • Developed hourly-indexed ARX models for robust cooling-load forecasting. • Proposed a two-stage weighted least-squares regression approach. • Considered the effect of outliers as well as trend of cooling load and weather patterns. • Included higher order terms and day type patterns in the forecasting models. • Demonstrated better accuracy compared with some ARX and ANN models. - Abstract: This paper presents a robust hourly cooling-load forecasting method based on time-indexed autoregressive with exogenous inputs (ARX) models, in which the coefficients are estimated through a two-stage weighted least squares regression. The prediction method includes a combination of two separate time-indexed ARX models to improve prediction accuracy of the cooling load over different forecasting periods. The two-stage weighted least-squares regression approach in this study is robust to outliers and suitable for fast and adaptive coefficient estimation. The proposed method is tested on a large-scale central cooling system in an academic institution. The numerical case studies show the proposed prediction method performs better than some ANN and ARX forecasting models for the given test data set

  15. Transcriptional and Genomic Targets of Neural Stem Cells for Functional Recovery after Hemorrhagic Stroke

    Directory of Open Access Journals (Sweden)

    Le Zhang

    2017-01-01

    Full Text Available Hemorrhagic stroke is a life-threatening disease characterized by a sudden rupture of cerebral blood vessels, and it is widely believed that neural cell death occurs after exposure to blood metabolites or subsequently damaged cells. Neural stem cells (NSCs, which maintain neurogenesis and are found in subgranular zone and subventricular zone, are thought to be an endogenous neuroprotective mechanism for these brain injuries. However, due to the complexity of NSCs and their microenvironment, current strategies cannot satisfactorily enhance functional recovery after hemorrhagic stroke. It is well known that transcriptional and genomic pathways play important roles in ensuring the normal functions of NSCs, including proliferation, migration, differentiation, and neural reconnection. Recently, emerging evidence from the use of new technologies such as next-generation sequencing and transcriptome profiling has provided insight into our understanding of genomic function and regulation of NSCs. In the present article, we summarize and present the current data on the control of NSCs at both the transcriptional and genomic levels. Using bioinformatics methods, we sought to predict novel therapeutic targets of endogenous neurogenesis and exogenous NSC transplantation for functional recovery after hemorrhagic stroke, which could also advance our understanding of its pathophysiology.

  16. Complexity of CNC transcription factors as revealed by gene targeting of the Nrf3 locus.

    Science.gov (United States)

    Derjuga, Anna; Gourley, Tania S; Holm, Teresa M; Heng, Henry H Q; Shivdasani, Ramesh A; Ahmed, Rafi; Andrews, Nancy C; Blank, Volker

    2004-04-01

    Cap'n'collar (CNC) family basic leucine zipper transcription factors play crucial roles in the regulation of mammalian gene expression and development. To determine the in vivo function of the CNC protein Nrf3 (NF-E2-related factor 3), we generated mice deficient in this transcription factor. We performed targeted disruption of two Nrf3 exons coding for CNC homology, basic DNA-binding, and leucine zipper dimerization domains. Nrf3 null mice developed normally and revealed no obvious phenotypic differences compared to wild-type animals. Nrf3(-/-) mice were fertile, and gross anatomy as well as behavior appeared normal. The mice showed normal age progression and did not show any apparent additional phenotype during their life span. We observed no differences in various blood parameters and chemistry values. We infected wild-type and Nrf3(-/-) mice with acute lymphocytic choriomeningitis virus and found no differences in these animals with respect to their number of virus-specific CD8 and CD4 T cells as well as their B-lymphocyte response. To determine whether the mild phenotype of Nrf3 null animals is due to functional redundancy, we generated mice deficient in multiple CNC factors. Contrary to our expectations, an absence of Nrf3 does not seem to cause additional lethality in compound Nrf3(-/-)/Nrf2(-/-) and Nrf3(-/-)/p45(-/-) mice. We hypothesize that the role of Nrf3 in vivo may become apparent only after appropriate challenge to the mice.

  17. MicroRNA-214 suppresses gluconeogenesis by targeting activating transcriptional factor 4.

    Science.gov (United States)

    Li, Kai; Zhang, Jin; Yu, Junjie; Liu, Bin; Guo, Yajie; Deng, Jiali; Chen, Shanghai; Wang, Chunxia; Guo, Feifan

    2015-03-27

    Although the gluconeogenesis pathway is already a target for the treatment of type 2 diabetes, the potential role of microRNAs (miRNAs) in gluconeogenesis remains unclear. Here, we investigated the physiological functions of miR-214 in gluconeogenesis. The expression of miR-214 was suppressed by glucagon via protein kinase A signaling in primary hepatocytes, and miR-214 was down-regulated in the livers of fasted, high fat diet-induced diabetic and leptin receptor-mutated (db/db) mice. The overexpression of miR-214 in primary hepatocytes suppressed glucose production, and silencing miR-214 reversed this effect. Gluconeogenesis was suppressed in the livers of mice injected with an adenovirus expressing miR-214 (Ad-miR-214). Additionally, Ad-miR-214 alleviated high fat diet-induced elevation of gluconeogenesis and hyperglycemia. Furthermore, we found that activating transcription factor 4 (ATF4), a reported target of miR-214, can reverse the suppressive effect of miR-214 on gluconeogenesis in primary hepatocytes, and this suppressive effect was blocked in liver-specific ATF4 knock-out mice. ATF4 regulated gluconeogenesis via affecting forkhead box protein O1 (FOXO1) transcriptional activity. Finally, liver-specific miR-214 transgenic mice exhibited suppressed gluconeogenesis and reduced expression of ATF4, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase in liver. Taken together, our results suggest that the miR-214-ATF4 axis is a novel pathway for the regulation of hepatic gluconeogenesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. MicroRNA-214 Suppresses Gluconeogenesis by Targeting Activating Transcriptional Factor 4*

    Science.gov (United States)

    Li, Kai; Zhang, Jin; Yu, Junjie; Liu, Bin; Guo, Yajie; Deng, Jiali; Chen, Shanghai; Wang, Chunxia; Guo, Feifan

    2015-01-01

    Although the gluconeogenesis pathway is already a target for the treatment of type 2 diabetes, the potential role of microRNAs (miRNAs) in gluconeogenesis remains unclear. Here, we investigated the physiological functions of miR-214 in gluconeogenesis. The expression of miR-214 was suppressed by glucagon via protein kinase A signaling in primary hepatocytes, and miR-214 was down-regulated in the livers of fasted, high fat diet-induced diabetic and leptin receptor-mutated (db/db) mice. The overexpression of miR-214 in primary hepatocytes suppressed glucose production, and silencing miR-214 reversed this effect. Gluconeogenesis was suppressed in the livers of mice injected with an adenovirus expressing miR-214 (Ad-miR-214). Additionally, Ad-miR-214 alleviated high fat diet-induced elevation of gluconeogenesis and hyperglycemia. Furthermore, we found that activating transcription factor 4 (ATF4), a reported target of miR-214, can reverse the suppressive effect of miR-214 on gluconeogenesis in primary hepatocytes, and this suppressive effect was blocked in liver-specific ATF4 knock-out mice. ATF4 regulated gluconeogenesis via affecting forkhead box protein O1 (FOXO1) transcriptional activity. Finally, liver-specific miR-214 transgenic mice exhibited suppressed gluconeogenesis and reduced expression of ATF4, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase in liver. Taken together, our results suggest that the miR-214-ATF4 axis is a novel pathway for the regulation of hepatic gluconeogenesis. PMID:25657009

  19. MGAT1 is a novel transcriptional target of Wnt/β-catenin signaling pathway.

    Science.gov (United States)

    Akiva, Izzet; Birgül Iyison, Necla

    2018-01-08

    The Wnt/β-catenin signaling pathway is an evolutionary conserved pathway, which has important functions in vertebrate early development, axis formation, cellular proliferation and morphogenesis. Additionally, Wnt/β-catenin signaling pathway is one of the most important intracellular pathways that controls cancer progression. To date most of the identified targets of this pathway are shown to harbor tumorigenic properties. We previously showed that Mannosyl glycoprotein acetylglucosaminyl-transferase (MGAT1) enzyme is among the Wnt/β-catenin signaling putative target genes in hepatocellular carcinoma cell lines (Huh7). MGAT1 protein levels were determined by Western Blotting from Huh7 cell lines in which Wnt/β-catenin pathway was activated by means of different approaches such as LiCl treatment and mutant β-catenin overexpression. Luciferase reporter assay was used to analyze the promoter activity of MGAT1. The mRNA levels of MGAT1 were determined by quantitative real-time PCR from Huh7 cells that were treated with either Wnt agonist or GSK-3β inhibitor. Wound healing and XTT cell proliferation assays were performed in order to determine the proliferation and migration capacities of MGAT1 overexpressing stable Huh7 cells. Finally, xenograft experiments were carried out to measure the tumor formation capacities in vivo. In this study we showed that the activation of Wnt/β-catenin pathway culminates in the upregulation of MGAT1 enzyme both at transcriptional and post-transcriptional levels. We also showed that overexpression of the β-catenin gene (CTNNB1) increased the promoter activity of MGAT1. We applied a set of complementary approaches to elucidate the functional importance of MGAT1 as a vital target of Wnt/β-catenin signaling in Huh7 cells. Our analyses related to cell proliferation and migration assays showed that in comparison to the control cells, MGAT1 expressing Huh7 cells have greater proliferative and invasive capabilities. Furthermore, the

  20. Visualization of the tire-soil interaction area by means of ObjectARX programming interface

    Science.gov (United States)

    Mueller, W.; Gruszczyński, M.; Raba, B.; Lewicki, A.; Przybył, K.; Zaborowicz, M.; Koszela, K.; Boniecki, P.

    2014-04-01

    The process of data visualization, important for their analysis, becomes problematic when large data sets generated via computer simulations are available. This problem concerns, among others, the models that describe the geometry of tire-soil interaction. For the purpose of a graphical representation of this area and implementation of various geometric calculations the authors have developed a plug-in application for AutoCAD, based on the latest technologies, including ObjectARX, LINQ and the use of Visual Studio platform. Selected programming tools offer a wide variety of IT structures that enable data visualization and data analysis and are important e.g. in model verification.

  1. Projection-based Bayesian recursive estimation of ARX model with uniform innovations

    Czech Academy of Sciences Publication Activity Database

    Kárný, Miroslav; Pavelková, Lenka

    2007-01-01

    Roč. 56, 9/10 (2007), s. 646-655 ISSN 0167-6911 R&D Projects: GA AV ČR 1ET100750401; GA MŠk 2C06001; GA MDS 1F43A/003/120 Institutional research plan: CEZ:AV0Z10750506 Keywords : ARX model * Bayesian recursive estimation * Uniform distribution Subject RIV: BC - Control Systems Theory Impact factor: 1.634, year: 2007 http://dx.doi.org/10.1016/j.sysconle.2007.03.005

  2. Genome-Wide Identification of the Target Genes of AP2-O, a Plasmodium AP2-Family Transcription Factor.

    Directory of Open Access Journals (Sweden)

    Izumi Kaneko

    2015-05-01

    Full Text Available Stage-specific transcription is a fundamental biological process in the life cycle of the Plasmodium parasite. Proteins containing the AP2 DNA-binding domain are responsible for stage-specific transcriptional regulation and belong to the only known family of transcription factors in Plasmodium parasites. Comprehensive identification of their target genes will advance our understanding of the molecular basis of stage-specific transcriptional regulation and stage-specific parasite development. AP2-O is an AP2 family transcription factor that is expressed in the mosquito midgut-invading stage, called the ookinete, and is essential for normal morphogenesis of this stage. In this study, we identified the genome-wide target genes of AP2-O by chromatin immunoprecipitation-sequencing and elucidate how this AP2 family transcription factor contributes to the formation of this motile stage. The analysis revealed that AP2-O binds specifically to the upstream genomic regions of more than 500 genes, suggesting that approximately 10% of the parasite genome is directly regulated by AP2-O. These genes are involved in distinct biological processes such as morphogenesis, locomotion, midgut penetration, protection against mosquito immunity and preparation for subsequent oocyst development. This direct and global regulation by AP2-O provides a model for gene regulation in Plasmodium parasites and may explain how these parasites manage to control their complex life cycle using a small number of sequence-specific AP2 transcription factors.

  3. A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

    Science.gov (United States)

    Sarris, Panagiotis F; Duxbury, Zane; Huh, Sung Un; Ma, Yan; Segonzac, Cécile; Sklenar, Jan; Derbyshire, Paul; Cevik, Volkan; Rallapalli, Ghanasyam; Saucet, Simon B; Wirthmueller, Lennart; Menke, Frank L H; Sohn, Kee Hoon; Jones, Jonathan D G

    2015-05-21

    Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Interactions between the R2R3-MYB transcription factor, AtMYB61, and target DNA binding sites.

    Directory of Open Access Journals (Sweden)

    Michael B Prouse

    Full Text Available Despite the prominent roles played by R2R3-MYB transcription factors in the regulation of plant gene expression, little is known about the details of how these proteins interact with their DNA targets. For example, while Arabidopsis thaliana R2R3-MYB protein AtMYB61 is known to alter transcript abundance of a specific set of target genes, little is known about the specific DNA sequences to which AtMYB61 binds. To address this gap in knowledge, DNA sequences bound by AtMYB61 were identified using cyclic amplification and selection of targets (CASTing. The DNA targets identified using this approach corresponded to AC elements, sequences enriched in adenosine and cytosine nucleotides. The preferred target sequence that bound with the greatest affinity to AtMYB61 recombinant protein was ACCTAC, the AC-I element. Mutational analyses based on the AC-I element showed that ACC nucleotides in the AC-I element served as the core recognition motif, critical for AtMYB61 binding. Molecular modelling predicted interactions between AtMYB61 amino acid residues and corresponding nucleotides in the DNA targets. The affinity between AtMYB61 and specific target DNA sequences did not correlate with AtMYB61-driven transcriptional activation with each of the target sequences. CASTing-selected motifs were found in the regulatory regions of genes previously shown to be regulated by AtMYB61. Taken together, these findings are consistent with the hypothesis that AtMYB61 regulates transcription from specific cis-acting AC elements in vivo. The results shed light on the specifics of DNA binding by an important family of plant-specific transcriptional regulators.

  5. MENA is a transcriptional target of the Wnt/beta-catenin pathway.

    Directory of Open Access Journals (Sweden)

    Ayaz Najafov

    Full Text Available Wnt/β-catenin signalling pathway plays important roles in embryonic development and carcinogenesis. Overactivation of the pathway is one of the most common driving forces in major cancers such as colorectal and breast cancers. The downstream effectors of the pathway and its regulation of carcinogenesis and metastasis are still not very well understood. In this study, which was based on two genome-wide transcriptomics screens, we identify MENA (ENAH, Mammalian enabled homologue as a novel transcriptional target of the Wnt/β-catenin signalling pathway. We show that the expression of MENA is upregulated upon overexpression of degradation-resistant β-catenin. Promoters of all mammalian MENA homologues contain putative binding sites for Tcf4 transcription factor--the primary effector of the Wnt/β-catenin pathway and we demonstrate functionality of these Tcf4-binding sites using luciferase reporter assays and overexpression of β-catenin, Tcf4 and dominant-negative Tcf4. In addition, lithium chloride-mediated inhibition of GSK3β also resulted in increase in MENA mRNA levels. Chromatin immunoprecipitation showed direct interaction between β-catenin and MENA promoter in Huh7 and HEK293 cells and also in mouse brain and liver tissues. Moreover, overexpression of Wnt1 and Wnt3a ligands increased MENA mRNA levels. Additionally, knock-down of MENA ortholog in D. melanogaster eyeful and sensitized eye cancer fly models resulted in increased tumor and metastasis formations. In summary, our study identifies MENA as novel nexus for the Wnt/β-catenin and the Notch signalling cascades.

  6. MENA is a transcriptional target of the Wnt/beta-catenin pathway.

    Science.gov (United States)

    Najafov, Ayaz; Seker, Tuncay; Even, Ipek; Hoxhaj, Gerta; Selvi, Osman; Ozel, Duygu Esen; Koman, Ahmet; Birgül-İyison, Necla

    2012-01-01

    Wnt/β-catenin signalling pathway plays important roles in embryonic development and carcinogenesis. Overactivation of the pathway is one of the most common driving forces in major cancers such as colorectal and breast cancers. The downstream effectors of the pathway and its regulation of carcinogenesis and metastasis are still not very well understood. In this study, which was based on two genome-wide transcriptomics screens, we identify MENA (ENAH, Mammalian enabled homologue) as a novel transcriptional target of the Wnt/β-catenin signalling pathway. We show that the expression of MENA is upregulated upon overexpression of degradation-resistant β-catenin. Promoters of all mammalian MENA homologues contain putative binding sites for Tcf4 transcription factor--the primary effector of the Wnt/β-catenin pathway and we demonstrate functionality of these Tcf4-binding sites using luciferase reporter assays and overexpression of β-catenin, Tcf4 and dominant-negative Tcf4. In addition, lithium chloride-mediated inhibition of GSK3β also resulted in increase in MENA mRNA levels. Chromatin immunoprecipitation showed direct interaction between β-catenin and MENA promoter in Huh7 and HEK293 cells and also in mouse brain and liver tissues. Moreover, overexpression of Wnt1 and Wnt3a ligands increased MENA mRNA levels. Additionally, knock-down of MENA ortholog in D. melanogaster eyeful and sensitized eye cancer fly models resulted in increased tumor and metastasis formations. In summary, our study identifies MENA as novel nexus for the Wnt/β-catenin and the Notch signalling cascades.

  7. Targeting RNA transcription and translation in ovarian cancer cells with pharmacological inhibitor CDKI-73.

    Science.gov (United States)

    Lam, Frankie; Abbas, Abdullahi Y; Shao, Hao; Teo, Theodosia; Adams, Julian; Li, Peng; Bradshaw, Tracey D; Fischer, Peter M; Walsby, Elisabeth; Pepper, Chris; Chen, Yi; Ding, Jian; Wang, Shudong

    2014-09-15

    Dysregulation of cellular transcription and translation is a fundamental hallmark of cancer. As CDK9 and Mnks play pivotal roles in the regulation of RNA transcription and protein synthesis, respectively, they are important targets for drug development. We herein report the cellular mechanism of a novel CDK9 inhibitor CDKI-73 in an ovarian cancer cell line (A2780). We also used shRNA-mediated CDK9 knockdown to investigate the importance of CDK9 in the maintenance of A2780 cells. This study revealed that CDKI-73 rapidly inhibited cellular CDK9 kinase activity and down-regulated the RNAPII phosphorylation. This subsequently caused a decrease in the eIF4E phosphorylation by blocking Mnk1 kinase activity. Consistently, CDK9 shRNA was also found to down-regulate the Mnk1 expression. Both CDKI-73 and CDK9 shRNA decreased anti-apoptotic proteins Mcl-1 and Bcl-2 and induced apoptosis. The study confirmed that CDK9 is required for cell survival and that ovarian cancer may be susceptible to CDK9 inhibition strategy. The data also implied a role of CDK9 in eIF4E-mediated translational control, suggesting that CDK9 may have important implication in the Mnk-eIF4E axis, the key determinants of PI3K/Akt/mTOR- and Ras/Raf/MAPK-mediated tumorigenic activity. As such, CDK9 inhibitor drug candidate CDKI-73 should have a major impact on these pathways in human cancers.

  8. Receptor-targeted aptamer-siRNA conjugate-directed transcriptional regulation of HIV-1

    Science.gov (United States)

    Zhou, Jiehua; Lazar, Daniel; Li, Haitang; Xia, Xin; Satheesan, Sangeetha; Charlins, Paige; O'Mealy, Denis; Akkina, Ramesh; Saayman, Sheena; Weinberg, Marc S.; Rossi, John J.; Morris, Kevin V.

    2018-01-01

    Gene-based therapies represent a promising therapeutic paradigm for the treatment of HIV-1, as they have the potential to maintain sustained viral inhibition with reduced treatment interventions. Such an option may represent a long-term treatment alternative to highly active antiretroviral therapy. Methods: We previously described a therapeutic approach, referred to as transcriptional gene silencing (TGS), whereby small noncoding RNAs directly inhibit the transcriptional activity of HIV-1 by targeting sites within the viral promoter, specifically the 5' long terminal repeat (LTR). TGS differs from traditional RNA interference (RNAi) in that it is characterized by concomitant silent-state epigenetic marks on histones and DNA. To deliver TGS-inducing RNAs, we developed functional RNA conjugates based on the previously reported dual function of the gp120 (A-1) aptamer conjugated to 27-mer Dicer-substrate anti-HIV-1 siRNA (dsiRNA), LTR-362. Results: We demonstrate here that high levels of processed guide RNAs localize to the nucleus in infected T lymphoblastoid CEM cell line and primary human CD4+ T-cells. Treatment of the aptamer-siRNA conjugates induced TGS with an ~10-fold suppression of viral p24 levels as measured at day 12 post infection. To explore the silencing efficacy of aptamer-siRNA conjugates in vivo, HIV-1-infected humanized NOD/SCID/IL2 rγnull mice (hu-NSG) were treated with the aptamer-siRNA conjugates. Systemic delivery of the A-1-stick-LTR-362 27-mer siRNA conjugates suppressed HIV-1 infection and protected CD4+ T cell levels in viremia hu-NSG mice. Principle conclusions: Collectively these data suggest that the gp120 aptamer-dsiRNA conjugate design is suitable for systemic delivery of small RNAs that can be used to suppress HIV-1. PMID:29556342

  9. Vorinostat in combination with bortezomib in patients with advanced malignancies directly alters transcription of target genes.

    Science.gov (United States)

    Kolesar, Jill M; Traynor, Anne M; Holen, Kyle D; Hoang, Tien; Seo, Songwon; Kim, Kyungmann; Alberti, Dona; Espinoza-Delgado, Igor; Wright, John J; Wilding, George; Bailey, Howard H; Schelman, William R

    2013-09-01

    Vorinostat is a small molecule inhibitor of class I and II histone deacetylase enzymes which alters the expression of target genes including the cell cycle gene p21, leading to cell cycle arrest and apoptosis. Patients enrolled in a phase I trial were treated with vorinostat alone on day 1 and vorinostat and bortezomib in combination on day 9. Paired biopsies were obtained in eleven subjects. Blood samples were obtained on days 1 and 9 of cycle 1 prior to dosing and 2 and 6 h post-dosing in all 60 subjects. Gene expression of p21, HSP70, AKT, Nur77, ERB1, and ERB2 was evaluated in peripheral blood mononuclear cells and tissue samples. Chromatin immunoprecipitation of p21, HSP70, and Nur77 was also performed in biopsy samples. In peripheral blood mononuclear cells, Nur77 was significantly and consistently decreased 2 h after vorinostat administration on both days 1 and 9, median ratio of gene expression relative to baseline of 0.69 with interquartile range 0.49-1.04 (p vorinostat and bortezomib. p21, a downstream target of Nur77, was significantly decreased on day 9, 2 and 6 h after administration of vorinostat and bortezomib, 0.67 (0.41-1.03) (p vorinostat in tissue biopsies in most patients. Vorinostat inhibits Nur77 expression, which in turn may decrease p21 and AKT expression in PBMCs. The influence of vorinostat on target gene expression in tumor tissue was variable; however, most patients demonstrated interaction of acetylated H3 with Nur77, HSP70, and p21 which provides evidence of interaction with the transcriptionally active acetylated H3.

  10. RBF–ARX model of an industrial furnace for drying olive pomace

    International Nuclear Information System (INIS)

    Casanova-Peláez, P.J.; Cruz-Peragón, F.; Palomar-Carnicero, J.M.; Dorado, R.; López-García, R.

    2012-01-01

    Highlights: ► We model a real furnace, fuelled with orujo, used to dry olive pomace. ► We apply a radial basic functions–auto-regression with exogenous variables (ARXs–RBFs) method. ► Root-mean-square error and r 2 are used to validate the ARX–RBF model. - Abstract: Drying operations are common in food industries. One of the main components in a drying system is the furnace. The furnace operation involves heat–mass transfer and combustion, thus it demands a complex mathematic representation. Since autoregressive methods are simple, and help to simulate rapidly a system, we model a drying furnace of olive pomace via an auto-regression with exogenous variables (ARXs) method. A neural network of radial basic functions (RBFs) defines the ARX experimental relation between the amounts of dry pomace (moisture content of 15%) used like fuel and the temperature of outlet gases. A real industrial furnace is studied to validate the proposed model, which can help to control the drying process.

  11. Modal Identification of A Tested Steel Frame using Linear ARX Model Structure

    Directory of Open Access Journals (Sweden)

    Yavuz Kaya

    2009-07-01

    Full Text Available This study contains the identification of modal dynamic properties of a 3-story large-scale steel test frame structure through shaking table measurements. Shaking table test is carried out to estimate the modal properties of the test frame such as natural frequencies, damping ratios and mode shapes. Among many different model structures, ARX (Auto Recursive Exogenous model structure is used for modal identification of the frame structure system. The unknown parameters in the obtained ARX model structure are estimated by Least-Square method by minimizing the AIC criteria with the help of a program coded in advanced computing software MATLAB®. The adopted model structure is then tested out in time domain to verify the validity of the model with the selected model parameters. Then the modal characteristics of test frame and the story stiffness are estimated using the white noise shakings. An attempt is done to determine the change of modal characteristics and the story stiffness of test frame according to the velocity, which the test frame structure experienced during the shaking schedule and also during the input shaking of El Centro 1940 NS. Results shows that there is an increase in damping ratio and a decrease in both story stiffness and natural frequency for all modes when the damage forms at cementitious device and the test frame structure itself during the shaking schedule.

  12. Activating transcription factor 3 is a target molecule linking hepatic steatosis to impaired glucose homeostasis.

    Science.gov (United States)

    Kim, Ji Yeon; Park, Keon Jae; Hwang, Joo-Yeon; Kim, Gyu Hee; Lee, DaeYeon; Lee, Yoo Jeong; Song, Eun Hyun; Yoo, Min-Gyu; Kim, Bong-Jo; Suh, Young Ho; Roh, Gu Seob; Gao, Bin; Kim, Won; Kim, Won-Ho

    2017-08-01

    Non-alcoholic fatty liver disease (NAFLD) contributes to impaired glucose tolerance, leading to type 2 diabetes (T2D); however, the precise mechanisms and target molecules that are involved remain unclear. Activating transcription factor 3 (ATF3) is associated with β-cell dysfunction that is induced by severe stress signals in T2D. We aimed to explore the exact functional role of ATF3 as a mechanistic link between hepatic steatosis and T2D development. Zucker diabetic fatty (ZDF) rats were utilized for animal experiments. An in vivo-jetPEI siRNA delivery system against ATF3 was used for loss-of-function experiments. We analyzed the baseline cross-sectional data derived from the biopsy-proven NAFLD registry (n=322). Human sera and liver tissues were obtained from 43 patients with biopsy-proven NAFLD and from seven healthy participants. ATF3 was highly expressed in the livers of ZDF rats and in human participants with NAFLD and/or T2D. Insulin resistance and hepatic steatosis were associated with increased ATF3 expression and decreased fatty acid oxidation via mitochondrial dysfunction and were attenuated by in vivo ATF3 silencing. Knockdown of ATF3 also ameliorated glucose intolerance, impaired insulin action, and inflammatory responses in ZDF rats. In patients with NAFLD and/or T2D, a significant positive correlation was observed between hepatic ATF3 expression and surrogate markers of T2D, mitochondrial dysfunction, and macrophage infiltration. Increased hepatic ATF3 expression is closely associated with hepatic steatosis and incident T2D; therefore, ATF3 may serve as a potential therapeutic target for NAFLD and hepatic steatosis-induced T2D. Hepatic activating transcription factor 3 (ATF3) may play an important role in oxidative stress-mediated hepatic steatosis and the development of type 2 diabetes (T2D) in a Zucker diabetic fatty (ZDF) rat model and in human patients with non-alcoholic fatty liver disease (NAFLD). Therefore, ATF3 may be a useful biomarker for

  13. Variable surface composition and radial interface formation in self-assembled free, mixed Ar/Xe clusters

    International Nuclear Information System (INIS)

    Tchaplyguine, M.; Maartensson, N.; Lundwall, M.; Oehrwall, G.; Feifel, R.; Svensson, S.; Bjoerneholm, O.; Gisselbrecht, M.; Sorensen, S.

    2004-01-01

    Using photoelectron spectroscopy, we demonstrate how the self-assembling process of cluster formation in an adiabatic expansion leads to radial segregation and layering as well as to variable surface composition for binary Ar/Xe clusters. The radial structuring can be qualitatively understood from the different interatomic bonding strengths of the two components

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

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    Evi Berchtold

    Full Text Available 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.

  15. Microphthalmia-associated transcription factor as the molecular target of cadmium toxicity in human melanocytes

    International Nuclear Information System (INIS)

    Chantarawong, Wipa; Takeda, Kazuhisa; Sangartit, Weerapon; Yoshizawa, Miki; Pradermwong, Kantimanee; Shibahara, Shigeki

    2014-01-01

    Highlights: • In human melanocytes, cadmium decreases the expression of MITF-M and tyrosinase and their mRNAs. • In human melanoma cells, cadmium decreases the expression of MITF-M protein and tyrosinase mRNA. • Expression of MITF-H is less sensitive to cadmium toxicity in melanocyte-linage cells. • Cadmium does not decrease the expression of MITF-H in retinal pigment epithelial cells. • MITF-M is the molecular target of cadmium toxicity in melanocytes. - Abstract: Dietary intake of cadmium is inevitable, causing age-related increase in cadmium accumulation in many organs, including hair, choroid and retinal pigment epithelium (RPE). Cadmium has been implicated in the pathogenesis of hearing loss and macular degeneration. The functions of cochlea and retina are maintained by melanocytes and RPE, respectively, and the differentiation of these pigment cells is regulated by microphthalmia-associated transcription factor (MITF). In the present study, we explored the potential toxicity of cadmium in the cochlea and retina by using cultured human melanocytes and human RPE cell lines. MITF consists of multiple isoforms, including melanocyte-specific MITF-M and widely expressed MITF-H. Levels of MITF-M protein and its mRNA in human epidermal melanocytes and HMV-II melanoma cells were decreased significantly by cadmium. In parallel with the MITF reduction, mRNA levels of tyrosinase, the key enzyme of melanin biosynthesis that is regulated by MITF-M, were also decreased. In RPE cells, however, the levels of total MITF protein, constituting mainly MITF-H, were not decreased by cadmium. We thus identify MITF-M as the molecular target of cadmium toxicity in melanocytes, thereby accounting for the increased risk of disability from melanocyte malfunction, such as hearing and vision loss among people with elevated cadmium exposure

  16. Microphthalmia-associated transcription factor as the molecular target of cadmium toxicity in human melanocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chantarawong, Wipa [Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai (Japan); Inter Departmental Multidisciplinary Graduate Program in Bioscience, Faculty of Science, Kasetsart University, Bangkok (Thailand); Takeda, Kazuhisa; Sangartit, Weerapon; Yoshizawa, Miki [Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai (Japan); Pradermwong, Kantimanee [Department of Zoology, Faculty of Science, Kasetsart University, Bangkok (Thailand); Shibahara, Shigeki, E-mail: shibahar@med.tohoku.ac.jp [Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai (Japan)

    2014-11-28

    Highlights: • In human melanocytes, cadmium decreases the expression of MITF-M and tyrosinase and their mRNAs. • In human melanoma cells, cadmium decreases the expression of MITF-M protein and tyrosinase mRNA. • Expression of MITF-H is less sensitive to cadmium toxicity in melanocyte-linage cells. • Cadmium does not decrease the expression of MITF-H in retinal pigment epithelial cells. • MITF-M is the molecular target of cadmium toxicity in melanocytes. - Abstract: Dietary intake of cadmium is inevitable, causing age-related increase in cadmium accumulation in many organs, including hair, choroid and retinal pigment epithelium (RPE). Cadmium has been implicated in the pathogenesis of hearing loss and macular degeneration. The functions of cochlea and retina are maintained by melanocytes and RPE, respectively, and the differentiation of these pigment cells is regulated by microphthalmia-associated transcription factor (MITF). In the present study, we explored the potential toxicity of cadmium in the cochlea and retina by using cultured human melanocytes and human RPE cell lines. MITF consists of multiple isoforms, including melanocyte-specific MITF-M and widely expressed MITF-H. Levels of MITF-M protein and its mRNA in human epidermal melanocytes and HMV-II melanoma cells were decreased significantly by cadmium. In parallel with the MITF reduction, mRNA levels of tyrosinase, the key enzyme of melanin biosynthesis that is regulated by MITF-M, were also decreased. In RPE cells, however, the levels of total MITF protein, constituting mainly MITF-H, were not decreased by cadmium. We thus identify MITF-M as the molecular target of cadmium toxicity in melanocytes, thereby accounting for the increased risk of disability from melanocyte malfunction, such as hearing and vision loss among people with elevated cadmium exposure.

  17. Peripheral CLOCK regulates target-tissue glucocorticoid receptor transcriptional activity in a circadian fashion in man.

    Directory of Open Access Journals (Sweden)

    Evangelia Charmandari

    Full Text Available Circulating cortisol fluctuates diurnally under the control of the "master" circadian CLOCK, while the peripheral "slave" counterpart of the latter regulates the transcriptional activity of the glucocorticoid receptor (GR at local glucocorticoid target tissues through acetylation. In this manuscript, we studied the effect of CLOCK-mediated GR acetylation on the sensitivity of peripheral tissues to glucocorticoids in humans.We examined GR acetylation and mRNA expression of GR, CLOCK-related and glucocorticoid-responsive genes in peripheral blood mononuclear cells (PBMCs obtained at 8 am and 8 pm from 10 healthy subjects, as well as in PBMCs obtained in the morning and cultured for 24 hours with exposure to 3-hour hydrocortisone pulses every 6 hours. We used EBV-transformed lymphocytes (EBVLs as non-synchronized controls.GR acetylation was higher in the morning than in the evening in PBMCs, mirroring the fluctuations of circulating cortisol in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone in non-synchronized EBVLs, however, some of these genes did not show the expected diurnal mRNA fluctuations in PBMCs in vivo. Instead, their mRNA oscillated in a Clock- and a GR acetylation-dependent fashion in naturally synchronized PBMCs cultured ex vivo in the absence of the endogenous glucocorticoid, suggesting that circulating cortisol might prevent circadian GR acetylation-dependent effects in some glucocorticoid-responsive genes in vivo.Peripheral CLOCK-mediated circadian acetylation of the human GR may function as a target-tissue, gene-specific counter regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it at night.

  18. Peripheral CLOCK Regulates Target-Tissue Glucocorticoid Receptor Transcriptional Activity in a Circadian Fashion in Man

    Science.gov (United States)

    Charmandari, Evangelia; Chrousos, George P.; Lambrou, George I.; Pavlaki, Aikaterini; Koide, Hisashi; Ng, Sinnie Sin Man; Kino, Tomoshige

    2011-01-01

    Context and Objective Circulating cortisol fluctuates diurnally under the control of the “master” circadian CLOCK, while the peripheral “slave” counterpart of the latter regulates the transcriptional activity of the glucocorticoid receptor (GR) at local glucocorticoid target tissues through acetylation. In this manuscript, we studied the effect of CLOCK-mediated GR acetylation on the sensitivity of peripheral tissues to glucocorticoids in humans. Design and Participants We examined GR acetylation and mRNA expression of GR, CLOCK-related and glucocorticoid-responsive genes in peripheral blood mononuclear cells (PBMCs) obtained at 8 am and 8 pm from 10 healthy subjects, as well as in PBMCs obtained in the morning and cultured for 24 hours with exposure to 3-hour hydrocortisone pulses every 6 hours. We used EBV-transformed lymphocytes (EBVLs) as non-synchronized controls. Results GR acetylation was higher in the morning than in the evening in PBMCs, mirroring the fluctuations of circulating cortisol in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone in non-synchronized EBVLs, however, some of these genes did not show the expected diurnal mRNA fluctuations in PBMCs in vivo. Instead, their mRNA oscillated in a Clock- and a GR acetylation-dependent fashion in naturally synchronized PBMCs cultured ex vivo in the absence of the endogenous glucocorticoid, suggesting that circulating cortisol might prevent circadian GR acetylation-dependent effects in some glucocorticoid-responsive genes in vivo. Conclusions Peripheral CLOCK-mediated circadian acetylation of the human GR may function as a target-tissue, gene-specific counter regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it at night. PMID:21980503

  19. Wnt-mediated down-regulation of Sp1 target genes by a transcriptional repressor Sp5

    Czech Academy of Sciences Publication Activity Database

    Fujimura, Naoko; Vacík, Tomáš; Machoň, Ondřej; Vlček, Čestmír; Scalabrin, S.; Speth, M.; Diep, D.; Krauss, S.; Kozmik, Zbyněk

    2007-01-01

    Roč. 282, č. 2 (2007), s. 1225-1237 ISSN 0021-9258 Institutional research plan: CEZ:AV0Z50520514 Keywords : Wnt -mediated signaling * Sp5 transcription factor * Sp1 target genes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.581, year: 2007

  20. Identification of the Drosophila Mes4 gene as a novel target of the transcription factor DREF

    Energy Technology Data Exchange (ETDEWEB)

    Suyari, Osamu; Ida, Hiroyuki [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Yoshioka, Yasuhide; Kato, Yasuko; Hashimoto, Reina [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Venture Laboratory, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Yamaguchi, Masamitsu, E-mail: myamaguc@kit.ac.jp [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan)

    2009-05-01

    The Mes4 gene has been identified as one of the maternal Dorsal target genes in Drosophila. In the present study, we found a DNA replication-related element (DRE, 5'-TATCGATA) in the Mes4 promoter recognized by the DRE-binding factor (DREF). Luciferase transient expression assays in S2 cells using Mes4 promoter-luciferase fusion plasmids revealed that the DRE sequence is essential for Mes4 promoter activity. Requirement of DRE for Mes4 promoter activity was further confirmed by anti-{beta}-galactosidase antibody-staining of various tissues from transgenic flies carrying Mes4 promoter-lacZ fusion genes. Furthermore, wild type Mes4 promoter activity was decreased by 40% in DREF-depleted S2 cells. These results indicate that DREF positively regulates Mes4 gene expression. Band mobility shift analyses using Kc cell nuclear extracts further indicated that the DRE sequence in the Mes4 promoter is especially important for binding to DREF. Moreover, specific binding of DREF to the involved genomic region could be demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. These results, taken together, indicate that the DRE/DREF system activates transcription of the Mes4 gene. In addition, knockdown of the Mes4 gene in wing imaginal discs using the GAL4-UAS system caused an atrophied wing phenotype, suggesting that Mes4 is required for wing morphogenesis.

  1. Identification of the Drosophila Mes4 gene as a novel target of the transcription factor DREF

    International Nuclear Information System (INIS)

    Suyari, Osamu; Ida, Hiroyuki; Yoshioka, Yasuhide; Kato, Yasuko; Hashimoto, Reina; Yamaguchi, Masamitsu

    2009-01-01

    The Mes4 gene has been identified as one of the maternal Dorsal target genes in Drosophila. In the present study, we found a DNA replication-related element (DRE, 5'-TATCGATA) in the Mes4 promoter recognized by the DRE-binding factor (DREF). Luciferase transient expression assays in S2 cells using Mes4 promoter-luciferase fusion plasmids revealed that the DRE sequence is essential for Mes4 promoter activity. Requirement of DRE for Mes4 promoter activity was further confirmed by anti-β-galactosidase antibody-staining of various tissues from transgenic flies carrying Mes4 promoter-lacZ fusion genes. Furthermore, wild type Mes4 promoter activity was decreased by 40% in DREF-depleted S2 cells. These results indicate that DREF positively regulates Mes4 gene expression. Band mobility shift analyses using Kc cell nuclear extracts further indicated that the DRE sequence in the Mes4 promoter is especially important for binding to DREF. Moreover, specific binding of DREF to the involved genomic region could be demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. These results, taken together, indicate that the DRE/DREF system activates transcription of the Mes4 gene. In addition, knockdown of the Mes4 gene in wing imaginal discs using the GAL4-UAS system caused an atrophied wing phenotype, suggesting that Mes4 is required for wing morphogenesis.

  2. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases

    Directory of Open Access Journals (Sweden)

    Xinxia Zhao

    2016-03-01

    Full Text Available Myostatin (MSTN is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs in tandem with single-stranded DNA oligonucleotides (ssODNs. We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

  3. In Vivo Chromatin Targets of the Transcription Factor Yin Yang 2 in Trophoblast Stem Cells

    Science.gov (United States)

    Pérez-Palacios, Raquel; Macías-Redondo, Sofía; Climent, María; Contreras-Moreira, Bruno; Muniesa, Pedro; Schoorlemmer, Jon

    2016-01-01

    Background Yin Yang 2 (YY2) is a zinc finger protein closely related to the well-characterized Yin Yang 1 (YY1). YY1 is a DNA-binding transcription factor, with defined functions in multiple developmental processes, such as implantation, cell differentiation, X inactivation, imprinting and organogenesis. Yy2 has been treated as a largely immaterial duplication of Yy1, as they share high homology in the Zinc Finger-region and similar if not identical in vitro binding sites. In contrast to these similarities, gene expression alterations in HeLa cells with attenuated levels of either Yy1 or Yy2 were to some extent gene-specific. Moreover, the chromatin binding sites for YY2, except for its association with transposable retroviral elements (RE) and Endogenous Retroviral Elements (ERVs), remain to be identified. As a first step towards defining potential Yy2 functions matching or complementary to Yy1, we considered in vivo DNA binding sites of YY2 in trophoblast stem (TS) cells. Results We report the presence of YY2 protein in mouse-derived embryonic stem (ES) and TS cell lines. Following up on our previous report on ERV binding by YY2 in TS cells, we investigated the tissue-specificity of REX1 and YY2 binding and confirm binding to RE/ERV targets in both ES cells and TS cells. Because of the higher levels of expression, we chose TS cells to understand the role of Yy2 in gene and chromatin regulation. We used in vivo YY2 association as a measure to identify potential target genes. Sequencing of chromatin obtained in chromatin-immunoprecipitation (ChIP) assays carried out with αYY2 serum allowed us to identify a limited number of chromatin targets for YY2. Some putative binding sites were validated in regular ChIP assays and gene expression of genes nearby was altered in the absence of Yy2. Conclusions YY2 binding to ERVs is not confined to TS cells. In vivo binding sites share the presence of a consensus binding motif. Selected sites were uniquely bound by YY2 as

  4. HIV-1 reverse transcription initiation: a potential target for novel antivirals?

    NARCIS (Netherlands)

    Abbink, Truus E. M.; Berkhout, Ben

    2008-01-01

    Reverse transcription is an essential step in the retroviral life cycle, as it converts the genomic RNA into DNA. In this review, we describe recent developments concerning the initiation step of this complex, multi-step reaction. During initiation of reverse transcription, a cellular tRNA primer is

  5. On-line leak detection method for OWL-1 loop by ARX modeling using dewpoint signals

    International Nuclear Information System (INIS)

    Oguma, Ritsuo; Hayashi, Koji; Kitajima, Toshio.

    1981-01-01

    Model identification technique based on ARX (autoregressive model with exogenous variable) process was applied to dewpoint data recorded at OWL-1 (Oarai Water Loop No. 1) loop cubicle in JMTR (Japan Materials Testing Reactor) and the dynamical interrelationship between the supply and exhaust dewpoints in the ventilation system of the cubicle was empirically determined. It was shown that the information so derived on the dewpoint dynamics can assist to enhance the sensitivity of leak detection, if it was incorporated into a leak monitoring system for the OWL-1 loop. A simple digital filter incorporating the dewpoint dynamics was designed in an attempt to develop an efficient leak monitor for the OWL-1 loop. This filter was applied to the dewpoint data recordings during an abnormal leak that had occurred at the OWL-1 loop in the 43 rd cycle of JMTR operation, which demonstrated the effectiveness of the present method for leak detection at its early stage. (author)

  6. Interatomic decay of inner-valence ionized states in ArXe clusters: Relativistic approach

    International Nuclear Information System (INIS)

    Fasshauer, Elke; Pernpointner, Markus; Gokhberg, Kirill

    2013-01-01

    In this work we investigate interatomic electronic decay processes taking place in mixed argon-xenon clusters upon the inner-valence ionization of an argon center. We demonstrate that both interatomic Coulombic decay and electron-transfer mediated decay (ETMD) are important in larger rare gas clusters as opposed to dimers. Calculated secondary electron spectra are shown to depend strongly on the spin-orbit coupling in the final states of the decay as well as the presence of polarizable environment. It follows from our calculations that ETMD is a pure interface process taking place between the argon-xenon layers. The interplay of all these effects is investigated in order to arrive at a suitable physical model for the decay of inner-valence vacancies taking place in mixed ArXe clusters.

  7. Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

    International Nuclear Information System (INIS)

    Wada, Takeyoshi; Asahi, Toru; Sawamura, Naoya

    2016-01-01

    The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. - Highlights: • We found that CRBN is a nucleocytoplasmic shutting protein and identified the key domain for nucleocytoplasmic shuttling. • CRBN associates with the transcription factor Ikaros via the N-terminal domain. • CRBN modulates Ikaros-mediated transcriptional regulation and its downstream target, enkephalin.

  8. Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Wada, Takeyoshi [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Asahi, Toru [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Research Organization for Nano & Life Innovation, Waseda University #03C309, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Sawamura, Naoya, E-mail: naoya.sawamura@gmail.com [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Research Organization for Nano & Life Innovation, Waseda University #03C309, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan)

    2016-08-26

    The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. - Highlights: • We found that CRBN is a nucleocytoplasmic shutting protein and identified the key domain for nucleocytoplasmic shuttling. • CRBN associates with the transcription factor Ikaros via the N-terminal domain. • CRBN modulates Ikaros-mediated transcriptional regulation and its downstream target, enkephalin.

  9. A-type nuclear lamins act as transcriptional repressors when targeted to promoters

    International Nuclear Information System (INIS)

    Lee, Damian C.; Welton, K. Linnea; Smith, Erica D.; Kennedy, Brian K.

    2009-01-01

    Regions of heterochromatin are often found at the periphery of the mammalian nucleus, juxtaposed to the nuclear lamina. Genes in these regions are likely maintained in a transcriptionally silent state, although other locations at the nuclear periphery associated with nuclear pores are sites of active transcription. As primary components of the nuclear lamina, A- and B-type nuclear lamins are intermediate filament proteins that interact with DNA, histones and known transcriptional repressors, leading to speculation that they may promote establishment of repressive domains. However, no direct evidence of a role for nuclear lamins in transcriptional repression has been reported. Here we find that human lamin A, when expressed in yeast and cultured human cells as a fusion protein to the Gal4 DNA-binding domain (DBD), can mediate robust transcriptional repression of promoters with Gal4 binding sites. Full repression by lamin A requires both the coiled-coil rod domain and the C-terminal tail domain. In human cells, other intermediate filament proteins such as lamin B and vimentin are unable to confer robust repression as Gal4-DBD fusions, indicating that this property is specific to A-type nuclear lamins. These findings indicate that A-type lamins can promote transcriptional repression when in proximity of a promoter

  10. XLMR in MRX families 29, 32, 33 and 38 results from the dup24 mutation in the ARX (Aristaless related homeobox gene

    Directory of Open Access Journals (Sweden)

    MacMillan Andrée

    2005-04-01

    Full Text Available Abstract Background X-linked mental retardation (XLMR is the leading cause of mental retardation in males. Mutations in the ARX gene in Xp22.1 have been found in numerous families with both nonsyndromic and syndromic XLMR. The most frequent mutation in this gene is a 24 bp duplication in exon 2. Based on this fact, a panel of XLMR families linked to Xp22 was tested for this particular ARX mutation. Methods Genomic DNA from XLMR families linked to Xp22.1 was amplified for exon 2 in ARX using a Cy5 labeled primer pair. The resulting amplicons were sized using the ALFexpress automated sequencer. Results A panel of 11 families with X-linked mental retardation was screened for the ARX 24dup mutation. Four nonsyndromic XLMR families – MRX29, MRX32, MRX33 and MRX38 – were found to have this particular gene mutation. Conclusion We have identified 4 additional XLMR families with the ARX dup24 mutation from a panel of 11 XLMR families linked to Xp22.1. This finding makes the ARX dup24 mutation the most common mutation in nonsyndromic XLMR families linked to Xp22.1. As this mutation can be readily tested for using an automated sequencer, screening should be considered for any male with nonsyndromic MR of unknown etiology.

  11. Decoupled ARX and RBF Neural Network Modeling Using PCA and GA Optimization for Nonlinear Distributed Parameter Systems.

    Science.gov (United States)

    Zhang, Ridong; Tao, Jili; Lu, Renquan; Jin, Qibing

    2018-02-01

    Modeling of distributed parameter systems is difficult because of their nonlinearity and infinite-dimensional characteristics. Based on principal component analysis (PCA), a hybrid modeling strategy that consists of a decoupled linear autoregressive exogenous (ARX) model and a nonlinear radial basis function (RBF) neural network model are proposed. The spatial-temporal output is first divided into a few dominant spatial basis functions and finite-dimensional temporal series by PCA. Then, a decoupled ARX model is designed to model the linear dynamics of the dominant modes of the time series. The nonlinear residual part is subsequently parameterized by RBFs, where genetic algorithm is utilized to optimize their hidden layer structure and the parameters. Finally, the nonlinear spatial-temporal dynamic system is obtained after the time/space reconstruction. Simulation results of a catalytic rod and a heat conduction equation demonstrate the effectiveness of the proposed strategy compared to several other methods.

  12. Modelling thermal comfort of visitors at urban squares in hot and arid climate using NN-ARX soft computing method

    Science.gov (United States)

    Kariminia, Shahab; Motamedi, Shervin; Shamshirband, Shahaboddin; Piri, Jamshid; Mohammadi, Kasra; Hashim, Roslan; Roy, Chandrabhushan; Petković, Dalibor; Bonakdari, Hossein

    2016-05-01

    Visitors utilize the urban space based on their thermal perception and thermal environment. The thermal adaptation engages the user's behavioural, physiological and psychological aspects. These aspects play critical roles in user's ability to assess the thermal environments. Previous studies have rarely addressed the effects of identified factors such as gender, age and locality on outdoor thermal comfort, particularly in hot, dry climate. This study investigated the thermal comfort of visitors at two city squares in Iran based on their demographics as well as the role of thermal environment. Assessing the thermal comfort required taking physical measurement and questionnaire survey. In this study, a non-linear model known as the neural network autoregressive with exogenous input (NN-ARX) was employed. Five indices of physiological equivalent temperature (PET), predicted mean vote (PMV), standard effective temperature (SET), thermal sensation votes (TSVs) and mean radiant temperature ( T mrt) were trained and tested using the NN-ARX. Then, the results were compared to the artificial neural network (ANN) and the adaptive neuro-fuzzy inference system (ANFIS). The findings showed the superiority of the NN-ARX over the ANN and the ANFIS. For the NN-ARX model, the statistical indicators of the root mean square error (RMSE) and the mean absolute error (MAE) were 0.53 and 0.36 for the PET, 1.28 and 0.71 for the PMV, 2.59 and 1.99 for the SET, 0.29 and 0.08 for the TSV and finally 0.19 and 0.04 for the T mrt.

  13. State of Charge Estimation Using the Extended Kalman Filter for Battery Management Systems Based on the ARX Battery Model

    Directory of Open Access Journals (Sweden)

    Hongjie Wu

    2013-01-01

    Full Text Available State of charge (SOC is a critical factor to guarantee that a battery system is operating in a safe and reliable manner. Many uncertainties and noises, such as fluctuating current, sensor measurement accuracy and bias, temperature effects, calibration errors or even sensor failure, etc. pose a challenge to the accurate estimation of SOC in real applications. This paper adds two contributions to the existing literature. First, the auto regressive exogenous (ARX model is proposed here to simulate the battery nonlinear dynamics. Due to its discrete form and ease of implemention, this straightforward approach could be more suitable for real applications. Second, its order selection principle and parameter identification method is illustrated in detail in this paper. The hybrid pulse power characterization (HPPC cycles are implemented on the 60AH LiFePO4 battery module for the model identification and validation. Based on the proposed ARX model, SOC estimation is pursued using the extended Kalman filter. Evaluation of the adaptability of the battery models and robustness of the SOC estimation algorithm are also verified. The results indicate that the SOC estimation method using the Kalman filter based on the ARX model shows great performance. It increases the model output voltage accuracy, thereby having the potential to be used in real applications, such as EVs and HEVs.

  14. Targeting artificial transcription factors to the utrophin A promoter: effects on dystrophic pathology and muscle function.

    Science.gov (United States)

    Lu, Yifan; Tian, Chai; Danialou, Gawiyou; Gilbert, Rénald; Petrof, Basil J; Karpati, George; Nalbantoglu, Josephine

    2008-12-12

    Duchenne muscular dystrophy is caused by a genetic defect in the dystrophin gene. The absence of dystrophin results in muscle fiber necrosis and regeneration, leading to progressive muscle fiber loss. Utrophin is a close analogue of dystrophin. A substantial, ectopic expression of utrophin in the extrasynaptic sarcolemma of dystrophin-deficient muscle fibers can prevent deleterious effects of dystrophin deficiency. An alternative approach for the extrasynaptic up-regulation of utrophin involves the augmentation of utrophin transcription via the endogenous utrophin A promoter using custom-designed transcriptional activator proteins with zinc finger (ZFP) motifs. We tested a panel of custom-designed ZFP for their ability to activate the utrophin A promoter. Expression of one such ZFP efficiently increased, in a time-dependent manner, utrophin transcript and protein levels both in vitro and in vivo. In dystrophic mouse (mdx) muscles, administration of adenoviral vectors expressing this ZFP led to significant enhancement of muscle function with decreased necrosis, restoration of the dystrophin-associated proteins, and improved resistance to eccentric contractions. These studies provide evidence that specifically designed ZFPs can act as strong transcriptional activators of the utrophin A promoter. These may thus serve as attractive therapeutic agents for dystrophin deficiency states such as Duchenne muscular dystrophy.

  15. Genome-wide strategies identify downstream target genes of chick connective tissue-associated transcription factors.

    Science.gov (United States)

    Orgeur, Mickael; Martens, Marvin; Leonte, Georgeta; Nassari, Sonya; Bonnin, Marie-Ange; Börno, Stefan T; Timmermann, Bernd; Hecht, Jochen; Duprez, Delphine; Stricker, Sigmar

    2018-03-29

    Connective tissues support organs and play crucial roles in development, homeostasis and fibrosis, yet our understanding of their formation is still limited. To gain insight into the molecular mechanisms of connective tissue specification, we selected five zinc-finger transcription factors - OSR1, OSR2, EGR1, KLF2 and KLF4 - based on their expression patterns and/or known involvement in connective tissue subtype differentiation. RNA-seq and ChIP-seq profiling of chick limb micromass cultures revealed a set of common genes regulated by all five transcription factors, which we describe as a connective tissue core expression set. This common core was enriched with genes associated with axon guidance and myofibroblast signature, including fibrosis-related genes. In addition, each transcription factor regulated a specific set of signalling molecules and extracellular matrix components. This suggests a concept whereby local molecular niches can be created by the expression of specific transcription factors impinging on the specification of local microenvironments. The regulatory network established here identifies common and distinct molecular signatures of limb connective tissue subtypes, provides novel insight into the signalling pathways governing connective tissue specification, and serves as a resource for connective tissue development. © 2018. Published by The Company of Biologists Ltd.

