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Sample records for human general transcription

  1. Structural motifs and potential sigma homologies in the large subunit of human general transcription factor TFIIE.

    Science.gov (United States)

    Ohkuma, Y; Sumimoto, H; Hoffmann, A; Shimasaki, S; Horikoshi, M; Roeder, R G

    1991-12-05

    The general transcription factor TFIIE has an essential role in eukaryotic transcription initiation together with RNA polymerase II and other general factors. Human TFIIE consists of two subunits of relative molecular mass 57,000 (TFIIE-alpha) and 34,000 (TFIIE-beta) and joins the preinitiation complex after RNA polymerase II and TFIIF. Here we report the cloning and structure of a complementary DNA encoding a functional human TFIIE-alpha. TFIIE-alpha is necessary for transcription initiation together with TFIIE-beta, and recombinant TFIIE-alpha can fully replace the natural subunit in an in vitro transcription assay. The sequence contains several interesting structural motifs (leucine repeat, zinc finger and helix-turn-helix) and sequence similarities to bacterial sigma factors that suggest direct involvement in the regulation of transcription initiation.

  2. Conserved sequence motifs in the small subunit of human general transcription factor TFIIE.

    Science.gov (United States)

    Sumimoto, H; Ohkuma, Y; Sinn, E; Kato, H; Shimasaki, S; Horikoshi, M; Roeder, R G

    1991-12-05

    A general initiation factor, TFIIE, is essential for transcription initiation by RNA polymerase II in conjunction with other general factors. TFIIE is a heterotetramer containing two subunits of relative molecular mass 57,000 (TFIIE-alpha) and two of 34,000 (TFIIE-beta). TFIIE-beta is required in conjunction with TFIIE-alpha for transcription initiation. Here we report the cloning and expression of a complementary DNA encoding a functional human TFIIE-beta. Recombinant TFIIE-beta could replace the natural TFIIE-beta for transcription in conjunction with TFIIE-alpha. Amino-acid sequence comparisons reveal regions with sequence similarities to: subregion 3 of bacterial sigma factors; a region of RAP30 (the small subunit of TFIIF) with sequence similarity to a sigma-factor subregion implicated in binding to RNA polymerase; and a portion of the basic region-helix-loop-helix motif found in several enhancer-binding proteins. These potential homologies have implications for the role of TFIIE in preinitiation complex assembly and function.

  3. Transcription of Satellite III non-coding RNAs is a general stress response in human cells

    Science.gov (United States)

    Valgardsdottir, Rut; Chiodi, Ilaria; Giordano, Manuela; Rossi, Antonio; Bazzini, Silvia; Ghigna, Claudia; Riva, Silvano; Biamonti, Giuseppe

    2008-01-01

    In heat-shocked human cells, heat shock factor 1 activates transcription of tandem arrays of repetitive Satellite III (SatIII) DNA in pericentromeric heterochromatin. Satellite III RNAs remain associated with sites of transcription in nuclear stress bodies (nSBs). Here we use real-time RT-PCR to study the expression of these genomic regions. Transcription is highly asymmetrical and most of the transcripts contain the G-rich strand of the repeat. A low level of G-rich RNAs is detectable in unstressed cells and a 104-fold induction occurs after heat shock. G-rich RNAs are induced by a wide range of stress treatments including heavy metals, UV-C, oxidative and hyper-osmotic stress. Differences exist among stressing agents both for the kinetics and the extent of induction (>100- to 80.000-fold). In all cases, G-rich transcripts are associated with nSBs. On the contrary, C-rich transcripts are almost undetectable in unstressed cells and modestly increase after stress. Production of SatIII RNAs after hyper-osmotic stress depends on the Tonicity Element Binding Protein indicating that activation of the arrays is triggered by different transcription factors. This is the first example of a non-coding RNA whose transcription is controlled by different transcription factors under different growth conditions. PMID:18039709

  4. Architecture of the Human and Yeast General Transcription and DNA Repair Factor TFIIH.

    Science.gov (United States)

    Luo, Jie; Cimermancic, Peter; Viswanath, Shruthi; Ebmeier, Christopher C; Kim, Bong; Dehecq, Marine; Raman, Vishnu; Greenberg, Charles H; Pellarin, Riccardo; Sali, Andrej; Taatjes, Dylan J; Hahn, Steven; Ranish, Jeff

    2015-09-03

    TFIIH is essential for both RNA polymerase II transcription and DNA repair, and mutations in TFIIH can result in human disease. Here, we determine the molecular architecture of human and yeast TFIIH by an integrative approach using chemical crosslinking/mass spectrometry (CXMS) data, biochemical analyses, and previously published electron microscopy maps. We identified four new conserved "topological regions" that function as hubs for TFIIH assembly and more than 35 conserved topological features within TFIIH, illuminating a network of interactions involved in TFIIH assembly and regulation of its activities. We show that one of these conserved regions, the p62/Tfb1 Anchor region, directly interacts with the DNA helicase subunit XPD/Rad3 in native TFIIH and is required for the integrity and function of TFIIH. We also reveal the structural basis for defects in patients with xeroderma pigmentosum and trichothiodystrophy, with mutations found at the interface between the p62 Anchor region and the XPD subunit. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Telomerase promoter reprogramming and interaction with general transcription factors in the human mesenchymal stem cell

    DEFF Research Database (Denmark)

    Serakinci, Nedime; Hoare, Stacey F.; Kassem, Moustapha

    2006-01-01

    The human adult mesenchymal stem cell (hMSC) does not express telomerase and has been shown to be the target for neoplastic transformation after transduction with hTERT. These findings lend support to the stem cell hypothesis of cancer development but by supplying hTERT, the molecular events...... and that modifications of the chromatin environment lead to reactivation of telomerase gene expression. It is shown that repression of hTERT expression in hMSCs is due to promoter-specific histone hypoacetylation coupled with low Pol II and TFIIB trafficking. This repression is overcome by treatment with Trichostatin...

  6. Transcriptional and post-transcriptional profile of human chromosome 21.

    Science.gov (United States)

    Nikolaev, Sergey I; Deutsch, Samuel; Genolet, Raphael; Borel, Christelle; Parand, Leila; Ucla, Catherine; Schütz, Frederic; Duriaux Sail, Genevieve; Dupré, Yann; Jaquier-Gubler, Pascale; Araud, Tanguy; Conne, Beatrice; Descombes, Patrick; Vassalli, Jean-Dominique; Curran, Joseph; Antonarakis, Stylianos E

    2009-08-01

    Recent studies have demonstrated extensive transcriptional activity across the human genome, a substantial fraction of which is not associated with any functional annotation. However, very little is known regarding the post-transcriptional processes that operate within the different classes of RNA molecules. To characterize the post-transcriptional properties of expressed sequences from human chromosome 21 (HSA21), we separated RNA molecules from three cell lines (GM06990, HeLa S3, and SK-N-AS) according to their ribosome content by sucrose gradient fractionation. Polyribosomal-associated RNA and total RNA were subsequently hybridized to genomic tiling arrays. We found that approximately 50% of the transcriptional signals were located outside of annotated exons and were considered as TARs (transcriptionally active regions). Although TARs were observed among polysome-associated RNAs, RT-PCR and RACE experiments revealed that approximately 40% were likely to represent nonspecific cross-hybridization artifacts. Bioinformatics discrimination of TARs according to conservation and sequence complexity allowed us to identify a set of high-confidence TARs. This set of TARs was significantly depleted in the polysomes, suggesting that it was not likely to be involved in translation. Analysis of polysome representation of RefSeq exons showed that at least 15% of RefSeq transcripts undergo significant post-transcriptional regulation in at least two of the three cell lines tested. Among the regulated transcripts, enrichment analysis revealed an over-representation of genes involved in Alzheimer's disease (AD), including APP and the BACE1 protease that cleaves APP to produce the pathogenic beta 42 peptide. We demonstrate that the combination of RNA fractionation and tiling arrays is a powerful method to assess the transcriptional and post-transcriptional properties of genomic regions.

  7. Accessorizing the human mitochondrial transcription machinery.

    Science.gov (United States)

    Bestwick, Megan L; Shadel, Gerald S

    2013-06-01

    The human genome comprises large chromosomes in the nucleus and mitochondrial DNA (mtDNA) housed in the dynamic mitochondrial network. Human cells contain up to thousands of copies of the double-stranded, circular mtDNA molecule that encodes essential subunits of the oxidative phosphorylation complexes and the rRNAs and tRNAs needed to translate these in the organelle matrix. Transcription of human mtDNA is directed by a single-subunit RNA polymerase, POLRMT, which requires two primary transcription factors, TFB2M (transcription factor B2, mitochondrial) and TFAM (transcription factor A, mitochondrial), to achieve basal regulation of the system. Here, we review recent advances in understanding the structure and function of the primary human transcription machinery and the other factors that facilitate steps in transcription beyond initiation and provide more intricate control over the system. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. General human activity patterns

    CERN Document Server

    Mollgaard, Anders; Mathiesen, Joachim

    2016-01-01

    We investigate the dynamics and interplay between human communication, movement, and social proximity by analyzing data collected from smartphones distributed among 638 individuals. The main question we consider is: to what extent do individuals act according to patterns shared across an entire population? Based on statistics of the entire population, we successfully predict 71\\% of the activity and 85\\% of the inactivity involved in communication, movement, and social proximity. We find that individual level statistics only result in marginally better predictions, indicating a high degree of shared activity patterns across the population. Finally, we predict short-term activity patterns using a generalized linear model, which suggests that a simple linear description might be sufficient to explain a wide range of actions, whether they be of social or of physical character.

  9. Human mediator subunit MED15 promotes transcriptional activation.

    Science.gov (United States)

    Nakatsubo, Takuya; Nishitani, Saori; Kikuchi, Yuko; Iida, Satoshi; Yamada, Kana; Tanaka, Aki; Ohkuma, Yoshiaki

    2014-10-01

    In eukaryotes, the Mediator complex is an essential transcriptional cofactor of RNA polymerase II (Pol II). In humans, it contains up to 30 subunits and consists of four modules: head, middle, tail, and CDK/Cyclin. One of the subunits, MED15, is located in the tail module, and was initially identified as Gal11 in budding yeast, where it plays an essential role in the transcriptional regulation of galactose metabolism with the potent transcriptional activator Gal4. For this reason, we investigated the function of the human MED15 subunit (hMED15) in transcriptional activation. First, we measured the effect of hMED15 knockdown on cell growth in HeLa cells. The growth rate was greatly reduced. By immunostaining, we observed the colocalization of hMED15 with the general transcription factors TFIIE and TFIIH in the nucleus. We measured the effects of siRNA-mediated knockdown of hMED15 on transcriptional activation using two different transcriptional activators, VP16 and SREBP1a. Treatment with siRNAs reduced transcriptional activation, and this reduction could be rescued by overexpression of HA/Flag-tagged, wild-type hMED15. To investigate hMED15 localization, we treated human MCF-7 cells with the MDM2 inhibitor Nutlin-3, thus inducing p21 transcription. We found that hMED15 localized to both the p53 binding site and the p21 promoter region, along with TFIIE and TFIIH. These results indicate that hMED15 promotes transcriptional activation.

  10. 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

    2017-08-02

    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.

  11. Accessorizing the human mitochondrial transcription machinery

    OpenAIRE

    Bestwick, Megan L; Shadel, Gerald S

    2013-01-01

    The human genome comprises large chromosomes in the nucleus and mitochondrial DNA (mtDNA) housed in the dynamic mitochondrial network. Human cells contain up to thousands of copies of the double-stranded, circular mtDNA molecule that encodes essential subunits of the oxidative phosphorylation complexes and the rRNAs and tRNAs needed to translate these in the organelle matrix. Transcription of human mtDNA is directed by a single-subunit RNA polymerase, POLRMT, which requires two primary transc...

  12. Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription.

    Science.gov (United States)

    Dumay-Odelot, Hélène; Durrieu-Gaillard, Stéphanie; El Ayoubi, Leyla; Parrot, Camila; Teichmann, Martin

    2014-01-01

    Human RNA polymerase III transcribes small untranslated RNAs that contribute to the regulation of essential cellular processes, including transcription, RNA processing and translation. Analysis of this transcription system by in vitro transcription techniques has largely contributed to the discovery of its transcription factors and to the understanding of the regulation of human RNA polymerase III transcription. Here we review some of the key steps that led to the identification of transcription factors and to the definition of minimal promoter sequences for human RNA polymerase III transcription.

  13. Transcriptional landscape of the prenatal human brain.

    Science.gov (United States)

    Miller, Jeremy A; Ding, Song-Lin; Sunkin, Susan M; Smith, Kimberly A; Ng, Lydia; Szafer, Aaron; Ebbert, Amanda; Riley, Zackery L; Royall, Joshua J; Aiona, Kaylynn; Arnold, James M; Bennet, Crissa; Bertagnolli, Darren; Brouner, Krissy; Butler, Stephanie; Caldejon, Shiella; Carey, Anita; Cuhaciyan, Christine; Dalley, Rachel A; Dee, Nick; Dolbeare, Tim A; Facer, Benjamin A C; Feng, David; Fliss, Tim P; Gee, Garrett; Goldy, Jeff; Gourley, Lindsey; Gregor, Benjamin W; Gu, Guangyu; Howard, Robert E; Jochim, Jayson M; Kuan, Chihchau L; Lau, Christopher; Lee, Chang-Kyu; Lee, Felix; Lemon, Tracy A; Lesnar, Phil; McMurray, Bergen; Mastan, Naveed; Mosqueda, Nerick; Naluai-Cecchini, Theresa; Ngo, Nhan-Kiet; Nyhus, Julie; Oldre, Aaron; Olson, Eric; Parente, Jody; Parker, Patrick D; Parry, Sheana E; Stevens, Allison; Pletikos, Mihovil; Reding, Melissa; Roll, Kate; Sandman, David; Sarreal, Melaine; Shapouri, Sheila; Shapovalova, Nadiya V; Shen, Elaine H; Sjoquist, Nathan; Slaughterbeck, Clifford R; Smith, Michael; Sodt, Andy J; Williams, Derric; Zöllei, Lilla; Fischl, Bruce; Gerstein, Mark B; Geschwind, Daniel H; Glass, Ian A; Hawrylycz, Michael J; Hevner, Robert F; Huang, Hao; Jones, Allan R; Knowles, James A; Levitt, Pat; Phillips, John W; Sestan, Nenad; Wohnoutka, Paul; Dang, Chinh; Bernard, Amy; Hohmann, John G; Lein, Ed S

    2014-04-10

    The anatomical and functional architecture of the human brain is mainly determined by prenatal transcriptional processes. We describe an anatomically comprehensive atlas of the mid-gestational human brain, including de novo reference atlases, in situ hybridization, ultra-high-resolution magnetic resonance imaging (MRI) and microarray analysis on highly discrete laser-microdissected brain regions. In developing cerebral cortex, transcriptional differences are found between different proliferative and post-mitotic layers, wherein laminar signatures reflect cellular composition and developmental processes. Cytoarchitectural differences between human and mouse have molecular correlates, including species differences in gene expression in subplate, although surprisingly we find minimal differences between the inner and outer subventricular zones even though the outer zone is expanded in humans. Both germinal and post-mitotic cortical layers exhibit fronto-temporal gradients, with particular enrichment in the frontal lobe. Finally, many neurodevelopmental disorder and human-evolution-related genes show patterned expression, potentially underlying unique features of human cortical formation. These data provide a rich, freely-accessible resource for understanding human brain development.

  14. Transcriptional network of p63 in human keratinocytes.

    Directory of Open Access Journals (Sweden)

    Silvia Pozzi

    Full Text Available p63 is a transcription factor required for the development and maintenance of ectodermal tissues in general, and skin keratinocytes in particular. The identification of its target genes is fundamental for understanding the complex network of gene regulation governing the development of epithelia. We report a list of almost 1000 targets derived from ChIP on chip analysis on two platforms; all genes analyzed changed in expression during differentiation of human keratinocytes. Functional annotation highlighted unexpected GO terms enrichments and confirmed that genes involved in transcriptional regulation are the most significant. A detailed analysis of these transcriptional regulators in condition of perturbed p63 levels confirmed the role of p63 in the regulatory network. Rather than a rigid master-slave hierarchical model, our data indicate that p63 connects different hubs involved in the multiple specific functions of the skin.

  15. New insight into transcription of human endogenous retroviral elements.

    Science.gov (United States)

    Pačes, Jan; Huang, Yao-Ting; Pačes, Václav; Rídl, Jakub; Chang, Chung-Ming

    2013-03-25

    It is generally assumed that human endogenous retroviral elements (HERVs) belong to the class of genomic repetitive nucleotide sequences often called 'junk DNA'. These elements were categorized to families, and members of some of these families (e.g. HERV-H, HERV-W and HERV-K) were shown to be transcribed. These transcriptions were associated with several severe diseases such as mental disorders, AIDS, autoimmune diseases and cancer. In this review we discuss several bioinformatics strategies for genome-wide scan of HERVs transcription using high-throughput RNA sequencing on several platforms. We show that many more HERVs than previously described are transcribed to various levels and we discuss possible implications of these transcriptions.

  16. Transcriptional Activity of Human Endogenous Retroviruses in Human Peripheral Blood Mononuclear Cells

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    Emanuela Balestrieri

    2015-01-01

    Full Text Available Human endogenous retroviruses (HERVs have been implicated in human physiology and in human pathology. A better knowledge of the retroviral transcriptional activity in the general population and during the life span would greatly help the debate on its pathologic potential. The transcriptional activity of four HERV families (H, K, W, and E was assessed, by qualitative and quantitative PCR, in PBMCs from 261 individuals aged from 1 to 80 years. Our results show that HERV-H, HERV-K, and HERV-W, but not HERV-E, are transcriptionally active in the test population already in the early childhood. In addition, the transcriptional levels of HERV-H, HERV-K, and HERV-W change significantly during the life span, albeit with distinct patterns. Our results, reinforce the hypothesis of a physiological correlation between HERVs activity and the different stages of life in humans. Studies aiming at identifying the factors, which are responsible for these changes during the individual’s life, are still needed. Although the observed phenomena are presumably subjected to great variability, the basal transcriptional activity of each individual, also depending on the different ages of life, must be carefully considered in all the studies involving HERVs as causative agents of disease.

  17. Transcriptional directionality of the human insulin-degrading enzyme promoter.

    Science.gov (United States)

    Zhang, Lang; Wang, Pan; Ding, Qingyang; Wang, Zhao

    2013-10-01

    Unidirectional promoters dominate among mammalian genomes. However, the mechanism through which the transcriptional directionality of promoters is accomplished remains to be clarified. Insulin-degrading enzyme (IDE) is a ubiquitously expressed zinc metalloprotease, whose promoter contains a CpG island. We previously showed that the basal promoter region of mouse IDE has bidirectional transcriptional activity, but an upstream promoter element blocks its antisense transcription. Therefore, we wonder whether the human IDE promoter contains an analogous element. Similarly, the basal promoter region of human IDE (-102 ~ +173 and -196 ~ +173 relative to the transcription start site) showed bidirectional transcriptional activity. However, the region from -348 to +173 could only be transcribed from the normal orientation, implying that an upstream promoter element between -348 and -196 blocks the antisense transcription of the human IDE promoter. Through promoter deletion and mutagenesis analysis, we mapped this element precisely and found that the upstream promoter element locates between -318 and -304. Furthermore, the transcription-blocking elements in the mouse and human IDE promoters inhibited the transcription of the SV40 promoter when put downstream of it. In conclusion, we identify an upstream promoter element which blocks the antisense transcription of the human IDE promoter. Our studies are helpful to clarify the transcriptional directionality of promoters.

  18. An environment-dependent transcriptional network specifies human microglia identity

    NARCIS (Netherlands)

    Gosselin, David; Skola, Dylan; Coufal, Nicole G.; Holtman, Inge R.; Schlachetzki, Johannes C. M.; Sajti, Eniko; Jaeger, Baptiste N.; O'Connor, Carolyn; Fitzpatrick, Conor; Pasillas, Martina P.; Pena, Monique; Adair, Amy; Gonda, David D.; Levy, Michael L.; Ransohoff, Richard M.; Gage, Fred H.; Glass, Christopher K.

    2017-01-01

    Microglia play essential roles in central nervous system (CNS) homeostasis and influence diverse aspects of neuronal function. However, the transcriptional mechanisms that specify human microglia phenotypes are largely unknown. We examined the transcriptomes and epigenetic landscapes of human microg

  19. The effects of cytosine methylation on general transcription factors

    Science.gov (United States)

    Jin, Jianshi; Lian, Tengfei; Gu, Chan; Yu, Kai; Gao, Yi Qin; Su, Xiao-Dong

    2016-07-01

    DNA methylation on CpG sites is the most common epigenetic modification. Recently, methylation in a non-CpG context was found to occur widely on genomic DNA. Moreover, methylation of non-CpG sites is a highly controlled process, and its level may vary during cellular development. To study non-CpG methylation effects on DNA/protein interactions, we have chosen three human transcription factors (TFs): glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK) and estrogen receptor (ER) with methylated or unmethylated DNA binding sequences, using single-molecule and isothermal titration calorimetry assays. The results demonstrated that these TFs interact with methylated DNA with different effects compared with their cognate DNA sequences. The effects of non-CpG methylation on transcriptional regulation were validated by cell-based luciferase assay at protein level. The mechanisms of non-CpG methylation influencing DNA-protein interactions were investigated by crystallographic analyses and molecular dynamics simulation. With BisChIP-seq assays in HEK-293T cells, we found that GR can recognize highly methylated sites within chromatin in cells. Therefore, we conclude that non-CpG methylation of DNA can provide a mechanism for regulating gene expression through directly affecting the binding of TFs.

  20. From reverse transcription to human brain tumors

    Directory of Open Access Journals (Sweden)

    Dmitrenko V. V.

    2013-05-01

    Full Text Available Reverse transcriptase from avian myeloblastosis virus (AMV was the subject of the study, from which the investi- gations of the Department of biosynthesis of nucleic acids were started. Production of AMV in grams quantities and isolation of AMV reverse transcriptase were established in the laboratory during the seventies of the past cen- tury and this initiated research on the cDNA synthesis, cloning and investigation of the structure and functions of the eukaryotic genes. Structures of salmon insulin and insulin-like growth factor (IGF family genes and their transcripts were determined during long-term investigations. Results of two modern techniques, microarray-ba- sed hybridization and SAGE, were used for the identification of the genes differentially expressed in astrocytic gliomas and human normal brain. Comparison of SAGE results on the genes overexpressed in glioblastoma with the results of microarray analysis revealed a limited number of common genes. 105 differentially expressed genes, common to both methods, can be included in the list of candidates for the molecular typing of glioblastoma. The first experiments on the classification of glioblastomas based on the data of the 20 genes expression were conducted by using of artificial neural network analysis. The results of these experiments showed that the expression profiles of these genes in 224 glioblastoma samples and 74 normal brain samples could be according to the Koho- nen’s maps. The CHI3L1 and CHI3L2 genes of chitinase-like cartilage protein were revealed among the most overexpressed genes in glioblastoma, which could have prognostic and diagnostic potential. Results of in vitro experiments demonstrated that both proteins, CHI3L1 and CHI3L2, may initiate the phosphorylation of ERK1/ ERK2 and AKT kinases leading to the activation of MAPK/ERK1/2 and PI3K/AKT signaling cascades in human embryonic kidney 293 cells, human glioblastoma U87MG, and U373 cells. The new human cell line

  1. Human cytomegalovirus IE2 protein interacts with transcription activating factors

    Institute of Scientific and Technical Information of China (English)

    XU; Jinping(徐进平); YE; Linbai(叶林柏)

    2002-01-01

    The human cytomegalovirus (HCMV) IE86 Cdna was cloned into Pgex-2T and fusion protein GST-IE86 was expressed in E. Coli. SDS-PAGE and Western blot assay indicated that fusion protein GST-IE86 with molecular weight of 92 ku is soluble in the supernatant of cell lysate. Protein GST and fusion protein GST-IE86 were purified by affinity chromatography. The technology of co-separation and specific affinity chromatography was used to study the interactions of HCMV IE86 protein with some transcriptional regulatory proteins and transcriptional factors. The results indicated that IE86 interacts separately with transcriptional factor TFIIB and promoter DNA binding transcription trans-activating factors SP1, AP1 and AP2 to form a heterogenous protein complex. These transcriptional trans-activating factors, transcriptional factor and IE86 protein were adsorbed and retained in the affinity chromatography simultaneously. But IE86 protein could not interact with NF-Кb, suggesting that the function of IE86 protein that can interact with transcriptional factor and transcriptional trans-activating factors has no relevance to protein glycosylation. IE86 protein probably has two domains responsible for binding transcriptional trans-activating regulatory proteins and transcriptional factors respectively, thus activating the transcription of many genes. The interactions accelerated the assembly of the transcriptional initiation complexes.

  2. Mitochondrial biology. Replication-transcription switch in human mitochondria.

    Science.gov (United States)

    Agaronyan, Karen; Morozov, Yaroslav I; Anikin, Michael; Temiakov, Dmitry

    2015-01-30

    Coordinated replication and expression of the mitochondrial genome is critical for metabolically active cells during various stages of development. However, it is not known whether replication and transcription can occur simultaneously without interfering with each other and whether mitochondrial DNA copy number can be regulated by the transcription machinery. We found that interaction of human transcription elongation factor TEFM with mitochondrial RNA polymerase and nascent transcript prevents the generation of replication primers and increases transcription processivity and thereby serves as a molecular switch between replication and transcription, which appear to be mutually exclusive processes in mitochondria. TEFM may allow mitochondria to increase transcription rates and, as a consequence, respiration and adenosine triphosphate production without the need to replicate mitochondrial DNA, as has been observed during spermatogenesis and the early stages of embryogenesis.

  3. Inhibition of RNA Polymerase II Transcription in Human Cells by Synthetic DNA-Binding Ligands

    Science.gov (United States)

    Dickinson, Liliane A.; Gulizia, Richard J.; Trauger, John W.; Baird, Eldon E.; Mosier, Donald E.; Gottesfeld, Joel M.; Dervan, Peter B.

    1998-10-01

    Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole--imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located with RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

  4. Genetic control of human brain transcript expression in Alzheimer disease.

    Science.gov (United States)

    Webster, Jennifer A; Gibbs, J Raphael; Clarke, Jennifer; Ray, Monika; Zhang, Weixiong; Holmans, Peter; Rohrer, Kristen; Zhao, Alice; Marlowe, Lauren; Kaleem, Mona; McCorquodale, Donald S; Cuello, Cindy; Leung, Doris; Bryden, Leslie; Nath, Priti; Zismann, Victoria L; Joshipura, Keta; Huentelman, Matthew J; Hu-Lince, Diane; Coon, Keith D; Craig, David W; Pearson, John V; Heward, Christopher B; Reiman, Eric M; Stephan, Dietrich; Hardy, John; Myers, Amanda J

    2009-04-01

    We recently surveyed the relationship between the human brain transcriptome and genome in a series of neuropathologically normal postmortem samples. We have now analyzed additional samples with a confirmed pathologic diagnosis of late-onset Alzheimer disease (LOAD; final n = 188 controls, 176 cases). Nine percent of the cortical transcripts that we analyzed had expression profiles correlated with their genotypes in the combined cohort, and approximately 5% of transcripts had SNP-transcript relationships that could distinguish LOAD samples. Two of these transcripts have been previously implicated in LOAD candidate-gene SNP-expression screens. This study shows how the relationship between common inherited genetic variants and brain transcript expression can be used in the study of human brain disorders. We suggest that studying the transcriptome as a quantitative endo-phenotype has greater power for discovering risk SNPs influencing expression than the use of discrete diagnostic categories such as presence or absence of disease.

  5. General trends in the evolution of prokaryotic transcriptional regulatory networks.

    Science.gov (United States)

    Madan Babu, M; Balaji, S; Aravind, L

    2007-01-01

    Gene expression in organisms is controlled by regulatory proteins termed transcription factors, which recognize and bind to specific nucleotide sequences. Over the years, considerable information has accumulated on the regulatory interactions between transcription factors and their target genes in various model prokaryotes, such as Escherichia coli and Bacillus subtilis. This has allowed the representation of this information in the form of a directed graph, which is commonly referred to as the transcriptional regulatory network. The network representation provides us with an excellent conceptual framework to understand the structure of the transcriptional regulation, both at local and global levels of organization. Several studies suggest that the transcriptional network inferred from model organisms may be approximated by a scale-free topology, which in turn implies the presence of a relatively small group of highly connected regulators (hubs or global regulators). While the graph theoretical principles have been applied to infer various properties of such networks, there have been few studies that have actually investigated the evolution of the transcriptional regulatory networks across diverse organisms. Using recently developed computational methods that exploit various evolutionary principles, we have attempted to reconstruct and compare these networks across a wide-range of prokaryotes. This has provided several insights on the modification and diversification of network structures of various organisms in course of evolution. Firstly, we observed that target genes show a much higher level of conservation than their transcriptional regulators. This in turn suggested that the same set of functions could be differently controlled across diverse organisms, contributing significantly to their adaptive radiations. In particular, at the local level of network structure, organism-specific optimization of the transcription network has evolved primarily via tinkering

  6. Characterization of the transcripts of human cytomegalovirus UL144

    Directory of Open Access Journals (Sweden)

    Sun Zhengrong

    2011-06-01

    Full Text Available Abstract Background The genome of human cytomegalovirus (HCMV has been studied extensively, particularly in the UL/b' region. In this study, transcripts of one of the UL/b' genes, UL144, were identified in 3 HCMV isolates obtained from urine samples of congenitally infected infants. Methods Northern blot hybridization, cDNA library screening, and RACE-PCR were used. Results We identified at least 4 differentially regulated 3'-coterminal transcripts of UL144 in infected cells of 1,300, 1,600, 1,700, and 3,500 nucleotides (nt. The 1600 nt transcript was the major form of UL144 mRNA. The largest transcript initiated from the region within the UL141 open reading frame (ORF and included UL141, UL142, UL143, UL144, and UL145 ORFs. Conclusions These findings reveal the complex nature of the transcription of the UL144 gene in clinical isolates.

  7. Transcriptional and post-transcriptional regulation of nucleotide excision repair genes in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Lefkofsky, Hailey B. [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Veloso, Artur [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Bioinformatics Program, Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI (United States); Ljungman, Mats, E-mail: ljungman@umich.edu [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI (United States)

    2015-06-15

    Nucleotide excision repair (NER) removes DNA helix-distorting lesions induced by UV light and various chemotherapeutic agents such as cisplatin. These lesions efficiently block the elongation of transcription and need to be rapidly removed by transcription-coupled NER (TC-NER) to avoid the induction of apoptosis. Twenty-nine genes have been classified to code for proteins participating in nucleotide excision repair (NER) in human cells. Here we explored the transcriptional and post-transcriptional regulation of these NER genes across 13 human cell lines using Bru-seq and BruChase-seq, respectively. Many NER genes are relatively large in size and therefore will be easily inactivated by UV-induced transcription-blocking lesions. Furthermore, many of these genes produce transcripts that are rather unstable. Thus, these genes are expected to rapidly lose expression leading to a diminished function of NER. One such gene is ERCC6 that codes for the CSB protein critical for TC-NER. Due to its large gene size and high RNA turnover rate, the ERCC6 gene may act as dosimeter of DNA damage so that at high levels of damage, ERCC6 RNA levels would be diminished leading to the loss of CSB expression, inhibition of TC-NER and the promotion of cell death.

  8. Human-specific transcriptional networks in the brain.

    Science.gov (United States)

    Konopka, Genevieve; Friedrich, Tara; Davis-Turak, Jeremy; Winden, Kellen; Oldham, Michael C; Gao, Fuying; Chen, Leslie; Wang, Guang-Zhong; Luo, Rui; Preuss, Todd M; Geschwind, Daniel H

    2012-08-23

    Understanding human-specific patterns of brain gene expression and regulation can provide key insights into human brain evolution and speciation. Here, we use next-generation sequencing, and Illumina and Affymetrix microarray platforms, to compare the transcriptome of human, chimpanzee, and macaque telencephalon. Our analysis reveals a predominance of genes differentially expressed within human frontal lobe and a striking increase in transcriptional complexity specific to the human lineage in the frontal lobe. In contrast, caudate nucleus gene expression is highly conserved. We also identify gene coexpression signatures related to either neuronal processes or neuropsychiatric diseases, including a human-specific module with CLOCK as its hub gene and another module enriched for neuronal morphological processes and genes coexpressed with FOXP2, a gene important for language evolution. These data demonstrate that transcriptional networks have undergone evolutionary remodeling even within a given brain region, providing a window through which to view the foundation of uniquely human cognitive capacities.

  9. Genetic characterization of general transcription factors TFIIF and TFIIB of Homo sapiens sapiens.

    Science.gov (United States)

    Purrello, M; Di Pietro, C; Rapisarda, A; Mirabile, E; Motta, S; Sichel, G; Grzeschik, K H

    1995-01-01

    Analysis of loci GTF2F1 and GTF2B, encoding Rap 74 (a subunit of TFIIF) and TFIIB, respectively, showed that they are present in a single copy in the human genome and are localized at 19p13.3 and 1p22, respectively. By using as probe a cDNA for Rap 30 (the other subunit of TFIIF), we localized the GTF2F2 locus to 13q14; the same probe also detected a cross-hybridizing sequence at 4q31 whose functional importance remains to be elucidated. These data and those previously published by our group demonstrate that genes coding for class II general transcription factors with reported sequence similarity to bacterial sigma proteins are scattered in different regions of the human genome, with no evidence of clustering. This dispersion and the identification of homologs of both TBP and TFIIB in Archaea suggest an early evolutionary origin of the general transcription apparatus of contemporary eukaryotes.

  10. Human brain evolution: transcripts, metabolites and their regulators.

    Science.gov (United States)

    Somel, Mehmet; Liu, Xiling; Khaitovich, Philipp

    2013-02-01

    What evolutionary events led to the emergence of human cognition? Although the genetic differences separating modern humans from both non-human primates (for example, chimpanzees) and archaic hominins (Neanderthals and Denisovans) are known, linking human-specific mutations to the cognitive phenotype remains a challenge. One strategy is to focus on human-specific changes at the level of intermediate phenotypes, such as gene expression and metabolism, in conjunction with evolutionary changes in gene regulation involving transcription factors, microRNA and proximal regulatory elements. In this Review we show how this strategy has yielded some of the first hints about the mechanisms of human cognition.

  11. Sensitive detection of viral transcripts in human tumor transcriptomes.

    Directory of Open Access Journals (Sweden)

    Sven-Eric Schelhorn

    Full Text Available In excess of 12% of human cancer incidents have a viral cofactor. Epidemiological studies of idiopathic human cancers indicate that additional tumor viruses remain to be discovered. Recent advances in sequencing technology have enabled systematic screenings of human tumor transcriptomes for viral transcripts. However, technical problems such as low abundances of viral transcripts in large volumes of sequencing data, viral sequence divergence, and homology between viral and human factors significantly confound identification of tumor viruses. We have developed a novel computational approach for detecting viral transcripts in human cancers that takes the aforementioned confounding factors into account and is applicable to a wide variety of viruses and tumors. We apply the approach to conducting the first systematic search for viruses in neuroblastoma, the most common cancer in infancy. The diverse clinical progression of this disease as well as related epidemiological and virological findings are highly suggestive of a pathogenic cofactor. However, a viral etiology of neuroblastoma is currently contested. We mapped 14 transcriptomes of neuroblastoma as well as positive and negative controls to the human and all known viral genomes in order to detect both known and unknown viruses. Analysis of controls, comparisons with related methods, and statistical estimates demonstrate the high sensitivity of our approach. Detailed investigation of putative viral transcripts within neuroblastoma samples did not provide evidence for the existence of any known human viruses. Likewise, de-novo assembly and analysis of chimeric transcripts did not result in expression signatures associated with novel human pathogens. While confounding factors such as sample dilution or viral clearance in progressed tumors may mask viral cofactors in the data, in principle, this is rendered less likely by the high sensitivity of our approach and the number of biological replicates

  12. Human DJ-1-specific Transcriptional Activation of Tyrosine Hydroxylase Gene*

    Science.gov (United States)

    Ishikawa, Shizuma; Taira, Takahiro; Takahashi-Niki, Kazuko; Niki, Takeshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M. M.

    2010-01-01

    Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-l-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice. PMID:20938049

  13. Transcriptional regulation of the human mitochondrial peptide deformylase (PDF).

    Science.gov (United States)

    Pereira-Castro, Isabel; Costa, Luís Teixeira da; Amorim, António; Azevedo, Luisa

    2012-05-18

    The last years of research have been particularly dynamic in establishing the importance of peptide deformylase (PDF), a protein of the N-terminal methionine excision (NME) pathway that removes formyl-methionine from mitochondrial-encoded proteins. The genomic sequence of the human PDF gene is shared with the COG8 gene, which encodes a component of the oligomeric golgi complex, a very unusual case in Eukaryotic genomes. Since PDF is crucial in maintaining mitochondrial function and given the atypical short distance between the end of COG8 coding sequence and the PDF initiation codon, we investigated whether the regulation of the human PDF is affected by the COG8 overlapping partner. Our data reveals that PDF has several transcription start sites, the most important of which only 18 bp from the initiation codon. Furthermore, luciferase-activation assays using differently-sized fragments defined a 97 bp minimal promoter region for human PDF, which is capable of very strong transcriptional activity. This fragment contains a potential Sp1 binding site highly conserved in mammalian species. We show that this binding site, whose mutation significantly reduces transcription activation, is a target for the Sp1 transcription factor, and possibly of other members of the Sp family. Importantly, the entire minimal promoter region is located after the end of COG8's coding region, strongly suggesting that the human PDF preserves an independent regulation from its overlapping partner. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. One novel transcript of human hereditary multipleexostoses 2 (EXT2)

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The encoding sequence of human hereditary multiple exostoses gene EXT2.1 is 30 bp longer than EXT2, and they differ in a sequence of 90 base pairs. In order to clarify EXT2.1 structure, this 90 bp sequence was analyzed with the Human Sequence Draft, a database provided by Celera Genomics. The result shows that EXT2.1 is a novel transcript of EXT2 gene, suggesting a rare event of alternative splicing.