  16. The RNA Exosome Adaptor ZFC3H1 Functionally Competes with Nuclear Export Activity to Retain Target Transcripts

    DEFF Research Database (Denmark)

    Silla, Toomas; Karadoulama, Evdoxia; Mąkosa, Dawid

    2018-01-01

    , containing polyadenylated (pA+) RNA secluded from nucleocytoplasmic export. We asked whether exosome co-factors could serve such nuclear retention. Co-localization studies revealed the enrichment of pA+ RNA foci with "pA-tail exosome targeting (PAXT) connection" components MTR4, ZFC3H1, and PABPN1......Mammalian genomes are promiscuously transcribed, yielding protein-coding and non-coding products. Many transcripts are short lived due to their nuclear degradation by the ribonucleolytic RNA exosome. Here, we show that abolished nuclear exosome function causes the formation of distinct nuclear foci...... but no overlap with known nuclear structures such as Cajal bodies, speckles, paraspeckles, or nucleoli. Interestingly, ZFC3H1 is required for foci formation, and in its absence, selected pA+ RNAs, including coding and non-coding transcripts, are exported to the cytoplasm in a process dependent on the mRNA export...

  17. Transcriptional targets shared by estrogen receptor- related receptors (ERRs) and estrogen receptor (ER) alpha, but not by ERbeta.

    Science.gov (United States)

    Vanacker, J M; Pettersson, K; Gustafsson, J A; Laudet, V

    1999-01-01

    The physiological activities of estrogens are thought to be mediated by specific nuclear receptors, ERalpha and ERbeta. However, certain tissues, such as the bone, that are highly responsive to estrogens only express a low level of these receptors. Starting from this apparent contradiction, we have evaluated the potentials of two related receptors ERRalpha and ERRbeta to intervene in estrogen signaling. ERalpha, ERRalpha and ERRbeta bind to and activate transcription through both the classical estrogen response element (ERE) and the SF-1 response element (SFRE). In contrast, ERbeta DNA-binding and transcriptional activity is restricted to the ERE. Accordingly, the osteopontin gene promoter is stimulated through SFRE sequences, by ERRalpha as well as by ERalpha, but not by ERbeta. Analysis of the cross-talk within the ER/ERR subgroup of nuclear receptors thus revealed common targets but also functional differences between the two ERs. PMID:10428965

  18. RUNX1 promotes cell growth in human T-cell acute lymphoblastic leukemia by transcriptional regulation of key target genes.

    Science.gov (United States)

    Jenkins, Catherine E; Gusscott, Samuel; Wong, Rachel J; Shevchuk, Olena O; Rana, Gurneet; Giambra, Vincenzo; Tyshchenko, Kateryna; Islam, Rashedul; Hirst, Martin; Weng, Andrew P

    2018-05-04

    RUNX1 is frequently mutated in T-cell acute lymphoblastic leukemia (T-ALL). The spectrum of RUNX1 mutations has led to the notion that it acts as a tumor suppressor in this context; however, other studies have placed RUNX1 along with transcription factors TAL1 and NOTCH1 as core drivers of an oncogenic transcriptional program. To reconcile these divergent roles, we knocked down RUNX1 in human T-ALL cell lines and deleted Runx1 or Cbfb in primary mouse T-cell leukemias. RUNX1 depletion consistently resulted in reduced cell proliferation and increased apoptosis. RUNX1 upregulated variable sets of target genes in each cell line, but consistently included a core set of oncogenic effectors including IGF1R and NRAS. Our results support the conclusion that RUNX1 has a net positive effect on cell growth in the context of established T-ALL. Copyright © 2018. Published by Elsevier Inc.

  19. The transcription factor Prep1 controls hepatic insulin sensitivity and gluconeogenesis by targeting nuclear localization of FOXO1.

    Science.gov (United States)

    Kulebyakin, Konstantin; Penkov, Dmitry; Blasi, Francesco; Akopyan, Zhanna; Tkachuk, Vsevolod

    2016-12-02

    Liver plays a key role in controlling body carbohydrate homeostasis by switching between accumulation and production of glucose and this way maintaining constant level of glucose in blood. Increased blood glucose level triggers release of insulin from pancreatic β-cells. Insulin represses hepatic glucose production and increases glucose accumulation. Insulin resistance is the main cause of type 2 diabetes and hyperglycemia. Currently thiazolidinediones (TZDs) targeting transcriptional factor PPARγ are used as insulin sensitizers for treating patients with type 2 diabetes. However, TZDs are reported to be associated with cardiovascular and liver problems and stimulate obesity. Thus, it is necessary to search new approaches to improve insulin sensitivity. A promising candidate is transcriptional factor Prep1, as it was shown earlier it could affect insulin sensitivity in variety of insulin-sensitive tissues. The aim of the present study was to evaluate a possible involvement of transcriptional factor Prep1 in control of hepatic glucose accumulation and production. We created mice with liver-specific Prep1 knockout and discovered that hepatocytes derived from these mice are much more sensitive to insulin, comparing to their WT littermates. Incubation of these cells with 100 nM insulin results in almost complete inhibition of gluconeogenesis, while in WT cells this repression is only partial. However, Prep1 doesn't affect gluconeogenesis in the absence of insulin. Also, we observed that nuclear content of gluconeogenic transcription factor FOXO1 was greatly reduced in Prep1 knockout hepatocytes. These findings suggest that Prep1 may control hepatic insulin sensitivity by targeting FOXO1 nuclear stability. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. The transcription factor Prep1 controls hepatic insulin sensitivity and gluconeogenesis by targeting nuclear localization of FOXO1

    International Nuclear Information System (INIS)

    Kulebyakin, Konstantin; Penkov, Dmitry; Blasi, Francesco; Akopyan, Zhanna; Tkachuk, Vsevolod

    2016-01-01

    Liver plays a key role in controlling body carbohydrate homeostasis by switching between accumulation and production of glucose and this way maintaining constant level of glucose in blood. Increased blood glucose level triggers release of insulin from pancreatic β-cells. Insulin represses hepatic glucose production and increases glucose accumulation. Insulin resistance is the main cause of type 2 diabetes and hyperglycemia. Currently thiazolidinediones (TZDs) targeting transcriptional factor PPARγ are used as insulin sensitizers for treating patients with type 2 diabetes. However, TZDs are reported to be associated with cardiovascular and liver problems and stimulate obesity. Thus, it is necessary to search new approaches to improve insulin sensitivity. A promising candidate is transcriptional factor Prep1, as it was shown earlier it could affect insulin sensitivity in variety of insulin-sensitive tissues. The aim of the present study was to evaluate a possible involvement of transcriptional factor Prep1 in control of hepatic glucose accumulation and production. We created mice with liver-specific Prep1 knockout and discovered that hepatocytes derived from these mice are much more sensitive to insulin, comparing to their WT littermates. Incubation of these cells with 100 nM insulin results in almost complete inhibition of gluconeogenesis, while in WT cells this repression is only partial. However, Prep1 doesn't affect gluconeogenesis in the absence of insulin. Also, we observed that nuclear content of gluconeogenic transcription factor FOXO1 was greatly reduced in Prep1 knockout hepatocytes. These findings suggest that Prep1 may control hepatic insulin sensitivity by targeting FOXO1 nuclear stability. - Highlights: • A novel model of liver-specific Prep1 knockout is established. • Ablation of Prep1 in hepatocytes increases insulin sensitivity. • Prep1 controls hepatic insulin sensitivity by regulating localization of FOXO1. • Prep1 regulates

  1. Reg IV is a direct target of intestinal transcriptional factor CDX2 in gastric cancer.

    Directory of Open Access Journals (Sweden)

    Yutaka Naito

    Full Text Available REG4, which encodes Reg IV protein, is a member of the calcium-dependent lectin superfamily and potent activator of the epidermal growth factor receptor/Akt/activator protein-1 signaling pathway. Several human cancers overexpress Reg IV, and Reg IV expression is associated with intestinal phenotype differentiation. However, regulation of REG4 transcription remains unclear. In the present study, we investigated whether CDX2 regulates Reg IV expression in gastric cancer (GC cells. Expression of Reg IV and CDX2 was analyzed by Western blot and quantitative reverse transcription-polymerase chain reaction in 9 GC cell lines and 2 colon cancer cell lines. The function of the 5'-flanking region of the REG4 gene was characterized by luciferase assay. In 9 GC cell lines, endogenous Reg IV and CDX2 expression were well correlated. Using an estrogen receptor-regulated form of CDX2, rapid induction of Reg IV expression was observed in HT-29 cells. Reporter gene assays revealed an important role in transcription for consensus CDX2 DNA binding elements in the 5'-flanking region of the REG4 gene. Chromatin immunoprecipitation assays showed that CDX2 binds directly to the 5'-flanking region of REG4. These results indicate that CDX2 protein directly regulates Reg IV expression.

  2. Transcriptional profiling uncovers a network of cholesterol-responsive atherosclerosis target genes.

    Directory of Open Access Journals (Sweden)

    Josefin Skogsberg

    2008-03-01

    Full Text Available Despite the well-documented effects of plasma lipid lowering regimes halting atherosclerosis lesion development and reducing morbidity and mortality of coronary artery disease and stroke, the transcriptional response in the atherosclerotic lesion mediating these beneficial effects has not yet been carefully investigated. We performed transcriptional profiling at 10-week intervals in atherosclerosis-prone mice with human-like hypercholesterolemia and a genetic switch to lower plasma lipoproteins (Ldlr(-/-Apo(100/100Mttp(flox/flox Mx1-Cre. Atherosclerotic lesions progressed slowly at first, then expanded rapidly, and plateaued after advanced lesions formed. Analysis of lesion expression profiles indicated that accumulation of lipid-poor macrophages reached a point that led to the rapid expansion phase with accelerated foam-cell formation and inflammation, an interpretation supported by lesion histology. Genetic lowering of plasma cholesterol (e.g., lipoproteins at this point all together prevented the formation of advanced plaques and parallel transcriptional profiling of the atherosclerotic arterial wall identified 37 cholesterol-responsive genes mediating this effect. Validation by siRNA-inhibition in macrophages incubated with acetylated-LDL revealed a network of eight cholesterol-responsive atherosclerosis genes regulating cholesterol-ester accumulation. Taken together, we have identified a network of atherosclerosis genes that in response to plasma cholesterol-lowering prevents the formation of advanced plaques. This network should be of interest for the development of novel atherosclerosis therapies.

  3. Hierarchical axon targeting of Drosophila olfactory receptor neurons specified by the proneural transcription factors Atonal and Amos.

    Science.gov (United States)

    Okumura, Misako; Kato, Tomoko; Miura, Masayuki; Chihara, Takahiro

    2016-01-01

    Sensory information is spatially represented in the brain to form a neural map. It has been suggested that axon-axon interactions are important for neural map formation; however, the underlying mechanisms are not fully understood. We used the Drosophila antennal lobe, the first olfactory center in the brain, as a model for studying neural map formation. Olfactory receptor neurons (ORNs) expressing the same odorant receptor target their axons to a single glomerulus out of approximately 50 glomeruli in the antennal lobe. Previous studies have showed that the axons of Atonal ORNs, specified by Atonal, a basic helix-loop-helix (bHLH) transcription factor, pioneer antennal lobe formation; however, the details remain to be elucidated. Here, we show that genetic ablation of Atonal ORNs affects antennal lobe structure and axon targeting of Amos ORNs, another type of ORN specified by the bHLH transcription factor Amos. During development, Atonal ORNs reach the antennal lobe and form the axon commissure before Amos ORNs. We also found that N-cadherin knockdown specifically in Atonal ORNs disrupts the glomerular boundary in the whole antennal lobe. Our results suggest that Atonal ORNs function as pioneer axons. Thus, correct axon targeting of Atonal ORNs is essential for formation of the whole antennal lobe. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  4. Targeting of chromatin readers: a novel strategy used by the Shigella flexneri virulence effector OspF to reprogram transcription

    Directory of Open Access Journals (Sweden)

    Habiba Harouz

    2014-12-01

    Full Text Available Shigella flexneri, a gram-negative bacterium responsible of bacillary dysentery, uses multiple strategies to overcome host immune defense. We have decrypted how this bacterium manipulates host-cell chromatin binders to take control of immune gene expression. We found that OspF, an injected virulence factor previously identified as a repressor of immune gene expression, targets the chromatin reader HP1γ. Heterochromatin Protein 1 family members specifically recognize and bind histone H3 methylated at Lys 9. Although initially identified as chromatin-associated transcriptional silencers in heterochromatin, their location in euchromatin indicates an active role in gene expression. Notably, HP1γ phosphorylation at Serine 83 defines a subpopulation exclusively located to euchromatin, targeted to the site of transcriptional elongation. We showed that OspF directly interacts with HP1γ, and causes HP1 dephosphorylation, suggesting a model in which this virulence effector “uses” HP1 proteins as beacons to target and repress immune gene expression (Harouz, et al. EMBO J (2014. OspF alters HP1γ phosphorylation mainly by inactivating the Erk-activated kinase MSK1, spotlighting it as a new HP1 kinase. In vivo, infectious stresses trigger HP1γ phosphorylation in the colon, principally in the lamina propria and the intestinal crypts. Several lines of evidence suggest that HP1 proteins are modified as extensively as histones, and decrypting the impact of these HP1 post-translational modifications on their transcriptional activities in vivo will be the next challenges to be taken up.

  5. Structural hierarchy controlling dimerization and target DNA recognition in the AHR transcriptional complex

    Energy Technology Data Exchange (ETDEWEB)

    Seok, Seung-Hyeon; Lee, Woojong; Jiang, Li; Molugu, Kaivalya; Zheng, Aiping; Li, Yitong; Park, Sanghyun; Bradfield, Christopher A.; Xing, Yongna (UW)

    2017-04-10

    he aryl hydrocarbon receptor (AHR) belongs to the PAS (PER-ARNT-SIM) family transcription factors and mediates broad responses to numerous environmental pollutants and cellular metabolites, modulating diverse biological processes from adaptive metabolism, acute toxicity, to normal physiology of vascular and immune systems. The AHR forms a transcriptionally active heterodimer with ARNT (AHR nuclear translocator), which recognizes the dioxin response element (DRE) in the promoter of downstream genes. We determined the crystal structure of the mammalian AHR–ARNT heterodimer in complex with the DRE, in which ARNT curls around AHR into a highly intertwined asymmetric architecture, with extensive heterodimerization interfaces and AHR interdomain interactions. Specific recognition of the DRE is determined locally by the DNA-binding residues, which discriminates it from the closely related hypoxia response element (HRE), and is globally affected by the dimerization interfaces and interdomain interactions. Changes at the interdomain interactions caused either AHR constitutive nuclear localization or failure to translocate to nucleus, underlying an allosteric structural pathway for mediating ligand-induced exposure of nuclear localization signal. These observations, together with the global higher flexibility of the AHR PAS-A and its loosely packed structural elements, suggest a dynamic structural hierarchy for complex scenarios of AHR activation induced by its diverse ligands.

  6. Human lactoferrin efficiently targeted into caprine beta-lactoglobulin locus with transcription activator-like effector nucleases

    Directory of Open Access Journals (Sweden)

    Yu-Guo Yuan

    2017-08-01

    Full Text Available Objective To create genetically modified goat as a biopharming source of recombinant human lacotoferrin (hLF with transcription activator-like effector nucleases. Methods TALENs and targeting vector were transferred into cultured fibroblasts to insert hLF cDNA in the goat beta-lactoglobulin (BLG locus with homology-directed repair. The gene targeted efficiency was checked using sequencing and TE7I assay. The bi-allelic gene targeted colonies were isolated and confirmed with polymerase chain reaction, and used as donor cells for somatic cell nuclear transfer (SCNT. Results The targeted efficiency for BLG gene was approximately 10%. Among 12 Bi-allelic gene targeted colonies, five were used in first round SCNT and 4 recipients (23% were confirmed pregnant at 30 d. In second round SCNT, 7 (53%, 4 (31%, and 3 (23% recipients were confirmed to be pregnant by ultrasound on 30 d, 60 d, and 90 d. Conclusion This finding signifies the combined use of TALENs and SCNT can generate bi-allelic knock-in fibroblasts that can be cloned in a fetus. Therefore, it might lay the foundation for transgenic hLF goat generation and possible use of their mammary gland as a bioreactor for large-scale production of recombinant hLF.

  7. Potential downstream target genes of aberrant ETS transcription factors are differentially affected in Ewing's sarcoma and prostate carcinoma.

    Directory of Open Access Journals (Sweden)

    Maria J Camões

    Full Text Available FLI1 and ERG, the major ETS transcription factors involved in rearrangements in the Ewing's sarcoma family of tumors (ESFT and in prostate carcinomas (PCa, respectively, belong to the same subfamily, having 98% sequence identity in the DNA binding domain. We therefore decided to investigate whether the aberrant transcription factors in both malignancies have some common downstream targets. We crossed a publicly available list of all putative EWSR1-FLI1 target genes in ESFT with our microarray expression data on 24 PCa and 6 non-malignant prostate tissues (NPT and choose four genes among the top-most differentially expressed between PCa with (PCa ERG+ and without (PCa ETS- ETS fusion genes (HIST1H4L, KCNN2, ECRG4 and LDOC1, as well as four well-validated direct targets of the EWSR1-FLI1 chimeric protein in ESFT (NR0B1, CAV1, IGFBP3 and TGFBR2. Using quantitative expression analysis in 16 ESFT and seven alveolar rhabdomyosarcomas (ARMS, we were able to validate the four genes previously described as direct targets of the EWSR1-FLI1 oncoprotein, showing overexpression of CAV1 and NR0B1 and underexpression of IGFBP3 and TGFBR2 in ESFT as compared to ARMS. Although none of these four genes showed significant expression differences between PCa ERG+ and PCa ETS-, CAV1, IGFBP3 and TGFBR2 were less expressed in PCa in an independent series of 56 PCa and 15 NPT, as also observed for ECRG4 and LDOC1, suggesting a role in prostate carcinogenesis in general. On the other hand, we demonstrate for the first time that both HIST1H4L and KCNN2 are significantly overexpressed in PCa ERG+ and that ERG binds to the promoter of these genes. Conversely, KCNN2 was found underexpressed in ESFT relative to ARMS, suggesting that the EWSR1-ETS oncoprotein may have the opposite effect of ERG rearrangements in PCa. We conclude that aberrant ETS transcription factors modulate target genes differently in ESFT and PCa.

  8. OVO homologue-like 1 (Ovol1) transcription factor: a novel target of neurogenin-3 in rodent pancreas.

    Science.gov (United States)

    Vetere, A; Li, W-C; Paroni, F; Juhl, K; Guo, L; Nishimura, W; Dai, X; Bonner-Weir, S; Sharma, A

    2010-01-01

    The basic helix-loop-helix transcription factor neurogenin-3 (NGN3) commits the fates of pancreatic progenitors to endocrine cell types, but knowledge of the mechanisms regulating the choice between proliferation and differentiation of these progenitors is limited. Using a chromatin immunoprecipitation cloning approach, we searched for direct targets of NGN3 and identified a zinc-finger transcription factor, OVO homologue-like 1 (OVOL1). Transactivation experiments were carried out to elucidate the functional role of NGN3 in Ovol1 gene expression. Embryonic and adult rodents pancreases were immunostained for OVOL1, Ki67 and NGN3. We showed that NGN3 negatively regulates transcription of Ovol1 in an E-box-dependent fashion. The presence of either NGN3 or NEUROD1, but not MYOD, reduced endogenous Ovol1 mRNA. OVOL1 was detected in pancreatic tissue around embryonic day 15.5, after which OVOL1 levels dramatically increased. In embryonic pancreas, OVOL1 protein levels were low in NGN3(+) or Ki67(+) cells, but high in quiescent differentiated cells. OVOL1 presence was maintained in adult pancreas, where it was detected in islets, pancreatic ducts and some acinar cells. Additionally OVOL1 presence was lacking in proliferating ductules in regenerating pancreas and induced in cells as they began to acquire their differentiated phenotype. The timing of OVOL1 appearance in pancreas and its increased levels in differentiated cells suggest that OVOL1 promotes the transition of cells from a proliferating, less-differentiated state to a quiescent more-differentiated state. We conclude that OVOL1, a downstream target of NGN3, may play an important role in regulating the balance between proliferation and differentiation of pancreatic cells.

  9. Transcription factors as targets for improving Aspergillus niger as cell factory

    DEFF Research Database (Denmark)

    Poulsen, Lars; Bruno, K.S.; Thykær, Jette

    for gene knockout. The resulting mutants were first exposed to screening experiments including morphological studies and investigation of acid profile and protease activity. Among others an interesting finding was that one mutant had an oxalic acid overproducing phenotype (OOP). In the screening...... experiments the OOP mutant showed a 30 % (± 5%) increase in oxalic acid titer. The OOP mutant was further characterized in 2L scale bioreactors, and a 90 % (±30%) increase of the overall yield coefficient of oxalic acid on glucose was seen. Further data on the OOP mutant will be presented and results from......). In the present study the effect of modulation of transcription factors in Aspergillus niger, which is an industrially important micro-organism used in various processes including organic acid and enzyme production, was investigated. The strategy described in this work focuses on regulation connected to p...

  10. Profiling ethanol-targeted transcription factors in human carcinoma cell-derived embryoid bodies.

    Science.gov (United States)

    Mandal, Chanchal; Halder, Debasish; Chai, Jin Choul; Lee, Young Seek; Jung, Kyoung Hwa; Chai, Young Gyu

    2016-01-15

    Fetal alcohol spectrum disorder is a collective term that represents fetal abnormalities associated with maternal alcohol consumption. Prenatal alcohol exposure and related anomalies are well characterized, but the molecular mechanism behind this phenomenon is not yet understood. Few insights have been gained from genetic and epigenetic studies of fetal alcohol spectrum disorder. Our aim was to profile the important molecular regulators of ethanol-related alterations of the genome. For this purpose, we have analyzed the gene expression pattern of human carcinoma cell-derived embryoid bodies in the absence or presence of ethanol. A cDNA microarray analysis was used to profile mRNA expression in embryoid bodies at day 7 with or without ethanol treatment. A total of 493 differentially expressed genes were identified in response to 50 mM ethanol exposure. Of these, 111 genes were up-regulated, and 382 were down-regulated. Gene ontology term enrichment analysis revealed that these genes are involved in important biological processes: neurological system processes, cognition, behavior, sensory perception of smell, taste and chemical stimuli and synaptic transmission. Similarly, the enrichment of disease-related genes included relevant categories such as neurological diseases, developmental disorders, skeletal and muscular disorders, and connective tissue disorders. Furthermore, we have identified a group of 26 genes that encode transcription factors. We validated the relative gene expression of several transcription factors using quantitative real time PCR. We hope that our study substantially contributes to the understanding of the molecular mechanisms underlying the pathology of alcohol-mediated anomalies and facilitates further research. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. A Novel ARX-Based Approach for the Steady-State Identification Analysis of Industrial Depropanizer Column Datasets

    Directory of Open Access Journals (Sweden)

    Franklin D. Rincón

    2015-04-01

    Full Text Available This paper introduces a novel steady-state identification (SSI method based on the auto-regressive model with exogenous inputs (ARX. This method allows the SSI with reduced tuning by analyzing the identifiability properties of the system. In particular, the singularity of the model matrices is used as an index for steady-state determination. In this contribution, the novel SSI method is compared to other available techniques, namely the F-like test, wavelet transform and a polynomial-based approach. These methods are implemented for SSI of three different case studies. In the first case, a simulated dataset is used for calibrating the output-based SSI methods. The second case corresponds to a literature nonlinear continuous stirred-tank reactor (CSTR example running at different steady states in which the ARX-based approach is tuned with the available input-output data. Finally, an industrial case with real data of a depropanizer column from PETROBRAS S.A. considering different pieces of equipment is analyzed. The results for a reflux drum case indicate that the wavelet and the F-like test can satisfactorily detect the steady-state periods after careful tuning and when respecting their hypothesis, i.e., smooth data for the wavelet method and the presence of variance in the data for the F-like test. Through a heat exchanger case with different measurement frequencies, we demonstrate the advantages of using the ARX-based method over the other techniques, which include the aspect of online implementation.

  12. Optimizations of siRNA design for the activation of gene transcription by targeting the TATA-box motif.

    Directory of Open Access Journals (Sweden)

    Miaomiao Fan

    Full Text Available Small interfering RNAs (siRNAs are widely used to repress gene expression by targeting mRNAs. Some reports reveal that siRNAs can also activate or inhibit gene expression through targeting the gene promoters. Our group has found that microRNAs (miRNAs could activate gene transcription via interaction with the TATA-box motif in gene promoters. To investigate whether siRNA targeting the same region could upregulate the promoter activity, we test the activating efficiency of siRNAs targeting the TATA-box motif of 16 genes and perform a systematic analysis to identify the common features of the functional siRNAs for effective activation of gene promoters. Further, we try various modifications to improve the activating efficiency of siRNAs and find that it is quite useful to design the promoter-targeting activating siRNA by following several rules such as (a complementary to the TATA-box-centered region; (b UA usage at the first two bases of the antisense strand; (c twenty-three nucleotides (nts in length; (d 2'-O-Methyl (2'-OMe modification at the 3' terminus of the antisense strand; (e avoiding mismatches at the 3' end of the antisense strand. The optimized activating siRNAs potently enhance the expression of interleukin-2 (IL-2 gene in human and mouse primary CD4+ T cells with a long-time effect. Taken together, our study provides a guideline for rational design the promoter-targeting siRNA to sequence-specifically enhance gene expression.

  13. Synthetic siRNAs effectively target cystein protease 12 and α-actinin transcripts in Trichomonas vaginalis.

    Science.gov (United States)

    Ravaee, Roya; Ebadi, Parimah; Hatam, Gholamreza; Vafafar, Arghavan; Ghahramani Seno, Mohammad Mahdi

    2015-10-01

    The flagellated protozoan Trichomonas vaginalis (T. vaginalis) causes trichomoniasis, a reproductive tract infection, in humans. Trichomoniasis is the most common non-viral sexually transmitted disease worldwide. In addition to direct consequences such as infertility and abortion, there are indications that trichomoniasis favours development of prostate cancer and it has also been associated with increased risk of spreading human immunodeficiency virus and papillomavirus infections. Reports from around the world show that the rate of drug resistance in T. vaginalis is increasing, and therefore new therapeutic approaches have to be developed. Studying molecular biology of T. vaginalis will be quite helpful in identifying new drugable targets. RNAi is a powerful technique which allows biologist to specifically target gene products (i.e. mRNA) helping them in unravelling gene functions and biology of systems. However, due to lack of some parts of the required intrinsic RNAi machinery, the RNAi system is not functional in all orders of life. Here, by using synthetic siRNAs targeting two genes, i.e. α-actinin and cystein protease 12 (cp12), we demonstrate T. vaginalis cells are amenable to RNAi experiments conducted by extrinsic siRNAs. Electroporation of siRNAs targeting α-actinin or cp12 into T. vaginalis cells resulted in, respectively, 48-67% and 33-72% downregulation of the cognate transcripts compared to the T. vaginalis cells received siRNAs targeting GL2 luciferase as a control. This finding is helpful in that it demonstrates the potential of using extrinsically induced RNAi in studies on molecular biology of T. vaginalis such as those aiming at identifying new drug targets. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Comparison of auditory evoked potentials and the A-line ARX Index for monitoring the hypnotic level during sevoflurane and propofol induction

    DEFF Research Database (Denmark)

    Litvan, H; Jensen, E W; Revuelta, M

    2002-01-01

    Extraction of the middle latency auditory evoked potentials (AEP) by an auto regressive model with exogenous input (ARX) enables extraction of the AEP within 1.7 s. In this way, the depth of hypnosis can be monitored at almost real-time. However, the identification and the interpretation of the a......Extraction of the middle latency auditory evoked potentials (AEP) by an auto regressive model with exogenous input (ARX) enables extraction of the AEP within 1.7 s. In this way, the depth of hypnosis can be monitored at almost real-time. However, the identification and the interpretation...

  15. Vibration-Based Damage Diagnosis in a Laboratory Cable-Stayed Bridge Model via an RCP-ARX Model Based Method

    International Nuclear Information System (INIS)

    Michaelides, P G; Apostolellis, P G; Fassois, S D

    2011-01-01

    Vibration-based damage detection and identification in a laboratory cable-stayed bridge model is addressed under inherent, environmental, and experimental uncertainties. The problem is challenging as conventional stochastic methods face difficulties due to uncertainty underestimation. A novel method is formulated based on identified Random Coefficient Pooled ARX (RCP-ARX) representations of the dynamics and statistical hypothesis testing. The method benefits from the ability of RCP models in properly capturing uncertainty. Its effectiveness is demonstrated via a high number of experiments under a variety of damage scenarios.

  16. Vibration-Based Damage Diagnosis in a Laboratory Cable-Stayed Bridge Model via an RCP-ARX Model Based Method

    Energy Technology Data Exchange (ETDEWEB)

    Michaelides, P G; Apostolellis, P G; Fassois, S D, E-mail: mixail@mech.upatras.gr, E-mail: fassois@mech.upatras.gr [Laboratory for Stochastic Mechanical Systems and Automation (SMSA), Department of Mechanical and Aeronautical Engineering, University of Patras, GR 265 00 Patras (Greece)

    2011-07-19

    Vibration-based damage detection and identification in a laboratory cable-stayed bridge model is addressed under inherent, environmental, and experimental uncertainties. The problem is challenging as conventional stochastic methods face difficulties due to uncertainty underestimation. A novel method is formulated based on identified Random Coefficient Pooled ARX (RCP-ARX) representations of the dynamics and statistical hypothesis testing. The method benefits from the ability of RCP models in properly capturing uncertainty. Its effectiveness is demonstrated via a high number of experiments under a variety of damage scenarios.

  17. Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR.

    Science.gov (United States)

    Hanning, Jennifer E; Groves, Ian J; Pett, Mark R; Coleman, Nicholas

    2013-05-21

    Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects.

  18. MDRL lncRNA regulates the processing of miR-484 primary transcript by targeting miR-361.

    Directory of Open Access Journals (Sweden)

    Kun Wang

    2014-07-01

    Full Text Available Long noncoding RNAs (lncRNAs are emerging as new players in gene regulation, but whether lncRNAs operate in the processing of miRNA primary transcript is unclear. Also, whether lncRNAs are involved in the regulation of the mitochondrial network remains to be elucidated. Here, we report that a long noncoding RNA, named mitochondrial dynamic related lncRNA (MDRL, affects the processing of miR-484 primary transcript in nucleus and regulates the mitochondrial network by targeting miR-361 and miR-484. The results showed that miR-361 that predominantly located in nucleus can directly bind to primary transcript of miR-484 (pri-miR-484 and prevent its processing by Drosha into pre-miR-484. miR-361 is able to regulate mitochondrial fission and apoptosis by regulating miR-484 levels. In exploring the underlying molecular mechanism by which miR-361 is regulated, we identified MDRL and demonstrated that it could directly bind to miR-361 and downregulate its expression levels, which promotes the processing of pri-miR-484. MDRL inhibits mitochondrial fission and apoptosis by downregulating miR-361, which in turn relieves inhibition of miR-484 processing by miR-361. Our present study reveals a novel regulating model of mitochondrial fission program which is composed of MDRL, miR-361 and miR-484. Our work not only expands the function of the lncRNA pathway in gene regulation but also establishes a new mechanism for controlling miRNA expression.

  19. Network motif-based identification of transcription factor-target gene relationships by integrating multi-source biological data

    Directory of Open Access Journals (Sweden)

    de los Reyes Benildo G

    2008-04-01

    Full Text Available Abstract Background Integrating data from multiple global assays and curated databases is essential to understand the spatio-temporal interactions within cells. Different experiments measure cellular processes at various widths and depths, while databases contain biological information based on established facts or published data. Integrating these complementary datasets helps infer a mutually consistent transcriptional regulatory network (TRN with strong similarity to the structure of the underlying genetic regulatory modules. Decomposing the TRN into a small set of recurring regulatory patterns, called network motifs (NM, facilitates the inference. Identifying NMs defined by specific transcription factors (TF establishes the framework structure of a TRN and allows the inference of TF-target gene relationship. This paper introduces a computational framework for utilizing data from multiple sources to infer TF-target gene relationships on the basis of NMs. The data include time course gene expression profiles, genome-wide location analysis data, binding sequence data, and gene ontology (GO information. Results The proposed computational framework was tested using gene expression data associated with cell cycle progression in yeast. Among 800 cell cycle related genes, 85 were identified as candidate TFs and classified into four previously defined NMs. The NMs for a subset of TFs are obtained from literature. Support vector machine (SVM classifiers were used to estimate NMs for the remaining TFs. The potential downstream target genes for the TFs were clustered into 34 biologically significant groups. The relationships between TFs and potential target gene clusters were examined by training recurrent neural networks whose topologies mimic the NMs to which the TFs are classified. The identified relationships between TFs and gene clusters were evaluated using the following biological validation and statistical analyses: (1 Gene set enrichment

  20. Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa.

    Science.gov (United States)

    Sonnleitner, Elisabeth; Valentini, Martina; Wenner, Nicolas; Haichar, Feth el Zahar; Haas, Dieter; Lapouge, Karine

    2012-01-01

    The opportunistic human pathogen Pseudomonas aeruginosa is able to utilize a wide range of carbon and nitrogen compounds, allowing it to grow in vastly different environments. The uptake and catabolism of growth substrates are organized hierarchically by a mechanism termed catabolite repression control (Crc) whereby the Crc protein establishes translational repression of target mRNAs at CA (catabolite activity) motifs present in target mRNAs near ribosome binding sites. Poor carbon sources lead to activation of the CbrAB two-component system, which induces transcription of the small RNA (sRNA) CrcZ. This sRNA relieves Crc-mediated repression of target mRNAs. In this study, we have identified novel targets of the CbrAB/Crc system in P. aeruginosa using transcriptome analysis in combination with a search for CA motifs. We characterized four target genes involved in the uptake and utilization of less preferred carbon sources: estA (secreted esterase), acsA (acetyl-CoA synthetase), bkdR (regulator of branched-chain amino acid catabolism) and aroP2 (aromatic amino acid uptake protein). Evidence for regulation by CbrAB, CrcZ and Crc was obtained in vivo using appropriate reporter fusions, in which mutation of the CA motif resulted in loss of catabolite repression. CbrB and CrcZ were important for growth of P. aeruginosa in cystic fibrosis (CF) sputum medium, suggesting that the CbrAB/Crc system may act as an important regulator during chronic infection of the CF lung.

  1. An effector of apple proliferation phytoplasma targets TCP transcription factors-a generalized virulence strategy of phytoplasma?

    Science.gov (United States)

    Janik, Katrin; Mithöfer, Axel; Raffeiner, Margot; Stellmach, Hagen; Hause, Bettina; Schlink, Katja

    2017-04-01

    The plant pathogen Candidatus Phytoplasma mali (P. mali) is the causative agent of apple proliferation, a disease of increasing importance in apple-growing areas within Europe. Despite its economic importance, little is known about the molecular mechanisms of disease manifestation within apple trees. In this study, we identified two TCP (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of Malus x domestica as binding partners of the P. mali SAP11-like effector ATP_00189. Phytohormone analyses revealed an effect of P. mali infection on jasmonates, salicylic acid and abscisic acid levels, showing that P. mali affects phytohormonal levels in apple trees, which is in line with the functions of the effector assumed from its binding to TCP transcription factors. To our knowledge, this is the first characterization of the molecular targets of a P. mali effector and thus provides the basis to better understand symptom development and disease progress during apple proliferation. As SAP11 homologues are found in several Phytoplasma species infecting a broad range of different plants, SAP11-like proteins seem to be key players in phytoplasmal infection. © 2016 BSPP AND JOHN WILEY & SONS LTD.

  2. Targeting the upstream transcriptional activator of PD-L1 as an alternative strategy in melanoma therapy.

    Science.gov (United States)

    Zhu, Bo; Tang, Liming; Chen, Shuyang; Yin, Chengqian; Peng, Shiguang; Li, Xin; Liu, Tongzheng; Liu, Wei; Han, Changpeng; Stawski, Lukasz; Xu, Zhi-Xiang; Zhou, Guangbiao; Chen, Xiang; Gao, Xiumei; Goding, Colin R; Xu, Nan; Cui, Rutao; Cao, Peng

    2018-05-22

    Programmed cell death ligand 1 (PD-L1) interacts with programmed cell death protein-1 (PD-1) as an immune checkpoint. Reactivating the immune response by inhibiting PD-L1 using therapeutic antibodies provides substantial clinical benefits in many, though not all, melanoma patients. However, transcriptional suppression of PD-L1 expression as an alternative therapeutic anti-melanoma strategy has not been exploited. Here we provide biochemical evidence demonstrating that ultraviolet radiation (UVR) induction of PD-L1 in skin is directly controlled by nuclear factor E2-related transcription factor 2 (NRF2). Depletion of NRF2 significantly induces tumor infiltration by both CD8 + and CD4 + T cells to suppress melanoma progression, and combining NRF2 inhibition with anti-PD-1 treatment enhanced its anti-tumor function. Our studies identify a critical and targetable PD-L1 upstream regulator and provide an alternative strategy to inhibit the PD-1/PD-L1 signaling in melanoma treatment.

  3. Transcription factor EB: from master coordinator of lysosomal pathways to candidate therapeutic target in degenerative storage diseases.

    Science.gov (United States)

    Sardiello, Marco

    2016-05-01

    The lysosome is the main catabolic hub of the cell. Owing to its role in fundamental processes such as autophagy, plasma membrane repair, mTOR signaling, and maintenance of cellular homeostasis, the lysosome has a profound influence on cellular metabolism and human health. Indeed, inefficient or impaired lysosomal function has been implicated in the pathogenesis of a number of degenerative diseases affecting various organs and tissues, most notably the brain, liver, and muscle. The discovery of the coordinated lysosomal expression and regulation (CLEAR) genetic program and its master controller, transcription factor EB (TFEB), has provided an unprecedented tool to study and manipulate lysosomal function. Most lysosome-based processes-including macromolecule degradation, autophagy, lysosomal exocytosis, and proteostasis-are under the transcriptional control of TFEB. Interestingly, impaired TFEB signaling has been suggested to be a contributing factor in the pathogenesis of several degenerative storage diseases. Preclinical studies based on TFEB exogenous expression to reinstate TFEB activity or promote CLEAR network-based lysosomal enhancement have highlighted TFEB as a candidate therapeutic target for the treatment of various degenerative storage diseases. © 2016 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals, Inc. on behalf of New York Academy of Sciences.

  4. Mediator complex cooperatively regulates transcription of retinoic acid target genes with Polycomb Repressive Complex 2 during neuronal differentiation.

    Science.gov (United States)

    Fukasawa, Rikiya; Iida, Satoshi; Tsutsui, Taiki; Hirose, Yutaka; Ohkuma, Yoshiaki

    2015-11-01

    The Mediator complex (Mediator) plays key roles in transcription and functions as the nexus for integration of various transcriptional signals. Previously, we screened for Mediator cyclin-dependent kinase (CDK)-interacting factors and identified three proteins related to chromatin regulation. One of them, SUZ12 is required for both stability and activity of Polycomb Repressive Complex 2 (PRC2). PRC2 primarily suppresses gene expression through histone H3 lysine 27 trimethylation, resulting in stem cell maintenance and differentiation; perturbation of this process leads to oncogenesis. Recent work showed that Mediator contributes to the embryonic stem cell state through DNA loop formation, which is strongly associated with chromatin architecture; however, it remains unclear how Mediator regulates gene expression in cooperation with chromatin regulators (i.e. writers, readers and remodelers). We found that Mediator CDKs interact directly with the PRC2 subunit EZH2, as well as SUZ12. Known PRC2 target genes were deregulated by Mediator CDK knockdown during neuronal differentiation, and both Mediator and PRC2 complexes co-occupied the promoters of developmental genes regulated by retinoic acid. Our results provide a mechanistic link between Mediator and PRC2 during neuronal differentiation. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  5. Upregulation of NKX2.2, a target of EWSR1/FLI1 fusion transcript, in primary renal Ewing sarcoma

    Directory of Open Access Journals (Sweden)

    Yoshinari Yamamoto

    2015-01-01

    Full Text Available Renal Ewing sarcoma (ES is a rare malignant tumor characterized by fusion of the EWSR1 gene with a member of the ETS family of oncogenes, arising at a specific chromosomal translocation. Diagnosis of ES can be problematic, especially from cytological or small bioptical specimens because the differential diagnoses comprising a diverse group of small round blue cell tumors (SRBCTs. We report a case of primary renal ES in a young male, which had a t(11;22 (q24;q12 chromosome translocation encoding a type2 EWSR1/FLI1 fusion transcript. The tumor cells showed diffuse cytoplasmic immunoreactivity for CD99 and diffuse nuclear immunoreactivity for NKX2.2, an important oncogenic transcriptional target of EWSR1/FLI1, not only in the histological, but also in the cytological specimens. From the results of this case, we speculate that NKX2.2, in combination with CD99, may be a useful immunocytochemical marker to distinguish renal ES from other SRBCTs of kidney.

  6. A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets.

    Science.gov (United States)

    Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

    2014-12-05

    Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.

  7. Retrotransposon-centered analysis of piRNA targeting shows a shift from active to passive retrotransposon transcription in developing mouse testes

    Directory of Open Access Journals (Sweden)

    Mourier Tobias

    2011-09-01

    Full Text Available Abstract Background Piwi-associated RNAs (piRNAs bind transcripts from retrotransposable elements (RTE in mouse germline cells and seemingly act as guides for genomic methylation, thereby repressing the activity of RTEs. It is currently unknown if and how Piwi proteins distinguish RTE transcripts from other cellular RNAs. During germline development, the main target of piRNAs switch between different types of RTEs. Using the piRNA targeting of RTEs as an indicator of RTE activity, and considering the entire population of genomic RTE loci along with their age and location, this study aims at further elucidating the dynamics of RTE activity during mouse germline development. Results Due to the inherent sequence redundancy between RTE loci, assigning piRNA targeting to specific loci is problematic. This limits the analysis, although certain features of piRNA targeting of RTE loci are apparent. As expected, young RTEs display a much higher level of piRNA targeting than old RTEs. Further, irrespective of age, RTE loci near protein-coding coding genes are targeted to a greater extent than RTE loci far from genes. During development, a shift in piRNA targeting is observed, with a clear increase in the relative piRNA targeting of RTEs residing within boundaries of protein-coding gene transcripts. Conclusions Reanalyzing published piRNA sequences and taking into account the features of individual RTE loci provide novel insight into the activity of RTEs during development. The obtained results are consistent with some degree of proportionality between what transcripts become substrates for Piwi protein complexes and the level by which the transcripts are present in the cell. A transition from active transcription of RTEs to passive co-transcription of RTE sequences residing within protein-coding transcripts appears to take place in postnatal development. Hence, the previously reported increase in piRNA targeting of SINEs in postnatal testis development

  8. Targeted gene disruption by use of transcription activator-like effector nuclease (TALEN) in the water flea Daphnia pulex.

    Science.gov (United States)

    Hiruta, Chizue; Ogino, Yukiko; Sakuma, Tetsushi; Toyota, Kenji; Miyagawa, Shinichi; Yamamoto, Takashi; Iguchi, Taisen

    2014-11-18

    The cosmopolitan microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have its complete genome sequenced, an unprecedented ca. 36% of which has no known homologs with any other species. Moreover, D. pulex is ideally suited for experimental manipulation because of its short reproductive cycle, large numbers of offspring, synchronization of oocyte maturation, and other life history characteristics. However, existing gene manipulation techniques are insufficient to accurately define gene functions. Although our previous investigations developed an RNA interference (RNAi) system in D. pulex, the possible time period of functional analysis was limited because the effectiveness of RNAi is transient. Thus, in this study, we developed a genome editing system for D. pulex by first microinjecting transcription activator-like effector nuclease (TALEN) mRNAs into early embryos and then evaluating TALEN activity and mutation phenotypes. We assembled a TALEN construct specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for distal limb development in invertebrates and vertebrates, and evaluated its activity in vitro by single-strand annealing assay. Then, we injected TALEN mRNAs into eggs within 1 hour post-ovulation. Injected embryos presented with defects in the second antenna and altered appendage development, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene. We succeeded, for the first time in D. pulex, in targeted mutagenesis by use of Platinum TALENs. This genome editing technique makes it possible to conduct reverse genetic analysis in D. pulex, making this species an even more appropriate model organism for environmental, evolutionary, and developmental genomics.

  9. The STAR protein QKI-7 recruits PAPD4 to regulate post-transcriptional polyadenylation of target mRNAs.

    Science.gov (United States)

    Yamagishi, Ryota; Tsusaka, Takeshi; Mitsunaga, Hiroko; Maehata, Takaharu; Hoshino, Shin-ichi

    2016-04-07

    Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27(kip1)and β-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27(kip1)mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells. © The Author(s) 2016. Published by Oxford

  10. Ref-1/APE1 as a Transcriptional Regulator and Novel Therapeutic Target in Pediatric T-cell Leukemia.

    Science.gov (United States)

    Ding, Jixin; Fishel, Melissa L; Reed, April M; McAdams, Erin; Czader, Magdalena B; Cardoso, Angelo A; Kelley, Mark R

    2017-07-01

    The increasing characterization of childhood acute lymphoblastic leukemia (ALL) has led to the identification of multiple molecular targets but has yet to translate into more effective targeted therapies, particularly for high-risk, relapsed T-cell ALL. Searching for master regulators controlling multiple signaling pathways in T-ALL, we investigated the multifunctional protein redox factor-1 (Ref-1/APE1), which acts as a signaling "node" by exerting redox regulatory control of transcription factors important in leukemia. Leukemia patients' transcriptome databases showed increased expression in T-ALL of Ref-1 and other genes of the Ref-1/SET interactome. Validation studies demonstrated that Ref-1 is expressed in high-risk leukemia T cells, including in patient biopsies. Ref-1 redox function is active in leukemia T cells, regulating the Ref-1 target NF-κB, and inhibited by the redox-selective Ref-1 inhibitor E3330. Ref-1 expression is not regulated by Notch signaling, but is upregulated by glucocorticoid treatment. E3330 disrupted Ref-1 redox activity in functional studies and resulted in marked inhibition of leukemia cell viability, including T-ALL lines representing different genotypes and risk groups. Potent leukemia cell inhibition was seen in primary cells from ALL patients, relapsed and glucocorticoid-resistant T-ALL cells, and cells from a murine model of Notch-induced leukemia. Ref-1 redox inhibition triggered leukemia cell apoptosis and downregulation of survival genes regulated by Ref-1 targets. For the first time, this work identifies Ref-1 as a novel molecular effector in T-ALL and demonstrates that Ref-1 redox inhibition results in potent inhibition of leukemia T cells, including relapsed T-ALL. These data also support E3330 as a specific Ref-1 small-molecule inhibitor for leukemia. Mol Cancer Ther; 16(7); 1401-11. ©2017 AACR . ©2017 American Association for Cancer Research.

  11. Dual targeting of gene delivery by genetic modification of adenovirus serotype 5 fibers and cell-selective transcriptional control.

    Science.gov (United States)

    Work, L M; Ritchie, N; Nicklin, S A; Reynolds, P N; Baker, A H

    2004-08-01

    Adenovirus (Ad)-mediated gene delivery is a promising approach for genetic manipulation of the vasculature and is being used in both preclinical models and clinical trials. However, safety concerns relating to infection of nontarget tissue and the poor infectivity of vascular cells compared to other cell types necessitates Ad vector refinement. Here, we combine a transductional targeting approach to improve vascular cell infectivity through RGD peptide insertion into adenovirus fibers, combined with transcriptional targeting to endothelial cells using a approximately 1 kb fragment of the fms-like tyrosine kinase receptor-1 (FLT-1) promoter. Single- and double-modified vectors were characterized in human cell lines that either support or have silenced FLT-1 expression. In rat hepatocytes and endothelial cells, the double modification substantially shifted transduction profiles toward vascular endothelial cells. Furthermore, in intact aortae derived from spontaneously hypertensive rats that display enhanced alphav integrin expression on dysfunctional endothelium, enhanced levels of transduction were observed using the double-modified vector but not in aortae derived from normotensive control rats. Our data indicate that Ad-mediated transduction can be beneficially modified in vitro and in vivo by combining fiber modification and a cell-selective promoter within a single-component vector system.

  12. Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

    Science.gov (United States)

    Quirin, Christina; Rohmer, Stanimira; Fernández-Ulibarri, Inés; Behr, Michael; Hesse, Andrea; Engelhardt, Sarah; Erbs, Philippe; Enk, Alexander H.

    2011-01-01

    Abstract Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show that promoter control of E1A facilitates highly selective expression of transgenes inserted into the late transcription unit. This, however, required multistep optimization of late transgene expression. Transgene insertion via internal ribosome entry site (IRES), splice acceptor (SA), or viral 2A sequences resulted in replication-dependent expression. Unexpectedly, analyses in appropriate substrates and with matching control viruses revealed that IRES and SA, but not 2A, facilitated indirect transgene targeting via tyrosinase promoter control of E1A. Transgene expression via SA was more selective (up to 1,500-fold) but less effective than via IRES. Notably, we also revealed transgene-dependent interference with splicing. Hence, the prodrug convertase FCU1 (a cytosine deaminase–uracil phosphoribosyltransferase fusion protein) was expressed only after optimizing the sequence surrounding the SA site and mutating a cryptic splice site within the transgene. The resulting tyrosinase promoter-regulated and FCU1-encoding adenovirus combined effective oncolysis with targeted prodrug activation therapy of melanoma. Thus, prodrug activation showed potent bystander killing and increased cytotoxicity of the virus up to 10-fold. We conclude that armed oncolytic viruses can be improved substantially by comparing and optimizing strategies for targeted transgene expression, thereby implementing selective and multimodal cancer therapies. PMID:20939692

  13. Genome-wide Analysis of RARβ Transcriptional Targets in Mouse Striatum Links Retinoic Acid Signaling with Huntington's Disease and Other Neurodegenerative Disorders.

    Science.gov (United States)

    Niewiadomska-Cimicka, Anna; Krzyżosiak, Agnieszka; Ye, Tao; Podleśny-Drabiniok, Anna; Dembélé, Doulaye; Dollé, Pascal; Krężel, Wojciech

    2017-07-01

    Retinoic acid (RA) signaling through retinoic acid receptors (RARs), known for its multiple developmental functions, emerged more recently as an important regulator of adult brain physiology. How RAR-mediated regulation is achieved is poorly known, partly due to the paucity of information on critical target genes in the brain. Also, it is not clear how reduced RA signaling may contribute to pathophysiology of diverse neuropsychiatric disorders. We report the first genome-wide analysis of RAR transcriptional targets in the brain. Using chromatin immunoprecipitation followed by high-throughput sequencing and transcriptomic analysis of RARβ-null mutant mice, we identified genomic targets of RARβ in the striatum. Characterization of RARβ transcriptional targets in the mouse striatum points to mechanisms through which RAR may control brain functions and display neuroprotective activity. Namely, our data indicate with statistical significance (FDR 0.1) a strong contribution of RARβ in controlling neurotransmission, energy metabolism, and transcription, with a particular involvement of G-protein coupled receptor (p = 5.0e -5 ), cAMP (p = 4.5e -4 ), and calcium signaling (p = 3.4e -3 ). Many identified RARβ target genes related to these pathways have been implicated in Alzheimer's, Parkinson's, and Huntington's disease (HD), raising the possibility that compromised RA signaling in the striatum may be a mechanistic link explaining the similar affective and cognitive symptoms in these diseases. The RARβ transcriptional targets were particularly enriched for transcripts affected in HD. Using the R6/2 transgenic mouse model of HD, we show that partial sequestration of RARβ in huntingtin protein aggregates may account for reduced RA signaling reported in HD.

  14. Identification of the Drosophila skpA gene as a novel target of the transcription factor DREF

    International Nuclear Information System (INIS)

    Dang Thi Phuong Thao; Ida, Hiroyuki; Yoshida, Hideki; Yamaguchi, Masamitsu

    2006-01-01

    SKPa is component of a Drosophila SCF complex that functions in combination with the ubiquitin-conjugating enzyme UbcD1. skpA null mutation results in centrosome overduplication, unusual chromatin condensation, defective endoreduplication and cell-cycle progression. While the molecular mechanisms that regulate expression of the skpA gene are poorly understood, the DNA replication-related element (DRE) and the DRE-binding factor (DREF) play important roles in regulating proliferation-related genes in Drosophila and DRE (5'-TATCGATA) and DRE-like (5'-CATCGATT) sequences were here found to be involved in skpA promoter activity. Thus both luciferase transient expression assays in cultured Drosophila S2 cells using skpA promoter-luciferase fusion plasmids and anti-lacZ immunostaining of various tissues from transgenic third instar larvae carrying the skpA promoter-lacZ fusion genes provided supportive evidence. Furthermore, anti-SKPa immunostaining of eye imaginal discs from flies overexpressing DREF showed ectopic expression of protein in the region posterior to the morphogenetic furrow where DREF is overexpressed. Knockdown of DREF in some tissues where SKPa distribution is well known almost completely abrogated the skpA gene expression. These findings, taken together, indicate that the Drosophila skpA gene is a novel target of the transcription factor DREF

  15. Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

    Science.gov (United States)

    Weyman, Philip D; Beeri, Karen; Lefebvre, Stephane C; Rivera, Josefa; McCarthy, James K; Heuberger, Adam L; Peers, Graham; Allen, Andrew E; Dupont, Christopher L

    2015-05-01

    Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle. © 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  16. Olean-18-ene triterpenoids from Celastraceae species inhibit HIV replication targeting NF-kB and Sp1 dependent transcription.

    Science.gov (United States)

    Osorio, Alex A; Muñóz, Alejandro; Torres-Romero, David; Bedoya, Luis M; Perestelo, Nayra R; Jiménez, Ignacio A; Alcamí, José; Bazzocchi, Isabel L

    2012-06-01

    In the present study we report the isolation of nine new olean-18-ene triterpenes (1-9), along with three known ones (10-12), from Cassine xylocarpa and Maytenus jelskii. Their stereostructures have been elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques (COSY, ROESY, HSQC and HMBC), and spectrometric methods. The natural compounds and derivatives 13-15 have been tested for their potential as inhibitors of human immunodeficiency virus type 1 replication. Five compounds from this series displayed potent antiviral activity with IC(50)s in the micromolar range (1, 3, 4, 7 and 8) being 1 and 8 the most active compounds. The target of these compounds was different from antiretroviral drugs currently licensed as they act as inhibitors of enhancer-dependent transcription. The structure-activity relationships were established based on the regiosubstitution and oxidation degree of the triterpene scaffold, revealing that these aspects were able to modulate the selectivity and intensity of HIV inhibition. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  17. Critical analysis of the potential for the therapeutic targeting of the Sp1 transcription factor in pancreatic cancer

    Directory of Open Access Journals (Sweden)

    Jutooru I

    2014-06-01

    Full Text Available Indira Jutooru,1 Gayathri Chadalapaka,1 Stephen Safe1,21Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX, USA; 2Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX, USAAbstract: Pancreatic ductal adenocarcinoma (PDAC is a major cause of cancer-related deaths in developed countries and, in 2013, it is estimated that in excess of 45,220 new cases were diagnosed in the United States. PDAC is a highly aggressive disease that invariably evades early diagnosis. The mean survival time for patients with metastatic disease is only 3–6 months, and only 20%–30% of pancreatic cancer patients are alive after 12 months. Because pancreatic cancers are frequently detected at an advanced stage, treatments have provided very limited improvements in tumor regression and overall survival times after diagnosis. 5-Fluorouracil alone or in combination with other drugs has been extensively used for treatment of advanced pancreatic cancer, and gemcitabine has partially replaced 5-fluorouracil as a treatment for pancreatic cancer. Gemcitabine provides increased clinical benefits in terms of response rate; however, future studies need to focus on developing treatment modalities that will improve the survival rate for pancreatic cancer patients. Specificity protein 1 (Sp1 is overexpressed in PDAC patients, and high expression is associated with poor prognosis, lymph node metastasis, and low survival. Knockdown studies have shown that Sp1 plays an important role in cell growth, angiogenesis, inflammation, survival, and metastasis. Sp1 expression is low in normal tissue when compared to tumor tissue, which makes Sp1 a potential target for development of new mechanism-based drugs for treatment of pancreatic cancer. Several drugs such as tolfenamic acid, betulinic acid, and methyl-2-cyano3,12-dioxooleana-1,9(11-dien-28-oate are shown to downregulate Sp1 expression through various pathways

  18. miR-17-5p targets the p300/CBP-associated factor and modulates androgen receptor transcriptional activity in cultured prostate cancer cells

    International Nuclear Information System (INIS)

    Gong, Ai-Yu; Eischeid, Alex N; Xiao, Jing; Zhao, Jian; Chen, Dongqing; Wang, Zhao-Yi; Young, Charles YF; Chen, Xian-Ming

    2012-01-01

    Androgen receptor (AR) signalling is critical to the initiation and progression of prostate cancer (PCa). Transcriptional activity of AR involves chromatin recruitment of co-activators, including the p300/CBP-associated factor (PCAF). Distinct miRNA expression profiles have been identified in PCa cells during the development and progression of the disease. Whether miRNAs regulate PCAF expression in PCa cells to regulate AR transcriptional activity is still unclear. Expression of PCAF was investigated in several PCa cell lines by qRT-PCR, Western blot, and immunocytochemistry. The effects of PCAF expression on AR-regulated transcriptional activity and cell growth in PCa cells were determined by chromatin immunoprecipitation, reporter gene construct analysis, and MTS assay. Targeting of PCAF by miR-17-5p was evaluated using the luciferase reporter assay. PCAF was upregulated in several PCa cell lines. Upregulation of PCAF promoted AR transcriptional activation and cell growth in cultured PCa cells. Expression of PCAF in PCa cells was associated with the downregulation of miR-17-5p. Targeting of the 3’-untranslated region of PCAF mRNA by miR-17-5p caused translational suppression and RNA degradation, and, consequently, modulation of AR transcriptional activity in PCa cells. PCAF is upregulated in cultured PCa cells, and upregulation of PCAF is associated with the downregulation of miR-17-5p. Targeting of PCAF by miR-17-5p modulates AR transcriptional activity and cell growth in cultured PCa cells

  19. Role of protein phosphatase 1 in dephosphorylation of Ebola virus VP30 protein and its targeting for the inhibition of viral transcription.

    Science.gov (United States)

    Ilinykh, Philipp A; Tigabu, Bersabeh; Ivanov, Andrey; Ammosova, Tatiana; Obukhov, Yuri; Garron, Tania; Kumari, Namita; Kovalskyy, Dmytro; Platonov, Maxim O; Naumchik, Vasiliy S; Freiberg, Alexander N; Nekhai, Sergei; Bukreyev, Alexander

    2014-08-15

    The filovirus Ebola (EBOV) causes the most severe hemorrhagic fever known. The EBOV RNA-dependent polymerase complex includes a filovirus-specific VP30, which is critical for the transcriptional but not replication activity of EBOV polymerase; to support transcription, VP30 must be in a dephosphorylated form. Here we show that EBOV VP30 is phosphorylated not only at the N-terminal serine clusters identified previously but also at the threonine residues at positions 143 and 146. We also show that host cell protein phosphatase 1 (PP1) controls VP30 dephosphorylation because expression of a PP1-binding peptide cdNIPP1 increased VP30 phosphorylation. Moreover, targeting PP1 mRNA by shRNA resulted in the overexpression of SIPP1, a cytoplasm-shuttling regulatory subunit of PP1, and increased EBOV transcription, suggesting that cytoplasmic accumulation of PP1 induces EBOV transcription. Furthermore, we developed a small molecule compound, 1E7-03, that targeted a non-catalytic site of PP1 and increased VP30 dephosphorylation. The compound inhibited the transcription but increased replication of the viral genome and completely suppressed replication of EBOV in cultured cells. Finally, mutations of Thr(143) and Thr(146) of VP30 significantly inhibited EBOV transcription and strongly induced VP30 phosphorylation in the N-terminal Ser residues 29-46, suggesting a novel mechanism of regulation of VP30 phosphorylation. Our findings suggest that targeting PP1 with small molecules is a feasible approach to achieve dysregulation of the EBOV polymerase activity. This novel approach may be used for the development of antivirals against EBOV and other filovirus species. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Aqp5 is a new transcriptional target of Dot1a and a regulator of Aqp2.

    Directory of Open Access Journals (Sweden)

    Hongyu Wu

    Full Text Available Dot1l encodes histone H3 K79 methyltransferase Dot1a. Mice with Dot1l deficiency in renal Aqp2-expressing cells (Dot1l(AC develop polyuria by unknown mechanisms. Here, we report that Aqp5 links Dot1l deletion to polyuria through Aqp2. cDNA array analysis revealed and real-time RT-qPCR validated Aqp5 as the most upregulated gene in Dot1l(AC vs. control mice. Aqp5 protein is barely detectable in controls, but robustly expressed in the Dot1l(AC kidneys, where it colocalizes with Aqp2. The upregulation of Aqp5 is coupled with reduced association of Dot1a and H3 dimethyl K79 with specific subregions in Aqp5 5' flanking region in Dot1l(AC vs. control mice. In vitro studies in IMCD3, MLE-15 and 293Tcells using multiple approaches including real-time RT-qPCR, luciferase reporter assay, cell surface biotinylation assay, colocalization, and co-immunoprecipitation uncovered that Dot1a represses Aqp5. Human AQP5 interacts with AQP2 and impairs its cell surface localization. The AQP5/AQP2 complex partially resides in the ER/Golgi. Consistently, AQP5 is expressed in none of 15 normal controls, but in all of 17 kidney biopsies from patients with diabetic nephropathy. In the patients with diabetic nephropathy, AQP5 colocalizes with AQP2 in the perinuclear region and AQP5 expression is associated with impaired cellular H3 dimethyl K79. Taken together, these data for the first time identify Aqp5 as a Dot1a potential transcriptional target, and an Aqp2 binding partner and regulator, and suggest that the upregulated Aqp5 may contribute to polyuria, possibly by impairing Aqp2 membrane localization, in Dot1l(AC mice and in patients with diabetic nephropathy.

  1. A titratable two-step transcriptional amplification strategy for targeted gene therapy based on ligand-induced intramolecular folding of a mutant human estrogen receptor

    DEFF Research Database (Denmark)

    Chen, Ian Y; Paulmurugan, Ramasamy; Nielsen, Carsten Haagen

    2014-01-01

    PURPOSE: The efficacy and safety of cardiac gene therapy depend critically on the level and the distribution of therapeutic gene expression following vector administration. We aimed to develop a titratable two-step transcriptional amplification (tTSTA) vector strategy, which allows modulation...... of transcriptionally targeted gene expression in the myocardium. PROCEDURES: We constructed a tTSTA plasmid vector (pcTnT-tTSTA-fluc), which uses the cardiac troponin T (cTnT) promoter to drive the expression of the recombinant transcriptional activator GAL4-mER(LBD)-VP2, whose ability to transactivate the downstream...... induction of myocardial fluc expression. HTV injection of pcTnT-tTSTA-fluc led to negligible long-term hepatic fluc expression, regardless of the raloxifene dose given. CONCLUSIONS: The tTSTA vector strategy can effectively modulate transgene expression in a tissue-specific manner. Further refinement...

  2. Hindsight regulates photoreceptor axon targeting through transcriptional control of jitterbug/Filamin and multiple genes involved in axon guidance in Drosophila.

    Science.gov (United States)

    Oliva, Carlos; Molina-Fernandez, Claudia; Maureira, Miguel; Candia, Noemi; López, Estefanía; Hassan, Bassem; Aerts, Stein; Cánovas, José; Olguín, Patricio; Sierralta, Jimena

    2015-09-01

    During axon targeting, a stereotyped pattern of connectivity is achieved by the integration of intrinsic genetic programs and the response to extrinsic long and short-range directional cues. How this coordination occurs is the subject of intense study. Transcription factors play a central role due to their ability to regulate the expression of multiple genes required to sense and respond to these cues during development. Here we show that the transcription factor HNT regulates layer-specific photoreceptor axon targeting in Drosophila through transcriptional control of jbug/Filamin and multiple genes involved in axon guidance and cytoskeleton organization.Using a microarray analysis we identified 235 genes whose expression levels were changed by HNT overexpression in the eye primordia. We analyzed nine candidate genes involved in cytoskeleton regulation and axon guidance, six of which displayed significantly altered gene expression levels in hnt mutant retinas. Functional analysis confirmed the role of OTK/PTK7 in photoreceptor axon targeting and uncovered Tiggrin, an integrin ligand, and Jbug/Filamin, a conserved actin- binding protein, as new factors that participate of photoreceptor axon targeting. Moreover, we provided in silico and molecular evidence that supports jbug/Filamin as a direct transcriptional target of HNT and that HNT acts partially through Jbug/Filamin in vivo to regulate axon guidance. Our work broadens the understanding of how HNT regulates the coordinated expression of a group of genes to achieve the correct connectivity pattern in the Drosophila visual system. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1018-1032, 2015. © 2015 Wiley Periodicals, Inc.

  3. The murine cytomegalovirus M35 protein antagonizes type I IFN induction downstream of pattern recognition receptors by targeting NF-κB mediated transcription.

    Directory of Open Access Journals (Sweden)

    Baca Chan

    2017-05-01

    Full Text Available The type I interferon (IFN response is imperative for the establishment of the early antiviral immune response. Here we report the identification of the first type I IFN antagonist encoded by murine cytomegalovirus (MCMV that shuts down signaling following pattern recognition receptor (PRR sensing. Screening of an MCMV open reading frame (ORF library identified M35 as a novel and strong negative modulator of IFNβ promoter induction following activation of both RNA and DNA cytoplasmic PRR. Additionally, M35 inhibits the proinflammatory cytokine response downstream of Toll-like receptors (TLR. Using a series of luciferase-based reporters with specific transcription factor binding sites, we determined that M35 targets NF-κB-, but not IRF-mediated, transcription. Expression of M35 upon retroviral transduction of immortalized bone marrow-derived macrophages (iBMDM led to reduced IFNβ transcription and secretion upon activation of stimulator of IFN genes (STING-dependent signaling. On the other hand, M35 does not antagonize interferon-stimulated gene (ISG 56 promoter induction or ISG transcription upon exogenous stimulation of the type I IFN receptor (IFNAR. M35 is present in the viral particle and, upon MCMV infection of fibroblasts, is immediately shuttled to the nucleus where it exerts its immunomodulatory effects. Deletion of M35 from the MCMV genome and hence from the viral particle resulted in elevated type I IFN transcription and secretion in vitro and in vivo. In the absence of M35, lower viral titers are observed during acute infection of the host, and productive infection in the salivary glands was not detected. In conclusion, the M35 protein is released by MCMV immediately upon infection in order to deftly inhibit the antiviral type I IFN response by targeting NF-κB-mediated transcription. The identification of this novel viral protein reinforces the importance of timely countermeasures in the complex relationship between virus and host.

  4. Preparation, crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the Ets transcription factor in complex with target DNA

    International Nuclear Information System (INIS)

    Suwa, Yoshiaki; Nakamura, Teruya; Toma, Sachiko; Ikemizu, Shinji; Kai, Hirofumi; Yamagata, Yuriko

    2008-01-01

    The complex between the Ets domain of Ets2 and its target DNA has been crystallized. The crystals diffracted to 3.0 Å resolution. The Ets2 transcription factor is a member of the Ets transcription-factor family. Ets2 plays a role in the malignancy of cancer and in Down’s syndrome by regulating the transcription of various genes. The DNA-binding domain of Ets2 (Ets domain; ETSD), which contains residues that are highly conserved among Ets transcription-factor family members, was expressed as a GST-fusion protein. The aggregation of ETSD produced after thrombin cleavage could be prevented by treatment with NDSB-195 (nondetergent sulfobetaine 195). ETSD was crystallized in complex with DNA containing the Ets2 target sequence (GGAA) by the hanging-drop vapour-diffusion method. The best crystals were grown using 25% PEG 3350, 80 mM magnesium acetate, 50 mM sodium cacodylate pH 5.0/5.5 as the reservoir at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 85.89, b = 95.52, c = 71.89 Å, β = 101.7° and a V M value of 3.56 Å 3 Da −1 . Diffraction data were collected to a resolution of 3.0 Å

  5. Preparation, crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the Ets transcription factor in complex with target DNA

    Energy Technology Data Exchange (ETDEWEB)

    Suwa, Yoshiaki; Nakamura, Teruya; Toma, Sachiko; Ikemizu, Shinji; Kai, Hirofumi; Yamagata, Yuriko, E-mail: yamagata@gpo.kumamoto-u.ac.jp [Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973 (Japan)

    2008-03-01

    The complex between the Ets domain of Ets2 and its target DNA has been crystallized. The crystals diffracted to 3.0 Å resolution. The Ets2 transcription factor is a member of the Ets transcription-factor family. Ets2 plays a role in the malignancy of cancer and in Down’s syndrome by regulating the transcription of various genes. The DNA-binding domain of Ets2 (Ets domain; ETSD), which contains residues that are highly conserved among Ets transcription-factor family members, was expressed as a GST-fusion protein. The aggregation of ETSD produced after thrombin cleavage could be prevented by treatment with NDSB-195 (nondetergent sulfobetaine 195). ETSD was crystallized in complex with DNA containing the Ets2 target sequence (GGAA) by the hanging-drop vapour-diffusion method. The best crystals were grown using 25% PEG 3350, 80 mM magnesium acetate, 50 mM sodium cacodylate pH 5.0/5.5 as the reservoir at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 85.89, b = 95.52, c = 71.89 Å, β = 101.7° and a V{sub M} value of 3.56 Å{sup 3} Da{sup −1}. Diffraction data were collected to a resolution of 3.0 Å.

  6. Carnitine palmitoyltransferase 1A (CPT1A): a transcriptional target of PAX3-FKHR and mediates PAX3-FKHR–dependent motility in alveolar rhabdomyosarcoma cells

    International Nuclear Information System (INIS)

    Liu, Lingling; Wang, Yong-Dong; Wu, Jing; Cui, Jimmy; Chen, Taosheng

    2012-01-01

    Alveolar rhabdomyosarcoma (ARMS) has a high propensity to metastasize, leading to its aggressiveness and a poor survival rate among those with the disease. More than 80% of aggressive ARMSs harbor a PAX3-FKHR fusion transcription factor, which regulates cell migration and promotes metastasis, most likely by regulating the fusion protein’s transcriptional targets. Therefore, identifying druggable transcription targets of PAX3-FKHR that are also downstream effectors of PAX3-FKHR–mediated cell migration and metastasis may lead to novel therapeutic approaches for treating ARMS. To identify genes whose expression is directly affected by the level of PAX3-FKHR in an ARMS cellular-context, we first developed an ARMS cell line in which PAX3-FKHR is stably down-regulated, and showed that stably downregulating PAX3-FKHR in ARMS cells significantly decreased the cells’ motility. We used microarray analysis to identify genes whose expression level decreased when PAX3-FKHR was downregulated. We used mutational analysis, promoter reporter assays, and electrophoretic mobility shift assays to determine whether PAX3-FKHR binds to the promoter region of the target gene. We used siRNA and pharmacologic inhibitor to downregulate the target gene of PAX3-FKHR and investigated the effect of such downregulation on cell motility. We found that when PAX3-FKHR was downregulated, the expression of carnitine palmitoyltransferase 1A (CPT1A) decreased. We showed that PAX3-FKHR binds to a paired-domain binding-site in the CPT1A promoter region, indicating that CPT1A is a novel transcriptional target of PAX3-FKHR. Furthermore, downregulating CPT1A decreased cell motility in ARMS cells, indicating that CPT1A is a downstream effector of PAX3-FKHR–mediated cell migration and metastasis. Taken together, we have identified CPT1A as a novel transcriptional target of PAX3-FKHR and revealed the novel function of CPT1A in promoting cell motility. CPT1A may represent a novel therapeutic target for

  7. The STAR protein QKI-7 recruits PAPD4 to regulate post-transcriptional polyadenylation of target mRNAs

    OpenAIRE

    Yamagishi, Ryota; Tsusaka, Takeshi; Mitsunaga, Hiroko; Maehata, Takaharu; Hoshino, Shin-ichi

    2016-01-01

    Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A)...

  8. State of Charge and State of Health Estimation of AGM VRLA Batteries by Employing a Dual Extended Kalman Filter and an ARX Model for Online Parameter Estimation

    Directory of Open Access Journals (Sweden)

    Ngoc-Tham Tran

    2017-01-01

    Full Text Available State of charge (SOC and state of health (SOH are key issues for the application of batteries, especially the absorbent glass mat valve regulated lead-acid (AGM VRLA type batteries used in the idle stop start systems (ISSs that are popularly integrated into conventional engine-based vehicles. This is due to the fact that SOC and SOH estimation accuracy is crucial for optimizing battery energy utilization, ensuring safety and extending battery life cycles. The dual extended Kalman filter (DEKF, which provides an elegant and powerful solution, is widely applied in SOC and SOH estimation based on a battery parameter model. However, the battery parameters are strongly dependent on operation conditions such as the SOC, current rate and temperature. In addition, battery parameters change significantly over the life cycle of a battery. As a result, many experimental pretests investigating the effects of the internal and external conditions of a battery on its parameters are required, since the accuracy of state estimation depends on the quality of the information regarding battery parameter changes. In this paper, a novel method for SOC and SOH estimation that combines a DEKF algorithm, which considers hysteresis and diffusion effects, and an auto regressive exogenous (ARX model for online parameters estimation is proposed. The DEKF provides precise information concerning the battery open circuit voltage (OCV to the ARX model. Meanwhile, the ARX model continues monitoring parameter variations and supplies information on them to the DEKF. In this way, the estimation accuracy can be maintained despite the changing parameters of a battery. Moreover, online parameter estimation from the ARX model can save the time and effort used for parameter pretests. The validation of the proposed algorithm is given by simulation and experimental results.

  9. Study of duplication 24bp of ARX gene among patients presenting a Mental Retardation with a syndromic and non syndromic forms

    International Nuclear Information System (INIS)

    Essouissi, Imen

    2006-01-01

    Mental Retardation (MR) is the most frequent handicap. It touches 3% of the general population. The genetic causes of this handicap account for 40% of these cases. ARX gene (Aristaless related homeobox gene) belongs to the family of the genes homeobox located in Xp22.1. It is considered as the most frequently muted gene after the FMR1 gene. It is implicated in various forms of syndromic and nonsyndromic MR. Several types of mutation were identified on the level of this gene, including deletions/insertions, duplications, missense and nonsense mutations, responsible for a wide spectrum of phenotypes. The goal of this work is to seek the most frequent change of gene ARX: duplication 24pb (at the origin of an expansion of the field poly has protein ARX in the position 144-155AA) among Tunisian boys presenting in particular family forms of non specific MR, sporadic forms of non specific MR like certain patients presenting a West syndrome.To prove the duplication of 24 Pb, we used in this work the Pcr technique. The change of duplication 24pb was not found in our series, this could be explained by the low number of cases family studied (38 families) and by the absence of connection studies accusing a mode of transmission related to X chromosome in particular for the sporadic cases. (Author)

  10. Differential developmental expression of transcription factors GATA-4 and GATA-6, their cofactor FOG-2 and downstream target genes in testicular carcinoma in situ and germ cell tumors

    DEFF Research Database (Denmark)

    Salonen, Jonna; Rajpert-De Meyts, E; Mannisto, Susanna

    2010-01-01

    Testicular germ cell cancer is the most common malignancy among young males. The pre-invasive precursor, carcinoma in situ testis (CIS), presumably originates from arrested and transformed fetal gonocytes. Given that GATA transcription factors have essential roles in embryonic and testicular deve...... development, we explored the expression of GATA-4, GATA-6, cofactor friend of GATA (FOG)-2, and downstream target genes during human testis development and addressed the question whether changes in this pathway may contribute to germ cell neoplasms....

  11. Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages reveals perturbation of transcriptional networks associated with interferon signalling

    Directory of Open Access Journals (Sweden)

    Craigon Marie

    2009-08-01

    Full Text Available Abstract Background Interferons (IFNs are potent antiviral cytokines capable of reprogramming the macrophage phenotype through the induction of interferon-stimulated genes (ISGs. Here we have used targeted RNA interference to suppress the expression of a number of key genes associated with IFN signalling in murine macrophages prior to stimulation with interferon-gamma. Genome-wide changes in transcript abundance caused by siRNA activity were measured using exon-level microarrays in the presence or absence of IFNγ. Results Transfection of murine bone-marrow derived macrophages (BMDMs with a non-targeting (control siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000 prior to stimulation with IFNγ. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. Network-based analysis of these data revealed six siRNAs to cause a marked shift in the macrophage transcriptome in the presence or absence IFNγ. These six siRNAs targeted the Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2 transcripts. The perturbation of the transcriptome by the six siRNAs was highly similar in each case and affected the expression of over 600 downstream transcripts. Regulated transcripts were clustered based on co-expression into five major groups corresponding to transcriptional networks associated with the type I and II IFN response, cell cycle regulation, and NF-KB signalling. In addition we have observed a significant non-specific immune stimulation of cells transfected with siRNA using Lipofectamine2000, suggesting use of this reagent in BMDMs, even at low concentrations, is enough to induce a type I IFN response. Conclusion Our results provide evidence that the type I IFN response in murine BMDMs is dependent on Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2, and that siRNAs targeted to these genes results in perturbation of key transcriptional networks associated

  12. Inference of expanded Lrp-like feast/famine transcription factor targets in a non-model organism using protein structure-based prediction.

    Science.gov (United States)

    Ashworth, Justin; Plaisier, Christopher L; Lo, Fang Yin; Reiss, David J; Baliga, Nitin S

    2014-01-01

    Widespread microbial genome sequencing presents an opportunity to understand the gene regulatory networks of non-model organisms. This requires knowledge of the binding sites for transcription factors whose DNA-binding properties are unknown or difficult to infer. We adapted a protein structure-based method to predict the specificities and putative regulons of homologous transcription factors across diverse species. As a proof-of-concept we predicted the specificities and transcriptional target genes of divergent archaeal feast/famine regulatory proteins, several of which are encoded in the genome of Halobacterium salinarum. This was validated by comparison to experimentally determined specificities for transcription factors in distantly related extremophiles, chromatin immunoprecipitation experiments, and cis-regulatory sequence conservation across eighteen related species of halobacteria. Through this analysis we were able to infer that Halobacterium salinarum employs a divergent local trans-regulatory strategy to regulate genes (carA and carB) involved in arginine and pyrimidine metabolism, whereas Escherichia coli employs an operon. The prediction of gene regulatory binding sites using structure-based methods is useful for the inference of gene regulatory relationships in new species that are otherwise difficult to infer.

  13. Tagging target genes of the mat1-2-1 transcription factor in Fusarium verticillioides (Gibberella fujikuroi MP-A)

    NARCIS (Netherlands)

    Keszthelyi, A.; Jeney, A.; Kerenyi, Z.; Mendes, O.; Waalwijk, C.; Hornok, L.

    2007-01-01

    Mating type in filamentous ascomycetes is controlled by idiomorphic alleles, named MAT1-1 and MAT1-2, which contain 1-3 genes. Of these genes MAT1-1-1 and MAT1-2-1 encode putative transcription factors and are thus considered to be the major regulators of sexual communication and mating. Fungi with

  14. The Populus ARBORKNOX1 homeodomain transcription factor regulates woody growth through binding to evolutionarily conserved target genes of diverse function

    Science.gov (United States)

    Lijun Liu; Matthew S. Zinkgraf; H. Earl Petzold; Eric P. Beers; Vladimir Filkov; Andrew Groover

    2014-01-01

    The class I KNOX homeodomain transcription factor ARBORKNOX1 (ARK1) is a key regulator of vascular cambium maintenance and cell differentiation in Populus. Currently, basic information is lacking concerning the distribution, functional characteristics, and evolution of ARK1 binding in the Populus genome.

  15. The regulatory mechanism of fruit ripening revealed by analyses of direct targets of the tomato MADS-box transcription factor RIPENING INHIBITOR

    Science.gov (United States)

    Fujisawa, Masaki; Ito, Yasuhiro

    2013-01-01

    The developmental process of ripening is unique to fleshy fruits and a key factor in fruit quality. The tomato (Solanum lycopersicum) MADS-box transcription factor RIPENING INHIBITOR (RIN), one of the earliest-acting ripening regulators, is required for broad aspects of ripening, including ethylene-dependent and -independent pathways. However, our knowledge of direct RIN target genes has been limited, considering the broad effects of RIN on ripening. In a recent work published in The Plant Cell, we identified 241 direct RIN target genes by chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) and transcriptome analysis. Functional classification of the targets revealed that RIN participates in the regulation of many biological processes including well-known ripening processes such as climacteric ethylene production and lycopene accumulation. In addition, we found that ethylene is required for the full expression of RIN and several RIN-targeting transcription factor genes at the ripening stage. Here, based on our recently published findings and additional data, we discuss the ripening processes regulated by RIN and the interplay between RIN and ethylene. PMID:23518588

  16. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

    Science.gov (United States)

    Gao, Xuefei; Tsang, Jason C.H.; Gaba, Fortis; Wu, Donghai; Lu, Liming; Liu, Pentao

    2014-01-01

    The transcription activator–like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas9) utlilize distinct molecular mechanisms in targeting site recognition. The two proteins can be modified to carry additional functional domains to regulate expression of genomic loci in mammalian cells. In this study, we have compared the two systems in activation and suppression of the Oct4 and Nanog loci by targeting their enhancers. Although both are able to efficiently activate the luciferase reporters, the CRISPR/dCas9 system is much less potent in activating the endogenous loci and in the application of reprogramming somatic cells to iPS cells. Nevertheless, repression by CRISPR/dCas9 is comparable to or even better than TALE repressors. We demonstrated that dCas9 protein binding results in significant physical interference to binding of native transcription factors at enhancer, less efficient active histone markers induction or recruitment of activating complexes in gene activation. This study thus highlighted the merits and drawbacks of transcription regulation by each system. A combined approach of TALEs and CRISPR/dCas9 should provide an optimized solution to regulate genomic loci and to study genetic elements such as enhancers in biological processes including somatic cell reprogramming and guided differentiation. PMID:25223790

  17. Modeling, molecular dynamics, and docking assessment of transcription factor rho: a potential drug target in Brucella melitensis 16M

    Directory of Open Access Journals (Sweden)

    Pradeepkiran JA

    2015-03-01

    Full Text Available Jangampalli Adi Pradeepkiran,1 Konidala Kranthi Kumar,1 Yellapu Nanda Kumar,2 Matcha Bhaskar11Division of Animal Biotechnology, Department of Zoology, Sri Venkateswara University, Tirupati, 2Biomedical Informatics Centre, Vector Control Research Centre, Indian Council of Medical Research, Pondicherry, India Abstract: The zoonotic disease brucellosis, a chronic condition in humans affecting renal and cardiac systems and causing osteoarthritis, is caused by Brucella, a genus of Gram-negative, facultative, intracellular pathogens. The mode of transmission and the virulence of the pathogens are still enigmatic. Transcription regulatory elements, such as rho proteins, play an important role in the termination of transcription and/or the selection of genes in Brucella. Adverse effects of the transcription inhibitors play a key role in the non-successive transcription challenges faced by the pathogens. In the investigation presented here, we computationally predicted the transcription termination factor rho (TtFRho inhibitors against Brucella melitensis 16M via a structure-based method. In view the unknown nature of its crystal structure, we constructed a robust three-dimensional homology model of TtFRho’s structure by comparative modeling with the crystal structure of the Escherichia coli TtFRho (Protein Data Bank ID: 1PVO as a template in MODELLER (v 9.10. The modeled structure was optimized by applying a molecular dynamics simulation for 2 ns with the CHARMM (Chemistry at HARvard Macromolecular Mechanics 27 force field in NAMD (NAnoscale Molecular Dynamics program; v 2.9 and then evaluated by calculating the stereochemical quality of the protein. The flexible docking for the interaction phenomenon of the template consists of ligand-related inhibitor molecules from the ZINC (ZINC Is Not Commercial database using a structure-based virtual screening strategy against minimized TtFRho. Docking simulations revealed two inhibitors compounds – ZINC

  18. Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes

    DEFF Research Database (Denmark)

    Pasini, Diego; Malatesta, Martina; Jung, Hye Ryung

    2010-01-01

    Polycomb group (PcG) proteins are transcriptional repressors, which regulate proliferation and cell fate decisions during development, and their deregulated expression is a frequent event in human tumours. The Polycomb repressive complex 2 (PRC2) catalyzes trimethylation (me3) of histone H3 lysine...... are poorly understood. To gain insight into these mechanisms, we have determined the global changes in histone modifications in embryonic stem (ES) cells lacking the PcG protein Suz12 that is essential for PRC2 activity. We show that loss of PRC2 activity results in a global increase in H3K27 acetylation....... The methylation to acetylation switch correlates with the transcriptional activation of PcG target genes, both during ES cell differentiation and in MLL-AF9-transduced hematopoietic stem cells. Moreover, we provide evidence that the acetylation of H3K27 is catalyzed by the acetyltransferases p300 and CBP. Based...

  19. Md-miR156ab and Md-miR395 Target WRKY Transcription Factors to Influence Apple Resistance to Leaf Spot Disease.

    Science.gov (United States)

    Zhang, Qiulei; Li, Yang; Zhang, Yi; Wu, Chuanbao; Wang, Shengnan; Hao, Li; Wang, Shengyuan; Li, Tianzhong

    2017-01-01

    MicroRNAs (miRNAs) are key regulators of gene expression that post-transcriptionally regulate transcription factors involved in plant physiological activities. Little is known about the effects of miRNAs in disease resistance in apple ( Malus × domestica ). We globally profiled miRNAs in the apple cultivar Golden Delicious (GD) infected or not with the apple leaf spot fungus Alternaria alternaria f. sp. mali (ALT1), and identified 58 miRNAs that exhibited more than a 2-fold upregulation upon ALT1 infection. We identified a pair of miRNAs that target protein-coding genes involved in the defense response against fungal pathogens; Md-miR156ab targets a novel WRKY transcription factor, MdWRKYN1, which harbors a TIR and a WRKY domain. Md-miR395 targets another transcription factor, MdWRKY26, which contains two WRKY domains. Real-time PCR analysis showed that Md-miR156ab and Md-miR395 levels increased, while MdWRKYN1 and MdWRKY26 expression decreased in ALT1-inoculated GD leaves; furthermore, the overexpression of Md-miR156ab and Md-miR395 resulted in a significant reduction in MdWRKYN1 and MdWRKY26 expression. To investigate whether these miRNAs and their targets play a crucial role in plant defense, we overexpressed MdWRKYN1 or knocked down Md-miR156ab activity, which in both cases enhanced the disease resistance of the plants by upregulating the expression of the WRKY-regulated pathogenesis-related (PR) protein-encoding genes MdPR3-1, MdPR3-2, MdPR4, MdPR5, MdPR10-1 , and MdPR10-2 . In a similar analysis, we overexpressed MdWRKY26 or suppressed Md-miR395 activity, and found that many PR protein-encoding genes were also regulated by MdWRKY26 . In GD, ALT-induced Md-miR156ab and Md-miR395 suppress MdWRKYN1 and MdWRKY26 expression, thereby decreasing the expression of some PR genes, and resulting in susceptibility to ALT1.

  20. Improved Detection of Lassa Virus by Reverse Transcription-PCR Targeting the 5′ Region of S RNA▿

    OpenAIRE

    Ölschläger, Stephan; Lelke, Michaela; Emmerich, Petra; Panning, Marcus; Drosten, Christian; Hass, Meike; Asogun, Danny; Ehichioya, Deborah; Omilabu, Sunday; Günther, Stephan

    2010-01-01

    The method of choice for the detection of Lassa virus is reverse transcription (RT)-PCR. However, the high degree of genetic variability of the virus poses a problem with the design of RT-PCR assays that will reliably detect all strains. Recently, we encountered difficulties in detecting some strains from Liberia and Nigeria in a commonly used glycoprotein precursor (GPC) gene-specific RT-PCR assay (A. H. Demby, J. Chamberlain, D. W. Brown, and C. S. Clegg, J. Clin. Microbiol. 32:2898-2903, 1...

  1. Curcumin and synthetic analogs induce reactive oxygen species and decreases specificity protein (Sp) transcription factors by targeting microRNAs

    International Nuclear Information System (INIS)

    Gandhy, Shruti U; Kim, KyoungHyun; Larsen, Lesley; Rosengren, Rhonda J; Safe, Stephen

    2012-01-01

    Curcumin inhibits growth of several cancer cell lines, and studies in this laboratory in bladder and pancreatic cancer cells show that curcumin downregulates specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and pro-oncogenic Sp-regulated genes. In this study, we investigated the anticancer activity of curcumin and several synthetic cyclohexanone and piperidine analogs in colon cancer cells. The effects of curcumin and synthetic analogs on colon cancer cell proliferation and apoptosis were determined using standardized assays. The changes in Sp proteins and Sp-regulated gene products were analysed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a), miR-20a, miR-17-5p and ZBTB10 and ZBTB4 mRNA expression. The IC 50 (half-maximal) values for growth inhibition (24 hr) of colon cancer cells by curcumin and synthetic cyclohexanone and piperidine analogs of curcumin varied from 10 μM for curcumin to 0.7 μM for the most active synthetic piperidine analog RL197, which was used along with curcumin as model agents in this study. Curcumin and RL197 inhibited RKO and SW480 colon cancer cell growth and induced apoptosis, and this was accompanied by downregulation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and Sp-regulated genes including the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET), survivin, bcl-2, cyclin D1 and NFκB (p65 and p50). Curcumin and RL197 also induced reactive oxygen species (ROS), and cotreatment with the antioxidant glutathione significantly attenuated curcumin- and RL197-induced growth inhibition and downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes. The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR)-27a, miR-20a and miR-17-5p that regulate these repressors. These results identify a new and highly potent

  2. Curcumin and synthetic analogs induce reactive oxygen species and decreases specificity protein (Sp transcription factors by targeting microRNAs

    Directory of Open Access Journals (Sweden)

    Gandhy Shruti U

    2012-11-01

    Full Text Available Abstract Background Curcumin inhibits growth of several cancer cell lines, and studies in this laboratory in bladder and pancreatic cancer cells show that curcumin downregulates specificity protein (Sp transcription factors Sp1, Sp3 and Sp4 and pro-oncogenic Sp-regulated genes. In this study, we investigated the anticancer activity of curcumin and several synthetic cyclohexanone and piperidine analogs in colon cancer cells. Methods The effects of curcumin and synthetic analogs on colon cancer cell proliferation and apoptosis were determined using standardized assays. The changes in Sp proteins and Sp-regulated gene products were analysed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a, miR-20a, miR-17-5p and ZBTB10 and ZBTB4 mRNA expression. Results The IC50 (half-maximal values for growth inhibition (24 hr of colon cancer cells by curcumin and synthetic cyclohexanone and piperidine analogs of curcumin varied from 10 μM for curcumin to 0.7 μM for the most active synthetic piperidine analog RL197, which was used along with curcumin as model agents in this study. Curcumin and RL197 inhibited RKO and SW480 colon cancer cell growth and induced apoptosis, and this was accompanied by downregulation of specificity protein (Sp transcription factors Sp1, Sp3 and Sp4 and Sp-regulated genes including the epidermal growth factor receptor (EGFR, hepatocyte growth factor receptor (c-MET, survivin, bcl-2, cyclin D1 and NFκB (p65 and p50. Curcumin and RL197 also induced reactive oxygen species (ROS, and cotreatment with the antioxidant glutathione significantly attenuated curcumin- and RL197-induced growth inhibition and downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes. The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR-27a, miR-20a and miR-17-5p that regulate these repressors

  3. Arabidopsis MYC Transcription Factors Are the Target of Hormonal Salicylic Acid/Jasmonic Acid Cross Talk in Response to Pieris brassicae Egg Extract.

    Science.gov (United States)

    Schmiesing, André; Emonet, Aurélia; Gouhier-Darimont, Caroline; Reymond, Philippe

    2016-04-01

    Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence, expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Cross talk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating this phenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of the JA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domain transcriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negative regulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYC protein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JA cross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation of SA/JA antagonism depending on the biological context in which the SA pathway is activated. © 2016 American Society of Plant Biologists. All Rights Reserved.

  4. The Mammalian "Obesogen" Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish.

    Directory of Open Access Journals (Sweden)

    Angeliki Lyssimachou

    Full Text Available Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT, which causes imposex in gastropod snails, induces an "obesogenic" phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR and peroxisome proliferator-activated receptor gamma (PPARγ. In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound.

  5. The Mammalian “Obesogen” Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish

    Science.gov (United States)

    Lyssimachou, Angeliki; Santos, Joana G.; André, Ana; Soares, Joana; Lima, Daniela; Guimarães, Laura; Almeida, C. Marisa R.; Teixeira, Catarina; Castro, L. Filipe C.; Santos, Miguel M.

    2015-01-01

    Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an “obesogenic” phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound. PMID:26633012

  6. Arabidopsis MYC Transcription Factors Are the Target of Hormonal Salicylic Acid/Jasmonic Acid Cross Talk in Response to Pieris brassicae Egg Extract1[OPEN

    Science.gov (United States)

    Schmiesing, André; Gouhier-Darimont, Caroline

    2016-01-01

    Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence, expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Cross talk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating this phenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of the JA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domain transcriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negative regulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYC protein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JA cross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation of SA/JA antagonism depending on the biological context in which the SA pathway is activated. PMID:26884488

  7. The Mammalian "Obesogen" Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish.

    Science.gov (United States)

    Lyssimachou, Angeliki; Santos, Joana G; André, Ana; Soares, Joana; Lima, Daniela; Guimarães, Laura; Almeida, C Marisa R; Teixeira, Catarina; Castro, L Filipe C; Santos, Miguel M

    2015-01-01

    Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an "obesogenic" phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound.

  8. The hetC Gene Is a Direct Target of the NtcA Transcriptional Regulator in Cyanobacterial Heterocyst Development

    Science.gov (United States)

    Muro-Pastor, Alicia M.; Valladares, Ana; Flores, Enrique; Herrero, Antonia

    1999-01-01

    The heterocyst is the site of nitrogen fixation in aerobically grown cultures of some filamentous cyanobacteria. Heterocyst development in Anabaena sp. strain PCC 7120 is dependent on the global nitrogen regulator NtcA and requires, among others, the products of the hetR and hetC genes. Expression of hetC, tested by RNA- DNA hybridization, was impaired in an ntcA mutant. A nitrogen-regulated, NtcA-dependent putative transcription start point was localized at nucleotide −571 with respect to the hetC translational start. Sequences upstream from this transcription start point exhibit the structure of the canonical cyanobacterial promoter activated by NtcA, and purified NtcA protein specifically bound to a DNA fragment containing this promoter. Activation of expression of hetC during heterocyst development appears thus to be directly operated by NtcA. NtcA-mediated activation of hetR expression was not impaired in a hetC mutant, indicating that HetC is not an NtcA-dependent element required for hetR induction. PMID:10542167

  9. Salicylic Acid Suppresses Jasmonic Acid Signaling Downstream of SCFCOI1-JAZ by Targeting GCC Promoter Motifs via Transcription Factor ORA59[C][W][OA

    Science.gov (United States)

    Van der Does, Dieuwertje; Leon-Reyes, Antonio; Koornneef, Annemart; Van Verk, Marcel C.; Rodenburg, Nicole; Pauwels, Laurens; Goossens, Alain; Körbes, Ana P.; Memelink, Johan; Ritsema, Tita; Van Wees, Saskia C.M.; Pieterse, Corné M.J.

    2013-01-01

    Antagonism between the defense hormones salicylic acid (SA) and jasmonic acid (JA) plays a central role in the modulation of the plant immune signaling network, but the molecular mechanisms underlying this phenomenon are largely unknown. Here, we demonstrate that suppression of the JA pathway by SA functions downstream of the E3 ubiquitin-ligase Skip-Cullin-F-box complex SCFCOI1, which targets JASMONATE ZIM-domain transcriptional repressor proteins (JAZs) for proteasome-mediated degradation. In addition, neither the stability nor the JA-induced degradation of JAZs was affected by SA. In silico promoter analysis of the SA/JA crosstalk transcriptome revealed that the 1-kb promoter regions of JA-responsive genes that are suppressed by SA are significantly enriched in the JA-responsive GCC-box motifs. Using GCC:GUS lines carrying four copies of the GCC-box fused to the β-glucuronidase reporter gene, we showed that the GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Using plants overexpressing the GCC-box binding APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors ERF1 or ORA59, we found that SA strongly reduces the accumulation of ORA59 but not that of ERF1. Collectively, these data indicate that the SA pathway inhibits JA signaling downstream of the SCFCOI1-JAZ complex by targeting GCC-box motifs in JA-responsive promoters via a negative effect on the transcriptional activator ORA59. PMID:23435661

  10. Salicylic acid suppresses jasmonic acid signaling downstream of SCFCOI1-JAZ by targeting GCC promoter motifs via transcription factor ORA59.

    Science.gov (United States)

    Van der Does, Dieuwertje; Leon-Reyes, Antonio; Koornneef, Annemart; Van Verk, Marcel C; Rodenburg, Nicole; Pauwels, Laurens; Goossens, Alain; Körbes, Ana P; Memelink, Johan; Ritsema, Tita; Van Wees, Saskia C M; Pieterse, Corné M J

    2013-02-01

    Antagonism between the defense hormones salicylic acid (SA) and jasmonic acid (JA) plays a central role in the modulation of the plant immune signaling network, but the molecular mechanisms underlying this phenomenon are largely unknown. Here, we demonstrate that suppression of the JA pathway by SA functions downstream of the E3 ubiquitin-ligase Skip-Cullin-F-box complex SCF(COI1), which targets JASMONATE ZIM-domain transcriptional repressor proteins (JAZs) for proteasome-mediated degradation. In addition, neither the stability nor the JA-induced degradation of JAZs was affected by SA. In silico promoter analysis of the SA/JA crosstalk transcriptome revealed that the 1-kb promoter regions of JA-responsive genes that are suppressed by SA are significantly enriched in the JA-responsive GCC-box motifs. Using GCC:GUS lines carrying four copies of the GCC-box fused to the β-glucuronidase reporter gene, we showed that the GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Using plants overexpressing the GCC-box binding APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors ERF1 or ORA59, we found that SA strongly reduces the accumulation of ORA59 but not that of ERF1. Collectively, these data indicate that the SA pathway inhibits JA signaling downstream of the SCF(COI1)-JAZ complex by targeting GCC-box motifs in JA-responsive promoters via a negative effect on the transcriptional activator ORA59.

  11. miR-25-3p, Positively Regulated by Transcription Factor AP-2α, Regulates the Metabolism of C2C12 Cells by Targeting Akt1

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    Feng Zhang

    2018-03-01

    Full Text Available miR-25, a member of the miR-106b-25 cluster, has been reported as playing an important role in many biological processes by numerous studies, while the role of miR-25 in metabolism and its transcriptional regulation mechanism remain unclear. In this study, gain-of-function and loss-of-function assays demonstrated that miR-25-3p positively regulated the metabolism of C2C12 cells by attenuating phosphoinositide 3-kinase (PI3K gene expression and triglyceride (TG content, and enhancing the content of adenosine triphosphate (ATP and reactive oxygen species (ROS. Furthermore, the results from bioinformatics analysis, dual luciferase assay, site-directed mutagenesis, qRT-PCR, and Western blotting demonstrated that miR-25-3p directly targeted the AKT serine/threonine kinase 1 (Akt1 3′ untranslated region (3′UTR. The core promoter of miR-25-3p was identified, and the transcription factor activator protein-2α (AP-2α significantly increased the expression of mature miR-25-3p by binding to its core promoter in vivo, as indicated by the chromatin immunoprecipitation (ChIP assay, and AP-2α binding also downregulated the expression of Akt1. Taken together, our findings suggest that miR-25-3p, positively regulated by the transcription factor AP-2α, enhances C2C12 cell metabolism by targeting the Akt1 gene.

  12. Transcriptome profiling confirmed correlations between symptoms and transcriptional changes in RDV infected rice and revealed nucleolus as a possible target of RDV manipulation.

    Science.gov (United States)

    Yang, Liang; Du, Zhenguo; Gao, Feng; Wu, Kangcheng; Xie, Lianhui; Li, Yi; Wu, Zujian; Wu, Jianguo

    2014-05-06

    Rice dwarf virus (RDV) is the causal agent of rice dwarf disease, which limits rice production in many areas of south East Asia. Transcriptional changes of rice in response to RDV infection have been characterized by Shimizu et al. and Satoh et al.. Both studies found induction of defense related genes and correlations between transcriptional changes and symptom development in RDV-infected rice. However, the same rice cultivar, namely Nipponbare belonging to the Japonic subspecies of rice was used in both studies. Gene expression changes of the indica subspecies of rice, namely Oryza sativa L. ssp. indica cv Yixiang2292 that show moderate resistance to RDV, in response to RDV infection were characterized using an Affymetrix Rice Genome Array. Differentially expressed genes (DEGs) were classified according to their Gene Ontology (GO) annotation. The effects of transient expression of Pns11 in Nicotiana benthaminana on the expression of nucleolar genes were studied using real-time PCR (RT-PCR). 856 genes involved in defense or other physiological processes were identified to be DEGs, most of which showed up-regulation. Ribosome- and nucleolus related genes were significantly enriched in the DEGs. Representative genes related to nucleolar function exhibited altered expression in N. benthaminana plants transiently expressing Pns11 of RDV. Induction of defense related genes is common for rice infected with RDV. There is a co-relation between symptom severity and transcriptional alteration in RDV infected rice. Besides ribosome, RDV may also target nucleolus to manipulate the translation machinery of rice. Given the tight links between nucleolus and ribosome, it is intriguing to speculate that RDV may enhance expression of ribosomal genes by targeting nucleolus through Pns11.

  13. Bioinformatic analysis of patient-derived ASPS gene expressions and ASPL-TFE3 fusion transcript levels identify potential therapeutic targets.

    Directory of Open Access Journals (Sweden)

    David G Covell

    Full Text Available Gene expression data, collected from ASPS tumors of seven different patients and from one immortalized ASPS cell line (ASPS-1, was analyzed jointly with patient ASPL-TFE3 (t(X;17(p11;q25 fusion transcript data to identify disease-specific pathways and their component genes. Data analysis of the pooled patient and ASPS-1 gene expression data, using conventional clustering methods, revealed a relatively small set of pathways and genes characterizing the biology of ASPS. These results could be largely recapitulated using only the gene expression data collected from patient tumor samples. The concordance between expression measures derived from ASPS-1 and both pooled and individual patient tumor data provided a rationale for extending the analysis to include patient ASPL-TFE3 fusion transcript data. A novel linear model was exploited to link gene expressions to fusion transcript data and used to identify a small set of ASPS-specific pathways and their gene expression. Cellular pathways that appear aberrantly regulated in response to the t(X;17(p11;q25 translocation include the cell cycle and cell adhesion. The identification of pathways and gene subsets characteristic of ASPS support current therapeutic strategies that target the FLT1 and MET, while also proposing additional targeting of genes found in pathways involved in the cell cycle (CHK1, cell adhesion (ARHGD1A, cell division (CDC6, control of meiosis (RAD51L3 and mitosis (BIRC5, and chemokine-related protein tyrosine kinase activity (CCL4.

  14. Heterogeneity of Metastatic Melanoma:  Correlation of MITF With Its Transcriptional Targets MLSN1, PEDF, HMB-45, and MART-1.

    Science.gov (United States)

    Zand, Sarvenaz; Buzney, Elizabeth; Duncan, Lyn M; Dadras, Soheil S

    2016-09-01

    Histologic and molecular heterogeneity is well recognized in malignant melanoma; however, the diversity of expression of new and classic melanoma markers has not been correlated in serial sections of metastases. We examined and correlated the expression of microphthalmia transcription factor (MITF) with its transcriptional targets, including melastatin (MLSN1/TRPM1), pigment epithelium-derived factor (SERPINF1/PEDF), SILV/PMEL17/GP100 (human melanoma black 45 [HMB-45]), and melanoma antigen recognized by T cells 1 (MART-1)/MLANA, in 13 melanoma metastases in lymph nodes of 13 patients. The expression levels and patterns of marker expression were recorded by a semiquantitative, 4-point ordinal reactivity method. Our results showed a consistently robust and diffuse expression of MITF protein in 12 (92%) of 13 metastatic tumors compared with variable expression of MLSN1 (46%) messenger RNA or PEDF (75%), HMB-45 (54%), and MART-1 (46%) proteins. Overall, in melanoma lymph node metastases, MITF protein expression was not tightly correlated with its gene targets. Moreover, the immunoreactivity for MITF, compared with MART-1 and HMB-45, was retained, supporting immunohistochemical detection of MITF as a more sensitive method of detecting metastatic melanoma. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. JC virus induces altered patterns of cellular gene expression: Interferon-inducible genes as major transcriptional targets

    International Nuclear Information System (INIS)

    Verma, Saguna; Ziegler, Katja; Ananthula, Praveen; Co, Juliene K.G.; Frisque, Richard J.; Yanagihara, Richard; Nerurkar, Vivek R.

    2006-01-01

    Human polyomavirus JC (JCV) infects 80% of the population worldwide. Primary infection, typically occurring during childhood, is asymptomatic in immunocompetent individuals and results in lifelong latency and persistent infection. However, among the severely immunocompromised, JCV may cause a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Virus-host interactions influencing persistence and pathogenicity are not well understood, although significant regulation of JCV activity is thought to occur at the level of transcription. Regulation of the JCV early and late promoters during the lytic cycle is a complex event that requires participation of both viral and cellular factors. We have used cDNA microarray technology to analyze global alterations in gene expression in JCV-permissive primary human fetal glial cells (PHFG). Expression of more than 400 cellular genes was altered, including many that influence cell proliferation, cell communication and interferon (IFN)-mediated host defense responses. Genes in the latter category included signal transducer and activator of transcription 1 (STAT1), interferon stimulating gene 56 (ISG56), myxovirus resistance 1 (MxA), 2'5'-oligoadenylate synthetase (OAS), and cig5. The expression of these genes was further confirmed in JCV-infected PHFG cells and the human glioblastoma cell line U87MG to ensure the specificity of JCV in inducing this strong antiviral response. Results obtained by real-time RT-PCR and Western blot analyses supported the microarray data and provide temporal information related to virus-induced changes in the IFN response pathway. Our data indicate that the induction of an antiviral response may be one of the cellular factors regulating/controlling JCV replication in immunocompetent hosts and therefore constraining the development of PML

  16. Ursodeoxycholic acid attenuates experimental autoimmune arthritis by targeting Th17 and inducing pAMPK and transcriptional corepressor SMILE.

    Science.gov (United States)

    Lee, Eun-Jung; Kwon, Jeong-Eun; Park, Min-Jung; Jung, Kyung-Ah; Kim, Da-Som; Kim, Eun-Kyung; Lee, Seung Hoon; Choi, Jong Young; Park, Sung-Hwan; Cho, Mi-La

    2017-08-01

    Ursodeoxycholic acid (UDCA) has been known that UDCA has prominent effects on liver, however, there is little known about its influence on autoimmune disease. Here, the benefit of UDCA on arthritis rheumatoid (RA) in vivo was tested. RA mouse were induced using collagen II (CIA, collagen induced arthritis) where the disease severity or UDCA-related signaling pathway such as AMP-activated protein kinase (AMPK) or small heterodimer partner interacting leucine zipper protein (SMILE) was evaluated by westerblot and immunohistochemical staining. Gene expression was measured by realtime-polymerase chain reaction (PCR). The administration of UDCA effectively alleviated the arthritic score and incidence with decreased cartilage damage and lipid metabolic parameters. UDCA also suppressed the secretion of pro-inflammatory cytokines. It was confirmed that UDCA upregulated the expression of SMILE and transcriptional activity of PPARγ via controlling AMPK or p38 activity. In the present study, the therapeutic effect of UDCA inducing SMILE through AMPK activation in rheumatoid arthritis mouse as well as other autoimmune disease was proposed. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  17. Downregulation of the Sonic Hedgehog/Gli pathway transcriptional target Neogenin-1 is associated with basal cell carcinoma aggressiveness.

    Science.gov (United States)

    Casas, Bárbara S; Adolphe, Christelle; Lois, Pablo; Navarrete, Nelson; Solís, Natalia; Bustamante, Eva; Gac, Patricio; Cabané, Patricio; Gallegos, Ivan; Wainwright, Brandon J; Palma, Verónica

    2017-10-13

    Basal Cell Carcinoma (BCC) is one of the most diagnosed cancers worldwide. It develops due to an unrestrained Sonic Hedgehog (SHH) signaling activity in basal cells of the skin. Certain subtypes of BCC are more aggressive than others, although the molecular basis of this phenomenon remains unknown. We have previously reported that Neogenin-1 (NEO1) is a downstream target gene of the SHH/GLI pathway in neural tissue. Given that SHH participates in epidermal homeostasis, here we analyzed the epidermal expression of NEO1 in order to identify whether it plays a role in adult epidermis or BCC. We describe the mRNA and protein expression profile of NEO1 and its ligands (Netrin-1 and RGMA) in human and mouse control epidermis and in a broad range of human BCCs. We identify in human BCC a significant positive correlation in the levels of NEO1 receptor, NTN-1 and RGMA ligands with respect to GLI1 , the main target gene of the canonical SHH pathway. Moreover, we show via cyclopamine inhibition of the SHH/GLI pathway of ex vivo cultures that NEO1 likely functions as a downstream target of SHH/GLI signaling in the skin. We also show how Neo1 expression decreases throughout BCC progression in the K14-Cre:Ptch1 lox/lox mouse model and that aggressive subtypes of human BCC exhibit lower levels of NEO1 than non-aggressive BCC samples. Taken together, these data suggest that NEO1 is a SHH/GLI target in epidermis. We propose that NEO1 may be important in tumor onset and is then down-regulated in advanced BCC or aggressive subtypes.

  18. iTAR: a web server for identifying target genes of transcription factors using ChIP-seq or ChIP-chip data.

    Science.gov (United States)

    Yang, Chia-Chun; Andrews, Erik H; Chen, Min-Hsuan; Wang, Wan-Yu; Chen, Jeremy J W; Gerstein, Mark; Liu, Chun-Chi; Cheng, Chao

    2016-08-12

    Chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) or microarray hybridization (ChIP-chip) has been widely used to determine the genomic occupation of transcription factors (TFs). We have previously developed a probabilistic method, called TIP (Target Identification from Profiles), to identify TF target genes using ChIP-seq/ChIP-chip data. To achieve high specificity, TIP applies a conservative method to estimate significance of target genes, with the trade-off being a relatively low sensitivity of target gene identification compared to other methods. Additionally, TIP's output does not render binding-peak locations or intensity, information highly useful for visualization and general experimental biological use, while the variability of ChIP-seq/ChIP-chip file formats has made input into TIP more difficult than desired. To improve upon these facets, here we present are fined TIP with key extensions. First, it implements a Gaussian mixture model for p-value estimation, increasing target gene identification sensitivity and more accurately capturing the shape of TF binding profile distributions. Second, it enables the incorporation of TF binding-peak data by identifying their locations in significant target gene promoter regions and quantifies their strengths. Finally, for full ease of implementation we have incorporated it into a web server ( http://syslab3.nchu.edu.tw/iTAR/ ) that enables flexibility of input file format, can be used across multiple species and genome assembly versions, and is freely available for public use. The web server additionally performs GO enrichment analysis for the identified target genes to reveal the potential function of the corresponding TF. The iTAR web server provides a user-friendly interface and supports target gene identification in seven species, ranging from yeast to human. To facilitate investigating the quality of ChIP-seq/ChIP-chip data, the web server generates the chart of the

  19. Epstein-Barr virus nuclear antigen 3C targets p53 and modulates its transcriptional and apoptotic activities

    International Nuclear Information System (INIS)

    Yi Fuming; Saha, Abhik; Murakami, Masanao; Kumar, Pankaj; Knight, Jason S.; Cai Qiliang; Choudhuri, Tathagata; Robertson, Erle S.