  15. Transcriptional responses of human epidermal keratinocytes to Oncostatin-M.

    Science.gov (United States)

    Finelt, Nika; Gazel, Alix; Gorelick, Steven; Blumenberg, Miroslav

    2005-08-21

    Oncostatin-M (OsM) plays an important role in inflammatory and oncogenic processes in skin, including psoriasis and Kaposi sarcoma. However, the molecular responses to OsM in keratinocytes have not been explored in depth. Here we show the results of transcriptional profiling in OsM-treated primary human epidermal keratinocytes, using high-density DNA microarrays. We find that OsM strongly and specifically affects the expression of many genes, in particular those involved with innate immunity, angiogenesis, adhesion, motility, tissue remodeling, cell cycle and transcription. The timing of the responses to OsM comprises two waves, early at 1h, and late at 48 h, with much fewer genes regulated in the intervening time points. Secreted cytokines and growth factors and their receptors, as well as nuclear transcription factors, are primary targets of OsM regulation, and these, in turn, effect the secondary changes.

  16. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    Directory of Open Access Journals (Sweden)

    Ralph D Hector

    Full Text Available Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5 cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR, which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  17. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    Science.gov (United States)

    Hector, Ralph D; Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  18. Uncovering layers of human RNA polymerase II transcription

    DEFF Research Database (Denmark)

    Jensen, Torben Heick

    In recent years DNA microarray and high-throughput sequencing technologies have challenged the “gene-centric” view that pre-mRNA is the only RNA species transcribed off protein-coding genes. Instead unorthodox transcription from within genic- and intergenic regions has been demonstrated to occur...... and unstable RNAs emitted from within, and immediately upstream human protein-coding genes. Mechanisms of their production and turn-over as well as their possible functions will be discussed...

  19. An expansive human regulatory lexicon encoded in transcription factor footprints.

    Science.gov (United States)

    Neph, Shane; Vierstra, Jeff; Stergachis, Andrew B; Reynolds, Alex P; Haugen, Eric; Vernot, Benjamin; Thurman, Robert E; John, Sam; Sandstrom, Richard; Johnson, Audra K; Maurano, Matthew T; Humbert, Richard; Rynes, Eric; Wang, Hao; Vong, Shinny; Lee, Kristen; Bates, Daniel; Diegel, Morgan; Roach, Vaughn; Dunn, Douglas; Neri, Jun; Schafer, Anthony; Hansen, R Scott; Kutyavin, Tanya; Giste, Erika; Weaver, Molly; Canfield, Theresa; Sabo, Peter; Zhang, Miaohua; Balasundaram, Gayathri; Byron, Rachel; MacCoss, Michael J; Akey, Joshua M; Bender, M A; Groudine, Mark; Kaul, Rajinder; Stamatoyannopoulos, John A

    2012-09-06

    Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency.

  20. Transcriptional divergence and conservation of human and mouse erythropoiesis.

    Science.gov (United States)

    Pishesha, Novalia; Thiru, Prathapan; Shi, Jiahai; Eng, Jennifer C; Sankaran, Vijay G; Lodish, Harvey F

    2014-03-18

    Mouse models have been used extensively for decades and have been instrumental in improving our understanding of mammalian erythropoiesis. Nonetheless, there are several examples of variation between human and mouse erythropoiesis. We performed a comparative global gene expression study using data from morphologically identical stage-matched sorted populations of human and mouse erythroid precursors from early to late erythroblasts. Induction and repression of major transcriptional regulators of erythropoiesis, as well as major erythroid-important proteins, are largely conserved between the species. In contrast, at a global level we identified a significant extent of divergence between the species, both at comparable stages and in the transitions between stages, especially for the 500 most highly expressed genes during development. This suggests that the response of multiple developmentally regulated genes to key erythroid transcriptional regulators represents an important modification that has occurred in the course of erythroid evolution. In developing a systematic framework to understand and study conservation and divergence between human and mouse erythropoiesis, we show how mouse models can fail to mimic specific human diseases and provide predictions for translating findings from mouse models to potential therapies for human disease.

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

    Directory of Open Access Journals (Sweden)

    Steven A L W Vanhoutvin

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

  2. Analysis of the heavy metal-responsive transcription factor MTF-1 from human and mouse

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, H.P.; Brugnera, E.; Georgiev, O. [Universitaet Zuerich (Switzerland)] [and others

    1995-09-01

    Heavy metal-induced transcription in mammalian cells is conferred by the metalresponsive 70 kDa transcription factor MTF-1 which contains six zinc fingers and at least three activation domains. In previous cell transfection experiments we have shown that the zinc finger region confers an about 3 fold metal inducibility of transcription, due to its differential zinc binding. However, we also noted that human MTF-1 was more metal-responsive than the mouse factor (about 10 fold versus 3 fold, respectively). Here we analyze this difference in more detail by using chimeric human-mouse factors and narrow the critical region to a 64 amino acid stretch immediately downstream of the zinc fingers, overlapping with the acidic activation domain. A short human segment of this region (aa 313-377) confers efficient metal induction to the mouse MTF-1 when replacing the corresponding mouse region. However, high metal inducibility requires an unaltered MTF-I and is lost when human MTF-I is fused to the general activation domain of herpesvirus VP16 Wild type and truncation mutants of MTF-1 fused to VPI6 yield chimeras of high transcriptional activity, some exceeding the wildtype regulator, but only limited (about 3 fold) heavy metal inducibility. 22 refs., 4 figs.

  3. Transcriptional signature and memory retention of human-induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Maria C N Marchetto

    Full Text Available Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs by over-expression of specific genes has been accomplished using mouse and human cells. However, it is still unclear how similar human iPSCs are to human Embryonic Stem Cells (hESCs. Here, we describe the transcriptional profile of human iPSCs generated without viral vectors or genomic insertions, revealing that these cells are in general similar to hESCs but with significant differences. For the generation of human iPSCs without viral vectors or genomic insertions, pluripotent factors Oct4 and Nanog were cloned in episomal vectors and transfected into human fetal neural progenitor cells. The transient expression of these two factors, or from Oct4 alone, resulted in efficient generation of human iPSCs. The reprogramming strategy described here revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Moreover, the episomal reprogramming strategy represents a safe way to generate human iPSCs for clinical purposes and basic research.

  4. A sensitive RNase protection assay to detect transcripts from potentially functional human endogenous L1 retrotransposons

    DEFF Research Database (Denmark)

    Woodcock, D M; Williamson, M R; Doherty, J P

    1996-01-01

    A high background of read-through transcripts from degenerate human L1 retrotransposons is present in almost all human cell types. This prevents the detection of RNA transcripts from potentially functional elements. To overcome this, we have developed an RNase protection assay based on the recons...... transcripts from divergent L1 families but are either discrete shorter transcripts or specifically processed products from longer initial transcripts....

  5. Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis

    Science.gov (United States)

    Zhang, Zhengjian; English, Brian P.; Grimm, Jonathan B.; Kazane, Stephanie A.; Hu, Wenxin; Tsai, Albert; Inouye, Carla; You, Changjiang; Piehler, Jacob; Schultz, Peter G.; Lavis, Luke D.; Revyakin, Andrey; Tjian, Robert

    2016-01-01

    Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A “step-wise” preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB–promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II–TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions. PMID:27798851

  6. Transcriptional regulation of human thromboxane synthase gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Lee, K.D.; Baek, S.J.; Fleischer, T [Univ. of Maryland Medical School, Baltimore, MD (United States)] [and others

    1994-09-01

    The human thromboxane synthase (TS) gene encodes a microsomal enzyme catalyzing the conversion of prostaglandin endoperoxide into thromboxane A{sub 2}(TxA{sub 2}), a potent inducer of vasoconstriction and platelet aggregation. A deficiency in platelet TS activity results in bleeding disorders, but the underlying molecular mechanism remains to be elucidated. Increased TxA{sub 2} has been associated with many pathophysiological conditions such as cardiovascular disease, pulmonary hypertension, pre-eclampsia, and thrombosis in sickle cell patients. Since the formation of TxA{sub 2} is dependent upon TS, the regulation of TS gene expression may presumably play a crucial role in vivo. Abrogation of the regulatory mechanism in TS gene expression might contribute, in part, to the above clinical manifestations. To gain insight into TS gene regulation, a 1.7 kb promoter of the human TS gene was cloned and sequenced. RNase protection assay and 5{prime} RACE protocols were used to map the transcription initiation site to nucleotide A, 30 bp downstream from a canonical TATA box. Several transcription factor binding sites, including AP-1, PU.1, and PEA3, were identified within this sequence. Transient expression studies in HL-60 cells transfected with constructs containing various lengths (0.2 to 5.5 kb) of the TS promoter/luciferase fusion gene indicated the presence of multiple repressor elements within the 5.5 kb TS promoter. However, a lineage-specific up-regulation of TS gene expression was observed in HL-60 cells induced by TPA to differentiate along the macrophage lineage. The increase in TS transcription was not detectable until 36 hr after addition of the inducer. These results suggest that expression of the human TS gene may be regulated by a mechanism involving repression and derepression of the TS promoter.

  7. Computational analysis of splicing errors and mutations in human transcripts

    Directory of Open Access Journals (Sweden)

    Gelfand Mikhail S

    2008-01-01

    Full Text Available Abstract Background Most retained introns found in human cDNAs generated by high-throughput sequencing projects seem to result from underspliced transcripts, and thus they capture intermediate steps of pre-mRNA splicing. On the other hand, mutations in splice sites cause exon skipping of the respective exon or activation of pre-existing cryptic sites. Both types of events reflect properties of the splicing mechanism. Results The retained introns were significantly shorter than constitutive ones, and skipped exons are shorter than exons with cryptic sites. Both donor and acceptor splice sites of retained introns were weaker than splice sites of constitutive introns. The authentic acceptor sites affected by mutations were significantly weaker in exons with activated cryptic sites than in skipped exons. The distance from a mutated splice site to the nearest equivalent site is significantly shorter in cases of activated cryptic sites compared to exon skipping events. The prevalence of retained introns within genes monotonically increased in the 5'-to-3' direction (more retained introns close to the 3'-end, consistent with the model of co-transcriptional splicing. The density of exonic splicing enhancers was higher, and the density of exonic splicing silencers lower in retained introns compared to constitutive ones and in exons with cryptic sites compared to skipped exons. Conclusion Thus the analysis of retained introns in human cDNA, exons skipped due to mutations in splice sites and exons with cryptic sites produced results consistent with the intron definition mechanism of splicing of short introns, co-transcriptional splicing, dependence of splicing efficiency on the splice site strength and the density of candidate exonic splicing enhancers and silencers. These results are consistent with other, recently published analyses.

  8. DNA-binding specificities of human transcription factors.

    Science.gov (United States)

    Jolma, Arttu; Yan, Jian; Whitington, Thomas; Toivonen, Jarkko; Nitta, Kazuhiro R; Rastas, Pasi; Morgunova, Ekaterina; Enge, Martin; Taipale, Mikko; Wei, Gonghong; Palin, Kimmo; Vaquerizas, Juan M; Vincentelli, Renaud; Luscombe, Nicholas M; Hughes, Timothy R; Lemaire, Patrick; Ukkonen, Esko; Kivioja, Teemu; Taipale, Jussi

    2013-01-17

    Although the proteins that read the gene regulatory code, transcription factors (TFs), have been largely identified, it is not well known which sequences TFs can recognize. We have analyzed the sequence-specific binding of human TFs using high-throughput SELEX and ChIP sequencing. A total of 830 binding profiles were obtained, describing 239 distinctly different binding specificities. The models represent the majority of human TFs, approximately doubling the coverage compared to existing systematic studies. Our results reveal additional specificity determinants for a large number of factors for which a partial specificity was known, including a commonly observed A- or T-rich stretch that flanks the core motifs. Global analysis of the data revealed that homodimer orientation and spacing preferences, and base-stacking interactions, have a larger role in TF-DNA binding than previously appreciated. We further describe a binding model incorporating these features that is required to understand binding of TFs to DNA.

  9. Gene transcriptional networks integrate microenvironmental signals in human breast cancer.

    Science.gov (United States)

    Xu, Ren; Mao, Jian-Hua

    2011-04-01

    A significant amount of evidence shows that microenvironmental signals generated from extracellular matrix (ECM) molecules, soluble factors, and cell-cell adhesion complexes cooperate at the extra- and intracellular level. This synergetic action of microenvironmental cues is crucial for normal mammary gland development and breast malignancy. To explore how the microenvironmental genes coordinate in human breast cancer at the genome level, we have performed gene co-expression network analysis in three independent microarray datasets and identified two microenvironment networks in human breast cancer tissues. Network I represents crosstalk and cooperation of ECM microenvironment and soluble factors during breast malignancy. The correlated expression of cytokines, chemokines, and cell adhesion proteins in Network II implicates the coordinated action of these molecules in modulating the immune response in breast cancer tissues. These results suggest that microenvironmental cues are integrated with gene transcriptional networks to promote breast cancer development.

  10. Mutations and binding sites of human transcription factors

    KAUST Repository

    Kamanu, Frederick Kinyua

    2012-06-01

    Mutations in any genome may lead to phenotype characteristics that determine ability of an individual to cope with adaptation to environmental challenges. In studies of human biology, among the most interesting ones are phenotype characteristics that determine responses to drug treatments, response to infections, or predisposition to specific inherited diseases. Most of the research in this field has been focused on the studies of mutation effects on the final gene products, peptides, and their alterations. Considerably less attention was given to the mutations that may affect regulatory mechanism(s) of gene expression, although these may also affect the phenotype characteristics. In this study we make a pilot analysis of mutations observed in the regulatory regions of 24,667 human RefSeq genes. Our study reveals that out of eight studied mutation types, insertions are the only one that in a statistically significant manner alters predicted transcription factor binding sites (TFBSs). We also find that 25 families of TFBSs have been altered by mutations in a statistically significant manner in the promoter regions we considered. Moreover, we find that the related transcription factors are, for example, prominent in processes related to intracellular signaling; cell fate; morphogenesis of organs and epithelium; development of urogenital system, epithelium, and tube; neuron fate commitment. Our study highlights the significance of studying mutations within the genes regulatory regions and opens way for further detailed investigations on this topic, particularly on the downstream affected pathways. 2012 Kamanu, Medvedeva, Schaefer, Jankovic, Archer and Bajic.

  11. Differences in transcriptional activity of cutaneous human papillomaviruses

    DEFF Research Database (Denmark)

    Vasiljevic, Natasa; Nielsen, Lone; Doherty, Geoff

    2008-01-01

    and 96 URR was up-regulated by cellular differentiation, linking the activity of these HPVs to the cellular state. UV-B irradiation activated HPV-8 but inhibited HPV-38 and HPV-93 whereas HPV-92 and 96 were not affected. As there are variable UV-B responses among the HPV types, further studies......The interaction between UV-B irradiation and cutaneous human papillomaviruses (HPV) has been suggested to be of relevance for the development of non-melanoma skin cancers. We investigated the activity within the upstream regulatory region (URR) of the HPV types 8, 38, 92, 93 and 96, as well...... as their responsiveness to UV-B irradiation and cellular differentiation. Promoter activities were higher in HaCaT than in SiHa cells, corresponding to the HPV tissue tropism. Transcriptional start sites were mapped at P(92) (HPV-38), P(45) (HPV-92), P(7439) (HPV-93) and P(256) (HPV-96). Transcription from HPV-8, 93...

  12. Fast transcription rates of RNA polymerase II in human cells

    Science.gov (United States)

    Maiuri, Paolo; Knezevich, Anna; De Marco, Alex; Mazza, Davide; Kula, Anna; McNally, Jim G; Marcello, Alessandro

    2011-01-01

    Averaged estimates of RNA polymerase II (RNAPII) elongation rates in mammalian cells have been shown to range between 1.3 and 4.3 kb min−1. In this work, nascent RNAs from an integrated human immunodeficiency virus type 1-derived vector were detectable at the single living cell level by fluorescent RNA tagging. At steady state, a constant number of RNAs was measured corresponding to a minimal density of polymerases with negligible fluctuations over time. Recovery of fluorescence after photobleaching was complete within seconds, indicating a high rate of RNA biogenesis. The calculated transcription rate above 50 kb min−1 points towards a wide dynamic range of RNAPII velocities in living cells. PMID:22015688

  13. TcoF-DB: dragon database for human transcription co-factors and transcription factor interacting proteins

    KAUST Repository

    Schaefer, Ulf

    2010-10-21

    The initiation and regulation of transcription in eukaryotes is complex and involves a large number of transcription factors (TFs), which are known to bind to the regulatory regions of eukaryotic DNA. Apart from TF-DNA binding, protein-protein interaction involving TFs is an essential component of the machinery facilitating transcriptional regulation. Proteins that interact with TFs in the context of transcription regulation but do not bind to the DNA themselves, we consider transcription co-factors (TcoFs). The influence of TcoFs on transcriptional regulation and initiation, although indirect, has been shown to be significant with the functionality of TFs strongly influenced by the presence of TcoFs. While the role of TFs and their interaction with regulatory DNA regions has been well-studied, the association between TFs and TcoFs has so far been given less attention. Here, we present a resource that is comprised of a collection of human TFs and the TcoFs with which they interact. Other proteins that have a proven interaction with a TF, but are not considered TcoFs are also included. Our database contains 157 high-confidence TcoFs and additionally 379 hypothetical TcoFs. These have been identified and classified according to the type of available evidence for their involvement in transcriptional regulation and their presence in the cell nucleus. We have divided TcoFs into four groups, one of which contains high-confidence TcoFs and three others contain TcoFs which are hypothetical to different extents. We have developed the Dragon Database for Human Transcription Co-Factors and Transcription Factor Interacting Proteins (TcoF-DB). A web-based interface for this resource can be freely accessed at http://cbrc.kaust.edu.sa/tcof/ and http://apps.sanbi.ac.za/tcof/. © The Author(s) 2010.

  14. Analysis of transcript and protein overlap in a human osteosarcoma cell line

    Directory of Open Access Journals (Sweden)

    Emanuelsson Olof

    2010-12-01

    Full Text Available Abstract Background An interesting field of research in genomics and proteomics is to compare the overlap between the transcriptome and the proteome. Recently, the tools to analyse gene and protein expression on a whole-genome scale have been improved, including the availability of the new generation sequencing instruments and high-throughput antibody-based methods to analyze the presence and localization of proteins. In this study, we used massive transcriptome sequencing (RNA-seq to investigate the transcriptome of a human osteosarcoma cell line and compared the expression levels with in situ protein data obtained in-situ from antibody-based immunohistochemistry (IHC and immunofluorescence microscopy (IF. Results A large-scale analysis based on 2749 genes was performed, corresponding to approximately 13% of the protein coding genes in the human genome. We found the presence of both RNA and proteins to a large fraction of the analyzed genes with 60% of the analyzed human genes detected by all three methods. Only 34 genes (1.2% were not detected on the transcriptional or protein level with any method. Our data suggest that the majority of the human genes are expressed at detectable transcript or protein levels in this cell line. Since the reliability of antibodies depends on possible cross-reactivity, we compared the RNA and protein data using antibodies with different reliability scores based on various criteria, including Western blot analysis. Gene products detected in all three platforms generally have good antibody validation scores, while those detected only by antibodies, but not by RNA sequencing, generally consist of more low-scoring antibodies. Conclusion This suggests that some antibodies are staining the cells in an unspecific manner, and that assessment of transcript presence by RNA-seq can provide guidance for validation of the corresponding antibodies.

  15. Structure and in vitro transcription of human globin genes.

    Science.gov (United States)

    Proudfoot, N J; Shander, M H; Manley, J L; Gefter, M L; Maniatis, T

    1980-09-19

    The alpha-like and beta-like subunits of human hemoglobin are encoded by a small family of genes that are differentially expressed during development. Through the use of molecular cloning procedures, each member of this gene family has been isolated and extensively characterized. Although the alpha-like and beta-like globin genes are located on different chromosomes, both sets of genes are arranged in closely linked clusters. In both clusters, each of the genes is transcribed from the same DNA strand, and the genes are arranged in the order of their expressions during development. Structural comparisons of immediately adjacent genes within each cluster have provided evidence for the occurrence of gene duplication and correction during evolution and have led to the discovery of pseudogenes, genes that have acquired numerous mutations that prevent their normal expression. Recently, in vivo and in vitro systems for studying the expression of cloned eukaryotic genes have been developed as a means of identifying DNA sequences that are necessary for normal gene function. This article describes the application of an in vitro transcription procedure to the study of human globin gene expression.

  16. Pluripotent stem cell transcription factors during human odontogenesis.

    Science.gov (United States)

    da Cunha, Juliana Malta; da Costa-Neves, Adriana; Kerkis, Irina; da Silva, Marcelo Cavenaghi Pereira

    2013-09-01

    Stem cells are capable of generating various cell lines and can be obtained from adult or embryonic tissues for clinical therapies. Stem cells from deciduous dental pulp are among those that are easily obtainable from adult tissues and have been widely studied because of their ability to differentiate into a variety of cell lines in the presence of various chemical mediators. We have analyze the expression of several proteins related to the differentiation and proliferative potential of cell populations that compose the tooth germ of human fetuses. We evaluate 20 human fetuses of both genders. After being paraffin-embedded, cap and bell stages of tooth germ development were subjected to immunohistochemistry for the following markers: Oct-4, Nanog, Stat-3 and Sox-2. The studied antibodies showed nuclear or cytoplasmic immunnostaining within various anatomical structures and with various degrees of expression, indicating the action of these proteins during tooth development. We conclude that the interrelationship between these transcription factors is complex and associated with self-renewal and cell differentiation. Our results suggest that the expression of Oct-4, Nanog, Sox-2 and Stat-3 are related to differentiation in ameloblasts and odontoblasts.

  17. The biological function of some human transcription factor binding motifs varies with position relative to the transcription start site.

    Science.gov (United States)

    Tharakaraman, Kannan; Bodenreider, Olivier; Landsman, David; Spouge, John L; Mariño-Ramírez, Leonardo

    2008-05-01

    A number of previous studies have predicted transcription factor binding sites (TFBSs) by exploiting the position of genomic landmarks like the transcriptional start site (TSS). The studies' methods are generally too computationally intensive for genome-scale investigation, so the full potential of 'positional regulomics' to discover TFBSs and determine their function remains unknown. Because databases often annotate the genomic landmarks in DNA sequences, the methodical exploitation of positional regulomics has become increasingly urgent. Accordingly, we examined a set of 7914 human putative promoter regions (PPRs) with a known TSS. Our methods identified 1226 eight-letter DNA words with significant positional preferences with respect to the TSS, of which only 608 of the 1226 words matched known TFBSs. Many groups of genes whose PPRs contained a common word displayed similar expression profiles and related biological functions, however. Most interestingly, our results included 78 words, each of which clustered significantly in two or three different positions relative to the TSS. Often, the gene groups corresponding to different positional clusters of the same word corresponded to diverse functions, e.g. activation or repression in different tissues. Thus, different clusters of the same word likely reflect the phenomenon of 'positional regulation', i.e. a word's regulatory function can vary with its position relative to a genomic landmark, a conclusion inaccessible to methods based purely on sequence. Further integrative analysis of words co-occurring in PPRs also yielded 24 different groups of genes, likely identifying cis-regulatory modules de novo. Whereas comparative genomics requires precise sequence alignments, positional regulomics exploits genomic landmarks to provide a 'poor man's alignment'. By exploiting the phenomenon of positional regulation, it uses position to differentiate the biological functions of subsets of TFBSs sharing a common sequence motif.

  18. VEGF promotes the transcription of the human PRL-3 gene in HUVEC through transcription factor MEF2C.

    Directory of Open Access Journals (Sweden)

    Jianliang Xu

    Full Text Available Phosphatase of regenerating liver 3 (PRL-3 is known to be overexpressed in many tumors, and its transcript level is high in the vasculature and endothelial cells of malignant tumor tissue. However, the mechanism(s underlying its enhanced expression and its function in endothelial cells remain unknown. Here, we report that vascular endothelial growth factor (VEGF can induce PRL-3 transcription in human umbilical vein endothelial cells (HUVEC. An analysis of its 5'UTR revealed that PRL-3 transcription is initiated from two distinct sites, which results in the formation of the two transcripts, PRL-3-iso1 and PRL-3-iso2, but only the latter is up-regulated in HUVEC by VEGF. The PRL-3-iso2 promoter region includes two functional MEF2 (myocyte enhancer factor2 binding sites. The over-expression of the constitutively active form of MEF2C promotes the abundance of the PRL-3-iso2 transcript in a number of human cell lines. The siRNA-induced knockdown of MEF2C abolished the stimulative effect of VEGF on PRL-3 transcript in HUVEC, indicating that the VEGF-induced promotion of PRL-3 expression requires the presence of MEF2C. Finally, blocking PRL-3 activity or expression suppresses tube formation by HUVEC. We suggest that PRL-3 functions downstream of the VEGF/MEF2C pathway in endothelial cells and may play an important role in tumor angiogenesis.

  19. VEGF promotes the transcription of the human PRL-3 gene in HUVEC through transcription factor MEF2C.

    Science.gov (United States)

    Xu, Jianliang; Cao, Shaoxian; Wang, Lu; Xu, Rui; Chen, Gong; Xu, Qiang

    2011-01-01

    Phosphatase of regenerating liver 3 (PRL-3) is known to be overexpressed in many tumors, and its transcript level is high in the vasculature and endothelial cells of malignant tumor tissue. However, the mechanism(s) underlying its enhanced expression and its function in endothelial cells remain unknown. Here, we report that vascular endothelial growth factor (VEGF) can induce PRL-3 transcription in human umbilical vein endothelial cells (HUVEC). An analysis of its 5'UTR revealed that PRL-3 transcription is initiated from two distinct sites, which results in the formation of the two transcripts, PRL-3-iso1 and PRL-3-iso2, but only the latter is up-regulated in HUVEC by VEGF. The PRL-3-iso2 promoter region includes two functional MEF2 (myocyte enhancer factor2) binding sites. The over-expression of the constitutively active form of MEF2C promotes the abundance of the PRL-3-iso2 transcript in a number of human cell lines. The siRNA-induced knockdown of MEF2C abolished the stimulative effect of VEGF on PRL-3 transcript in HUVEC, indicating that the VEGF-induced promotion of PRL-3 expression requires the presence of MEF2C. Finally, blocking PRL-3 activity or expression suppresses tube formation by HUVEC. We suggest that PRL-3 functions downstream of the VEGF/MEF2C pathway in endothelial cells and may play an important role in tumor angiogenesis.

  20. Human BLCAP transcript: new editing events in normal and cancerous tissues.

    Science.gov (United States)

    Galeano, Federica; Leroy, Anne; Rossetti, Claudia; Gromova, Irina; Gautier, Philippe; Keegan, Liam P; Massimi, Luca; Di Rocco, Concezio; O'Connell, Mary A; Gallo, Angela

    2010-07-01

    Bladder cancer-associated protein (BLCAP) is a highly conserved protein among species, and it is considered a novel candidate tumor suppressor gene originally identified from human bladder carcinoma. However, little is known about the regulation or the function of this protein. Here, we show that the human BLCAP transcript undergoes multiple A-to-I editing events. Some of the new editing events alter the highly conserved amino terminus of the protein creating alternative protein isoforms by changing the genetically coded amino acids. We found that both ADAR1 and ADAR2-editing enzymes cooperate to edit this transcript and that different tissues displayed distinctive ratios of edited and unedited BLCAP transcripts. Moreover, we observed a general decrease in BLCAP-editing level in astrocytomas, bladder cancer and colorectal cancer when compared with the related normal tissues. The newly identified editing events, found to be downregulated in cancers, could be useful for future studies as a diagnostic tool to distinguish malignancies or epigenetic changes in different tumors.

  1. Transcription-induced CAG repeat contraction in human cells is mediated in part by transcription-coupled nucleotide excision repair.

    Science.gov (United States)

    Lin, Yunfu; Wilson, John H

    2007-09-01

    Expansions of CAG repeat tracts in the germ line underlie several neurological diseases. In human patients and mouse models, CAG repeat tracts display an ongoing instability in neurons, which may exacerbate disease symptoms. It is unclear how repeats are destabilized in nondividing cells, but it cannot involve DNA replication. We showed previously that transcription through CAG repeats induces their instability (Y. Lin, V. Dion, and J. H. Wilson, Nat. Struct. Mol. Biol. 13:179-180). Here, we present a genetic analysis of the link between transcription-induced repeat instability and nucleotide excision repair (NER) in human cells. We show that short interfering RNA-mediated knockdown of CSB, a component specifically required for transcription-coupled NER (TC-NER), and knockdowns of ERCC1 and XPG, which incise DNA adjacent to damage, stabilize CAG repeat tracts. These results suggest that TC-NER is involved in the pathway for transcription-induced CAG repeat instability. In contrast, knockdowns of OGG1 and APEX1, key components involved in base excision repair, did not affect repeat instability. In addition, repeats are stabilized by knockdown of transcription factor IIS, consistent with a requirement for RNA polymerase II (RNAPII) to backtrack from a transcription block. Repeats also are stabilized by knockdown of either BRCA1 or BARD1, which together function as an E3 ligase that can ubiquitinate arrested RNAPII. Treatment with the proteasome inhibitor MG132, which stabilizes repeats, confirms proteasome involvement. We integrate these observations into a tentative pathway for transcription-induced CAG repeat instability that can account for the contractions observed here and potentially for the contractions and expansions seen with human diseases.

  2. Intergenic and repeat transcription in human, chimpanzee and macaque brains measured by RNA-Seq.

    Directory of Open Access Journals (Sweden)

    Augix Guohua Xu

    Full Text Available Transcription is the first step connecting genetic information with an organism's phenotype. While expression of annotated genes in the human brain has been characterized extensively, our knowledge about the scope and the conservation of transcripts located outside of the known genes' boundaries is limited. Here, we use high-throughput transcriptome sequencing (RNA-Seq to characterize the total non-ribosomal transcriptome of human, chimpanzee, and rhesus macaque brain. In all species, only 20-28% of non-ribosomal transcripts correspond to annotated exons and 20-23% to introns. By contrast, transcripts originating within intronic and intergenic repetitive sequences constitute 40-48% of the total brain transcriptome. Notably, some repeat families show elevated transcription. In non-repetitive intergenic regions, we identify and characterize 1,093 distinct regions highly expressed in the human brain. These regions are conserved at the RNA expression level across primates studied and at the DNA sequence level across mammals. A large proportion of these transcripts (20% represents 3'UTR extensions of known genes and may play roles in alternative microRNA-directed regulation. Finally, we show that while transcriptome divergence between species increases with evolutionary time, intergenic transcripts show more expression differences among species and exons show less. Our results show that many yet uncharacterized evolutionary conserved transcripts exist in the human brain. Some of these transcripts may play roles in transcriptional regulation and contribute to evolution of human-specific phenotypic traits.

  3. Triiodothyronine inhibits transcription from the human growth hormone promoter.

    Science.gov (United States)

    Morin, A; Louette, J; Voz, M L; Tixier-Vidal, A; Belayew, A; Martial, J A

    1990-07-09

    Three DNA constructs, the natural human growth hormone gene (hGH-hGH) its 500 bp promoter linked to the chloramphenicol acetyl transferase reporter gene (hGH-CAT), and its structural part linked to the herpes virus thymidine kinase promoter (TK-hGH) were introduced into rat pituitary GC cells by DEAE-dextran transfection. Transient expression was followed as a function of triiodothyronine (T3) concentration. The hGH-CAT expression was specifically inhibited by T3 following a typical dose-response curve while hGH-GH gene expression was not significantly modified. The transient expression of TK-hGH increased as a function of T3 concentration. These results indicate that T3 exerts two opposite effects on hGH gene expression. First, it down-regulates expression by acting on the promoter; second, it up-regulates expression by acting on the structural part of the gene. These action could be due to regulation of transcription and mRNA stabilization, respectively.

  4. Characterization of the transcriptional activation domains of human TEF3-1 (transcription enhancer factor 3 isoform 1).

    Science.gov (United States)

    Qiao, Cheng; Jiang, Yajie; Deng, Cuilan; Huang, Zebo; Teng, Kaixuan; Chen, Lan; Liu, Xin

    2015-03-01

    TEF3-1 (transcription enhancer factor 3 isoform 1) is a human transcriptional factor, which has a N-terminal TEA/ATTS domain supposedly for DNA binding and C-terminal PRD and STY domains for transcriptional activation. Taking advantage of the efficient reporter design of yeast two-hybrid system, we characterized the TEF3-1 domains in activating gene expression. Previously study usually mentioned that the C-terminal domain of TEF3-1 has the transcriptional activity, however, our data shows that the peptides TEF3-11-66 and TEF3-1197-434 functioned as two independent activation domains, suggesting that N-terminal domain of TEF3-1 also has transcriptional activation capacity. Additionally, more deletions of amino acids 197-434 showed that only the peptides TEF3-1197-265 contained the minimum sequences for the C-terminal transcriptional activation domain. The protein structure is predicted to contain a helix-turn-helix structure in TEF3-11-66 and four β sheets in TEF3-1197-265. Finally, after the truncated fragments of TEF3-1 were expressed in HUVEC cells, the whole TEF3-1 and the two activation domains could increase F-actin stress fiber, cell proliferation, migration and targeted gene expression. Further analysis and characterization of the activation domains in TEF3-1 may broaden our understanding of the gene involved in angiogenesis and other pathological processes.

  5. Interaction with general transcription factor IIF (TFIIF) is required for the suppression of activated transcription by RPB5-mediating protein(RMP)

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    RMP was reported to regulate transcription via competing with HBx to bind the general transcription factor IIB (TFIIB) and interacting with RPB5 subunit of RNA polymerase Ⅱ as a corepressor of transcription regulator. However, our present research uncovered that RMP also regulates the transcription through interaction with the general transcription factors IIF (TFIIF), which assemble in the preinitiation complex and function in both transcription initiation and elongation. With in vitro pull-down assay and Far-Western analysis, we demonstrated that RMP could bind with bacterially expressed recombinant RAP30 and RAP74of TFIIF subunits. In the immunoprecipitation assay in COS 1 cells cotransfected with FLAG-tagged RMP or its mutants, GST-fused RAP30 and RAP74 were co-immunoprecipitated with RMP in approximately equal molar ratio, which suggests that RAP30 and RAP74 interact with RMP as a TFIIF complex. Interestingly both RAP30 and RAP74 interact with the same domain (D5) of the C-terminal RMP of 118-amino-acid residuals which overlaps with its TFIIB-binding domain. Internal deletion of D5 region of RMP abolished its binding ability with both subunits of TFIIF, while D5 domain alone was sufficient to interact with TFIIF subunits. The result of luciferase assay showed that overexpression of RMP, but not the mutant RMP lacking D5 region, suppressed the transcription activated by Gal-VP16, suggesting that interaction with TFIIF is required for RMP to suppress the activated transcription. The interaction between RMP and TFIIF may be an additional passway for RMP to regulate the transcription, or alternatively TFIIF may cooperate with RPB5 and TFIIB for the corepressor function of RMP.

  6. Inhibition of human insulin gene transcription and MafA transcriptional activity by the dual leucine zipper kinase.

    Science.gov (United States)

    Stahnke, Marie-Jeannette; Dickel, Corinna; Schröder, Sabine; Kaiser, Diana; Blume, Roland; Stein, Roland; Pouponnot, Celio; Oetjen, Elke

    2014-09-01

    Insulin biosynthesis is an essential β-cell function and inappropriate insulin secretion and biosynthesis contribute to the pathogenesis of diabetes mellitus type 2. Previous studies showed that the dual leucine zipper kinase (DLK) induces β-cell apoptosis. Since β-cell dysfunction precedes β-cell loss, in the present study the effect of DLK on insulin gene transcription was investigated in the HIT-T15 β-cell line. Downregulation of endogenous DLK increased whereas overexpression of DLK decreased human insulin gene transcription. 5'- and 3'-deletion human insulin promoter analyses resulted in the identification of a DLK responsive element that mapped to the DNA binding-site for the β-cell specific transcription factor MafA. Overexpression of DLK wild-type but not its kinase-dead mutant inhibited MafA transcriptional activity conferred by its transactivation domain. Furthermore, in the non-β-cell line JEG DLK inhibited MafA overexpression-induced human insulin promoter activity. Overexpression of MafA and DLK or its kinase-dead mutant into JEG cells revealed that DLK but not its mutant reduced MafA protein content. Inhibition of the down-stream DLK kinase c-Jun N-terminal kinase (JNK) by SP600125 attenuated DLK-induced MafA loss. Furthermore, mutation of the serine 65 to alanine, shown to confer MafA protein stability, increased MafA-dependent insulin gene transcription and prevented DLK-induced MafA loss in JEG cells. These data suggest that DLK by activating JNK triggers the phosphorylation and degradation of MafA thereby attenuating insulin gene transcription. Given the importance of MafA for β-cell function, the inhibition of DLK might preserve β-cell function and ultimately retard the development of diabetes mellitus type 2.

  7. Transcriptional profile of isoproterenol-induced cardiomyopathy and comparison to exercise-induced cardiac hypertrophy and human cardiac failure

    Directory of Open Access Journals (Sweden)

    McIver Lauren J

    2009-12-01

    Full Text Available Abstract Background Isoproterenol-induced cardiac hypertrophy in mice has been used in a number of studies to model human cardiac disease. In this study, we compared the transcriptional response of the heart in this model to other animal models of heart failure, as well as to the transcriptional response of human hearts suffering heart failure. Results We performed microarray analyses on RNA from mice with isoproterenol-induced cardiac hypertrophy and mice with exercise-induced physiological hypertrophy and identified 865 and 2,534 genes that were significantly altered in pathological and physiological cardiac hypertrophy models, respectively. We compared our results to 18 different microarray data sets (318 individual arrays representing various other animal models and four human cardiac diseases and identified a canonical set of 64 genes that are generally altered in failing hearts. We also produced a pairwise similarity matrix to illustrate relatedness of animal models with human heart disease and identified ischemia as the human condition that most resembles isoproterenol treatment. Conclusion The overall patterns of gene expression are consistent with observed structural and molecular differences between normal and maladaptive cardiac hypertrophy and support a role for the immune system (or immune cell infiltration in the pathology of stress-induced hypertrophy. Cross-study comparisons such as the results presented here provide targets for further research of cardiac disease that might generally apply to maladaptive cardiac stresses and are also a means of identifying which animal models best recapitulate human disease at the transcriptional level.