    2009-01-01

    The p53 tumor suppressor gene is one of the most commonly mutated genes in human cancers and the corresponding encoded protein induces apoptosis or cell-cycle arrest at the G1/S checkpoint in response to DNA damage. To date, previous studies have shown that antigens encoded by human tumor viruses such as SV40 large T antigen, adenovirus E1A and HPV E6 interact with p53 and disrupt its functional activity. In a similar fashion, we now show that EBNA3C, one of the EBV latent antigens essential for the B-cell immortalization in vitro, interacts directly with p53. Additionally, we mapped the interaction of EBNA3C with p53 to the C-terminal DNA-binding and the tetramerization domain of p53, and the region of EBNA3C responsible for binding to p53 was mapped to the N-terminal domain of EBNA3C (residues 130-190), previously shown to interact with a number of important cell-cycle components, specifically SCF Skp2 , cyclin A, and cMyc. Furthermore, we demonstrate that EBNA3C substantially represses the transcriptional activity of p53 in luciferase based reporter assays, and rescues apoptosis induced by ectopic p53 expression in SAOS-2 (p53 -/- ) cells. Interestingly, we also show that the DNA-binding ability of p53 is diminished in the presence of EBNA3C. Thus, the interaction between the p53 and EBNA3C provides new insights into the mechanism(s) by which the EBNA3C oncoprotein can alter cellular gene expression in EBV associated human cancers.

  20. The Bacterial Effector HopX1 Targets JAZ Transcriptional Repressors to Activate Jasmonate Signaling and Promote Infection in Arabidopsis

    Science.gov (United States)

    Gimenez-Ibanez, Selena; Boter, Marta; Fernández-Barbero, Gemma; Chini, Andrea; Rathjen, John P.; Solano, Roberto

    2014-01-01

    Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR), which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile). Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA)-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto) DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome. PMID:24558350

  1. Hsa-miR-11181 regulates Wnt signaling pathway through targeting of APC2 transcripts in SW480 cell line.

    Science.gov (United States)

    Dokanehiifard, Sadat; Soltani, Bahram M

    2018-01-30

    Wnt signaling plays important roles in differentiation, morphogenesis and development. This signaling pathway is highly regulated at all levels and microRNAs are small noncoding RNAs regulating Wnt signaling. Here, we intended to investigate hsa-miR-11181 (a novel miRNA located in TrkC gene) effect on Wnt signaling pathway in SW480 cell line. TOP/FOP flash assay indicated up-regulation of Wnt signaling, following the overexpression of hsa-miR-11181, verified through RT-qPCR. Bioinformatics analysis predicted APC1, APC2 and Axin1 might be targeted by hsa-miR-11181. Then, RT-qPCR analysis indicated that APC2 and Axin1 have been significantly down-regulated following the hsa-miR-11181 overexpression. However dual luciferase assay analysis supported only APC2 3'-UTR is directly targeted by this miRNA. Then, treatment of SW480 cells with Wnt-inhibitory small molecules supported the effect of hsa-miR-11181 at the inhibitory complex level containing APC2 protein. Consistently, viability of SW480 cells overexpressing hsa-miR-11181 was significantly elevated, measured through MTT assay. Overall, these results suggest that hsa-miR-11181 may play a crucial role in Wnt signaling regulation and confirmed that APC2 3'-UTR is targeted by hsa-miR-11181 and propose the presence of its recognition sites in the promoter or coding regions of Axin1 gene. Copyright © 2017. Published by Elsevier B.V.

  2. Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

    Directory of Open Access Journals (Sweden)

    Motoharu Ono

    Full Text Available We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

  3. Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

    Science.gov (United States)

    Ono, Motoharu; Yamada, Kayo; Avolio, Fabio; Afzal, Vackar; Bensaddek, Dalila; Lamond, Angus I

    2015-01-01

    We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

  4. MicroR-146 blocks the activation of M1 macrophage by targeting signal transducer and activator of transcription 1 in hepatic schistosomiasis

    Directory of Open Access Journals (Sweden)

    Xing He

    2016-11-01

    Full Text Available Schistosomiasis is a chronic disease caused by the parasite of the Schistosoma genus and is characterized by egg-induced hepatic granulomas and fibrosis. Macrophages play a central role in schistosomiasis with several studies highlighting their differentiation into M2 cells involved in the survival of infected mice through limitation of immunopathology. However, little is known regarding the mechanisms of regulating macrophage differentiation. Here, we showed that the early stage of infection by Schistosoma japonicum induced expression of type 1 T-helper-cell (Th1 cytokine, interferon-γ (IFN-γ, leading to increase in M1 cells. However, the presence of liver-trapped eggs induced the expression of Th2 cytokines including interleukin-4 (IL-4, IL-10, and IL-13 that upregulated the transcription of miR-146b by activating signal transducer and activator of transcription 3/6 (STAT3/6 that bind to the promoter of the pre-miR-146b gene. We found that the miR-146a/b was significantly upregulated in macrophages during the progression of hepatic schistosomiasis. The elevated miR-146a/b inhibited the IFN-γ-induced differentiation of macrophages to M1 cells through targeting STAT1. Our data indicate the protective roles of miR-146a/b in hepatic schistosomiasis through regulating the differentiation of macrophages into M2 cells.

  5. The role of Forkhead-box Class O (FoxO) transcription factors in cancer: A target for the management of cancer

    International Nuclear Information System (INIS)

    Reagan-Shaw, Shannon; Ahmad, Nihal

    2007-01-01

    Human Forkhead-Box Class O (FoxO) transcription factors are primarily regulated through the phosphoinositide-3-kinase (PI3k)-Akt pathway via phosphorylation and nuclear exclusion. Acetylation and ubiquitination represent another level of regulation for FoxO proteins and FoxO-regulated gene expression. FoxO factors can act as tumor suppressors; however, the loss of FoxO function leads to increased cellular survival and a predisposition to neoplasia, especially of epithelial cancers. Based on the critical role of FoxO signaling, this family of transcription factors appears to be a promising target for future drug discovery for epithelial cancers. This review describes mechanism of the regulation of FoxO proteins and their role in epithelial cancers. Based on the current knowledge and studies in the past decade, we suggest that the development of novel agents which specifically activate FoxO members could be useful in the prevention as well as treatment of cancer in general and epithelial cancers in particular

  6. The transcription factor Ace2 and its paralog Swi5 regulate ethanol production during static fermentation through their targets Cts1 and Rps4a in Saccharomyces cerevisiae.

    Science.gov (United States)

    Wu, Yao; Du, Jie; Xu, Guoqiang; Jiang, Linghuo

    2016-05-01

    Saccharomyces cerevisiae is the most widely used fermentation organism for ethanol production. However, the gene expression regulatory networks behind the ethanol fermentation are still not fully understood. Using a static fermentation model, we examined the ethanol yields on biomass of deletion mutants for 77 yeast genes encoding nonessential transcription factors, and found that deletion mutants for ACE2 and SWI5 showed dramatically increased ethanol yields. Overexpression of ACE2 or SWI5 in wild type cells reduced their ethanol yields. Furthermore, among the 34 target genes regulated by Ace2 and Swi5, deletion of CTS1,RPS4a,SIC1,EGT2,DSE2, or SCP160 led to increased ethanol yields, with the former two showing higher effects. Overexpression of CTS1 or RPS4a in both ace2/ace2 and swi5/swi5 mutants reduced their ethanol yields. In contrast, deletion of MCR1 or HO significantly decreased ethanol yields, with the former one showing the highest effect. Therefore, Ace2 and Swi5 are two negative regulators of ethanol yield during static fermentation of yeast cells, and both CTS1 and RPS4a are major effectors mediating these two transcription factors in regulating ethanol production. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Genome-wide transcriptional profiling of Botrytis cinerea genes targeting plant cell walls during infections of different hosts

    Directory of Open Access Journals (Sweden)

    Barbara eBlanco-Ulate

    2014-09-01

    Full Text Available Cell walls are barriers that impair colonization of host tissues, but also are important reservoirs of energy-rich sugars. Growing hyphae of necrotrophic fungal pathogens, such as Botrytis cinerea (Botrytis, henceforth, secrete enzymes that disassemble cell wall polysaccharides. In this work we describe the annotation of 275 putative secreted Carbohydrate-Active enZymes (CAZymes identified in the Botrytis B05.10 genome. Using RNAseq we determined which Botrytis CAZymes were expressed during infections of lettuce leaves, ripe tomato fruit, and grape berries. On the three hosts, Botrytis expressed a common group of 229 potentially secreted CAZymes, including 28 pectin backbone-modifying enzymes, 21 hemicellulose-modifying proteins, 18 enzymes that might target pectin and hemicellulose side-branches, and 16 enzymes predicted to degrade cellulose. The diversity of the Botrytis CAZymes may be partly responsible for its wide host range. Thirty-six candidate CAZymes with secretion signals were found exclusively when Botrytis interacted with ripe tomato fruit and grape berries. Pectin polysaccharides are notably abundant in grape and tomato cell walls, but lettuce leaf walls have less pectin and are richer in hemicelluloses and cellulose. The results of this study not only suggest that Botrytis targets similar wall polysaccharide networks on fruit and leaves, but also that it may selectively attack host wall polysaccharide substrates depending on the host tissue.

  8. The transcription factor Swi4 is target for PKA regulation of cell size at the G1 to S transition in Saccharomyces cerevisiae.

    Science.gov (United States)

    Amigoni, Loredana; Colombo, Sonia; Belotti, Fiorella; Alberghina, Lilia; Martegani, Enzo

    2015-08-03

    To investigate the specific target of PKA in the regulation of cell cycle progression and cell size we developed a new approach using the yeast strain GG104 bearing a deletion in adenylate cyclase gene and permeable to cAMP ( cyr1Δ, pde2Δ, msn2Δ, msn4Δ). In this strain the PKA activity is absent and can be activated by addition of cAMP in the medium, without any other change of the growth conditions. In the present work we show that the activation of PKA by exogenous cAMP in the GG104 strain exponentially growing in glucose medium caused a marked increase of cell size and perturbation of cell cycle with a transient arrest of cells in G1, followed by an accumulation of cells in G2/M phase with a minimal change in the growth rate. Deletion of CLN1 gene, but not of CLN2, abolished the transient G1 phase arrest. Consistently we found that PKA activation caused a transcriptional repression of CLN1 gene. Transcription of CLN1 is controlled by SBF and MBF dual-regulated promoter. We found that also the deletion of SWI4 gene abolished the transient G1 arrest suggesting that Swi4 is a target responsible for PKA modulation of G1/S phase transition. We generated a SWI4 allele mutated in the consensus site for PKA (Swi4(S159A)) and we found that expression of Swi4(S159A) protein in the GG104-Swi4Δ strain did not restore the transient G1 arrest induced by PKA activation, suggesting that Swi4 phosphorylation by PKA regulates CLN1 gene expression and G1/S phase transition.

  9. ChIP-on-chip analysis identifies IL-22 as direct target gene of ectopically expressed FOXP3 transcription factor in human T cells

    Directory of Open Access Journals (Sweden)

    Jeron Andreas

    2012-12-01

    Full Text Available Abstract Background The transcription factor (TF forkhead box P3 (FOXP3 is constitutively expressed at high levels in naturally occurring CD4+CD25+ regulatory T cells (nTregs. It is not only the most accepted marker for that cell population but is also considered lineage determinative. Chromatin immunoprecipitation (ChIP of TFs in combination with genomic tiling microarray analysis (ChIP-on-chip has been shown to be an appropriate tool for identifying FOXP3 transcription factor binding sites (TFBSs on a genome-wide scale. In combination with microarray expression analysis, the ChIP-on-chip technique allows identification of direct FOXP3 target genes. Results ChIP-on-chip analysis of the human FOXP3 expressed in resting and PMA/ionomycin–stimulated Jurkat T cells revealed several thousand putative FOXP3 binding sites and demonstrated the importance of intronic regions for FOXP3 binding. The analysis of expression data showed that the stimulation-dependent down-regulation of IL-22 was correlated with direct FOXP3 binding in the IL-22 promoter region. This association was confirmed by real-time PCR analysis of ChIP-DNA. The corresponding ChIP-region also contained a matching FOXP3 consensus sequence. Conclusions Knowledge of the general distribution patterns of FOXP3 TFBSs in the human genome under resting and activated conditions will contribute to a better understanding of this TF and its influence on direct target genes, as well as its importance for the phenotype and function of Tregs. Moreover, FOXP3-dependent repression of Th17-related IL-22 may be relevant to an understanding of the phenomenon of Treg/Th17 cell plasticity.

  10. Rice and Bean Targets for Biofortification Combined with High Carotenoid Content Crops Regulate Transcriptional Mechanisms Increasing Iron Bioavailability

    Science.gov (United States)

    Dias, Desirrê Morais; de Castro Moreira, Maria Eliza; Gomes, Mariana Juste Contin; Lopes Toledo, Renata Celi; Nutti, Marilia Regini; Pinheiro Sant’Ana, Helena Maria; Martino, Hércia Stampini Duarte

    2015-01-01

    Iron deficiency affects thousands of people worldwide. Biofortification of staple food crops aims to support the reduction of this deficiency. This study evaluates the effect of combinations of common beans and rice, targets for biofortification, with high carotenoid content crops on the iron bioavailability, protein gene expression, and antioxidant effect. Iron bioavailability was measured by the depletion/repletion method. Seven groups were tested (n = 7): Pontal bean (PB); rice + Pontal bean (R + BP); Pontal bean + sweet potato (PB + SP); Pontal bean + pumpkin (PB + P); Pontal bean + rice + sweet potato (PB + R + P); Pontal bean + rice + sweet potato (PB + R + SP); positive control (Ferrous Sulfate). The evaluations included: hemoglobin gain, hemoglobin regeneration efficiency (HRE), gene expression of divalente metal transporter 1 (DMT-1), duodenal citocromo B (DcytB), ferroportin, hephaestin, transferrin and ferritin and total plasma antioxidant capacity (TAC). The test groups, except the PB, showed higher HRE (p bioavailability and antioxidant capacity. PMID:26610564

  11. Concomitant targeting of multiple key transcription factors effectively disrupts cancer stem cells enriched in side population of human pancreatic cancer cells.

    Directory of Open Access Journals (Sweden)

    Xiyan Wang

    Full Text Available A major challenge in the treatment of pancreatic ductal adenocarcinoma is the failure of chemotherapy, which is likely due to the presence of the cancer stem cells (CSCs.To identify side population (SP cells and characterize s-like properties in human pancreatic cancer cell lines (h-PCCLs and to exploit the efficacy of concomitant targeting of multiple key transcription factors governing the stemness of pancreatic CSCs in suppressing CSC-like phenotypes.Flow cytometry and Hoechst 33342 DNA-binding dye efflux assay were used to sort SP and non-SP (NSP cells from three h-PCCLs: PANC-1, SW1990, and BxPc-3. The self-renewal ability, invasiveness, migration and drug resistance of SP cells were evaluated. Expression of CSC marker genes was analyzed. Tumorigenicity was assessed using a xenograft model in nude mice. Effects of a complex decoy oligonucleotide (cdODN-SCO designed to simultaneously targeting Sox2, Oct4 and c-Myc were assessed.CSCs were enriched in the side proportion (SP cells contained in the h-PCCLs and they possessed aggressive growth, invasion, migration and drug-resistance properties, compared with NSP cells. SP cells overexpressed stem cell markers CD133 and ALDH1, pluripotency maintaining factors Nanog, Sox2 and Oct4, oncogenic transcription factor c-Myc, signaling molecule Notch1, and drug resistant gene ABCG2. Moreover, SP cells consistently demonstrated significantly greater tumorigenicity than NSP cells in xenograft model of nude mice. CdODN-SOC efficiently suppressed all CSC properties and phenotypes, and minimized the tumorigenic capability of the SP cells and the resistance to chemotherapy. By comparison, the negative control failed to do so.The findings indicate that targeting the key genes conferring the stemness of CSCs can efficiently eliminate CSC-like phenotypes, and thus may be considered a new approach for cancer therapy. Specifically, the present study establishes the combination of Sox2/Oct4/c-Myc targeting as a

  12. Pyrosequencing the transcriptome of the greenhouse whitefly, Trialeurodes vaporariorum reveals multiple transcripts encoding insecticide targets and detoxifying enzymes

    Directory of Open Access Journals (Sweden)

    Gorman Kevin

    2011-01-01

    Full Text Available Abstract Background The whitefly Trialeurodes vaporariorum is an economically important crop pest in temperate regions that has developed resistance to most classes of insecticides. However, the molecular mechanisms underlying resistance have not been characterised and, to date, progress has been hampered by a lack of nucleotide sequence data for this species. Here, we use pyrosequencing on the Roche 454-FLX platform to produce a substantial and annotated EST dataset. This 'unigene set' will form a critical reference point for quantitation of over-expressed messages via digital transcriptomics. Results Pyrosequencing produced around a million sequencing reads that assembled into 54,748 contigs, with an average length of 965 bp, representing a dramatic expansion of existing cDNA sequences available for T. vaporariorum (only 43 entries in GenBank at the time of this publication. BLAST searching of non-redundant databases returned 20,333 significant matches and those gene families potentially encoding gene products involved in insecticide resistance were manually curated and annotated. These include, enzymes potentially involved in the detoxification of xenobiotics and those encoding the targets of the major chemical classes of insecticides. A total of 57 P450s, 17 GSTs and 27 CCEs were identified along with 30 contigs encoding the target proteins of six different insecticide classes. Conclusion Here, we have developed new transcriptomic resources for T. vaporariorum. These include a substantial and annotated EST dataset that will serve the community studying this important crop pest and will elucidate further the molecular mechanisms underlying insecticide resistance.

  13. The E1A proteins of all six human adenovirus subgroups target the p300/CBP acetyltransferases and the SAGA transcriptional regulatory complex

    International Nuclear Information System (INIS)

    Shuen, Michael; Avvakumov, Nikita; Torchia, Joe; Mymryk, Joe S.

    2003-01-01

    The N-terminal/conserved region 1 (CR1) portion of the human adenovirus (Ad) 5 E1A protein was previously shown to inhibit growth in the simple eukaryote Saccharomyces cerevisiae. We now demonstrate that the corresponding regions of the E1A proteins of Ad3,-4,-9,-12, and -40, which represent the remaining five Ad subgroups, also inhibit yeast growth. These results suggest that the E1A proteins of all six human Ad subgroups share a common cellular target(s) conserved in yeast. Growth inhibition induced by either full-length or the N-terminal/CR1 portion of Ad5 E1A was relieved by coexpression of the E1A binding portions of the mammalian p300, CBP, and pCAF acetyltransferases. Similarly, growth inhibition by the N-terminal/CR1 portions of the other Ad E1A proteins was suppressed by expression of the same regions of CBP or pCAF known to bind Ad5 E1A. The physical interaction of each of the different Ad E1A proteins with CBP, p300, and pCAF was confirmed in vitro. Furthermore, deletion of the gene encoding yGcn5, the yeast homolog of pCAF and a subunit of the SAGA transcriptional regulatory complex, restored growth in yeast expressing each of the different Ad E1A proteins. This indicates that the SAGA complex is a conserved target of all Ad E1A proteins. Our results demonstrate for the first time that the p300, CBP, and pCAF acetyltransferases are common targets for the E1A proteins of all six human Ad subgroups, highlighting the importance of these interactions for E1A function

  14. Rice and Bean Targets for Biofortification Combined with High Carotenoid Content Crops Regulate Transcriptional Mechanisms Increasing Iron Bioavailability

    Directory of Open Access Journals (Sweden)

    Desirrê Morais Dias

    2015-11-01

    Full Text Available Iron deficiency affects thousands of people worldwide. Biofortification of staple food crops aims to support the reduction of this deficiency. This study evaluates the effect of combinations of common beans and rice, targets for biofortification, with high carotenoid content crops on the iron bioavailability, protein gene expression, and antioxidant effect. Iron bioavailability was measured by the depletion/repletion method. Seven groups were tested (n = 7: Pontal bean (PB; rice + Pontal bean (R + BP; Pontal bean + sweet potato (PB + SP; Pontal bean + pumpkin (PB + P; Pontal bean + rice + sweet potato (PB + R + P; Pontal bean + rice + sweet potato (PB + R + SP; positive control (Ferrous Sulfate. The evaluations included: hemoglobin gain, hemoglobin regeneration efficiency (HRE, gene expression of divalente metal transporter 1 (DMT-1, duodenal citocromo B (DcytB, ferroportin, hephaestin, transferrin and ferritin and total plasma antioxidant capacity (TAC. The test groups, except the PB, showed higher HRE (p < 0.05 than the control. Gene expression of DMT-1, DcytB and ferroportin increased (p < 0.05 in the groups fed with high content carotenoid crops (sweet potato or pumpkin. The PB group presented lower (p < 0.05 TAC than the other groups. The combination of rice and common beans, and those with high carotenoid content crops increased protein gene expression, increasing the iron bioavailability and antioxidant capacity.

  15. Transcription factor Nrf1 is topologically repartitioned across membranes to enable target gene transactivation through its acidic glucose-responsive domains.

    Science.gov (United States)

    Zhang, Yiguo; Ren, Yonggang; Li, Shaojun; Hayes, John D

    2014-01-01

    The membrane-bound Nrf1 transcription factor regulates critical homeostatic and developmental genes. The conserved N-terminal homology box 1 (NHB1) sequence in Nrf1 targets the cap'n'collar (CNC) basic basic-region leucine zipper (bZIP) factor to the endoplasmic reticulum (ER), but it is unknown how its activity is controlled topologically within membranes. Herein, we report a hitherto unknown mechanism by which the transactivation activity of Nrf1 is controlled through its membrane-topology. Thus after Nrf1 is anchored within ER membranes, its acidic transactivation domains (TADs), including the Asn/Ser/Thr-rich (NST) glycodomain situated between acidic domain 1 (AD1) and AD2, are transiently translocated into the lumen of the ER, where NST is glycosylated in the presence of glucose to yield an inactive 120-kDa Nrf1 glycoprotein. Subsequently, portions of the TADs partially repartition across membranes into the cyto/nucleoplasmic compartments, whereupon an active 95-kDa form of Nrf1 accumulates, a process that is more obvious in glucose-deprived cells and may involve deglycosylation. The repartitioning of Nrf1 out of membranes is monitored within this protein by its acidic-hydrophobic amphipathic glucose-responsive domains, particularly the Neh5L subdomain within AD1. Therefore, the membrane-topological organization of Nrf1 dictates its post-translational modifications (i.e. glycosylation, the putative deglycosylation and selective proteolysis), which together control its ability to transactivate target genes.

  16. DNA Binding Drugs Targeting the Regulatory DNA Binding Site of the ETS Domain Family Transcription Factor Associated With Human Breast Cancer

    National Research Council Canada - National Science Library

    Wang, Yong-Dong

    1999-01-01

    .... The key approach is to prevent the binding of two transcription factors, ESX and AP-2, to the consensus DNA binding sites contained within the Her2/neu promoter resulting in inhibition of transcription factor function...

  17. miR-320a regulates cell proliferation and apoptosis in multiple myeloma by targeting pre-B-cell leukemia transcription factor 3

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Yinghao [Jiangsu Institute of Hematology, First Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis and Hemostasis Under Ministry of Health, Collaborative Innovation Center of Hematology, Suzhou, 215006 (China); Department of Hematology, Affiliated Hospital of Guizhou Medical University, The Hematopoietic Stem Cell Transplant Center of Guizhou Province, Blood Diseases Diagnosis and Treatment Center of Guizhou Province, Guiyang, 550004, Guizhou Province (China); Wu, Depei, E-mail: wudepei@medmail.com.cn [Jiangsu Institute of Hematology, First Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis and Hemostasis Under Ministry of Health, Collaborative Innovation Center of Hematology, Suzhou, 215006 (China); Wang, Jishi, E-mail: lgylhlyh@aliyun.com [Department of Hematology, Affiliated Hospital of Guizhou Medical University, The Hematopoietic Stem Cell Transplant Center of Guizhou Province, Blood Diseases Diagnosis and Treatment Center of Guizhou Province, Guiyang, 550004, Guizhou Province (China); Li, Yan; Chai, Xiao; Kang, Qian [Department of Hematology, Affiliated Hospital of Guizhou Medical University, The Hematopoietic Stem Cell Transplant Center of Guizhou Province, Blood Diseases Diagnosis and Treatment Center of Guizhou Province, Guiyang, 550004, Guizhou Province (China)

    2016-05-13

    Aberrant expression of microRNAs (miRNAs) is implicated in cancer development and progression. While miR-320a is reported to be deregulated in many malignancy types, its biological role in multiple myeloma (MM) remains unclear. Here, we observed reduced expression of miR-320a in MM samples and cell lines. Ectopic expression of miR-320a dramatically suppressed cell viability and clonogenicity and induced apoptosis in vitro. Mechanistic investigation led to the identification of Pre-B-cellleukemia transcription factor 3 (PBX3) as a novel and direct downstream target of miR-320a. Interestingly, reintroduction of PBX3 abrogated miR-320a-induced MM cell growth inhibition and apoptosis. In a mouse xenograft model, miR-320a overexpression inhibited tumorigenicity and promoted apoptosis. Our findings collectively indicate that miR-320a inhibits cell proliferation and induces apoptosis in MM cells by directly targeting PBX3, supporting its utility as a novel and potential therapeutic agent for miRNA-based MM therapy. -- Highlights: •Expression of miR-320a in MM cell induces apoptosis in vitro. •miR-320a represses PBX3 via targeting specific sequences in the 3′UTR region. •Exogenous expression of PBX3 reverses the effects of miR-320a in inhibiting MM cell growth and promoting apoptosis. •Overexpression of miR-320a inhibits tumor growth and increases apoptosis in vivo.

  18. Combined serial analysis of gene expression and transcription factor binding site prediction identifies novel-candidate-target genes of Nr2e1 in neocortex development.

    Science.gov (United States)

    Schmouth, Jean-François; Arenillas, David; Corso-Díaz, Ximena; Xie, Yuan-Yun; Bohacec, Slavita; Banks, Kathleen G; Bonaguro, Russell J; Wong, Siaw H; Jones, Steven J M; Marra, Marco A; Simpson, Elizabeth M; Wasserman, Wyeth W

    2015-07-24

    Nr2e1 (nuclear receptor subfamily 2, group e, member 1) encodes a transcription factor important in neocortex development. Previous work has shown that nuclear receptors can have hundreds of target genes, and bind more than 300 co-interacting proteins. However, recognition of the critical role of Nr2e1 in neural stem cells and neocortex development is relatively recent, thus the molecular mechanisms involved for this nuclear receptor are only beginning to be understood. Serial analysis of gene expression (SAGE), has given researchers both qualitative and quantitative information pertaining to biological processes. Thus, in this work, six LongSAGE mouse libraries were generated from laser microdissected tissue samples of dorsal VZ/SVZ (ventricular zone and subventricular zone) from the telencephalon of wild-type (Wt) and Nr2e1-null embryos at the critical development ages E13.5, E15.5, and E17.5. We then used a novel approach, implementing multiple computational methods followed by biological validation to further our understanding of Nr2e1 in neocortex development. In this work, we have generated a list of 1279 genes that are differentially expressed in response to altered Nr2e1 expression during in vivo neocortex development. We have refined this list to 64 candidate direct-targets of NR2E1. Our data suggested distinct roles for Nr2e1 during different neocortex developmental stages. Most importantly, our results suggest a possible novel pathway by which Nr2e1 regulates neurogenesis, which includes Lhx2 as one of the candidate direct-target genes, and SOX9 as a co-interactor. In conclusion, we have provided new candidate interacting partners and numerous well-developed testable hypotheses for understanding the pathways by which Nr2e1 functions to regulate neocortex development.

  19. The unique and cooperative roles of the Grainy head-like transcription factors in epidermal development reflect unexpected target gene specificity.

    Science.gov (United States)

    Boglev, Yeliz; Wilanowski, Tomasz; Caddy, Jacinta; Parekh, Vishwas; Auden, Alana; Darido, Charbel; Hislop, Nikki R; Cangkrama, Michael; Ting, Stephen B; Jane, Stephen M

    2011-01-15

    The Grainy head-like 3 (Grhl3) gene encodes a transcription factor that plays essential roles in epidermal morphogenesis during embryonic development, with deficient mice exhibiting failed skin barrier formation, defective wound repair, and loss of eyelid fusion. Despite sharing significant sequence homology, overlapping expression patterns, and an identical core consensus DNA binding site, the other members of the Grhl family (Grhl1 and -2) fail to compensate for the loss of Grhl3 in these processes. Here, we have employed diverse genetic models, coupled with biochemical studies, to define the inter-relationships of the Grhl factors in epidermal development. We show that Grhl1 and Grhl3 have evolved complete functional independence, as evidenced by a lack of genetic interactions in embryos carrying combinations of targeted alleles of these genes. In contrast, compound heterozygous Grhl2/Grhl3 embryos displayed failed wound repair, and loss of a single Grhl2 allele in Grhl3-null embryos results in fully penetrant eyes open at birth. Expression of Grhl2 from the Grhl3 locus in homozygous knock-in mice corrects the wound repair defect, but these embryos still display a complete failure of skin barrier formation. This functional dissociation is due to unexpected differences in target gene specificity, as both GRHL2 and GRHL3 bind to and regulate expression of the wound repair gene Rho GEF 19, but regulation of the barrier forming gene, Transglutaminase 1 (TGase1), is unique to GRHL3. Our findings define the mechanisms underpinning the unique and cooperative roles of the Grhl genes in epidermal development. Copyright © 2010 Elsevier Inc. All rights reserved.

  20. Establishment of a sensitive system for analysis of human vaginal microbiota on the basis of rRNA-targeted reverse transcription-quantitative PCR.

    Science.gov (United States)

    Kurakawa, Takashi; Ogata, Kiyohito; Tsuji, Hirokazu; Kado, Yukiko; Takahashi, Takuya; Kida, Yumi; Ito, Masahiro; Okada, Nobuhiko; Nomoto, Koji

    2015-04-01

    Ten specific primer sets, for Lactobacillus gasseri, Lactobacillus crispatus, Atopobium vaginae, Gardnerella vaginalis, Mobiluncus curtisii, Chlamydia trachomatis/muridarum, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, and Bifidobacterium angulatum, were developed for quantitative analysis of vaginal microbiota. rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) analysis of the vaginal samples from 12 healthy Japanese volunteers using the new primer sets together with 25 existing primer sets revealed the diversity of their vaginal microbiota: Lactobacilli such as L. crispatus, L. gasseri, Lactobacillus jensenii, Lactobacillus iners, and Lactobacillus vaginalis, as the major populations at 10(7) cells/ml vaginal fluid, were followed by facultative anaerobes such as Streptococcus and strict anaerobes at lower population levels of 10(4) cells/ml or less. Certain bacterial vaginosis (BV)-related bacteria, such as G. vaginalis, A. vaginae, M. curtisii, and Prevotella, were also detected in some subjects. Especially in one subject, both G. vaginalis and A. vaginae were detected at high population levels of 10(8.8) and 10(8.9) cells/ml vaginal fluid, suggesting that she is an asymptomatic BV patient. These results suggest that the RT-qPCR system is effective for accurate analysis of major vaginal commensals and diagnosis of several vaginal infections. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. β-Catenin transcriptional activity is minimal in canine osteosarcoma and its targeted inhibition results in minimal changes to cell line behaviour.

    Science.gov (United States)

    Piskun, Caroline M; Stein, Timothy J

    2016-06-01

    Canine osteosarcoma (OS) is an aggressive malignancy associated with poor outcomes. Therapeutic improvements are likely to develop from an improved understanding of signalling pathways contributing to OS development and progression. The Wnt signalling pathway is of interest for its role in osteoblast differentiation, its dysregulation in numerous cancer types, and the relative frequency of cytoplasmic accumulation of β-catenin in canine OS. This study aimed to determine the biological impact of inhibiting canonical Wnt signalling in canine OS, by utilizing either β-catenin siRNA or a dominant-negative T-cell factor (TCF) construct. There were no consistent, significant changes in cell line behaviour with either method compared to parental cell lines. Interestingly, β-catenin transcriptional activity was three-fold higher in normal canine primary osteoblasts compared to canine OS cell lines. These results suggest canonical Wnt signalling is minimally active in canine OS and its targeted inhibition is not a relevant therapeutic strategy. © 2013 John Wiley & Sons Ltd.

  2. microProtein Prediction Program (miP3) : A Software for Predicting microProteins and Their Target Transcription Factors

    NARCIS (Netherlands)

    de Klein, Niek; Magnani, Enrico; Banf, Michael; Rhee, Seung Yon

    2015-01-01

    An emerging concept in transcriptional regulation is that a class of truncated transcription factors (TFs), called microProteins (miPs), engages in protein-protein interactions with TF complexes and provides feedback controls. A handful of miP examples have been described in the literature but the

  3. MIR@NT@N: a framework integrating transcription factors, microRNAs and their targets to identify sub-network motifs in a meta-regulation network model

    Directory of Open Access Journals (Sweden)

    Wasserman Wyeth W

    2011-03-01

    Full Text Available Abstract Background To understand biological processes and diseases, it is crucial to unravel the concerted interplay of transcription factors (TFs, microRNAs (miRNAs and their targets within regulatory networks and fundamental sub-networks. An integrative computational resource generating a comprehensive view of these regulatory molecular interactions at a genome-wide scale would be of great interest to biologists, but is not available to date. Results To identify and analyze molecular interaction networks, we developed MIR@NT@N, an integrative approach based on a meta-regulation network model and a large-scale database. MIR@NT@N uses a graph-based approach to predict novel molecular actors across multiple regulatory processes (i.e. TFs acting on protein-coding or miRNA genes, or miRNAs acting on messenger RNAs. Exploiting these predictions, the user can generate networks and further analyze them to identify sub-networks, including motifs such as feedback and feedforward loops (FBL and FFL. In addition, networks can be built from lists of molecular actors with an a priori role in a given biological process to predict novel and unanticipated interactions. Analyses can be contextualized and filtered by integrating additional information such as microarray expression data. All results, including generated graphs, can be visualized, saved and exported into various formats. MIR@NT@N performances have been evaluated using published data and then applied to the regulatory program underlying epithelium to mesenchyme transition (EMT, an evolutionary-conserved process which is implicated in embryonic development and disease. Conclusions MIR@NT@N is an effective computational approach to identify novel molecular regulations and to predict gene regulatory networks and sub-networks including conserved motifs within a given biological context. Taking advantage of the M@IA environment, MIR@NT@N is a user-friendly web resource freely available at http

  4. Selective estrogen receptor modulators and betulinic acid act synergistically to target ERα and SP1 transcription factor dependent Pygopus expression in breast cancer.

    Science.gov (United States)

    Tzenov, Youlian R; Andrews, Phillip; Voisey, Kim; Gai, Luis; Carter, Beverley; Whelan, Kathryn; Popadiuk, Catherine; Kao, Kenneth R

    2016-06-01

    Estrogen and progesterone hormone receptor (ER and PR) expression in invasive breast cancer predicts response to hormone disruptive therapy. Pygopus2 (hPYGO2) encodes a chromatin remodelling protein important for breast cancer growth and cell cycle progression. The aims of this study were to determine the mechanism of expression of hPYGO2 in breast cancer and to examine how this expression is affected therapeutically. hPYGO2 and ER protein expression was examined in a breast tumour microarray by immunohistochemistry. hPYGO2 RNA and protein expression was examined in ER+ and ER- breast cancer cell lines in the presence of selective estrogen hormone receptor modulator drugs and the specificity protein-1 (SP1) inhibitor, betulinic acid (BA). The effects of these drugs on the ability for ER and SP1 to bind the hPYGO2 promoter and affect cell cycle progression were studied using chromatin immunoprecipitation assays. hPYGO2 was expressed in seven of eight lines and in nuclei of 98% of 65 breast tumours, including 3 Ductal carcinoma in situ and 62 invasive specimens representing ER-negative (22%) and ER-positive (78%) cases. Treatment with either 4-Hydroxytamoxifen (OHT) or fulvestrant reduced hPYGO2 mRNA 10-fold and protein 5-10-fold within 4 h. Promoter analysis indicated an ER/SP1 binding site at nt -225 to -531 of hPYGO2. SP1 RNA interference and BA reduced hPYGO2 protein and RNA expression by fivefold in both ER- and ER+ cells. Further attenuation was achieved by combining BA and 4-OHT resulting in eightfold reduction in cell growth. Our findings reveal a mechanistic link between hormone signalling and the growth transcriptional programme. The activation of its expression by ERα and/or SP1 suggests hPYGO2 as a theranostic target for hormone therapy responsive and refractory breast cancer. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  5. Reciprocal occupancy of BCL6 and STAT5 on Growth Hormone target genes: contrasting transcriptional outcomes and promoter-specific roles of p300 and HDAC3.

    Science.gov (United States)

    Lin, Grace; LaPensee, Christopher R; Qin, Zhaohui S; Schwartz, Jessica

    2014-09-01

    Expression of the Growth Hormone (GH)-stimulated gene Socs2 (Suppressor of Cytokine Signaling 2) is mediated by the transcription activator STAT5 (Signal Transducer and Activator of Transcription 5) and the transcription repressor BCL6 (B-Cell Lymphoma 6). ChIP-Sequencing identified Cish (Cytokine-Inducible SH2-containing protein) and Bcl6 as having similar patterns of reciprocal occupancy by BCL6 and STAT5 in response to GH, though GH stimulates Cish and inhibits Bcl6 expression. The co-activator p300 occupied Socs2, Cish and Bcl6 promoters, and enhanced STAT5-mediated activation of Socs2 and Cish. In contrast, on Bcl6, p300 functioned as a repressor and inhibited in conjunction with STAT5 or BCL6. The co-repressor HDAC3 (Histone deacetylase 3) inhibited the Socs2, Cish and Bcl6 promoters in the presence of STAT5. Thus transcriptional outcomes on GH-regulated genes occupied by BCL6 and STAT5 are determined in a promoter-specific fashion by co-regulatory proteins which mediate the distinction between activating and repressive transcription factors. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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

  7. Arx: a toolset for the efficient simulation and direct synthesis of high-performance signal processing algorithms

    NARCIS (Netherlands)

    Hofstra, K.L.; Gerez, Sabih H.

    2007-01-01

    This paper addresses the efficient implementation of highperformance signal-processing algorithms. In early stages of such designs many computation-intensive simulations may be necessary. This calls for hardware description formalisms targeted for efficient simulation (such as the programming

  8. Evolutionary dynamics of DNA-binding sites and direct target genes of a floral master regulatory transcription factor [ChIP-Seq

    NARCIS (Netherlands)

    Muiño, J.M.; Bruijn, de S.A.; Vingron, Martin; Angenent, G.C.; Kaufmann, K.

    2015-01-01

    Plant development is controlled by transcription factors (TFs) which form complex gene-regulatory networks. Genome-wide TF DNA-binding studies revealed that these TFs have several thousands of binding sites in the Arabidopsis genome, and may regulate the expression of many genes directly. Given the

  9. Evolutionary dynamics of DNA-binding sites and direct target genes of a floral master regulatory transcription factor [RNA-Seq

    NARCIS (Netherlands)

    Muiño, J.M.; Bruijn, de S.A.; Vingron, Martin; Angenent, G.C.; Kaufmann, Kerstin

    2015-01-01

    Plant development is controlled by transcription factors (TFs) which form complex gene-regulatory networks. Genome-wide TF DNA-binding studies revealed that these TFs have several thousands of binding sites in the Arabidopsis genome, and may regulate the expression of many genes directly. Given the

  10. miR156-Targeted SBP-Box Transcription Factors Interact with DWARF53 to Regulate TEOSINTE BRANCHED1 and BARREN STALK1 Expression in Bread Wheat.

    Science.gov (United States)

    Liu, Jie; Cheng, Xiliu; Liu, Pan; Sun, Jiaqiang

    2017-07-01

    Genetic and environmental factors affect bread wheat ( Triticum aestivum ) plant architecture, which determines grain yield. In this study, we demonstrate that miR156 controls bread wheat plant architecture. We show that overexpression of tae-miR156 in bread wheat cultivar Kenong199 leads to increased tiller number and severe defects in spikelet formation, probably due to the tae-miR156-mediated repression of a group of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE ( SPL ) genes. Furthermore, we found that the expression of two genes TEOSINTE BRANCHED1 ( TaTB1 ) and BARREN STALK1 ( TaBA1 ), whose orthologous genes in diverse plant species play conserved roles in regulating plant architecture, is markedly reduced in the tae-miR156-OE bread wheat plants. Significantly, we demonstrate that the strigolactone (SL) signaling repressor DWARF53 (TaD53), which physically associates with the transcriptional corepressor TOPLESS, can directly interact with the N-terminal domains of miR156-controlled TaSPL3/17. Most importantly, TaSPL3/17-mediated transcriptional activation of TaBA1 and TaTB1 can be largely repressed by TaD53 in the transient expression system. Our results reveal potential association between miR156-TaSPLs and SL signaling pathways during bread wheat tillering and spikelet development. © 2017 American Society of Plant Biologists. All Rights Reserved.

  11. Effects of exposure to Prestige-like heavy fuel oil and to perfluorooctane sulfonate on conventional biomarkers and target gene transcription in the thicklip grey mullet Chelon labrosus

    Energy Technology Data Exchange (ETDEWEB)

    Bilbao, E.; Raingeard, D.; Diaz de Cerio, O.; Ortiz-Zarragoitia, M.; Ruiz, P.; Izagirre, U.; Orbea, A.; Marigomez, I.; Cajaraville, M.P. [Laboratory of Cell Biology and Histology, Department of Zoology and Animal Cell Biology, School of Science and Technology, University of the Basque Country, E-48080 Bilbao, Basque Country (Spain); Cancio, I., E-mail: ibon.cancio@ehu.es [Laboratory of Cell Biology and Histology, Department of Zoology and Animal Cell Biology, School of Science and Technology, University of the Basque Country, E-48080 Bilbao, Basque Country (Spain)

    2010-07-01

    Thicklip grey mullets Chelon labrosus inhabit coastal and estuarine areas where they can be chronically exposed to commonly released pollutants such as polycyclic aromatic hydrocarbons (PAHs) and perfluorinated compounds. These pollutants can also originate from accidental spills, such as the Prestige oil spill in 2002, which resulted in the release of a heavy fuel oil that affected coastal ecosystems in the Bay of Biscay. Peroxisome proliferation (PP), induced biotransformation metabolism, immunosuppression and endocrine disruption are some of the possible biological effects caused by such chemicals. With the aim of studying the effects of organic toxic chemicals on such biological processes at the transcriptional and at the cell/tissue level, juvenile mullets were exposed to the typical mammalian peroxisome proliferator perfluorooctane sulfonate (PFOS), and to fresh (F) and weathered (WF) Prestige-like heavy fuel oil for 2 and 16 days. First, fragments of genes relevant to biotransformation, immune/inflammatory and endocrine disruption processes were cloned using degenerate primers. Fuel oil elicited a significant PP response as proved by the transcriptional upregulation of palmitoyl-CoA oxidase (aox1), peroxisome proliferator activated receptor {alpha} (ppar{alpha}) and retinoic X receptor, by the AOX1 activity induction and by the increased peroxisomal volume density. PFOS only elicited a significant induction of AOX1 activity at day 2 and of PPAR{alpha} mRNA expression at day 16. All treatments significantly increased catalase mRNA expression at day 16 in liver and at day 2 in gill. Cyp1a transcription (liver and gill) and EROD activity were induced in fuel oil treated organisms. In the case of phase II metabolism only hepatic glutathione S-transferase mRNA was overexpressed in mullets exposed to WF for 16 days. Functionally, this response was reflected in a significant accumulation of bile PAH metabolites. WF treated fish accumulated mainly high molecular

  12. Effects of exposure to Prestige-like heavy fuel oil and to perfluorooctane sulfonate on conventional biomarkers and target gene transcription in the thicklip grey mullet Chelon labrosus

    International Nuclear Information System (INIS)

    Bilbao, E.; Raingeard, D.; Diaz de Cerio, O.; Ortiz-Zarragoitia, M.; Ruiz, P.; Izagirre, U.; Orbea, A.; Marigomez, I.; Cajaraville, M.P.; Cancio, I.

    2010-01-01

    Thicklip grey mullets Chelon labrosus inhabit coastal and estuarine areas where they can be chronically exposed to commonly released pollutants such as polycyclic aromatic hydrocarbons (PAHs) and perfluorinated compounds. These pollutants can also originate from accidental spills, such as the Prestige oil spill in 2002, which resulted in the release of a heavy fuel oil that affected coastal ecosystems in the Bay of Biscay. Peroxisome proliferation (PP), induced biotransformation metabolism, immunosuppression and endocrine disruption are some of the possible biological effects caused by such chemicals. With the aim of studying the effects of organic toxic chemicals on such biological processes at the transcriptional and at the cell/tissue level, juvenile mullets were exposed to the typical mammalian peroxisome proliferator perfluorooctane sulfonate (PFOS), and to fresh (F) and weathered (WF) Prestige-like heavy fuel oil for 2 and 16 days. First, fragments of genes relevant to biotransformation, immune/inflammatory and endocrine disruption processes were cloned using degenerate primers. Fuel oil elicited a significant PP response as proved by the transcriptional upregulation of palmitoyl-CoA oxidase (aox1), peroxisome proliferator activated receptor α (pparα) and retinoic X receptor, by the AOX1 activity induction and by the increased peroxisomal volume density. PFOS only elicited a significant induction of AOX1 activity at day 2 and of PPARα mRNA expression at day 16. All treatments significantly increased catalase mRNA expression at day 16 in liver and at day 2 in gill. Cyp1a transcription (liver and gill) and EROD activity were induced in fuel oil treated organisms. In the case of phase II metabolism only hepatic glutathione S-transferase mRNA was overexpressed in mullets exposed to WF for 16 days. Functionally, this response was reflected in a significant accumulation of bile PAH metabolites. WF treated fish accumulated mainly high molecular weight metabolites

  13. Effects of exposure to Prestige-like heavy fuel oil and to perfluorooctane sulfonate on conventional biomarkers and target gene transcription in the thicklip grey mullet Chelon labrosus.

    Science.gov (United States)

    Bilbao, E; Raingeard, D; de Cerio, O Diaz; Ortiz-Zarragoitia, M; Ruiz, P; Izagirre, U; Orbea, A; Marigómez, I; Cajaraville, M P; Cancio, I

    2010-07-01

    Thicklip grey mullets Chelon labrosus inhabit coastal and estuarine areas where they can be chronically exposed to commonly released pollutants such as polycyclic aromatic hydrocarbons (PAHs) and perfluorinated compounds. These pollutants can also originate from accidental spills, such as the Prestige oil spill in 2002, which resulted in the release of a heavy fuel oil that affected coastal ecosystems in the Bay of Biscay. Peroxisome proliferation (PP), induced biotransformation metabolism, immunosuppression and endocrine disruption are some of the possible biological effects caused by such chemicals. With the aim of studying the effects of organic toxic chemicals on such biological processes at the transcriptional and at the cell/tissue level, juvenile mullets were exposed to the typical mammalian peroxisome proliferator perfluorooctane sulfonate (PFOS), and to fresh (F) and weathered (WF) Prestige-like heavy fuel oil for 2 and 16 days. First, fragments of genes relevant to biotransformation, immune/inflammatory and endocrine disruption processes were cloned using degenerate primers. Fuel oil elicited a significant PP response as proved by the transcriptional upregulation of palmitoyl-CoA oxidase (aox1), peroxisome proliferator activated receptor alpha (pparalpha) and retinoic X receptor, by the AOX1 activity induction and by the increased peroxisomal volume density. PFOS only elicited a significant induction of AOX1 activity at day 2 and of PPARalpha mRNA expression at day 16. All treatments significantly increased catalase mRNA expression at day 16 in liver and at day 2 in gill. Cyp1a transcription (liver and gill) and EROD activity were induced in fuel oil treated organisms. In the case of phase II metabolism only hepatic glutathione S-transferase mRNA was overexpressed in mullets exposed to WF for 16 days. Functionally, this response was reflected in a significant accumulation of bile PAH metabolites. WF treated fish accumulated mainly high molecular weight

  14. RNA-guided transcriptional regulation

    Science.gov (United States)

    Church, George M.; Mali, Prashant G.; Esvelt, Kevin M.

    2016-02-23

    Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

  15. Small interfering RNA targeting HIF-1{alpha} reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Staab, Adrian [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Paul Scherrer Institute (PSI), Villigen (Switzerland); Fleischer, Markus [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Wuerzburg Univ. (Germany). Medical Clinic II; Loeffler, Juergen; Einsele, Herrmann [Wuerzburg Univ. (Germany). Medical Clinic II; Said, Harun M.; Katzer, Astrid; Flentje, Michael [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Plathow, Christian [Freiburg Univ. (Germany). Dept. of Nuclear Medicine; Vordermark, Dirk [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Halle-Wittenberg Univ. (Germany). Dept. of Radiation Oncology

    2011-04-15

    Background: Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. Material and Methods: HIF-1{alpha} expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1{alpha} siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1{alpha}. HIF-1{alpha} protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O{sub 2} (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1{alpha}-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER') was calculated as the ratio of the doses to achieve the same survival at 0.1% O{sub 2} as at ambient oxygen tensions. OER' was obtained at cell survival levels of 50%, 37%, and 10%. Results: HIF-1{alpha}-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1{alpha}-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. Conclusion: Inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro. (orig.)

  16. Small interfering RNA targeting HIF-1α reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

    International Nuclear Information System (INIS)

    Staab, Adrian; Fleischer, Markus; Wuerzburg Univ.; Loeffler, Juergen; Einsele, Herrmann; Said, Harun M.; Katzer, Astrid; Flentje, Michael; Plathow, Christian; Vordermark, Dirk; Halle-Wittenberg Univ.

    2011-01-01

    Background: Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1α (HIF-1α) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. Material and Methods: HIF-1α expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1α siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1α. HIF-1α protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O 2 (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1α-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER') was calculated as the ratio of the doses to achieve the same survival at 0.1% O 2 as at ambient oxygen tensions. OER' was obtained at cell survival levels of 50%, 37%, and 10%. Results: HIF-1α-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1α-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1α-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. Conclusion: Inhibition of HIF-1 activation by using HIF-1α-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro. (orig.)

  17. [Results transferability on RXL, ARX, X-Pand, BN2 (Dade Behring) and modular DP (Roche Diagnostics) analysers: application to component assays of fibrotest and Actitest].

    Science.gov (United States)

    Imbert-Bismut, F; Messous, D; Raoult, A; Poynard, T; Bertrand, J J; Marie, P A; Louis, V; Audy, C; Thouy, J M; Hainque, B; Piton, A

    2005-01-01

    The follow up of patients with chronic liver diseases and the data from multicentric clinical studies are affected by the variability of assay results for the same parameter between the different laboratories. Today, the main objective in clinical chemistry throughout the world is to harmonise the assay results between the laboratories after the confirmation of their traceability, in relation to defined reference systems. In this context, the purpose of our study was to verify the homogeneity of haptoglobin, apolipoprotein A1, total bilirubin, GGT activity, ALAT activity results, which are combined in Fibrotest and Actitest, between Dimension Analysers RXL, ARX and X-PAND (Dade Behring Society). Moreover, we verified the transferability of Fibrotest and Actitest results between the RXL, and either the BN2 (haptoglobin and apolipoprotein A1) or the Modular DP (total bilirubin, GGT and ALAT activity concentrations). The serum samples from 150 hospitalised patients were analysed on the different analysers. Specific protein assays were calibrated using solutions standardised against reference material on Dimension and BN2 analysers. Total bilirubin assays were performed by a diazoreaction on Dimension and Modular DP analysers. The GGT and ALAT activity measurements on the Dimension analysers were performed in accordance with the reference methods defined by the International Federation of Clinical Chemisty and Laboratory Medicine (IFCC). On the Modular, enzyme activity measurements were performed according to the Szasz method (L-gamma- glutamyl-4-nitroanilide as substrate) modified by Persijn and van der Slik (L-gamma- glutamyl-3-carboxy- 4-nitroanilide as substrat) for GGT and according to the IFCC specifications for ALAT. The methods of enzymatic activity measurement were calibrated on the Modular only. Liver fibrosis and necroinflammatory activity indices were determined using calculation algorithms, after having adjusted each component's result of Fibrotest and

  18. Method to determine transcriptional regulation pathways in organisms

    Science.gov (United States)

    Gardner, Timothy S.; Collins, James J.; Hayete, Boris; Faith, Jeremiah

    2012-11-06

    The invention relates to computer-implemented methods and systems for identifying regulatory relationships between expressed regulating polypeptides and targets of the regulatory activities of such regulating polypeptides. More specifically, the invention provides a new method for identifying regulatory dependencies between biochemical species in a cell. In particular embodiments, provided are computer-implemented methods for identifying a regulatory interaction between a transcription factor and a gene target of the transcription factor, or between a transcription factor and a set of gene targets of the transcription factor. Further provided are genome-scale methods for predicting regulatory interactions between a set of transcription factors and a corresponding set of transcriptional target substrates thereof.

  19. The MSX1 homeobox transcription factor is a downstream target of PHOX2B and activates the Delta-Notch pathway in neuroblastoma

    International Nuclear Information System (INIS)

    Revet, Ingrid; Huizenga, Gerda; Chan, Alvin; Koster, Jan; Volckmann, Richard; Sluis, Peter van; Ora, Ingrid; Versteeg, Rogier; Geerts, Dirk

    2008-01-01

    Neuroblastoma is an embryonal tumour of the peripheral sympathetic nervous system (SNS). One of the master regulator genes for peripheral SNS differentiation, the homeobox transcription factor PHOX2B, is mutated in familiar and sporadic neuroblastomas. Here we report that inducible expression of PHOX2B in the neuroblastoma cell line SJNB-8 down-regulates MSX1, a homeobox gene important for embryonic neural crest development. Inducible expression of MSX1 in SJNB-8 caused inhibition of both cell proliferation and colony formation in soft agar. Affymetrix micro-array and Northern blot analysis demonstrated that MSX1 strongly up-regulated the Delta-Notch pathway genes DLK1, NOTCH3, and HEY1. In addition, the proneural gene NEUROD1 was down-regulated. Western blot analysis showed that MSX1 induction caused cleavage of the NOTCH3 protein to its activated form, further confirming activation of the Delta-Notch pathway. These experiments describe for the first time regulation of the Delta-Notch pathway by MSX1, and connect these genes to the PHOX2B oncogene, indicative of a role in neuroblastoma biology. Affymetrix micro-array analysis of a neuroblastic tumour series consisting of neuroblastomas and the more benign ganglioneuromas showed that MSX1, NOTCH3 and HEY1 are more highly expressed in ganglioneuromas. This suggests a block in differentiation of these tumours at distinct developmental stages or lineages

  20. Transcription factor HIF1A: downstream targets, associated pathways, polymorphic hypoxia response element (HRE) sites, and initiative for standardization of reporting in scientific literature.

    Science.gov (United States)

    Slemc, Lucija; Kunej, Tanja

    2016-11-01

    Hypoxia-inducible factor-1α (HIF-1α) has crucial role in adapting cells to hypoxia through expression regulation of many genes. Identification of HIF-1α target genes (HIF-1α-TGs) is important for understanding the adapting mechanism. The aim of the present study was to collect known HIF-1α-TGs and identify their associated pathways. Targets and associated genomics data were retrieved using PubMed, WoS ( http://apps.webofknowledge.com/ ), HGNC ( http://www.genenames.org/ ), NCBI ( http://www.ncbi.nlm.nih.gov/ ), Ensemblv.84 ( http://www.ensembl.org/index.html ), DAVID Bioinformatics Resources ( https://david.ncifcrf.gov /), and Disease Ontology database ( http://disease-ontology.org/ ). From 51 papers, we collected 98 HIF-1α TGs found to be associated with 20 pathways, including metabolism of carbohydrates and pathways in cancer. Reanalysis of genomic coordinates of published HREs (hypoxia response elements) revealed six polymorphisms within HRE sites (HRE-SNPs): ABCG2, ACE, CA9, and CP. Due to large heterogeneity of results presentation in scientific literature, we also propose a first step towards reporting standardization of HIF-1α-target interactions consisting of ten relevant data types. Suggested minimal checklist for reporting will enable faster development of a complete catalog of HIF-1α-TGs, data sharing, bioinformatics analyses, and setting novel more targeted hypotheses. The proposed format for data standardization is not yet complete but presents a baseline for further optimization of the protocol with additional details, for example, regarding the experimental validation.

  1. Identification of the feedforward component in manual control with predictable target signals.

    Science.gov (United States)

    Drop, Frank M; Pool, Daan M; Damveld, Herman J; van Paassen, Marinus M; Mulder, Max

    2013-12-01

    In the manual control of a dynamic system, the human controller (HC) often follows a visible and predictable reference path. Compared with a purely feedback control strategy, performance can be improved by making use of this knowledge of the reference. The operator could effectively introduce feedforward control in conjunction with a feedback path to compensate for errors, as hypothesized in literature. However, feedforward behavior has never been identified from experimental data, nor have the hypothesized models been validated. This paper investigates human control behavior in pursuit tracking of a predictable reference signal while being perturbed by a quasi-random multisine disturbance signal. An experiment was done in which the relative strength of the target and disturbance signals were systematically varied. The anticipated changes in control behavior were studied by means of an ARX model analysis and by fitting three parametric HC models: two different feedback models and a combined feedforward and feedback model. The ARX analysis shows that the experiment participants employed control action on both the error and the target signal. The control action on the target was similar to the inverse of the system dynamics. Model fits show that this behavior can be modeled best by the combined feedforward and feedback model.

  2. Analysis of phage Mu DNA transposition by whole-genome Escherichia coli tiling arrays reveals a complex relationship to distribution of target selection protein B, transcription and chromosome architectural elements.

    Science.gov (United States)

    Ge, Jun; Lou, Zheng; Cui, Hong; Shang, Lei; Harshey, Rasika M

    2011-09-01

    Of all known transposable elements, phage Mu exhibits the highest transposition efficiency and the lowest target specificity. In vitro, MuB protein is responsible for target choice. In this work, we provide a comprehensive assessment of the genome-wide distribution of MuB and its relationship to Mu target selection using high-resolution Escherichia coli tiling DNA arrays. We have also assessed how MuB binding and Mu transposition are influenced by chromosome-organizing elements such as AT-rich DNA signatures, or the binding of the nucleoid-associated protein Fis, or processes such as transcription. The results confirm and extend previous biochemical and lower resolution in vivo data. Despite the generally random nature of Mu transposition and MuB binding, there were hot and cold insertion sites and MuB binding sites in the genome, and differences between the hottest and coldest sites were large. The new data also suggest that MuB distribution and subsequent Mu integration is responsive to DNA sequences that contribute to the structural organization of the chromosome.

  3. MicroRNA-20b-5p inhibits platelet-derived growth factor-induced proliferation of human fetal airway smooth muscle cells by targeting signal transducer and activator of transcription 3.

    Science.gov (United States)

    Tang, Jin; Luo, Lingying

    2018-06-01

    Pediatric asthma is still a health threat to the pediatric population in recent years. The airway remodeling induced by abnormal airway smooth muscle (ASM) cell proliferation is an important cause of asthma. MicroRNAs (miRNAs) are important regulators of ASM cell proliferation. Numerous studies have reported that miR-20b-5p is a critical regulator for cell proliferation. However, whether miR-20b-5p is involved in regulating ASM cell proliferation remains unknown. In this study, we aimed to investigate the potential role of miR-20b-5p in regulating the proliferation of fetal ASM cell induced by platelet-derived growth factor (PDGF). Here, we showed that miR-20b-5p was significantly decreased in fetal ASM cells treated with PDGF. Biological experiments showed that the overexpression of miR-20b-5p inhibited the proliferation while miR-20b-5p inhibition markedly promoted the proliferation of fetal ASM cells. Bioinformatics analysis and luciferase reporter assay showed that miR-20b-5p directly targeted the 3'-UTR of signal transducer and activator of transcription 3 (STAT3). Further data showed that miR-20b-5p negatively regulated the expression of STAT3 in fetal ASM cells. Moreover, miR-20b-5p regulates the transcriptional activity of STAT3 in fetal ASM cells. Overexpression of STAT3 reversed the inhibitory effect of miR-20b-5p overexpression on fetal ASM cell proliferation while the knockdown of STAT3 abrogated the promoted effect of miR-20b-5p inhibition on fetal ASM cell proliferation. Overall, our results show that miR-20b-5p impedes PDGF-induced proliferation of fetal ASM cells through targeting STAT3. Our study suggests that miR-20b-5p may play an important role in airway remodeling during asthma and suggests that miR-20b-5p may serve as a potential therapeutic target for pediatric asthma. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  4. TaHsfA6f is a transcriptional activator that regulates a suite of heat stress protection genes in wheat (Triticum aestivum L.) including previously unknown Hsf targets.

    Science.gov (United States)

    Xue, Gang-Ping; Drenth, Janneke; McIntyre, C Lynne

    2015-02-01

    Heat stress is a significant environmental factor adversely affecting crop yield. Crop adaptation to high-temperature environments requires transcriptional reprogramming of a suite of genes involved in heat stress protection. This study investigated the role of TaHsfA6f, a member of the A6 subclass of heat shock transcription factors, in the regulation of heat stress protection genes in Triticum aestivum (bread wheat), a poorly understood phenomenon in this crop species. Expression analysis showed that TaHsfA6f was expressed constitutively in green organs but was markedly up-regulated during heat stress. Overexpression of TaHsfA6f in transgenic wheat using a drought-inducible promoter resulted in up-regulation of heat shock proteins (HSPs) and a number of other heat stress protection genes that included some previously unknown Hsf target genes such as Golgi anti-apoptotic protein (GAAP) and the large isoform of Rubisco activase. Transgenic wheat plants overexpressing TaHsfA6f showed improved thermotolerance. Transactivation assays showed that TaHsfA6f activated the expression of reporter genes driven by the promoters of several HSP genes (TaHSP16.8, TaHSP17, TaHSP17.3, and TaHSP90.1-A1) as well as TaGAAP and TaRof1 (a co-chaperone) under non-stress conditions. DNA binding analysis revealed the presence of high-affinity TaHsfA6f-binding heat shock element-like motifs in the promoters of these six genes. Promoter truncation and mutagenesis analyses identified TaHsfA6f-binding elements that were responsible for transactivation of TaHSP90.1-A1 and TaGAAP by TaHsfA6f. These data suggest that TaHsfA6f is a transcriptional activator that directly regulates TaHSP, TaGAAP, and TaRof1 genes in wheat and its gene regulatory network has a positive impact on thermotolerance. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  5. An ex vivo evaluation of the efficacy of andrographolide in modulating differential expression of transcription factors and target genes in periodontal cells and its potential role in treating periodontal diseases.

    Science.gov (United States)

    Ambili R; Janam, Prasanthila; Saneesh Babu, P S; Prasad, Manu; Vinod, D; Anil Kumar, P R; Kumary, T V; Asha Nair, S; Radhakrishna Pillai, M

    2017-01-20

    Andrographolide is a herbal extract traditionally used in South Asian countries for treating inflammatory diseases. To evaluate the efficacy of andrographolide in management of periodontal disease which is a highly prevalent oral disease. Periodontal ligament fibroblasts (PDLF) were cultured from healthy and diseased periodontium using explant culture methods. The safe dose of AG was determined using MTT assay. LPS (lipopolysaccharide) of the most important periodontopathogen, P gingivalis was used to activate NF-κB and STAT3 in PDLF. The efficacy of AG in inhibiting NF-κB and STAT3 was analyzed using immunofluorescence. Down regulation of expression of target genes of these transcription factors related to inflammation and bone resorption were analyzed using real time PCR. AG up to the concentration of 25μM was found to be safe as determined by MTT assay. Statistically significant activation of NF-κB and STAT3 in cultured PDLF was observed in diseased group compared to healthy controls before and after LPS challenge. 5μM AG pretreatment significantly inhibited activation of NF-κB and STAT3 and down regulated expression of inflammatory and bone resorptive genes in cultured PDLF. The findings of the present study propose the adjunctive use of a novel herbal drug andrographolide as a promising host modulation agent for periodontal therapy by inhibiting NF-κB and STAT3 activation and inhibition of inflammation and bone resorption related genes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. Transcriptional mapping of rabies virus in vivo

    International Nuclear Information System (INIS)

    Flamand, A.; Delagneau, J.F.

    1978-01-01

    Synthesis of the proteins of rabies virus was studied in hamster cell infected with uv-irradiated virus. The uv target size of genes L, N, M 1 , and M 2 was measured during primary transcription. Except for N, the target size of the remaining genes was considerably larger than that of their physical sizes. The data fit the hypothesis that four genes occupy a single transcriptional unit and that transcription of rabies virus proceeds in the order N, M 1 , M 2 , and L

  7. 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 coding...... regions, the map of transcripts is very complex due to small transcripts from the flanking ends of the transcription unit, the use of multiple start and stop sites for the main transcript, production of multiple functional RNA molecules from the same primary transcript, and RNA molecules made...... by independent transcription from within the unit. In genomic regions separating those that encode proteins or highly abundant RNA molecules with known function, transcripts are generally of low abundance and short-lived. In most of these cases, it is unclear to what extent a function is related to transcription...

  8. Successive composition of two laser channels upon excitation of He-Ar-Xe (2.03 μm) and Ar-Xe (1.73 μm) mixtures by uranium fission fragments

    International Nuclear Information System (INIS)

    Pikulev, A A; Tsvetkov, V M; Sosnin, P V; Sinyanskii, A A

    2009-01-01

    The operation efficiency of the scheme with successive composition of two laser channels upon excitation of the active medium by uranium-235 fission fragments is studied experimentally and numerically. For the He:Ar:Xe = 380:380:1 mixture (at a pressure of 1 atm and the lasing wavelength λ = 2.03 μm) the maximum lasing power of a double channel (1 kW) is almost twice that of a single channel (540 W). Calculations show that in the case of ideal composition (without losses on mirrors) the lasing power of the double channel can be increased to 1.2 kW. For the Ar:Xe = 380:1 mixture (the pressure is 0.5 atm, λ = 1.73 μm) the maximum lasing power of the double channel (620 W) is slightly above that of the single channel (520 W), which is caused by the losses on aluminum mirrors employed for channel doubling and by a negative effect of optical inhomogeneities. In the case of ideal composition, the lasing power can be increased to 830 W. (lasers)

  9. Transcription regulation by the Mediator complex.

    Science.gov (United States)

    Soutourina, Julie

    2018-04-01

    Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.

  10. Numeric definition of the clinical performance of the nested reverse transcription-PCR for detection of hematogenous epithelial cells and correction for specific mRNA of non-target cell origin as evaluated for prostate cancer cells

    NARCIS (Netherlands)

    Schamhart, Denis; Swinnen, Johannes; Kurth, Karl-Heinz; Westerhof, Alex; Kusters, Ron; Borchers, Holger; Sternberg, Cora

    2003-01-01

    Background: Inappropriate quality management,of reverse transcription-PCR (RT-PCR) assays for the detection of blood-borne prostate cancer (PCa) cells hampers clinical conclusions. Improvement of the RT-PCR-methodology for prostate-specific, antigen (PSA) mRNA should focus on an appropriate numeric.

  11. Transcriptional regulation by competing transcription factor modules.

    Directory of Open Access Journals (Sweden)

    Rutger Hermsen

    2006-12-01

    Full Text Available Gene regulatory networks lie at the heart of cellular computation. In these networks, intracellular and extracellular signals are integrated by transcription factors, which control the expression of transcription units by binding to cis-regulatory regions on the DNA. The designs of both eukaryotic and prokaryotic cis-regulatory regions are usually highly complex. They frequently consist of both repetitive and overlapping transcription factor binding sites. To unravel the design principles of these promoter architectures, we have designed in silico prokaryotic transcriptional logic gates with predefined input-output relations using an evolutionary algorithm. The resulting cis-regulatory designs are often composed of modules that consist of tandem arrays of binding sites to which the transcription factors bind cooperatively. Moreover, these modules often overlap with each other, leading to competition between them. Our analysis thus identifies a new signal integration motif that is based upon the interplay between intramodular cooperativity and intermodular competition. We show that this signal integration mechanism drastically enhances the capacity of cis-regulatory domains to integrate signals. Our results provide a possible explanation for the complexity of promoter architectures and could be used for the rational design of synthetic gene circuits.

  12. Fatty Acid–Regulated Transcription Factors in the Liver

    Science.gov (United States)

    Jump, Donald B.; Tripathy, Sasmita; Depner, Christopher M.

    2014-01-01

    Fatty acid regulation of hepatic gene transcription was first reported in the early 1990s. Several transcription factors have been identified as targets of fatty acid regulation. This regulation is achieved by direct fatty acid binding to the transcription factor or by indirect mechanisms where fatty acids regulate signaling pathways controlling the expression of transcription factors or the phosphorylation, ubiquitination, or proteolytic cleavage of the transcription factor. Although dietary fatty acids are well-established regulators of hepatic transcription factors, emerging evidence indicates that endogenously generated fatty acids are equally important in controlling transcription factors in the context of glucose and lipid homeostasis. Our first goal in this review is to provide an up-to-date examination of the molecular and metabolic bases of fatty acid regulation of key transcription factors controlling hepatic metabolism. Our second goal is to link these mechanisms to nonalcoholic fatty liver disease (NAFLD), a growing health concern in the obese population. PMID:23528177

  13. WRKY transcription factors

    Science.gov (United States)

    Bakshi, Madhunita; Oelmüller, Ralf

    2014-01-01

    WRKY transcription factors are one of the largest families of transcriptional regulators found exclusively in plants. They have diverse biological functions in plant disease resistance, abiotic stress responses, nutrient deprivation, senescence, seed and trichome development, embryogenesis, as well as additional developmental and hormone-controlled processes. WRKYs can act as transcriptional activators or repressors, in various homo- and heterodimer combinations. Here we review recent progress on the function of WRKY transcription factors in Arabidopsis and other plant species such as rice, potato, and parsley, with a special focus on abiotic, developmental, and hormone-regulated processes. PMID:24492469

  14. Expression of the potential therapeutic target CXXC5 in primary acute myeloid leukemia cells - high expression is associated with adverse prognosis as well as altered intracellular signaling and transcriptional regulation.

    Science.gov (United States)

    Bruserud, Øystein; Reikvam, Håkon; Fredly, Hanne; Skavland, Jørn; Hagen, Karen-Marie; van Hoang, Tuyen Thy; Brenner, Annette K; Kadi, Amir; Astori, Audrey; Gjertsen, Bjørn Tore; Pendino, Frederic

    2015-02-20

    The CXXC5 gene encodes a transcriptional activator with a zinc-finger domain, and high expression in human acute myeloid leukemia (AML) cells is associated with adverse prognosis. We now characterized the biological context of CXXC5 expression in primary human AML cells. The global gene expression profile of AML cells derived from 48 consecutive patients was analyzed; cells with high and low CXXC5 expression then showed major differences with regard to extracellular communication and intracellular signaling. We observed significant differences in the phosphorylation status of several intracellular signaling mediators (CREB, PDK1, SRC, STAT1, p38, STAT3, rpS6) that are important for PI3K-Akt-mTOR signaling and/or transcriptional regulation. High CXXC5 expression was also associated with high mRNA expression of several stem cell-associated transcriptional regulators, the strongest associations being with WT1, GATA2, RUNX1, LYL1, DNMT3, SPI1, and MYB. Finally, CXXC5 knockdown in human AML cell lines caused significantly increased expression of the potential tumor suppressor gene TSC22 and genes encoding the growth factor receptor KIT, the cytokine Angiopoietin 1 and the selenium-containing glycoprotein Selenoprotein P. Thus, high CXXC5 expression seems to affect several steps in human leukemogenesis, including intracellular events as well as extracellular communication.

  15. Colon cancer associated transcripts in human cancers.

    Science.gov (United States)

    Chen, Yincong; Xie, Haibiao; Gao, Qunjun; Zhan, Hengji; Xiao, Huizhong; Zou, Yifan; Zhang, Fuyou; Liu, Yuchen; Li, Jianfa

    2017-10-01

    Long non-coding RNAs serve as important regulators in complicated cellular activities, including cell differentiation, proliferation and death. Dysregulation of long non-coding RNAs occurs in the formation and progression of cancers. The family of colon cancer associated transcripts, long non-coding RNAs colon cancer associated transcript-1 and colon cancer associated transcript-2 are known as oncogenes involved in various cancers. Colon cancer associated transcript-1 is a novel lncRNA located in 8q24.2, and colon cancer associated transcript-2 maps to the 8q24.21 region encompassing rs6983267. Colon cancer associated transcripts have close associations with clinical characteristics, such as lymph node metastasis, high TNM stage and short overall survival. Knockdown of them can reverse the malignant phenotypes of cancer cells, including proliferation, migration, invasion and apoptosis. Moreover, they can increase the expression level of c-MYC and oncogenic microRNAs via activating a series of complex mechanisms. In brief, the family of colon cancer associated transcripts may serve as potential biomarkers or therapeutic targets for human cancers. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  16. Corticosteroid receptors adopt distinct cyclical transcriptional signatures.

    Science.gov (United States)

    Le Billan, Florian; Amazit, Larbi; Bleakley, Kevin; Xue, Qiong-Yao; Pussard, Eric; Lhadj, Christophe; Kolkhof, Peter; Viengchareun, Say; Fagart, Jérôme; Lombès, Marc

    2018-05-07

    Mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) are two closely related hormone-activated transcription factors that regulate major pathophysiologic functions. High homology between these receptors accounts for the crossbinding of their corresponding ligands, MR being activated by both aldosterone and cortisol and GR essentially activated by cortisol. Their coexpression and ability to bind similar DNA motifs highlight the need to investigate their respective contributions to overall corticosteroid signaling. Here, we decipher the transcriptional regulatory mechanisms that underlie selective effects of MRs and GRs on shared genomic targets in a human renal cellular model. Kinetic, serial, and sequential chromatin immunoprecipitation approaches were performed on the period circadian protein 1 ( PER1) target gene, providing evidence that both receptors dynamically and cyclically interact at the same target promoter in a specific and distinct transcriptional signature. During this process, both receptors regulate PER1 gene by binding as homo- or heterodimers to the same promoter region. Our results suggest a novel level of MR-GR target gene regulation, which should be considered for a better and integrated understanding of corticosteroid-related pathophysiology.-Le Billan, F., Amazit, L., Bleakley, K., Xue, Q.-Y., Pussard, E., Lhadj, C., Kolkhof, P., Viengchareun, S., Fagart, J., Lombès, M. Corticosteroid receptors adopt distinct cyclical transcriptional signatures.

  17. TALE-mediated modulation of transcriptional enhancers in vivo.

    Science.gov (United States)

    Crocker, Justin; Stern, David L

    2013-08-01

    We tested whether transcription activator-like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.

  18. mRNA Transcript Diversity Creates New Opportunities for Pharmacological Intervention

    OpenAIRE

    Barrie, Elizabeth S.; Smith, Ryan M.; Sanford, Jonathan C.; Sadee, Wolfgang

    2012-01-01

    Most protein coding genes generate multiple RNA transcripts through alternative splicing, variable 3′ and 5′UTRs, and RNA editing. Although drug design typically targets the main transcript, alternative transcripts can have profound physiological effects, encoding proteins with distinct functions or regulatory properties. Formation of these alternative transcripts is tissue-selective and context-dependent, creating opportunities for more effective and targeted therapies with reduced adverse e...

  19. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors

    KAUST Repository

    Piatek, Agnieszka Anna

    2014-11-14

    Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3

  20. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors

    KAUST Repository

    Piatek, Agnieszka Anna; Ali, Zahir; Baazim, Hatoon; Li, Lixin; Abulfaraj, Aala A.; Alshareef, Sahar; Aouida, Mustapha; Mahfouz, Magdy M.

    2014-01-01

    Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3

  1. The transcriptional activator GAL4-VP16 regulates the intra ...

    Indian Academy of Sciences (India)

    Activator also reduced the TBP dimer levels both in vitro and in vivo, suggesting the dimer may be a direct target of transcriptional activators. The transcriptional activator facilitated the dimer to monomer transition and activated monomers further to help TBP bind even the weaker TATA boxes stably. The overall stimulatory ...

  2. The putative Agrobacterium transcriptional activator-like virulence protein VirD5 may target T-complex to prevent the degradation of coat proteins in the plant cell nucleus.

    Science.gov (United States)

    Wang, Yafei; Peng, Wei; Zhou, Xu; Huang, Fei; Shao, Lingyun; Luo, Meizhong

    2014-09-01

    Agrobacterium exports at least five virulence proteins (VirE2, VirE3, VirF, VirD2, VirD5) into host cells and hijacks some host plant factors to facilitate its transformation process. Random DNA binding selection assays (RDSAs), electrophoretic mobility shift assays (EMSAs) and yeast one-hybrid systems were used to identify protein-bound DNA elements. Bimolecular fluorescence complementation, glutathione S-transferase pull-down and yeast two-hybrid assays were used to detect protein interactions. Protoplast transformation, coprecipitation, competitive binding and cell-free degradation assays were used to analyze the relationships among proteins. We found that Agrobacterium VirD5 exhibits transcriptional activation activity in yeast, is located in the plant cell nucleus, and forms homodimers. A specific VirD5-bound DNA element designated D5RE (VirD5 response element) was identified. VirD5 interacted directly with Arabidopsis VirE2 Interacting Protein 1 (AtVIP1). However, the ternary complex of VirD5-AtVIP1-VirE2 could be detected, whereas that of VirD5-AtVIP1-VBF (AtVIP1 Binding F-box protein) could not. We demonstrated that VirD5 competes with VBF for binding to AtVIP1 and stabilizes AtVIP1 and VirE2 in the cell-free degradation system. Our results indicated that VirD5 may act as both a transcriptional activator-like effector to regulate host gene expression and a protector preventing the coat proteins of the T-complex from being quickly degraded by the host's ubiquitin proteasome system (UPS). © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  3. The Transcription Factor Encyclopedia

    DEFF Research Database (Denmark)

    Yusuf, Dimas; Butland, Stefanie L; Swanson, Magdalena I

    2012-01-01

    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......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...

  4. Characterization of human mesothelin transcripts in ovarian and pancreatic cancer

    International Nuclear Information System (INIS)

    Muminova, Zhanat E; Strong, Theresa V; Shaw, Denise R

    2004-01-01

    Mesothelin is an attractive target for cancer immunotherapy due to its restricted expression in normal tissues and high level expression in several tumor types including ovarian and pancreatic adenocarcinomas. Three mesothelin transcript variants have been reported, but their relative expression in normal tissues and tumors has been poorly characterized. The goal of the present study was to clarify which mesothelin transcript variants are commonly expressed in human tumors. Human genomic and EST nucleotide sequences in the public databases were used to evaluate sequences reported for the three mesothelin transcript variants in silico. Subsequently, RNA samples from normal ovary, ovarian and pancreatic carcinoma cell lines, and primary ovarian tumors were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing to directly identify expressed transcripts. In silico comparisons of genomic DNA sequences with available EST sequences supported expression of mesothelin transcript variants 1 and 3, but there were no sequence matches for transcript variant 2. Newly-derived nucleotide sequences of RT-PCR products from tissues and cell lines corresponded to mesothelin transcript variant 1. Mesothelin transcript variant 2 was not detected. Transcript variant 3 was observed as a small percentage of total mesothelin amplification products from all studied cell lines and tissues. Fractionation of nuclear and cytoplasmic RNA indicated that variant 3 was present primarily in the nuclear fraction. Thus, mesothelin transcript variant 3 may represent incompletely processed hnRNA. Mesothelin transcript variant 1 represents the predominant mature mRNA species expressed by both normal and tumor cells. This conclusion should be important for future development of cancer immunotherapies, diagnostic tests, and gene microarray studies targeting mesothelin

  5. TPL2 (Therapeutic Targeting Tumor Progression Locus-2)/ATF4 (Activating Transcription Factor-4)/SDF1α (Chemokine Stromal Cell-Derived Factor-α) Axis Suppresses Diabetic Retinopathy.

    Science.gov (United States)

    Lai, De-Wei; Lin, Keng-Hung; Sheu, Wayne Huey-Herng; Lee, Maw-Rong; Chen, Chung-Yu; Lee, Wen-Jane; Hung, Yi-Wen; Shen, Chin-Chang; Chung, Tsung-Ju; Liu, Shing-Hwa; Sheu, Meei-Ling

    2017-09-01

    Diabetic retinopathy is characterized by vasopermeability, vascular leakage, inflammation, blood-retinal barrier breakdown, capillary degeneration, and neovascularization. However, the mechanisms underlying the association between diabetes mellitus and progression retinopathy remain unclear. TPL2 (tumor progression locus 2), a serine-threonine protein kinase, exerts a pathological effect on vascular angiogenesis. This study investigated the role of N ε -(carboxymethyl)lysine, a major advanced glycation end products, and the involved TPL2-related molecular signals in diabetic retinopathy using models of in vitro and in vivo and human samples. Serum N ε -(carboxymethyl)lysine levels and TPL2 kinase activity were significantly increased in clinical patients and experimental animals with diabetic retinopathy. Intravitreal administration of pharmacological blocker or neutralizing antibody inhibited TPL2 and effectively suppressed the pathological characteristics of retinopathy in streptozotocin-induced diabetic animal models. Intravitreal VEGF (vascular endothelial growth factor) neutralization also suppressed the diabetic retinopathy in diabetic animal models. Mechanistic studies in primary human umbilical vein endothelial cells and primary retinal microvascular endothelial cells from streptozotocin-diabetic rats, db/db mice, and samples from patients with diabetic retinopathy revealed a positive parallel correlation between N ε -(carboxymethyl)lysine and the TPL2/chemokine SDF1α (stromal cell-derived factor-α) axis that is dependent on endoplasmic reticulum stress-related molecules, especially ATF4 (activating transcription factor-4). This study demonstrates that inhibiting the N ε -(carboxymethyl)lysine-induced TPL2/ATF4/SDF1α axis can effectively prevent diabetes mellitus-mediated retinal microvascular dysfunction. This signaling axis may include the therapeutic potential for other diseases involving pathological neovascularization or macular edema. © 2017

  6. Basal transcription machinery

    Indian Academy of Sciences (India)

    2007-03-29

    Mar 29, 2007 ... The holoenzyme of prokaryotic RNA polymerase consists of the core enzyme, made of two , , ' and subunits, which lacks promoter selectivity and a sigma () subunit which enables the core enzyme to initiate transcription in a promoter dependent fashion. A stress sigma factor s, in prokaryotes ...

  7. Machine Dictation and Transcription.

    Science.gov (United States)

    Harvey, Evelyn; And Others

    This instructional package contains both an instructor's manual and a student's manual for a course in machine dictation and transcription. The instructor's manual contains an overview with tips on teaching the course, letters for dictation, and a key to the letters. The student's manual contains an overview of the course and of the skills needed…

  8. Transcriptional Regulation in Haematopoiesis:

    DEFF Research Database (Denmark)

    Lauridsen, Felicia K B

    with the capacity to both self-renew and differentiate. This thesis is built upon two studies, which investigate two different aspects of the haematopoietic system; heterogeneity within the HSC compartment (presented in manuscript I), and the interplay between transcription factors controlling granulocyte/ monocyte...

  9. The ascorbate peroxidase APX1 is a direct target of a zinc finger transcription factor ZFP36 and a late embryogenesis abundant protein OsLEA5 interacts with ZFP36 to co-regulate OsAPX1 in seed germination in rice.

    Science.gov (United States)

    Huang, Liping; Jia, Jing; Zhao, Xixi; Zhang, MengYao; Huang, Xingxiu; E Ji; Ni, Lan; Jiang, Mingyi

    2018-01-01

    Seed germination is a vital developmental process. Abscisic acid (ABA) is an essential repressor of seed germination, while ROS (reactive oxygen species) also plays a vital role in regulating seed germination. ABA could inhibit the production of ROS in seed germination, but the mechanism of ABA reduced ROS production in seed germination was hitherto unknown. Here, by ChIP (chromatin immunoprecipitation)-seq, we found that ZFP36, a rice zinc finger transcription factor, could directly bind to the promoter of OsAPX1, coding an ascorbate peroxidase (APX) which has the most affinity for H 2 O 2 (substrate; a type of ROS), and act as a transcriptional activator of OsAPX1 promoter. Moreover, ZFP36 could interact with a late embryogenesis abundant protein OsLEA5 to co-regulate the promoter activity of OsAPX1. The seed germination is highly inhibited in ZFP36 overexpression plants under ABA treatment, while an RNA interference (RNAi) mutant of OsLEA5 rice seeds were less sensitive to ABA, and exogenous ASC (ascorbate acid) could alleviate the inhibition induced by ABA. Thus, our conclusion is that OsAPX1 is a direct target of ZFP36 and OsLEA5 could interact with ZFP36 to co-regulate ABA-inhibited seed germination by controlling the expression of OsAPX1. Copyright © 2017. Published by Elsevier Inc.

  10. Polyphenol Compound as a Transcription Factor Inhibitor

    Directory of Open Access Journals (Sweden)

    Seyeon Park

    2015-10-01

    Full Text Available A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor–DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein–protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1, c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB and β-catenin/T cell factor (Tcf.

  11. Polyphenol Compound as a Transcription Factor Inhibitor.

    Science.gov (United States)

    Park, Seyeon

    2015-10-30

    A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor-DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein-protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)).

  12. Transcriptional regulation by Polycomb group proteins

    DEFF Research Database (Denmark)

    Di Croce, Luciano; Helin, Kristian

    2013-01-01

    Polycomb group (PcG) proteins are epigenetic regulators of transcription that have key roles in stem-cell identity, differentiation and disease. Mechanistically, they function within multiprotein complexes, called Polycomb repressive complexes (PRCs), which modify histones (and other proteins......) and silence target genes. The dynamics of PRC1 and PRC2 components has been the focus of recent research. Here we discuss our current knowledge of the PRC complexes, how they are targeted to chromatin and how the high diversity of the PcG proteins allows these complexes to influence cell identity....

  13. 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....

  14. Deciphering Transcriptional Regulation

    DEFF Research Database (Denmark)

    Valen, Eivind

    The myriad of cells in the human body are all made from the same blueprint: the human genome. At the heart of this diversity lies the concept of gene regulation, the process in which it is decided which genes are used where and when. Genes do not function as on/off buttons, but more like a volume...... mostly near the start of the gene known as the promoter. This region contains patterns scattered in the DNA that the TFs can recognize and bind to. Such binding can prompt the assembly of the pre-initiation complex which ultimately leads to transcription of the gene. In order to achieve the regulation...... on what characterizes a hippocampus promoter. Pairing CAGE with TF binding site prediction we identi¿ed a likely key regulator of hippocampus. Finally, we developed a method for CAGE exploration. While the DeepCAGE library characterized a full 1.4 million transcription initiation events it did not capture...

  15. Pax4 acts as a key player in pancreas development and plasticity.

    Science.gov (United States)

    Napolitano, Tiziana; Avolio, Fabio; Courtney, Monica; Vieira, Andhira; Druelle, Noémie; Ben-Othman, Nouha; Hadzic, Biljana; Navarro, Sergi; Collombat, Patrick

    2015-08-01

    The embryonic development of the pancreas is orchestrated by a complex and coordinated transcription factor network. Neurogenin3 (Neurog3) initiates the endocrine program by activating the expression of additional transcription factors driving survival, proliferation, maturation and lineage allocation of endocrine precursors. Among the direct targets of Neurog3, Pax4 appears as one of the key regulators of β-cell specification. Indeed, mice lacking Pax4 die a few days postpartum, as they develop severe hyperglycemia due to the absence of mature pancreatic β-cells. Pax4 also directly regulates the expression of Arx, a gene that plays a crucial role in α-cell specification. Comparative analysis of Pax4 and Arx mutants, as well as Arx/Pax4 double mutants, showed that islet subtype destiny is mainly directed by cross-repression of the Pax4 and Arx factors. Importantly, the ectopic expression of Pax4 in α-cells was found sufficient to induce their neogenesis and conversion into β-like cells, not only during development but also in adult rodents. Therefore, differentiated endocrine α-cells can be considered as a putative source for insulin-producing β-like cells. These findings have clearly widened our understanding regarding pancreatic development, but they also open new research avenues in the context of diabetes research. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Transcriptional networks controlling adipocyte differentiation

    DEFF Research Database (Denmark)

    Siersbæk, R; Mandrup, Susanne

    2011-01-01

    " of the transcription factor networks operating at specific time points during adipogenesis. Using such global "snapshots," we have demonstrated that dramatic remodeling of the chromatin template occurs within the first few hours following adipogenic stimulation and that many of the early transcription factors bind...... in a cooperative fashion to transcription factor hotspots. Such hotspots are likely to represent key chromatin nodes, where many adipogenic signaling pathways converge to drive the adipogenic transcriptional reprogramming....

  17. How salicylic acid takes transcriptional control over jasmonic acid signaling

    Directory of Open Access Journals (Sweden)

    Lotte eCaarls

    2015-03-01

    Full Text Available Transcriptional regulation is a central process in plant immunity. The induction or repression of defense genes is orchestrated by signaling networks that are directed by plant hormones of which salicylic acid (SA and jasmonic acid (JA are the major players. Extensive cross-communication between the hormone signaling pathways allows for fine tuning of transcriptional programs, determining resistance to invaders and trade-offs with plant development. Here, we give an overview of how SA can control transcriptional reprogramming of JA-induced genes in Arabidopsis thaliana. SA can influence activity and/or localization of transcriptional regulators by post-translational modifications of transcription factors and co-regulators. SA-induced redox changes, mediated by thioredoxins and glutaredoxins, modify transcriptional regulators that are involved in suppression of JA-dependent genes, such as NPR1 and TGA transcription factors, which affects their localization or DNA binding activity. Furthermore, SA can mediate sequestering of JA-responsive transcription factors away from their target genes by stalling them in the cytosol or in complexes with repressor proteins in the nucleus. SA also affects JA-induced transcription by inducing degradation of transcription factors with an activating role in JA signaling, as was shown for the ERF transcription factor ORA59. Additionally, SA can induce negative regulators, among which WRKY transcription factors, that can directly or indirectly inhibit JA-responsive gene expression. Finally, at the DNA level, modification of histones by SA-dependent factors can result in repression of JA-responsive genes. These diverse and complex regulatory mechanisms affect important signaling hubs in the integration of hormone signaling networks. Some pathogens have evolved effectors that highjack hormone crosstalk mechanisms for their own good, which are described in this review as well.

  18. Emerging properties and functional consequences of noncoding transcription

    DEFF Research Database (Denmark)

    Ard, Ryan; Allshire, Robin C; Marquardt, Sebastian

    2017-01-01

    specific lncRNAs, support grows for the notion that the act of transcription rather than the RNA product itself is functionally important in many cases. Indeed, this alternative mechanism might better explain how low-abundance lncRNAs transcribed from noncoding DNA function in organisms. Here, we highlight......Eukaryotic genomes are rich in transcription units encoding "long noncoding RNAs" (lncRNAs). The purpose of all this transcription is unclear since most lncRNAs are quickly targeted for destruction during synthesis or shortly thereafter. As debates continue over the functional significance of many...... some of the recently emerging features that distinguish coding from noncoding transcription and discuss how these differences might have important implications for the functional consequences of noncoding transcription....

  19. Dissection of combinatorial control by the Met4 transcriptional complex.

    Science.gov (United States)

    Lee, Traci A; Jorgensen, Paul; Bognar, Andrew L; Peyraud, Caroline; Thomas, Dominique; Tyers, Mike

    2010-02-01

    Met4 is the transcriptional activator of the sulfur metabolic network in Saccharomyces cerevisiae. Lacking DNA-binding ability, Met4 must interact with proteins called Met4 cofactors to target promoters for transcription. Two types of DNA-binding cofactors (Cbf1 and Met31/Met32) recruit Met4 to promoters and one cofactor (Met28) stabilizes the DNA-bound Met4 complexes. To dissect this combinatorial system, we systematically deleted each category of cofactor(s) and analyzed Met4-activated transcription on a genome-wide scale. We defined a core regulon for Met4, consisting of 45 target genes. Deletion of both Met31 and Met32 eliminated activation of the core regulon, whereas loss of Met28 or Cbf1 interfered with only a subset of targets that map to distinct sectors of the sulfur metabolic network. These transcriptional dependencies roughly correlated with the presence of Cbf1 promoter motifs. Quantitative analysis of in vivo promoter binding properties indicated varying levels of cooperativity and interdependency exists between members of this combinatorial system. Cbf1 was the only cofactor to remain fully bound to target promoters under all conditions, whereas other factors exhibited different degrees of regulated binding in a promoter-specific fashion. Taken together, Met4 cofactors use a variety of mechanisms to allow differential transcription of target genes in response to various cues.

  20. MULTI-KEV X-Ray Yields From High-Z Gas Targets Fielded At Omega

    International Nuclear Information System (INIS)

    Kane, J.O.; Fournier, K.B.; May, M.J.; Colvin, J.D.; Thomas, C.A.; Marrs, R.E.; Compton, S.M.; Moody, J.D.; Bond, E.J.; Davis, J.F.

    2010-01-01

    The authors report on modeling of x-ray yield from gas-filled targets shot at the OMEGA laser facility. The OMEGA targets were 1.8 mm long, 1.95 mm in diameter Be cans filled with either a 50:50 Ar:Xe mixture, pure Ar, pure Kr or pure Xe at ∼ 1 atm. The OMEGA experiments heated the gas with 20 kJ of 3ω (∼ 350 nm) laser energy delivered in a 1 ns square pulse. the emitted x-ray flux was monitored with the x-ray diode based DANTE instruments in the sub-keV range. Two-dimensional x-ray images (for energies 3-5 keV) of the targets were recorded with gated x-ray detectors. The x-ray spectra were recorded with the HENWAY crystal spectrometer at OMEGA. Predictions are 2D r-z cylindrical with DCA NLTE atomic physics. Models generally: (1) underpredict the Xe L-shell yields; (2) overpredict the Ar K-shell yields; (3) correctly predict the Xe thermal yields; and (4) greatly underpredict the Ar thermal yields. However, there are spreads within the data, e.g. the DMX Ar K-shell yields are correctly predicted. The predicted thermal yields show strong angular dependence.

  1. 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.

  2. Peroxisome proliferator-activated receptor gamma recruits the positive transcription elongation factor b complex to activate transcription and promote adipogenesis

    DEFF Research Database (Denmark)

    Iankova, Irena; Petersen, Rasmus K; Annicotte, Jean-Sébastien

    2006-01-01

    Positive transcription elongation factor b (P-TEFb) phosphorylates the C-terminal domain of RNA polymerase II, facilitating transcriptional elongation. In addition to its participation in general transcription, P-TEFb is recruited to specific promoters by some transcription factors such as c......-Myc or MyoD. The P-TEFb complex is composed of a cyclin-dependent kinase (cdk9) subunit and a regulatory partner (cyclin T1, cyclin T2, or cyclin K). Because cdk9 has been shown to participate in differentiation processes, such as muscle cell differentiation, we studied a possible role of cdk9...... with and phosphorylation of peroxisome proliferator-activated receptor gamma (PPARgamma), which is the master regulator of this process, on the promoter of PPARgamma target genes. PPARgamma-cdk9 interaction results in increased transcriptional activity of PPARgamma and therefore increased adipogenesis....

  3. Intergenic disease-associated regions are abundant in novel transcripts.

    Science.gov (United States)

    Bartonicek, N; Clark, M B; Quek, X C; Torpy, J R; Pritchard, A L; Maag, J L V; Gloss, B S; Crawford, J; Taft, R J; Hayward, N K; Montgomery, G W; Mattick, J S; Mercer, T R; Dinger, M E

    2017-12-28

    Genotyping of large populations through genome-wide association studies (GWAS) has successfully identified many genomic variants associated with traits or disease risk. Unexpectedly, a large proportion of GWAS single nucleotide polymorphisms (SNPs) and associated haplotype blocks are in intronic and intergenic regions, hindering their functional evaluation. While some of these risk-susceptibility regions encompass cis-regulatory sites, their transcriptional potential has never been systematically explored. To detect rare tissue-specific expression, we employed the transcript-enrichment method CaptureSeq on 21 human tissues to identify 1775 multi-exonic transcripts from 561 intronic and intergenic haploblocks associated with 392 traits and diseases, covering 73.9 Mb (2.2%) of the human genome. We show that a large proportion (85%) of disease-associated haploblocks express novel multi-exonic non-coding transcripts that are tissue-specific and enriched for GWAS SNPs as well as epigenetic markers of active transcription and enhancer activity. Similarly, we captured transcriptomes from 13 melanomas, targeting nine melanoma-associated haploblocks, and characterized 31 novel melanoma-specific transcripts that include fusion proteins, novel exons and non-coding RNAs, one-third of which showed allelically imbalanced expression. This resource of previously unreported transcripts in disease-associated regions ( http://gwas-captureseq.dingerlab.org ) should provide an important starting point for the translational community in search of novel biomarkers, disease mechanisms, and drug targets.

  4. Designed Transcriptional Regulation in Mammalian Cells Based on TALE- and CRISPR/dCas9.

    Science.gov (United States)

    Lebar, Tina; Jerala, Roman

    2018-01-01

    Transcriptional regulation lies at the center of many cellular processes and is the result of cellular response to different external and internal signals. Control of transcription of selected genes enables an unprecedented access to shape the cellular response. While orthogonal transcription factors from bacteria, yeast, plants, or other cells have been used to introduce new cellular logic into mammalian cells, the discovery of designable modular DNA binding domains, such as Transcription Activator-Like Effectors (TALEs) and the CRISPR system, enable targeting of almost any selected DNA sequence. Fusion or conditional association of DNA targeting domain with transcriptional effector domains enables controlled regulation of almost any endogenous or ectopic gene. Moreover, the designed regulators can be linked into genetic circuits to implement complex responses, such as different types of Boolean functions and switches. In this chapter, we describe the protocols for achieving efficient transcriptional regulation with TALE- and CRISPR-based designed transcription factors in mammalian cells.

  5. Transcript structure and domain display: a customizable transcript visualization tool.

    Science.gov (United States)

    Watanabe, Kenneth A; Ma, Kaiwang; Homayouni, Arielle; Rushton, Paul J; Shen, Qingxi J

    2016-07-01

    Transcript Structure and Domain Display (TSDD) is a publicly available, web-based program that provides publication quality images of transcript structures and domains. TSDD is capable of producing transcript structures from GFF/GFF3 and BED files. Alternatively, the GFF files of several model organisms have been pre-loaded so that users only needs to enter the locus IDs of the transcripts to be displayed. Visualization of transcripts provides many benefits to researchers, ranging from evolutionary analysis of DNA-binding domains to predictive function modeling. TSDD is freely available for non-commercial users at http://shenlab.sols.unlv.edu/shenlab/software/TSD/transcript_display.html : jeffery.shen@unlv.nevada.edu. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. 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.

  7. Transcription control engineering and applications in synthetic biology

    Directory of Open Access Journals (Sweden)

    Michael D. Engstrom

    2017-09-01

    Full Text Available In synthetic biology, researchers assemble biological components in new ways to produce systems with practical applications. One of these practical applications is control of the flow of genetic information (from nucleic acid to protein, a.k.a. gene regulation. Regulation is critical for optimizing protein (and therefore activity levels and the subsequent levels of metabolites and other cellular properties. The central dogma of molecular biology posits that information flow commences with transcription, and accordingly, regulatory tools targeting transcription have received the most attention in synthetic biology. In this mini-review, we highlight many past successes and summarize the lessons learned in developing tools for controlling transcription. In particular, we focus on engineering studies where promoters and transcription terminators (cis-factors were directly engineered and/or isolated from DNA libraries. We also review several well-characterized transcription regulators (trans-factors, giving examples of how cis- and trans-acting factors have been combined to create digital and analogue switches for regulating transcription in response to various signals. Last, we provide examples of how engineered transcription control systems have been used in metabolic engineering and more complicated genetic circuits. While most of our mini-review focuses on the well-characterized bacterium Escherichia coli, we also provide several examples of the use of transcription control engineering in non-model organisms. Similar approaches have been applied outside the bacterial kingdom indicating that the lessons learned from bacterial studies may be generalized for other organisms.

  8. Transcription control engineering and applications in synthetic biology.

    Science.gov (United States)

    Engstrom, Michael D; Pfleger, Brian F

    2017-09-01

    In synthetic biology, researchers assemble biological components in new ways to produce systems with practical applications. One of these practical applications is control of the flow of genetic information (from nucleic acid to protein), a.k.a. gene regulation. Regulation is critical for optimizing protein (and therefore activity) levels and the subsequent levels of metabolites and other cellular properties. The central dogma of molecular biology posits that information flow commences with transcription, and accordingly, regulatory tools targeting transcription have received the most attention in synthetic biology. In this mini-review, we highlight many past successes and summarize the lessons learned in developing tools for controlling transcription. In particular, we focus on engineering studies where promoters and transcription terminators ( cis -factors) were directly engineered and/or isolated from DNA libraries. We also review several well-characterized transcription regulators ( trans- factors), giving examples of how cis- and trans -acting factors have been combined to create digital and analogue switches for regulating transcription in response to various signals. Last, we provide examples of how engineered transcription control systems have been used in metabolic engineering and more complicated genetic circuits. While most of our mini-review focuses on the well-characterized bacterium Escherichia coli , we also provide several examples of the use of transcription control engineering in non-model organisms. Similar approaches have been applied outside the bacterial kingdom indicating that the lessons learned from bacterial studies may be generalized for other organisms.

  9. 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...

  10. 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...

  11. 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. PMID:24755522

  12. Eukaryotic transcription factors

    DEFF Research Database (Denmark)

    Staby, Lasse; O'Shea, Charlotte; Willemoës, Martin

    2017-01-01

    Gene-specific transcription factors (TFs) are key regulatory components of signaling pathways, controlling, for example, cell growth, development, and stress responses. Their biological functions are determined by their molecular structures, as exemplified by their structured DNA-binding domains...... regions with function-related, short sequence motifs and molecular recognition features with structural propensities. This review focuses on molecular aspects of TFs, which represent paradigms of ID-related features. Through specific examples, we review how the ID-associated flexibility of TFs enables....... It is furthermore emphasized how classic biochemical concepts like allostery, conformational selection, induced fit, and feedback regulation are undergoing a revival with the appreciation of ID. The review also describes the most recent advances based on computational simulations of ID-based interaction mechanisms...

  13. Harnessing CRISPR/Cas systems for programmable transcriptional and post-transcriptional regulation

    KAUST Repository

    Mahas, Ahmed

    2017-11-29

    Genome editing has enabled broad advances and novel approaches in studies of gene function and structure; now, emerging methods aim to precisely engineer post-transcriptional processes. Developing precise, efficient molecular tools to alter the transcriptome holds great promise for biotechnology and synthetic biology applications. Different approaches have been employed for targeted degradation of RNA species in eukaryotes, but they lack programmability and versatility, thereby limiting their utility for diverse applications. The CRISPR/Cas9 system has been harnessed for genome editing in many eukaryotic species and, using a catalytically inactive Cas9 variant, the CRISPR/dCas9 system has been repurposed for transcriptional regulation. Recent studies have used other CRISPR/Cas systems for targeted RNA degradation and RNA-based manipulations. For example, Cas13a, a Type VI-A endonuclease, has been identified as an RNA-guided RNA ribonuclease and used for manipulation of RNA. Here, we discuss different modalities for targeted RNA interference with an emphasis on the potential applications of CRISPR/Cas systems as programmable transcriptional regulators for broad uses, including functional biology, biotechnology, and synthetic biology applications.

  14. Harnessing CRISPR/Cas systems for programmable transcriptional and post-transcriptional regulation

    KAUST Repository

    Mahas, Ahmed; Neal Stewart, C.; Mahfouz, Magdy M.

    2017-01-01

    Genome editing has enabled broad advances and novel approaches in studies of gene function and structure; now, emerging methods aim to precisely engineer post-transcriptional processes. Developing precise, efficient molecular tools to alter the transcriptome holds great promise for biotechnology and synthetic biology applications. Different approaches have been employed for targeted degradation of RNA species in eukaryotes, but they lack programmability and versatility, thereby limiting their utility for diverse applications. The CRISPR/Cas9 system has been harnessed for genome editing in many eukaryotic species and, using a catalytically inactive Cas9 variant, the CRISPR/dCas9 system has been repurposed for transcriptional regulation. Recent studies have used other CRISPR/Cas systems for targeted RNA degradation and RNA-based manipulations. For example, Cas13a, a Type VI-A endonuclease, has been identified as an RNA-guided RNA ribonuclease and used for manipulation of RNA. Here, we discuss different modalities for targeted RNA interference with an emphasis on the potential applications of CRISPR/Cas systems as programmable transcriptional regulators for broad uses, including functional biology, biotechnology, and synthetic biology applications.

  15. Targeting Transcription Elongation Machinery for Breast Cancer Therapy

    Science.gov (United States)

    2016-05-01

    Luo CONTRACTING ORGANIZATION: University of California, Berkeley Berkeley, CA 94704 REPORT DATE: May 2016 TYPE OF REPORT: Annual PREPARED FOR...ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER University of California, Berkeley BERKELEY, CA 94704 9. SPONSORING...molecules. We have employed the CRISPR /Cas9 genome-editing tool to knock out the gene encoding the SEC component AFF4 or knock in a mutant cyclin T1 (AAG

  16. Genome-Wide Chromosomal Targets of Oncogenic Transcription Factors

    Science.gov (United States)

    2008-04-01

    Altman, W.E., Attiya, S., Bader, J.S., Bemben, L.A., Berka , J., Braverman, M.S., Chen, Y.J., Chen, Z., et al. 2005. Genome sequencing in microfabricated...software after filtering to exclude bad spots. qPCR validation. Primer pairs used in Figure 1 were designed to cover three peaks and three troughs in

  17. PPARα-independent transcriptional targets of perfluoroalkyl acids revealed by transcript profiling

    Data.gov (United States)

    U.S. Environmental Protection Agency — Microarray datasets used in the analysis. This dataset is associated with the following publication: Rosen, M., K. Das, J. Rooney, B. Abbott, C. Lau, and C. Corton....

  18. Sputum is a surrogate for bronchoalveolar lavage for monitoring Mycobacterium tuberculosis transcriptional profiles in TB patients.

    Science.gov (United States)

    Garcia, Benjamin J; Loxton, Andre G; Dolganov, Gregory M; Van, Tran T; Davis, J Lucian; de Jong, Bouke C; Voskuil, Martin I; Leach, Sonia M; Schoolnik, Gary K; Walzl, Gerhard; Strong, Michael; Walter, Nicholas D

    2016-09-01

    Pathogen-targeted transcriptional profiling in human sputum may elucidate the physiologic state of Mycobacterium tuberculosis (M. tuberculosis) during infection and treatment. However, whether M. tuberculosis transcription in sputum recapitulates transcription in the lung is uncertain. We therefore compared M. tuberculosis transcription in human sputum and bronchoalveolar lavage (BAL) samples from 11 HIV-negative South African patients with pulmonary tuberculosis. We additionally compared these clinical samples with in vitro log phase aerobic growth and hypoxic non-replicating persistence (NRP-2). Of 2179 M. tuberculosis transcripts assayed in sputum and BAL via multiplex RT-PCR, 194 (8.9%) had a p-value <0.05, but none were significant after correction for multiple testing. Categorical enrichment analysis indicated that expression of the hypoxia-responsive DosR regulon was higher in BAL than in sputum. M. tuberculosis transcription in BAL and sputum was distinct from both aerobic growth and NRP-2, with a range of 396-1020 transcripts significantly differentially expressed after multiple testing correction. Collectively, our results indicate that M. tuberculosis transcription in sputum approximates M. tuberculosis transcription in the lung. Minor differences between M. tuberculosis transcription in BAL and sputum suggested lower oxygen concentrations or higher nitric oxide concentrations in BAL. M. tuberculosis-targeted transcriptional profiling of sputa may be a powerful tool for understanding M. tuberculosis pathogenesis and monitoring treatment responses in vivo. Published by Elsevier Ltd.

  19. A glyphosate-based pesticide impinges on transcription

    International Nuclear Information System (INIS)

    Marc, Julie; Le Breton, Magali; Cormier, Patrick; Morales, Julia; Belle, Robert; Mulner-Lorillon, Odile

    2005-01-01

    Widely spread chemicals used for human benefits may exert adverse effects on health or the environment, the identification of which are a major challenge. The early development of the sea urchin constitutes an appropriate model for the identification of undesirable cellular and molecular targets of pollutants. The widespread glyphosate-based pesticide affected sea urchin development by impeding the hatching process at millimolar range concentration of glyphosate. Glyphosate, the active herbicide ingredient of Roundup, by itself delayed hatching as judged from the comparable effect of different commercial glyphosate-based pesticides and from the effect of pure glyphosate addition to a threshold concentration of Roundup. The surfactant polyoxyethylene amine (POEA), the major component of commercial Roundup, was found to be highly toxic to the embryos when tested alone and therefore could contribute to the inhibition of hatching. Hatching, a landmark of early development, is a transcription-dependent process. Correlatively, the herbicide inhibited the global transcription, which follows fertilization at the 16-cell stage. Transcription inhibition was dose-dependent in the millimolar glyphosate range concentration. A 1257-bp fragment of the hatching enzyme transcript from Sphaerechinus granularis was cloned and sequenced; its transcription was delayed by 2 h in the pesticide-treated embryos. Because transcription is a fundamental basic biological process, the pesticide may be of health concern by inhalation near herbicide spraying at a concentration 25 times the adverse transcription concentration in the sprayed microdroplets

  20. Modeling of Slovak Language for Broadcast News Transcription

    Directory of Open Access Journals (Sweden)

    STAŠ Ján

    2015-10-01

    Full Text Available The paper describes recent progress in the development the Slovak language models for transcription of spontaneous speech such as broadcast news, educational talks and lectures, or meetings. This work extends previous research oriented on the automatic transcription of dictated speech and brings some new extensions for improving perplexity and robustness of the Slovak language models trained on the web-based and electronic language resources for being more precise in recognition of spontaneous speech. These improvements include better text preprocessing, document classification, class-based and filled pauses modeling, web-data augmentation and fast model adaptation to the target domain. Experiments have been performed on the four different evaluation data sets, including judicial and newspaper readings, broadcast news recordings and parliament proceedings with the Slovak transcription system. Preliminary results show significant decrease of the word error rate for multiple transcription system configurations of acoustic and language models.

  1. The Mediator complex and transcription regulation

    Science.gov (United States)

    Poss, Zachary C.; Ebmeier, Christopher C.

    2013-01-01

    The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module. PMID:24088064

  2. Combinatorial microRNA target predictions

    DEFF Research Database (Denmark)

    Krek, Azra; Grün, Dominic; Poy, Matthew N.