  8. A cDNA encoding RAP74, a general initiation factor for transcription by RNA polymerase II.

    Science.gov (United States)

    Finkelstein, A; Kostrub, C F; Li, J; Chavez, D P; Wang, B Q; Fang, S M; Greenblatt, J; Burton, Z F

    1992-01-30

    RAP30/74 (also known as TFIIF, beta gamma and FC is one of several general factors required for initiation by RNA polymerase II. The small RAP30 subunit of RAP30/74 binds directly to polymerase and appears structurally and functionally homologous to bacterial sigma factors in their RNA polymerase-binding region. RAP30/74 or recombinant RAP30 suppresses nonspecific binding of RNA polymerase II to DNA and is required for RNA polymerase II to assemble stably into a preinitiation complex containing promoter DNA and the general factors TFIID, TFIIA and TFIIB; both RAP30 and RAP74 are physical components of the preinitiation complex. A complementary DNA encoding human RAP30 has been isolated, and here we report the isolation of a cDNA encoding human RAP74. RAP30 and RAP74 produced in Escherichia coli can be used in place of natural human RAP30/74 to direct accurate transcription initiation by RNA polymerase II in vitro.

  9. A general strategy for cellular reprogramming: the importance of transcription factor cross-repression.

    Science.gov (United States)

    Crespo, Isaac; Del Sol, Antonio

    2013-10-01

    Transcription factor cross-repression is an important concept in cellular differentiation. A bistable toggle switch constitutes a molecular mechanism that determines cellular commitment and provides stability to transcriptional programs of binary cell fate choices. Experiments support that perturbations of these toggle switches can interconvert these binary cell fate choices, suggesting potential reprogramming strategies. However, more complex types of cellular transitions could involve perturbations of combinations of different types of multistable motifs. Here, we introduce a method that generalizes the concept of transcription factor cross-repression to systematically predict sets of genes, whose perturbations induce cellular transitions between any given pair of cell types. Furthermore, to our knowledge, this is the first method that systematically makes these predictions without prior knowledge of potential candidate genes and pathways involved, providing guidance on systems where little is known. Given the increasing interest of cellular reprogramming in medicine and basic research, our method represents a useful computational methodology to assist researchers in the field in designing experimental strategies.

  10. Automated discovery of functional generality of human gene expression programs.

    Directory of Open Access Journals (Sweden)

    Georg K Gerber

    2007-08-01

    Full Text Available An important research problem in computational biology is the identification of expression programs, sets of co-expressed genes orchestrating normal or pathological processes, and the characterization of the functional breadth of these programs. The use of human expression data compendia for discovery of such programs presents several challenges including cellular inhomogeneity within samples, genetic and environmental variation across samples, uncertainty in the numbers of programs and sample populations, and temporal behavior. We developed GeneProgram, a new unsupervised computational framework based on Hierarchical Dirichlet Processes that addresses each of the above challenges. GeneProgram uses expression data to simultaneously organize tissues into groups and genes into overlapping programs with consistent temporal behavior, to produce maps of expression programs, which are sorted by generality scores that exploit the automatically learned groupings. Using synthetic and real gene expression data, we showed that GeneProgram outperformed several popular expression analysis methods. We applied GeneProgram to a compendium of 62 short time-series gene expression datasets exploring the responses of human cells to infectious agents and immune-modulating molecules. GeneProgram produced a map of 104 expression programs, a substantial number of which were significantly enriched for genes involved in key signaling pathways and/or bound by NF-kappaB transcription factors in genome-wide experiments. Further, GeneProgram discovered expression programs that appear to implicate surprising signaling pathways or receptor types in the response to infection, including Wnt signaling and neurotransmitter receptors. We believe the discovered map of expression programs involved in the response to infection will be useful for guiding future biological experiments; genes from programs with low generality scores might serve as new drug targets that exhibit minimal

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

    Directory of Open Access Journals (Sweden)

    Marc Santolini

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

  12. Technical Advance: Transcription factor, promoter, and enhancer utilization in human myeloid cells

    Science.gov (United States)

    Joshi, Anagha; Pooley, Christopher; Freeman, Tom C.; Lennartsson, Andreas; Babina, Magda; Schmidl, Christian; Geijtenbeek, Teunis; Michoel, Tom; Severin, Jessica; Itoh, Masayoshi; Lassmann, Timo; Kawaji, Hideya; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R. R.; Rehli, Michael; Hume, David A.

    2015-01-01

    The generation of myeloid cells from their progenitors is regulated at the level of transcription by combinatorial control of key transcription factors influencing cell-fate choice. To unravel the global dynamics of this process at the transcript level, we generated transcription profiles for 91 human cell types of myeloid origin by use of CAGE profiling. The CAGE sequencing of these samples has allowed us to investigate diverse aspects of transcription control during myelopoiesis, such as identification of novel transcription factors, miRNAs, and noncoding RNAs specific to the myeloid lineage. We further reconstructed a transcription regulatory network by clustering coexpressed transcripts and associating them with enriched cis-regulatory motifs. With the use of the bidirectional expression as a proxy for enhancers, we predicted over 2000 novel enhancers, including an enhancer 38 kb downstream of IRF8 and an intronic enhancer in the KIT gene locus. Finally, we highlighted relevance of these data to dissect transcription dynamics during progressive maturation of granulocyte precursors. A multifaceted analysis of the myeloid transcriptome is made available (www.myeloidome.roslin.ed.ac.uk). This high-quality dataset provides a powerful resource to study transcriptional regulation during myelopoiesis and to infer the likely functions of unannotated genes in human innate immunity. PMID:25717144

  13. Technical Advance: Transcription factor, promoter, and enhancer utilization in human myeloid cells.

    Science.gov (United States)

    Joshi, Anagha; Pooley, Christopher; Freeman, Tom C; Lennartsson, Andreas; Babina, Magda; Schmidl, Christian; Geijtenbeek, Teunis; Michoel, Tom; Severin, Jessica; Itoh, Masayoshi; Lassmann, Timo; Kawaji, Hideya; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R R; Rehli, Michael; Hume, David A

    2015-05-01

    The generation of myeloid cells from their progenitors is regulated at the level of transcription by combinatorial control of key transcription factors influencing cell-fate choice. To unravel the global dynamics of this process at the transcript level, we generated transcription profiles for 91 human cell types of myeloid origin by use of CAGE profiling. The CAGE sequencing of these samples has allowed us to investigate diverse aspects of transcription control during myelopoiesis, such as identification of novel transcription factors, miRNAs, and noncoding RNAs specific to the myeloid lineage. We further reconstructed a transcription regulatory network by clustering coexpressed transcripts and associating them with enriched cis-regulatory motifs. With the use of the bidirectional expression as a proxy for enhancers, we predicted over 2000 novel enhancers, including an enhancer 38 kb downstream of IRF8 and an intronic enhancer in the KIT gene locus. Finally, we highlighted relevance of these data to dissect transcription dynamics during progressive maturation of granulocyte precursors. A multifaceted analysis of the myeloid transcriptome is made available (www.myeloidome.roslin.ed.ac.uk). This high-quality dataset provides a powerful resource to study transcriptional regulation during myelopoiesis and to infer the likely functions of unannotated genes in human innate immunity.

  14. Substrate availability and transcriptional regulation of metabolic genes in human skeletal muscle during recovery from exercise

    DEFF Research Database (Denmark)

    Pilegaard, Henriette; Osada, Takuya; Andersen, Lisbeth Tingsted

    2005-01-01

    In skeletal muscle of humans, transcription of several metabolic genes is transiently induced during recovery from exercise when no food is consumed. To determine the potential influence of substrate availability on the transcriptional regulation of metabolic genes during recovery from exercise, ...

  15. Genomic structure and cloning of two transcript isoforms of human Sp8.

    NARCIS (Netherlands)

    M.A. Milona (Maria-athina); J.E. Gough (Julie); A.J. Edgar (Alasdair)

    2004-01-01

    textabstractBACKGROUND: The Specificity proteins (Sp) are a family of transcription factors that have three highly conserved zinc-fingers located towards the carboxy-terminal that bind GC-boxes and assist in the initiation of gene transcription. Human Sp1-7 genes have been characte

  16. A transcript finishing initiative for closing gaps in the human transcriptome

    DEFF Research Database (Denmark)

    Sogayar, Mari Cleide; Camargo, Anamaria A; Bettoni, Fabiana

    2004-01-01

    We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experi...

  17. Identification of cross-species shared transcriptional networks of diabetic nephropathy in human and mouse glomeruli.

    Science.gov (United States)

    Hodgin, Jeffrey B; Nair, Viji; Zhang, Hongyu; Randolph, Ann; Harris, Raymond C; Nelson, Robert G; Weil, E Jennifer; Cavalcoli, James D; Patel, Jignesh M; Brosius, Frank C; Kretzler, Matthias

    2013-01-01

    Murine models are valuable instruments in defining the pathogenesis of diabetic nephropathy (DN), but they only partially recapitulate disease manifestations of human DN, limiting their utility. To define the molecular similarities and differences between human and murine DN, we performed a cross-species comparison of glomerular transcriptional networks. Glomerular gene expression was profiled in patients with early type 2 DN and in three mouse models (streptozotocin DBA/2, C57BLKS db/db, and eNOS-deficient C57BLKS db/db mice). Species-specific transcriptional networks were generated and compared with a novel network-matching algorithm. Three shared human-mouse cross-species glomerular transcriptional networks containing 143 (Human-DBA STZ), 97 (Human-BKS db/db), and 162 (Human-BKS eNOS(-/-) db/db) gene nodes were generated. Shared nodes across all networks reflected established pathogenic mechanisms of diabetes complications, such as elements of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and vascular endothelial growth factor receptor (VEGFR) signaling pathways. In addition, novel pathways not previously associated with DN and cross-species gene nodes and pathways unique to each of the human-mouse networks were discovered. The human-mouse shared glomerular transcriptional networks will assist DN researchers in selecting mouse models most relevant to the human disease process of interest. Moreover, they will allow identification of new pathways shared between mice and humans.

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

    Directory of Open Access Journals (Sweden)

    Yuval Tabach

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

  19. Regulation of human protein S gene (PROS1) transcription

    NARCIS (Netherlands)

    Wolf, Cornelia de

    2006-01-01

    This thesis describes the investigation of the transcriptional regulation of the gene for anticoagulant plasma Protein S, PROS1. Protein S is a cofactor for Protein C in the Protein C anticoagulant pathway. The coagulation cascade is negatively regulated by this pathway through inactivation of activ

  20. Uncovering layers of human RNA polymerase II transcription

    DEFF Research Database (Denmark)

    Jensen, Torben Heick

    In recent years DNA microarray and high-throughput sequencing technologies have challenged the “gene-centric” view that pre-mRNA is the only RNA species transcribed off protein-coding genes. Instead unorthodox transcription from within genic- and intergenic regions has been demonstrated to occur...

  1. Regulation of human protein S gene (PROS1) transcription

    NARCIS (Netherlands)

    Wolf, Cornelia de

    2006-01-01

    This thesis describes the investigation of the transcriptional regulation of the gene for anticoagulant plasma Protein S, PROS1. Protein S is a cofactor for Protein C in the Protein C anticoagulant pathway. The coagulation cascade is negatively regulated by this pathway through inactivation of

  2. Small RNAs targeting transcription start site induce heparanase silencing through interference with transcription initiation in human cancer cells.

    Directory of Open Access Journals (Sweden)

    Guosong Jiang

    Full Text Available Heparanase (HPA, an endo-h-D-glucuronidase that cleaves the heparan sulfate chain of heparan sulfate proteoglycans, is overexpressed in majority of human cancers. Recent evidence suggests that small interfering RNA (siRNA induces transcriptional gene silencing (TGS in human cells. In this study, transfection of siRNA against -9/+10 bp (siH3, but not -174/-155 bp (siH1 or -134/-115 bp (siH2 region relative to transcription start site (TSS locating at 101 bp upstream of the translation start site, resulted in TGS of heparanase in human prostate cancer, bladder cancer, and gastric cancer cells in a sequence-specific manner. Methylation-specific PCR and bisulfite sequencing revealed no DNA methylation of CpG islands within heparanase promoter in siH3-transfected cells. The TGS of heparanase did not involve changes of epigenetic markers histone H3 lysine 9 dimethylation (H3K9me2, histone H3 lysine 27 trimethylation (H3K27me3 or active chromatin marker acetylated histone H3 (AcH3. The regulation of alternative splicing was not involved in siH3-mediated TGS. Instead, siH3 interfered with transcription initiation via decreasing the binding of both RNA polymerase II and transcription factor II B (TFIIB, but not the binding of transcription factors Sp1 or early growth response 1, on the heparanase promoter. Moreover, Argonaute 1 and Argonaute 2 facilitated the decreased binding of RNA polymerase II and TFIIB on heparanase promoter, and were necessary in siH3-induced TGS of heparanase. Stable transfection of the short hairpin RNA construct targeting heparanase TSS (-9/+10 bp into cancer cells, resulted in decreased proliferation, invasion, metastasis and angiogenesis of cancer cells in vitro and in athymic mice models. These results suggest that small RNAs targeting TSS can induce TGS of heparanase via interference with transcription initiation, and significantly suppress the tumor growth, invasion, metastasis and angiogenesis of cancer cells.

  3. Transcriptional control of yeast ribosome biogenesis: A multifaceted role for general regulatory factors.

    Science.gov (United States)

    Bosio, Maria Cristina; Fermi, Beatrice; Dieci, Giorgio

    2017-08-08

    In Saccharomyces cerevisiae, a group of more than 200 co-regulated genes (Ribi genes) is involved in ribosome biogenesis. This regulon has recently been shown to rely on a small set of transcriptional regulators (mainly Abf1, but also Reb1, Tbf1 and Rap1) previously referred to as general regulatory factors (GRFs) because of their widespread binding and action at many promoters and other specialized genomic regions. Intriguingly, Abf1 binding to Ribi genes is differentially modulated in response to distinct nutrition signaling pathways. Such a dynamic promoter association has the potential to orchestrate both activation and repression of Ribi genes in synergy with neighboring regulatory sites and through the functional interplay of histone acetyltransferases and deacetylases.

  4. Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro

    Science.gov (United States)

    2013-03-11

    Bacillus cereus group of bacteria, are attributed to poly- γ-D-glutamate acid (PGA) capsule, lethal toxin (LT) and edema toxin (ET) [10-12]. These toxins...M, Hellman M, Muhie S, et al. (2013) Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro...author and source are credited. Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro Rasha

  5. Transcriptional analysis of the 5'-noncoding region of the human involucrin gene.

    Science.gov (United States)

    Lopez-Bayghen, E; Vega, A; Cadena, A; Granados, S E; Jave, L F; Gariglio, P; Alvarez-Salas, L M

    1996-01-01

    Human involucrin whose gene transcription is directed by a 2456-nucleotide (nt) 5'-noncoding region is a structural component of the epithelial cornified layer. Transient transfection assays demonstrated that this region is transcriptionally active in multiplying keratinocytes and is enhanced by 2 mM CaCl2 treatment. Calcium-independent transcriptional activity and the interaction with the AP-1 transcriptional factor was located on the proximal part (nt -159 to -1) of the 5'-noncoding region. However, CaCl2 responsiveness was mapped to a distal 1185-nt fragment (nt -2456 to -1272). Moreover, this fragment potentiated the Herpes simplex thymidine kinase promoter in normal keratinocytes and is responsive to calcium treatment in a cell type-specific manner. Interestingly, the absence of a 491-nt fragment located between the two enhancer domains (nt -651 to -160) resulted in transcriptional activation in multiplying keratinocytes. This fragment interacts with AP-1 and the YY1 transcriptional silencer. It is concluded that human involucrin 5'-noncoding region contains at least three regulatory domains, a distal CaCl2-responsive enhancer, a putative transcriptional silencer (that interacts with AP-1 and YY1), and a proximal enhancer/promoter (that interacts with AP-1). Thus, this study demonstrates the presence of particular transcriptional factors can potentially regulate the human involucrin expression.

  6. Transcription factor ZNF25 is associated with osteoblast differentiation of human skeletal stem cells

    DEFF Research Database (Denmark)

    Twine, Natalie A.; Harkness, Linda; Kassem, Moustapha;

    2016-01-01

    Background The differentiation of human bone marrow derived skeletal stem cells (known as human bone marrow stromal or mesenchymal stem cells, hMSCs) into osteoblasts involves the activation of a small number of well-described transcription factors. To identify additional osteoblastic transcription...... containing G protein-coupled receptor 5 and RAN-binding protein 3-like. We also observed enrichment in extracellular matrix organization, skeletal system development and regulation of ossification in the entire upregulated set of genes. Consistent with its function as a transcription factor during osteoblast...

  7. Structure and associated DNA-helicase activity of a general transcription initiation factor that binds to RNA polymerase II.

    Science.gov (United States)

    Sopta, M; Burton, Z F; Greenblatt, J

    1989-10-05

    RAP30/74 is a heteromeric general transcription initiation factor which binds to RNA polymerase II. Here we report that preparations of RAP30/74 contain an ATP-dependent DNA helicase whose probable function is to melt the DNA at transcriptional start sites. The sequence of the RAP30 subunit of RAP30/74 indicates that RAP30 may be distantly related to bacterial sigma factors.

  8. Transcriptional networks implicated in human nonalcoholic fatty liver disease.

    Science.gov (United States)

    Ye, Hua; Liu, Wei

    2015-10-01

    The transcriptome of nonalcoholic fatty liver disease (NAFLD) was investigated in several studies. However, the implications of transcriptional networks in progressive NAFLD are not clear and mechanisms inducing transition from nonalcoholic simple fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) are still elusive. The aims of this study were to (1) construct networks for progressive NAFLD, (2) identify hub genes and functional modules in these networks and (3) infer potential linkages among hub genes, transcription factors and microRNAs (miRNA) for NAFLD progression. A systems biology approach by combining differential expression analysis and weighted gene co-expression network analysis (WGCNA) was utilized to dissect transcriptional profiles in 19 normal, 10 NAFL and 16 NASH patients. Based on this framework, 3 modules related to chromosome organization, proteasomal ubiquitin-dependent protein degradation and immune response were identified in NASH network. Furthermore, 9 modules of co-expressed genes associated with NAFL/NASH transition were found. Further characterization of these modules defined 13 highly connected hub genes in NAFLD progression network. Interestingly, 11 significantly changed miRNAs were predicted to target 10 of the 13 hub genes. Characterization of modules and hub genes that may be regulated by miRNAs could facilitate the identification of candidate genes and pathways responsible for NAFL/NASH transition and lead to a better understanding of NAFLD pathogenesis. The identified modules and hub genes may point to potential targets for therapeutic interventions.

  9. Human Factors Problems in General Aviation

    Science.gov (United States)

    1980-04-01

    of general aviation pilots, and, vhen relevant, pilots in general. This information was acquired through the use of both automated and manual searches... Bergey , 1978; Bolz and Eisele, 1979; FAA, 1979b; Smyth, 1980). These changes will result from a number of forces that are acting in concert to speed...not have been adequately addressed in the documentation. Literature Search. Both automated and manual search procedures were used in pursuit of a

  10. Large-scale identification of sequence variants influencing human transcription factor occupancy in vivo.

    Science.gov (United States)

    Maurano, Matthew T; Haugen, Eric; Sandstrom, Richard; Vierstra, Jeff; Shafer, Anthony; Kaul, Rajinder; Stamatoyannopoulos, John A

    2015-12-01

    The function of human regulatory regions depends exquisitely on their local genomic environment and on cellular context, complicating experimental analysis of common disease- and trait-associated variants that localize within regulatory DNA. We use allelically resolved genomic DNase I footprinting data encompassing 166 individuals and 114 cell types to identify >60,000 common variants that directly influence transcription factor occupancy and regulatory DNA accessibility in vivo. The unprecedented scale of these data enables systematic analysis of the impact of sequence variation on transcription factor occupancy in vivo. We leverage this analysis to develop accurate models of variation affecting the recognition sites for diverse transcription factors and apply these models to discriminate nearly 500,000 common regulatory variants likely to affect transcription factor occupancy across the human genome. The approach and results provide a new foundation for the analysis and interpretation of noncoding variation in complete human genomes and for systems-level investigation of disease-associated variants.

  11. Computational Analysis of Transcriptional Circuitries in Human Embryonic Stem Cells Reveals Multiple and Independent Networks

    Directory of Open Access Journals (Sweden)

    Xiaosheng Wang

    2014-01-01

    Full Text Available It has been known that three core transcription factors (TFs, NANOG, OCT4, and SOX2, collaborate to form a transcriptional circuitry to regulate pluripotency and self-renewal of human embryonic stem (ES cells. Similarly, MYC also plays an important role in regulating pluripotency and self-renewal of human ES cells. However, the precise mechanism by which the transcriptional regulatory networks control the activity of ES cells remains unclear. In this study, we reanalyzed an extended core network, which includes the set of genes that are cobound by the three core TFs and additional TFs that also bind to these cobound genes. Our results show that beyond the core transcriptional network, additional transcriptional networks are potentially important in the regulation of the fate of human ES cells. Several gene families that encode TFs play a key role in the transcriptional circuitry of ES cells. We also demonstrate that MYC acts independently of the core module in the regulation of the fate of human ES cells, consistent with the established argument. We find that TP53 is a key connecting molecule between the core-centered and MYC-centered modules. This study provides additional insights into the underlying regulatory mechanisms involved in the fate determination of human ES cells.

  12. Human Cytomegalovirus Immediate Early Interaction with Host Nuclear Structures: Definition of an Immediate Transcript Environment

    Science.gov (United States)

    Ishov, Alexander M.; Stenberg, Richard M.; Maul, Gerd G.

    1997-01-01

    The development of an induced transcript environment was investigated at the supramolecular level through comparative localization of the human cytomegalovirus immediate early (IE) transcripts and specific nuclear domains shortly after infection. Compact aggregates of IE transcripts form only adjacent to nuclear domain 10 (ND10), and the viral protein IE86 accumulates exclusively juxtaposed to the subpopulation of ND10 with transcripts. The stream of transcripts is funneled from ND10 into the spliceosome assembly factor SC35 domain through the accumulation of IE86 protein, which recruits some components of the basal transcription machinery. Concomitantly the IE72 protein binds to ND10 and later disperses them. The domain containing the zinc finger region of IE72 is essential for this dispersal. Positional analysis of proteins IE86 and IE72, IE transcripts, ND10, the spliceosome assembly factor SC35, and basal transcription factors defines spatially and temporally an immediate transcript environment, the basic components of which exist in the cell before viral infection, providing the structural environment for the virus to usurp. PMID:9214377

  13. Cocoa procyanidins and human cytokine transcription and secretion.

    Science.gov (United States)

    Mao, T; Van De Water, J; Keen, C L; Schmitz, H H; Gershwin, M E

    2000-08-01

    We examined whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate cytokine production at the levels of transcription and protein secretion in both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). In resting cells, interleukin (IL)-1beta and IL-4 gene expression from cocoa-treated cells varied markedly among the subjects tested. However, at the protein level, the larger fractions (pentamer through decamer) stimulated a dramatic increase in IL-1beta concentration (up to ninefold) with increasing degree of polymerization. Similarly, these larger fractions augmented IL-4 concentration by as much as 2 pg/ml, whereas the control displayed levels nearly undetectable. In the presence of PHA, gene expression also seemed to be most affected by the larger procyanidin fractions. The pentameric through decameric fractions increased IL-1 beta expression by 7-19% compared with PHA control, whereas the hexameric through decameric fractions significantly inhibited PHA-induced IL-4 transcription in the range of 71-86%. This observation at the transcription level for IL-1 beta was reflected at the protein level in PHA-stimulated PBMC. Significant reductions in mitogen-induced IL-4 production were also seen at the protein level with the hexamer, heptamer and octamer. Individual oligomeric cocoa fractions were unstimulatory for IL-2 in resting PBMC. However, when induced with PHA, the pentamer, hexamer and heptamer fractions caused a 61-73% inhibition in IL-2 gene expression. This study offers additional data for the consideration of the health benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

  14. Transcriptional dysregulation in NIPBL and cohesin mutant human cells.

    Directory of Open Access Journals (Sweden)

    Jinglan Liu

    2009-05-01

    Full Text Available Cohesin regulates sister chromatid cohesion during the mitotic cell cycle with Nipped-B-Like (NIPBL facilitating its loading and unloading. In addition to this canonical role, cohesin has also been demonstrated to play a critical role in regulation of gene expression in nondividing cells. Heterozygous mutations in the cohesin regulator NIPBL or cohesin structural components SMC1A and SMC3 result in the multisystem developmental disorder Cornelia de Lange Syndrome (CdLS. Genome-wide assessment of transcription in 16 mutant cell lines from severely affected CdLS probands has identified a unique profile of dysregulated gene expression that was validated in an additional 101 samples and correlates with phenotypic severity. This profile could serve as a diagnostic and classification tool. Cohesin binding analysis demonstrates a preference for intergenic regions suggesting a cis-regulatory function mimicking that of a boundary/insulator interacting protein. However, the binding sites are enriched within the promoter regions of the dysregulated genes and are significantly decreased in CdLS proband, indicating an alternative role of cohesin as a transcription factor.

  15. Identification of the Human Zinc Transcriptional Regulatory Element (ZTRE)

    Science.gov (United States)

    Coneyworth, Lisa J.; Jackson, Kelly A.; Tyson, John; Bosomworth, Helen J.; van der Hagen, Eline; Hann, Georgia M.; Ogo, Ogo A.; Swann, Daniel C.; Mathers, John C.; Valentine, Ruth A.; Ford, Dianne

    2012-01-01

    Many genes with crucial roles in zinc homeostasis in mammals respond to fluctuating zinc supply through unknown mechanisms, and uncovering these mechanisms is essential to understanding the process at cellular and systemic levels. We detected zinc-dependent binding of a zinc-induced protein to a specific sequence, the zinc transcriptional regulatory element (ZTRE), in the SLC30A5 (zinc transporter ZnT5) promoter and showed that substitution of the ZTRE abrogated the repression of a reporter gene in response to zinc. We identified the ZTRE in other genes, including (through an unbiased search) the CBWD genes and (through targeted analysis) in multiple members of the SLC30 family, including SLC30A10, which is repressed by zinc. The function of the CBWD genes is currently unknown, but roles for homologs in metal homeostasis are being uncovered in bacteria. We demonstrated that CBWD genes are repressed by zinc and that substitution of the ZTRE in SLC30A10 and CBWD promoter-reporter constructs abrogates this response. Other metals did not affect expression of the transcriptional regulator, binding to the ZTRE or promoter-driven reporter gene expression. These findings provide the basis for elucidating how regulation of a network of genes through this novel mechanism contributes to zinc homeostasis and how the cell orchestrates this response. PMID:22902622

  16. Identification of Novel Short C-Terminal Transcripts of Human SERPINA1 Gene

    Science.gov (United States)

    Matamala, Nerea; Aggarwal, Nupur; Iadarola, Paolo; Fumagalli, Marco; Gomez-Mariano, Gema; Lara, Beatriz; Martinez, Maria Teresa; Cuesta, Isabel; Stolk, Jan

    2017-01-01

    Human SERPINA1 gene is located on chromosome 14q31-32.3 and is organized into three (IA, IB, and IC) non-coding and four (II, III, IV, V) coding exons. This gene produces α1-antitrypsin (A1AT), a prototypical member of the serpin superfamily of proteins. We demonstrate that human peripheral blood leukocytes express not only a product corresponding to the transcript coding for the full-length A1AT protein but also two short transcripts (ST1C4 and ST1C5) of A1AT. In silico sequence analysis revealed that the last exon of the short transcripts contains an Open Reading Frame (ORF) and thus putatively can produce peptides. We found ST1C4 expression across different human tissues whereas ST1C5 was mainly restricted to leukocytes, specifically neutrophils. A high up-regulation (10-fold) of short transcripts was observed in isolated human blood neutrophils after activation with lipopolysaccharide. Parallel analyses by liquid chromatography-mass spectrometry identified peptides corresponding to C-terminal region of A1AT in supernatants of activated but not naïve neutrophils. Herein we report for the first time a tissue specific expression and regulation of short transcripts of SERPINA1 gene, and the presence of C-terminal peptides in supernatants from activated neutrophils, in vitro. This gives a novel insight into the studies on the transcription of SERPINA1 gene. PMID:28107454

  17. Evaluation of hypothalamic murine and human melanocortin 3 receptor transcript structure.

    Science.gov (United States)

    Taylor-Douglas, Dezmond C; Basu, Arunabha; Gardner, Ryan M; Aspelund, Sender; Wen, Xin; Yanovski, Jack A

    2014-11-07

    The melanocortin 3 receptor (MC3R) is involved in regulation of energy homeostasis. However, its transcript structure is not well understood. We therefore studied initiation and termination sites for hypothalamic murine Mc3r and human MC3R transcripts. Rapid Amplification of cDNA Ends (RACE) was performed for the 5' and 3' ends of murine and human hypothalamic RNA. 5' RACE experiments using hypothalamic murine RNA indicated mouse hypothalamus expresses two major Mc3r transcription start sites: one with a 5' UTR approximately 368 bases in length and another previously unknown transcript with a 5' UTR approximately 440 bases in length. 5' RACE experiments using human hypothalamic RNA identified a 5' UTR beginning 533 bases upstream of the start codon with a 248 base splice. 3' RACE experiments using hypothalamic murine RNA indicated the 3' UTR terminates approximately 1286 bases after the translational stop codon, with a previously unknown 787 base splice between consensus splice donor and acceptor sites. 3' RACE experiments using human MC3R transcript indicated the 3' UTR terminates approximately 115-160 bases after the translational stop codon. These data provide insight into melanocortin 3 receptor transcript structure.

  18. Whole genome expression profiling shows that BRG1 transcriptionally regulates UV inducible genes and other novel targets in human cells.

    Science.gov (United States)

    Zhang, Ling; Nemzow, Leah; Chen, Hua; Hu, Jennifer J; Gong, Feng

    2014-01-01

    UV irradiation is known to cause cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), and plays a large role in the development of cancer. Tumor suppression, through DNA repair and proper cell cycle regulation, is an integral factor in maintaining healthy cells and preventing development of cancer. Transcriptional regulation of the genes involved in the various tumor suppression pathways is essential for them to be expressed when needed and to function properly. BRG1, an ATPase catalytic subunit of the SWI/SNF chromatin remodeling complex, has been identified as a tumor suppressor protein, as it has been shown to play a role in Nucleotide Excision Repair (NER) of CPDs, suppress apoptosis, and restore checkpoint deficiency, in response to UV exposure. Although BRG1 has been shown to regulate transcription of some genes that are instrumental in proper DNA damage repair and cell cycle maintenance in response to UV, its role in transcriptional regulation of the whole genome in response to UV has not yet been elucidated. With whole genome expression profiling in SW13 cells, we show that upon UV induction, BRG1 regulates transcriptional expression of many genes involved in cell stress response. Additionally, our results also highlight BRG1's general role as a master regulator of the genome, as it transcriptionally regulates approximately 4.8% of the human genome, including expression of genes involved in many pathways. RT-PCR and ChIP were used to validate our genome expression analysis. Importantly, our study identifies several novel transcriptional targets of BRG1, such as ATF3. Thus, BRG1 has a larger impact on human genome expression than previously thought, and our studies will provide inroads for future analysis of BRG1's role in gene regulation.

  19. ISOBUTYRAMIDE ACTIVATES TRANSCRIPTION OF HUMAN FETAL γ- AND MURINE EMBRYONIC εy-GLOBIN GENES

    Institute of Scientific and Technical Information of China (English)

    张俊武; 张雪青; 陈平

    2001-01-01

    Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globingene expression and to test cell toxicity of the drug.Methods. ME L cells were transfected with the recombinant construct μLCRAγψβδβ3 and the stable transfor-mants were cultured in the medium with different concentrations of isobutyramide. The experimental mice and rab-bit were injected with different doses of isobutyramide. The globin mRNAs were analyzed by RNase protection as-say. The hematologdcal toxicity and electrolyte toxicity of the drug were tested.Results. An inducible and dose-dependent expression of the human γγ-, ββ- and mouse Aa-globin gene was ob-served in the transfected MEL cells. The induction of the human γ-globin gene is significant stronger than that ofthe ββ-globin gene. With 2.5 ~ 5 mmol/L isobutyramide, the induction of the human γ-globin gene is even moreeffective than that of mouse aa-globin gene. After a 15-day injection under the doses of 500 ~ 900mg * kg- 1 * d-1,the level of the mouse embryonic εy-globin mRNA could be significantly induced up to 3 ~ 4 fold of that of uninject-ed controls. The changes of hemoglobin(Hb), RBC, hematocrit(HCT), WBC, derived from mice injected withdifferent doses of isobutyramide at the interval of 24 hours for 2 ~4 weeks, were generally within the normalrange. In rabbits injected with isobutyramide in the same regiment for 2 weeks, the concentration of blood K+,Na +, C1- and CO.2 were all within normal range and serum ionic osmotic pressure remained stable as well.Conclusion. Our results suggested that isobutyramide is a weak inducer of cell differentiation, but it canselectively activate transcription of human γy-globin gene at a certain degree, and it can act on early stages of ery-throid progenitor differentiation in adult mice and activate transcription of embryonic εy-globin gene and have nohematological toxicity. Our results have further proved the potential value of

  20. Potential transcriptional regulatory regions exist upstream of the human ezrin gene promoter in esophageal carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Shuying Gao; Yanpeng Dai; Meijun Yin; Jing Ye; Gang Li; Jie Yu

    2011-01-01

    We previously demonstrated that the region -87/+ 134 of the human ezrin gene (VIL2) exhibited promoter activity in human esophageal carcinoma EC109 cells, and a further upstream region -1324/-890 positively regulated transcription.In this study, to identify the transcriptional regulatory regions upstream of the VIL2 promoter, we cloned VIL2 - 1541/- 706 segment containing the -1324/-890, and investigated its transcriptional regulatory properties via luciferase assays in transiently transfected cells.In EC109 cells, it was found that VIL2 -1541/-706 possessed promoter and enhancer activities.We also localized transcriptional regulatory regions by fusing 5′- or 3′-deletion segments of VIL2 -1541/-706 to a luciferase reporter.We found that there were three positive and one negative transcriptional regulatory regions ithin VIL2 -1541/-706 in EC109 cells.When these regions were separately located upstream of the luciferase gene without promoter, or located upstream of the VIL2 promoter or SV40 promoter directing the luciferase gene, only VIL2 -1297/-1186 exhibited considerable promoter and enhancer activities, which were lower than those of -1541/-706.In addition, transient expression of Sp1 increased ezrin expression and the transcriptional activation of VIL2 -1297/-1186.Other three regions,although exhibiting significantly positive or negative transcriptional regulation in deletion experiments, showed a weaker or absent regulation.These data suggested that more than one region upstream of the VIL2 promoter participated in VIL2 transcription, and the VIL2 -1297/-1186, probably as a key transcriptional regulatory region, regulated VIL2 transcription in company with other potential regulatory regions.

  1. A transcriptional profile of aging in the human kidney.

    Directory of Open Access Journals (Sweden)

    Graham E J Rodwell

    2004-12-01

    Full Text Available In this study, we found 985 genes that change expression in the cortex and the medulla of the kidney with age. Some of the genes whose transcripts increase in abundance with age are known to be specifically expressed in immune cells, suggesting that immune surveillance or inflammation increases with age. The age-regulated genes show a similar aging profile in the cortex and the medulla, suggesting a common underlying mechanism for aging. Expression profiles of these age-regulated genes mark not only age, but also the relative health and physiology of the kidney in older individuals. Finally, the set of aging-regulated kidney genes suggests specific mechanisms and pathways that may play a role in kidney degeneration with age.

  2. A transcriptional profile of aging in the human kidney.

    Science.gov (United States)

    Rodwell, Graham E J; Sonu, Rebecca; Zahn, Jacob M; Lund, James; Wilhelmy, Julie; Wang, Lingli; Xiao, Wenzhong; Mindrinos, Michael; Crane, Emily; Segal, Eran; Myers, Bryan D; Brooks, James D; Davis, Ronald W; Higgins, John; Owen, Art B; Kim, Stuart K

    2004-12-01

    In this study, we found 985 genes that change expression in the cortex and the medulla of the kidney with age. Some of the genes whose transcripts increase in abundance with age are known to be specifically expressed in immune cells, suggesting that immune surveillance or inflammation increases with age. The age-regulated genes show a similar aging profile in the cortex and the medulla, suggesting a common underlying mechanism for aging. Expression profiles of these age-regulated genes mark not only age, but also the relative health and physiology of the kidney in older individuals. Finally, the set of aging-regulated kidney genes suggests specific mechanisms and pathways that may play a role in kidney degeneration with age.

  3. Structural basis for converting a general transcription factor into an operon-specific virulence regulator.

    Science.gov (United States)

    Belogurov, Georgiy A; Vassylyeva, Marina N; Svetlov, Vladimir; Klyuyev, Sergiy; Grishin, Nick V; Vassylyev, Dmitry G; Artsimovitch, Irina

    2007-04-13

    RfaH, a paralog of the general transcription factor NusG, is recruited to elongating RNA polymerase at specific regulatory sites. The X-ray structure of Escherichia coli RfaH reported here reveals two domains. The N-terminal domain displays high similarity to that of NusG. In contrast, the alpha-helical coiled-coil C domain, while retaining sequence similarity, is strikingly different from the beta barrel of NusG. To our knowledge, such an all-beta to all-alpha transition of the entire domain is the most extreme example of protein fold evolution known to date. Both N domains possess a vast hydrophobic cavity that is buried by the C domain in RfaH but is exposed in NusG. We propose that this cavity constitutes the RNA polymerase-binding site, which becomes unmasked in RfaH only upon sequence-specific binding to the nontemplate DNA strand that triggers domain dissociation. Finally, we argue that RfaH binds to the beta' subunit coiled coil, the major target site for the initiation sigma factors.