    2005-01-01

    MicroRNAs are small noncoding RNAs that recognize and bind to partially complementary sites in the 3' untranslated regions of target genes in animals and, by unknown mechanisms, regulate protein production of the target transcript1, 2, 3. Different combinations of microRNAs are expressed...... in different cell types and may coordinately regulate cell-specific target genes. Here, we present PicTar, a computational method for identifying common targets of microRNAs. Statistical tests using genome-wide alignments of eight vertebrate genomes, PicTar's ability to specifically recover published micro......RNA targets, and experimental validation of seven predicted targets suggest that PicTar has an excellent success rate in predicting targets for single microRNAs and for combinations of microRNAs. We find that vertebrate microRNAs target, on average, roughly 200 transcripts each. Furthermore, our results...

  3. CERN: Fixed target targets

    Energy Technology Data Exchange (ETDEWEB)

    Anon.

    1993-03-15

    Full text: While the immediate priority of CERN's research programme is to exploit to the full the world's largest accelerator, the LEP electron-positron collider and its concomitant LEP200 energy upgrade (January, page 1), CERN is also mindful of its long tradition of diversified research. Away from LEP and preparations for the LHC proton-proton collider to be built above LEP in the same 27-kilometre tunnel, CERN is also preparing for a new generation of heavy ion experiments using a new source, providing heavier ions (April 1992, page 8), with first physics expected next year. CERN's smallest accelerator, the LEAR Low Energy Antiproton Ring continues to cover a wide range of research topics, and saw a record number of hours of operation in 1992. The new ISOLDE on-line isotope separator was inaugurated last year (July, page 5) and physics is already underway. The remaining effort concentrates around fixed target experiments at the SPS synchrotron, which formed the main thrust of CERN's research during the late 1970s. With the SPS and LEAR now approaching middle age, their research future was extensively studied last year. Broadly, a vigorous SPS programme looks assured until at least the end of 1995. Decisions for the longer term future of the West Experimental Area of the SPS will have to take into account the heavy demand for test beams from work towards experiments at big colliders, both at CERN and elsewhere. The North Experimental Area is the scene of larger experiments with longer lead times. Several more years of LEAR exploitation are already in the pipeline, but for the longer term, the ambitious Superlear project for a superconducting ring (January 1992, page 7) did not catch on. Neutrino physics has a long tradition at CERN, and this continues with the preparations for two major projects, the Chorus and Nomad experiments (November 1991, page 7), to start next year in the West Area. Delicate neutrino oscillation effects could become visible for the first

  4. WRKY transcription factor superfamily: Structure, origin and functions

    African Journals Online (AJOL)

    terminal ends contain the WRKYGQR amino acid sequence and a zinc-finger motif. WRKY transcription factors can regulate the expression of target genes that contain the W-box elements (C/T)TGAC(C/T) in the promoter regions by specifically ...

  5. The Wnt Transcriptional Switch: TLE Removal or Inactivation?

    Science.gov (United States)

    Ramakrishnan, Aravinda-Bharathi; Sinha, Abhishek; Fan, Vinson B; Cadigan, Ken M

    2018-02-01

    Many targets of the Wnt/β-catenin signaling pathway are regulated by TCF transcription factors, which play important roles in animal development, stem cell biology, and oncogenesis. TCFs can regulate Wnt targets through a "transcriptional switch," repressing gene expression in unstimulated cells and promoting transcription upon Wnt signaling. However, it is not clear whether this switch mechanism is a general feature of Wnt gene regulation or limited to a subset of Wnt targets. Co-repressors of the TLE family are known to contribute to the repression of Wnt targets in the absence of signaling, but how they are inactivated or displaced by Wnt signaling is poorly understood. In this mini-review, we discuss several recent reports that address the prevalence and molecular mechanisms of the Wnt transcription switch, including the finding of Wnt-dependent ubiquitination/inactivation of TLEs. Together, these findings highlight the growing complexity of the regulation of gene expression by the Wnt pathway. © 2017 WILEY Periodicals, Inc.

  6. Selection Shapes Transcriptional Logic and Regulatory Specialization in Genetic Networks.

    Science.gov (United States)

    Fogelmark, Karl; Peterson, Carsten; Troein, Carl

    2016-01-01

    Living organisms need to regulate their gene expression in response to environmental signals and internal cues. This is a computational task where genes act as logic gates that connect to form transcriptional networks, which are shaped at all scales by evolution. Large-scale mutations such as gene duplications and deletions add and remove network components, whereas smaller mutations alter the connections between them. Selection determines what mutations are accepted, but its importance for shaping the resulting networks has been debated. To investigate the effects of selection in the shaping of transcriptional networks, we derive transcriptional logic from a combinatorially powerful yet tractable model of the binding between DNA and transcription factors. By evolving the resulting networks based on their ability to function as either a simple decision system or a circadian clock, we obtain information on the regulation and logic rules encoded in functional transcriptional networks. Comparisons are made between networks evolved for different functions, as well as with structurally equivalent but non-functional (neutrally evolved) networks, and predictions are validated against the transcriptional network of E. coli. We find that the logic rules governing gene expression depend on the function performed by the network. Unlike the decision systems, the circadian clocks show strong cooperative binding and negative regulation, which achieves tight temporal control of gene expression. Furthermore, we find that transcription factors act preferentially as either activators or repressors, both when binding multiple sites for a single target gene and globally in the transcriptional networks. This separation into positive and negative regulators requires gene duplications, which highlights the interplay between mutation and selection in shaping the transcriptional networks.

  7. CERN: Fixed target targets

    International Nuclear Information System (INIS)

    Anon.

    1993-01-01

    Full text: While the immediate priority of CERN's research programme is to exploit to the full the world's largest accelerator, the LEP electron-positron collider and its concomitant LEP200 energy upgrade (January, page 1), CERN is also mindful of its long tradition of diversified research. Away from LEP and preparations for the LHC proton-proton collider to be built above LEP in the same 27-kilometre tunnel, CERN is also preparing for a new generation of heavy ion experiments using a new source, providing heavier ions (April 1992, page 8), with first physics expected next year. CERN's smallest accelerator, the LEAR Low Energy Antiproton Ring continues to cover a wide range of research topics, and saw a record number of hours of operation in 1992. The new ISOLDE on-line isotope separator was inaugurated last year (July, page 5) and physics is already underway. The remaining effort concentrates around fixed target experiments at the SPS synchrotron, which formed the main thrust of CERN's research during the late 1970s. With the SPS and LEAR now approaching middle age, their research future was extensively studied last year. Broadly, a vigorous SPS programme looks assured until at least the end of 1995. Decisions for the longer term future of the West Experimental Area of the SPS will have to take into account the heavy demand for test beams from work towards experiments at big colliders, both at CERN and elsewhere. The North Experimental Area is the scene of larger experiments with longer lead times. Several more years of LEAR exploitation are already in the pipeline, but for the longer term, the ambitious Superlear project for a superconducting ring (January 1992, page 7) did not catch on. Neutrino physics has a long tradition at CERN, and this continues with the preparations for two major projects, the Chorus and Nomad experiments (November 1991, page 7), to start next year in the West Area. Delicate neutrino oscillation effects could become

  8. A Transcription and Translation Protocol for Sensitive Cross-Cultural Team Research.

    Science.gov (United States)

    Clark, Lauren; Birkhead, Ana Sanchez; Fernandez, Cecilia; Egger, Marlene J

    2017-10-01

    Assurance of transcript accuracy and quality in interview-based qualitative research is foundational for data accuracy and study validity. Based on our experience in a cross-cultural ethnographic study of women's pelvic organ prolapse, we provide practical guidance to set up step-by-step interview transcription and translation protocols for team-based research on sensitive topics. Beginning with team decisions about level of detail in transcription, completeness, and accuracy, we operationalize the process of securing vendors to deliver the required quality of transcription and translation. We also share rubrics for assessing transcript quality and the team protocol for managing transcripts (assuring consistency of format, insertion of metadata, anonymization, and file labeling conventions) and procuring an acceptable initial translation of Spanish-language interviews. Accurate, complete, and systematically constructed transcripts in both source and target languages respond to the call for more transparency and reproducibility of scientific methods.

  9. Regulation of circadian clock transcriptional output by CLOCK:BMAL1

    Science.gov (United States)

    Trott, Alexandra J.

    2018-01-01

    The mammalian circadian clock relies on the transcription factor CLOCK:BMAL1 to coordinate the rhythmic expression of 15% of the transcriptome and control the daily regulation of biological functions. The recent characterization of CLOCK:BMAL1 cistrome revealed that although CLOCK:BMAL1 binds synchronously to all of its target genes, its transcriptional output is highly heterogeneous. By performing a meta-analysis of several independent genome-wide datasets, we found that the binding of other transcription factors at CLOCK:BMAL1 enhancers likely contribute to the heterogeneity of CLOCK:BMAL1 transcriptional output. While CLOCK:BMAL1 rhythmic DNA binding promotes rhythmic nucleosome removal, it is not sufficient to generate transcriptionally active enhancers as assessed by H3K27ac signal, RNA Polymerase II recruitment, and eRNA expression. Instead, the transcriptional activity of CLOCK:BMAL1 enhancers appears to rely on the activity of ubiquitously expressed transcription factors, and not tissue-specific transcription factors, recruited at nearby binding sites. The contribution of other transcription factors is exemplified by how fasting, which effects several transcription factors but not CLOCK:BMAL1, either decreases or increases the amplitude of many rhythmically expressed CLOCK:BMAL1 target genes. Together, our analysis suggests that CLOCK:BMAL1 promotes a transcriptionally permissive chromatin landscape that primes its target genes for transcription activation rather than directly activating transcription, and provides a new framework to explain how environmental or pathological conditions can reprogram the rhythmic expression of clock-controlled genes. PMID:29300726

  10. Characterization of senscence-associated NAC transcription factors in Barley (Hordeum Vulgare L.)

    DEFF Research Database (Denmark)

    Podzimska, Dagmara Agata

    , such as yield, biomass production and nutrient quality, and NAC (NAM, ATAF1/2 and CUC2) transcription factors are promising targets for the breeding. The aim of this thesis was thus to assess the role of NAC transcription factors in regulation of senescence in barley (Hordeum vulgare L.) and to contribute...

  11. MADS interactomics : towards understanding the molecular mechanisms of plant MADS-domain transcription factor function

    NARCIS (Netherlands)

    Smaczniak, C.D.

    2013-01-01

    Protein-protein and protein-DNA interactions are essential for the molecular action of transcription factors. By combinatorial binding to target gene promoters, transcription factors are able to up- or down-regulate the expression of these genes. MADS-domain proteins comprise a large family of

  12. Transcriptional control of megakaryocyte development.

    Science.gov (United States)

    Goldfarb, A N

    2007-10-15

    Megakaryocytes are highly specialized cells that arise from a bipotent megakaryocytic-erythroid progenitor (MEP). This developmental leap requires coordinated activation of megakaryocyte-specific genes, radical changes in cell cycle properties, and active prevention of erythroid differentiation. These programs result from upregulation of megakaryocyte-selective transcription factors, downregulation of erythroid-selective transcription factors and ongoing mediation of common erythro-megakaryocytic transcription factors. Unlike most developmental programs, no single lineage-unique family of master regulators exerts executive control over the megakaryocytic plan. Rather, an assemblage of non-unique factors and signals converge to determine lineage and differentiation. In human megakaryopoiesis, hereditary disorders of platelet production have confirmed contributions from three distinct transcription factor families. Murine models have extended this repertoire to include multiple additional factors. At a mechanistic level, the means by which these non-unique factors collaborate in the establishment of a perfectly unique cell type remains a central question.

  13. Target laboratory

    International Nuclear Information System (INIS)

    Ephraim, D.C.; Pednekar, A.R.

    1993-01-01

    A target laboratory to make stripper foils for the accelerator and various targets for use in the experiments is set up in the pelletron accelerator facility. The facilities available in the laboratory are: (1) D.C. glow discharge setup, (2) carbon arc set up, and (3) vacuum evaporation set up (resistance heating), electron beam source, rolling mill - all for target preparation. They are described. Centrifugal deposition technique is used for target preparation. (author). 3 figs

  14. Screening Driving Transcription Factors in the Processing of Gastric Cancer

    Directory of Open Access Journals (Sweden)

    Guangzhong Xu

    2016-01-01

    Full Text Available Background. Construction of the transcriptional regulatory network can provide additional clues on the regulatory mechanisms and therapeutic applications in gastric cancer. Methods. Gene expression profiles of gastric cancer were downloaded from GEO database for integrated analysis. All of DEGs were analyzed by GO enrichment and KEGG pathway enrichment. Transcription factors were further identified and then a global transcriptional regulatory network was constructed. Results. By integrated analysis of the six eligible datasets (340 cases and 43 controls, a bunch of 2327 DEGs were identified, including 2100 upregulated and 227 downregulated DEGs. Functional enrichment analysis of DEGs showed that digestion was a significantly enriched GO term for biological process. Moreover, there were two important enriched KEGG pathways: cell cycle and homologous recombination. Furthermore, a total of 70 differentially expressed TFs were identified and the transcriptional regulatory network was constructed, which consisted of 566 TF-target interactions. The top ten TFs regulating most downstream target genes were BRCA1, ARID3A, EHF, SOX10, ZNF263, FOXL1, FEV, GATA3, FOXC1, and FOXD1. Most of them were involved in the carcinogenesis of gastric cancer. Conclusion. The transcriptional regulatory network can help researchers to further clarify the underlying regulatory mechanisms of gastric cancer tumorigenesis.

  15. Ice targets

    International Nuclear Information System (INIS)

    Pacheco, C.; Stark, C.; Tanaka, N.; Hodgkins, D.; Barnhart, J.; Kosty, J.

    1979-12-01

    This report presents a description of ice targets that were constructed for research work at the High Resolution Spectrometer (HRS) and at the Energetic Pion Channel and Spectrometer (EPICS). Reasons for using these ice targets and the instructions for their construction are given. Results of research using ice targets will be published at a later date

  16. National Capital Planning Commission Meeting Transcripts

    Data.gov (United States)

    National Capital Planning Commission — Transcripts of the monthly (with the exception of August) National Capital Planning Commission meeting transcripts are provided for research to confirm actions taken...

  17. Regulatory hotspots in the malaria parasite genome dictate transcriptional variation.

    Directory of Open Access Journals (Sweden)

    Joseph M Gonzales

    2008-09-01

    Full Text Available The determinants of transcriptional regulation in malaria parasites remain elusive. The presence of a well-characterized gene expression cascade shared by different Plasmodium falciparum strains could imply that transcriptional regulation and its natural variation do not contribute significantly to the evolution of parasite drug resistance. To clarify the role of transcriptional variation as a source of stain-specific diversity in the most deadly malaria species and to find genetic loci that dictate variations in gene expression, we examined genome-wide expression level polymorphisms (ELPs in a genetic cross between phenotypically distinct parasite clones. Significant variation in gene expression is observed through direct co-hybridizations of RNA from different P. falciparum clones. Nearly 18% of genes were regulated by a significant expression quantitative trait locus. The genetic determinants of most of these ELPs resided in hotspots that are physically distant from their targets. The most prominent regulatory locus, influencing 269 transcripts, coincided with a Chromosome 5 amplification event carrying the drug resistance gene, pfmdr1, and 13 other genes. Drug selection pressure in the Dd2 parental clone lineage led not only to a copy number change in the pfmdr1 gene but also to an increased copy number of putative neighboring regulatory factors that, in turn, broadly influence the transcriptional network. Previously unrecognized transcriptional variation, controlled by polymorphic regulatory genes and possibly master regulators within large copy number variants, contributes to sweeping phenotypic evolution in drug-resistant malaria parasites.

  18. Oncogenes Activate an Autonomous Transcriptional Regulatory Circuit That Drives Glioblastoma

    Directory of Open Access Journals (Sweden)

    Dinesh K. Singh

    2017-01-01

    Full Text Available Efforts to identify and target glioblastoma (GBM drivers have primarily focused on receptor tyrosine kinases (RTKs. Clinical benefits, however, have been elusive. Here, we identify an SRY-related box 2 (SOX2 transcriptional regulatory network that is independent of upstream RTKs and capable of driving glioma-initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2 and zinc-finger E-box binding homeobox 1 (ZEB1, which are frequently co-expressed irrespective of driver mutations, as potential SOX2 targets. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor-suppressor-deficient astrocytes into glioma-initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo.

  19. Transcriptional regulatory networks downstream of TAL1/SCL in T-cell acute lymphoblastic leukemia

    OpenAIRE

    Palomero, Teresa; Odom, Duncan T.; O'Neil, Jennifer; Ferrando, Adolfo A.; Margolin, Adam; Neuberg, Donna S.; Winter, Stuart S.; Larson, Richard S.; Li, Wei; Liu, X. Shirley; Young, Richard A.; Look, A. Thomas

    2006-01-01

    Aberrant expression of 1 or more transcription factor oncogenes is a critical component of the molecular pathogenesis of human T-cell acute lymphoblastic leukemia (T-ALL); however, oncogenic transcriptional programs downstream of T-ALL oncogenes are mostly unknown. TAL1/SCL is a basic helix-loop-helix (bHLH) transcription factor oncogene aberrantly expressed in 60% of human T-ALLs. We used chromatin immunoprecipitation (ChIP) on chip to identify 71 direct transcriptional targets of TAL1/SCL. ...

  20. Analysis of a Plant Transcriptional Regulatory Network Using Transient Expression Systems.

    Science.gov (United States)

    Díaz-Triviño, Sara; Long, Yuchen; Scheres, Ben; Blilou, Ikram

    2017-01-01

    In plant biology, transient expression systems have become valuable approaches used routinely to rapidly study protein expression, subcellular localization, protein-protein interactions, and transcriptional activity prior to in vivo studies. When studying transcriptional regulation, luciferase reporter assays offer a sensitive readout for assaying promoter behavior in response to different regulators or environmental contexts and to confirm and assess the functional relevance of predicted binding sites in target promoters. This chapter aims to provide detailed methods for using luciferase reporter system as a rapid, efficient, and versatile assay to analyze transcriptional regulation of target genes by transcriptional regulators. We describe a series of optimized transient expression systems consisting of Arabidopsis thaliana protoplasts, infiltrated Nicotiana benthamiana leaves, and human HeLa cells to study the transcriptional regulations of two well-characterized transcriptional regulators SCARECROW (SCR) and SHORT-ROOT (SHR) on one of their targets, CYCLIN D6 (CYCD6).Here, we illustrate similarities and differences in outcomes when using different systems. The plant-based systems revealed that the SCR-SHR complex enhances CYCD6 transcription, while analysis in HeLa cells showed that the complex is not sufficient to strongly induce CYCD6 transcription, suggesting that additional, plant-specific regulators are required for full activation. These results highlight the importance of the system and suggest that including heterologous systems, such as HeLa cells, can provide a more comprehensive analysis of a complex gene regulatory network.

  1. The DNA replication checkpoint directly regulates MBF-dependent G1/S transcription.

    Science.gov (United States)

    Dutta, Chaitali; Patel, Prasanta K; Rosebrock, Adam; Oliva, Anna; Leatherwood, Janet; Rhind, Nicholas

    2008-10-01

    The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G(1)/S transcriptional program by directly regulating MBF, the G(1)/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G(1)/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitro phosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Replacement of two potential phosphorylation sites with phosphomimetic amino acids suffices to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggests that checkpoint regulation of the MBF transcription factor is a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G(1)/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes.

  2. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Pedersen, Marianne Terndrup

    2011-01-01

    a role in transcriptional repression. TET1 binds a significant proportion of Polycomb group target genes. Furthermore, TET1 associates and colocalizes with the SIN3A co-repressor complex. We propose that TET1 fine-tunes transcription, opposes aberrant DNA methylation at CpG-rich sequences and thereby...... throughout the genome of embryonic stem cells, with the majority of binding sites located at transcription start sites (TSSs) of CpG-rich promoters and within genes. The hmC modification is found in gene bodies and in contrast to mC is also enriched at CpG-rich TSSs. We provide evidence further that TET1 has...... contributes to the regulation of DNA methylation fidelity....

  3. Uncovering Transcriptional Regulatory Networks by Sparse Bayesian Factor Model

    Directory of Open Access Journals (Sweden)

    Qi Yuan(Alan

    2010-01-01

    Full Text Available Abstract The problem of uncovering transcriptional regulation by transcription factors (TFs based on microarray data is considered. A novel Bayesian sparse correlated rectified factor model (BSCRFM is proposed that models the unknown TF protein level activity, the correlated regulations between TFs, and the sparse nature of TF-regulated genes. The model admits prior knowledge from existing database regarding TF-regulated target genes based on a sparse prior and through a developed Gibbs sampling algorithm, a context-specific transcriptional regulatory network specific to the experimental condition of the microarray data can be obtained. The proposed model and the Gibbs sampling algorithm were evaluated on the simulated systems, and results demonstrated the validity and effectiveness of the proposed approach. The proposed model was then applied to the breast cancer microarray data of patients with Estrogen Receptor positive ( status and Estrogen Receptor negative ( status, respectively.

  4. Targets of curcumin

    Science.gov (United States)

    Zhou, Hongyu; Beevers, Christopher S.; Huang, Shile

    2010-01-01

    Curcumin (diferuloylmethane), an orange-yellow component of turmeric or curry powder, is a polyphenol natural product isolated from the rhizome of the plant Curcuma longa. For centuries, curcumin has been used in some medicinal preparation or used as a food-coloring agent. In recent years, extensive in vitro and in vivo studies suggested curcumin has anticancer, antiviral, antiarthritic, anti-amyloid, antioxidant, and anti-inflammatory properties. The underlying mechanisms of these effects are diverse and appear to involve the regulation of various molecular targets, including transcription factors (such as nuclear factor-κB), growth factors (such as vascular endothelial cell growth factor), inflammatory cytokines (such as tumor necrosis factor, interleukin 1 and interleukin 6), protein kinases (such as mammalian target of rapamycin, mitogen-activated protein kinases, and Akt) and other enzymes (such as cyclooxygenase 2 and 5 lipoxygenase). Thus, due to its efficacy and regulation of multiple targets, as well as its safety for human use, curcumin has received considerable interest as a potential therapeutic agent for the prevention and/or treatment of various malignant diseases, arthritis, allergies, Alzheimer’s disease, and other inflammatory illnesses. This review summarizes various in vitro and in vivo pharmacological aspects of curcumin as well as the underlying action mechanisms. The recently identified molecular targets and signaling pathways modulated by curcumin are also discussed here. PMID:20955148

  5. The targets of curcumin.

    Science.gov (United States)

    Zhou, Hongyu; Beevers, Christopher S; Huang, Shile

    2011-03-01

    Curcumin (diferuloylmethane), an orange-yellow component of turmeric or curry powder, is a polyphenol natural product isolated from the rhizome of the plant Curcuma longa. For centuries, curcumin has been used in some medicinal preparation or used as a food-coloring agent. In recent years, extensive in vitro and in vivo studies suggested curcumin has anticancer, antiviral, antiarthritic, anti-amyloid, antioxidant, and anti-inflammatory properties. The underlying mechanisms of these effects are diverse and appear to involve the regulation of various molecular targets, including transcription factors (such as nuclear factor-kB), growth factors (such as vascular endothelial cell growth factor), inflammatory cytokines (such as tumor necrosis factor, interleukin 1 and interleukin 6), protein kinases (such as mammalian target of rapamycin, mitogen-activated protein kinases, and Akt) and other enzymes (such as cyclooxygenase 2 and 5 lipoxygenase). Thus, due to its efficacy and regulation of multiple targets, as well as its safety for human use, curcumin has received considerable interest as a potential therapeutic agent for the prevention and/or treatment of various malignant diseases, arthritis, allergies, Alzheimer's disease, and other inflammatory illnesses. This review summarizes various in vitro and in vivo pharmacological aspects of curcumin as well as the underlying action mechanisms. The recently identified molecular targets and signaling pathways modulated by curcumin are also discussed here.

  6. Metabolic and Transcriptional Reprogramming in Developing Soybean (Glycine max Embryos

    Directory of Open Access Journals (Sweden)

    Ruth Grene

    2013-05-01

    Full Text Available Soybean (Glycine max seeds are an important source of seed storage compounds, including protein, oil, and sugar used for food, feed, chemical, and biofuel production. We assessed detailed temporal transcriptional and metabolic changes in developing soybean embryos to gain a systems biology view of developmental and metabolic changes and to identify potential targets for metabolic engineering. Two major developmental and metabolic transitions were captured enabling identification of potential metabolic engineering targets specific to seed filling and to desiccation. The first transition involved a switch between different types of metabolism in dividing and elongating cells. The second transition involved the onset of maturation and desiccation tolerance during seed filling and a switch from photoheterotrophic to heterotrophic metabolism. Clustering analyses of metabolite and transcript data revealed clusters of functionally related metabolites and transcripts active in these different developmental and metabolic programs. The gene clusters provide a resource to generate predictions about the associations and interactions of unknown regulators with their targets based on “guilt-by-association” relationships. The inferred regulators also represent potential targets for future metabolic engineering of relevant pathways and steps in central carbon and nitrogen metabolism in soybean embryos and drought and desiccation tolerance in plants.

  7. Transcriptional regulation of c-fos

    International Nuclear Information System (INIS)

    Prywes, R.; Fisch, T.M.; Roeder, R.G.

    1988-01-01

    Expression of the c-fos proto-oncogene is induced rapidly and transiently by serum and other mitogenic agents. This rapid induction is therefore likely to involve posttranslational modifications and provides an excellent model for an early nuclear target of the signal transduction process, growth factors that bind to tyrosine kinase receptors. The authors have sought to understand the mechanism of transcriptional induction by each of these agents. The first step in this process was to identify the sequence elements in the c-fos gene responsible for induction by each of these agents. A specific element, termed serum response element (SRE), has been identified by transfection experiments of c-fos promoter constructs. To study regulation via SRE, a nuclear factor that binds to the SRE, termed serum response factor (SRF), has been identified with the gel mobility shift assay

  8. Exploring the utility of organo-polyoxometalate hybrids to inhibit SOX transcription factors.

    Science.gov (United States)

    Narasimhan, Kamesh; Micoine, Kevin; Lacôte, Emmanuel; Thorimbert, Serge; Cheung, Edwin; Hasenknopf, Bernold; Jauch, Ralf

    2014-01-01

    SOX transcription factors constitute an attractive target class for intervention with small molecules as they play a prominent role in the field of regenerative biomedicine and cancer biology. However, rationally engineering specific inhibitors that interfere with transcription factor DNA interfaces continues to be a monumental challenge in the field of transcription factor chemical biology. Polyoxometalates (POMs) are inorganic compounds that were previously shown to target the high-mobility group (HMG) of SOX proteins at nanomolar concentrations. In continuation of this work, we carried out an assessment of the selectivity of a panel of newly synthesized organo-polyoxometalate hybrids in targeting different transcription factor families to enable the usage of polyoxometalates as specific SOX transcription factor drugs. The residual DNA-binding activities of 15 different transcription factors were measured after treatment with a panel of diverse polyoxometalates. Polyoxometalates belonging to the Dawson structural class were found to be more potent inhibitors than the Keggin class. Further, organically modified Dawson polyoxometalates were found to be the most potent in inhibiting transcription factor DNA binding activity. The size of the polyoxometalates and its derivitization were found to be the key determinants of their potency. Polyoxometalates are highly potent, nanomolar range inhibitors of the DNA binding activity of the Sox-HMG family. However, binding assays involving a limited subset of structurally diverse polyoxometalates revealed a low selectivity profile against different transcription factor families. Further progress in achieving selectivity and deciphering structure-activity relationship of POMs require the identification of POM binding sites on transcription factors using elaborate approaches like X-ray crystallography and multidimensional NMR. In summary, our report reaffirms that transcription factors are challenging molecular architectures

  9. Assessing quality and completeness of human transcriptional regulatory pathways on a genome-wide scale

    Directory of Open Access Journals (Sweden)

    Aifantis Iannis

    2011-02-01

    Full Text Available Abstract Background Pathway databases are becoming increasingly important and almost omnipresent in most types of biological and translational research. However, little is known about the quality and completeness of pathways stored in these databases. The present study conducts a comprehensive assessment of transcriptional regulatory pathways in humans for seven well-studied transcription factors: MYC, NOTCH1, BCL6, TP53, AR, STAT1, and RELA. The employed benchmarking methodology first involves integrating genome-wide binding with functional gene expression data to derive direct targets of transcription factors. Then the lists of experimentally obtained direct targets are compared with relevant lists of transcriptional targets from 10 commonly used pathway databases. Results The results of this study show that for the majority of pathway databases, the overlap between experimentally obtained target genes and targets reported in transcriptional regulatory pathway databases is surprisingly small and often is not statistically significant. The only exception is MetaCore pathway database which yields statistically significant intersection with experimental results in 84% cases. Additionally, we suggest that the lists of experimentally derived direct targets obtained in this study can be used to reveal new biological insight in transcriptional regulation and suggest novel putative therapeutic targets in cancer. Conclusions Our study opens a debate on validity of using many popular pathway databases to obtain transcriptional regulatory targets. We conclude that the choice of pathway databases should be informed by solid scientific evidence and rigorous empirical evaluation. Reviewers This article was reviewed by Prof. Wing Hung Wong, Dr. Thiago Motta Venancio (nominated by Dr. L Aravind, and Prof. Geoff J McLachlan.

  10. Perfluorooctanoic acid stimulated mitochondrial biogenesis and gene transcription in rats

    International Nuclear Information System (INIS)

    Walters, M.W.; Bjork, J.A.; Wallace, K.B.

    2009-01-01

    Perfluorooctanoic acid (PFOA), used in the production of non-stick surface compounds, exhibits a worldwide distribution in the serum of humans and wildlife. In rodents PFOA transactivates PPARα and PPARγ nuclear receptors and increases mitochondrial DNA (mtDNA) copy number, which may be critical to the altered metabolic state of affected animals. A key regulator of mitochondrial biogenesis and transcription of mitochondrial genes is the PPARγ coactivator-1α (Pgc-1α) protein. The purpose of this study was to determine if Pgc-1α is implicated in the stimulation of mitochondrial biogenesis that occurs following the treatment of rats with PFOA. Livers from adult male Sprague-Dawley rats that received a 30 mg/kg daily oral dose of PFOA for 28 days were used for all experiments. Analysis of mitochondrial replication and transcription was performed by real time PCR, and proteins were detected using western blotting. PFOA treatment caused a transcriptional activation of the mitochondrial biogenesis pathway leading to a doubling of mtDNA copy number. Further, transcription of OXPHOS genes encoded by mtDNA was 3-4 times greater than that of nuclear encoded genes, suggestive of a preferential induction of mtDNA transcription. Western blot analysis revealed an increase in Pgc-1α, unchanged Tfam and decreased Cox II and Cox IV subunit protein expression. We conclude that PFOA treatment in rats induces mitochondrial biogenesis at the transcriptional level with a preferential stimulation of mtDNA transcription and that this occurs by way of activation of the Pgc-1α pathway. Implication of the Pgc-1α pathway is consistent with PPARγ transactivation by PFOA and reveals new understanding and possibly new critical targets for assessing or averting the associated metabolic disease.

  11. Transcriptional regulation of hepatic lipogenesis.

    Science.gov (United States)

    Wang, Yuhui; Viscarra, Jose; Kim, Sun-Joong; Sul, Hei Sook

    2015-11-01

    Fatty acid and fat synthesis in the liver is a highly regulated metabolic pathway that is important for very low-density lipoprotein (VLDL) production and thus energy distribution to other tissues. Having common features at their promoter regions, lipogenic genes are coordinately regulated at the transcriptional level. Transcription factors, such as upstream stimulatory factors (USFs), sterol regulatory element-binding protein 1C (SREBP1C), liver X receptors (LXRs) and carbohydrate-responsive element-binding protein (ChREBP) have crucial roles in this process. Recently, insights have been gained into the signalling pathways that regulate these transcription factors. After feeding, high blood glucose and insulin levels activate lipogenic genes through several pathways, including the DNA-dependent protein kinase (DNA-PK), atypical protein kinase C (aPKC) and AKT-mTOR pathways. These pathways control the post-translational modifications of transcription factors and co-regulators, such as phosphorylation, acetylation or ubiquitylation, that affect their function, stability and/or localization. Dysregulation of lipogenesis can contribute to hepatosteatosis, which is associated with obesity and insulin resistance.

  12. Structural insights into transcription complexes

    NARCIS (Netherlands)

    Berger, I.; Blanco, A.G.; Boelens, R.; Cavarelli, J.; Coll, M.; Folkers, G.E.; Nie, Y.; Pogenberg, V.; Schultz, P.; Wilmanns, M.; Moras, D.; Poterszman, A.

    2011-01-01

    Control of transcription allows the regulation of cell activity in response to external stimuli and research in the field has greatly benefited from efforts in structural biology. In this review, based on specific examples from the European SPINE2-COMPLEXES initiative, we illustrate the impact of

  13. Transcription factor-based biosensor

    Science.gov (United States)

    Dietrich, Jeffrey A; Keasling, Jay D

    2013-10-08

    The present invention provides for a system comprising a BmoR transcription factor, a .sigma..sup.54-RNA polymerase, and a pBMO promoter operatively linked to a reporter gene, wherein the pBMO promoter is capable of expression of the reporter gene with an activated form of the BmoR and the .sigma..sup.54-RNA polymerase.

  14. Transcriptional regulation by nonclassical action of thyroid hormone

    Directory of Open Access Journals (Sweden)

    Moeller Lars C

    2011-08-01

    Full Text Available Abstract Thyroid hormone (TH is essential for normal development, growth and metabolism. Its effects were thought to be principally mediated through triiodothyronine (T3, acting as a ligand for the nuclear TH receptors (TRs α and β residing on thyroid hormone response elements (TREs in the promoter of TH target genes. In this classical model of TH action, T3 binding to TRs leads to recruitment of basal transcription factors and increased transcription of TH responsive genes. Recently, the concept of TH action on gene expression has become more diverse and now includes nonclassical actions of T3 and T4: T3 has been shown to activate PI3K via the TRs, which ultimately increases transcription of certain genes, e.g. HIF-1α. Additionally, both T3 and thyroxine (T4 can bind to a membrane integrin, αvβ3, which leads to activation of the PI3K and MAPK signal transduction pathways and finally also increases gene transcription, e.g. of the FGF2 gene. Therefore, these initially nongenomic, nonclassical actions seem to serve as additional interfaces for transcriptional regulation by TH. Aim of this perspective is to summarize the genes that are currently known to be induced by nonclassical TH action and the mechanisms involved.

  15. Cooperative activation of transcription by autoimmune regulator AIRE and CBP

    International Nuclear Information System (INIS)

    Pitkaenen, J.; Rebane, A.; Rowell, J.; Murumaegi, A.; Stroebel, P.; Moell, K.; Saare, M.; Heikkilae, J.; Doucas, V.; Marx, A.; Peterson, P.

    2005-01-01

    Autoimmune regulator (AIRE) is a transcriptional regulator that is believed to control the expression of tissue-specific genes in the thymus. Mutated AIRE is responsible for onset of the hereditary autoimmune disease APECED. AIRE is able to form nuclear bodies (NBs) and interacts with the ubiquitous transcriptional coactivator CBP. In this paper, we show that CBP and AIRE synergistically activate transcription on different promoter reporters whereas AIRE gene mutation R257X, found in APECED patients, interferes with this coactivation effect. Furthermore, the overexpression of AIRE and CBP collaboratively enhance endogenous IFNβ mRNA expression. The immunohistochemical studies suggest that CBP, depending on the balance of nuclear proteins, is a component of AIRE NBs. We also show that AIRE NBs are devoid of active chromatin and, therefore, not sites of transcription. In addition, we demonstrate by 3D analyses that AIRE and CBP, when colocalizing, are located spatially differently within AIRE NBs. In conclusion, our data suggest that AIRE activates transcription of the target genes, i.e., autoantigens in collaboration with CBP and that this activation occurs outside of AIRE NBs

  16. Antiproton Target

    CERN Multimedia

    1980-01-01

    Antiproton target used for the AA (antiproton accumulator). The first type of antiproton production target used from 1980 to 1982 comprised a rod of copper 3mm diameter and 120mm long embedded in a graphite cylinder that was itself pressed into a finned aluminium container. This assembly was air-cooled and it was used in conjunction with the Van der Meer magnetic horn. In 1983 Fermilab provided us with lithium lenses to replace the horn with a view to increasing the antiproton yield by about 30%. These lenses needed a much shorter target made of heavy metal - iridium was chosen for this purpose. The 50 mm iridium rod was housed in an extension to the original finned target container so that it could be brought very close to the entrance to the lithium lens. Picture 1 shows this target assembly and Picture 2 shows it mounted together with the lithium lens. These target containers had a short lifetime due to a combination of beam heating and radiation damage. This led to the design of the water-cooled target in...

  17. Longitudinal evaluation of leukocyte transcripts in killer whales (Orcinus Orca)

    Science.gov (United States)

    Sitt, Tatjana; Bowen, Lizabeth; Lee, Chia-Shan; Blanchard, Myra; McBain, James; Dold, Christopher; Stott, Jeffrey L.

    2016-01-01

    Early identification of illness and/or presence of environmental and/or social stressors in free-ranging and domestic cetaceans is a priority for marine mammal health care professionals. Incorporation of leukocyte gene transcript analysis into the diagnostic tool kit has the potential to augment classical diagnostics based upon ease of sample storage and shipment, inducible nature and well-defined roles of transcription and associated downstream actions. Development of biomarkers that could serve to identify “insults” and potentially differentiate disease etiology would be of great diagnostic value. To this end, a modest number of peripheral blood leukocyte gene transcripts were selected for application to a domestic killer whale population with a focus on broad representation of inducible immunologically relevant genes. Normalized leukocyte transcript values, longitudinally acquired from 232 blood samples derived from 26 clinically healthy whales, were not visibly influenced temporally nor by sex or the specific Park in which they resided. Stability in leukocyte transcript number during periods of health enhances their potential use in diagnostics through identification of outliers. Transcript levels of two cytokine genes, IL-4 and IL-17, were highly variable within the group as compared to the other transcripts. IL-4 transcripts were typically absent. Analysis of transcript levels on the other genes of interest, on an individual animal basis, identified more outliers than were visible when analyzed in the context of the entire population. The majority of outliers (9 samples) were low, though elevated transcripts were identified for IL-17 from 2 animals and one each for Cox-2 and IL-10. The low number of outliers was not unexpected as sample selection was intentionally directed towards animals that were clinically healthy at the time of collection. Outliers may reflect animals experiencing subclinical disease that is transient and self-limiting. The

  18. A role for the transcription factor HEY1 in glioblastoma

    DEFF Research Database (Denmark)

    Hulleman, Esther; Quarto, Micaela; Vernell, Richard

    2009-01-01

    Glioblastoma multiforme (GBM), the highest-grade glioma, is the most frequent tumour of the brain with a very poor prognosis and limited therapeutic options. Although little is known about the molecular mechanisms that underlie glioblastoma formation, a number of signal transduction routes......, such as the Notch and Ras signalling pathways, seem to play an important role in the formation of GBM. In the present study, we show by in situ hybridization on primary tumour material that the transcription factor HEY1, a target of the Notch signalling pathway, is specifically upregulated in glioma...... and that expression of HEY1 in GBM correlates with tumour-grade and survival. In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways. Finally...

  19. 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.

  20. Hydrogen peroxide sensing, signaling and regulation of transcription factors

    Directory of Open Access Journals (Sweden)

    H. Susana Marinho

    2014-01-01

    Full Text Available The regulatory mechanisms by which hydrogen peroxide (H2O2 modulates the activity of transcription factors in bacteria (OxyR and PerR, lower eukaryotes (Yap1, Maf1, Hsf1 and Msn2/4 and mammalian cells (AP-1, NRF2, CREB, HSF1, HIF-1, TP53, NF-κB, NOTCH, SP1 and SCREB-1 are reviewed. The complexity of regulatory networks increases throughout the phylogenetic tree, reaching a high level of complexity in mammalians. Multiple H2O2 sensors and pathways are triggered converging in the regulation of transcription factors at several levels: (1 synthesis of the transcription factor by upregulating transcription or increasing both mRNA stability and translation; (ii stability of the transcription factor by decreasing its association with the ubiquitin E3 ligase complex or by inhibiting this complex; (iii cytoplasm–nuclear traffic by exposing/masking nuclear localization signals, or by releasing the transcription factor from partners or from membrane anchors; and (iv DNA binding and nuclear transactivation by modulating transcription factor affinity towards DNA, co-activators or repressors, and by targeting specific regions of chromatin to activate individual genes. We also discuss how H2O2 biological specificity results from diverse thiol protein sensors, with different reactivity of their sulfhydryl groups towards H2O2, being activated by different concentrations and times of exposure to H2O2. The specific regulation of local H2O2 concentrations is also crucial and results from H2O2 localized production and removal controlled by signals. Finally, we formulate equations to extract from typical experiments quantitative data concerning H2O2 reactivity with sensor molecules. Rate constants of 140 M−1 s−1 and ≥1.3 × 103 M−1 s−1 were estimated, respectively, for the reaction of H2O2 with KEAP1 and with an unknown target that mediates NRF2 protein synthesis. In conclusion, the multitude of H2O2 targets and mechanisms provides an opportunity for

  1. Serotonin transporter evolution and impact of polymorphic transcriptional regulation

    DEFF Research Database (Denmark)

    Søeby, Karen; Larsen, Svend Ask; Olsen, Line

    2005-01-01

    The serotonin transporter (SERT) is the primary drug target in the current antidepressant therapy. A functional polymorphism in the 2nd intron of the 5HTT gene encoding the SERT has been identified and associated with susceptibility to affective disorders and treatment response to antidepressants...... in the VNTRs of all mammalian SERT genes. The number of these putative binding sites varies proportionally to the length of the VNTR. We propose that the intronic VNTR have been selectively targeted through mammalian evolution to finetune transcriptional regulation of the serotonin expression....

  2. Alternative staffing services. Contract transcription.

    Science.gov (United States)

    Tessier, C

    1992-03-01

    Contract medical transcription services can be of great assistance in meeting the demands for transcription, without jeopardizing patient, physician, or institutional confidentiality. You simply must require the contract service to provide at least the same degree of protection and preservation of confidentiality that you should require inhouse. To achieve this you must make these requirements explicit, comprehensive, comprehensible, believable, and enforceable. Discuss the requirements with prospective contractors. Review them at least annually with existing contractors and when contracts are due for renewal. Be sure to specify the consequence of breaching confidentiality, and if there are breaches, enforce the terms of the contract. Consult your institution's legal counsel both in developing the contract and in enforcing its provisions. Take into consideration your department's and institution's policies, AHIMA's statement on confidentiality, as well as local, state, and federal laws. Above all, never lose sight of the patient. Ultimately, it is not patient information that you are obligated to protect. It is the patient.

  3. PAX3-FOXO1: Zooming in on an "undruggable" target.

    Science.gov (United States)

    Wachtel, Marco; Schäfer, Beat W

    2018-06-01

    Driver oncogenes are prime targets for therapy in tumors many of which, including leukemias and sarcomas, express recurrent fusion transcription factors. One specific example for such a cancer type is alveolar rhabdomyosarcoma, which is associated in the majority of cases with the fusion protein PAX3-FOXO1. Since fusion transcription factors are challenging targets for development of small molecule inhibitors, indirect inhibitory strategies for this type of oncogenes represent a more promising approach. One can envision strategies at different molecular levels including upstream modifiers and activators, epigenetic and transcriptional co-regulators, and downstream effector targets. In this review, we will discuss the current knowledge regarding potential therapeutic targets that might contribute to indirect interference with PAX3-FOXO1 activity in alveolar rhabdomyosarcoma at the different molecular levels and extrapolate these findings to fusion transcription factors in general. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  4. The post-transcriptional operon

    DEFF Research Database (Denmark)

    Tenenbaum, Scott A.; Christiansen, Jan; Nielsen, Henrik

    2011-01-01

    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...... of the model in identifying new participants in cellular pathways as well as in deepening our understanding of cellular responses....

  5. E2F1-Mediated Induction of NFYB Attenuates Apoptosis via Joint Regulation of a Pro-Survival Transcriptional Program.

    Directory of Open Access Journals (Sweden)

    Xiaolei Jiang

    Full Text Available The E2F1 transcription factor regulates cell proliferation and apoptosis through the control of a considerable variety of target genes. Previous work has detailed the role of other transcription factors in mediating the specificity of E2F function. Here we identify the NF-YB transcription factor as a novel direct E2F1 target. Genome-wide expression analysis of the effects of NFYB knockdown on E2F1-mediated transcription identified a large group of genes that are co-regulated by E2F1 and NFYB. We also provide evidence that knockdown of NFYB enhances E2F1-induced apoptosis, suggesting a pro-survival function of the NFYB/E2F1 joint transcriptional program. Bioinformatic analysis suggests that deregulation of these NFY-dependent E2F1 target genes might play a role in sarcomagenesis as well as drug resistance.

  6. Nascent-Seq reveals novel features of mouse circadian transcriptional regulation

    Science.gov (United States)

    Menet, Jerome S; Rodriguez, Joseph; Abruzzi, Katharine C; Rosbash, Michael

    2012-01-01

    A substantial fraction of the metazoan transcriptome undergoes circadian oscillations in many cells and tissues. Based on the transcription feedback loops important for circadian timekeeping, it is commonly assumed that this mRNA cycling reflects widespread transcriptional regulation. To address this issue, we directly measured the circadian dynamics of mouse liver transcription using Nascent-Seq (genome-wide sequencing of nascent RNA). Although many genes are rhythmically transcribed, many rhythmic mRNAs manifest poor transcriptional rhythms, indicating a prominent contribution of post-transcriptional regulation to circadian mRNA expression. This analysis of rhythmic transcription also showed that the rhythmic DNA binding profile of the transcription factors CLOCK and BMAL1 does not determine the transcriptional phase of most target genes. This likely reflects gene-specific collaborations of CLK:BMAL1 with other transcription factors. These insights from Nascent-Seq indicate that it should have broad applicability to many other gene expression regulatory issues. DOI: http://dx.doi.org/10.7554/eLife.00011.001 PMID:23150795

  7. DNA replication initiator Cdc6 also regulates ribosomal DNA transcription initiation.

    Science.gov (United States)

    Huang, Shijiao; Xu, Xiaowei; Wang, Guopeng; Lu, Guoliang; Xie, Wenbing; Tao, Wei; Zhang, Hongyin; Jiang, Qing; Zhang, Chuanmao

    2016-04-01

    RNA-polymerase-I-dependent ribosomal DNA (rDNA) transcription is fundamental to rRNA processing, ribosome assembly and protein synthesis. However, how this process is initiated during the cell cycle is not fully understood. By performing a proteomic analysis of transcription factors that bind RNA polymerase I during rDNA transcription initiation, we identified that the DNA replication initiator Cdc6 interacts with RNA polymerase I and its co-factors, and promotes rDNA transcription in G1 phase in an ATPase-activity-dependent manner. We further showed that Cdc6 is targeted to the nucleolus during late mitosis and G1 phase in a manner that is dependent on B23 (also known as nucleophosmin, NPM1), and preferentially binds to the rDNA promoter through its ATP-binding domain. Overexpression of Cdc6 increases rDNA transcription, whereas knockdown of Cdc6 results in a decreased association of both RNA polymerase I and the RNA polymerase I transcription factor RRN3 with rDNA, and a reduction of rDNA transcription. Furthermore, depletion of Cdc6 impairs the interaction between RRN3 and RNA polymerase I. Taken together, our data demonstrate that Cdc6 also serves as a regulator of rDNA transcription initiation, and indicate a mechanism by which initiation of rDNA transcription and DNA replication can be coordinated in cells. © 2016. Published by The Company of Biologists Ltd.

  8. Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

    Energy Technology Data Exchange (ETDEWEB)

    Tennyson, C.N.; Worton, R.G. [Univ. of Toronto and the Hospital for Sick Children, Ontario (Canada)

    1994-09-01

    The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

  9. Mutual interdependence of splicing and transcription elongation.

    Science.gov (United States)

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

    Transcription and splicing are intrinsically linked, as splicing needs a pre-mRNA substrate to commence. The more nuanced view is that the rate of transcription contributes to splicing regulation. On the other hand there is accumulating evidence that splicing has an active role in controlling transcription elongation by DNA-dependent RNA polymerase II (RNAP II). We briefly review those mechanisms and propose a unifying model where splicing controls transcription elongation to provide an optimal timing for successive rounds of splicing.

  10. Targeted Learning

    CERN Document Server

    van der Laan, Mark J

    2011-01-01

    The statistics profession is at a unique point in history. The need for valid statistical tools is greater than ever; data sets are massive, often measuring hundreds of thousands of measurements for a single subject. The field is ready to move towards clear objective benchmarks under which tools can be evaluated. Targeted learning allows (1) the full generalization and utilization of cross-validation as an estimator selection tool so that the subjective choices made by humans are now made by the machine, and (2) targeting the fitting of the probability distribution of the data toward the targe

  11. Target preparation

    International Nuclear Information System (INIS)

    Hinn, G.M.

    1984-01-01

    A few of the more interesting of the 210 targets prepared in the Laboratory last year are listed. In addition the author continues to use powdered silver mixed with /sup 9,10/BeO to produce sources for accelerator radio dating of Alaskan and South Polar snow. Currently, he is trying to increase production by multiple sample processing. Also the author routinely makes 3 μg/cm 2 cracked slacked carbon stripper foils and is continuing research with some degree of success in making enriched 28 Si targets starting with the oxide

  12. Interplay between DNA supercoiling and transcription elongation.

    Science.gov (United States)

    Ma, Jie; Wang, Michelle

    2014-01-01

    Transcription-coupled DNA supercoiling has been shown to be an important regulator of transcription that is broadly present in the cell. Here we review experimental work which shows that RNA polymerase is a powerful torsional motor that can alter DNA topology and structure, and DNA supercoiling in turn directly affects transcription elongation.

  13. Global Analysis of Photosynthesis Transcriptional Regulatory Networks

    Science.gov (United States)

    Imam, Saheed; Noguera, Daniel R.; Donohue, Timothy J.

    2014-01-01

    Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis. PMID:25503406

  14. Global analysis of photosynthesis transcriptional regulatory networks.

    Directory of Open Access Journals (Sweden)

    Saheed Imam

    2014-12-01

    Full Text Available Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888, which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

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

    Directory of Open Access Journals (Sweden)

    Xiang Zhou

    2010-03-01

    Full Text Available 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.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.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-interaction detection and TFBS-discovery accuracy. We estimated the accuracy

  16. Metabolic Network Topology Reveals Transcriptional Regulatory Signatures of Type 2 Diabetes

    DEFF Research Database (Denmark)

    Zelezniak, Aleksej; Pers, Tune Hannes; Pinho Soares, Simao Pedro

    2010-01-01

    mechanisms underlying these transcriptional changes and their impact on the cellular metabolic phenotype is a challenging task due to the complexity of transcriptional regulation and the highly interconnected nature of the metabolic network. In this study we integrate skeletal muscle gene expression datasets...... with human metabolic network reconstructions to identify key metabolic regulatory features of T2DM. These features include reporter metabolites—metabolites with significant collective transcriptional response in the associated enzyme-coding genes, and transcription factors with significant enrichment...... factor regulatory network connecting several parts of metabolism. The identified transcription factors include members of the CREB, NRF1 and PPAR family, among others, and represent regulatory targets for further experimental analysis. Overall, our results provide a holistic picture of key metabolic...

  17. The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line

    DEFF Research Database (Denmark)

    Suzuki, Harukazu; Forrest, Alistair R R; van Nimwegen, Erik

    2009-01-01

    , we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks......Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites...... involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process....

  18. Directing traffic on DNA-How transcription factors relieve or induce transcriptional interference.

    Science.gov (United States)

    Hao, Nan; Palmer, Adam C; Dodd, Ian B; Shearwin, Keith E

    2017-03-15

    Transcriptional interference (TI) is increasingly recognized as a widespread mechanism of gene control, particularly given the pervasive nature of transcription, both sense and antisense, across all kingdoms of life. Here, we discuss how transcription factor binding kinetics strongly influence the ability of a transcription factor to relieve or induce TI.

  19. Uncovering transcriptional interactions via an adaptive fuzzy logic approach

    Directory of Open Access Journals (Sweden)

    Chen Chung-Ming

    2009-12-01

    Full Text Available Abstract Background To date, only a limited number of transcriptional regulatory interactions have been uncovered. In a pilot study integrating sequence data with microarray data, a position weight matrix (PWM performed poorly in inferring transcriptional interactions (TIs, which represent physical interactions between transcription factors (TF and upstream sequences of target genes. Inferring a TI means that the promoter sequence of a target is inferred to match the consensus sequence motifs of a potential TF, and their interaction type such as AT or RT is also predicted. Thus, a robust PWM (rPWM was developed to search for consensus sequence motifs. In addition to rPWM, one feature extracted from ChIP-chip data was incorporated to identify potential TIs under specific conditions. An interaction type classifier was assembled to predict activation/repression of potential TIs using microarray data. This approach, combining an adaptive (learning fuzzy inference system and an interaction type classifier to predict transcriptional regulatory networks, was named AdaFuzzy. Results AdaFuzzy was applied to predict TIs using real genomics data from Saccharomyces cerevisiae. Following one of the latest advances in predicting TIs, constrained probabilistic sparse matrix factorization (cPSMF, and using 19 transcription factors (TFs, we compared AdaFuzzy to four well-known approaches using over-representation analysis and gene set enrichment analysis. AdaFuzzy outperformed these four algorithms. Furthermore, AdaFuzzy was shown to perform comparably to 'ChIP-experimental method' in inferring TIs identified by two sets of large scale ChIP-chip data, respectively. AdaFuzzy was also able to classify all predicted TIs into one or more of the four promoter architectures. The results coincided with known promoter architectures in yeast and provided insights into transcriptional regulatory mechanisms. Conclusion AdaFuzzy successfully integrates multiple types of

  20. Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

    International Nuclear Information System (INIS)

    Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha; Jeong, Jae Hoon; Pak, Youngmi Kim

    2014-01-01

    Highlights: • TFAM localizes in nuclei and mitochondria of neuronal cells. • Nuclear TFAM does not bind the Tfam promoter. • Nuclear TFAM reduced the Tfam promoter activity via suppressing NRF-1 activity. • A novel self-negative feedback regulation of Tfam gene expression is explored. • FAM may play different roles depending on its subcellular localizations. - Abstract: The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated that TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations

  1. Scaling proprioceptor gene transcription by retrograde NT3 signaling.

    Directory of Open Access Journals (Sweden)

    Jun Lee

    Full Text Available Cell-type specific intrinsic programs instruct neuronal subpopulations before target-derived factors influence later neuronal maturation. Retrograde neurotrophin signaling controls neuronal survival and maturation of dorsal root ganglion (DRG sensory neurons, but how these potent signaling pathways intersect with transcriptional programs established at earlier developmental stages remains poorly understood. Here we determine the consequences of genetic alternation of NT3 signaling on genome-wide transcription programs in proprioceptors, an important sensory neuron subpopulation involved in motor reflex behavior. We find that the expression of many proprioceptor-enriched genes is dramatically altered by genetic NT3 elimination, independent of survival-related activities. Combinatorial analysis of gene expression profiles with proprioceptors isolated from mice expressing surplus muscular NT3 identifies an anticorrelated gene set with transcriptional levels scaled in opposite directions. Voluntary running experiments in adult mice further demonstrate the maintenance of transcriptional adjustability of genes expressed by DRG neurons, pointing to life-long gene expression plasticity in sensory neurons.