  4. Egyptian human babesiosis and general review.

    Science.gov (United States)

    El-Bahnasawy, Mamdouh M; Morsy, Tosson A

    2008-04-01

    Babesiosis is tick-borne malaria-like disease. Man is an opportuneistic host for Babesia species. This paper presented the second Egyptian human babesiosis. The signs and symptoms, CBC, liver functions and kidney functions tests and all other serologic tests did not give any definite diagnosis. Also, he was sero-negative for malaria infection. The patient was critically diagnosed by the demonstration of the typical ring forms of Babesia species in stained blood smears. He was successfully treated with Quinine and Clindamycin, and was discharged from the hospital after the clinical and parasitological improvement. The epidemiology of zoonotic babesiosis was discussed.

  5. Sp1 and KLF15 regulate basal transcription of the human LRP5 gene

    Directory of Open Access Journals (Sweden)

    Zou Yongxin

    2010-02-01

    Full Text Available Abstract Background LRP5, a member of the low density lipoprotein receptor superfamily, regulates diverse developmental processes in embryogenesis and maintains physiological homeostasis in adult organisms. However, how the expression of human LRP5 gene is regulated remains unclear. Results In order to characterize the transcriptional regulation of human LRP5 gene, we cloned the 5' flanking region and evaluated its transcriptional activity in a luciferase reporter system. We demonstrated that both KLF15 and Sp1 binding sites between -72 bp and -53 bp contribute to the transcriptional activation of human LRP5 promoter. Chromatin immunoprecipitation assay demonstrated that the ubiquitous transcription factors KLF15 and Sp1 bind to this region. Using Drosophila SL2 cells, we showed that KLF15 and Sp1 trans-activated the LRP5 promoter in a manner dependent on the presence of Sp1-binding and KLF15-binding motifs. Conclusions Both KLF15 and Sp1 binding sites contribute to the basal activity of human LRP5 promoter. This study provides the first insight into the mechanisms by which transcription of human LRP5 gene is regulated.

  6. Transcription of human respiratory syncytial virus genome RNA in vitro: requirement of cellular factor(s).

    OpenAIRE

    Barik, S

    1992-01-01

    Extracts made from human respiratory syncytial virus (RSV)-infected Hep-2 cells synthesized mRNAs encoded by all known viral genes. In contrast, RSV ribonucleoproteins purified from infected cells failed to transcribe in vitro; transcription was restored by addition of a cytoplasmic extract of uninfected Hep-2 cells, demonstrating that a cellular factor(s) has a role in RSV gene expression. Quantitation of the individual gene mRNAs transcribed in vitro revealed polarity of transcription of th...

  7. An antisense transcript in the human cytomegalovirus UL87 gene region

    Directory of Open Access Journals (Sweden)

    Ma Yanping

    2011-11-01

    Full Text Available Abstract Background Rapid advances in research on antisense transcripts are gradually changing our comprehension of genomic and gene expression aspects of the Herpesviridae. One such herpesvirus is the human cytomegalovirus (HCMV. Although transcription of the HCMV UL87 gene has not been specifically investigated, cDNA clones of UL87 antisense transcripts were found in HCMV cDNA libraries previously. In this study, the transcription of the UL87 antisense strand was investigated in three clinically isolated HCMV strains. Results First, an 800 nucleotides transcript having an antisense orientation to the UL87 gene was found in a late HCMV cDNA library. Then, the UL87 antisense transcript was confirmed by Rapid amplification of cDNA ends (RACE and Northern blot in three HCMV clinical strains. Two ORFs were predicted in the antisense transcript. The putative protein of ORF 1 showed a high degree of conservation among HCMV and other CMV strains. Conclusion An 800nt antisense transcript in the UL87 gene region exists in HCMV clinical strains.

  8. The Adenovirus E4-ORF3 Protein Stimulates SUMOylation of General Transcription Factor TFII-I to Direct Proteasomal Degradation

    Directory of Open Access Journals (Sweden)

    Rebecca G. Bridges

    2016-01-01

    Full Text Available Modulation of host cell transcription, translation, and posttranslational modification processes is critical for the ability of many viruses to replicate efficiently within host cells. The human adenovirus (Ad early region 4 open reading frame 3 (E4-ORF3 protein forms unique inclusions throughout the nuclei of infected cells and inhibits the antiviral Mre11-Rad50-Nbs1 DNA repair complex through relocalization. E4-ORF3 also induces SUMOylation of Mre11 and Nbs1. We recently identified additional cellular targets of E4-ORF3 and found that E4-ORF3 stimulates ubiquitin-like modification of 41 cellular proteins involved in a wide variety of processes. Among the proteins most abundantly modified in an E4-ORF3-dependent manner was the general transcription factor II–I (TFII-I. Analysis of Ad-infected cells revealed that E4-ORF3 induces TFII-I relocalization and SUMOylation early during infection. In the present study, we explored the relationship between E4-ORF3 and TFII-I. We found that Ad infection or ectopic E4-ORF3 expression leads to SUMOylation of TFII-I that precedes a rapid decline in TFII-I protein levels. We also show that E4-ORF3 is required for ubiquitination of TFII-I and subsequent proteasomal degradation. This is the first evidence that E4-ORF3 regulates ubiquitination. Interestingly, we found that E4-ORF3 modulation of TFII-I occurs in diverse cell types but only E4-ORF3 of Ad species C regulates TFII-I, providing critical insight into the mechanism by which E4-ORF3 targets TFII-I. Finally, we show that E4-ORF3 stimulates the activity of a TFII-I-repressed viral promoter during infection. Our results characterize a novel mechanism of TFII-I regulation by Ad and highlight how a viral protein can modulate a critical cellular transcription factor during infection.

  9. Comprehensive analysis of human endogenous retrovirus group HERV-W locus transcription in multiple sclerosis brain lesions by high-throughput amplicon sequencing.

    Science.gov (United States)

    Schmitt, Katja; Richter, Christin; Backes, Christina; Meese, Eckart; Ruprecht, Klemens; Mayer, Jens

    2013-12-01

    Human endogenous retroviruses (HERVs) of the HERV-W group comprise hundreds of loci in the human genome. Deregulated HERV-W expression and HERV-W locus ERVWE1-encoded Syncytin-1 protein have been implicated in the pathogenesis of multiple sclerosis (MS). However, the actual transcription of HERV-W loci in the MS context has not been comprehensively analyzed. We investigated transcription of HERV-W in MS brain lesions and white matter brain tissue from healthy controls by employing next-generation amplicon sequencing of HERV-W env-specific reverse transcriptase (RT) PCR products, thus revealing transcribed HERV-W loci and the relative transcript levels of those loci. We identified more than 100 HERV-W loci that were transcribed in the human brain, with a limited number of loci being predominantly transcribed. Importantly, relative transcript levels of HERV-W loci were very similar between MS and healthy brain tissue samples, refuting deregulated transcription of HERV-W env in MS brain lesions, including the high-level-transcribed ERVWE1 locus encoding Syncytin-1. Quantitative RT-PCR likewise did not reveal differences in MS regarding HERV-W env general transcript or ERVWE1- and ERVWE2-specific transcript levels. However, we obtained evidence for interindividual differences in HERV-W transcript levels. Reporter gene assays indicated promoter activity of many HERV-W long terminal repeats (LTRs), including structurally incomplete LTRs. Our comprehensive analysis of HERV-W transcription in the human brain thus provides important information on the biology of HERV-W in MS lesions and normal human brain, implications for study design, and mechanisms by which HERV-W may (or may not) be involved in MS.

  10. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    Science.gov (United States)

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes. PMID:23530060

  11. From GWAS to Function: Transcriptional regulation of pigmentation genes in humans Transcriptional regulation of pigmentation genes in humans

    NARCIS (Netherlands)

    M. Visser (Mijke)

    2015-01-01

    markdownabstractHuman pigmentation is one of the most explicit visual traits, which therefore has been subject of many research studies. With the emergence of large-scale genetic association studies like GWASs, numerous SNPs have been associated with a phenotype of interest, such as human eye, hair

  12. Hes-1, a known transcriptional repressor, acts as a transcriptional activator for the human acid alpha-glucosidase gene in human fibroblast cells.

    Science.gov (United States)

    Yan, Bo; Raben, Nina; Plotz, Paul H

    2002-03-01

    Hes-1, the mammalian homologue 1 of Drosophila hairy and Enhancer of split proteins, belongs to a family of basic helix-loop-helix proteins that are essential to neurogenesis, myogenesis, hematopoiesis, and sex determination. Hes-1 is a transcriptional repressor for a number of known genes including the human acid alpha-glucosidase (GAA) gene as we have previously shown in Hep G2 cells. The human GAA gene encodes the enzyme for glycogen breakdown in lysosomes, deficiency of which results in Glycogen Storage Disease type II (Pompe syndrome). Using constructs containing the DNA element that demonstrates repressive activity in Hep G2 cells and conditions in which the same transcription factors, Hes-1 and YY1, bind, we have shown that this element functions as an enhancer in human fibroblasts. Site-directed mutagenesis and overexpression of Hes-1 showed that Hes-1 functions as a transcriptional activator. The dual function of Hes-1 we have found is likely to contribute to the subtle tissue-specific control of this housekeeping gene.

  13. GAD2 Alternative Transcripts in the Human Prefrontal Cortex, and in Schizophrenia and Affective Disorders.

    Directory of Open Access Journals (Sweden)

    Kasey N Davis

    Full Text Available Genetic variation and early adverse environmental events work together to increase risk for schizophrenia. γ-aminobutyric acid (GABA, the major inhibitory neurotransmitter in adult mammalian brain, plays a major role in normal brain development, and has been strongly implicated in the pathobiology of schizophrenia. GABA synthesis is controlled by two glutamic acid decarboxylase (GAD genes, GAD1 and GAD2, both of which produce a number of alternative transcripts. Genetic variants in the GAD1 gene are associated with increased risk for schizophrenia, and reduced expression of its major transcript in the human dorsolateral prefrontal cortex (DLPFC. No consistent changes in GAD2 expression have been found in brains from patients with schizophrenia. In this work, with the use of RNA sequencing and PCR technologies, we confirmed and tracked the expression of an alternative truncated transcript of GAD2 (ENST00000428517 in human control DLPFC homogenates across lifespan besides the well-known full length transcript of GAD2. In addition, using quantitative RT-PCR, expression of GAD2 full length and truncated transcripts were measured in the DLPFC of patients with schizophrenia, bipolar disorder and major depression. The expression of GAD2 full length transcript is decreased in the DLPFC of schizophrenia and bipolar disorder patients, while GAD2 truncated transcript is increased in bipolar disorder patients but decreased in schizophrenia patients. Moreover, the patients with schizophrenia with completed suicide or positive nicotine exposure showed significantly higher expression of GAD2 full length transcript. Alternative transcripts of GAD2 may be important in the growth and development of GABA-synthesizing neurons as well as abnormal GABA signaling in the DLPFC of patients with schizophrenia and affective disorders.

  14. Transcriptional specialization of human dendritic cell subsets in response to microbial vaccines.

    Science.gov (United States)

    Banchereau, Romain; Baldwin, Nicole; Cepika, Alma-Martina; Athale, Shruti; Xue, Yaming; Yu, Chun I; Metang, Patrick; Cheruku, Abhilasha; Berthier, Isabelle; Gayet, Ingrid; Wang, Yuanyuan; Ohouo, Marina; Snipes, LuAnn; Xu, Hui; Obermoser, Gerlinde; Blankenship, Derek; Oh, Sangkon; Ramilo, Octavio; Chaussabel, Damien; Banchereau, Jacques; Palucka, Karolina; Pascual, Virginia

    2014-10-22

    The mechanisms by which microbial vaccines interact with human APCs remain elusive. Herein, we describe the transcriptional programs induced in human DCs by pathogens, innate receptor ligands and vaccines. Exposure of DCs to influenza, Salmonella enterica and Staphylococcus aureus allows us to build a modular framework containing 204 transcript clusters. We use this framework to characterize the responses of human monocytes, monocyte-derived DCs and blood DC subsets to 13 vaccines. Different vaccines induce distinct transcriptional programs based on pathogen type, adjuvant formulation and APC targeted. Fluzone, Pneumovax and Gardasil, respectively, activate monocyte-derived DCs, monocytes and CD1c+ blood DCs, highlighting APC specialization in response to vaccines. Finally, the blood signatures from individuals vaccinated with Fluzone or infected with influenza reveal a signature of adaptive immunity activation following vaccination and symptomatic infections, but not asymptomatic infections. These data, offered with a web interface, may guide the development of improved vaccines.

  15. The Human Mind As General Problem Solver

    Science.gov (United States)

    Gurr, Henry

    2011-10-01

    Since leaving U Cal Irvine Neutrino Research, I have been a University Physics Teacher, and an Informal Researcher Of Human Functionality. My talk will share what I discovered about the best ways to learn, many of which are regularities that are to be expected from the Neuronal Network Properties announced in the publications of physicist John Joseph Hopfield. Hopfield's Model of mammalian brain-body, provides solid instructive understanding of how best Learn, Solve Problems, Live! With it we understand many otherwise puzzling features of our intellect! Examples Why 1) Analogies and metaphors powerful in class instruction, ditto poems. 2) Best learning done in physical (Hands-On) situations with tight immediate dynamical feedback such as seen in learning to ride bike, drive car, speak language, etc. 3) Some of the best learning happens in seeming random exploration, bump around, trial and error. 4) Scientific discoveries happen, with no apparent effort, at odd moments. 5) Important discoveries DEPEND on considerable frustrating effort, then Flash of Insight AHA EURIKA.

  16. Six human cyclosporiasis: with general review.

    Science.gov (United States)

    Bourée, Patrice; Lancon, Aurelia; Bisaro, Francine; Bonnot, Gilles

    2007-08-01

    Cyclospora cayetanensis is an emerging coccidian pathogen known in animal since a long time, but recognized in human in last two decade. The present study of cyclosporiasis specified its characteristic features. An epidemiological, clinical and biological retrospective study was carried out on 6 patients, 24 to 67 years old, who complained of diarrhea and weight loss, after a travel abroad. The only pathogenic agent found in every patient was Cyclospora cayetanensis. The patients returned back from Asian countries and from Madagascar. They were cured by Trimethoprim-sulfamethoxazole (TMZ). Diarrhoea was the main symptom of infection by C. cayetanensis. Undoubtedly, transmission occurred by the oro-fecal route, and/or by consumption of infected water or foods. Diagnosis was asserted, recovery of this protozoan parasite in the stools samples. TMZ proved to be the most effective and possibly replaced Ciprofloxacine. The prophylaxis recommended control measure of water supply and foods. Cyclospora should be considered in the assessments of patients with unexplained prolonged diarrhoea, if the investigation for bacterial and parasitological diarrhoea or the traveler's diarrhoea were negative.

  17. GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy.

    Science.gov (United States)

    Kuschal, Christiane; Botta, Elena; Orioli, Donata; Digiovanna, John J; Seneca, Sara; Keymolen, Kathelijn; Tamura, Deborah; Heller, Elizabeth; Khan, Sikandar G; Caligiuri, Giuseppina; Lanzafame, Manuela; Nardo, Tiziana; Ricotti, Roberta; Peverali, Fiorenzo A; Stephens, Robert; Zhao, Yongmei; Lehmann, Alan R; Baranello, Laura; Levens, David; Kraemer, Kenneth H; Stefanini, Miria

    2016-04-01

    The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.

  18. GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy

    Science.gov (United States)

    Kuschal, Christiane; Botta, Elena; Orioli, Donata; Digiovanna, John J.; Seneca, Sara; Keymolen, Kathelijn; Tamura, Deborah; Heller, Elizabeth; Khan, Sikandar G.; Caligiuri, Giuseppina; Lanzafame, Manuela; Nardo, Tiziana; Ricotti, Roberta; Peverali, Fiorenzo A.; Stephens, Robert; Zhao, Yongmei; Lehmann, Alan R.; Baranello, Laura; Levens, David; Kraemer, Kenneth H.; Stefanini, Miria

    2016-01-01

    The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP. PMID:26996949

  19. Adenoviral vectors stimulate glucagon transcription in human mesenchymal stem cells expressing pancreatic transcription factors.

    Directory of Open Access Journals (Sweden)

    Arnaud Zaldumbide

    Full Text Available Viral gene carriers are being widely used as gene transfer systems in (transdifferentiation and reprogramming strategies. Forced expression of key regulators of pancreatic differentiation in stem cells, liver cells, pancreatic duct cells, or cells from the exocrine pancreas, can lead to the initiation of endocrine pancreatic differentiation. While several viral vector systems have been employed in such studies, the results reported with adenovirus vectors have been the most promising in vitro and in vivo. In this study, we examined whether the viral vector system itself could impact the differentiation capacity of human bone-marrow derived mesenchymal stem cells (hMSCs toward the endocrine lineage. Lentivirus-mediated expression of Pdx-1, Ngn-3, and Maf-A alone or in combination does not lead to robust expression of any of the endocrine hormones (i.e. insulin, glucagon and somatostatin in hMSCs. Remarkably, subsequent transduction of these genetically modified cells with an irrelevant early region 1 (E1-deleted adenoviral vector potentiates the differentiation stimulus and promotes glucagon gene expression in hMSCs by affecting the chromatin structure. This adenovirus stimulation was observed upon infection with an E1-deleted adenovirus vector, but not after exposure to helper-dependent adenovirus vectors, pointing at the involvement of genes retained in the E1-deleted adenovirus vector in this phenomenon. Lentivirus mediated expression of the adenovirus E4-ORF3 mimics the adenovirus effect. From these data we conclude that E1-deleted adenoviral vectors are not inert gene-transfer vectors and contribute to the modulation of the cellular differentiation pathways.

  20. Genome-wide analysis of alternative transcripts in human breast cancer

    Science.gov (United States)

    Wen, Ji; Toomer, Kevin H.

    2016-01-01

    Transcript variants play a critical role in diversifying gene expression. Alternative splicing is a major mechanism for generating transcript variants. A number of genes have been implicated in breast cancer pathogenesis with their aberrant expression of alternative transcripts. In this study, we performed genome-wide analyses of transcript variant expression in breast cancer. With RNA-Seq data from 105 patients, we characterized the transcriptome of breast tumors, by pairwise comparison of gene expression in the breast tumor versus matched healthy tissue from each patient. We identified 2839 genes, ~10 % of protein-coding genes in the human genome, that had differential expression of transcript variants between tumors and healthy tissues. The validity of the computational analysis was confirmed by quantitative RT-PCR assessment of transcript variant expression from four top candidate genes. The alternative transcript profiling led to classification of breast cancer into two subgroups and yielded a novel molecular signature that could be prognostic of patients’ tumor burden and survival. We uncovered nine splicing factors (FOX2, MBNL1, QKI, PTBP1, ELAVL1, HNRNPC, KHDRBS1, SFRS2, and TIAR) that were involved in aberrant splicing in breast cancer. Network analyses for the coordinative patterns of transcript variant expression identified twelve “hub” genes that differentiated the cancerous and normal transcriptomes. Dysregulated expression of alternative transcripts may reveal novel biomarkers for tumor development. It may also suggest new therapeutic targets, such as the “hub” genes identified through the network analyses of transcript variant expression, or splicing factors implicated in the formation of the tumor transcriptome. PMID:25913416

  1. Targeting the Central Pocket in Human Transcription Factor TEAD as a Potential Cancer Therapeutic Strategy

    OpenAIRE

    Pobbati, Ajaybabu V.; Han, Xiao; Hung, Alvin W.; Weiguang, Seetoh; Huda, Nur; Chen, Guo-Ying; Kang, Congbao; Chia, Cheng San Brian; Luo, Xuelian; Hong, Wanjin; Poulsen, Anders

    2015-01-01

    The human TEAD family of transcription factors (TEAD1-4) is required for YAP-mediated transcription in the Hippo pathway. Hyperactivation of TEAD’s co-activator YAP contributes to tissue overgrowth and human cancers, suggesting that pharmacological interference of TEAD-YAP activity may be an effective strategy for anticancer therapy. Here we report the discovery of a central pocket in the YAP-binding domain (YBD) of TEAD that is targetable by small molecule inhibitors. Our X-ray crystallograp...

  2. Dissecting the nascent human transcriptome by analysing the RNA content of transcription factories

    Science.gov (United States)

    Caudron-Herger, Maïwen; Cook, Peter R.; Rippe, Karsten; Papantonis, Argyris

    2015-01-01

    While mapping total and poly-adenylated human transcriptomes has now become routine, characterizing nascent transcripts remains challenging, largely because nascent RNAs have such short half-lives. Here, we describe a simple, fast and cost-effective method to isolate RNA associated with transcription factories, the sites responsible for the majority of nuclear transcription. Following stimulation of human endothelial cells with the pro-inflammatory cytokine TNFα, we isolate and analyse the RNA content of factories by sequencing. Comparison with total, poly(A)+ and chromatin RNA fractions reveals that sequencing of purified factory RNA maps the complete nascent transcriptome; it is rich in intronic unprocessed transcript, as well as long intergenic non-coding (lincRNAs) and enhancer-associated RNAs (eRNAs), micro-RNA precursors and repeat-derived RNAs. Hence, we verify that transcription factories produce most nascent RNA and confer a regulatory role via their association with a set of specifically-retained non-coding transcripts. PMID:25897132

  3. Transcription pattern of UL131A-128 mRNA in clinical strains of human cytomegalovirus

    Indian Academy of Sciences (India)

    Zhengrong Sun; Gaowei Ren; Yanping Ma; Ning Wang; Yaohua Ji; Ying Qi; Mali Li; Rong He; Qiang Ruan

    2010-09-01

    Human cytomegalovirus (HCMV) mRNA was obtained from human embryonic lung fibroblast cells infected by HCMV clinical strains from urine samples of infants at different kinetic periods. The cDNA of UL131A-128 mRNAs was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and analysed by sequencing. Mean while, clones containing UL131A-128 transcripts in an HCMV cDNA library of a clinical strain were selected and sequenced. It was demonstrated that UL131A-128 mRNA was expressed with immediately early, early and late kinetics. Sequences obtained by RT-PCR showed that the UL131A gene consisted of two exons and the coding region of the UL130 gene was not interrupted by any intron in the region as reported earlier. However, the transcript of the UL128 gene showed two patterns: one pattern consisted of three exons as reported earlier; the other contained the three exons and also the first intron. Moreover, the above characteristics of UL131A-128 spliced transcripts were confirmed by the sequences of clones selected from the HCMV cDNA library. Our results demonstrated that the UL131A, UL130 and UL128 genes were transcribed with the 3′-coterminal, although the initiation points of their mRNA may be different. The variation in the transcripts found in our study indicated the complex nature of transcription of UL131A-128 genes in clinical strains of HCMV.

  4. Transcriptional Dynamics of Immortalized Human Mesenchymal Stem Cells during Transformation.

    Directory of Open Access Journals (Sweden)

    Masao Takeuchi

    Full Text Available Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type 16 E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN; these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent growth and tumorigenicity in nude mice, (sarcoma and exhibited increased expression of genes encoding growth factor and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5 (GPC5, which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1, the cell surface receptor for hedgehog (Hh signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in growth in U3-DT cells (transformants derived from UE6E7T-3 cells through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional

  5. Distinct DNA-based epigenetic switches trigger transcriptional activation of silent genes in human dermal fibroblasts.

    Science.gov (United States)

    Pandian, Ganesh N; Taniguchi, Junichi; Junetha, Syed; Sato, Shinsuke; Han, Le; Saha, Abhijit; AnandhaKumar, Chandran; Bando, Toshikazu; Nagase, Hiroki; Vaijayanthi, Thangavel; Taylor, Rhys D; Sugiyama, Hiroshi

    2014-01-24

    The influential role of the epigenome in orchestrating genome-wide transcriptional activation instigates the demand for the artificial genetic switches with distinct DNA sequence recognition. Recently, we developed a novel class of epigenetically active small molecules called SAHA-PIPs by conjugating selective DNA binding pyrrole-imidazole polyamides (PIPs) with the histone deacetylase inhibitor SAHA. Screening studies revealed that certain SAHA-PIPs trigger targeted transcriptional activation of pluripotency and germ cell genes in mouse and human fibroblasts, respectively. Through microarray studies and functional analysis, here we demonstrate for the first time the remarkable ability of thirty-two different SAHA-PIPs to trigger the transcriptional activation of exclusive clusters of genes and noncoding RNAs. QRT-PCR validated the microarray data, and some SAHA-PIPs activated therapeutically significant genes like KSR2. Based on the aforementioned results, we propose the potential use of SAHA-PIPs as reagents capable of targeted transcriptional activation.

  6. General organisational principles of the transcriptional regulation system: a tree or a circle?

    Science.gov (United States)

    Muskhelishvili, Georgi; Sobetzko, Patrick; Geertz, Marcel; Berger, Michael

    2010-04-01

    Recent advances of systemic approaches to gene expression and cellular metabolism provide unforeseen opportunities for relating and integrating extensive datasets describing the transcriptional regulation system as a whole. However, due to the multifaceted nature of the phenomenon, these datasets often contain logically distinct types of information determined by underlying approach and adopted methodology of data analysis. Consequently, to integrate the datasets comprising information on the states of chromatin structure, transcriptional regulatory network and cellular metabolism, a novel methodology enabling interconversion of logically distinct types of information is required. Here we provide a holistic conceptual framework for analysis of global transcriptional regulation as a system coordinated by structural coupling between the transcription machinery and DNA topology, acting as interdependent sensors and determinants of metabolic functions. In this operationally closed system any transition in physiological state represents an emergent property determined by shifts in structural coupling, whereas genetic regulation acts as a genuine device converting one logical type of information into the other.

  7. Identification of Claudin 1 Transcript Variants in Human Invasive Breast Cancer

    Science.gov (United States)

    Zelinski, Teresa; Xie, Jiuyong; Cooper, Steven; Penner, Carla; Leygue, Etienne; Myal, Yvonne

    2016-01-01

    Background The claudin 1 tight junction protein, solely responsible for the barrier function of epithelial cells, is frequently down regulated in invasive human breast cancer. The underlying mechanism is largely unknown, and no obvious mutations in the claudin 1 gene (CLDN1) have been identified to date in breast cancer. Since many genes have been shown to undergo deregulation through splicing and mis-splicing events in cancer, the current study was undertaken to investigate the occurrence of transcript variants for CLDN1 in human invasive breast cancer. Methods RT-PCR analysis of CLDN1 transcripts was conducted on RNA isolated from 12 human invasive breast tumors. The PCR products from each tumor were resolved by agarose gel electrophoresis, cloned and sequenced. Genomic DNA was also isolated from each of the 12 tumors and amplified using PCR CLDN1 specific primers. Sanger sequencing and single nucleotide polymorphism (SNP) analyses were conducted. Results A number of CLDN1 transcript variants were identified in these breast tumors. All variants were shorter than the classical CLDN1 transcript. Sequence analysis of the PCR products revealed several splice variants, primarily in exon 1 of CLDN1; resulting in truncated proteins. One variant, V1, resulted in a premature stop codon and thus likely led to nonsense mediated decay. Interestingly, another transcript variant, V2, was not detected in normal breast tissue samples. Further, sequence analysis of the tumor genomic DNA revealed SNPs in 3 of the 4 coding exons, including a rare missense SNP (rs140846629) in exon 2 which represents an Ala124Thr substitution. To our knowledge this is the first report of CLDN1 transcript variants in human invasive breast cancer. These studies suggest that alternate splicing may also be a mechanism by which claudin 1 is down regulated at both the mRNA and protein levels in invasive breast cancer and may provide novel insights into how CLDN1 is reduced or silenced in human breast

  8. A dynamic network of transcription in LPS-treated human subjects

    Directory of Open Access Journals (Sweden)

    Moldawer Lyle L

    2009-07-01

    Full Text Available Abstract Background Understanding the transcriptional regulatory networks that map out the coordinated dynamic responses of signaling proteins, transcription factors and target genes over time would represent a significant advance in the application of genome wide expression analysis. The primary challenge is monitoring transcription factor activities over time, which is not yet available at the large scale. Instead, there have been several developments to estimate activities computationally. For example, Network Component Analysis (NCA is an approach that can predict transcription factor activities over time as well as the relative regulatory influence of factors on each target gene. Results In this study, we analyzed a gene expression data set in blood leukocytes from human subjects administered with lipopolysaccharide (LPS, a prototypical inflammatory challenge, in the context of a reconstructed regulatory network including 10 transcription factors, 99 target genes and 149 regulatory interactions. We found that the computationally estimated activities were well correlated to their coordinated action. Furthermore, we found that clustering the genes in the context of regulatory influences greatly facilitated interpretation of the expression data, as clusters of gene expression corresponded to the activity of specific factors or more interestingly, factor combinations which suggest coordinated regulation of gene expression. The resulting clusters were therefore more biologically meaningful, and also led to identification of additional genes under the same regulation. Conclusion Using NCA, we were able to build a network that accounted for between 8–11% genes in the known transcriptional response to LPS in humans. The dynamic network illustrated changes of transcription factor activities and gene expressions as well as interactions of signaling proteins, transcription factors and target genes.

  9. Deciphering human heat shock transcription factor 1 regulation via post-translational modification in yeast.

    Directory of Open Access Journals (Sweden)

    Liliana Batista-Nascimento

    Full Text Available Heat shock transcription factor 1 (HSF1 plays an important role in the cellular response to proteotoxic stresses. Under normal growth conditions HSF1 is repressed as an inactive monomer in part through post-translation modifications that include protein acetylation, sumoylation and phosphorylation. Upon exposure to stress HSF1 homotrimerizes, accumulates in nucleus, binds DNA, becomes hyper-phosphorylated and activates the expression of stress response genes. While HSF1 and the mechanisms that regulate its activity have been studied for over two decades, our understanding of HSF1 regulation remains incomplete. As previous studies have shown that HSF1 and the heat shock response promoter element (HSE are generally structurally conserved from yeast to metazoans, we have made use of the genetically tractable budding yeast as a facile assay system to further understand the mechanisms that regulate human HSF1 through phosphorylation of serine 303. We show that when human HSF1 is expressed in yeast its phosphorylation at S303 is promoted by the MAP-kinase Slt2 independent of a priming event at S307 previously believed to be a prerequisite. Furthermore, we show that phosphorylation at S303 in yeast and mammalian cells occurs independent of GSK3, the kinase primarily thought to be responsible for S303 phosphorylation. Lastly, while previous studies have suggested that S303 phosphorylation represses HSF1-dependent transactivation, we now show that S303 phosphorylation also represses HSF1 multimerization in both yeast and mammalian cells. Taken together, these studies suggest that yeast cells will be a powerful experimental tool for deciphering aspects of human HSF1 regulation by post-translational modifications.

  10. O-GlcNAc transferase regulates transcriptional activity of human Oct4.

    Science.gov (United States)

    Constable, Sandii; Lim, Jae-Min; Vaidyanathan, Krithika; Wells, Lance

    2017-10-01

    O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Cross-species transcriptional network analysis defines shared inflammatory responses in murine and human lupus nephritis.

    Science.gov (United States)

    Berthier, Celine C; Bethunaickan, Ramalingam; Gonzalez-Rivera, Tania; Nair, Viji; Ramanujam, Meera; Zhang, Weijia; Bottinger, Erwin P; Segerer, Stephan; Lindenmeyer, Maja; Cohen, Clemens D; Davidson, Anne; Kretzler, Matthias

    2012-07-15

    Lupus nephritis (LN) is a serious manifestation of systemic lupus erythematosus. Therapeutic studies in mouse LN models do not always predict outcomes of human therapeutic trials, raising concerns about the human relevance of these preclinical models. In this study, we used an unbiased transcriptional network approach to define, in molecular terms, similarities and differences among three lupus models and human LN. Genome-wide gene-expression networks were generated using natural language processing and automated promoter analysis and compared across species via suboptimal graph matching. The three murine models and human LN share both common and unique features. The 20 commonly shared network nodes reflect the key pathologic processes of immune cell infiltration/activation, endothelial cell activation/injury, and tissue remodeling/fibrosis, with macrophage/dendritic cell activation as a dominant cross-species shared transcriptional pathway. The unique nodes reflect differences in numbers and types of infiltrating cells and degree of remodeling among the three mouse strains. To define mononuclear phagocyte-derived pathways in human LN, gene sets activated in isolated NZB/W renal mononuclear cells were compared with human LN kidney profiles. A tissue compartment-specific macrophage-activation pattern was seen, with NF-κB1 and PPARγ as major regulatory nodes in the tubulointerstitial and glomerular networks, respectively. Our study defines which pathologic processes in murine models of LN recapitulate the key transcriptional processes active in human LN and suggests that there are functional differences between mononuclear phagocytes infiltrating different renal microenvironments.

  12. High resolution analysis of the human transcriptome: detection of extensive alternative splicing independent of transcriptional activity

    Directory of Open Access Journals (Sweden)

    Rouet Fabien

    2009-10-01

    Full Text Available Abstract Background Commercially available microarrays have been used in many settings to generate expression profiles for a variety of applications, including target selection for disease detection, classification, profiling for pharmacogenomic response to therapeutics, and potential disease staging. However, many commercially available microarray platforms fail to capture transcript diversity produced by alternative splicing, a major mechanism for driving proteomic diversity through transcript heterogeneity. Results The human Genome-Wide SpliceArray™ (GWSA, a novel microarray platform, utilizes an existing probe design concept to monitor such transcript diversity on a genome scale. The human GWSA allows the detection of alternatively spliced events within the human genome through the use of exon body and exon junction probes to provide a direct measure of each transcript, through simple calculations derived from expression data. This report focuses on the performance and validation of the array when measured against standards recently published by the Microarray Quality Control (MAQC Project. The array was shown to be highly quantitative, and displayed greater than 85% correlation with the HG-U133 Plus 2.0 array at the gene level while providing more extensive coverage of each gene. Almost 60% of splice events among genes demonstrating differential expression of greater than 3 fold also contained extensive splicing alterations. Importantly, almost 10% of splice events within the gene set displaying constant overall expression values had evidence of transcript diversity. Two examples illustrate the types of events identified: LIM domain 7 showed no differential expression at the gene level, but demonstrated deregulation of an exon skip event, while erythrocyte membrane protein band 4.1 -like 3 was differentially expressed and also displayed deregulation of a skipped exon isoform. Conclusion Significant changes were detected independent of

  13. CRITERIA FOR AN UPDATED CLASSIFICATION OF HUMAN TRANSCRIPTION FACTOR DNA-BINDING DOMAINS

    NARCIS (Netherlands)

    Wingender, Edgar

    2013-01-01

    By binding to cis-regulatory elements in a sequence-specific manner, transcription factors regulate the activity of nearby genes. Here, we discuss the criteria for a comprehensive classification of human TFs based on their DNA-binding domains. In particular, classification of basic leucine zipper (b

  14. CRITERIA FOR AN UPDATED CLASSIFICATION OF HUMAN TRANSCRIPTION FACTOR DNA-BINDING DOMAINS

    NARCIS (Netherlands)

    Wingender, Edgar

    By binding to cis-regulatory elements in a sequence-specific manner, transcription factors regulate the activity of nearby genes. Here, we discuss the criteria for a comprehensive classification of human TFs based on their DNA-binding domains. In particular, classification of basic leucine zipper

  15. Transcriptional dynamics during human adipogenesis and its link to adipose morphology and distribution

    DEFF Research Database (Denmark)

    Ehrlund, Anna; Mejhert, Niklas; Björk, Christel

    2017-01-01

    White adipose tissue (WAT) can develop into several phenotypes with different pathophysiological impact on type 2 diabetes. To better understand the adipogenic process, the transcriptional events that occur during in vitro differentiation of human adipocytes were investigated and the findings lin...... such as glucocorticosteroids, insulin, cyclic AMP and thyroid hormones. Taken together, our results suggest a complex, but highly coordinated regulation of adipogenesis....

  16. 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

    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...

  17. Restricted varicella-zoster virus transcription in human trigeminal ganglia obtained soon after death

    NARCIS (Netherlands)

    W.J.D. Ouwendijk (Werner ); A. Choe (Alexander); M.A. Nagel (Maria ); D. Gilden (Don); A.D.M.E. Osterhaus (Albert); R.J. Cohrs (Randall ); G.M.G.M. Verjans (George)

    2012-01-01

    textabstractWe analyzed the varicella-zoster virus (VZV) transcriptome in 43 latently infected human trigeminal ganglia (TG) with postmortem intervals (PMIs) ranging from 3.7 to 24 h. Multiplex reverse transcriptase PCR (RT-PCR) revealed no VZV transcripts with a PMI of <9 h. Real-time PCR indicated

  18. Transcriptional Analysis of Normal Human Fibroblast Responses to Microgravity Stress

    Institute of Scientific and Technical Information of China (English)

    Yongqing Liu; Eugenia Wang

    2008-01-01

    To understand the molecular mechanism (s) of how spaceflight affects cellular signaling pathways, quiescent normal human WI-38 fibroblasts were flown on the STS-93 space shuttle mission. Subsequently, RNA samples from the space flown and ground-control cells were used to construct two cDNA libraries, which were then processed for suppression subtractive hybridization (SSH) to identify spaceflight-specific gene expression. The SSH data show that key genes related to oxidative stress, DNA repair, and fatty acid oxidation are activated by spaceflight, suggesting the induction of cellular oxidative stress. This is further substantiated by the up-regulation of neuregulin 1 and the calcium-binding protein calmodulin 2. Another obvious stress sign is that spaceflight evokes the Ras/mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling pathways, along with up-regulating several G1-phase cell cycle traverse genes. Other genes showing up regulation of expression are involved in protein synthesis and pro-apoptosis, as well as pro-survival. Interactome analysis of functionally related genes shows that c-Myc is the "hub" for those genes showing significant changes. Hence, our results suggest that microgravity travel may impact changes in gene expression mostly associated with cellular stress signaling, directing cells to either apoptotic death or premature senescence.

  19. Detection of Human Picornaviruses by Multiplex Reverse Transcription-PCR and Liquid Hybridization

    OpenAIRE

    Jokela, Pia; Joki-Korpela, Päivi; Maaronen, Marita; Glumoff, Virpi; Hyypiä, Timo

    2005-01-01

    A qualitative multiplex reverse transcription (RT)-PCR and liquid hybridization assay for the detection of human enteroviruses, rhinoviruses, parechoviruses, and Aichi virus was developed. Furthermore, a separate assay for the recognition of hepatitis A virus was established to complement the test pattern so that all human picornaviruses were covered. The amplicons, which represented the 5′ untranslated regions of the viral RNA genomes, were identified in liquid hybridization reactions with g...

  20. Categories, concepts, and conditioning: how humans generalize fear.

    Science.gov (United States)

    Dunsmoor, Joseph E; Murphy, Gregory L

    2015-02-01

    During the past century, Pavlovian conditioning has served as the predominant experimental paradigm and theoretical framework to understand how humans learn to fear and avoid real or perceived dangers. Animal models for translational research offer insight into basic behavioral and neurophysiological factors mediating the acquisition, expression, inhibition, and generalization of fear. However, it is important to consider the limits of traditional animal models when applied to humans. Here, we focus on the question of how humans generalize fear. We propose that to understand fear generalization in humans requires taking into account research on higher-level cognition such as category-based induction, inferential reasoning, and representation of conceptual knowledge. Doing so will open the door for productive avenues of new research.

  1. Overlapping transcription structure of human cytomegalovirus UL140 and UL141 genes

    Indian Academy of Sciences (India)

    Yanping Ma; Mali Li; Bo Zheng; Ning Wang; Shuang Gao; Lin Wang; Qi Ying; Zhengrong Sun; Qiang Ruan

    2013-03-01

    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 transcripts with length of 2800, 2400 and 1700 nt, as well as a group of transcripts of about 1000–1300 nt, were found in this gene region with an accordant 3′ ends. Among the transcripts, two initiated upstream of the start code of the UL140 gene and contained the UL140 and UL141 open reading frame (ORF), one initiated in the middle of the UL140 gene, and could encode short ORFs upstream of the UL141 ORF. A group of transcripts initiated upstream or downstream of the start code of the UL141 gene, and could encode `nested’ ORFs, including the UL141 ORF. These `nested’ ORFs possess different initiation sites but the same termination site as that of the UL141 ORF.

  2. Novel transcripts of carbonic anhydrase II in mouse and human testis.

    Science.gov (United States)

    Mezquita, P; Mezquita, C; Mezquita, J

    1999-03-01

    Intracellular and extracellular sources of bicarbonate are essential for sperm motility, sperm binding to the zona pellucida and the acrosome reaction. Carbonic anhydrase II, catalysing the synthesis of bicarbonate within spermatozoa, must play a significant role in these mechanisms. We report here the expression of carbonic anhydrase II during mouse spermatogenesis and the primary structure of testicular transcripts coding for carbonic anhydrase II isolated from adult mouse and human testes. The mouse carbonic anhydrase II (Car2) mRNA displays a 5' untranslated region (UTR) larger than the corresponding somatic sequence. The additional 5' sequence contains the 'TATA box' used in somatic tissues and other promoter sequences, suggesting the use of testis-specific promoters further upstream with read-through of downstream promoters. The 3'UTR of the Car2 mRNA is shorter in mature testicular cells than in somatic cells. Polysomal gradient analysis of carbonic anhydrase II transcripts isolated from adult mouse testis and kidney revealed different translation potential: most of the testicular transcripts were present in the non-polysomal fractions, whereas a considerable fraction of kidney transcripts were polysome-associated. These results suggest that specific transcriptional and post-transcriptional mechanisms regulate the expression of carbonic anhydrase II during mammalian spermatogenesis.

  3. Monocyte human leukocyte antigen-DR transcriptional downregulation by cortisol during septic shock.

    Science.gov (United States)

    Le Tulzo, Yves; Pangault, Celine; Amiot, Laurence; Guilloux, Valérie; Tribut, Olivier; Arvieux, Cédric; Camus, Christophe; Fauchet, Renée; Thomas, Rémi; Drénou, Bernard

    2004-05-15

    Monocyte deactivation has been identified as a major factor of immunosuppression in sepsis and is associated with a loss of surface human leukocyte antigen-DR (HLA-DR) expression on circulating monocytes. Using flow cytometry, quantitative reverse transcription-polymerase chain reaction, we investigated this phenomenon in septic patients. We confirmed the early loss of monocyte HLA-DR expression in all infected patients and demonstrated that this persistent lowered expression at Day 6 correlated with severity scores, secondary infection, and death. This phenomenon occurred at a transcriptional level via a decrease in the class II transactivator A (CIITA) transcription. Furthermore, these abnormalities correlated with the high cortisol levels observed in sepsis and not with those of other putative factors such as catecholamines or interleukin-10. Finally, in vitro studies evidenced that glucocorticoids decrease HLA-DR expression at a transcriptional level via a decrease in CIITA mRNA levels, mainly by down modulating its isoforms I and III. We conclude that in human sepsis, the loss of HLA-DR expression on circulating monocytes is associated with a poor outcome. We suggest that the high endogenous cortisol level observed in septic shock may be a possible new factor involved in the loss of HLA-DR expression on monocytes via its effect on HLA-DR and CIITA transcription.

  4. A novel intermediate in transcription initiation by human mitochondrial RNA polymerase.

    Science.gov (United States)

    Morozov, Yaroslav I; Agaronyan, Karen; Cheung, Alan C M; Anikin, Michael; Cramer, Patrick; Temiakov, Dmitry

    2014-04-01

    The mitochondrial genome is transcribed by a single-subunit T7 phage-like RNA polymerase (mtRNAP), structurally unrelated to cellular RNAPs. In higher eukaryotes, mtRNAP requires two transcription factors for efficient initiation-TFAM, a major nucleoid protein, and TFB2M, a transient component of mtRNAP catalytic site. The mechanisms behind assembly of the mitochondrial transcription machinery and its regulation are poorly understood. We isolated and identified a previously unknown human mitochondrial transcription intermediate-a pre-initiation complex that includes mtRNAP, TFAM and promoter DNA. Using protein-protein cross-linking, we demonstrate that human TFAM binds to the N-terminal domain of mtRNAP, which results in bending of the promoter DNA around mtRNAP. The subsequent recruitment of TFB2M induces promoter melting and formation of an open initiation complex. Our data indicate that the pre-initiation complex is likely to be an important target for transcription regulation and provide basis for further structural, biochemical and biophysical studies of mitochondrial transcription.

  5. Gastrointestinal Fibroblasts Have Specialized, Diverse Transcriptional Phenotypes: A Comprehensive Gene Expression Analysis of Human Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Youichi Higuchi

    Full Text Available Fibroblasts are the principal stromal cells that exist in whole organs and play vital roles in many biological processes. Although the functional diversity of fibroblasts has been estimated, a comprehensive analysis of fibroblasts from the whole body has not been performed and their transcriptional diversity has not been sufficiently explored. The aim of this study was to elucidate the transcriptional diversity of human fibroblasts within the whole body.Global gene expression analysis was performed on 63 human primary fibroblasts from 13 organs. Of these, 32 fibroblasts from gastrointestinal organs (gastrointestinal fibroblasts: GIFs were obtained from a pair of 2 anatomical sites: the submucosal layer (submucosal fibroblasts: SMFs and the subperitoneal layer (subperitoneal fibroblasts: SPFs. Using hierarchical clustering analysis, we elucidated identifiable subgroups of fibroblasts and analyzed the transcriptional character of each subgroup.In unsupervised clustering, 2 major clusters that separate GIFs and non-GIFs were observed. Organ- and anatomical site-dependent clusters within GIFs were also observed. The signature genes that discriminated GIFs from non-GIFs, SMFs from SPFs, and the fibroblasts of one organ from another organ consisted of genes associated with transcriptional regulation, signaling ligands, and extracellular matrix remodeling.GIFs are characteristic fibroblasts with specific gene expressions from transcriptional regulation, signaling ligands, and extracellular matrix remodeling related genes. In addition, the anatomical site- and organ-dependent diversity of GIFs was also discovered. These features of GIFs contribute to their specific physiological function and homeostatic maintenance, and create a functional diversity of the gastrointestinal tract.

  6. Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise.

    Science.gov (United States)

    Pilegaard, H; Ordway, G A; Saltin, B; Neufer, P D

    2000-10-01

    Exercise training elicits a number of adaptive changes in skeletal muscle that result in an improved metabolic efficiency. The molecular mechanisms mediating the cellular adaptations to exercise training in human skeletal muscle are unknown. To test the hypothesis that recovery from exercise is associated with transcriptional activation of specific genes, six untrained male subjects completed 60-90 min of exhaustive one-legged knee extensor exercise for five consecutive days. On day 5, nuclei were isolated from biopsies of the vastus lateralis muscle of the untrained and the trained leg before exercise and from the trained leg immediately after exercise and after 15 min, 1 h, 2 h, and 4 h of recovery. Transcriptional activity of the uncoupling protein 3 (UCP3), pyruvate dehydrogenase kinase 4 (PDK4), and heme oxygenase-1 (HO-1) genes (relative to beta-actin) increased by three- to sevenfold in response to exercise, peaking after 1-2 h of recovery. Increases in mRNA levels followed changes in transcription, peaking between 2 and 4 h after exercise. Lipoprotein lipase and carnitine pamitoyltransferase I gene transcription and mRNA levels showed similar but less dramatic induction patterns, with increases ranging from two- to threefold. In a separate study, a single 4-h bout of cycling exercise (n = 4) elicited from 5 to >20-fold increases in UCP3, PDK4, and HO-1 transcription, suggesting that activation of these genes may be related to the duration or intensity of exercise. These data demonstrate that exercise induces transient increases in transcription of metabolic genes in human skeletal muscle. Moreover, the findings suggest that the cumulative effects of transient increases in transcription during recovery from consecutive bouts of exercise may represent the underlying kinetic basis for the cellular adaptations associated with exercise training.

  7. ISL1 directly regulates FGF10 transcription during human cardiac outflow formation.

    Directory of Open Access Journals (Sweden)

    Christelle Golzio

    Full Text Available The LIM homeodomain gene Islet-1 (ISL1 encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations.

  8. Large-scale identification of sequence variants impacting human transcription factor occupancy in vivo

    Science.gov (United States)

    Maurano, Matthew T.; Haugen, Eric; Sandstrom, Richard; Vierstra, Jeff; Shafer, Anthony; Kaul, Rajinder; Stamatoyannopoulos, John A.

    2015-01-01

    The function of human regulatory regions depends exquisitely on their local genomic environment and cellular context, complicating experimental analysis of the expanding pool of common disease- and trait-associated variants that localize within regulatory DNA. We leverage allelically resolved genomic DNaseI footprinting data encompassing 166 individuals and 114 cell types to identify >60,000 common variants that directly impact transcription factor occupancy and regulatory DNA accessibility in vivo. The unprecedented scale of these data enable systematic analysis of the impact of sequence variation on transcription factor occupancy in vivo. We leverage this analysis to develop accurate models of variation affecting the recognition sites for diverse transcription factors, and apply these models to discriminate nearly 500,000 common regulatory variants likely to affect transcription factor occupancy across the human genome. The approach and results provide a novel foundation for analysis and interpretation of noncoding variation in complete human genomes, and for systems-level investigation of disease-associated variants. PMID:26502339

  9. Disease Model of GATA4 Mutation Reveals Transcription Factor Cooperativity in Human Cardiogenesis.

    Science.gov (United States)

    Ang, Yen-Sin; Rivas, Renee N; Ribeiro, Alexandre J S; Srivas, Rohith; Rivera, Janell; Stone, Nicole R; Pratt, Karishma; Mohamed, Tamer M A; Fu, Ji-Dong; Spencer, C Ian; Tippens, Nathaniel D; Li, Molong; Narasimha, Anil; Radzinsky, Ethan; Moon-Grady, Anita J; Yu, Haiyuan; Pruitt, Beth L; Snyder, Michael P; Srivastava, Deepak

    2016-12-15

    Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions, leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling, and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. STAT4-mediated transcriptional repression of the IL5 gene in human memory Th2 cells.

    Science.gov (United States)

    Gonzales-van Horn, Sarah R; Estrada, Leonardo D; van Oers, Nicolai S C; Farrar, J David

    2016-06-01

    Type I interferon (IFN-α/β) plays a critical role in suppressing viral replication by driving the transcription of hundreds of interferon-sensitive genes (ISGs). While many ISGs are transcriptionally activated by the ISGF3 complex, the significance of other signaling intermediates in IFN-α/β-mediated gene regulation remains elusive, particularly in rare cases of gene silencing. In human Th2 cells, IFN-α/β signaling suppressed IL5 and IL13 mRNA expression during recall responses to T-cell receptor (TCR) activation. This suppression occurred through a rapid reduction in the rate of nascent transcription, independent of de novo expression of ISGs. Further, IFN-α/β-mediated STAT4 activation was required for repressing the human IL5 gene, and disrupting STAT4 dimerization reversed this effect. This is the first demonstration of STAT4 acting as a transcriptional repressor in response to IFN-α/β signaling and highlights the unique activity of this cytokine to acutely block the expression of an inflammatory cytokine in human T cells.

  11. Targeting the Central Pocket in Human Transcription Factor TEAD as a Potential Cancer Therapeutic Strategy.

    Science.gov (United States)

    Pobbati, Ajaybabu V; Han, Xiao; Hung, Alvin W; Weiguang, Seetoh; Huda, Nur; Chen, Guo-Ying; Kang, CongBao; Chia, Cheng San Brian; Luo, Xuelian; Hong, Wanjin; Poulsen, Anders

    2015-11-03

    The human TEAD family of transcription factors (TEAD1-4) is required for YAP-mediated transcription in the Hippo pathway. Hyperactivation of TEAD's co-activator YAP contributes to tissue overgrowth and human cancers, suggesting that pharmacological interference of TEAD-YAP activity may be an effective strategy for anticancer therapy. Here we report the discovery of a central pocket in the YAP-binding domain (YBD) of TEAD that is targetable by small-molecule inhibitors. Our X-ray crystallography studies reveal that flufenamic acid, a non-steroidal anti-inflammatory drug (NSAID), binds to the central pocket of TEAD2 YBD. Our biochemical and functional analyses further demonstrate that binding of NSAIDs to TEAD inhibits TEAD-YAP-dependent transcription, cell migration, and proliferation, indicating that the central pocket is important for TEAD function. Therefore, our studies discover a novel way of targeting TEAD transcription factors and set the stage for therapeutic development of specific TEAD-YAP inhibitors against human cancers. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Detection of a common chimeric transcript between human chromosomes 7 and 16

    Directory of Open Access Journals (Sweden)

    Fang Wenwen

    2012-12-01

    Full Text Available Abstract Interchromosomal chimeric RNA molecules are often transcription products from genomic rearrangement in cancerous cells. Here we report the computational detection of an interchromosomal RNA fusion between ZC3HAV1L and CHMP1A from RNA-seq data of normal human mammary epithelial cells, and experimental confirmation of the chimeric transcript in multiple human cells and tissues. Our experimental characterization also detected three variants of the ZC3HAV1L-CHMP1A chimeric RNA, suggesting that these genes are involved in complex splicing. The fusion sequence at the novel exon-exon boundary, and the absence of corresponding DNA rearrangement suggest that this chimeric RNA is likely produced by trans-splicing in human cells. Reviewers This article was reviewed by Rory Johnson (nominated by Fyodor Kondrashov; Gal Avital and Itai Yanai

  13. Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest

    DEFF Research Database (Denmark)

    Sylvestersen, Kathrine B; Horn, Heiko; Jungmichel, Stephanie

    2014-01-01

    contain regulated functions on their own. Collectively, we present a site-specific MMA dataset in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase......., transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared to the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers......The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein...

  14. Dissecting the interface between signaling and transcriptional regulation in human B cells

    DEFF Research Database (Denmark)

    Wang, Kai; Alvarez, Mariano J; Bisikirska, Brygida C

    2009-01-01

    A key role of signal transduction pathways is to control transcriptional programs in the nucleus as a function of signals received by the cell via complex post-translational modification cascades. This determines cell-context specific responses to environmental stimuli. Given the difficulty...... of quantitating protein concentration and post-translational modifications, signaling pathway studies are still for the most part conducted one interaction at the time. Thus, genome-wide, cell-context specific dissection of signaling pathways is still an open challenge in molecular systems biology....... In this manuscript we extend the MINDy algorithm for the identification of posttranslational modulators of transcription factor activity, to produce a first genome-wide map of the interface between signaling and transcriptional regulatory programs in human B cells. We show that the serine-threonine kinase STK38...

  15. Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

    Science.gov (United States)

    Mercer, Andrew C; Gaj, Thomas; Sirk, Shannon J; Lamb, Brian M; Barbas, Carlos F

    2014-10-17

    The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.

  16. A Progenitor Cell Expressing Transcription Factor RORγt Generates All Human Innate Lymphoid Cell Subsets.

    Science.gov (United States)

    Scoville, Steven D; Mundy-Bosse, Bethany L; Zhang, Michael H; Chen, Li; Zhang, Xiaoli; Keller, Karen A; Hughes, Tiffany; Chen, Luxi; Cheng, Stephanie; Bergin, Stephen M; Mao, Hsiaoyin C; McClory, Susan; Yu, Jianhua; Carson, William E; Caligiuri, Michael A; Freud, Aharon G

    2016-05-17

    The current model of murine innate lymphoid cell (ILC) development holds that mouse ILCs are derived downstream of the common lymphoid progenitor through lineage-restricted progenitors. However, corresponding lineage-restricted progenitors in humans have yet to be discovered. Here we identified a progenitor population in human secondary lymphoid tissues (SLTs) that expressed the transcription factor RORγt and was unique in its ability to generate all known ILC subsets, including natural killer (NK) cells, but not other leukocyte populations. In contrast to murine fate-mapping data, which indicate that only ILC3s express Rorγt, these human progenitor cells as well as human peripheral blood NK cells and all mature ILC populations expressed RORγt. Thus, all human ILCs can be generated through an RORγt(+) developmental pathway from a common progenitor in SLTs. These findings help establish the developmental signals and pathways involved in human ILC development.

  17. Transcriptional profiling of ectoderm specification to keratinocyte fate in human embryonic stem cells.

    Science.gov (United States)

    Tadeu, Ana Mafalda Baptista; Lin, Samantha; Hou, Lin; Chung, Lisa; Zhong, Mei; Zhao, Hongyu; Horsley, Valerie

    2015-01-01

    In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ-secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions.

  18. Genetic factors affecting gene transcription and catalytic activity of UDP-glucuronosyltransferases in human liver.

    Science.gov (United States)

    Liu, Wanqing; Ramírez, Jacqueline; Gamazon, Eric R; Mirkov, Snezana; Chen, Peixian; Wu, Kehua; Sun, Chang; Cox, Nancy J; Cook, Edwin; Das, Soma; Ratain, Mark J

    2014-10-15

    The aim of this study was to discover cis- and trans-acting factors significantly affecting mRNA expression and catalytic activity of human hepatic UDP-glucuronosyltransferases (UGTs). Transcription levels of five major hepatic UGT1A (UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9) and five UGT2B (UGT2B4, UGT2B7, UGT2B10, UGT2B15 and UGT2B17) genes were quantified in human liver tissue samples (n = 125) using real-time PCR. Glucuronidation activities of 14 substrates were measured in 47 livers. We genotyped 167 tagSNPs (single-nucleotide polymorphisms) in UGT1A (n = 43) and UGT2B (n = 124), as well as the known functional UGT1A1*28 and UGT2B17 CNV (copy number variation) polymorphisms. Transcription levels of 15 transcription factors (TFs) known to regulate these UGTs were quantified. We found that UGT expression and activity were highly variable among the livers (median and range of coefficient of variations: 135%, 74-217% and 52%, 39-105%, respectively). CAR, PXR and ESR1 were found to be the most important trans-regulators of UGT transcription (median and range of correlation coefficients: 46%, 6-58%; 47%, 9-58%; and 52%, 24-75%, respectively). Hepatic UGT activities were mainly determined by UGT gene transcription levels. Twenty-one polymorphisms were significantly (FDR-adjusted P transcription and testosterone glucuronidation rate, in addition to that attributable to the UGT2B17 CNV. Our study discovered novel pharmacogenetic markers and provided detailed insight into the genetic network regulating hepatic UGTs.

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Zach K O'Brown

    2015-12-01

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

  1. NF-Y recruits both transcription activator and repressor to modulate tissue- and developmental stage-specific expression of human γ-globin gene.

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    Xingguo Zhu

    Full Text Available The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.

  2. Transcript Regulation of Human Telomerase Reverse Transcriptase by c-myc and mad1

    Institute of Scientific and Technical Information of China (English)

    Lin ZOU; Peng-Hui ZHANG; Chun-Li LUO; Zhi-Guang TU

    2005-01-01

    Telomerase activity is highly positive correlated to most malignant neoplasms. Human telomerase reverse transcriptase (hTERT) is the rate-limiting factor of telomerase activity. Recent studies have shown that the expression of hTERT is mainly determined by its transcript regulation. Among the transcript regulation factors of hTERT, c-myc and mad1 are well known. Here, we constructed c-myc and mad1 eukaryotic expression vectors, then co-transfected them with the wild-type (Tw) or mutant hTERT promoter (Td)luciferase reporter plasmid, which were double-mutated in the E-box sequences from CACGTG to CACCTG of Tw. The change of luciferase activity in different cells was detected. The results showed that Tw was obviously activated in T24 and EJ bladder cancer cells, but not in normal fibrocytes. c-myc and mad1 had positive and negative effects respectively on the Tw transcript in a dose-dependent manner, while the roles of c-myc and mad1 in regulating the Td transcript were reversed. c-myc combined with mad1 can downregulate Tw but not Td. These observations indicate that c-myc and mad1 can regulate the hTERT transcript in a different manner in hTERT positive cells, but not in normal cells. This may provide an insight into some telomerase-related carcinogenesis mechanisms.

  3. Distinguishing the Transcription Regulation Patterns in Promoters of Human Genes with Different Function or Evolutionary Age

    KAUST Repository

    Alam, Tanvir

    2012-07-01

    Distinguishing transcription regulatory patterns of different gene groups is a common problem in various bioinformatics studies. In this work we developed a methodology to deal with such a problem based on machine learning techniques. We applied our method to two biologically important problems related to detecting a difference in transcription regulation of: a/ protein-coding and long non-coding RNAs (lncRNAs) in human, as well as b/ a difference between primate-specific and non-primate-specific long non-coding RNAs. Our method is capable to classify RNAs using various regulatory features of genes that transcribe into these RNAs, such as nucleotide frequencies, transcription factor binding sites, de novo sequence motifs, CpG islands, repetitive elements, histone modification marks, and others. Ten-fold cross-validation tests suggest that our model can distinguish protein-coding and non-coding RNAs with accuracy above 80%. Twenty-fold cross-validation tests suggest that our model can distinguish primate-specific from non-primate-specific promoters of lncRNAs with accuracy above 80%. Consequently, we can hypothesize that transcription of the groups of genes mentioned above are regulated by different mechanisms. Feature selection techniques allowed us to reduce the number of features significantly while keeping the accuracy around 80%. Consequently, we can conclude that selected features play significant role in transcription regulation of coding and non-coding genes, as well as primate-specific and non-primate-specific lncRNA genes.

  4. A network of paralogous stress response transcription factors in the human pathogen Candida glabrata.

    Directory of Open Access Journals (Sweden)

    Jawad eMerhej

    2016-05-01

    Full Text Available The yeast Candida glabrata has become the second cause of systemic candidemia in humans. However, relatively few genome-wide studies have been conducted in this organism and our knowledge of its transcriptional regulatory network is quite limited. In the present work, we combined genome-wide chromatin immunoprecipitation (ChIP-seq, transcriptome analyses and DNA binding motif predictions to describe the regulatory interactions of the seven Yap (Yeast AP1 transcription factors of C. glabrata. We described a transcriptional network containing 255 regulatory interactions and 309 potential target genes. We predicted with high confidence the preferred DNA binding sites for 5 of the 7 CgYaps and showed a strong conservation of the Yap DNA binding properties between S. cerevisiae and C. glabrata. We provided reliable functional annotation for 3 of the 7 Yaps and identified for Yap1 and Yap5 a core regulon which is conserved in S. cerevisiae, C. glabrata and C. albicans. We uncovered new roles for CgYap7 in the regulation of iron-sulfur cluster biogenesis, for CgYap1 in the regulation of heme biosynthesis and for CgYap5 in the repression of GRX4 in response to iron starvation. These transcription factors define an interconnected transcriptional network at the cross-roads between redox homeostasis, oxygen consumption and iron metabolism.

  5. PPARG in Human Adipogenesis: Differential Contribution of Canonical Transcripts and Dominant Negative Isoforms

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    M. Aprile

    2014-01-01

    Full Text Available The nuclear receptor PPARγ is a key regulator of adipogenesis, and alterations of its function are associated with different pathological processes related to metabolic syndrome. We recently identified two PPARG transcripts encoding dominant negative PPARγ isoforms. The existence of different PPARG variants suggests that alternative splicing is crucial to modulate PPARγ function, underlying some underestimated aspects of its regulation. Here we investigate PPARG expression in different tissues and cells affected in metabolic syndrome and, in particular, during adipocyte differentiation of human mesenchymal stem cells. We defined the transcript-specific expression pattern of PPARG variants encoding both canonical and dominant negative isoforms and identified a novel PPARG transcript, γ1ORF4. Our analysis indicated that, during adipogenesis, the transcription of alternative PPARG variants is regulated in a time-specific manner through differential usage of distinct promoters. In addition, our analysis describes—for the first time—the differential contribution of three ORF4 variants to this process, suggesting a still unexplored role for these dominant negative isoforms during adipogenesis. Therefore, our results highlight crucial aspects of PPARG regulation, suggesting the need of further investigation to rule out the differential impact of all PPARG transcripts in both physiologic and pathologic conditions, such as metabolism-related disorders.

  6. Human population-specific gene expression and transcriptional network modification with polymorphic transposable elements.

    Science.gov (United States)

    Wang, Lu; Rishishwar, Lavanya; Mariño-Ramírez, Leonardo; Jordan, I King

    2016-12-19

    Transposable element (TE) derived sequences are known to contribute to the regulation of the human genome. The majority of known TE-derived regulatory sequences correspond to relatively ancient insertions, which are fixed across human populations. The extent to which human genetic variation caused by recent TE activity leads to regulatory polymorphisms among populations has yet to be thoroughly explored. In this study, we searched for associations between polymorphic TE (polyTE) loci and human gene expression levels using an expression quantitative trait loci (eQTL) approach. We compared locus-specific polyTE insertion genotypes to B cell gene expression levels among 445 individuals from 5 human populations. Numerous human polyTE loci correspond to both cis and trans eQTL, and their regulatory effects are directly related to cell type-specific function in the immune system. PolyTE loci are associated with differences in expression between European and African population groups, and a single polyTE loci is indirectly associated with the expression of numerous genes via the regulation of the B cell-specific transcription factor PAX5 The polyTE-gene expression associations we found indicate that human TE genetic variation can have important phenotypic consequences. Our results reveal that TE-eQTL are involved in population-specific gene regulation as well as transcriptional network modification.

  7. Human population-specific gene expression and transcriptional network modification with polymorphic transposable elements

    Science.gov (United States)

    Wang, Lu; Mariño-Ramírez, Leonardo

    2017-01-01

    Abstract Transposable element (TE) derived sequences are known to contribute to the regulation of the human genome. The majority of known TE-derived regulatory sequences correspond to relatively ancient insertions, which are fixed across human populations. The extent to which human genetic variation caused by recent TE activity leads to regulatory polymorphisms among populations has yet to be thoroughly explored. In this study, we searched for associations between polymorphic TE (polyTE) loci and human gene expression levels using an expression quantitative trait loci (eQTL) approach. We compared locus-specific polyTE insertion genotypes to B cell gene expression levels among 445 individuals from 5 human populations. Numerous human polyTE loci correspond to both cis and trans eQTL, and their regulatory effects are directly related to cell type-specific function in the immune system. PolyTE loci are associated with differences in expression between European and African population groups, and a single polyTE loci is indirectly associated with the expression of numerous genes via the regulation of the B cell-specific transcription factor PAX5. The polyTE-gene expression associations we found indicate that human TE genetic variation can have important phenotypic consequences. Our results reveal that TE-eQTL are involved in population-specific gene regulation as well as transcriptional network modification. PMID:27998931

  8. Evidence for transcript networks composed of chimeric RNAs in human cells.

    Directory of Open Access Journals (Sweden)

    Sarah Djebali

    Full Text Available The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5' and 3' transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1 the non-random interconnections of genes involved, (2 the greater phylogenetic depth of the genes involved in many chimeric interactions, (3 the coordination of the expression of connected genes and (4 the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.

  9. Principle Generalized Net Model of a Human Stress Reaction

    Directory of Open Access Journals (Sweden)

    Anthony Shannon

    2008-04-01

    Full Text Available The present study was aimed at investigating the mechanism of a human stress reaction by means of Generalized Nets (GNs. A principle GN-model of the main structures, organs and systems of the human body taking part in the acute and chronic reaction of the organism to a stress stimulus is generated. A possible application of the GN-model of the human stress reaction for testing the effect of known or newly synthesized pharmacological products as well as of food supplements is discussed.

  10. TcoF-DB v2: update of the database of human and mouse transcription co-factors and transcription factor interactions

    KAUST Repository

    Schmeier, Sebastian

    2016-10-17

    Transcription factors (TFs) play a pivotal role in transcriptional regulation, making them crucial for cell survival and important biological functions. For the regulation of transcription, interactions of different regulatory proteins known as transcription co-factors (TcoFs) and TFs are essential in forming necessary protein complexes. Although TcoFs themselves do not bind DNA directly, their influence on transcriptional regulation and initiation, although indirect, has been shown to be significant, with the functionality of TFs strongly influenced by the presence of TcoFs. In the TcoF-DB v2 database, we collect information on TcoFs. In this article, we describe updates and improvements implemented in TcoF-DB v2. TcoF-DB v2 provides several new features that enables exploration of the roles of TcoFs. The content of the database has significantly expanded, and is enriched with information from Gene Ontology, biological pathways, diseases and molecular signatures. TcoF-DB v2 now includes many more TFs; has substantially increased the number of human TcoFs to 958, and now includes information on mouse (418 new TcoFs). TcoF-DB v2 enables the exploration of information on TcoFs and allows investigations into their influence on transcriptional regulation in humans and mice. TcoF-DB v2 can be accessed at http://tcofdb.org/.

  11. Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution.

    Science.gov (United States)

    Hu, Jinchuan; Adar, Sheera; Selby, Christopher P; Lieb, Jason D; Sancar, Aziz

    2015-05-01

    We developed a method for genome-wide mapping of DNA excision repair named XR-seq (excision repair sequencing). Human nucleotide excision repair generates two incisions surrounding the site of damage, creating an ∼30-mer. In XR-seq, this fragment is isolated and subjected to high-throughput sequencing. We used XR-seq to produce stranded, nucleotide-resolution maps of repair of two UV-induced DNA damages in human cells: cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4)PPs]. In wild-type cells, CPD repair was highly associated with transcription, specifically with the template strand. Experiments in cells defective in either transcription-coupled excision repair or general excision repair isolated the contribution of each pathway to the overall repair pattern and showed that transcription-coupled repair of both photoproducts occurs exclusively on the template strand. XR-seq maps capture transcription-coupled repair at sites of divergent gene promoters and bidirectional enhancer RNA (eRNA) production at enhancers. XR-seq data also uncovered the repair characteristics and novel sequence preferences of CPDs and (6-4)PPs. XR-seq and the resulting repair maps will facilitate studies of the effects of genomic location, chromatin context, transcription, and replication on DNA repair in human cells.

  12. Transcription factor binding sites are genetic determinants of retroviral integration in the human genome.

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    Barbara Felice

    Full Text Available Gamma-retroviruses and lentiviruses integrate non-randomly in mammalian genomes, with specific preferences for active chromatin, promoters and regulatory regions. Gene transfer vectors derived from gamma-retroviruses target at high frequency genes involved in the control of growth, development and differentiation of the target cell, and may induce insertional tumors or pre-neoplastic clonal expansions in patients treated by gene therapy. The gene expression program of the target cell is apparently instrumental in directing gamma-retroviral integration, although the molecular basis of this phenomenon is poorly understood. We report a bioinformatic analysis of the distribution of transcription factor binding sites (TFBSs flanking >4,000 integrated proviruses in human hematopoietic and non-hematopoietic cells. We show that gamma-retroviral, but not lentiviral vectors, integrate in genomic regions enriched in cell-type specific subsets of TFBSs, independently from their relative position with respect to genes and transcription start sites. Analysis of sequences flanking the integration sites of Moloney leukemia virus (MLV- and human immunodeficiency virus (HIV-derived vectors carrying mutations in their long terminal repeats (LTRs, and of HIV vectors packaged with an MLV integrase, indicates that the MLV integrase and LTR enhancer are the viral determinants of the selection of TFBS-rich regions in the genome. This study identifies TFBSs as differential genomic determinants of retroviral target site selection in the human genome, and suggests that transcription factors binding the LTR enhancer may synergize with the integrase in tethering retroviral pre-integration complexes to transcriptionally active regulatory regions. Our data indicate that gamma-retroviruses and lentiviruses have evolved dramatically different strategies to interact with the host cell chromatin, and predict a higher risk in using gamma-retroviral vs. lentiviral vectors for human

  13. Mapping of liver-enriched transcription factors in the human intestine

    Institute of Scientific and Technical Information of China (English)

    Frank; Lehner; Ulf; Kulik; Juergen; Klempnauer; Juergen; Borlak

    2010-01-01

    AIM: To investigate the gene expression pattern of hepatocyte nuclear factor 6 (HNF6) and other liverenriched transcription factors in various segments of the human intestine to better understand the differentiation of the gut epithelium. METHODS: Samples of healthy duodenum and jejunum were obtained from patients with pancreatic cancer whereas ileum and colon was obtained from patients undergoing right or left hemicolectomy or (recto)sigmoid or rectal resection. All surgical specimens were subjected to his...

  14. Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms

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    Mueller Nancy

    2005-10-01

    Full Text Available Abstract Background Human T-cell leukemia virus type I (HTLV-I causes adult T-cell leukemia (ATL after a long latent period. Among accessory genes encoded by HTLV-I, the tax gene is thought to play a central role in oncogenesis. However, Tax expression is disrupted by several mechanims including genetic changes of the tax gene, deletion/hypermethylation of 5'-LTR. To clarify the role of epigenetic changes, we analyzed DNA methylation and histone modification in the whole HTLV-I provirus genome. Results The gag, pol and env genes of HTLV-I provirus were more methylated than pX region, whereas methylation of 5'-LTR was variable and 3'-LTR was not methylated at all. In ATL cell lines, complete DNA methylation of 5'-LTR was associated with transcriptional silencing of viral genes. HTLV-I provirus was more methylated in primary ATL cells than in carrier state, indicating the association with disease progression. In seroconvertors, DNA methylation was already observed in internal sequences of provirus just after seroconversion. Taken together, it is speculated that DNA methylation first occurs in the gag, pol and env regions and then extends in the 5' and 3' directions in vivo, and when 5'-LTR becomes methylated, viral transcription is silenced. Analysis of histone modification in the HTLV-I provirus showed that the methylated provirus was associated with hypoacetylation. However, the tax gene transcript could not be detected in fresh ATL cells regardless of hyperacetylated histone H3 in 5'-LTR. The transcription rapidly recovered after in vitro culture in such ATL cells. Conclusion These results showed that epigenetic changes of provirus facilitated ATL cells to evade host immune system by suppressing viral gene transcription. In addition, this study shows the presence of another reversible mechanism that suppresses the tax gene transcription without DNA methylation and hypoacetylated histone.

  15. The Impact of Endurance Training on Human Skeletal Muscle Memory, Global Isoform Expression and Novel Transcripts.

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    Maléne E Lindholm

    2016-09-01

    Full Text Available Regularly performed endurance training has many beneficial effects on health and skeletal muscle function, and can be used to prevent and treat common diseases e.g. cardiovascular disease, type II diabetes and obesity. The molecular adaptation mechanisms regulating these effects are incompletely understood. To date, global transcriptome changes in skeletal muscles have been studied at the gene level only. Therefore, global isoform expression changes following exercise training in humans are unknown. Also, the effects of repeated interventions on transcriptional memory or training response have not been studied before. In this study, 23 individuals trained one leg for three months. Nine months later, 12 of the same subjects trained both legs in a second training period. Skeletal muscle biopsies were obtained from both legs before and after both training periods. RNA sequencing analysis of all 119 skeletal muscle biopsies showed that training altered the expression of 3,404 gene isoforms, mainly associated with oxidative ATP production. Fifty-four genes had isoforms that changed in opposite directions. Training altered expression of 34 novel transcripts, all with protein-coding potential. After nine months of detraining, no training-induced transcriptome differences were detected between the previously trained and untrained legs. Although there were several differences in the physiological and transcriptional responses to repeated training, no coherent evidence of an endurance training induced transcriptional skeletal muscle memory was found. This human lifestyle intervention induced differential expression of thousands of isoforms and several transcripts from unannotated regions of the genome. It is likely that the observed isoform expression changes reflect adaptational mechanisms and processes that provide the functional and health benefits of regular physical activity.

  16. Dissecting Interferon-Induced Transcriptional Programs in Human Peripheral Blood Cells

    Science.gov (United States)

    Waddell, Simon J.; Popper, Stephen J.; Rubins, Kathleen H.; Griffiths, Michael J.; Brown, Patrick O.; Levin, Michael; Relman, David A.