  2. Transcriptional analysis of exopolysaccharides biosynthesis gene clusters in Lactobacillus plantarum.

    Science.gov (United States)

    Vastano, Valeria; Perrone, Filomena; Marasco, Rosangela; Sacco, Margherita; Muscariello, Lidia

    2016-04-01

    Exopolysaccharides (EPS) from lactic acid bacteria contribute to specific rheology and texture of fermented milk products and find applications also in non-dairy foods and in therapeutics. Recently, four clusters of genes (cps) associated with surface polysaccharide production have been identified in Lactobacillus plantarum WCFS1, a probiotic and food-associated lactobacillus. These clusters are involved in cell surface architecture and probably in release and/or exposure of immunomodulating bacterial molecules. Here we show a transcriptional analysis of these clusters. Indeed, RT-PCR experiments revealed that the cps loci are organized in five operons. Moreover, by reverse transcription-qPCR analysis performed on L. plantarum WCFS1 (wild type) and WCFS1-2 (ΔccpA), we demonstrated that expression of three cps clusters is under the control of the global regulator CcpA. These results, together with the identification of putative CcpA target sequences (catabolite responsive element CRE) in the regulatory region of four out of five transcriptional units, strongly suggest for the first time a role of the master regulator CcpA in EPS gene transcription among lactobacilli.

  3. In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters.

    OpenAIRE

    Buckley, SM; Delhove, JM; Perocheau, DP; Karda, R; Rahim, AA; Howe, SJ; Ward, NJ; Birrell, MA; Belvisi, MG; Arbuthnot, P; Johnson, MR; Waddington, SN; McKay, TR

    2015-01-01

    The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruse...

  4. Orphan nuclear receptor TLX recruits histone deacetylases to repress transcription and regulate neural stem cell proliferation

    OpenAIRE

    Sun, GuoQiang; Yu, Ruth T.; Evans, Ronald M.; Shi, Yanhong

    2007-01-01

    TLX is a transcription factor that is essential for neural stem cell proliferation and self-renewal. However, the molecular mechanism of TLX-mediated neural stem cell proliferation and self-renewal is largely unknown. We show here that TLX recruits histone deacetylases (HDACs) to its downstream target genes to repress their transcription, which in turn regulates neural stem cell proliferation. TLX interacts with HDAC3 and HDAC5 in neural stem cells. The HDAC5-interaction domain was mapped to ...

  5. Targeting of microRNAs for therapeutics

    DEFF Research Database (Denmark)

    Stenvang, Jan; Lindow, Morten; Kauppinen, Sakari

    2008-01-01

    miRNAs (microRNAs) comprise a class of small endogenous non-coding RNAs that post-transcriptionally repress gene expression by base-pairing with their target mRNAs. Recent evidence has shown that miRNAs play important roles in a wide variety of human diseases, such as viral infections, cancer...

  6. The Journey of a Transcription Factor

    DEFF Research Database (Denmark)

    Pireyre, Marie

    Plants have developed astonishing networks regulating their metabolism to adapt to their environment. The complexity of these networks is illustrated by the expansion of families of regulators such as transcription factors in the plant kingdom. Transcription factors specifically impact...... transcriptional networks by integrating exogenous and endogenous stimuli and regulating gene expression accordingly. Regulation of transcription factors and their activation is thus highly important to modulate the transcriptional programs and increase fitness of the plant in a given environment. Plant metabolism....... The biosynthetic machinery of GLS is governed by interplay of six MYB and three bHLH transcription factors. MYB28, MYB29 and MYB76 regulate methionine-derived GLS, and MYB51, MYB34 and MYB122 regulate tryptophan-derived GLS. The three bHLH transcription factors MYC2, MYC3 and MYC4 physically interact with all six...

  7. Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe

    OpenAIRE

    Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.

    2012-01-01

    In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription ...

  8. Exploring the utility of organo-polyoxometalate hybrids to inhibit SOX transcription factors

    Directory of Open Access Journals (Sweden)

    Kamesh Narasimhan

    2014-01-01

    Conclusion: Polyoxometalates are highly potent, nanomolar range inhibitors of the DNA binding activity of the Sox-HMG family. However, binding assays involving a limited subset of structurally diverse polyoxometalates revealed a low selectivity profile against different transcription factor families. Further progress in achieving selectivity and deciphering structure-activity relationship of POMs require the identification of POM binding sites on transcription factors using elaborate approaches like X-ray crystallography and multidimensional NMR. In summary, our report reaffirms that transcription factors are challenging molecular architectures and that future polyoxometalate chemistry must consider further modification strategies, to address the substantial challenges involved in achieving target selectivity.

  9. Post-translational regulation of Oct4 transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Jonathan P Saxe

    Full Text Available Oct4 is a key component of the molecular circuitry which regulates embryonic stem cell proliferation and differentiation. It is essential for maintenance of undifferentiated, pluripotent cell populations, and accomplishes these tasks by binding DNA in multiple heterodimer and homodimer configurations. Very little is known about how formation of these complexes is regulated, or the mechanisms through which Oct4 proteins respond to complex extracellular stimuli which regulate pluripotency. Here, we provide evidence for a phosphorylation-based mechanism which regulates specific Oct4 homodimer conformations. Point mutations of a putative phosphorylation site can specifically abrogate transcriptional activity of a specific homodimer assembly, with little effect on other configurations. Moreover, we performed bioinformatic predictions to identify a subset of Oct4 target genes which may be regulated by this specific assembly, and show that altering Oct4 protein levels affects transcription of Oct4 target genes which are regulated by this assembly but not others. Finally, we identified several signaling pathways which may mediate this phosphorylation and act in combination to regulate Oct4 transcriptional activity and protein stability. These results provide a mechanism for rapid and reversible alteration of Oct4 transactivation potential in response to extracellular signals.

  10. Silencing of IFN-stimulated gene transcription is regulated by histone H1 and its chaperone TAF-I.

    Science.gov (United States)

    Kadota, Shinichi; Nagata, Kyosuke

    2014-07-01

    Chromatin structure and its alteration play critical roles in the regulation of transcription. However, the transcriptional silencing mechanism with regard to the chromatin structure at an unstimulated state of the interferon (IFN)-stimulated gene (ISG) remains unclear. Here we investigated the role of template activating factor-I (TAF-I, also known as SET) in ISG transcription. Knockdown (KD) of TAF-I increased ISG transcript and simultaneously reduced the histone H1 level on the ISG promoters during the early stages of transcription after IFN stimulation from the unstimulated state. The transcription factor levels on the ISG promoters were increased in TAF-I KD cells only during the early stages of transcription. Furthermore, histone H1 KD also increased ISG transcript. TAF-I and histone H1 double KD did not show the additive effect in ISG transcription, suggesting that TAF-I and histone H1 may act on the same regulatory pathway to control ISG transcription. In addition, TAF-I KD and histone H1 KD affected the chromatin structure near the ISG promoters. On the basis of these findings, we propose that TAF-I and its target histone H1 are key regulators of the chromatin structure at the ISG promoter to maintain the silent state of ISG transcription. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. The metabolic activator FOXO1 binds hepatitis B virus DNA and activates its transcription

    International Nuclear Information System (INIS)

    Shlomai, Amir; Shaul, Yosef

    2009-01-01

    Hepatitis B virus (HBV) is a small DNA virus that targets the liver and infects humans worldwide. Recently we have shown that the metabolic regulator PGC-1α coactivates HBV transcription thereby rendering the virus susceptible to fluctuations in the nutritional status of the liver. PGC-1α coactivation of HBV is mediated through the liver-enriched nuclear receptor HNF4α and through another yet unknown transcription factor(s). Here we show that the forkhead transcription factor FOXO1, a known target for PGC-1α coactivation and a central mediator of glucose metabolism in the liver, binds HBV core promoter and activates its transcription. This activation is further enhanced in the presence of PGC-1α, implying that FOXO1 is a target for PGC-1α coactivation of HBV transcription. Thus, our results identify another key metabolic regulator as an activator of HBV transcription, thereby supporting the principle that HBV gene expression is regulated in a similar way to key hepatic metabolic genes.

  12. Transcriptional Regulatory Network Analysis of MYB Transcription Factor Family Genes in Rice

    Directory of Open Access Journals (Sweden)

    Shuchi eSmita

    2015-12-01

    Full Text Available MYB transcription factor (TF is one of the largest TF families and regulates defense responses to various stresses, hormone signaling as well as many metabolic and developmental processes in plants. Understanding these regulatory hierarchies of gene expression networks in response to developmental and environmental cues is a major challenge due to the complex interactions between the genetic elements. Correlation analyses are useful to unravel co-regulated gene pairs governing biological process as well as identification of new candidate hub genes in response to these complex processes. High throughput expression profiling data are highly useful for construction of co-expression networks. In the present study, we utilized transcriptome data for comprehensive regulatory network studies of MYB TFs by top down and guide gene approaches. More than 50% of OsMYBs were strongly correlated under fifty experimental conditions with 51 hub genes via top down approach. Further, clusters were identified using Markov Clustering (MCL. To maximize the clustering performance, parameter evaluation of the MCL inflation score (I was performed in terms of enriched GO categories by measuring F-score. Comparison of co-expressed cluster and clads analyzed from phylogenetic analysis signifies their evolutionarily conserved co-regulatory role. We utilized compendium of known interaction and biological role with Gene Ontology enrichment analysis to hypothesize function of coexpressed OsMYBs. In the other part, the transcriptional regulatory network analysis by guide gene approach revealed 40 putative targets of 26 OsMYB TF hubs with high correlation value utilizing 815 microarray data. The putative targets with MYB-binding cis-elements enrichment in their promoter region, functional co-occurrence as well as nuclear localization supports our finding. Specially, enrichment of MYB binding regions involved in drought-inducibility implying their regulatory role in drought

  13. Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

    DEFF Research Database (Denmark)

    Damgaard Jensen, Emil; Ferreira, Raphael; Jakociunas, Tadas

    2017-01-01

    on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene...... transcription start site. In this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker's yeast Saccharomyces cerevisiae. By testing 101...... production and increases in TAG. Taken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also...

  14. A transcription activator-like effector (TALE) induction system mediated by proteolysis.

    Science.gov (United States)

    Copeland, Matthew F; Politz, Mark C; Johnson, Charles B; Markley, Andrew L; Pfleger, Brian F

    2016-04-01

    Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic.

  15. Chemically Induced Degradation of the Oncogenic Transcription Factor BCL6

    Directory of Open Access Journals (Sweden)

    Nina Kerres

    2017-09-01

    Full Text Available The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL. Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.

  16. Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

    Science.gov (United States)

    Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

    2015-11-05

    Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Transcriptional landscapes of Axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    Caballero-Pérez, Juan; Espinal-Centeno, Annie; Falcon, Francisco; García-Ortega, Luis F; Curiel-Quesada, Everardo; Cruz-Hernández, Andrés; Bako, Laszlo; Chen, Xuemei; Martínez, Octavio; Alberto Arteaga-Vázquez, Mario; Herrera-Estrella, Luis; Cruz-Ramírez, Alfredo

    2018-01-15

    The axolotl (Ambystoma mexicanum) is the vertebrate model system with the highest regeneration capacity. Experimental tools established over the past 100 years have been fundamental to start unraveling the cellular and molecular basis of tissue and limb regeneration. In the absence of a reference genome for the Axolotl, transcriptomic analysis become fundamental to understand the genetic basis of regeneration. Here we present one of the most diverse transcriptomic data sets for Axolotl by profiling coding and non-coding RNAs from diverse tissues. We reconstructed a population of 115,906 putative protein coding mRNAs as full ORFs (including isoforms). We also identified 352 conserved miRNAs and 297 novel putative mature miRNAs. Systematic enrichment analysis of gene expression allowed us to identify tissue-specific protein-coding transcripts. We also found putative novel and conserved microRNAs which potentially target mRNAs which are reported as important disease candidates in heart and liver. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Suppression of PTEN transcription by UVA

    Science.gov (United States)

    Zhao, Baozhong; Ming, Mei; He, Yu-Ying

    2012-01-01

    Although UVA has different physical and biological targets than UVB, the contribution of UVA to skin cancer susceptibility and its molecular basis remain largely unknown. Here we show that chronic UVA radiation suppresses PTEN expression at the mRNA level. Subchronic and acute UVA radiation also down-regulated PTEN in normal human epidermal keratinocytes, skin culture and mouse skin. At the molecular level, chronic UVA radiation decreased the transcriptional activity of the PTEN promoter in a methylation-independent manner, while it had no effect on the protein stability or mRNA stability of PTEN. In contrast, we found that UVA-induced activation of the Ras/ERK/AKT and NF-κB pathways plays an important role in UV-induced PTEN down-regulation. Inhibiting ERK or AKT increases PTEN expression. Our findings may provide unique insights into PTEN down-regulation as a critical component of UVA’s molecular impact during keratinocyte transformation. PMID:23129115

  19. Repressive effects of resveratrol on androgen receptor transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Wen-feng Shi

    2009-10-01

    Full Text Available The chemopreventive effects of resveratrol (RSV on prostate cancer have been well established; the androgen receptor (AR plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity.The AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+ cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(- cells serving as controls. AR(+ cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE.AR in the AR (+ stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment.We demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding.

  20. Promoter proximal polyadenylation sites reduce transcription activity

    DEFF Research Database (Denmark)

    Andersen, Pia Kjølhede; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site......, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites...... on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500...

  1. Transcription and recombination: when RNA meets DNA.

    Science.gov (United States)

    Aguilera, Andrés; Gaillard, Hélène

    2014-08-01

    A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription-replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  2. Specificity and robustness in transcription control networks.

    Science.gov (United States)

    Sengupta, Anirvan M; Djordjevic, Marko; Shraiman, Boris I

    2002-02-19

    Recognition by transcription factors of the regulatory DNA elements upstream of genes is the fundamental step in controlling gene expression. How does the necessity to provide stability with respect to mutation constrain the organization of transcription control networks? We examine the mutation load of a transcription factor interacting with a set of n regulatory response elements as a function of the factor/DNA binding specificity and conclude on theoretical grounds that the optimal specificity decreases with n. The predicted correlation between variability of binding sites (for a given transcription factor) and their number is supported by the genomic data for Escherichia coli. The analysis of E. coli genomic data was carried out using an algorithm suggested by the biophysical model of transcription factor/DNA binding. Complete results of the search for candidate transcription factor binding sites are available at http://www.physics.rockefeller.edu/~boris/public/search_ecoli.

  3. NF-Y loss triggers p53 stabilization and apoptosis in HPV18-positive cells by affecting E6 transcription.

    Science.gov (United States)

    Benatti, Paolo; Basile, Valentina; Dolfini, Diletta; Belluti, Silvia; Tomei, Margherita; Imbriano, Carol

    2016-07-19

    The expression of the high risk HPV18 E6 and E7 oncogenic proteins induces the transformation of epithelial cells, through the disruption of p53 and Rb function. The binding of cellular transcription factors to cis-regulatory elements in the viral Upstream Regulatory Region (URR) stimulates E6/E7 transcription. Here, we demonstrate that the CCAAT-transcription factor NF-Y binds to a non-canonical motif within the URR and activates viral gene expression. In addition, NF-Y indirectly up-regulates HPV18 transcription through the transactivation of multiple cellular transcription factors. NF-YA depletion inhibits the expression of E6 and E7 genes and re-establishes functional p53. The activation of p53 target genes in turn leads to apoptotic cell death. Finally, we show that NF-YA loss sensitizes HPV18-positive cells toward the DNA damaging agent Doxorubicin, via p53-mediated transcriptional response.

  4. ARX MPC for people with type 1 diabetes

    DEFF Research Database (Denmark)

    Boiroux, Dimitri; Finan, Daniel Aaron; Jørgensen, John Bagterp

    Type 1 diabetes is a chronic disease characterized by a lack of production of pancreatic insulin, consequently leading to high blood glucose concentrations (hyperglycemia). Hyperglycemia has negative health effects in the long term such as eye, nerve, and kidney disease. Exogenous insulin must......, or even death. Currently, insulin administration is performed by the subject with type 1 diabetes based on infrequent glucose measurements (in the form of finger-sticks), often resulting in an unsatisfactory blood glucose control. An artificial pancreas is a medical device that injects exogenous insulin...... and insulin injection information to compute the optimal insulin administration for the current conditions. We use model predictive control (MPC) to compute the optimal insulin administration for 20 virtual type 1 diabetes subjects. The system (i.e., subject) has one manipulated input (insulin infusion rate...

  5. 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.

  6. Transcriptional networks and chromatin remodeling controlling adipogenesis

    DEFF Research Database (Denmark)

    Siersbæk, Rasmus; Nielsen, Ronni; Mandrup, Susanne

    2012-01-01

    Adipocyte differentiation is tightly controlled by a transcriptional cascade, which directs the extensive reprogramming of gene expression required to convert fibroblast-like precursor cells into mature lipid-laden adipocytes. Recent global analyses of transcription factor binding and chromatin...... remodeling have revealed 'snapshots' of this cascade and the chromatin landscape at specific time-points of differentiation. These studies demonstrate that multiple adipogenic transcription factors co-occupy hotspots characterized by an open chromatin structure and specific epigenetic modifications....... Such transcription factor hotspots are likely to represent key signaling nodes which integrate multiple adipogenic signals at specific chromatin sites, thereby facilitating coordinated action on gene expression....

  7. NAC transcription factors in senescence

    DEFF Research Database (Denmark)

    Podzimska-Sroka, Dagmara; O'Shea, Charlotte; Gregersen, Per L.

    2015-01-01

    involving the hormone abscisic acid, Arabidopsis NAP promotes chlorophyll degradation, a hallmark of senescence. Furthermore, studies of the functional rice ortholog, OsNAP, suggest that NAC genes can be targeted to obtain specific changes in lifespan control and nutrient remobilization in crop plants...

  8. RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins

    KAUST Repository

    Baazim, Hatoon

    2014-05-01

    Developing targeted genome regulation approaches holds much promise for accelerating trait discovery and development in agricultural biotechnology. Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) system provides bacteria and archaea with an adaptive molecular immunity mechanism against invading nucleic acids through phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing purposes across a variety of cell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) protein combined with guide RNAs (gRNAs) were used as a DNA-targeting platform to modulate the expression patterns in bacterial, yeast and human cells. Here, we employed this DNA-targeting system for targeted transcriptional regulation in planta by developing chimeric dCas9-based activators and repressors. For example, we fused to the C-terminus of dCas9 with the activation domains of EDLL and TAL effectors, respectively, to generate transcriptional activators, and the SRDX repression domain to generate transcriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TAD activators, guided by gRNAs complementary to promoter elements, induce strong transcriptional activation on episomal targets in plant cells. Moreover, our data suggest that the dCas9:SRDX repressor and the dCas9:EDLL and dCas9:TAD activators are capable of markedly repressing or activating, respectively, the transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9:TFs DNA targeting system can be used in plants as a functional genomic tool and for biotechnological applications.

  9. Transcriptional repression of BODENLOS by HD-ZIP transcription factor HB5 in Arabidopsis thaliana.

    NARCIS (Netherlands)

    Smet, De I.; Lau, S.; Ehrismann, J.S.; Axiotis, I.; Kolb, M.; Kientz, M.; Weijers, D.; Jürgens, G.

    2013-01-01

    In Arabidopsis thaliana, the phytohormone auxin is an important patterning agent during embryogenesis and post-embryonic development, exerting effects through transcriptional regulation. The main determinants of the transcriptional auxin response machinery are AUXIN RESPONSE FACTOR (ARF)

  10. Serotonin transporter evolution and impact of polymorphic transcriptional regulation

    DEFF Research Database (Denmark)

    Søeby, Karen; Larsen, Svend Ask; Olsen, Line

    2005-01-01

    The serotonin transporter (SERT) is the primary drug target in the current antidepressant therapy. A functional polymorphism in the 2nd intron of the 5HTT gene encoding the SERT has been identified and associated with susceptibility to affective disorders and treatment response to antidepressants....... This study addresses the possible impact of the variable number of tandem repeats (VNTR) to behavior and disease by examining the evolutionary origin and mechanisms of differential transcriptional regulation of SERT. We trace the evolutionary origin of the VNTR and show that it is present and varies...

  11. EBF factors drive expression of multiple classes of target genes governing neuronal development.

    Science.gov (United States)

    Green, Yangsook S; Vetter, Monica L

    2011-04-30

    Early B cell factor (EBF) family members are transcription factors known to have important roles in several aspects of vertebrate neurogenesis, including commitment, migration and differentiation. Knowledge of how EBF family members contribute to neurogenesis is limited by a lack of detailed understanding of genes that are transcriptionally regulated by these factors. We performed a microarray screen in Xenopus animal caps to search for targets of EBF transcriptional activity, and identified candidate targets with multiple roles, including transcription factors of several classes. We determined that, among the most upregulated candidate genes with expected neuronal functions, most require EBF activity for some or all of their expression, and most have overlapping expression with ebf genes. We also found that the candidate target genes that had the most strongly overlapping expression patterns with ebf genes were predicted to be direct transcriptional targets of EBF transcriptional activity. The identification of candidate targets that are transcription factor genes, including nscl-1, emx1 and aml1, improves our understanding of how EBF proteins participate in the hierarchy of transcription control during neuronal development, and suggests novel mechanisms by which EBF activity promotes migration and differentiation. Other candidate targets, including pcdh8 and kcnk5, expand our knowledge of the types of terminal differentiated neuronal functions that EBF proteins regulate.

  12. EBF factors drive expression of multiple classes of target genes governing neuronal development

    Directory of Open Access Journals (Sweden)

    Vetter Monica L

    2011-04-01

    Full Text Available Abstract Background Early B cell factor (EBF family members are transcription factors known to have important roles in several aspects of vertebrate neurogenesis, including commitment, migration and differentiation. Knowledge of how EBF family members contribute to neurogenesis is limited by a lack of detailed understanding of genes that are transcriptionally regulated by these factors. Results We performed a microarray screen in Xenopus animal caps to search for targets of EBF transcriptional activity, and identified candidate targets with multiple roles, including transcription factors of several classes. We determined that, among the most upregulated candidate genes with expected neuronal functions, most require EBF activity for some or all of their expression, and most have overlapping expression with ebf genes. We also found that the candidate target genes that had the most strongly overlapping expression patterns with ebf genes were predicted to be direct transcriptional targets of EBF transcriptional activity. Conclusions The identification of candidate targets that are transcription factor genes, including nscl-1, emx1 and aml1, improves our understanding of how EBF proteins participate in the hierarchy of transcription control during neuronal development, and suggests novel mechanisms by which EBF activity promotes migration and differentiation. Other candidate targets, including pcdh8 and kcnk5, expand our knowledge of the types of terminal differentiated neuronal functions that EBF proteins regulate.

  13. Enhanceosomes as integrators of hypoxia inducible factor (HIF) and other transcription factors in the hypoxic transcriptional response.

    Science.gov (United States)

    Pawlus, Matthew R; Hu, Cheng-Jun

    2013-09-01

    Hypoxia is a prevalent attribute of the solid tumor microenvironment that promotes the expression of genes through posttranslational modifications and stabilization of alpha subunits (HIF1α and HIF2α) of hypoxia-inducible factors (HIFs). Despite significant similarities, HIF1 (HIF1α/ARNT) and HIF2 (HIF2α/ARNT) activate common as well as unique target genes and exhibit different functions in cancer biology. More surprisingly, accumulating data indicates that the HIF1- and/or HIF2-mediated hypoxia responses can be oncogenic as well as tumor suppressive. While the role of HIF in the hypoxia response is well established, recent data support the concept that HIF is necessary, but not sufficient for the hypoxic response. Other transcription factors that are activated by hypoxia are also required for the HIF-mediated hypoxia response. HIFs, other transcription factors, co-factors and RNA poll II recruited by HIF and other transcription factors form multifactorial enhanceosome complexes on the promoters of HIF target genes to activate hypoxia inducible genes. Importantly, HIF1 or HIF2 requires distinct partners in activating HIF1 or HIF2 target genes. Because HIF enhanceosome formation is required for the gene activation and distinct functions of HIF1 and HIF2 in tumor biology, disruption of the HIF1 or HIF2 specific enhanceosome complex may prove to be a beneficial strategy in tumor treatment in which tumor growth is specifically dependent upon HIF1 or HIF2 activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Overlapping transcription structure of human cytomegalovirus

    Indian Academy of Sciences (India)

    Transcription of human cytomegalovirus UL/b′ region has been studied extensively for some genes. In this study, transcripts of the UL140 and UL141, two of the UL/b′ genes, were identified in late RNAs of three HCMV isolates using Northern blot hybridization, cDNA library screening and RACE-PCR. At least three ...

  15. Overlapping transcription structure of human cytomegalovirus ...

    Indian Academy of Sciences (India)

    2013-01-21

    Jan 21, 2013 ... Transcription of human cytomegalovirus UL/b′ region has been studied extensively for some genes. In this study, transcripts of the UL140 and UL141, two of the UL/b′ genes, were identified in late RNAs of three HCMV isolates using Northern blot hybridization, cDNA library screening and RACE-PCR.

  16. Transcription of Byzantine Chant - Problems, Possibilities, Formats

    DEFF Research Database (Denmark)

    Troelsgård, Christian

    2007-01-01

    Discusses the problems and possibilities for transsription of Byzantine chant on the basis of medieval musical manuscripts. A relatively 'neutral' style of transcription is suggested for musicological purposes.......Discusses the problems and possibilities for transsription of Byzantine chant on the basis of medieval musical manuscripts. A relatively 'neutral' style of transcription is suggested for musicological purposes....

  17. Regulation of transcription in hyperthermophilic archaea

    NARCIS (Netherlands)

    Brinkman, A.B.

    2002-01-01

    The aim of the research presented here was to insight in the mechanisms by which transcription in hyperthermophilic archaea is regulated. To accomplish this, we have aimed (I) to identify transcriptional regulatory proteins from hyperthermophilic archaea, (II) to characterize these

  18. 45 CFR 99.27 - Official transcript.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Official transcript. 99.27 Section 99.27 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION PROCEDURE FOR HEARINGS FOR THE CHILD CARE AND DEVELOPMENT FUND Hearing Procedures § 99.27 Official transcript. The Department will...

  19. RNA polymerase II collision interrupts convergent transcription

    DEFF Research Database (Denmark)

    Hobson, David J; Wei, Wu; Steinmetz, Lars M

    2012-01-01

    Antisense noncoding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical...

  20. Temporally Regulated Neural Crest Transcription Factors Distinguish Neuroectodermal Tumors of Varying Malignancy and Differentiation

    Directory of Open Access Journals (Sweden)

    Timothy R. Gershon

    2005-06-01

    Full Text Available Neuroectodermal tumor cells, like neural crest (NC cells, are pluripotent, proliferative, and migratory. We tested the hypothesis that genetic programs essential to NC development are activated in neuroectodermal tumors. We examined the expression of transcription factors PAX3, PAX7, AP-2α, and SOX10 in human embryos and neuroectodermal tumors: neurofibroma, schwannoma, neuroblastoma, malignant nerve sheath tumor, melanoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, and Ewing's sarcoma. We also examined the expression of P0, ERBB3, and STX, targets of SOX10, AP-2α, and PAX3, respectively. PAX3, AP-2α, and SOX10 were expressed sequentially in human NC development, whereas PAX7 was restricted to mesoderm. Tumors expressed PAX3, AP-2α, SOX10, and PAX7 in specific combinations. SOX10 and AP-2α were expressed in relatively differentiated neoplasms. The early NC marker, PAX3, and its homologue, PAX7, were detected in poorly differentiated tumors and tumors with malignant potential. Expression of NC transcription factors and target genes correlated. Transcription factors essential to NC development are thus present in neuroectodermal tumors. Correlation of specific NC transcription factors with phenotype, and with expression of specific downstream genes, provides evidence that these transcription factors actively influence gene expression and tumor behavior. These findings suggest that PAX3, PAX7, AP-2α, and SOX10 are potential markers of prognosis and targets for therapeutic intervention.

  1. Transcriptional profiling of the dose response: a more powerful approach for characterizing drug activities.

    Directory of Open Access Journals (Sweden)

    Rui-Ru Ji

    2009-09-01

    Full Text Available The dose response curve is the gold standard for measuring the effect of a drug treatment, but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns, but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose, calculate classical pharmacological parameters such as the EC50, and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib, nilotinib, dasatinib and PD0325901 across a six-logarithm dose range, using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets, dose-dependent effects on cellular processes were identified using automated pathway analysis, and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover, these methods are automated, robust to non-optimized assays, and could be applied to other sources of quantitative data.

  2. CREB and FoxO1: two transcription factors for the regulation of hepatic gluconeogenesis

    Science.gov (United States)

    Oh, Kyoung-Jin; Han, Hye-Sook; Kim, Min-Jung; Koo, Seung-Hoi

    2013-01-01

    Liver plays a major role in maintaining glucose homeostasis in mammals. Under fasting conditions, hepatic glucose production is critical as a source of fuel to maintain the basic functions in other tissues, including skeletal muscle, red blood cells, and the brain. Fasting hormones glucagon and cortisol play major roles during the process, in part by activating the transcription of key enzyme genes in the gluconeogenesis such as phosphoenol pyruvate carboxykinase (PEPCK) and glucose 6 phosphatase catalytic subunit (G6Pase). Conversely, gluconeogenic transcription is repressed by pancreatic insulin under feeding conditions, which effectively inhibits transcriptional activator complexes by either promoting post-translational modifications or activating transcriptional inhibitors in the liver, resulting in the reduction of hepatic glucose output. The transcriptional regulatory machineries have been highlighted as targets for type 2 diabetes drugs to control glycemia, so understanding of the complex regulatory mechanisms for transcription circuits for hepatic gluconeogenesis is critical in the potential development of therapeutic tools for the treatment of this disease. In this review, the current understanding regarding the roles of two key transcriptional activators, CREB and FoxO1, in the regulation of hepatic gluconeogenic program is discussed. [BMB Reports 2013; 46(12): 567-574] PMID:24238363

  3. Controlling transcription in human pluripotent stem cells using CRISPR-effectors.

    Science.gov (United States)

    Genga, Ryan M; Kearns, Nicola A; Maehr, René

    2016-05-15

    The ability to manipulate transcription in human pluripotent stem cells (hPSCs) is fundamental for the discovery of key genes and mechanisms governing cellular state and differentiation. Recently developed CRISPR-effector systems provide a systematic approach to rapidly test gene function in mammalian cells, including hPSCs. In this review, we discuss recent advances in CRISPR-effector technologies that have been employed to control transcription through gene activation, gene repression, and epigenome engineering. We describe an application of CRISPR-effector mediated transcriptional regulation in hPSCs by targeting a synthetic promoter driving a GFP transgene, demonstrating the ease and effectiveness of CRISPR-effector mediated transcriptional regulation in hPSCs. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    International Nuclear Information System (INIS)

    Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-01

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

  5. Comprehensive Interrogation of Natural TALE DNA Binding Modules and Transcriptional Repressor Domains

    Science.gov (United States)

    Cong, Le; Zhou, Ruhong; Kuo, Yu-chi; Cunniff, Margaret; Zhang, Feng

    2012-01-01

    Transcription activator-like effectors (TALE) are sequence-specific DNA binding proteins that harbor modular, repetitive DNA binding domains. TALEs have enabled the creation of customizable designer transcriptional factors and sequence-specific nucleases for genome engineering. Here we report two improvements of the TALE toolbox for achieving efficient activation and repression of endogenous gene expression in mammalian cells. We show that the naturally occurring repeat variable diresidue (RVD) Asn-His (NH) has high biological activity and specificity for guanine, a highly prevalent base in mammalian genomes. We also report an effective TALE transcriptional repressor architecture for targeted inhibition of transcription in mammalian cells. These findings will improve the precision and effectiveness of genome engineering that can be achieved using TALEs. PMID:22828628

  6. DOT1L and H3K79 Methylation in Transcription and Genomic Stability.

    Science.gov (United States)

    Wood, Katherine; Tellier, Michael; Murphy, Shona

    2018-02-27

    The organization of eukaryotic genomes into chromatin provides challenges for the cell to accomplish basic cellular functions, such as transcription, DNA replication and repair of DNA damage. Accordingly, a range of proteins modify and/or read chromatin states to regulate access to chromosomal DNA. Yeast Dot1 and the mammalian homologue DOT1L are methyltransferases that can add up to three methyl groups to histone H3 lysine 79 (H3K79). H3K79 methylation is implicated in several processes, including transcription elongation by RNA polymerase II, the DNA damage response and cell cycle checkpoint activation. DOT1L is also an important drug target for treatment of mixed lineage leukemia (MLL)-rearranged leukemia where aberrant transcriptional activation is promoted by DOT1L mislocalisation. This review summarizes what is currently known about the role of Dot1/DOT1L and H3K79 methylation in transcription and genomic stability.

  7. DOT1L and H3K79 Methylation in Transcription and Genomic Stability

    Directory of Open Access Journals (Sweden)

    Katherine Wood

    2018-02-01

    Full Text Available The organization of eukaryotic genomes into chromatin provides challenges for the cell to accomplish basic cellular functions, such as transcription, DNA replication and repair of DNA damage. Accordingly, a range of proteins modify and/or read chromatin states to regulate access to chromosomal DNA. Yeast Dot1 and the mammalian homologue DOT1L are methyltransferases that can add up to three methyl groups to histone H3 lysine 79 (H3K79. H3K79 methylation is implicated in several processes, including transcription elongation by RNA polymerase II, the DNA damage response and cell cycle checkpoint activation. DOT1L is also an important drug target for treatment of mixed lineage leukemia (MLL-rearranged leukemia where aberrant transcriptional activation is promoted by DOT1L mislocalisation. This review summarizes what is currently known about the role of Dot1/DOT1L and H3K79 methylation in transcription and genomic stability.

  8. The transcriptional corepressor MTGR1 regulates intestinal secretory lineage allocation.

    Science.gov (United States)

    Parang, Bobak; Rosenblatt, Daniel; Williams, Amanda D; Washington, Mary K; Revetta, Frank; Short, Sarah P; Reddy, Vishruth K; Hunt, Aubrey; Shroyer, Noah F; Engel, Michael E; Hiebert, Scott W; Williams, Christopher S

    2015-03-01

    Notch signaling largely determines intestinal epithelial cell fate. High Notch activity drives progenitors toward absorptive enterocytes by repressing secretory differentiation programs, whereas low Notch permits secretory cell assignment. Myeloid translocation gene-related 1 (MTGR1) is a transcriptional corepressor in the myeloid translocation gene/Eight-Twenty-One family. Given that Mtgr1(-/-) mice have a dramatic reduction of intestinal epithelial secretory cells, we hypothesized that MTGR1 is a key repressor of Notch signaling. In support of this, transcriptome analysis of laser capture microdissected Mtgr1(-/-) intestinal crypts revealed Notch activation, and secretory markers Mucin2, Chromogranin A, and Growth factor-independent 1 (Gfi1) were down-regulated in Mtgr1(-/-) whole intestines and Mtgr1(-/-) enteroids. We demonstrate that MTGR1 is in a complex with Suppressor of Hairless Homolog, a key Notch effector, and represses Notch-induced Hairy/Enhancer of Split 1 activity. Moreover, pharmacologic Notch inhibition using a γ-secretase inhibitor (GSI) rescued the hyperproliferative baseline phenotype in the Mtgr1(-/-) intestine and increased production of goblet and enteroendocrine lineages in Mtgr1(-/-) mice. GSI increased Paneth cell production in wild-type mice but failed to do so in Mtgr1(-/-) mice. We determined that MTGR1 can interact with GFI1, a transcriptional corepressor required for Paneth cell differentiation, and repress GFI1 targets. Overall, the data suggest that MTGR1, a transcriptional corepressor well characterized in hematopoiesis, plays a critical role in intestinal lineage allocation. © FASEB.

  9. Integrative modeling of transcriptional regulation in response to antirheumatic therapy

    Directory of Open Access Journals (Sweden)

    Thiesen Hans-Juergen

    2009-08-01

    Full Text Available Abstract Background The investigation of gene regulatory networks is an important issue in molecular systems biology and significant progress has been made by combining different types of biological data. The purpose of this study was to characterize the transcriptional program induced by etanercept therapy in patients with rheumatoid arthritis (RA. Etanercept is known to reduce disease symptoms and progression in RA, but the underlying molecular mechanisms have not been fully elucidated. Results Using a DNA microarray dataset providing genome-wide expression profiles of 19 RA patients within the first week of therapy we identified significant transcriptional changes in 83 genes. Most of these genes are known to control the human body's immune response. A novel algorithm called TILAR was then applied to construct a linear network model of the genes' regulatory interactions. The inference method derives a model from the data based on the Least Angle Regression while incorporating DNA-binding site information. As a result we obtained a scale-free network that exhibits a self-regulating and highly parallel architecture, and reflects the pleiotropic immunological role of the therapeutic target TNF-alpha. Moreover, we could show that our integrative modeling strategy performs much better than algorithms using gene expression data alone. Conclusion We present TILAR, a method to deduce gene regulatory interactions from gene expression data by integrating information on transcription factor binding sites. The inferred network uncovers gene regulatory effects in response to etanercept and thus provides useful hypotheses about the drug's mechanisms of action.

  10. ERK-dependent phosphorylation of the transcription initiation factor TIF-IA is required for RNA polymerase I transcription and cell growth

    DEFF Research Database (Denmark)

    Zhao, Jian; Yuan, Xuejun; Frödin, Morten

    2003-01-01

    -specific transcription initiation factor TIF-IA. Activation of TIF-IA and ribosomal gene transcription is sensitive to PD98059, indicating that TIF-IA is targeted by MAPK in vivo. Phosphopeptide mapping and mutational analysis reveals two serine residues (S633 and S649) that are phosphorylated by ERK and RSK kinases....... Replacement of S649 by alanine inactivates TIF-IA, inhibits pre-rRNA synthesis, and retards cell growth. The results provide a link between growth factor signaling, ribosome production, and cell growth, and may have a major impact on the mechanism of cell transformation....

  11. Large-scale analysis of antisense transcription in wheat using the Affymetrix GeneChip Wheat Genome Array

    Directory of Open Access Journals (Sweden)

    Settles Matthew L

    2009-05-01

    Full Text Available Abstract Background Natural antisense transcripts (NATs are transcripts of the opposite DNA strand to the sense-strand either at the same locus (cis-encoded or a different locus (trans-encoded. They can affect gene expression at multiple stages including transcription, RNA processing and transport, and translation. NATs give rise to sense-antisense transcript pairs and the number of these identified has escalated greatly with the availability of DNA sequencing resources and public databases. Traditionally, NATs were identified by the alignment of full-length cDNAs or expressed sequence tags to genome sequences, but an alternative method for large-scale detection of sense-antisense transcript pairs involves the use of microarrays. In this study we developed a novel protocol to assay sense- and antisense-strand transcription on the 55 K Affymetrix GeneChip Wheat Genome Array, which is a 3' in vitro transcription (3'IVT expression array. We selected five different tissue types for assay to enable maximum discovery, and used the 'Chinese Spring' wheat genotype because most of the wheat GeneChip probe sequences were based on its genomic sequence. This study is the first report of using a 3'IVT expression array to discover the expression of natural sense-antisense transcript pairs, and may be considered as proof-of-concept. Results By using alternative target preparation schemes, both the sense- and antisense-strand derived transcripts were labeled and hybridized to the Wheat GeneChip. Quality assurance verified that successful hybridization did occur in the antisense-strand assay. A stringent threshold for positive hybridization was applied, which resulted in the identification of 110 sense-antisense transcript pairs, as well as 80 potentially antisense-specific transcripts. Strand-specific RT-PCR validated the microarray observations, and showed that antisense transcription is likely to be tissue specific. For the annotated sense

  12. Hypoxia-Inducible Factor 3 Is an Oxygen-Dependent Transcription Activator and Regulates a Distinct Transcriptional Response to Hypoxia

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    Peng Zhang

    2014-03-01

    Full Text Available Hypoxia-inducible factors (HIFs play key roles in the cellular response to hypoxia. It is widely accepted that whereas HIF-1 and HIF-2 function as transcriptional activators, HIF-3 inhibits HIF-1/2α action. Contrary to this idea, we show that zebrafish Hif-3α has strong transactivation activity. Hif-3α is degraded under normoxia. Mutation of P393, P493, and L503 inhibits this oxygen-dependent degradation. Transcriptomics and chromatin immunoprecipitation analyses identify genes that are regulated by Hif-3α, Hif-1α, or both. Under hypoxia or when overexpressed, Hif-3α binds to its target gene promoters and upregulates their expression. Dominant-negative inhibition and knockdown of Hif-3α abolish hypoxia-induced Hif-3α-promoter binding and gene expression. Hif-3α not only mediates hypoxia-induced growth and developmental retardation but also possesses hypoxia-independent activities. Importantly, transactivation activity is conserved and human HIF-3α upregulates similar genes in human cells. These findings suggest that Hif-3 is an oxygen-dependent transcription factor and activates a distinct transcriptional response to hypoxia.

  13. Recent advances in functional assays of transcriptional enhancers.

    Science.gov (United States)

    Babbitt, Courtney C; Markstein, Michele; Gray, Jesse M

    2015-09-01

    In this special edition of Genomics, we present reviews of the current state of the field in identifying and functionally understanding transcriptional enhancers in cells and developing tissues. Typically several enhancers coordinate the expression of an individual target gene, each controlling that gene's expression in specific cell types at specific times. Until recently, identifying each gene's enhancers had been challenging because enhancers do not occupy prescribed locations relative to their target genes. Recently there have been powerful advances in DNA sequencing and other technologies that make it possible to identify the majority of enhancers in virtually any cell type of interest. The reviews in this edition of Genomics highlight some of these new and powerful approaches. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. In silico transcriptional regulatory networks involved in tomato fruit ripening

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    Stilianos Arhondakis

    2016-08-01

    Full Text Available ABSTRACTTomato fruit ripening is a complex developmental programme partly mediated by transcriptional regulatory networks. Several transcription factors (TFs which are members of gene families such as MADS-box and ERF were shown to play a significant role in ripening through interconnections into an intricate network. The accumulation of large datasets of expression profiles corresponding to different stages of tomato fruit ripening and the availability of bioinformatics tools for their analysis provide an opportunity to identify TFs which might regulate gene clusters with similar co-expression patterns. We identified two TFs, a SlWRKY22-like and a SlER24 transcriptional activator which were shown to regulate modules by using the LeMoNe algorithm for the analysis of our microarray datasets representing four stages of fruit ripening, breaker, turning, pink and red ripe. The WRKY22-like module comprised a subgroup of six various calcium sensing transcripts with similar to the TF expression patterns according to real time PCR validation. A promoter motif search identified a cis acting element, the W-box, recognized by WRKY TFs that was present in the promoter region of all six calcium sensing genes. Moreover, publicly available microarray datasets of similar ripening stages were also analyzed with LeMoNe resulting in TFs such as SlERF.E1, SlERF.C1, SlERF.B2, SLERF.A2, SlWRKY24, SLWRKY37 and MADS-box/TM29 which might also play an important role in regulation of ripening. These results suggest that the SlWRKY22-like might be involved in the coordinated regulation of expression of the six calcium sensing genes. Conclusively the LeMoNe tool might lead to the identification of putative TF targets for further physiological analysis as regulators of tomato fruit ripening.

  15. Transcriptional regulation of genes related to progesterone production.

    Science.gov (United States)

    Mizutani, Tetsuya; Ishikane, Shin; Kawabe, Shinya; Umezawa, Akihiro; Miyamoto, Kaoru

    2015-01-01

    Steroid hormones are synthesized from cholesterol in various tissues, mainly in the adrenal glands and gonads. Because these lipid-soluble steroid hormones immediately diffuse through the cells in which they are produced, their secretion directly reflects the activity of the genes related to their production. Progesterone is important not only for luteinization and maintenance of pregnancy, but also as a substrate for most other steroids. Steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3β-HSD) are well-known proteins essential for progesterone production. In addition to them, glutathione S-transferase A1-1 and A3-3 are shown to exert Δ(5)-Δ(4) isomerization activity to produce progesterone in a cooperative fashion with 3β-HSD. 5-Aminolevulinic acid synthase 1, ferredoxin 1, and ferredoxin reductase also play a role in steroidogenesis as accessory factors. Members of the nuclear receptor 5A (NR5A) family (steroidogenic factor 1 and liver receptor homolog 1) play a crucial role in the transcriptional regulation of these genes. The NR5A family activates these genes by binding to NR5A responsive elements present within their promoter regions, as well as to the elements far from their promoters. In addition, various NR5A-interacting proteins including peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear receptor subfamily 0, group B, member 1 (DAX-1), and CCAAT/enhancer-binding proteins (C/EBP) are involved in the transcription of NR5A target genes and regulate the transcription either positively or negatively under both basal and tropic hormone-stimulated conditions. In this review, we describe the transcriptional regulation of genes related to progesterone production.

  16. Functional analysis of jasmonate-responsive transcription factors in Arabidopsis thaliana

    NARCIS (Netherlands)

    Zarei, Adel

    2007-01-01

    The aim of the studies described in this thesis was the functional analysis of JA-responsive transcription factors in Arabidopsis with an emphasis on the interaction with the promoters of their target genes. In short, the following new results were obtained. The promoter of the PDF1.2 gene contains

  17. CRISPR-Cas9-Mediated Genome Editing and Transcriptional Control in Yarrowia lipolytica.

    Science.gov (United States)

    Schwartz, Cory; Wheeldon, Ian

    2018-01-01

    The discovery and adaptation of RNA-guided nucleases has resulted in the rapid development of efficient, scalable, and easily accessible synthetic biology tools for targeted genome editing and transcriptional control. In these systems, for example CRISPR-Cas9 from Streptococcus pyogenes, a protein with nuclease activity is targeted to a specific nucleotide sequence by a short RNA molecule, whereupon binding it cleaves the targeted nucleotide strand. To extend this genome-editing ability to the industrially important oleaginous yeast Yarrowia lipolytica, we developed a set of easily usable and effective CRISPR-Cas9 episomal vectors. In this protocols chapter, we first present a method by which arbitrary protein-coding genes can be disrupted via indel formation after CRISPR-Cas9 targeting. A second method demonstrates how the same CRISPR-Cas9 system can be used to induce markerless gene cassette integration into the genome by inducing homologous recombination after DNA cleavage by Cas9. Finally, we describe how a catalytically inactive form of Cas9 fused to a transcriptional repressor can be used to control transcription of native genes in Y. lipolytica. The CRISPR-Cas9 tools and strategies described here greatly increase the types of genome editing and transcriptional control that can be achieved in Y. lipolytica, and promise to facilitate more advanced engineering of this important oleaginous host.

  18. Transcriptional blockages in a cell-free system by sequence-selective DNA alkylating agents.

    Science.gov (United States)

    Ferguson, L R; Liu, A P; Denny, W A; Cullinane, C; Talarico, T; Phillips, D R

    2000-04-14

    There is considerable interest in DNA sequence-selective DNA-binding drugs as potential inhibitors of gene expression. Five compounds with distinctly different base pair specificities were compared in their effects on the formation and elongation of the transcription complex from the lac UV5 promoter in a cell-free system. All were tested at drug levels which killed 90% of cells in a clonogenic survival assay. Cisplatin, a selective alkylator at purine residues, inhibited transcription, decreasing the full-length transcript, and causing blockage at a number of GG or AG sequences, making it probable that intrastrand crosslinks are the blocking lesions. A cyclopropylindoline known to be an A-specific alkylator also inhibited transcription, with blocks at adenines. The aniline mustard chlorambucil, that targets primarily G but also A sequences, was also effective in blocking the formation of full-length transcripts. It produced transcription blocks either at, or one base prior to, AA or GG sequences, suggesting that intrastrand crosslinks could again be involved. The non-alkylating DNA minor groove binder Hoechst 33342 (a bisbenzimidazole) blocked formation of the full-length transcript, but without creating specific blockage sites. A bisbenzimidazole-linked aniline mustard analogue was a more effective transcription inhibitor than either chlorambucil or Hoechst 33342, with different blockage sites occurring immediately as compared with 2 h after incubation. The blockages were either immediately prior to AA or GG residues, or four to five base pairs prior to such sites, a pattern not predicted from in vitro DNA-binding studies. Minor groove DNA-binding ligands are of particular interest as inhibitors of gene expression, since they have the potential ability to bind selectively to long sequences of DNA. The results suggest that the bisbenzimidazole-linked mustard does cause alkylation and transcription blockage at novel DNA sites. in addition to sites characteristic of

  19. Modeling post-transcriptional regulation activity of small non-coding RNAs in Escherichia coli.

    Science.gov (United States)

    Wang, Rui-Sheng; Jin, Guangxu; Zhang, Xiang-Sun; Chen, Luonan

    2009-04-29

    Transcriptional regulation is a fundamental process in biological systems, where transcription factors (TFs) have been revealed to play crucial roles. In recent years, in addition to TFs, an increasing number of non-coding RNAs (ncRNAs) have been shown to mediate post-transcriptional processes and regulate many critical pathways in both prokaryotes and eukaryotes. On the other hand, with more and more high-throughput biological data becoming available, it is possible and imperative to quantitatively study gene regulation in a systematic and detailed manner. Most existing studies for inferring transcriptional regulatory interactions and the activity of TFs ignore the possible post-transcriptional effects of ncRNAs. In this work, we propose a novel framework to infer the activity of regulators including both TFs and ncRNAs by exploring the expression profiles of target genes and (post)transcriptional regulatory relationships. We model the integrated regulatory system by a set of biochemical reactions which lead to a log-bilinear problem. The inference process is achieved by an iterative algorithm, in which two linear programming models are efficiently solved. In contrast to available related studies, the effects of ncRNAs on transcription process are considered in this work, and thus more reasonable and accurate reconstruction can be expected. In addition, the approach is suitable for large-scale problems from the viewpoint of computation. Experiments on two synthesized data sets and a model system of Escherichia coli (E. coli) carbon source transition from glucose to acetate illustrate the effectiveness of our model and algorithm. Our results show that incorporating the post-transcriptional regulation of ncRNAs into system model can mine the hidden effects from the regulation activity of TFs in transcription processes and thus can uncover the biological mechanisms in gene regulation in a more accurate manner. The software for the algorithm in this paper is available

  20. Early transcriptional response of soybean contrasting accessions to root dehydration.

    Directory of Open Access Journals (Sweden)

    José Ribamar Costa Ferreira Neto

    Full Text Available Drought is a significant constraint to yield increase in soybean. The early perception of water deprivation is critical for recruitment of genes that promote plant tolerance. DeepSuperSAGE libraries, including one control and a bulk of six stress times imposed (from 25 to 150 min of root dehydration for drought-tolerant and sensitive soybean accessions, allowed to identify new molecular targets for drought tolerance. The survey uncovered 120,770 unique transcripts expressed by the contrasting accessions. Of these, 57,610 aligned with known cDNA sequences, allowing the annotation of 32,373 unitags. A total of 1,127 unitags were up-regulated only in the tolerant accession, whereas 1,557 were up-regulated in both as compared to their controls. An expression profile concerning the most representative Gene Ontology (GO categories for the tolerant accession revealed the expression "protein binding" as the most represented for "Molecular Function", whereas CDPK and CBL were the most up-regulated protein families in this category. Furthermore, particular genes expressed different isoforms according to the accession, showing the potential to operate in the distinction of physiological behaviors. Besides, heat maps comprising GO categories related to abiotic stress response and the unitags regulation observed in the expression contrasts covering tolerant and sensitive accessions, revealed the unitags potential for plant breeding. Candidate genes related to "hormone response" (LOX, ERF1b, XET, "water response" (PUB, BMY, "salt stress response" (WRKY, MYB and "oxidative stress response" (PER figured among the most promising molecular targets. Additionally, nine transcripts (HMGR, XET, WRKY20, RAP2-4, EREBP, NAC3, PER, GPX5 and BMY validated by RT-qPCR (four different time points confirmed their differential expression and pointed that already after 25 minutes a transcriptional reorganization started in response to the new condition, with important

  1. The global transcriptional response of fission yeast to hydrogen sulfide.

    Directory of Open Access Journals (Sweden)

    Xu Jia

    Full Text Available BACKGROUND: Hydrogen sulfide (H(2S is a newly identified member of the small family of gasotransmitters that are endogenous gaseous signaling molecules that have a fundamental role in human biology and disease. Although it is a relatively recent discovery and the mechanism of H(2S activity is not completely understood, it is known to be involved in a number of cellular processes; H(2S can affect ion channels, transcription factors and protein kinases in mammals. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we have used fission yeast as a model organism to study the global gene expression profile in response to H(2S by microarray. We initially measured the genome-wide transcriptional response of fission yeast to H(2S. Through the functional classification of genes whose expression profile changed in response to H(2S, we found that H(2S mainly influences genes that encode putative or known stress proteins, membrane transporters, cell cycle/meiotic proteins, transcription factors and respiration protein in the mitochondrion. Our analysis showed that there was a significant overlap between the genes affected by H(2S and the stress response. We identified that the target genes of the MAPK pathway respond to H(2S; we also identified that a number of transporters respond to H(2S, these include sugar/carbohydrate transporters, ion transporters, and amino acid transporters. We found many mitochondrial genes to be down regulated upon H(2S treatment and that H(2S can reduce mitochondrial oxygen consumption. CONCLUSION/SIGNIFICANCE: This study identifies potential molecular targets of the signaling molecule H(2S in fission yeast and provides clues about the identity of homologues human proteins and will further the understanding of the cellular role of H(2S in human diseases.

  2. Targeted drugs in radiation therapy

    International Nuclear Information System (INIS)

    Favaudon, V.; Hennequin, C.; Hennequin, C.

    2004-01-01

    New drugs aiming at the development of targeted therapies have been assayed in combination with ionizing radiation over the past few years. The rationale of this concept comes from the fact that the cytotoxic potential of targeted drugs is limited, thus requiring concomitant association with a cytotoxic agent for the eradication of tumor cells. Conversely a low level of cumulative toxicity is expected from targeted drugs. Most targeted drugs act through inhibition of post-translational modifications of proteins, such as dimerization of growth factor receptors, prenylation reactions, or phosphorylation of tyrosine or serine-threonine residues. Many systems involving the proteasome, neo-angiogenesis promoters, TGF-β, cyclooxygenase or the transcription factor NF-κB, are currently under investigation in hopes they will allow a control of cell proliferation, apoptosis, cell cycle progression, tumor angiogenesis and inflammation. A few drugs have demonstrated an antitumor potential in particular phenotypes. In most instances, however, radiation-drug interactions proved to be strictly additive in terms of cell growth inhibition or induced cell death. Strong potentiation of the response to radiotherapy is expected to require interaction with DNA repair mechanisms. (authors)

  3. CHD chromatin remodelers and the transcription cycle

    Science.gov (United States)

    Murawska, Magdalena

    2011-01-01

    It is well established that ATP-dependent chromatin remodelers modulate DNA access of transcription factors and RNA polymerases by “opening” or “closing” chromatin structure. However, this view is far too simplistic. Recent findings have demonstrated that these enzymes not only set the stage for the transcription machinery to act but also are actively involved at every step of the transcription process. As a consequence, they affect initiation, elongation, termination and RNA processing. In this review we will use the CHD family as a paradigm to illustrate the progress that has been made in revealing these new concepts. PMID:22223048

  4. NAC transcription factors: structurally distinct, functionally diverse

    DEFF Research Database (Denmark)

    Olsen, Addie Nina; Ernst, Heidi A; Leggio, Leila Lo

    2005-01-01

    level and localization, and to the first indications of NAC participation in transcription factor networks. The recent determination of the DNA and protein binding NAC domain structure offers insight into the molecular functions of the protein family. Research into NAC transcription factors has......NAC proteins constitute one of the largest families of plant-specific transcription factors, and the family is present in a wide range of land plants. Here, we summarize the biological and molecular functions of the NAC family, paying particular attention to the intricate regulation of NAC protein...