    2010-01-01

    Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, and of homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in understanding pathophysiology. The transcriptional programs of cell populations in health therefore provide a paradigm for deconvoluting disease-associated gene expression profiles. We used human cDNA microarrays to (1) compare the gene expression programs in human peripheral blood mononuclear cells (PBMCs) elicited by 6 major mediators of the immune response: interferons α, β, ω and γ, IL12 and TNFα; and (2) characterize the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes) to IFNγ stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNγ and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFα stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNγ, and emergent properties associated with IFN-mediated activation of mixed cell populations. This information provides a detailed view of cellular activation by immune mediators, and contributes an interpretive framework for the definition of host immune responses in a variety of disease settings. PMID:20339534

  17. Commensal Streptococcus salivarius Modulates PPARγ Transcriptional Activity in Human Intestinal Epithelial Cells.

    Science.gov (United States)

    Couvigny, Benoît; de Wouters, Tomas; Kaci, Ghalia; Jacouton, Elsa; Delorme, Christine; Doré, Joël; Renault, Pierre; Blottière, Hervé M; Guédon, Eric; Lapaque, Nicolas

    2015-01-01

    The impact of commensal bacteria in eukaryotic transcriptional regulation has increasingly been demonstrated over the last decades. A multitude of studies have shown direct effects of commensal bacteria from local transcriptional activity to systemic impact. The commensal bacterium Streptococcus salivarius is one of the early bacteria colonizing the oral and gut mucosal surfaces. It has been shown to down-regulate nuclear transcription factor (NF-кB) in human intestinal cells, a central regulator of the host mucosal immune system response to the microbiota. In order to evaluate its impact on a further important transcription factor shown to link metabolism and inflammation in the intestine, namely PPARγ (peroxisome proliferator-activated receptor), we used human intestinal epithelial cell-lines engineered to monitor PPARγ transcriptional activity in response to a wide range of S. salivarius strains. We demonstrated that different strains from this bacterial group share the property to inhibit PPARγ activation independently of the ligand used. First attempts to identify the nature of the active compounds showed that it is a low-molecular-weight, DNase-, proteases- and heat-resistant metabolite secreted by S. salivarius strains. Among PPARγ-targeted metabolic genes, I-FABP and Angptl4 expression levels were dramatically reduced in intestinal epithelial cells exposed to S. salivarius supernatant. Both gene products modulate lipid accumulation in cells and down-regulating their expression might consequently affect host health. Our study shows that species belonging to the salivarius group of streptococci impact both host inflammatory and metabolic regulation suggesting a possible role in the host homeostasis and health.

  18. Commensal Streptococcus salivarius Modulates PPARγ Transcriptional Activity in Human Intestinal Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Benoît Couvigny

    Full Text Available The impact of commensal bacteria in eukaryotic transcriptional regulation has increasingly been demonstrated over the last decades. A multitude of studies have shown direct effects of commensal bacteria from local transcriptional activity to systemic impact. The commensal bacterium Streptococcus salivarius is one of the early bacteria colonizing the oral and gut mucosal surfaces. It has been shown to down-regulate nuclear transcription factor (NF-кB in human intestinal cells, a central regulator of the host mucosal immune system response to the microbiota. In order to evaluate its impact on a further important transcription factor shown to link metabolism and inflammation in the intestine, namely PPARγ (peroxisome proliferator-activated receptor, we used human intestinal epithelial cell-lines engineered to monitor PPARγ transcriptional activity in response to a wide range of S. salivarius strains. We demonstrated that different strains from this bacterial group share the property to inhibit PPARγ activation independently of the ligand used. First attempts to identify the nature of the active compounds showed that it is a low-molecular-weight, DNase-, proteases- and heat-resistant metabolite secreted by S. salivarius strains. Among PPARγ-targeted metabolic genes, I-FABP and Angptl4 expression levels were dramatically reduced in intestinal epithelial cells exposed to S. salivarius supernatant. Both gene products modulate lipid accumulation in cells and down-regulating their expression might consequently affect host health. Our study shows that species belonging to the salivarius group of streptococci impact both host inflammatory and metabolic regulation suggesting a possible role in the host homeostasis and health.

  19. Discovering multiple transcripts of human hepatocytes using massively parallel signature sequencing (MPSS

    Directory of Open Access Journals (Sweden)

    Li Yi-Xue

    2007-07-01

    Full Text Available Abstract Background The liver is the largest human internal organ – it is composed of multiple cell types and plays a vital role in fulfilling the body's metabolic needs and maintaining homeostasis. Of these cell types the hepatocytes, which account for three-quarters of the liver's volume, perform its main functions. To discover the molecular basis of hepatocyte function, we employed Massively Parallel Signature Sequencing (MPSS to determine the transcriptomic profile of adult human hepatocytes obtained by laser capture microdissection (LCM. Results 10,279 UniGene clusters, representing 7,475 known genes, were detected in human hepatocytes. In addition, 1,819 unique MPSS signatures matching the antisense strand of 1,605 non-redundant UniGene clusters (such as APOC1, APOC2, APOB and APOH were highly expressed in hepatocytes. Conclusion Apart from a large number of protein-coding genes, some of the antisense transcripts expressed in hepatocytes could play important roles in transcriptional interference via a cis-/trans-regulation mechanism. Our result provided a comprehensively transcriptomic atlas of human hepatocytes using MPSS technique, which could be served as an available resource for an in-depth understanding of human liver biology and diseases.

  20. Exercise induces transient transcriptional activation of the PGC-1α gene in human skeletal muscle

    Science.gov (United States)

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P Darrell

    2003-01-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1α (PGC-1α) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell culture and rodent skeletal muscle. To determine whether PGC-1α transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one-legged knee extensor exercise training. At the end of training, subjects completed 3 h of two-legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3-hydroxyacyl-CoA dehydrogenase mRNA, were higher in the trained than the untrained leg prior to exercise. Exercise induced a marked transient increase (P 40-fold) and mRNA content (7- to 10-fold), peaking within 2 h after exercise. Activation of PGC-1α was greater in the trained leg despite the lower relative workload. Interestingly, exercise did not affect nuclear respiratory factor 1 (NRF-1) mRNA, a gene induced by PGC-1α in cell culture. HKII, mitochondrial transcription factor A, peroxisome proliferator activated receptor α, and calcineurin Aα and Aβ mRNA were elevated (≈2- to 6-fold; P < 0.05) at 6 h of recovery in the untrained leg but did not change in the trained leg. The present data demonstrate that exercise induces a dramatic transient increase in PGC-1α transcription and mRNA content in human skeletal muscle. Consistent with its role as a transcriptional coactivator, these findings suggest that PGC-1α may coordinate the activation of metabolic genes in human muscle in response to exercise. PMID:12563009

  1. Exercise induces transient transcriptional activation of the PGC-1alpha gene in human skeletal muscle.

    Science.gov (United States)

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P Darrell

    2003-02-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1alpha (PGC-1alpha) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell culture and rodent skeletal muscle. To determine whether PGC-1alpha transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one-legged knee extensor exercise training. At the end of training, subjects completed 3 h of two-legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3-hydroxyacyl-CoA dehydrogenase mRNA, were higher in the trained than the untrained leg prior to exercise. Exercise induced a marked transient increase (P 40-fold) and mRNA content (7- to 10-fold), peaking within 2 h after exercise. Activation of PGC-1alpha was greater in the trained leg despite the lower relative workload. Interestingly, exercise did not affect nuclear respiratory factor 1 (NRF-1) mRNA, a gene induced by PGC-1alpha in cell culture. HKII, mitochondrial transcription factor A, peroxisome proliferator activated receptor alpha, and calcineurin Aalpha and Abeta mRNA were elevated (approximately 2- to 6-fold; P < 0.05) at 6 h of recovery in the untrained leg but did not change in the trained leg. The present data demonstrate that exercise induces a dramatic transient increase in PGC-1alpha transcription and mRNA content in human skeletal muscle. Consistent with its role as a transcriptional coactivator, these findings suggest that PGC-1alpha may coordinate the activation of metabolic genes in human muscle in response to exercise.

  2. Nurr1 enhances transcription of the human dopamine transporter gene through a novel mechanism.

    Science.gov (United States)

    Sacchetti, P; Mitchell, T R; Granneman, J G; Bannon, M J

    2001-03-01

    The importance of the nuclear receptor nurr1 for the appropriate development of mesencephalic dopamine-synthesizing neurons has been clearly demonstrated through the targeted disruption of the nurr1 gene. The persistence of nurr1 expression in adult tissue suggests a possible role for this transcription factor in the maintenance, as well as development, of the dopaminergic phenotype. To address this issue, we analyzed the effects of nurr1 on the transcriptional expression of the human dopamine transporter gene (hDAT), one of the most specific phenotypic markers for dopaminergic neurons. Nurr1 enhanced the transcriptional activity of hDAT gene constructs transiently transfected into a newly described cell line (SN4741) that expresses a dopaminergic phenotype, whereas other members of the NGFI-B subfamily of nuclear receptors had lesser or no effects. Nurr1 activation of hDAT was not dependent upon heterodimerization with the retinoid X receptor. Unexpectedly, functional analysis of a series of gene constructs revealed that a region of the hDAT 5'-flanking sequence devoid of NGFI-B response element (NBRE)-like sites mediated nurr1 activation. Additional experiments using a nurr1 mutant construct suggest that nurr1 activates hDAT transcription via a novel NBRE-independent mechanism.

  3. High DNA melting temperature predicts transcription start site location in human and mouse.

    LENUS (Irish Health Repository)

    Dineen, David G

    2009-12-01

    The accurate computational prediction of transcription start sites (TSS) in vertebrate genomes is a difficult problem. The physicochemical properties of DNA can be computed in various ways and a many combinations of DNA features have been tested in the past for use as predictors of transcription. We looked in detail at melting temperature, which measures the temperature, at which two strands of DNA separate, considering the cooperative nature of this process. We find that peaks in melting temperature correspond closely to experimentally determined transcription start sites in human and mouse chromosomes. Using melting temperature alone, and with simple thresholding, we can predict TSS with accuracy that is competitive with the most accurate state-of-the-art TSS prediction methods. Accuracy is measured using both experimentally and manually determined TSS. The method works especially well with CpG island containing promoters, but also works when CpG islands are absent. This result is clear evidence of the important role of the physical properties of DNA in the process of transcription. It also points to the importance for TSS prediction methods to include melting temperature as prior information.

  4. Structural basis of human transcription factor Sry-related box 17 binding to DNA.

    Science.gov (United States)

    Gao, Nana; Jiang, Wei; Gao, Hai; Cheng, Zhong; Qian, Huolian; Si, Shuyi; Xie, Yong

    2013-04-01

    Sry-related box (Sox) transcription factors share a conserved high-mobility-group box domain (HMG-domain) that binds DNA in the minor groove and bends DNA for further assembly of transcriptional machineries. During organogenesis, each member of the Sox family triggers a specific cell lineage differentiation, indicating that their interactions with DNA are different from each other. Therefore, investigating structural rearrangement of each Sox transcription factor HMG-domain upon binding to DNA would help to elucidate the distinctive molecular mechanism by which they interact with DNA. Previous studies have determined the crystal structures of Sox2 HMG-domain/DNA, Sox4 HMGdomain/ DNA, Sox9 HMG-domain/DNA and Sox17 HMG-domain/DNA complexes. However, major gaps remain in the structural information on the Sox transcription factor HMG-domains. Here, we report the crystal structure of the human Sox17 HMG-domain alone at 2.4 A resolution. Comparing this structure and the structure of the mouse Sox17 HMGdomain/ DNA complex provides structural understanding of the mechanism of Sox17 binding to DNA. Specifically, after electrostatic interactions attract Sox17 to DNA, Asn73, Ser99, and Trp106 form hydrogen bonds with DNA, Arg70, Lys80, Arg83, His94, and Asn95 on Sox17 undergo conformational changes and form hydrogen bonds with DNA, contributing to the electrostatic interaction between Sox17 and DNA.

  5. The transcriptional program of a human B cell line in response to Myc

    Science.gov (United States)

    Schuhmacher, Marino; Kohlhuber, Franz; Hölzel, Michael; Kaiser, Carmen; Burtscher, Helmut; Jarsch, Michael; Bornkamm, Georg W.; Laux, Gerhard; Polack, Axel; Weidle, Ulrich H.; Eick, Dirk

    2001-01-01

    The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes growth, proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition, we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis, lipid metabolism, protein turnover/folding, nucleotide/DNA synthesis, transport, nucleolus function/RNA binding, transcription and splicing, oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes. PMID:11139609

  6. Comprehensive analysis of the transcriptional profile of the Mediator complex across human cancer types.

    Science.gov (United States)

    Syring, Isabella; Klümper, Niklas; Offermann, Anne; Braun, Martin; Deng, Mario; Boehm, Diana; Queisser, Angela; von Mässenhausen, Anne; Brägelmann, Johannes; Vogel, Wenzel; Schmidt, Doris; Majores, Michael; Schindler, Anne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg; Shaikhibrahim, Zaki; Perner, Sven

    2016-04-26

    The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking.We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

  7. Characterization of human mitochondrial ferritin promoter: identification of transcription factors and evidences of epigenetic control

    Science.gov (United States)

    Guaraldo, Michela; Santambrogio, Paolo; Rovelli, Elisabetta; di Savino, Augusta; Saglio, Giuseppe; Cittaro, Davide; Roetto, Antonella; Levi, Sonia

    2016-09-01

    Mitochondrial ferritin (FtMt) is an iron storage protein belonging to the ferritin family but, unlike the cytosolic ferritin, it has an iron-unrelated restricted tissue expression. FtMt appears to be preferentially expressed in cell types characterized by high metabolic activity and oxygen consumption, suggesting a role in protecting mitochondria from iron-dependent oxidative damage. The human gene (FTMT) is intronless and its promoter region has not been described yet. To analyze the regulatory mechanisms controlling FTMT expression, we characterized the 5‧ flanking region upstream the transcriptional starting site of FTMT by in silico enquiry of sequences conservation, DNA deletion analysis, and ChIP assay. The data revealed a minimal promoter region and identified the presence of SP1, CREB and YY1 as positive regulators, and GATA2, FoxA1 and C/EBPβ as inhibitors of the transcriptional regulation. Furthermore, the FTMT transcription is increased by acetylating and de-methylating agent treatments in K562 and HeLa cells. These treatments up-regulate FtMt expression even in fibroblasts derived from a Friedreich ataxia patient, where it might exert a beneficial effect against mitochondrial oxidative damage. The expression of FTMT appears regulated by a complex mechanism involving epigenetic events and interplay between transcription factors.

  8. Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart.

    Science.gov (United States)

    Tarradas, Anna; Pinsach-Abuin, Mel Lina; Mackintosh, Carlos; Llorà-Batlle, Oriol; Pérez-Serra, Alexandra; Batlle, Montserrat; Pérez-Villa, Félix; Zimmer, Thomas; Garcia-Bassets, Ivan; Brugada, Ramon; Beltran-Alvarez, Pedro; Pagans, Sara

    2017-01-01

    Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. Developing a General Framework for Human Autonomy Teaming

    Science.gov (United States)

    Lachter, Joel; Brandt, Summer; Shively, Jay

    2017-01-01

    Automation has entered nearly every aspect of our lives, but it often remains hard to understand. Why is this? Automation is often brittle, requiring constant human oversight to assure it operates as intended. This oversight has become harder as automation has become more complicated. To resolve this problem, Human-Autonomy Teaming (HAT) has been proposed. HAT looks to make automation act as more of a teammate, by having it communicate with human operators in a more human, goal-directed, manner which provides transparency into the reasoning behind automated recommendations and actions. This, in turn, permits more trust in the automation when it is appropriate, and less when it is not, allowing a more targeted supervision of automated functions. This paper proposes a framework for HAT, incorporating two key tenets: bi-directional communication, and operator directed authority. We have successfully applied these tenets to integrating the autonomous constrained flight planner (an aide for planning diverts) into a dispatch station. We propose the development of general design patterns that may allow these results to be generalized to domains such as photography and automotive navigation. While these domains are very different, we find application of our HAT tenets provides a number of opportunities for improving interaction between human operators and automation.

  10. Transcriptional profiles of supragranular-enriched genes associate with corticocortical network architecture in the human brain

    Science.gov (United States)

    Krienen, Fenna M.; Yeo, B. T. Thomas; Ge, Tian; Buckner, Randy L.; Sherwood, Chet C.

    2016-01-01

    The human brain is patterned with disproportionately large, distributed cerebral networks that connect multiple association zones in the frontal, temporal, and parietal lobes. The expansion of the cortical surface, along with the emergence of long-range connectivity networks, may be reflected in changes to the underlying molecular architecture. Using the Allen Institute’s human brain transcriptional atlas, we demonstrate that genes particularly enriched in supragranular layers of the human cerebral cortex relative to mouse distinguish major cortical classes. The topography of transcriptional expression reflects large-scale brain network organization consistent with estimates from functional connectivity MRI and anatomical tracing in nonhuman primates. Microarray expression data for genes preferentially expressed in human upper layers (II/III), but enriched only in lower layers (V/VI) of mouse, were cross-correlated to identify molecular profiles across the cerebral cortex of postmortem human brains (n = 6). Unimodal sensory and motor zones have similar molecular profiles, despite being distributed across the cortical mantle. Sensory/motor profiles were anticorrelated with paralimbic and certain distributed association network profiles. Tests of alternative gene sets did not consistently distinguish sensory and motor regions from paralimbic and association regions: (i) genes enriched in supragranular layers in both humans and mice, (ii) genes cortically enriched in humans relative to nonhuman primates, (iii) genes related to connectivity in rodents, (iv) genes associated with human and mouse connectivity, and (v) 1,454 gene sets curated from known gene ontologies. Molecular innovations of upper cortical layers may be an important component in the evolution of long-range corticocortical projections. PMID:26739559

  11. Transcriptional profiles of supragranular-enriched genes associate with corticocortical network architecture in the human brain.

    Science.gov (United States)

    Krienen, Fenna M; Yeo, B T Thomas; Ge, Tian; Buckner, Randy L; Sherwood, Chet C

    2016-01-26

    The human brain is patterned with disproportionately large, distributed cerebral networks that connect multiple association zones in the frontal, temporal, and parietal lobes. The expansion of the cortical surface, along with the emergence of long-range connectivity networks, may be reflected in changes to the underlying molecular architecture. Using the Allen Institute's human brain transcriptional atlas, we demonstrate that genes particularly enriched in supragranular layers of the human cerebral cortex relative to mouse distinguish major cortical classes. The topography of transcriptional expression reflects large-scale brain network organization consistent with estimates from functional connectivity MRI and anatomical tracing in nonhuman primates. Microarray expression data for genes preferentially expressed in human upper layers (II/III), but enriched only in lower layers (V/VI) of mouse, were cross-correlated to identify molecular profiles across the cerebral cortex of postmortem human brains (n = 6). Unimodal sensory and motor zones have similar molecular profiles, despite being distributed across the cortical mantle. Sensory/motor profiles were anticorrelated with paralimbic and certain distributed association network profiles. Tests of alternative gene sets did not consistently distinguish sensory and motor regions from paralimbic and association regions: (i) genes enriched in supragranular layers in both humans and mice, (ii) genes cortically enriched in humans relative to nonhuman primates, (iii) genes related to connectivity in rodents, (iv) genes associated with human and mouse connectivity, and (v) 1,454 gene sets curated from known gene ontologies. Molecular innovations of upper cortical layers may be an important component in the evolution of long-range corticocortical projections.

  12. Distinct regulatory mechanisms of the human ferritin gene by hypoxia and hypoxia mimetic cobalt chloride at the transcriptional and post-transcriptional levels.

    Science.gov (United States)

    Huang, Bo-Wen; Miyazawa, Masaki; Tsuji, Yoshiaki

    2014-12-01

    Cobalt chloride has been used as a hypoxia mimetic because it stabilizes hypoxia inducible factor-1α (HIF1-α) and activates gene transcription through a hypoxia responsive element (HRE). However, differences between hypoxia and hypoxia mimetic cobalt chloride in gene regulation remain elusive. Expression of ferritin, the major iron storage protein, is regulated at the transcriptional and posttranscriptional levels through DNA and RNA regulatory elements. Here we demonstrate that hypoxia and cobalt chloride regulate ferritin heavy chain (ferritin H) expression by two distinct mechanisms. Both hypoxia and cobalt chloride increased HIF1-α but a putative HRE in the human ferritin H gene was not activated. Instead, cobalt chloride but not hypoxia activated ferritin H transcription through an antioxidant responsive element (ARE), to which Nrf2 was recruited. Intriguingly, cobalt chloride downregulated ferritin H protein expression while it upregulated other ARE-regulated antioxidant genes in K562 cells. Further characterization demonstrated that cobalt chloride increased interaction between iron regulatory proteins (IRP1 and IRP2) and iron responsive element (IRE) in the 5'UTR of ferritin H mRNA, resulting in translational block of the accumulated ferritin H mRNA. In contrast, hypoxia had marginal effect on ferritin H transcription but increased its translation through decreased IRP1-IRE interaction. These results suggest that hypoxia and hypoxia mimetic cobalt chloride employ distinct regulatory mechanisms through the interplay between DNA and mRNA elements at the transcriptional and post-transcriptional levels.

  13. Transcriptional activation of human CDCA8 gene regulated by transcription factor NF-Y in embryonic stem cells and cancer cells.

    Science.gov (United States)

    Dai, Can; Miao, Cong-Xiu; Xu, Xiao-Ming; Liu, Lv-Jun; Gu, Yi-Fan; Zhou, Di; Chen, Lian-Sheng; Lin, Ge; Lu, Guang-Xiu

    2015-09-11

    The cell division cycle associated 8 (CDCA8) gene plays an important role in mitosis. Overexpression of CDCA8 was reported in some human cancers and is required for cancer growth and progression. We found CDCA8 expression was also high in human ES cells (hESCs) but dropped significantly upon hESC differentiation. However, the regulation of CDCA8 expression has not yet been studied. Here, we characterized the CDCA8 promoter and identified its cis-elements and transcription factors. Three transcription start sites were identified. Reporter gene assays revealed that the CDCA8 promoter was activated in hESCs and cancer cell lines. The promoter drove the reporter expression specifically to pluripotent cells during early mouse embryo development and to tumor tissues in tumor-bearing mice. These results indicate that CDCA8 is transcriptionally activated in hESCs and cancer cells. Mechanistically, two key activation elements, bound by transcription factor NF-Y and CREB1, respectively, were identified in the CDCA8 basic promoter by mutation analyses and electrophoretic motility shift assays. NF-Y binding is positively correlated with promoter activities in different cell types. Interestingly, the NF-YA subunit, binding to the promoter, is primarily a short isoform in hESCs and a long isoform in cancer cells, indicating a different activation mechanism of the CDCA8 transcription between hESCs and cancer cells. Finally, enhanced CDCA8 promoter activities by NF-Y overexpression and reduced CDCA8 transcription by NF-Y knockdown further verified that NF-Y is a positive regulator of CDCA8 transcription. Our study unearths the molecular mechanisms underlying the activation of CDCA8 expression in hESCs and cancer cells, which provides a better understanding of its biological functions.

  14. Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

    DEFF Research Database (Denmark)

    Ferreira, Elisa N; Pires, Lilian C; Parmigiani, Raphael B;

    2004-01-01

    The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification...... can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within...

  15. A transcriptional roadmap to the senescence and differentiation of human oral keratinocytes.

    Science.gov (United States)

    Jang, Da Hyun; Bhawal, Ujjal K; Min, Hong-Ki; Kang, Hyun Ki; Abiko, Yoshimitsu; Min, Byung-Moo

    2015-01-01

    Human epithelial cells undergo morphological and molecular changes leading to terminal differentiation and replicative senescence after a finite number of cell divisions during serial subculture. However, the target genes and their functional significance in the senescence and differentiation in normal human oral keratinocytes have been poorly defined. Here, we demonstrated normal human oral keratinocytes transcriptional signature profiling to senescence and differentiation. Using microarray analysis, our findings indicated that the gene expression profiles induced by serial subculture are distinct classes of gene. The greatest number of these altered genes was identified as being related to biological pathways of transport, cell proliferation, cell cycle, defense and immune response, cell death, transcription, apoptosis, and inflammatory response, suggesting that the serial subculture is able to induce a multitude of specific gene expression changes during senescence and differentiation. Several highly upregulated genes (IL-1β, S100A8, S100A9, MMP1, MMP9, IL-8, BHLHB2, HES1, and TWIST1) in response to the serial subculture in normal human oral keratinocytes were observed. In vitro and in vivo studies also exhibited a close relationship between senescence and differentiation of primary oral keratinocytes and expression of inflammatory molecules. These results suggest a new approach to determine the biological events underlying the pathogenesis of oral keratinocyte aging. © The Author 2014. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Transcription Activity of Ectogenic Human Carcinoembryonic Antigen Promoter in Lung Adenocarcinoma Cells A549

    Institute of Scientific and Technical Information of China (English)

    XIONG Weining; FANG Huijuan; XU Yongjian; XIONG Shendao; CAO Yong; SONG Qingfeng; ZENG Daxiong; ZHANG Huilan

    2006-01-01

    The transcription activity of ectogenic human carcinoembryonic antigen (CEA) promoter in lung adenocarcinoma cells A549 was investigated for the further gene-targeting therapy. The reporter gene green fluorescent protein (GFP) driven by CEA promoter and human cytomegalovirus (CMV) promoter were relatively constructed and named plasmid pCEA-EGFP and pCMV-GFP respectively. The intensity of fluorescence was detected by fluorescence microscope and flow cytometry analysis after the pCEA-GFP and pSNAV-GFP plasmids were transfected into A549 cells through liposome respectively. The results showed (4.08±0.63) % of the A549 cells transfected with pCEA-AFP plasmid expressed, significantly lower than that of the A549 cells transfected with pCMV-GFP [(43.27±3.54) %]. It was suggested that ectogenic human CEA promoter in lung adenocarcinoma cells A549 was weakly expressed. The distinct specificity of CEA promoter in CEA high expression cells was regarded as a tool in selective gene therapy, but the transcription activity of ectogenic human CEA promoter was needed to increase in the future.

  17. Characterization of hematopoietic GATA transcription factor expression in mouse and human dendritic cells.

    Science.gov (United States)

    Scheenstra, Maaike R; Salunkhe, Vishal; De Cuyper, Iris M; Hoogenboezem, Mark; Li, Eveline; Kuijpers, Taco W; van den Berg, Timo K; Gutiérrez, Laura

    2015-12-01

    Dendritic cells (DCs) are key initiators and regulators of the immune response. The development of the DC lineage and their subsets requires an orchestrated regulation of their transcriptional program. Gata1, a transcription factor expressed in several hematopoietic cell lineages, has been recently reported to be required for mouse DC development and function. In humans, GATA1 is involved in the lineage separation between monocyte-derived DCs and Langerhans cells (LC) and loss of GATA1 results in differentiation arrest at the monocyte stage. The hematopoietic GATA factors (i.e. Gata1, Gata2, Gata3) are known to regulate each other's expression and to function consecutively throughout lineage commitment (so-called GATA switch). In humans, mutations in GATA2 are causative of MonoMAC disease, a human immunodeficiency syndrome characterized by loss of DCs, monocytes, B and NK cells. However, additional data on the expression of hematopoietic GATA factors in the DC lineage is missing. In this study, we have characterized the expression of hematopoietic GATA factors in murine and human DCs and their expression dynamics upon TLR stimulation. We found that all hematopoietic GATA factors are expressed in DCs, but identified species-specific differences in the relative expression of each GATA factor, and how their expression fluctuates upon stimulation.

  18. Genome-wide transcriptional profiling of human glioblastoma cells in response to ITE treatment.

    Science.gov (United States)

    Kang, Bo; Zhou, Yanwen; Zheng, Min; Wang, Ying-Jie

    2015-09-01

    A ligand-activated transcription factor aryl hydrocarbon receptor (AhR) is recently revealed to play a key role in embryogenesis and tumorigenesis (Feng et al. [1], Safe et al. [2]) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) (Song et al. [3]) is an endogenous AhR ligand that possesses anti-tumor activity. In order to gain insights into how ITE acts via the AhR in embryogenesis and tumorigenesis, we analyzed the genome-wide transcriptional profiles of the following three groups of cells: the human glioblastoma U87 parental cells, U87 tumor sphere cells treated with vehicle (DMSO) and U87 tumor sphere cells treated with ITE. Here, we provide the details of the sample gathering strategy and show the quality controls and the analyses associated with our gene array data deposited into the Gene Expression Omnibus (GEO) under the accession code of GSE67986.

  19. Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication

    Directory of Open Access Journals (Sweden)

    Candolfi Ermanno

    2011-07-01

    Full Text Available Abstract The Human Phosphate-Binding protein (HPBP is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.

  20. Atomic force microscope observation of branching in single transcript molecules derived from human cardiac muscle

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Jason; Hsueh, Carlin; Gimzewski, James K [Department of Chemistry and Biochemistry, UCLA, 607 Charles Young Drive East, Los Angeles, CA 90095 (United States); Mishra, Bud [Courant Institute of Mathematical Sciences, NYU, 251 Mercer Street, New York, NY 10012 (United States)], E-mail: jreed@chem.ucla.edu, E-mail: gim@chem.ucla.edu

    2008-09-24

    We have used an atomic force microscope to examine a clinically derived sample of single-molecule gene transcripts, in the form of double-stranded cDNA, (c: complementary) obtained from human cardiac muscle without the use of polymerase chain reaction (PCR) amplification. We observed a log-normal distribution of transcript sizes, with most molecules being in the range of 0.4-7.0 kilobase pairs (kb) or 130-2300 nm in contour length, in accordance with the expected distribution of mRNA (m: messenger) sizes in mammalian cells. We observed novel branching structures not previously known to exist in cDNA, and which could have profound negative effects on traditional analysis of cDNA samples through cloning, PCR and DNA sequencing.

  1. Human transcriptional coactivator PC4 stimulates DNA end joining and activates DSB repair activity.

    Science.gov (United States)

    Batta, Kiran; Yokokawa, Masatoshi; Takeyasu, Kunio; Kundu, Tapas K

    2009-01-23

    Human transcriptional coactivator PC4 is a highly abundant nuclear protein that is involved in diverse cellular processes ranging from transcription to chromatin organization. Earlier, we have shown that PC4, a positive activator of p53, overexpresses upon genotoxic insult in a p53-dependent manner. In the present study, we show that PC4 stimulates ligase-mediated DNA end joining irrespective of the source of DNA ligase. Pull-down assays reveal that PC4 helps in the association of DNA ends through its C-terminal domain. In vitro nonhomologous end-joining assays with cell-free extracts show that PC4 enhances the joining of noncomplementary DNA ends. Interestingly, we found that PC4 activates double-strand break (DSB) repair activity through stimulation of DSB rejoining in vivo. Together, these findings demonstrate PC4 as an activator of nonhomologous end joining and DSB repair activity.

  2. Site-Specific Oligonucleotide Binding Represses Transcription of the Human c-myc Gene in vitro

    Science.gov (United States)

    Cooney, Michael; Czernuszewicz, Graznya; Postel, Edith H.; Flint, S. Jane; Hogan, Michael E.

    1988-07-01

    A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.

  3. KeyGenes, a Tool to Probe Tissue Differentiation Using a Human Fetal Transcriptional Atlas

    NARCIS (Netherlands)

    Roost, M.S.; Iperen, L. van; Ariyurek, Y.; Buermans, H.P.; Arindrarto, W.; Devalla, H.D.; Passier, R.; Mummery, C.L.; Carlotti, F.; Koning, E.J. de; Zwet, E.W. van; Goeman, J.J.; Chuva de Sousa Lopes, S.M.

    2015-01-01

    Differentiated derivatives of human pluripotent stem cells in culture are generally phenotypically immature compared to their adult counterparts. Their identity is often difficult to determine with certainty because little is known about their human fetal equivalents in vivo. Cellular identity and s

  4. KeyGenes, a Tool to Probe Tissue Differentiation Using a Human Fetal Transcriptional Atlas

    NARCIS (Netherlands)

    Roost, Matthias S; van Iperen, Liesbeth; Ariyurek, Yavuz; Buermans, Henk P; Arindrarto, Wibowo; Devalla, Harsha D; Passier, Robert; Mummery, Christine L; Carlotti, Françoise; de Koning, Eelco J P; van Zwet, Erik W; Goeman, Jelle J; Chuva de Sousa Lopes, Susana M

    2015-01-01

    Differentiated derivatives of human pluripotent stem cells in culture are generally phenotypically immature compared to their adult counterparts. Their identity is often difficult to determine with certainty because little is known about their human fetal equivalents in vivo. Cellular identity and

  5. Transcriptional and epigenetic regulation of KIAA1199 gene expression in human breast cancer.

    Directory of Open Access Journals (Sweden)

    Cem Kuscu

    Full Text Available Emerging evidence has demonstrated that upregulated expression of KIAA1199 in human cancer bodes for poor survival. The regulatory mechanism controlling KIAA1199 expression in cancer remains to be characterized. In the present study, we have isolated and characterized the human KIAA1199 promoter in terms of regulation of KIAA1199 gene expression. A 3.3 kb fragment of human genomic DNA containing the 5'-flanking sequence of the KIAA1199 gene possesses both suppressive and activating elements. Employing a deletion mutagenesis approach, a 1.4 kb proximal region was defined as the basic KIAA1199 promoter containing a TATA-box close to the transcription start site. A combination of 5'-primer extension study with 5'RACE DNA sequencing analysis revealed one major transcription start site that is utilized in the human KIAA1199 gene. Bioinformatics analysis suggested that the 1.4 kb KIAA1199 promoter contains putative activating regulatory elements, including activator protein-1(AP-1, Twist-1, and NF-κB sites. Sequential deletion and site-direct mutagenesis analysis demonstrated that the AP-1 and distal NF-κB sites are required for KIAA1199 gene expression. Further analyses using an electrophoretic mobility-shift assay and chromatin immunoprecipitation confirmed the requirement of these cis- and trans-acting elements in controlling KIAA1199 gene expression. Finally, we found that upregulated KIAA1199 expression in human breast cancer specimens correlated with hypomethylation of the regulatory region. Involvement of DNA methylation in regulation of KIAA1199 expression was recapitulated in human breast cancer cell lines. Taken together, our study unraveled the regulatory mechanisms controlling KIAA1199 gene expression in human cancer.

  6. WWP2 promotes degradation of transcription factor OCT4 in human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    Huiming Xu; Weicheng Wang; Chunliang Li; Hongyao Yu; Acong Yang; Beibei Wang; Ying Jin

    2009-01-01

    POU transcription factor OCT4 not only plays an essential role in maintaining the pluripotent and self-renewing state of embryonic stem (ES) cells but also acts as a cell fate determinant through a gene dosage effect. However, the molecular mechanisms that control the intracellular OCT4 protein level remain elusive. Here, we report that human WWP2, an E3 ubiquitin (Ub)-protein ligase, interacts with OCT4 specifically through its WW domain and enhances Ub modification of OCT4 both in vitro and in vivo. We first demonstrated that endogenous OCT4 in hu-man ES cells can be post-translationally modified by Ub. Furthermore, we found that WWP2 promoted degradation of OCT4 through the 26S proteasome in a dosage-dependent manner, and the active site cysteine residue of WWP2 was required for both its enzymatic activity and proteolytic effect on OCT4. Remarkably, our data show that the en-dogenous OCT4 protein level was significantly elevated when WWP2 expression was downregulated by specific RNA interference (RNAi), suggesting that WWP2 is an important regulator for maintaining a proper OCT4 protein level in human ES cells. Moreover, northern blot analysis showed that the WWP2 transcript was widely present in diverse human tissues/organs and highly expressed in undifferentiated human ES cells. However, its expression level was quickly decreased after human ES cells differentiated, indicating that WWP2 expression might be developmentally regulated. Our findings demonstrate that WWP2 is an important regulator of the OCT4 protein level in human ES cells.

  7. Three promoters regulate the transcriptional activity of the human holocarboxylase synthetase gene.

    Science.gov (United States)

    Xia, Mengna; Malkaram, Sridhar A; Zempleni, Janos

    2013-11-01

    Holocarboxylase synthetase (HLCS) is the only protein biotin ligase in the human proteome. HLCS-dependent biotinylation of carboxylases plays crucial roles in macronutrient metabolism. HLCS appears to be an essential part of multiprotein complexes in the chromatin that cause gene repression and contribute toward genome stability. Consistent with these essential functions, HLCS knockdown causes strong phenotypes including shortened life span and low stress resistance in Drosophila melanogaster, and de-repression of long-terminal repeats in humans, other mammalian cell lines and Drosophila. Despite previous observations that the expression of HLCS depends on biotin status in rats and in human cell lines, little is known about the regulation of HLCS expression. The goal of this study was to identify promoters that regulate the expression of the human HLCS gene. Initially, the human HLCS locus was interrogated in silico using predictors of promoters including sequences of HLCS mRNA and expressed sequence tags, CpG islands, histone marks denoting transcriptionally poised chromatin, transcription factor binding sites and DNaseI hypersensitive regions. Our predictions revealed three putative HLCS promoters, denoted P1, P2 and P3. Promoters lacked a TATA box, which is typical for housekeeping genes. When the three promoters were cloned into a luciferase reporter plasmid, reporter gene activity was at least three times background noise in human breast, colon and kidney cell lines; activities consistently followed the pattern P1>P3>P2. Promoter activity depended on the concentration of biotin in culture media, but the effect was moderate. We conclude that we have identified promoters in the human HLCS gene.

  8. Transcriptional repression in normal human keratinocytes by wild-type and mutant p53.

    Science.gov (United States)

    Alvarez-Salas, L M; Velazquez, A; Lopez-Bayghen, E; Woodworth, C D; Garrido, E; Gariglio, P; DiPaolo, J A

    1995-05-01

    Wild-type p53 is a nuclear phosphoprotein that inhibits cell proliferation and represses transcriptionally most TATA box-containing promoters in transformed or tumor-derived cell lines. This study demonstrates that p53 alters transcription of the long control region (LCR) of human papillomavirus type 18 (HPV-18). Wild-type and mutant p53 143Val to Ala repressed the HPV-18 LCR promoter in normal human keratinocytes, the natural host cell for HPV infections. Repression by wild-type p53 was also observed in C-33A cells and in an HPV-16-immortalized cell line with an inducible wild-type p53. However, when C-33A cells were cotransfected with the HPV-18 LCR and mutant 143Val to Ala, repression did not occur. Mutant p53 135Cys to Ser did not induce repression in either normal human keratinocytes or in the C-33A line; although like 143Val to Ala, it is thought to affect the DNA binding activity of the wild-type protein. The ability of mutant p53 143Val to Ala to inactivate the HPV early promoter in normal cells (by approximately 60% reduction) suggests that this mutant may be able to associate with wild-type p53 and interact with TATA box-binding proteins. Therefore, these results demonstrate that the transcriptional activities of p53 mutants may be dependent upon the cell type assayed and the form of its endogenous p53. Furthermore, normal human keratinocytes represent an alternative model for determining the activities of p53 mutants.

  9. HBV X PROTEIN (HBX) INTERACTS WITH GENERAL TRANSCRIPTION FACTOR TFIIB BOTH IN VITRO AND IN VIVO

    Institute of Scientific and Technical Information of China (English)

    杨益大; 马亦林; 郑临; 陈亚岗; 马伟杭; 村上清史

    1999-01-01

    Objective. In order to demonstrate the binding ot HBV X protein (HBX) with the general transcription factor TFIIB. Methods. In vitro glutathion S-transferase (GST) resin Pull Down assay and Far-Western Blotting assay, in vivo Co-immunoprecipition assay were used. Results. The X199 (51-99) domain of HBX is reponsibIe for HBX binding to TFIIB. While the dl0 domain (J25-295) of TFIIB is required tor TFIIB binding to HBX. When the two basic amino acids (K) at position 178 and 189 of TFIIB were substituted by neutral amino acids (L), the binding of TFIIBKI78L and KI89L to HBX was siginificantly reduced. When the the basic amino acids were substituted by the acidic amino acids (E), the binding of TFIIB K178E and K189E to HBX were almost lost. In vitro results of HBX binding to TFIIB were further confirmed by in vivo co-immunoprecipitation assay. Our results also indicated that the Woodchuck.hepatitis virus X protein (WHX) interacts with TFIIB. Canclusion. These results suggested that the communication between HBX and general transcription tactor TFIIR is crae of the mechanisms which account for its transeriptional transactivation.

  10. Analysis of the human intestinal epithelial cell transcriptional response to Lactobacillus acidophilus, Lactobacillus salivarius, Bifidobacterium lactis and Escherichia coli

    DEFF Research Database (Denmark)

    Putaala, H; Barrangou, R; Leyer, G J

    2010-01-01

    The complex microbial population residing in the human gastrointestinal tract consists of commensal, potential pathogenic and beneficial species, which are probably perceived differently by the host and consequently could be expected to trigger specific transcriptional responses. Here, we provide...... insights into the relationship between probiotics and human intestinal epithelial cells, notably with regard to strain-specific responses, and highlight the differences between transcriptional responses to pathogenic and probiotic bacteria.......The complex microbial population residing in the human gastrointestinal tract consists of commensal, potential pathogenic and beneficial species, which are probably perceived differently by the host and consequently could be expected to trigger specific transcriptional responses. Here, we provide...... a comparative analysis of the global in vitro transcriptional response of human intestinal epithelial cells to Lactobacillus acidophilus NCFM™, Lactobacillus salivarius Ls-33, Bifidobacterium animalis subsp. lactis 420, and enterohaemorrhagic Escherichia coli O157:H7 (EHEC). Interestingly, L. salivarius Ls-33...

  11. Substrate availability and transcriptional regulation of metabolic genes in human skeletal muscle during recovery from exercise.

    Science.gov (United States)

    Pilegaard, Henriette; Osada, Takuya; Andersen, Lisbeth T; Helge, Jørn W; Saltin, Bengt; Neufer, P Darrell

    2005-08-01

    In skeletal muscle of humans, transcription of several metabolic genes is transiently induced during recovery from exercise when no food is consumed. To determine the potential influence of substrate availability on the transcriptional regulation of metabolic genes during recovery from exercise, 9 male subjects (aged 22-27) completed 75 minutes of cycling exercise at 75% Vo2 max on 2 occasions, consuming either a high-carbohydrate (HC) or low-carbohydrate (LC) diet during the subsequent 24 hours of recovery. Nuclei were isolated and tissue frozen from vastus lateralis muscle biopsies obtained before exercise and 2, 5, 8, and 24 hours after exercise. Muscle glycogen was restored to near resting levels within 5 hours in the HC trial, but remained depressed through 24 hours in the LC trial. During the 2- to 8-hour recovery period, leg glucose uptake was 5- to 15-fold higher with HC ingestion, whereas arterial plasma free fatty acid levels were approximately 3- to 7-fold higher with LC ingestion. Exercise increased (P < .05) transcription and/or mRNA content of the pyruvate dehydrogenase kinase 4, uncoupling protein 3, lipoprotein lipase, carnitine palmitoyltransferase I, hexokinase II, peroxisome proliferator activated receptor gamma coactivator-1 alpha, and peroxisome proliferator activated receptor alpha. Providing HC during recovery reversed the activation of pyruvate dehydrogenase kinase 4, uncoupling protein 3, lipoprotein lipase, and carnitine palmitoyltransferase I within 5 to 8 hours after exercise, whereas providing LC during recovery elicited a sustained/enhanced increase in activation of these genes through 8 to 24 hours of recovery. These findings provide evidence that factors associated with substrate availability and/or cellular metabolic recovery (eg, muscle glycogen restoration) influence the transcriptional regulation of metabolic genes in skeletal muscle of humans during recovery from exercise.

  12. The Promyelocytic Leukemia Zinc Finger Transcription Factor Is Critical for Human Endometrial Stromal Cell Decidualization.

    Directory of Open Access Journals (Sweden)

    Ramakrishna Kommagani

    2016-04-01

    Full Text Available Progesterone, via the progesterone receptor (PGR, is essential for endometrial stromal cell decidualization, a cellular transformation event in which stromal fibroblasts differentiate into decidual cells. Uterine decidualization supports embryo implantation and placentation as well as subsequent events, which together ensure a successful pregnancy. Accordingly, impaired decidualization results not only in implantation failure or early fetal miscarriage, but also may lead to potential adverse outcomes in all three pregnancy trimesters. Transcriptional reprogramming on a genome-wide scale underlies progesterone dependent decidualization of the human endometrial stromal cell (hESC. However, identification of the functionally essential signals encoded by these global transcriptional changes remains incomplete. Importantly, this knowledge-gap undercuts future efforts to improve diagnosis and treatment of implantation failure based on a dysfunctional endometrium. By integrating genome-wide datasets derived from decidualization of hESCs in culture, we reveal that the promyelocytic leukemia zinc finger (PLZF transcription factor is rapidly induced by progesterone and that this induction is indispensable for progesterone-dependent decidualization. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq identified at least ten progesterone response elements within the PLZF gene, indicating that PLZF may act as a direct target of PGR signaling. The spatiotemporal expression profile for PLZF in both the human and mouse endometrium offers further support for stromal PLZF as a mediator of the progesterone decidual signal. To identify functional targets of PLZF, integration of PLZF ChIP-Seq and RNA Pol II RNA-Seq datasets revealed that the early growth response 1 (EGR1 transcription factor is a PLZF target for which its level of expression must be reduced to enable progesterone dependent hESC decidualization. Apart from furnishing

  13. Interferon-λ4 (IFNL4 transcript expression in human liver tissue samples.

    Directory of Open Access Journals (Sweden)

    Ahmad Amanzada

    Full Text Available Eradication of hepatitis C virus (HCV infection, both spontaneous and treatment-induced, is marked by the wildtype allele C of a single nucleotide polymorphism upstream of the IL28B gene, rs12979860. This favorable allele was recently described to be in linkage disequilibrium with the wildtype allele TT of a dinucleotide polymorphism, ss469415590, located within a new protein-coding gene. While the TT allele introduces a frame-shift and disrupts the open reading frame, only the variant allele, ΔG, creates a novel type III interferon (IFN protein, IFN-λ4/IFNL4. Absence of IFNL4 is thus supposed to favor resolution of HCV infection. As to date IFNL4 mRNA transcription has only been investigated in polyI:C-stimulated primary human hepatocytes and not yet in HCV infection in vivo, this study analyzed IFNL4 mRNA expression in human liver biopsy specimens. Samples were obtained from patients with a broad panel of disorders including no liver disease, liver diseases of non-viral etiology, chronic hepatitis B and chronic hepatitis C. Hepatic IFNL4 transcripts were detectable exclusively in a subgroup of chronic hepatitis C patients (24/45. Their amounts were positively related to liver HCV RNA copy numbers (p = 0.0023, r = 0.56 suggesting that the hepatic viral load influences IFNL4 transcription irrespective of IFNL4 governing genotype. Both, the IFNL4 creating allele ΔG (p<0.0001 and actual IFNL4 transcription (p = 0.0015 were found to be correlated to the activation of IFN stimulatory genes (ISGs. By contrast, IFNL4 ss469415590 genotypes were not found to be related to IFN-λ2/3/IL28 or IFN-λ1/IL29 gene expression. In conclusion, this study is the first report on intrahepatic transcript levels of the recently discovered IFNL4 gene. Data indicate that HCV infection in particular might activate IFNL4 transcription in the liver. It provides a possible explanation as to why hepatitis C patients show ISG stimulation in their livers in the

  14. LncRNA profiling of human lymphoid progenitors reveals transcriptional divergence of B and T lineages

    Science.gov (United States)

    Casero, David; Sandoval, Salemiz; Seet, Christopher S.; Scholes, Jessica; Zhu, Yuhua; Ha, Vi Luan; Luong, Annie; Parekh, Chintan; Crooks, Gay M.

    2015-01-01

    To elucidate the transcriptional landscape that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitors spanning the earliest stages of B and T lymphoid specification. Over 3000 novel long non-coding RNA genes (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage-specific and more lineage-specific than protein coding patterns. Protein-coding genes co-expressed with neighboring lncRNA genes were enriched for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships between the earliest progenitors in the human bone marrow and thymus. PMID:26502406

  15. Aging affects the transcriptional regulation of human skeletal muscle disuse atrophy

    DEFF Research Database (Denmark)

    Suetta, Charlotte; Frandsen, Ulrik; Nielsen, Line;

    2012-01-01

    Important insights concerning the molecular basis of skeletal muscle disuse-atrophy and aging related muscle loss have been obtained in cell culture and animal models, but these regulatory signaling pathways have not previously been studied in aging human muscle. In the present study, muscle...... atrophy was induced by immobilization in healthy old and young individuals to study the time-course and transcriptional factors underlying human skeletal muscle atrophy. The results reveal that irrespectively of age, mRNA expression levels of MuRF-1 and Atrogin-1 increased in the very initial phase (2......-4 days) of human disuse-muscle atrophy along with a marked reduction in PGC-1a and PGC-1ß (1-4 days) and a ~10% decrease in myofiber size (4 days). Further, an age-specific decrease in Akt and S6 phosphorylation was observed in young muscle within the first days (1-4 days) of immobilization. In contrast...

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

    Directory of Open Access Journals (Sweden)

    Kim Seon-Young

    2006-07-01

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

  17. Identification of the transcriptional promoters in the proximal regions of human microRNA genes.

    Science.gov (United States)

    Long, Yue-Sheng; Deng, Guang-Fei; Sun, Xun-Sha; Yi, Yong-Hong; Su, Tao; Zhao, Qi-Hua; Liao, Wei-Ping

    2011-08-01

    To identify the transcriptional promoters in the proximal regions of human microRNA (miRNA) genes, we analyzed the 5' flanking regions of intergenic miRNAs and intronic miRNAs. With the TSSG program prediction, we found that the ratio of intronic-s miRNA genes with a least one promoter was significantly lower than those of intergenic miRNA genes and intronic-a miRNA genes. More than half of the miRNA genes have only one promoter and less than 20% of the miRNA genes have more than three promoters in the 5-kb upstream regions. All potential promoters are randomly distributed within these regions. Approximately 60% of the miRNA promoters have a TATA-like box, being significantly higher than that of all human promoters. Luciferase reporter assays showed that 22 of the 30 promoters drove gene expression in HEK-293 cells, indicating a high accuracy of the promoter prediction. This study lays a foundation for future investigation into the transcriptional regulatory mechanisms of human miRNA genes.

  18. Asian dust storm particles induce a broad toxicological transcriptional program in human epidermal keratinocytes.

    Science.gov (United States)

    Choi, Hyun; Shin, Dong Wook; Kim, Wonnyon; Doh, Seong-Jae; Lee, Soo Hwan; Noh, Minsoo

    2011-01-15

    Exposure to airborne dust particles originated from seasonal Asian dust storms in Chinese and Mongolian deserts results in increased incidence of a range of diseases including asthma, contact dermatitis and conjunctivitis. The areas affected by Asian dust particles extend from East China to the west coast of North America. In order to study toxicological mechanisms in human skin, we evaluated the effects of dust particles collected during Asian dust storms (Asian dust particles) on gene expression in human epidermal keratinocytes (HEK). In HEK, exposure to Asian dust particles significantly increased gene expressions of cytochrome P450 1A1 (CYP1A1), CYP1A2, and CYP1B1, which is an indication of aryl hydrocarbon receptor (AHR) activation. In addition, Asian dust particles increased gene transcription of the cytokines IL-6, IL-8, and GM-CSF, which have broad pro-inflammatory and immunomodulatory properties. Asian dust particles significantly up-regulated expression of caspase 14 in HEK, suggesting that Asian dust particles directly affect keratinocyte differentiation. We also demonstrated that protein extract of pollen, a material frequently adsorbed onto Asian dust particles, potentially contributes to the increased transcription of IL-6, CYP1A1, CYP1A2, and CYP1B1. Taken together, these studies suggest that Asian dust particles can exert toxicological effects on human skin through the activation of the cellular detoxification system, the production of pro-inflammatory and immunomodulatory cytokines, and changes in the expression of proteins essential in normal epidermal differentiation.

  19. A novel transcriptional regulator of L-arabinose utilization in human gut bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Changsoo; Tesar, Christine; Li, Xiaoqing; Kim, Youngchang; Rodionov, Dmitry A.; Joachimiak, Andrzej

    2015-12-02

    Carbohydrate metabolism plays a crucial role in the ecophysiology of human gut microbiota. Mechanisms of transcriptional regulation of sugar catabolism in commensal and prevalent human gut bacteria such as Bacteroides thetaiotaomicron remain mostly unknown. By a combination of bioinformatics and experimental approaches, we have identified an NrtR family transcription factor (BT0354 in B. thetaiotaomicron, BtAraR) as a novel regulator controlling the arabinose utilization genes. L-arabinose was confirmed to be a negative effector of BtAraR. We have solved the crystal structures of the apo and L-arabinose-bound BtAraR proteins, as well as the complex of apo-protein with a specific DNA operator. BtAraR forms a homodimer with each subunit comprised of the ligand-binding Nudix hydrolase-like domain and the DNA-binding winged-helix-turn-helix (wHTH) domain. We have identified the residues involved in binding of L-arabinose and recognition of DNA. The majority of these residues are well conserved in the AraR orthologs in Bacteroidetes. In the structure of the BtAraR-DNA complex, we found the unique interaction of arginine intercalating its guanidinum moiety into the base pair stacking of B-DNA. L-arabinose binding induces movement of wHTH domains, resulting in a conformation unsuitable for DNA binding. Our analysis facilitates reconstruction of the metabolic and regulatory networks involved in carbohydrate utilization in human gut Bacteroides.

  20. YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors.

    Science.gov (United States)

    Marti, Patricia; Stein, Claudia; Blumer, Tanja; Abraham, Yann; Dill, Michael T; Pikiolek, Monika; Orsini, Vanessa; Jurisic, Giorgia; Megel, Philippe; Makowska, Zuzanna; Agarinis, Claudia; Tornillo, Luigi; Bouwmeester, Tewis; Ruffner, Heinz; Bauer, Andreas; Parker, Christian N; Schmelzle, Tobias; Terracciano, Luigi M; Heim, Markus H; Tchorz, Jan S

    2015-11-01

    The Yes-associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down-regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug-induced apoptosis. We further show that the proangiogenic microfibrillar-associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31(+) vasculature. These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins. © 2015 by the American Association for the Study of Liver Diseases.

  1. Characterization of human constitutive photomorphogenesis protein 1, a RING finger ubiquitin ligase that interacts with Jun transcription factors and modulates their transcriptional activity.

    Science.gov (United States)

    Bianchi, Elisabetta; Denti, Simona; Catena, Raffaella; Rossetti, Grazisa; Polo, Simona; Gasparian, Sona; Putignano, Stella; Rogge, Lars; Pardi, Ruggero

    2003-05-30

    RING finger proteins have been implicated in many fundamental cellular processes, including the control of gene expression. A key regulator of light-dependent development in Arabidopsis thaliana is the constitutive photomorphogenesis protein 1 (atCOP1), a RING finger protein that plays an essential role in translating light/dark signals into specific changes in gene transcription. atCOP1 binds the basic leucine zipper factor HY5 and suppresses its transcriptional activity through a yet undefined mechanism that results in HY5 degradation in response to darkness. Furthermore, the pleiotropic phenotype of atCOP1 mutants indicates that atCOP1 may be a central regulator of several transcriptional pathways. Here we report the cloning and characterization of the human orthologue of atCOP1. Human COP1 (huCOP1) distributes both to the cytoplasm and the nucleus of cells and shows a striking degree of sequence conservation with atCOP1, suggesting the possibility of a functional conservation as well. In co-immunoprecipitation assays huCOP1 specifically binds basic leucine zipper factors of the Jun family. As a functional consequence of this interaction, expression of huCOP1 in mammalian cells down-regulates c-Jun-dependent transcription and the expression of the AP-1 target genes, urokinase and matrix metalloproteinase 1. The RING domain of huCOP1 displays ubiquitin ligase activity in an autoubiquitination assay in vitro; however, suppression of AP-1-dependent transcription by huCOP1 occurs in the absence of changes in c-Jun protein levels, suggesting that this inhibitory effect is independent of c-Jun degradation. Our findings indicate that huCOP1 is a novel regulator of AP-1-dependent transcription sharing the important properties of Arabidopsis COP1 in the control of gene expression.

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

    Science.gov (United States)

    Strakova, Zuzana; Reed, Jennifer; Ihnatovych, Ivanna

    2010-06-01

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

  3. Identification of transcription factors regulating CTNNAL1 expression in human bronchial epithelial cells.

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    Yang Xiang

    Full Text Available Adhesion molecules play important roles in airway hyperresponsiveness or airway inflammation. Our previous study indicated catenin alpha-like 1 (CTNNAL1, an alpha-catenin-related protein, was downregulated in asthma patients and animal model. In this study, we observed that the expression of CTNNAL1 was increased in lung tissue of the ozone-stressed Balb/c mice model and in acute ozone stressed human bronchial epithelial cells (HBEC. In order to identify the possible DNA-binding proteins regulating the transcription of CTNNAL1 gene in HBEC, we designed 8 oligo- nucleotide probes corresponding to various regions of the CTNNAL1 promoter in electrophoretic mobility shift assays (EMSA. We detected 5 putative transcription factors binding sites within CTNNAL1 promoter region that can recruit LEF-1, AP-2α and CREB respectively by EMSA and antibody supershift assay. Chromatin immunoprecipitation (ChIP assay verified that AP-2 α and LEF-1 could be recruited to the CTNNAL1 promoter. Therefore we further analyzed the functions of putative AP-2 and LEF-1 sites within CTNNAL1 promoter by site-directed mutagenesis of those sites within pGL3/FR/luc. We observed a reduction in human CTNNAL1 promoter activity of mutants of both AP-2α and LEF-1 sites. Pre-treatment with ASOs targeting LEF-1and AP-2α yielded significant reduction of ozone-stress-induced CTNNAL1 expression. The activation of AP-2α and LEF-1, followed by CTNNAL1 expression, showed a correlation during a 16-hour time course. Our data suggest that a robust transcriptional CTNNAL1 up-regulation occurs during acute ozone-induced stress and is mediated at least in part by ozone-induced recruitments of LEF-1 and AP-2α to the human CTNNAL1 promoter.

  4. SREBP-2 negatively regulates FXR-dependent transcription of FGF19 in human intestinal cells.

    Science.gov (United States)

    Miyata, Masaaki; Hata, Tatsuya; Yamazoe, Yasushi; Yoshinari, Kouichi

    2014-01-10

    Sterol regulatory element-binding protein-2 (SREBP-2) is a basic helix-loop-helix-leucine zipper transcription factor that positively regulates transcription of target genes involved in cholesterol metabolism. In the present study, we have investigated a possible involvement of SREBP-2 in human intestinal expression of fibroblast growth factor (FGF)19, which is an endocrine hormone involved in the regulation of lipid and glucose metabolism. Overexpression of constitutively active SREBP-2 decreased FGF19 mRNA levels in human colon-derived LS174T cells. In reporter assays, active SREBP-2 overexpression suppressed GW4064/FXR-mediated increase in reporter activities in regions containing the IR-1 motif (+848 to +5200) in the FGF19 gene. The suppressive effect disappeared in reporter activities in the region containing the IR-1 motif when the mutation was introduced into the IR-1 motif. In electrophoretic mobility shift assays, binding of the FXR/retinoid X receptor α heterodimer to the IR-1 motif was attenuated by adding active SREBP-2, but SREBP-2 binding to the IR-1 motif was not observed. In chromatin immunoprecipitation assays, specific binding of FXR to the IR-1-containing region of the FGF19 gene (+3214 to +3404) was increased in LS174T cells by treatment with cholesterol and 25-hydroxycholesterol. Specific binding of SREBP-2 to FXR was observed in glutathione-S-transferase (GST) pull-down assays. These results suggest that SREBP-2 negatively regulates the FXR-mediated transcriptional activation of the FGF19 gene in human intestinal cells.

  5. Transcriptional Factor PU.1 Regulates Decidual C1q Expression in Early Pregnancy in Human.

    Science.gov (United States)

    Madhukaran, Shanmuga Priyaa; Kishore, Uday; Jamil, Kaiser; Teo, Boon Heng Dennis; Choolani, Mahesh; Lu, Jinhua

    2015-01-01

    C1q is the first recognition subcomponent of the complement classical pathway, which in addition to being synthesized in the liver, is also expressed by macrophages and dendritic cells (DCs). Trophoblast invasion during early placentation results in accumulation of debris that triggers the complement system. Hence, both early and late components of the classical pathway are widely distributed in the placenta and decidua. In addition, C1q has recently been shown to significantly contribute to feto-maternal tolerance, trophoblast migration, and spiral artery remodeling, although the exact mechanism remains unknown. Pregnancy in mice, genetically deficient in C1q, mirrors symptoms similar to that of human preeclampsia. Thus, regulated complement activation has been proposed as an essential requirement for normal successful pregnancy. Little is known about the molecular pathways that regulate C1q expression in pregnancy. PU.1, an Ets-family transcription factor, is required for the development of hematopoietic myeloid lineage immune cells, and its expression is tissue-specific. Recently, PU.1 has been shown to regulate C1q gene expression in DCs and macrophages. Here, we have examined if PU.1 transcription factor regulates decidual C1q expression. We used immune-histochemical analysis, PCR, and immunostaining to localize and study the gene expression of PU.1 transcription factor in early human decidua. PU.1 was highly expressed at gene and protein level in early human decidual cells including trophoblast and stromal cells. Surprisingly, nuclear as well as cytoplasmic PU.1 expression was observed. Decidual cells with predominantly nuclear PU.1 expression had higher C1q expression. It is likely that nuclear and cytoplasmic PU.1 localization has a role to play in early pregnancy via regulating C1q expression in the decidua during implantation.

  6. Identification of the Imprinted KLF14 Transcription Factor Undergoing Human-Specific Accelerated Evolution

    OpenAIRE

    Layla Parker-Katiraee; Carson, Andrew R.; Takahiro Yamada; Philippe Arnaud; Robert Feil; Abu-Amero, Sayeda N.; Moore, Gudrun E; Masahiro Kaneda; Perry, George H.; Stone, Anne C.; Charles Lee; Makiko Meguro-Horike; Hiroyuki Sasaki; Keiko Kobayashi; Kazuhiko Nakabayashi

    2007-01-01

    Imprinted genes are expressed in a parent-of-origin manner and are located in clusters throughout the genome. Aberrations in the expression of imprinted genes on human Chromosome 7 have been suggested to play a role in the etiologies of Russell-Silver Syndrome and autism. We describe the imprinting of KLF14, an intronless member of the Krüppel-like family of transcription factors located at Chromosome 7q32. We show that it has monoallelic maternal expression in all embryonic and extra-embryon...

  7. Identification of the imprinted KLF14 transcription factor undergoing human-specific accelerated evolution

    OpenAIRE

    Parker-Katiraee, L.; Carson, A.R.; Yamada, T; Meguro-Horike, M.; Nakabayashi, K.; Scherer, S.W.; Arnaud, P.; Feil, R; Abu-Amero, S. N.; Moore, G.E.; Kaneda, M.; Sasaki, H.; Perry, G. H.; Stone, A C; Lee, C

    2007-01-01

    Imprinted genes are expressed in a parent-of-origin manner and are located in clusters throughout the genome. Aberrations in the expression of imprinted genes on human Chromosome 7 have been suggested to play a role in the etiologies of Russell-Silver Syndrome and autism. We describe the imprinting of KLF14, an intronless member of the Krüppel-like family of transcription factors located at Chromosome 7q32. We show that it has monoallelic maternal expression in all embryonic and extra-embryon...

  8. Evidence for evolutionary and nonevolutionary forces shaping the distribution of human genetic variants near transcription start sites.

    Science.gov (United States)

    Scala, Giovanni; Affinito, Ornella; Miele, Gennaro; Monticelli, Antonella; Cocozza, Sergio

    2014-01-01

    The regions surrounding transcription start sites (TSSs) of genes play a critical role in the regulation of gene expression. At the same time, current evidence indicates that these regions are particularly stressed by transcription-related mutagenic phenomena. In this work we performed a genome-wide analysis of the distribution of single nucleotide polymorphisms (SNPs) inside the 10 kb region flanking human TSSs by dividing SNPs into four classes according to their frequency (rare, two intermediate classes, and common). We found that, in this 10 kb region, the distribution of variants depends on their frequency and on their localization relative to the TSS. We found that the distribution of variants is generally different for TSSs located inside or outside of CpG islands. We found a significant relationship between the distribution of rare variants and nucleosome occupancy scores. Furthermore, our analysis suggests that evolutionary (purifying selection) and nonevolutionary (biased gene conversion) forces both play a role in determining the relative SNP frequency around TSSs. Finally, we analyzed the potential pathogenicity of each class of variant using the Combined Annotation Dependent Depletion score. In conclusion, this study provides a novel and detailed view of the distribution of genomic variants around TSSs, providing insight into the forces that instigate and maintain variability in such critical regions.

  9. A systems biological approach to identify key transcription factors and their genomic neighborhoods in human sarcomas

    Institute of Scientific and Technical Information of China (English)

    Antti Ylip(a)(a); Olli Yli-Harja; Wei Zhang; Matti Nykter

    2011-01-01

    Identification of genetic signatures is the main objective for many computational oncology studies. The signature usually consists of numerous genes that are differentially expressed between two clinically distinct groups of samples, such as tumor subtypes. Prospectively, many signatures have been found to generalize poorly to other datasets and, thus, have rarely been accepted into clinical use. Recognizing the limited success of traditionally generated signatures, we developed a systems biology-based framework for robust identification of key transcription factors and their genomic regulatory neighborhoods. Application of the framework to study the differences between gastrointestinal stromal tumor (GIST) and leiomyosarcoma (LMS) resulted in the identification of nine transcription factors (SRF, NKX2-5, CCDC6, LEF1, VDR, ZNF250, TRIM63, MAF, and MYC). Functional annotations of the obtained neighborhoods identified the biological processes which the key transcription factors regulate differently between the tumor types. Analyzing the differences in the expression patterns using our approach resulted in a more robust genetic signature and more biological insight into the diseases compared to a traditional genetic signature.

  10. The Transcription and Translation Landscapes during Human Cytomegalovirus Infection Reveal Novel Host-Pathogen Interactions.

    Science.gov (United States)

    Tirosh, Osnat; Cohen, Yifat; Shitrit, Alina; Shani, Odem; Le-Trilling, Vu Thuy Khanh; Trilling, Mirko; Friedlander, Gilgi; Tanenbaum, Marvin; Stern-Ginossar, Noam

    2015-01-01

    Viruses are by definition fully dependent on the cellular translation machinery, and develop diverse mechanisms to co-opt this machinery for their own benefit. Unlike many viruses, human cytomegalovirus (HCMV) does suppress the host translation machinery, and the extent to which translation machinery contributes to the overall pattern of viral replication and pathogenesis remains elusive. Here, we combine RNA sequencing and ribosomal profiling analyses to systematically address this question. By simultaneously examining the changes in transcription and translation along HCMV infection, we uncover extensive transcriptional control that dominates the response to infection, but also diverse and dynamic translational regulation for subsets of host genes. We were also able to show that, at late time points in infection, translation of viral mRNAs is higher than that of cellular mRNAs. Lastly, integration of our translation measurements with recent measurements of protein abundance enabled comprehensive identification of dozens of host proteins that are targeted for degradation during HCMV infection. Since targeted degradation indicates a strong biological importance, this approach should be applicable for discovering central host functions during viral infection. Our work provides a framework for studying the contribution of transcription, translation and degradation during infection with any virus.

  11. The Transcription and Translation Landscapes during Human Cytomegalovirus Infection Reveal Novel Host-Pathogen Interactions.

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    Osnat Tirosh

    Full Text Available Viruses are by definition fully dependent on the cellular translation machinery, and develop diverse mechanisms to co-opt this machinery for their own benefit. Unlike many viruses, human cytomegalovirus (HCMV does suppress the host translation machinery, and the extent to which translation machinery contributes to the overall pattern of viral replication and pathogenesis remains elusive. Here, we combine RNA sequencing and ribosomal profiling analyses to systematically address this question. By simultaneously examining the changes in transcription and translation along HCMV infection, we uncover extensive transcriptional control that dominates the response to infection, but also diverse and dynamic translational regulation for subsets of host genes. We were also able to show that, at late time points in infection, translation of viral mRNAs is higher than that of cellular mRNAs. Lastly, integration of our translation measurements with recent measurements of protein abundance enabled comprehensive identification of dozens of host proteins that are targeted for degradation during HCMV infection. Since targeted degradation indicates a strong biological importance, this approach should be applicable for discovering central host functions during viral infection. Our work provides a framework for studying the contribution of transcription, translation and degradation during infection with any virus.

  12. Transcription Regulation of the Human Telomerase Reverse Transcriptase (hTERT Gene

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    Muhammad Khairul Ramlee

    2016-08-01

    Full Text Available Embryonic stem cells and induced pluripotent stem cells have the ability to maintain their telomere length via expression of an enzymatic complex called telomerase. Similarly, more than 85%–90% of cancer cells are found to upregulate the expression of telomerase, conferring them with the potential to proliferate indefinitely. Telomerase Reverse Transcriptase (TERT, the catalytic subunit of telomerase holoenzyme, is the rate-limiting factor in reconstituting telomerase activity in vivo. To date, the expression and function of the human Telomerase Reverse Transcriptase (hTERT gene are known to be regulated at various molecular levels (including genetic, mRNA, protein and subcellular localization by a number of diverse factors. Among these means of regulation, transcription modulation is the most important, as evident in its tight regulation in cancer cell survival as well as pluripotent stem cell maintenance and differentiation. Here, we discuss how hTERT gene transcription is regulated, mainly focusing on the contribution of trans-acting factors such as transcription factors and epigenetic modifiers, as well as genetic alterations in hTERT proximal promoter.

  13. General anesthesia suppresses normal heart rate variability in humans

    Science.gov (United States)

    Matchett, Gerald; Wood, Philip

    2014-06-01

    The human heart normally exhibits robust beat-to-beat heart rate variability (HRV). The loss of this variability is associated with pathology, including disease states such as congestive heart failure (CHF). The effect of general anesthesia on intrinsic HRV is unknown. In this prospective, observational study we enrolled 100 human subjects having elective major surgical procedures under general anesthesia. We recorded continuous heart rate data via continuous electrocardiogram before, during, and after anesthesia, and we assessed HRV of the R-R intervals. We assessed HRV using several common metrics including Detrended Fluctuation Analysis (DFA), Multifractal Analysis, and Multiscale Entropy Analysis. Each of these analyses was done in each of the four clinical phases for each study subject over the course of 24 h: Before anesthesia, during anesthesia, early recovery, and late recovery. On average, we observed a loss of variability on the aforementioned metrics that appeared to correspond to the state of general anesthesia. Following the conclusion of anesthesia, most study subjects appeared to regain their normal HRV, although this did not occur immediately. The resumption of normal HRV was especially delayed on DFA. Qualitatively, the reduction in HRV under anesthesia appears similar to the reduction in HRV observed in CHF. These observations will need to be validated in future studies, and the broader clinical implications of these observations, if any, are unknown.

  14. The general transcriptional repressor Tup1 is required for dimorphism and virulence in a fungal plant pathogen.

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    Alberto Elías-Villalobos

    2011-09-01

    Full Text Available A critical step in the life cycle of many fungal pathogens is the transition between yeast-like growth and the formation of filamentous structures, a process known as dimorphism. This morphological shift, typically triggered by multiple environmental signals, is tightly controlled by complex genetic pathways to ensure successful pathogenic development. In animal pathogenic fungi, one of the best known regulators of dimorphism is the general transcriptional repressor, Tup1. However, the role of Tup1 in fungal dimorphism is completely unknown in plant pathogens. Here we show that Tup1 plays a key role in orchestrating the yeast to hypha transition in the maize pathogen Ustilago maydis. Deletion of the tup1 gene causes a drastic reduction in the mating and filamentation capacity of the fungus, in turn leading to a reduced virulence phenotype. In U. maydis, these processes are controlled by the a and b mating-type loci, whose expression depends on the Prf1 transcription factor. Interestingly, Δtup1 strains show a critical reduction in the expression of prf1 and that of Prf1 target genes at both loci. Moreover, we observed that Tup1 appears to regulate Prf1 activity by controlling the expression of the prf1 transcriptional activators, rop1 and hap2. Additionally, we describe a putative novel prf1 repressor, named Pac2, which seems to be an important target of Tup1 in the control of dimorphism and virulence. Furthermore, we show that Tup1 is required for full pathogenic development since tup1 deletion mutants are unable to complete the sexual cycle. Our findings establish Tup1 as a key factor coordinating dimorphism in the phytopathogen U. maydis and support a conserved role for Tup1 in the control of hypha-specific genes among animal and plant fungal pathogens.

  15. Transcriptional and post-transcriptional control of DNA methyltransferase 3B is regulated by phosphatidylinositol 3 kinase/Akt pathway in human hepatocellular carcinoma cell lines.

    Science.gov (United States)

    Mei, Chuanzhong; Sun, Lidong; Liu, Yonglei; Yang, Yong; Cai, Xiumei; Liu, Mingzhu; Yao, Wantong; Wang, Can; Li, Xin; Wang, Liying; Li, Zengxia; Shi, Yinghong; Qiu, Shuangjian; Fan, Jia; Zha, Xiliang

    2010-09-01

    DNA methyltransferases (DNMTs) are essential for maintenance of aberrant methylation in cancer cells and play important roles in the development of cancers. Unregulated activation of PI3K/Akt pathway is a prominent feature of many human cancers including human hepatocellular carcinoma (HCC). In present study, we found that DNMT3B mRNA and protein levels were decreased in a dose- and time-dependent manner in HCC cell lines with LY294002 treatment. However, we detected that LY294002 treatment did not induce increase of the degradation of DNMT3B protein using protein decay assay. Moreover we found that Akt induced alteration of the expression of DNMT3B in cells transfected with myristylated variants of Akt2 or cells transfected with small interfering RNA respectively. Based on DNMT3B promoter dual-luciferase reporter assay, we found PI3K pathway regulates DNMT3B expression at transcriptional level. And DNMT3B mRNA decay analysis suggested that down-regulation of DNMT3B by LY294002 is also post-transcriptional control. Furthermore, we demonstrated that LY294002 down-regulated HuR expression in a time-dependent manner in BEL-7404. In summary, we have, for the first time, demonstrate that PI3K/Akt pathway regulates the expression of DNMT3B at transcriptional and post-transcriptional levels, which is particularly important to understand the effects of PI3K/Akt and DNMT3B on hepatocarcinogenesis.

  16. An atlas of transcriptional, chromatin accessibility, and surface marker changes in human mesoderm development.

    Science.gov (United States)

    Koh, Pang Wei; Sinha, Rahul; Barkal, Amira A; Morganti, Rachel M; Chen, Angela; Weissman, Irving L; Ang, Lay Teng; Kundaje, Anshul; Loh, Kyle M

    2016-12-20

    Mesoderm is the developmental precursor to myriad human tissues including bone, heart, and skeletal muscle. Unravelling the molecular events through which these lineages become diversified from one another is integral to developmental biology and understanding changes in cellular fate. To this end, we developed an in vitro system to differentiate human pluripotent stem cells through primitive streak intermediates into paraxial mesoderm and its derivatives (somites, sclerotome, dermomyotome) and separately, into lateral mesoderm and its derivatives (cardiac mesoderm). Whole-population and single-cell analyses of these purified populations of human mesoderm lineages through RNA-seq, ATAC-seq, and high-throughput surface marker screens illustrated how transcriptional changes co-occur with changes in open chromatin and surface marker landscapes throughout human mesoderm development. This molecular atlas will facilitate study of human mesoderm development (which cannot be interrogated in vivo due to restrictions on human embryo studies) and provides a broad resource for the study of gene regulation in development at the single-cell level, knowledge that might one day be exploited for regenerative medicine.