  5. Characterization of herpes simplex virus 2 primary microRNA Transcript regulation.

    Science.gov (United States)

    Tang, Shuang; Bosch-Marce, Marta; Patel, Amita; Margolis, Todd P; Krause, Philip R

    2015-05-01

    In order to understand factors that may influence latency-associated transcription and latency-associated transcript (LAT) phenotypes, we studied the expression of the herpes simplex virus 2 (HSV-2) LAT-associated microRNAs (miRNAs). We mapped the transcription initiation sites of all three primary miRNA transcripts and identified the ICP4-binding sequences at the transcription initiation sites of both HSV-2 LAT (pri-miRNA for miR-I and miR-II, which target ICP34.5, and miR-III, which targets ICP0) and L/ST (a pri-miRNA for miR-I and miR-II) but not at that of the primary miR-H6 (for which the target is unknown). We confirmed activity of the putative HSV-2 L/ST promoter and found that ICP4 trans-activates the L/ST promoter when the ICP4-binding site at its transcription initiation site is mutated, suggesting that ICP4 may play a dual role in regulating transcription of L/ST and, consequently, of miR-I and miR-II. LAT exon 1 (containing LAT enhancer sequences), together with the LAT promoter region, comprises a bidirectional promoter required for the expression of both LAT-encoded miRNAs and miR-H6 in latently infected mouse ganglia. The ability of ICP4 to suppress ICP34.5-targeting miRNAs and to activate lytic viral genes suggests that ICP4 could play a key role in the switch between latency and reactivation. The HSV-2 LAT and viral miRNAs expressed in the LAT region are the most abundant viral transcripts during HSV latency. The balance between the expression of LAT and LAT-associated miRNAs and the expression of lytic viral transcripts from the opposite strand appears to influence whether individual HSV-infected neurons will be latently or productively infected. The outcome of neuronal infection may thus depend on regulation of gene expression of the corresponding primary miRNAs. In the present study, we characterize promoter sequences responsible for miRNA expression, including identification of the primary miRNA 5' ends and evaluation of ICP4 response. These

  6. Dynamics of co-transcriptional pre-mRNA folding influences the induction of dystrophin exon skipping by antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Keng Boon Wee

    Full Text Available Antisense oligonucleotides (AONs mediated exon skipping offers potential therapy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, a "window of analysis" that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structures were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered "engaged" if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localisation of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency of 94% of 176 previously reported AONs. Four novel insights are inferred: (1 the lowest frequencies of engaged nucleotides are associated with the most efficient AONs; (2 engaged nucleotides at 3' or 5' ends of the target site attenuate AON performance more than at other sites; (3 the performance of longer AONs is less attenuated by engaged nucleotides at 3' or 5' ends of the target site compared to shorter AONs; (4 engaged nucleotides at 3' end of a short target site attenuates AON efficiency more than at 5' end.

  7. Repression of meiotic genes by antisense transcription and by Fkh2 transcription factor in Schizosaccharomyces pombe.

    Science.gov (United States)

    Chen, Huei-Mei; Rosebrock, Adam P; Khan, Sohail R; Futcher, Bruce; Leatherwood, Janet K

    2012-01-01

    In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.

  8. Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe

    Science.gov (United States)

    Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.

    2012-01-01

    In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the “unspliced” signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression. PMID:22238674

  9. Repression of meiotic genes by antisense transcription and by Fkh2 transcription factor in Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Huei-Mei Chen

    Full Text Available In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.

  10. 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

  11. A critique on nuclear factor-kappa B and signal transducer and activator of transcription 3: The key transcription factors in periodontal pathogenesis

    Directory of Open Access Journals (Sweden)

    Ranjith Ambili

    2017-01-01

    Full Text Available Periodontal disease is initiated by microorganisms in dental plaque, and host immunoinflammatory response to the microbial challenge helps in disease progression. Conventional periodontal therapy was mainly targeted on the elimination of microbial component. However, a better understanding of molecular aspects in host response will enable the clinicians to formulate effective host modulation therapy (HMT for the periodontal management. Inflammatory mediators were the main targets for HMT in the past. Transcription factors can regulate the production of multiple mediators simultaneously, and inhibition of these factors will be more beneficial than blocking individual molecule. Two important transcription factors implicated in chronic inflammatory diseases are nuclear factor kappa B (NF-κB and signal transducers and activators of transcription 3. The role of these factors in periodontal disease is a less explored area. This comprehensive review is aimed at unveiling the critical role of NF-κB and signal transducers and activators of transcription 3 in periodontal pathogenesis. An online search was performed using MEDLINE/PubMed database. All publications till 2016 related to NF-κB, signal transducer and activator of transcription 3 (STAT3, and inflammation were included in writing this review. A total of 27,390 references were published based on the search terms used. Out of these, 507 were related to the periodontal research published in English till 2016. Relevant papers were chosen after carefully reading the abstract. This review has attempted to comprehend the existing knowledge regarding the role of transcription factors NF-κB and STAT3 in periodontal disease. Moreover, it also provides a connecting molecular link for the periodontal medicine concept.

  12. 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.

  13. Battles and hijacks: Noncoding transcription in plants

    KAUST Repository

    Ariel, Federico; Romero-Barrios, Natali; Jé gu, Teddy; Benhamed, Moussa; Crespi, Martin

    2015-01-01

    splicing, fine-tuning of miRNA activity, and the control of mRNA translation or accumulation. Recently, dual noncoding transcription by alternative RNA polymerases was implicated in epigenetic and chromatin conformation dynamics. This review integrates

  14. Salmonella Typhimurium transcription profiles in space flight

    Data.gov (United States)

    National Aeronautics and Space Administration — Salmonella transcription profiles were obtained from samples flown on space shuttle mission STS-115 and compared to profiles from Salmonella grown under identical...

  15. Transcript for Understanding Medical Words: A Tutorial

    Science.gov (United States)

    ... medlineplus.gov/medicalwordstranscript.html Transcript for Understanding Medical Words: A Tutorial To use the sharing features on ... get to what those mean in a minute. Word Roots Word Roots. Let's begin with body parts. ...

  16. Inhibition of FoxO1 acetylation by INHAT subunit SET/TAF-Iβ induces p21 transcription.

    Science.gov (United States)

    Chae, Yun-Cheol; Kim, Kee-Beom; Kang, Joo-Young; Kim, Se-Ryeon; Jung, Hyeon-Soo; Seo, Sang-Beom

    2014-08-25

    Post-translational modification of forkhead family transcription factor, FoxO1, is an important regulatory mode for its diverse activities. FoxO1 is acetylated by HAT coactivators and its transcriptional activity is decreased via reduced DNA binding affinity. Here, we report that SET/TAF-Iβ inhibited p300-mediated FoxO1 acetylation in an INHAT domain-dependent manner. SET/TAF-Iβ interacted with FoxO1 and activated transcription of FoxO1 target gene, p21. Moreover, SET/TAF-Iβ inhibited acetylation of FoxO1 and increased p21 transcription induced by oxidative stress. Our results suggest that SET/TAF-Iβ inhibits FoxO1 acetylation and activates its transcriptional activity toward p21. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. Histone H4 Lys 20 methyltransferase SET8 promotes androgen receptor-mediated transcription activation in prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Lushuai [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Yanyan; Du, Fengxia [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Han, Xiao [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Xiaohua [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Niu, Yuanjie [Chawnshang Chang Sex Hormone Research Center, Tianjin Institute of Urology, Tianjin Medical University, Tianjin 300070 (China); Ren, Shancheng, E-mail: renshancheng@gmail.com [Department of Urology, Shanghai Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Sun, Yingli, E-mail: sunyl@big.ac.cn [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

    Highlights: • Dihydrotestosterone stimulates H4K20me1 enrichment at the PSA promoter. • SET8 promotes AR-mediated transcription activation. • SET8 interacts with AR and promotes cell proliferation. - Abstract: Histone methylation status in different lysine residues has an important role in transcription regulation. The effect of H4K20 monomethylation (H4K20me1) on androgen receptor (AR)-mediated gene transcription remains unclear. Here we show that AR agonist stimulates the enrichment of H4K20me1 and SET8 at the promoter of AR target gene PSA in an AR dependent manner. Furthermore, SET8 is crucial for the transcription activation of PSA. Co-immunoprecipitation analyses demonstrate that SET8 interacts with AR. Therefore, we conclude that SET8 is involved in AR-mediated transcription activation, possibly through its interaction with AR and H4K20me1 modification.

  18. A DNA-binding-site landscape and regulatory network analysis for NAC transcription factors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Lindemose, Søren; Jensen, Michael Krogh; de Velde, Jan Van

    2014-01-01

    regulatory networks of 12 NAC transcription factors. Our data offer specific single-base resolution fingerprints for most TFs studied and indicate that NAC DNA-binding specificities might be predicted from their DNA-binding domain's sequence. The developed methodology, including the application......Target gene identification for transcription factors is a prerequisite for the systems wide understanding of organismal behaviour. NAM-ATAF1/2-CUC2 (NAC) transcription factors are amongst the largest transcription factor families in plants, yet limited data exist from unbiased approaches to resolve...... the DNA-binding preferences of individual members. Here, we present a TF-target gene identification workflow based on the integration of novel protein binding microarray data with gene expression and multi-species promoter sequence conservation to identify the DNA-binding specificities and the gene...

  19. A unified architecture of transcriptional regulatory elements

    DEFF Research Database (Denmark)

    Andersson, Robin; Sandelin, Albin Gustav; Danko, Charles G.

    2015-01-01

    Gene expression is precisely controlled in time and space through the integration of signals that act at gene promoters and gene-distal enhancers. Classically, promoters and enhancers are considered separate classes of regulatory elements, often distinguished by histone modifications. However...... and enhancers are considered a single class of functional element, with a unified architecture for transcription initiation. The context of interacting regulatory elements and the surrounding sequences determine local transcriptional output as well as the enhancer and promoter activities of individual elements....

  20. Transcriptional Waves in the Yeast Cell Cycle

    OpenAIRE

    Oliva, Anna; Rosebrock, Adam; Ferrezuelo, Francisco; Pyne, Saumyadipta; Chen, Haiying; Skiena, Steve; Futcher, Bruce; Leatherwood, Janet

    2005-01-01

    Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillat...

  1. A nanobody modulates the p53 transcriptional program without perturbing its functional architecture

    Science.gov (United States)

    Bethuyne, Jonas; De Gieter, Steven; Zwaenepoel, Olivier; Garcia-Pino, Abel; Durinck, Kaat; Verhelle, Adriaan; Hassanzadeh-Ghassabeh, Gholamreza; Speleman, Frank; Loris, Remy; Gettemans, Jan

    2014-01-01

    The p53 transcription factor plays an important role in genome integrity. To perform this task, p53 regulates the transcription of genes promoting various cellular outcomes including cell cycle arrest, apoptosis or senescence. The precise regulation of this activity remains elusive as numerous mechanisms, e.g. posttranslational modifications of p53 and (non-)covalent p53 binding partners, influence the p53 transcriptional program. We developed a novel, non-invasive tool to manipulate endogenous p53. Nanobodies (Nb), raised against the DNA-binding domain of p53, allow us to distinctively target both wild type and mutant p53 with great specificity. Nb3 preferentially binds ‘structural’ mutant p53, i.e. R175H and R282W, while a second but distinct nanobody, Nb139, binds both mutant and wild type p53. The co-crystal structure of the p53 DNA-binding domain in complex with Nb139 (1.9 Å resolution) reveals that Nb139 binds opposite the DNA-binding surface. Furthermore, we demonstrate that Nb139 does not disturb the functional architecture of the p53 DNA-binding domain using conformation-specific p53 antibody immunoprecipitations, glutaraldehyde crosslinking assays and chromatin immunoprecipitation. Functionally, the binding of Nb139 to p53 allows us to perturb the transactivation of p53 target genes. We propose that reduced recruitment of transcriptional co-activators or modulation of selected post-transcriptional modifications account for these observations. PMID:25324313

  2. Identification of novel small molecules that inhibit STAT3-dependent transcription and function.

    Directory of Open Access Journals (Sweden)

    Iryna Kolosenko

    Full Text Available Activation of Signal Transducer and Activator of Transcription 3 (STAT3 has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z' = 0,8. This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set. Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents.

  3. Characterization and Improvement of RNA-Seq Precision in Quantitative Transcript Expression Profiling

    Energy Technology Data Exchange (ETDEWEB)

    Labaj, Pawel P.; Leparc, German G.; Linggi, Bryan E.; Markillie, Lye Meng; Wiley, H. S.; Kreil, David P.

    2011-07-01

    Measurement precision determines the power of any analysis to reliably identify significant signals, such as in screens for differential expression, independent of whether the experimental design incorporates replicates or not. With the compilation of large scale RNA-Seq data sets with technical replicate samples, however, we can now, for the first time, perform a systematic analysis of the precision of expression level estimates from massively parallel sequencing technology. This then allows considerations for its improvement by computational or experimental means. Results: We report on a comprehensive study of target coverage and measurement precision, including their dependence on transcript expression levels, read depth and other parameters. In particular, an impressive target coverage of 84% of the estimated true transcript population could be achieved with 331 million 50 bp reads, with diminishing returns from longer read lengths and even less gains from increased sequencing depths. Most of the measurement power (75%) is spent on only 7% of the known transcriptome, however, making less strongly expressed transcripts harder to measure. Consequently, less than 30% of all transcripts could be quantified reliably with a relative error < 20%. Based on established tools, we then introduce a new approach for mapping and analyzing sequencing reads that yields substantially improved performance in gene expression profiling, increasing the number of transcripts that can reliably be quantified to over 40%. Extrapolations to higher sequencing depths highlight the need for efficient complementary steps. In discussion we outline possible experimental and computational strategies for further improvements in quantification precision.

  4. The Brakeless co-regulator can directly activate and repress transcription in early Drosophila embryos.

    Science.gov (United States)

    Crona, Filip; Holmqvist, Per-Henrik; Tang, Min; Singla, Bhumica; Vakifahmetoglu-Norberg, Helin; Fantur, Katrin; Mannervik, Mattias

    2015-11-01

    The Brakeless protein performs many important functions during Drosophila development, but how it controls gene expression is poorly understood. We previously showed that Brakeless can function as a transcriptional co-repressor. In this work, we perform transcriptional profiling of brakeless mutant embryos. Unexpectedly, the majority of affected genes are down-regulated in brakeless mutants. We demonstrate that genomic regions in close proximity to some of these genes are occupied by Brakeless, that over-expression of Brakeless causes a reciprocal effect on expression of these genes, and that Brakeless remains an activator of the genes upon fusion to an activation domain. Together, our results show that Brakeless can both repress and activate gene expression. A yeast two-hybrid screen identified the Mediator complex subunit Med19 as interacting with an evolutionarily conserved part of Brakeless. Both down- and up-regulated Brakeless target genes are also affected in Med19-depleted embryos, but only down-regulated targets are influenced in embryos depleted of both Brakeless and Med19. Our data provide support for a Brakeless activator function that regulates transcription by interacting with Med19. We conclude that the transcriptional co-regulator Brakeless can either activate or repress transcription depending on context. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Linking Core Promoter Classes to Circadian Transcription.

    Directory of Open Access Journals (Sweden)

    Pål O Westermark

    2016-08-01

    Full Text Available Circadian rhythms in transcription are generated by rhythmic abundances and DNA binding activities of transcription factors. Propagation of rhythms to transcriptional initiation involves the core promoter, its chromatin state, and the basal transcription machinery. Here, I characterize core promoters and chromatin states of genes transcribed in a circadian manner in mouse liver and in Drosophila. It is shown that the core promoter is a critical determinant of circadian mRNA expression in both species. A distinct core promoter class, strong circadian promoters (SCPs, is identified in mouse liver but not Drosophila. SCPs are defined by specific core promoter features, and are shown to drive circadian transcriptional activities with both high averages and high amplitudes. Data analysis and mathematical modeling further provided evidence for rhythmic regulation of both polymerase II recruitment and pause release at SCPs. The analysis provides a comprehensive and systematic view of core promoters and their link to circadian mRNA expression in mouse and Drosophila, and thus reveals a crucial role for the core promoter in regulated, dynamic transcription.

  6. TAF(II)250: a transcription toolbox.

    Science.gov (United States)

    Wassarman, D A; Sauer, F

    2001-08-01

    Activation of RNA-polymerase-II-dependent transcription involves conversion of signals provided by gene-specific activator proteins into the synthesis of messenger RNA. This conversion requires dynamic structural changes in chromatin and assembly of general transcription factors (GTFs) and RNA polymerase II at core promoter sequence elements surrounding the transcription start site of genes. One hallmark of transcriptional activation is the interaction of DNA-bound activators with coactivators such as the TATA-box binding protein (TBP)-associated factors (TAF(II)s) within the GTF TFIID. TAF(II)250 possesses a variety of activities that are likely to contribute to the initial steps of RNA polymerase II transcription. TAF(II)250 is a scaffold for assembly of other TAF(II)s and TBP into TFIID, TAF(II)250 binds activators to recruit TFIID to particular promoters, TAF(II)250 regulates binding of TBP to DNA, TAF(II)250 binds core promoter initiator elements, TAF(II)250 binds acetylated lysine residues in core histones, and TAF(II)250 possesses protein kinase, ubiquitin-activating/conjugating and acetylase activities that modify histones and GTFs. We speculate that these activities achieve two goals--(1) they aid in positioning and stabilizing TFIID at particular promoters, and (2) they alter chromatin structure at the promoter to allow assembly of GTFs--and we propose a model for how TAF(II)250 converts activation signals into active transcription.

  7. A biophysical model for transcription factories

    International Nuclear Information System (INIS)

    Canals-Hamann, Ana Z; Neves, Ricardo Pires das; Reittie, Joyce E; Iñiguez, Carlos; Soneji, Shamit; Enver, Tariq; Buckle, Veronica J; Iborra, Francisco J

    2013-01-01

    Transcription factories are nuclear domains where gene transcription takes place although the molecular basis for their formation and maintenance are unknown. In this study, we explored how the properties of chromatin as a polymer may contribute to the structure of transcription factories. We found that transcriptional active chromatin contains modifications like histone H4 acetylated at Lysine 16 (H4K16ac). Single fibre analysis showed that this modification spans the entire body of the gene. Furthermore, H4K16ac genes cluster in regions up to 500 Kb alternating active and inactive chromatin. The introduction of H4K16ac in chromatin induces stiffness in the chromatin fibre. The result of this change in flexibility is that chromatin could behave like a multi-block copolymer with repetitions of stiff-flexible (active-inactive chromatin) components. Copolymers with such structure self-organize through spontaneous phase separation into microdomains. Consistent with such model H4K16ac chromatin form foci that associates with nascent transcripts. We propose that transcription factories are the result of the spontaneous concentration of H4K16ac chromatin that are in proximity, mainly in cis

  8. Intrinsic terminators in Mycoplasma hyopneumoniae transcription.

    Science.gov (United States)

    Fritsch, Tiago Ebert; Siqueira, Franciele Maboni; Schrank, Irene Silveira

    2015-04-08

    Mycoplasma hyopneumoniae, an important pathogen of swine, exhibits a low guanine and cytosine (GC) content genome. M. hyopneumoniae genome is organised in long transcriptional units and promoter sequences have been mapped upstream of all transcription units. These analysis provided insights into the gene organisation and transcription initiation at the genome scale. However, the presence of transcriptional terminator sequences in the M. hyopneumoniae genome is poorly understood. In silico analyses demonstrated the presence of putative terminators in 82% of the 33 monocistronic units (mCs) and in 74% of the 116 polycistronic units (pCs) considering different classes of terminators. The functional activity of 23 intrinsic terminators was confirmed by RT-PCR and qPCR. Analysis of all terminators found by three software algorithms, combined with experimental results, allowed us to propose a pattern of RNA hairpin formation during the termination process and to predict the location of terminators in the M. hyopneumoniae genome sequence. The stem-loop structures of intrinsic terminators of mycoplasma diverge from the pattern of terminators found in other bacteria due the low content of guanine and cytosine. In M. hyopneumoniae, transcription can end after a transcriptional unit and before its terminator sequence and can also continue past the terminator sequence with RNA polymerases gradually releasing the RNA.

  9. MADS-box gene evolution - structure and transcription patterns

    DEFF Research Database (Denmark)

    Johansen, Bo; Pedersen, Louise Buchholt; Skipper, Martin

    2002-01-01

    Mads-box genes, ABC model, Evolution, Phylogeny, Transcription patterns, Gene structure, Conserved motifs......Mads-box genes, ABC model, Evolution, Phylogeny, Transcription patterns, Gene structure, Conserved motifs...

  10. EWS/FLI mediates transcriptional repression via NKX2.2 during oncogenic transformation in Ewing's sarcoma.

    Directory of Open Access Journals (Sweden)

    Leah A Owen

    2008-04-01

    Full Text Available EWS/FLI is a master regulator of Ewing's sarcoma formation. Gene expression studies in A673 Ewing's sarcoma cells have demonstrated that EWS/FLI downregulates more genes than it upregulates, suggesting that EWS/FLI, and/or its targets, function as transcriptional repressors. One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature. We now report that microarray analysis demonstrated that the transcriptional profile of NKX2.2 consists solely of downregulated genes, and overlaps with the EWS/FLI downregulated signature, suggesting that NKX2.2 mediates oncogenic transformation via transcriptional repression. Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable. Furthermore, blockade of TLE or HDAC function, two protein families thought to mediate the repressive function of NKX2.2, inhibited the transformed phenotype and reversed the NKX2.2 transcriptional profile in Ewing's sarcoma cells. Whole genome localization studies (ChIP-chip revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding. These data demonstrate that the transcriptional repressive function of NKX2.2 is necessary, and sufficient, for the oncogenic phenotype of Ewing's sarcoma, and suggest a therapeutic approach to this disease.

  11. Frequency Modulation of Transcriptional Bursting Enables Sensitive and Rapid Gene Regulation.

    Science.gov (United States)

    Li, Congxin; Cesbron, François; Oehler, Michael; Brunner, Michael; Höfer, Thomas

    2018-04-25

    Gene regulation is a complex non-equilibrium process. Here, we show that quantitating the temporal regulation of key gene states (transcriptionally inactive, active, and refractory) provides a parsimonious framework for analyzing gene regulation. Our theory makes two non-intuitive predictions. First, for transcription factors (TFs) that regulate transcription burst frequency, as opposed to amplitude or duration, weak TF binding is sufficient to elicit strong transcriptional responses. Second, refractoriness of a gene after a transcription burst enables rapid responses to stimuli. We validate both predictions experimentally by exploiting the natural, optogenetic-like responsiveness of the Neurospora GATA-type TF White Collar Complex (WCC) to blue light. Further, we demonstrate that differential regulation of WCC target genes is caused by different gene activation rates, not different TF occupancy, and that these rates are tuned by both the core promoter and the distance between TF-binding site and core promoter. In total, our work demonstrates the relevance of a kinetic, non-equilibrium framework for understanding transcriptional regulation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Transcriptional Regulation and the Diversification of Metabolism in Wine Yeast Strains

    Science.gov (United States)

    Rossouw, Debra; Jacobson, Dan; Bauer, Florian F.

    2012-01-01

    Transcription factors and their binding sites have been proposed as primary targets of evolutionary adaptation because changes to single transcription factors can lead to far-reaching changes in gene expression patterns. Nevertheless, there is very little concrete evidence for such evolutionary changes. Industrial wine yeast strains, of the species Saccharomyces cerevisiae, are a geno- and phenotypically diverse group of organisms that have adapted to the ecological niches of industrial winemaking environments and have been selected to produce specific styles of wine. Variation in transcriptional regulation among wine yeast strains may be responsible for many of the observed differences and specific adaptations to different fermentative conditions in the context of commercial winemaking. We analyzed gene expression profiles of wine yeast strains to assess the impact of transcription factor expression on metabolic networks. The data provide new insights into the molecular basis of variations in gene expression in industrial strains and their consequent effects on metabolic networks important to wine fermentation. We show that the metabolic phenotype of a strain can be shifted in a relatively predictable manner by changing expression levels of individual transcription factors, opening opportunities to modify transcription networks to achieve desirable outcomes. PMID:22042577

  13. Inhibition of transcriptional activity of c-JUN by SIRT1

    International Nuclear Information System (INIS)

    Gao Zhanguo; Ye Jianping

    2008-01-01

    c-JUN is a major component of heterodimer transcription factor AP-1 (Activator Protein-1) that activates gene transcription in cell proliferation, inflammation and stress responses. SIRT1 (Sirtuin 1) is a histone deacetylase that controls gene transcription through modification of chromatin structure. However, it is not clear if SIRT1 regulates c-JUN activity in the control of gene transcription. Here, we show that SIRT1 associated with c-JUN in co-immunoprecipitation of whole cell lysate, and inhibited the transcriptional activity of c-JUN in the mammalian two hybridization system. SIRT1 was found in the AP-1 response element in the matrix metalloproteinase-9 (MMP9) promoter DNA leading to inhibition of histone 3 acetylation as shown in a ChIP assay. The SIRT1 signal was reduced by the AP-1 activator PMA, and induced by the SIRT1 activator Resveratrol in the promoter DNA. SIRT1-mediaetd inhibition of AP-1 was demonstrated in the MMP9 gene expression at the gene promoter, mRNA and protein levels. In mouse embryonic fibroblast (MEF) with SIRT1 deficiency (SIRT1 -/- ), mRNA and protein of MMP9 were increased in the basal condition, and the inhibitory activity of Resveratrol was significantly attenuated. Glucose-induced MMP9 expression was also inhibited by SIRT1 in response to Resveratrol. These data consistently suggest that SIRT1 directly inhibits the transcriptional activity of AP-1 by targeting c-JUN

  14. Regulation of gene expression by manipulating transcriptional repressor activity using a novel CoSRI technology.

    Science.gov (United States)

    Xu, Yue; Li, Song Feng; Parish, Roger W

    2017-07-01

    Targeted gene manipulation is a central strategy for studying gene function and identifying related biological processes. However, a methodology for manipulating the regulatory motifs of transcription factors is lacking as these factors commonly possess multiple motifs (e.g. repression and activation motifs) which collaborate with each other to regulate multiple biological processes. We describe a novel approach designated conserved sequence-guided repressor inhibition (CoSRI) that can specifically reduce or abolish the repressive activities of transcription factors in vivo. The technology was evaluated using the chimeric MYB80-EAR transcription factor and subsequently the endogenous WUS transcription factor. The technology was employed to develop a reversible male sterility system applicable to hybrid seed production. In order to determine the capacity of the technology to regulate the activity of endogenous transcription factors, the WUS repressor was chosen. The WUS repression motif could be inhibited in vivo and the transformed plants exhibited the wus-1 phenotype. Consequently, the technology can be used to manipulate the activities of transcriptional repressor motifs regulating beneficial traits in crop plants and other eukaryotic organisms. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  15. Dynamic EBF1 occupancy directs sequential epigenetic and transcriptional events in B-cell programming.

    Science.gov (United States)

    Li, Rui; Cauchy, Pierre; Ramamoorthy, Senthilkumar; Boller, Sören; Chavez, Lukas; Grosschedl, Rudolf

    2018-01-15

    B-cell fate determination requires the action of transcription factors that operate in a regulatory network to activate B-lineage genes and repress lineage-inappropriate genes. However, the dynamics and hierarchy of events in B-cell programming remain obscure. To uncouple the dynamics of transcription factor expression from functional consequences, we generated induction systems in developmentally arrested Ebf1 -/- pre-pro-B cells to allow precise experimental control of EBF1 expression in the genomic context of progenitor cells. Consistent with the described role of EBF1 as a pioneer transcription factor, we show in a time-resolved analysis that EBF1 occupancy coincides with EBF1 expression and precedes the formation of chromatin accessibility. We observed dynamic patterns of EBF1 target gene expression and sequential up-regulation of transcription factors that expand the regulatory network at the pro-B-cell stage. A continuous EBF1 function was found to be required for Cd79a promoter activity and for the maintenance of an accessible chromatin domain that is permissive for binding of other transcription factors. Notably, transient EBF1 occupancy was detected at lineage-inappropriate genes prior to their silencing in pro-B cells. Thus, persistent and transient functions of EBF1 allow for an ordered sequence of epigenetic and transcriptional events in B-cell programming. © 2018 Li et al.; Published by Cold Spring Harbor Laboratory Press.

  16. Using network component analysis to dissect regulatory networks mediated by transcription factors in yeast.

    Directory of Open Access Journals (Sweden)

    Chun Ye

    2009-03-01

    Full Text Available Understanding the relationship between genetic variation and gene expression is a central question in genetics. With the availability of data from high-throughput technologies such as ChIP-Chip, expression, and genotyping arrays, we can begin to not only identify associations but to understand how genetic variations perturb the underlying transcription regulatory networks to induce differential gene expression. In this study, we describe a simple model of transcription regulation where the expression of a gene is completely characterized by two properties: the concentrations and promoter affinities of active transcription factors. We devise a method that extends Network Component Analysis (NCA to determine how genetic variations in the form of single nucleotide polymorphisms (SNPs perturb these two properties. Applying our method to a segregating population of Saccharomyces cerevisiae, we found statistically significant examples of trans-acting SNPs located in regulatory hotspots that perturb transcription factor concentrations and affinities for target promoters to cause global differential expression and cis-acting genetic variations that perturb the promoter affinities of transcription factors on a single gene to cause local differential expression. Although many genetic variations linked to gene expressions have been identified, it is not clear how they perturb the underlying regulatory networks that govern gene expression. Our work begins to fill this void by showing that many genetic variations affect the concentrations of active transcription factors in a cell and their affinities for target promoters. Understanding the effects of these perturbations can help us to paint a more complete picture of the complex landscape of transcription regulation. The software package implementing the algorithms discussed in this work is available as a MATLAB package upon request.

  17. Hippocampal CA1 transcriptional profile of sleep deprivation: relation to aging and stress.

    Directory of Open Access Journals (Sweden)

    Nada M Porter

    Full Text Available Many aging changes seem similar to those elicited by sleep-deprivation and psychosocial stress. Further, sleep architecture changes with age suggest an age-related loss of sleep. Here, we hypothesized that sleep deprivation in young subjects would elicit both stress and aging-like transcriptional responses.F344 rats were divided into control and sleep deprivation groups. Body weight, adrenal weight, corticosterone level and hippocampal CA1 transcriptional profiles were measured. A second group of animals was exposed to novel environment stress (NES, and their hippocampal transcriptional profiles measured. A third cohort exposed to control or SD was used to validate transcriptional results with Western blots. Microarray results were statistically contrasted with prior transcriptional studies. Microarray results pointed to sleep pressure signaling and macromolecular synthesis disruptions in the hippocampal CA1 region. Animals exposed to NES recapitulated nearly one third of the SD transcriptional profile. However, the SD-aging relationship was more complex. Compared to aging, SD profiles influenced a significant subset of genes. mRNA associated with neurogenesis and energy pathways showed agreement between aging and SD, while immune, glial, and macromolecular synthesis pathways showed SD profiles that opposed those seen in aging.We conclude that although NES and SD exert similar transcriptional changes, selective presynaptic release machinery and Homer1 expression changes are seen in SD. Among other changes, the marked decrease in Homer1 expression with age may represent an important divergence between young and aged brain response to SD. Based on this, it seems reasonable to conclude that therapeutic strategies designed to promote sleep in young subjects may have off-target effects in the aged. Finally, this work identifies presynaptic vesicular release and intercellular adhesion molecular signatures as novel therapeutic targets to counter

  18. Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors | Office of Cancer Genomics

    Science.gov (United States)

    Targeted and inducible regulation of mammalian gene expression is a broadly important research capability that may also enable development of novel therapeutics for treating human diseases. Here we demonstrate that a catalytically inactive RNA-guided CRISPR-Cpf1 nuclease fused to transcriptional activation domains can up-regulate endogenous human gene expression. We engineered drug-inducible Cpf1-based activators and show how this system can be used to tune the regulation of endogenous gene transcription in human cells.

  19. Dynamic analysis of stochastic transcription cycles.

    Directory of Open Access Journals (Sweden)

    Claire V Harper

    2011-04-01

    Full Text Available In individual mammalian cells the expression of some genes such as prolactin is highly variable over time and has been suggested to occur in stochastic pulses. To investigate the origins of this behavior and to understand its functional relevance, we quantitatively analyzed this variability using new mathematical tools that allowed us to reconstruct dynamic transcription rates of different reporter genes controlled by identical promoters in the same living cell. Quantitative microscopic analysis of two reporter genes, firefly luciferase and destabilized EGFP, was used to analyze the dynamics of prolactin promoter-directed gene expression in living individual clonal and primary pituitary cells over periods of up to 25 h. We quantified the time-dependence and cyclicity of the transcription pulses and estimated the length and variation of active and inactive transcription phases. We showed an average cycle period of approximately 11 h and demonstrated that while the measured time distribution of active phases agreed with commonly accepted models of transcription, the inactive phases were differently distributed and showed strong memory, with a refractory period of transcriptional inactivation close to 3 h. Cycles in transcription occurred at two distinct prolactin-promoter controlled reporter genes in the same individual clonal or primary cells. However, the timing of the cycles was independent and out-of-phase. For the first time, we have analyzed transcription dynamics from two equivalent loci in real-time in single cells. In unstimulated conditions, cells showed independent transcription dynamics at each locus. A key result from these analyses was the evidence for a minimum refractory period in the inactive-phase of transcription. The response to acute signals and the result of manipulation of histone acetylation was consistent with the hypothesis that this refractory period corresponded to a phase of chromatin remodeling which significantly

  20. Wild type p53 transcriptionally represses the SALL2 transcription factor under genotoxic stress.

    Directory of Open Access Journals (Sweden)

    Carlos Farkas

    Full Text Available SALL2- a member of the Spalt gene family- is a poorly characterized transcription factor found deregulated in various cancers, which suggests it plays a role in the disease. We previously identified SALL2 as a novel interacting protein of neurotrophin receptors and showed that it plays a role in neuronal function, which does not necessarily explain why or how SALL2 is deregulated in cancer. Previous evidences indicate that SALL2 gene is regulated by the WT1 and AP4 transcription factors. Here, we identified SALL2 as a novel downstream target of the p53 tumor suppressor protein. Bioinformatic analysis of the SALL2 gene revealed several putative p53 half sites along the promoter region. Either overexpression of wild-type p53 or induction of the endogenous p53 by the genotoxic agent doxorubicin repressed SALL2 promoter activity in various cell lines. However R175H, R249S, and R248W p53 mutants, frequently found in the tumors of cancer patients, were unable to repress SALL2 promoter activity, suggesting that p53 specific binding to DNA is important for the regulation of SALL2. Electrophoretic mobility shift assay demonstrated binding of p53 to one of the identified p53 half sites in the Sall2 promoter, and chromatin immunoprecipitation analysis confirmed in vivo interaction of p53 with the promoter region of Sall2 containing this half site. Importantly, by using a p53ER (TAM knockin model expressing a variant of p53 that is completely dependent on 4-hydroxy-tamoxifen for its activity, we show that p53 activation diminished SALL2 RNA and protein levels during genotoxic cellular stress in primary mouse embryo fibroblasts (MEFs and radiosensitive tissues in vivo. Thus, our finding indicates that p53 represses SALL2 expression in a context-specific manner, adding knowledge to the understanding of SALL2 gene regulation, and to a potential mechanism for its deregulation in cancer.

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

    KAUST Repository

    Kulakovskiy, Ivan V.; Belostotsky, A. A.; Kasianov, Artem S.; Esipova, Natalia G.; Medvedeva, Yulia; Eliseeva, Irina A.; Makeev, Vsevolod J.

    2011-01-01

    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

  2. Controlling cellular P-TEFb activity by the HIV-1 transcriptional transactivator Tat.

    Directory of Open Access Journals (Sweden)

    Lisa Muniz

    Full Text Available The human immunodeficiency virus 1 (HIV-1 transcriptional transactivator (Tat is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II. Tat recruits the host positive transcription elongation factor b (P-TEFb to the HIV-1 promoter through binding to the transactivator RNA (TAR at the 5'-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5'-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR.

  3. Mga2 transcription factor regulates an oxygen-responsive lipid homeostasis pathway in fission yeast

    DEFF Research Database (Denmark)

    Burr, Risa; Stewart, Emerson V; Shao, Wei

    2016-01-01

    -binding protein (SREBP) transcription factors regulate lipid homeostasis. In mammals, SREBP-2 controls cholesterol biosynthesis, whereas SREBP-1 controls triacylglycerol and glycerophospholipid biosynthesis. In the fission yeast Schizosaccharomyces pombe, the SREBP-2 homolog Sre1 regulates sterol homeostasis....... In the absence of mga2, fission yeast exhibited growth defects under both normoxia and low oxygen conditions. Mga2 transcriptional targets were enriched for lipid metabolism genes, and mga2Δ cells showed disrupted triacylglycerol and glycerophospholipid homeostasis, most notably with an increase in fatty acid...

  4. SUMOylation of the ING1b tumor suppressor regulates gene transcription

    DEFF Research Database (Denmark)

    Satpathy, Shankha; Guérillon, Claire; Kim, Tae-Sun

    2014-01-01

    members of histone deacetylase complexes, whereas ING3-5 are stoichiometric components of different histone acetyltransferase complexes. The INGs target these complexes to histone marks, thus acting as epigenetic regulators. ING proteins affect angiogenesis, apoptosis, DNA repair, metastasis......1b E195A), we further demonstrate that ING1b SUMOylation regulates the binding of ING1b to the ISG15 and DGCR8 promoters, consequently regulating ISG15 and DGCR8 transcription. These results suggest a role for ING1b SUMOylation in the regulation of gene transcription....

  5. Can AtTZF1 act as a transcriptional activator or repressor in plants?

    OpenAIRE

    Pomeranz, Marcelo; Zhang, Li; Finer, John; Jang, Jyan-Chyun

    2011-01-01

    In animals, Tandem CCCH Zinc Finger (TZF) proteins can affect gene expression at both transcriptional and post-transcriptional levels. In Arabidopsis thaliana, AtTZF1 is a member of the TZF family characterized by a plant-unique tandem zinc finger motif. AtTZF1 can bind both DNA and RNA in vitro, and it can traffic between the nucleus and cytoplasmic foci. However, no in vivo DNA/RNA targets have been identified so far, and little is known about the molecular mechanisms underlying AtTZF1's pr...

  6. Heavy-ion targets

    International Nuclear Information System (INIS)

    Adair, H.L.; Kobisk, E.H.

    1985-01-01

    This chapter examines the characteristics of targets required in heavy-ion accelerator physics experiments. The effects of target parameters on heavy-ion experimental results are reviewed. The target fabrication and characterization techniques used to minimize experimental problems during heavy-ion bombardment are described. Topics considered include target thickness and uniformity, target lifetime, target purity, substrate materials, Doppler shift effects, metal preparations, and target preparation methods

  7. A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system.

    Science.gov (United States)

    D'Souza, Alicia D; Belotserkovskii, Boris P; Hanawalt, Philip C

    2018-02-01

    The selective inhibition of transcription of a chosen gene by an artificial agent has numerous applications. Usually, these agents are designed to bind a specific nucleotide sequence in the promoter or within the transcribed region of the chosen gene. However, since optimal binding sites might not exist within the gene, it is of interest to explore the possibility of transcription inhibition when the agent is designed to bind at other locations. One of these possibilities arises when an additional transcription initiation site (e.g. secondary promoter) is present upstream from the primary promoter of the target gene. In this case, transcription inhibition might be achieved by inducing the formation of an RNA-DNA hybrid (R-loop) upon transcription from the secondary promoter. The R-loop could extend into the region of the primary promoter, to interfere with promoter recognition by RNA polymerase and thereby inhibit transcription. As a sequence-specific R-loop-inducing agent, a peptide nucleic acid (PNA) could be designed to facilitate R-loop formation by sequestering the non-template DNA strand. To investigate this mode for transcription inhibition, we have employed a model system in which a PNA binding site is localized between the T3 and T7 phage RNA polymerase promoters, which respectively assume the roles of primary and secondary promoters. In accord with our model, we have demonstrated that with PNA-bound DNA substrates, transcription from the T7 promoter reduces transcription from the T3 promoter by 30-fold, while in the absence of PNA binding there is no significant effect of T7 transcription upon T3 transcription. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Dissection of TALE-dependent gene activation reveals that they induce transcription cooperatively and in both orientations.

    Science.gov (United States)

    Streubel, Jana; Baum, Heidi; Grau, Jan; Stuttman, Johannes; Boch, Jens

    2017-01-01

    Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators.

  9. Decoupling of ammonium regulation and ntcA transcription in the diazotrophic marine cyanobacterium Trichodesmium sp. IMS101.

    Science.gov (United States)

    Post, Anton F; Rihtman, Branko; Wang, Qingfeng

    2012-03-01

    Nitrogen (N) physiology in the marine cyanobacterium Trichodesmium IMS101 was studied along with transcript accumulation of the N-regulatory gene ntcA and of two of its target genes: napA (nitrate assimilation) and nifH (N(2) fixation). N(2) fixation was impaired in the presence of nitrite, nitrate and urea. Strain IMS101 was capable of growth on these combined N sources at ammonium. Whereas ecologically relevant N concentrations (2-20 μM) suppressed growth and assimilation, much higher concentrations were required to affect transcript levels. Transcripts of nifH accumulated under nitrogen-fixing conditions; these transcript levels were maintained in the presence of nitrate (100 μM) and ammonium (20 μM). However, nifH transcript levels were below detection at ammonium concentrations >20 μM. napA mRNA was found at low levels in both N(2)-fixing and ammonium-utilizing filaments, and it accumulated in filaments grown with nitrate. The positive effect of nitrate on napA transcription was abolished by ammonium additions of >200 μM. This effect was restored upon addition of the glutamine synthetase inhibitor L-methionin-DL-sulfoximine. Surprisingly, ntcA transcript levels remained high in the presence of ammonium, even at elevated concentrations. These findings indicate that ammonium repression is decoupled from transcriptional activation of ntcA in Trichodesmium IMS101.

  10. Manuscript Transcription by Crowdsourcing: Transcribe Bentham

    Directory of Open Access Journals (Sweden)

    Martin Moyle

    2011-02-01

    Full Text Available Transcribe Bentham is testing the feasibility of outsourcing the work of manuscript transcription to members of the public. UCL Library Services holds 60,000 folios of manuscripts of the philosopher and jurist Jeremy Bentham (1748–1832. Transcribe Bentham will digitise 12,500 Bentham folios, and, through a wiki-based interface, allow volunteer transcribers to take temporary ownership of manuscript images and to create TEI-encoded transcription text for final approval by UCL experts. Approved transcripts will be stored and preserved, with the manuscript images, in UCL’s public Digital Collections repository. The project makes innovative use of traditional library material. It will stimulate public engagement with UCL’s scholarly archive collections and the challenges of palaeography and manuscript transcription; it will raise the profile of the work and thought of Jeremy Bentham; and it will create new digital resources for future use by professional researchers. Towards the end of the project, the transcription tool will be made available to other projects and services. This paper is based on a presentation given by the lead author at LIBER’s 39th Annual General Conference in Aarhus, Denmark, 2010.

  11. Structural Basis of Mitochondrial Transcription Initiation.

    Science.gov (United States)

    Hillen, Hauke S; Morozov, Yaroslav I; Sarfallah, Azadeh; Temiakov, Dmitry; Cramer, Patrick

    2017-11-16

    Transcription in human mitochondria is driven by a single-subunit, factor-dependent RNA polymerase (mtRNAP). Despite its critical role in both expression and replication of the mitochondrial genome, transcription initiation by mtRNAP remains poorly understood. Here, we report crystal structures of human mitochondrial transcription initiation complexes assembled on both light and heavy strand promoters. The structures reveal how transcription factors TFAM and TFB2M assist mtRNAP to achieve promoter-dependent initiation. TFAM tethers the N-terminal region of mtRNAP to recruit the polymerase to the promoter whereas TFB2M induces structural changes in mtRNAP to enable promoter opening and trapping of the DNA non-template strand. Structural comparisons demonstrate that the initiation mechanism in mitochondria is distinct from that in the well-studied nuclear, bacterial, or bacteriophage transcription systems but that similarities are found on the topological and conceptual level. These results provide a framework for studying the regulation of gene expression and DNA replication in mitochondria. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Malleable machines in transcription regulation: the mediator complex.

    Directory of Open Access Journals (Sweden)

    Agnes Tóth-Petróczy

    2008-12-01

    Full Text Available The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II. The complex has a modular architecture (Head, Middle, and Tail and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs distinguished by a high protein-protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the

  13. Systematic analysis of transcription start sites in avian development.

    Directory of Open Access Journals (Sweden)

    Marina Lizio

    2017-09-01

    Full Text Available Cap Analysis of Gene Expression (CAGE in combination with single-molecule sequencing technology allows precision mapping of transcription start sites (TSSs and genome-wide capture of promoter activities in differentiated and steady state cell populations. Much less is known about whether TSS profiling can characterize diverse and non-steady state cell populations, such as the approximately 400 transitory and heterogeneous cell types that arise during ontogeny of vertebrate animals. To gain such insight, we used the chick model and performed CAGE-based TSS analysis on embryonic samples covering the full 3-week developmental period. In total, 31,863 robust TSS peaks (>1 tag per million [TPM] were mapped to the latest chicken genome assembly, of which 34% to 46% were active in any given developmental stage. ZENBU, a web-based, open-source platform, was used for interactive data exploration. TSSs of genes critical for lineage differentiation could be precisely mapped and their activities tracked throughout development, suggesting that non-steady state and heterogeneous cell populations are amenable to CAGE-based transcriptional analysis. Our study also uncovered a large set of extremely stable housekeeping TSSs and many novel stage-specific ones. We furthermore demonstrated that TSS mapping could expedite motif-based promoter analysis for regulatory modules associated with stage-specific and housekeeping genes. Finally, using Brachyury as an example, we provide evidence that precise TSS mapping in combination with Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR-on technology enables us, for the first time, to efficiently target endogenous avian genes for transcriptional activation. Taken together, our results represent the first report of genome-wide TSS mapping in birds and the first systematic developmental TSS analysis in any amniote species (birds and mammals. By facilitating promoter-based molecular analysis and genetic

  14. The transcript release factor PTRF augments ribosomal gene transcription by facilitating reinitiation of RNA polymerase I

    Czech Academy of Sciences Publication Activity Database

    Jansa, Petr; Burek, C.; Sander, E. E.; Grummt, I.

    2001-01-01

    Roč. 29, č. 2 (2001), s. 423-429 ISSN 0305-1048 Institutional research plan: CEZ:AV0Z5052915 Keywords : rDNA transcription * PTRF * transcription reinitiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.373, year: 2001

  15. Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription.

    Science.gov (United States)

    Gerasimova, N S; Pestov, N A; Kulaeva, O I; Clark, D J; Studitsky, V M

    2016-05-26

    RNA polymerase II (Pol II) transcription through chromatin is accompanied by formation of small intranucleosomal DNA loops. Pol II captured within a small loop drives accumulation of DNA supercoiling, facilitating further transcription. DNA breaks relieve supercoiling and induce Pol II arrest, allowing detection of DNA damage hidden in chromatin structure.

  16. The Intertwined Roles of DNA Damage and Transcription

    OpenAIRE

    Di Palo, Giacomo

    2016-01-01

    DNA damage and transcription are two interconnected events. Transcription can induce damage and scheduled DNA damage can be required for transcription. Here, we analyzed genome-wide distribution of 8oxodG-marked oxidative DNA damage obtained by OxiDIP-Seq, and we found a correlation with transcription of protein coding genes.

  17. The Demethylase JMJD2C Localizes to H3K4me3 Positive Transcription Start Sites and Is Dispensable for Embryonic Development

    DEFF Research Database (Denmark)

    Pedersen, Marianne Terndrup; Agger, Karl; Laugesen, Anne

    2014-01-01

    cell (ESC) self-renewal and embryonic development. Moreover, we report that JMJD2C localizes to H3K4me3 positive transcription start sites in both primary cells and in the human carcinoma KYSE150 cell line, containing an amplification of the JMJD2C locus. Binding is dependent on the double Tudor domain...... expression of a subset of target genes involved in cell cycle progression. Taken together, we show that JMJD2C is targeted to H3K4me3 positive transcription start sites, where it can contribute to transcriptional regulation, and report that the putative oncogene, JMJD2C, is not generally required...

  18. Towards understanding the lifespan extension by reduced insulin signaling: bioinformatics analysis of DAF-16/FOXO direct targets in Caenorhabditis elegans.

    Science.gov (United States)

    Li, Yan-Hui; Zhang, Gai-Gai

    2016-04-12

    DAF-16, the C. elegans FOXO transcription factor, is an important determinant in aging and longevity. In this work, we manually curated FOXODB http://lyh.pkmu.cn/foxodb/, a database of FOXO direct targets. It now covers 208 genes. Bioinformatics analysis on 109 DAF-16 direct targets in C. elegans found interesting results. (i) DAF-16 and transcription factor PQM-1 co-regulate some targets. (ii) Seventeen targets directly regulate lifespan. (iii) Four targets are involved in lifespan extension induced by dietary restriction. And (iv) DAF-16 direct targets might play global roles in lifespan regulation.

  19. Battles and hijacks: Noncoding transcription in plants

    KAUST Repository

    Ariel, Federico

    2015-06-01

    Noncoding RNAs have emerged as major components of the eukaryotic transcriptome. Genome-wide analyses revealed the existence of thousands of long noncoding RNAs (lncRNAs) in several plant species. Plant lncRNAs are transcribed by the plant-specific RNA polymerases Pol IV and Pol V, leading to transcriptional gene silencing, as well as by Pol II. They are involved in a wide range of regulatory mechanisms impacting on gene expression, including chromatin remodeling, modulation of alternative splicing, fine-tuning of miRNA activity, and the control of mRNA translation or accumulation. Recently, dual noncoding transcription by alternative RNA polymerases was implicated in epigenetic and chromatin conformation dynamics. This review integrates the current knowledge on the regulatory mechanisms acting through plant noncoding transcription. © 2015 Elsevier Ltd.

  20. Phonemic Transcriptions in British and American Dictionaries

    Directory of Open Access Journals (Sweden)

    Rastislav Šuštaršič

    2005-06-01

    Full Text Available In view of recent criticisms concerning vowel symbols in some British English dictionaries (in particular by J. Windsor Lewis in JIPA (Windsor Lewis, 2003, with regard to the Oxford Dictionary of Pronunciation (Upton, 2001, this article extends the discussion on English phonemic transcriptions by including those that typically occur in standard American dictionaries, and by comparing the most common conventions of British and American dictionaries. In addition to symbols for both vowels and consonants, the paper also deals with the different representations of word accentuation and the issue of consistency regarding application of phonemic (systemic, broad, rather than phonetic (allophonic, narrow transcription. The different transcriptions are assessed from the points of view of their departures from the International Phonetic Alphabet, their overlapping with orthographic representation (spelling and their appropriateness in terms of reflecting actual pronunciation in standard British and/or American pronunciation.

  1. Crowdsourcing for quantifying transcripts: An exploratory study.

    Science.gov (United States)

    Azzam, Tarek; Harman, Elena

    2016-02-01

    This exploratory study attempts to demonstrate the potential utility of crowdsourcing as a supplemental technique for quantifying transcribed interviews. Crowdsourcing is the harnessing of the abilities of many people to complete a specific task or a set of tasks. In this study multiple samples of crowdsourced individuals were asked to rate and select supporting quotes from two different transcripts. The findings indicate that the different crowdsourced samples produced nearly identical ratings of the transcripts, and were able to consistently select the same supporting text from the transcripts. These findings suggest that crowdsourcing, with further development, can potentially be used as a mixed method tool to offer a supplemental perspective on transcribed interviews. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Transcription regulatory networks analysis using CAGE

    KAUST Repository

    Tegnér, Jesper N.

    2009-10-01

    Mapping out cellular networks in general and transcriptional networks in particular has proved to be a bottle-neck hampering our understanding of biological processes. Integrative approaches fusing computational and experimental technologies for decoding transcriptional networks at a high level of resolution is therefore of uttermost importance. Yet, this is challenging since the control of gene expression in eukaryotes is a complex multi-level process influenced by several epigenetic factors and the fine interplay between regulatory proteins and the promoter structure governing the combinatorial regulation of gene expression. In this chapter we review how the CAGE data can be integrated with other measurements such as expression, physical interactions and computational prediction of regulatory motifs, which together can provide a genome-wide picture of eukaryotic transcriptional regulatory networks at a new level of resolution. © 2010 by Pan Stanford Publishing Pte. Ltd. All rights reserved.

  3. Runx transcription factors in neuronal development

    Directory of Open Access Journals (Sweden)

    Shiga Takashi

    2008-08-01

    Full Text Available Abstract Runt-related (Runx transcription factors control diverse aspects of embryonic development and are responsible for the pathogenesis of many human diseases. In recent years, the functions of this transcription factor family in the nervous system have just begun to be understood. In dorsal root ganglion neurons, Runx1 and Runx3 play pivotal roles in the development of nociceptive and proprioceptive sensory neurons, respectively. Runx appears to control the transcriptional regulation of neurotrophin receptors, numerous ion channels and neuropeptides. As a consequence, Runx contributes to diverse aspects of the sensory system in higher vertebrates. In this review, we summarize recent progress in determining the role of Runx in neuronal development.

  4. Transcriptional inhibition by the retinoblastoma protein

    DEFF Research Database (Denmark)

    Fattaey, A; Helin, K; Harlow, E

    1993-01-01

    The retinoblastoma protein, pRB, appears to play a key role in coordinating the regulation of cell cycle position and transcriptional events. pRB undergoes specific cell-cycle-dependent phosphorylation, being underphosphorylated in G1 and heavily phosphorylated in S, G2, and M. The underphosphory......The retinoblastoma protein, pRB, appears to play a key role in coordinating the regulation of cell cycle position and transcriptional events. pRB undergoes specific cell-cycle-dependent phosphorylation, being underphosphorylated in G1 and heavily phosphorylated in S, G2, and M......-mediated transcription would be lost by mutation in the retinoblastoma gene in human tumours, by pRB's interaction with DNA tumour virus oncoproteins, or by phosphorylation during the cell cycle....

  5. Deciphering the Innate Lymphoid Cell Transcriptional Program

    Directory of Open Access Journals (Sweden)

    Cyril Seillet

    2016-10-01

    Full Text Available Innate lymphoid cells (ILCs are enriched at mucosal surfaces, where they provide immune surveillance. All ILC subsets develop from a common progenitor that gives rise to pre-committed progenitors for each of the ILC lineages. Currently, the temporal control of gene expression that guides the emergence of these progenitors is poorly understood. We used global transcriptional mapping to analyze gene expression in different ILC progenitors. We identified PD-1 to be specifically expressed in PLZF+ ILCp and revealed that the timing and order of expression of the transcription factors NFIL3, ID2, and TCF-1 was critical. Importantly, induction of ILC lineage commitment required only transient expression of NFIL3 prior to ID2 and TCF-1 expression. These findings highlight the importance of the temporal program that permits commitment of progenitors to the ILC lineage, and they expand our understanding of the core transcriptional program by identifying potential regulators of ILC development.

  6. Transcription as a Threat to Genome Integrity.

    Science.gov (United States)

    Gaillard, Hélène; Aguilera, Andrés

    2016-06-02

    Genomes undergo different types of sporadic alterations, including DNA damage, point mutations, and genome rearrangements, that constitute the basis for evolution. However, these changes may occur at high levels as a result of cell pathology and trigger genome instability, a hallmark of cancer and a number of genetic diseases. In the last two decades, evidence has accumulated that transcription constitutes an important natural source of DNA metabolic errors that can compromise the integrity of the genome. Transcription can create the conditions for high levels of mutations and recombination by its ability to open the DNA structure and remodel chromatin, making it more accessible to DNA insulting agents, and by its ability to become a barrier to