  17. DDX11L: a novel transcript family emerging from human subtelomeric regions

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    D'Urso Michele

    2009-05-01

    Full Text Available Abstract Background The subtelomeric regions of human chromosomes exhibit an extraordinary plasticity. To date, due to the high GC content and to the presence of telomeric repeats, the subtelomeric sequences are underrepresented in the genomic libraries and consequently their sequences are incomplete in the finished human genome sequence, and still much remains to be learned about subtelomere organization, evolution and function. Indeed, only in recent years, several studies have disclosed, within human subtelomeres, novel gene family members. Results During a project aimed to analyze genes located in the telomeric region of the long arm of the human X chromosome, we have identified a novel transcript family, DDX11L, members of which map to 1pter, 2q13/14.1, 2qter, 3qter, 6pter, 9pter/9qter, 11pter, 12pter, 15qter, 16pter, 17pter, 19pter, 20pter/20qter, Xpter/Xqter and Yqter. Furthermore, we partially sequenced the underrepresented subtelomeres of human chromosomes showing a common evolutionary origin. Conclusion Our data indicate that an ancestral gene, originated as a rearranged portion of the primate DDX11 gene, and propagated along many subtelomeric locations, is emerging within subtelomeres of human chromosomes, defining a novel gene family. These findings support the possibility that the high plasticity of these regions, sites of DNA exchange among different chromosomes, could trigger the emergence of new genes.

  18. UVB-induced cell death signaling is associated with G1-S progression and transcription inhibition in primary human fibroblasts.

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    Tatiana Grohmann Ortolan

    Full Text Available DNA damage induced by ultraviolet (UV radiation can be removed by nucleotide excision repair through two sub-pathways, one general (GGR and the other specific for transcribed DNA (TCR, and the processing of unrepaired lesions trigger signals that may lead to cell death. These signals involve the tumor suppressor p53 protein, a central regulator of cell responses to DNA damage, and the E3 ubiquitin ligase Mdm2, that forms a feedback regulatory loop with p53. The involvement of cell cycle and transcription on the signaling to apoptosis was investigated in UVB-irradiated synchronized, DNA repair proficient, CS-B (TCR-deficient and XP-C (GGR-deficient primary human fibroblasts. Cells were irradiated in the G1 phase of the cell cycle, with two doses with equivalent levels of apoptosis (low and high, defined for each cell line. In the three cell lines, the low doses of UVB caused only a transient delay in progression to the S phase, whereas the high doses induced permanent cell cycle arrest. However, while accumulation of Mdm2 correlated well with the recovery from transcription inhibition at the low doses for normal and CS-B fibroblasts, for XP-C cells this protein was shown to be accumulated even at UVB doses that induced high levels of apoptosis. Thus, UVB-induced accumulation of Mdm2 is critical for counteracting p53 activation and apoptosis avoidance, but its effect is limited due to transcription inhibition. However, in the case of XP-C cells, an excess of unrepaired DNA damage would be sufficient to block S phase progression, which would signal to apoptosis, independent of Mdm2 accumulation. The data clearly discriminate DNA damage signals that lead to cell death, depending on the presence of UVB-induced DNA damage in replicating or transcribing regions.

  19. Identification of the imprinted KLF14 transcription factor undergoing human-specific accelerated evolution.

    Science.gov (United States)

    Parker-Katiraee, Layla; Carson, Andrew R; Yamada, Takahiro; Arnaud, Philippe; Feil, Robert; Abu-Amero, Sayeda N; Moore, Gudrun E; Kaneda, Masahiro; Perry, George H; Stone, Anne C; Lee, Charles; Meguro-Horike, Makiko; Sasaki, Hiroyuki; Kobayashi, Keiko; Nakabayashi, Kazuhiko; Scherer, Stephen W

    2007-05-04

    Imprinted genes are expressed in a parent-of-origin manner and are located in clusters throughout the genome. Aberrations in the expression of imprinted genes on human Chromosome 7 have been suggested to play a role in the etiologies of Russell-Silver Syndrome and autism. We describe the imprinting of KLF14, an intronless member of the Krüppel-like family of transcription factors located at Chromosome 7q32. We show that it has monoallelic maternal expression in all embryonic and extra-embryonic tissues studied, in both human and mouse. We examine epigenetic modifications in the KLF14 CpG island in both species and find this region to be hypomethylated. In addition, we perform chromatin immunoprecipitation and find that the murine Klf14 CpG island lacks allele-specific histone modifications. Despite the absence of these defining features, our analysis of Klf14 in offspring from DNA methyltransferase 3a conditional knockout mice reveals that the gene's expression is dependent upon a maternally methylated region. Due to the intronless nature of Klf14 and its homology to Klf16, we suggest that the gene is an ancient retrotransposed copy of Klf16. By sequence analysis of numerous species, we place the timing of this event after the divergence of Marsupialia, yet prior to the divergence of the Xenarthra superclade. We identify a large number of sequence variants in KLF14 and, using several measures of diversity, we determine that there is greater variability in the human lineage with a significantly increased number of nonsynonymous changes, suggesting human-specific accelerated evolution. Thus, KLF14 may be the first example of an imprinted transcript undergoing accelerated evolution in the human lineage.

  20. Identification of the imprinted KLF14 transcription factor undergoing human-specific accelerated evolution.

    Directory of Open Access Journals (Sweden)

    Layla Parker-Katiraee

    2007-05-01

    Full Text Available Imprinted genes are expressed in a parent-of-origin manner and are located in clusters throughout the genome. Aberrations in the expression of imprinted genes on human Chromosome 7 have been suggested to play a role in the etiologies of Russell-Silver Syndrome and autism. We describe the imprinting of KLF14, an intronless member of the Krüppel-like family of transcription factors located at Chromosome 7q32. We show that it has monoallelic maternal expression in all embryonic and extra-embryonic tissues studied, in both human and mouse. We examine epigenetic modifications in the KLF14 CpG island in both species and find this region to be hypomethylated. In addition, we perform chromatin immunoprecipitation and find that the murine Klf14 CpG island lacks allele-specific histone modifications. Despite the absence of these defining features, our analysis of Klf14 in offspring from DNA methyltransferase 3a conditional knockout mice reveals that the gene's expression is dependent upon a maternally methylated region. Due to the intronless nature of Klf14 and its homology to Klf16, we suggest that the gene is an ancient retrotransposed copy of Klf16. By sequence analysis of numerous species, we place the timing of this event after the divergence of Marsupialia, yet prior to the divergence of the Xenarthra superclade. We identify a large number of sequence variants in KLF14 and, using several measures of diversity, we determine that there is greater variability in the human lineage with a significantly increased number of nonsynonymous changes, suggesting human-specific accelerated evolution. Thus, KLF14 may be the first example of an imprinted transcript undergoing accelerated evolution in the human lineage.

  1. Accurate Gene Expression-Based Biodosimetry Using a Minimal Set of Human Gene Transcripts

    Energy Technology Data Exchange (ETDEWEB)

    Tucker, James D., E-mail: jtucker@biology.biosci.wayne.edu [Department of Biological Sciences, Wayne State University, Detroit, Michigan (United States); Joiner, Michael C. [Department of Radiation Oncology, Wayne State University, Detroit, Michigan (United States); Thomas, Robert A.; Grever, William E.; Bakhmutsky, Marina V. [Department of Biological Sciences, Wayne State University, Detroit, Michigan (United States); Chinkhota, Chantelle N.; Smolinski, Joseph M. [Department of Electrical and Computer Engineering, Wayne State University, Detroit, Michigan (United States); Divine, George W. [Department of Public Health Sciences, Henry Ford Hospital, Detroit, Michigan (United States); Auner, Gregory W. [Department of Electrical and Computer Engineering, Wayne State University, Detroit, Michigan (United States)

    2014-03-15

    Purpose: Rapid and reliable methods for conducting biological dosimetry are a necessity in the event of a large-scale nuclear event. Conventional biodosimetry methods lack the speed, portability, ease of use, and low cost required for triaging numerous victims. Here we address this need by showing that polymerase chain reaction (PCR) on a small number of gene transcripts can provide accurate and rapid dosimetry. The low cost and relative ease of PCR compared with existing dosimetry methods suggest that this approach may be useful in mass-casualty triage situations. Methods and Materials: Human peripheral blood from 60 adult donors was acutely exposed to cobalt-60 gamma rays at doses of 0 (control) to 10 Gy. mRNA expression levels of 121 selected genes were obtained 0.5, 1, and 2 days after exposure by reverse-transcriptase real-time PCR. Optimal dosimetry at each time point was obtained by stepwise regression of dose received against individual gene transcript expression levels. Results: Only 3 to 4 different gene transcripts, ASTN2, CDKN1A, GDF15, and ATM, are needed to explain ≥0.87 of the variance (R{sup 2}). Receiver-operator characteristics, a measure of sensitivity and specificity, of 0.98 for these statistical models were achieved at each time point. Conclusions: The actual and predicted radiation doses agree very closely up to 6 Gy. Dosimetry at 8 and 10 Gy shows some effect of saturation, thereby slightly diminishing the ability to quantify higher exposures. Analyses of these gene transcripts may be advantageous for use in a field-portable device designed to assess exposures in mass casualty situations or in clinical radiation emergencies.

  2. Type 2 diabetes and obesity induce similar transcriptional reprogramming in human myocytes.

    Science.gov (United States)

    Väremo, Leif; Henriksen, Tora Ida; Scheele, Camilla; Broholm, Christa; Pedersen, Maria; Uhlén, Mathias; Pedersen, Bente Klarlund; Nielsen, Jens

    2017-05-25

    Skeletal muscle is one of the primary tissues involved in the development of type 2 diabetes (T2D). The close association between obesity and T2D makes it difficult to isolate specific effects attributed to the disease alone. Therefore, here we set out to identify and characterize intrinsic properties of myocytes, associated independently with T2D or obesity. We generated and analyzed RNA-seq data from primary differentiated myotubes from 24 human subjects, using a factorial design (healthy/T2D and non-obese/obese), to determine the influence of each specific factor on genome-wide transcription. This setup enabled us to identify intrinsic properties, originating from muscle precursor cells and retained in the corresponding myocytes. Bioinformatic and statistical methods, including differential expression analysis, gene-set analysis, and metabolic network analysis, were used to characterize the different myocytes. We found that the transcriptional program associated with obesity alone was strikingly similar to that induced specifically by T2D. We identified a candidate epigenetic mechanism, H3K27me3 histone methylation, mediating these transcriptional signatures. T2D and obesity were independently associated with dysregulated myogenesis, down-regulated muscle function, and up-regulation of inflammation and extracellular matrix components. Metabolic network analysis identified that in T2D but not obesity a specific metabolite subnetwork involved in sphingolipid metabolism was transcriptionally regulated. Our findings identify inherent characteristics in myocytes, as a memory of the in vivo phenotype, without the influence from a diabetic or obese extracellular environment, highlighting their importance in the development of T2D.

  3. Reconstructing Generalized Logical Networks of Transcriptional Regulation in Mouse Brain from Temporal Gene Expression Data

    Energy Technology Data Exchange (ETDEWEB)

    Song, Mingzhou (Joe) [New Mexico State University, Las Cruces; Lewis, Chris K. [New Mexico State University, Las Cruces; Lance, Eric [New Mexico State University, Las Cruces; Chesler, Elissa J [ORNL; Kirova, Roumyana [Bristol-Myers Squibb Pharmaceutical Research & Development, NJ; Langston, Michael A [University of Tennessee, Knoxville (UTK); Bergeson, Susan [Texas Tech University, Lubbock

    2009-01-01

    The problem of reconstructing generalized logical networks to account for temporal dependencies among genes and environmental stimuli from high-throughput transcriptomic data is addressed. A network reconstruction algorithm was developed that uses the statistical significance as a criterion for network selection to avoid false-positive interactions arising from pure chance. Using temporal gene expression data collected from the brains of alcohol-treated mice in an analysis of the molecular response to alcohol, this algorithm identified genes from a major neuronal pathway as putative components of the alcohol response mechanism. Three of these genes have known associations with alcohol in the literature. Several other potentially relevant genes, highlighted and agreeing with independent results from literature mining, may play a role in the response to alcohol. Additional, previously-unknown gene interactions were discovered that, subject to biological verification, may offer new clues in the search for the elusive molecular mechanisms of alcoholism.

  4. Whole genome transcription profiling of Anaplasma phagocytophilum in human and tick host cells by tiling array analysis

    Directory of Open Access Journals (Sweden)

    Chavez Adela

    2008-07-01

    Full Text Available Abstract Background Anaplasma phagocytophilum (Ap is an obligate intracellular bacterium and the agent of human granulocytic anaplasmosis, an emerging tick-borne disease. Ap alternately infects ticks and mammals and a variety of cell types within each. Understanding the biology behind such versatile cellular parasitism may be derived through the use of tiling microarrays to establish high resolution, genome-wide transcription profiles of the organism as it infects cell lines representative of its life cycle (tick; ISE6 and pathogenesis (human; HL-60 and HMEC-1. Results Detailed, host cell specific transcriptional behavior was revealed. There was extensive differential Ap gene transcription between the tick (ISE6 and the human (HL-60 and HMEC-1 cell lines, with far fewer differentially transcribed genes between the human cell lines, and all disproportionately represented by membrane or surface proteins. There were Ap genes exclusively transcribed in each cell line, apparent human- and tick-specific operons and paralogs, and anti-sense transcripts that suggest novel expression regulation processes. Seven virB2 paralogs (of the bacterial type IV secretion system showed human or tick cell dependent transcription. Previously unrecognized genes and coding sequences were identified, as were the expressed p44/msp2 (major surface proteins paralogs (of 114 total, through elevated signal produced to the unique hypervariable region of each – 2/114 in HL-60, 3/114 in HMEC-1, and none in ISE6. Conclusion Using these methods, whole genome transcription profiles can likely be generated for Ap, as well as other obligate intracellular organisms, in any host cells and for all stages of the cell infection process. Visual representation of comprehensive transcription data alongside an annotated map of the genome renders complex transcription into discernable patterns.

  5. Interaction of human dipeptidyl peptidase IV and human immunodeficiency virus type-1 transcription transactivator in Sf9 cells

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    Reutter Werner

    2010-10-01

    Full Text Available Abstract Background Dipeptidyl peptidase IV (DPPIV also known as the T cell activation marker CD26 is a multifunctional protein which is involved in various biological processes. The association of human-DPPIV with components of the human immunodeficiency virus type-1 (HIV1 is well documented and raised some discussions. Several reports implicated the interaction of human-DPPIV with the HIV1 transcription transactivator protein (HIV1-Tat and the inhibition of the dipeptidyl peptidase activity of DPPIV by the HIV1-Tat protein. Furthermore, enzyme kinetic data implied another binding site for the HIV1-Tat other than the active centre of DPPIV. However, the biological significance of this interaction of the HIV1-Tat protein and human-DPPIV has not been studied, yet. Therefore, we focused on the interaction of HIV1-Tat protein with DPPIV and investigated the subsequent biological consequences of this interaction in Spodoptera frugiperda cells, using the BAC-TO-BAC baculovirus system. Results The HIV1-Tat protein (Tat-BRU co-localized and co-immunoprecipitated with human-DPPIV protein, following co-expression in the baculovirus-driven Sf9 cell expression system. Furthermore, tyrosine phosphorylation of DPPIV protein was up-regulated in Tat/DPPIV-co-expressing cells after 72 h culturing and also in DPPIV-expressing Sf9 cells after application of purified recombinant Tat protein. As opposed to the expression of Tat alone, serine phosphorylation of the Tat protein was decreased when co-expressed with human-DPPIV protein. Conclusions We show for the first time that human-DPPIV and HIV1-Tat co-immunoprecipitate. Furthermore, our findings indicate that the interaction of HIV1-Tat and human-DPPIV may be involved in signalling platforms that regulate the biological function of both human-DPPIV and HIV1-Tat.

  6. Isolation and characterization of csbB, a gene controlled by Bacillus subtilis general stress transcription factor sigma B.

    Science.gov (United States)

    Akbar, S; Price, C W

    1996-10-24

    In the bacterium Bacillus subtilis (Bs), the alternative transcription factor sigma B is activated by environmental stresses to control the expression of a large set of unlinked genes. However, the range of physiological functions mediated by these sigma B-controlled genes is presently unknown. We report here that the newly identified gene csbB is under the dual control of a sigma B-dependent and a sigma B-independent promoter. The predicted product of csbB is a 329 residue protein containing two potential membrane-spanning segments in its C-terminal region, leading us to speculate that one class of sigma B-controlled genes acts to modify the cell envelope as part of the general stress response.

  7. Cyclosporin A Decreases Human Macrophage Interleukin-6 Synthesis at Post-Transcriptional Level

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    Juan E. Losa García

    1999-01-01

    Full Text Available In addition to its well-established effect on T cells, cyclosporin A (CsA also inhibits inflammatory cytokine production by macrophages. However, little is known about the mechanism of action of CsA on macrophage cytokine production. We measured the effect of CsA on basal and phorbol-myristate-acetate (PMA-stimulated production of interleukin-6 using the human monocyte cell line U937 differentiated with dimethylsulfoxide (DMSO. Interleukin-6 levels were measured in supernatant and cell lysates using specific enzyme-linked immunosorbent assays. We found that CsA decreases not only IL-6 release but also cytokine synthesis. The concentration of CsA used did not affect either cell viability or proliferation. Three possibilities may be advanced to explain the CsA-due decrease in IL-6 production by macrophages: (a inhibition of the synthesis of an early common regulatory protein, (b inhibition of cytokine gene transcription, or (c modulation of post-transcriptional events. The first possibility was tested by measuring the effect of cycloheximide on the experimental system during the first 3 hours of culture. Although cycloheximide decreased total cytokine synthesis, the pattern of cytokine modulation by CsA persisted. These data suggest that CsA-mediated macrophage cytokine inhibition is not mediated by an early common regulatory protein. To further explore the inhibition mechanism, we measured IL-6 mRNA levels by Northern blot. IL-6 mRNA levels were unaffected by CsA both in resting and PMA-stimulated cells. We conclude that in human macrophages CsA diminishes IL-6 production at post-transcriptional level.

  8. Inherited human sex reversal due to impaired nucleocytoplasmic trafficking of SRY defines a male transcriptional threshold.

    Science.gov (United States)

    Chen, Yen-Shan; Racca, Joseph D; Phillips, Nelson B; Weiss, Michael A

    2013-09-17

    Human testis determination is initiated by SRY (sex determining region on Y chromosome). Mutations in SRY cause gonadal dysgenesis with female somatic phenotype. Two subtle variants (V60L and I90M in the high-mobility group box) define inherited alleles shared by an XY sterile daughter and fertile father. Whereas specific DNA binding and bending are unaffected in a rat embryonic pre-Sertoli cell line, the variants exhibited selective defects in nucleocytoplasmic shuttling due to impaired nuclear import (V60L; mediated by Exportin-4) or export (I90M; mediated by chromosome region maintenance 1). Decreased shuttling limits nuclear accumulation of phosphorylated (activated) SRY, in turn reducing occupancy of DNA sites regulating Sertoli-cell differentiation [the testis-specific SRY-box 9 (Sox9) enhancer]. Despite distinct patterns of biochemical and cell-biological perturbations, V60L and I90M each attenuated Sox9 expression in transient transfection assays by twofold. Such attenuation was also observed in studies of V60A, a clinical variant associated with ovotestes and hence ambiguity between divergent cell fates. This shared twofold threshold is reminiscent of autosomal syndromes of transcription-factor haploinsufficiency, including XY sex reversal associated with mutations in SOX9. Our results demonstrate that nucleocytoplasmic shuttling of SRY is necessary for robust initiation of testicular development. Although also characteristic of ungulate orthologs, such shuttling is not conserved among rodents wherein impaired nuclear export of the high-mobility group box and import-dependent phosphorylation are compensated by a microsatellite-associated transcriptional activation domain. Human sex reversal due to subtle defects in the nucleocytoplasmic shuttling of SRY suggests that its transcriptional activity lies near the edge of developmental ambiguity.

  9. Transcription-induced mutational strand bias and its effect on substitution rates in human genes.

    Science.gov (United States)

    Mugal, Carina F; von Grünberg, Hans-Hennig; Peifer, Martin

    2009-01-01

    If substitution rates are not the same on the two complementary DNA strands, a substitution is considered strand asymmetric. Such substitutional strand asymmetries are determined here for the three most frequent types of substitution on the human genome (C --> T, A --> G, and G --> T). Substitution rate differences between both strands are estimated for 4,590 human genes by aligning all repeats occurring within the introns with their ancestral consensus sequences. For 1,630 of these genes, both coding strand and noncoding strand rates could be compared with rates in gene-flanking regions. All three rates considered are found to be on average higher on the coding strand and lower on the transcribed strand in comparison to their values in the gene-flanking regions. This finding points to the simultaneous action of rate-increasing effects on the coding strand--such as increased adenine and cytosine deamination--and transcription-coupled repair as a rate-reducing effect on the transcribed strand. The common behavior of the three rates leads to strong correlations of the rate asymmetries: Whenever one rate is strand biased, the other two rates are likely to show the same bias. Furthermore, we determine all three rate asymmetries as a function of time: the A --> G and G --> T rate asymmetries are both found to be constant in time, whereas the C --> T rate asymmetry shows a pronounced time dependence, an observation that explains the difference between our results and those of an earlier work by Green et al. (2003. Transcription-associated mutational asymmetry in mammalian evolution. Nat Genet. 33:514-517.). Finally, we show that in addition to transcription also the replication process biases the substitution rates in genes.

  10. Transcription factor ATF-3 regulates allele variation phenotypes of the human SLC11A1 gene.

    Science.gov (United States)

    Taka, Styliani; Gazouli, Maria; Politis, Panagotis K; Pappa, Kalliopi I; Anagnou, Nicholas P

    2013-03-01

    Genetic polymorphisms in the human solute carrier family 11 member 1 (SLC11A1) gene predispose to susceptibility to infectious/inflammatory diseases and cancer. Human susceptibility to these diseases exhibits allelic association with a polymorphic regulatory Z-DNA-forming microsatellite of a (GT/AC)n repeat. The carriage of different alleles may influence chromatin remodeling and accessibility by transcription factors. Of particular importance is the binding site for the Activating Protein 1 (AP-1) elements, (ATF-3 and c-Jun), adjacent to the 5' sequence of the Z-DNA-forming polymorphism. The aim of the study was to characterize the transcriptional mechanisms controlling different alleles of SLC11A1 expression by ATF-3 and c-Jun. Allele 2, [T(GT)5AC(GT)5AC(GT)10GGCAGA(G)6], and Allele 3, [T(GT)5AC(GT)5AC(GT)9GGCAGA(G)6], were subcloned into the PGL2Basic vector. Transient transfections of THP-1 cells with the constructs, in the presence or absence of pATF-3 were preformed. Luciferase expression was determined. To document the recruitment of ATF-3 and c-Jun, to the polymorphic promoter alleles in vivo, we performed ChIP assays with transient transfected THP-1 cells treated with or without lipopolyssacharides. Our data documented that ATF-3 suppresses the transcriptional activation of Allele-3, and this suppression is enhanced in the presence of lipopolyssacharides. Our findings suggest that ATF-3 and c-Jun may influence heritable variation in SLC11A1-dependent innate resistance to infection and inflammation both within and between populations.

  11. CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription

    Science.gov (United States)

    Tang, Zhonghui; Luo, Oscar Junhong; Li, Xingwang; Zheng, Meizhen; Zhu, Jacqueline Jufen; Szalaj, Przemyslaw; Trzaskoma, Pawel; Magalska, Adriana; Wlodarczyk, Jakub; Ruszczycki, Blazej; Michalski, Paul; Piecuch, Emaly; Wang, Ping; Wang, Danjuan; Tian, Simon Zhongyuan; Penrad-Mobayed, May; Sachs, Laurent M.; Ruan, Xiaoan; Wei, Chia-Lin; Liu, Edison T.; Wilczynski, Grzegorz M.; Plewczynski, Dariusz; Li, Guoliang; Ruan, Yijun

    2015-01-01

    Summary Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced ChIA-PET strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CTCF and RNAPII with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes towards CTCF-foci for coordinated transcription. Furthermore, we show that haplotype-variants and allelic-interactions have differential effects on chromosome configuration influencing gene expression and may provide mechanistic insights into functions associated with disease susceptibility. 3D-genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D-genome strategy thus provides unique insights in the topological mechanism of human variations and diseases. PMID:26686651

  12. CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription.

    Science.gov (United States)

    Tang, Zhonghui; Luo, Oscar Junhong; Li, Xingwang; Zheng, Meizhen; Zhu, Jacqueline Jufen; Szalaj, Przemyslaw; Trzaskoma, Pawel; Magalska, Adriana; Wlodarczyk, Jakub; Ruszczycki, Blazej; Michalski, Paul; Piecuch, Emaly; Wang, Ping; Wang, Danjuan; Tian, Simon Zhongyuan; Penrad-Mobayed, May; Sachs, Laurent M; Ruan, Xiaoan; Wei, Chia-Lin; Liu, Edison T; Wilczynski, Grzegorz M; Plewczynski, Dariusz; Li, Guoliang; Ruan, Yijun

    2015-12-17

    Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.

  13. Distinct Contributions of Replication and Transcription to Mutation Rate Variation of Human Genomes

    KAUST Repository

    Cui, Peng

    2012-03-23

    Here, we evaluate the contribution of two major biological processes—DNA replication and transcription—to mutation rate variation in human genomes. Based on analysis of the public human tissue transcriptomics data, high-resolution replicating map of Hela cells and dbSNP data, we present significant correlations between expression breadth, replication time in local regions and SNP density. SNP density of tissue-specific (TS) genes is significantly higher than that of housekeeping (HK) genes. TS genes tend to locate in late-replicating genomic regions and genes in such regions have a higher SNP density compared to those in early-replication regions. In addition, SNP density is found to be positively correlated with expression level among HK genes. We conclude that the process of DNA replication generates stronger mutational pressure than transcription-associated biological processes do, resulting in an increase of mutation rate in TS genes while having weaker effects on HK genes. In contrast, transcription-associated processes are mainly responsible for the accumulation of mutations in highly-expressed HK genes.

  14. CD161 Defines a Transcriptional and Functional Phenotype across Distinct Human T Cell Lineages

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    Joannah R. Fergusson

    2014-11-01

    Full Text Available The C-type lectin CD161 is expressed by a large proportion of human T lymphocytes of all lineages, including a population known as mucosal-associated invariant T (MAIT cells. To understand whether different T cell subsets expressing CD161 have similar properties, we examined these populations in parallel using mass cytometry and mRNA microarray approaches. The analysis identified a conserved CD161++/MAIT cell transcriptional signature enriched in CD161+CD8+ T cells, which can be extended to CD161+ CD4+ and CD161+TCRγδ+ T cells. Furthermore, this led to the identification of a shared innate-like, TCR-independent response to interleukin (IL-12 plus IL-18 by different CD161-expressing T cell populations. This response was independent of regulation by CD161, which acted as a costimulatory molecule in the context of T cell receptor stimulation. Expression of CD161 hence identifies a transcriptional and functional phenotype, shared across human T lymphocytes and independent of both T cell receptor (TCR expression and cell lineage.

  15. Predicting novel histopathological microlesions in human epileptic brain through transcriptional clustering.

    Science.gov (United States)

    Dachet, Fabien; Bagla, Shruti; Keren-Aviram, Gal; Morton, Andrew; Balan, Karina; Saadat, Laleh; Valyi-Nagy, Tibor; Kupsky, William; Song, Fei; Dratz, Edward; Loeb, Jeffrey A

    2015-02-01

    Although epilepsy is associated with a variety of abnormalities, exactly why some brain regions produce seizures and others do not is not known. We developed a method to identify cellular changes in human epileptic neocortex using transcriptional clustering. A paired analysis of high and low spiking tissues recorded in vivo from 15 patients predicted 11 cell-specific changes together with their 'cellular interactome'. These predictions were validated histologically revealing millimetre-sized 'microlesions' together with a global increase in vascularity and microglia. Microlesions were easily identified in deeper cortical layers using the neuronal marker NeuN, showed a marked reduction in neuronal processes, and were associated with nearby activation of MAPK/CREB signalling, a marker of epileptic activity, in superficial layers. Microlesions constitute a common, undiscovered layer-specific abnormality of neuronal connectivity in human neocortex that may be responsible for many 'non-lesional' forms of epilepsy. The transcriptional clustering approach used here could be applied more broadly to predict cellular differences in other brain and complex tissue disorders.

  16. Reconstructing Generalized Logical Networks of Transcriptional Regulation in Mouse Brain from Temporal Gene Expression Data

    Directory of Open Access Journals (Sweden)

    2009-03-01

    Full Text Available Gene expression time course data can be used not only to detect differentially expressed genes but also to find temporal associations among genes. The problem of reconstructing generalized logical networks to account for temporal dependencies among genes and environmental stimuli from transcriptomic data is addressed. A network reconstruction algorithm was developed that uses statistical significance as a criterion for network selection to avoid false-positive interactions arising from pure chance. The multinomial hypothesis testing-based network reconstruction allows for explicit specification of the false-positive rate, unique from all extant network inference algorithms. The method is superior to dynamic Bayesian network modeling in a simulation study. Temporal gene expression data from the brains of alcohol-treated mice in an analysis of the molecular response to alcohol are used for modeling. Genes from major neuronal pathways are identified as putative components of the alcohol response mechanism. Nine of these genes have associations with alcohol reported in literature. Several other potentially relevant genes, compatible with independent results from literature mining, may play a role in the response to alcohol. Additional, previously unknown gene interactions were discovered that, subject to biological verification, may offer new clues in the search for the elusive molecular mechanisms of alcoholism.

  17. Reconstructing Generalized Logical Networks of Transcriptional Regulation in Mouse Brain from Temporal Gene Expression Data

    Directory of Open Access Journals (Sweden)

    Lodowski Kerrie H

    2009-01-01

    Full Text Available Gene expression time course data can be used not only to detect differentially expressed genes but also to find temporal associations among genes. The problem of reconstructing generalized logical networks to account for temporal dependencies among genes and environmental stimuli from transcriptomic data is addressed. A network reconstruction algorithm was developed that uses statistical significance as a criterion for network selection to avoid false-positive interactions arising from pure chance. The multinomial hypothesis testing-based network reconstruction allows for explicit specification of the false-positive rate, unique from all extant network inference algorithms. The method is superior to dynamic Bayesian network modeling in a simulation study. Temporal gene expression data from the brains of alcohol-treated mice in an analysis of the molecular response to alcohol are used for modeling. Genes from major neuronal pathways are identified as putative components of the alcohol response mechanism. Nine of these genes have associations with alcohol reported in literature. Several other potentially relevant genes, compatible with independent results from literature mining, may play a role in the response to alcohol. Additional, previously unknown gene interactions were discovered that, subject to biological verification, may offer new clues in the search for the elusive molecular mechanisms of alcoholism.

  18. Transcription factors link mouse WAP-T mammary tumors with human breast cancer.

    Science.gov (United States)

    Otto, Benjamin; Streichert, Thomas; Wegwitz, Florian; Gevensleben, Heidrun; Klätschke, Kristin; Wagener, Christoph; Deppert, Wolfgang; Tolstonog, Genrich V

    2013-03-15

    Mouse models are important tools to decipher the molecular mechanisms of mammary carcinogenesis and to mimic the respective human disease. Despite sharing common phenotypic and genetic features, the proper translation of murine models to human breast cancer remains a challenging task. In a previous study we showed that in the SV40 transgenic WAP-T mice an active Met-pathway and epithelial-mesenchymal characteristics distinguish low- and high-grade mammary carcinoma. To assign these murine tumors to corresponding human tumors we here incorporated the analysis of expression of transcription factor (TF) coding genes and show that thereby a more accurate interspecies translation can be achieved. We describe a novel cross-species translation procedure and demonstrate that expression of unsupervised selected TFs, such as ELF5, HOXA5 and TFCP2L1, can clearly distinguish between the human molecular breast cancer subtypes--or as, for example, expression of TFAP2B between yet unclassified subgroups. By integrating different levels of information like histology, gene set enrichment, expression of differentiation markers and TFs we conclude that tumors in WAP-T mice exhibit similarities to both, human basal-like and non-basal-like subtypes. We furthermore suggest that the low- and high-grade WAP-T tumor phenotypes might arise from distinct cells of tumor origin. Our results underscore the importance of TFs as common cross-species denominators in the regulatory networks underlying mammary carcinogenesis.

  19. Prognostic values of tissue factor and its alternatively splice transcripts in human gastric cancer tissues.

    Science.gov (United States)

    Wu, Min; Chen, Lujun; Xu, Ting; Xu, Bin; Jiang, Jingting; Wu, Changping

    2017-08-08

    We have previously reported that the higher expression of TF in human esophageal cancer tissues was significantly associated with tumor invasion, intratumoral microvessel density and patients' postoperative prognoses. Besides its trans-membranous form, TF also has alternatively spliced transcripts. In the present study, the transcripts of the two TF isoforms, flTF and asTF, in human gastric cancer tissues were determined by real-time PCR, and the correlation between the expression of TF isoforms and patient's clinicopathological features was also analyzed. Our results showed that the relative mRNA expression levels of flTF and asTF in human gastric cancer tissues was significantly higher than those in normal tissues (P=0.035 and P=0.006, respectively). The relative mRNA expression level of asTF was significantly associated with age (P=0.018), meanwhile, we could not find that flTF or asTF expression level was correlated with any other characteristics of the patients, including gender, TNM stage, pathological grade, tumor size, histological type, or chemotherapy sensitivity. Univariate analysis demonstrated that the overall survival rate of gastric cancer patients with lower flTF or asTF expression level was greater than those with higher expression level (P=0.018 and =0.038, respectively). Multivariate COX model analysis also demonstrated that flTF expression (P=0.048) or asTF expression (P=0.002) could be used as independent prognostic predictors in human gastric cancer. Thus, both flTF and asTF mRNA expression levels in cancer tissues could be used as useful risk factors for evaluating the prognoses of patients suffering from gastric cancer.

  20. Lysophosphatidic acid (LPA 18:1 transcriptional regulation of primary human gingival fibroblasts

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    D. Roselyn Cerutis

    2014-12-01

    Full Text Available The pleiotropic, bioactive lipid lysophosphatidic acid [(LPA, 1-acyl-sn-glycerol-3-phosphate] exerts critical regulatory actions in physiology and pathophysiology in many systems. It is present in normal bodily fluids, and is elevated in pathology (1. In vivo, “LPA” exists as distinct molecular species, each having a single fatty acid of varying chain length and degree of unsaturation covalently attached to the glycerol backbone via an acyl, alkyl, or alkenyl link. These species differ in affinities for the individual LPA receptors [(LPARs, LPA1-6] and coupling to G proteins (2. However, LPA 18:1 has been and continues to be the most commonly utilized species in reported studies. The actions of “LPA” remain poorly defined in oral biology and pathophysiology. Our laboratory has addressed this knowledge gap by studying in vitro the actions of the major human salivary LPA species [18:1, 18:0, and 16:0 (3] in human oral cells (4–7. This includes gingival fibroblasts (GF, which our flow cytometry data from multiple donors found that they express LPA1-5 (6. We have also reported that these species are ten-fold elevated to pharmacologic levels in the saliva and gingival crevicular fluid obtained from patients with moderate–severe periodontitis (8. As the potential of LPA to regulate transcriptional activity had not been examined in the oral system, this study used whole human genome microarray analysis to test the hypothesis that LPA 18:1-treated human GF would show significant changes in gene transcripts relevant to their biology, wound-healing, and inflammatory responses. LPA 18:1 was found to significantly regulate a large, complex set of genes critical to GF biology in these categories and to periodontal disease. The raw data has been deposited at NCBI's GEO database as record GSE57496.

  1. Sp110 transcription is induced and required by Anaplasma phagocytophilum for infection of human promyelocytic cells

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    Naranjo Victoria

    2007-09-01

    Full Text Available Abstract Background The tick-borne intracellular pathogen, Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae causes human granulocytic anaplasmosis after infection of polymorphonuclear leucocytes. The human Sp110 gene is a member of the nuclear body (NB components that functions as a nuclear hormone receptor transcriptional coactivator and plays an important role in immunoprotective mechanisms against pathogens in humans. In this research, we hypothesized that Sp110 may be involved in the infection of human promyelocytic HL-60 cells with A. phagocytophilum. Methods The human Sp110 and A. phagocytophilum msp4 mRNA levels were evaluated by real-time RT-PCR in infected human HL-60 cells sampled at 0, 12, 24, 48, 72 and 96 hours post-infection. The effect of Sp110 expression on A. phagocytophilum infection was determined by RNA interference (RNAi. The expression of Sp110 was silenced in HL-60 cells by RNAi using pre-designed siRNAs using the Nucleofector 96-well shuttle system (Amaxa Biosystems, Gaithersburg, MD, USA. The A. phagocytophilum infection levels were evaluated in HL-60 cells after RNAi by real-time PCR of msp4 and normalizing against human Alu sequences. Results While Sp110 mRNA levels increased concurrently with A. phagocytophilum infections in HL-60 cells, the silencing of Sp110 expression by RNA interference resulted in decreased infection levels. Conclusion These results demonstrated that Sp110 expression is required for A. phagocytophilum infection and multiplication in HL-60 cells, and suggest a previously undescribed mechanism by which A. phagocytophilum modulates Sp110 mRNA levels to facilitate establishment of infection of human HL-60 cells.

  2. Deciphering the transcriptional circuitry of microRNA genes expressed during human monocytic differentiation

    KAUST Repository

    Schmeier, Sebastian

    2009-12-10

    Background: Macrophages are immune cells involved in various biological processes including host defence, homeostasis, differentiation, and organogenesis. Disruption of macrophage biology has been linked to increased pathogen infection, inflammation and malignant diseases. Differential gene expression observed in monocytic differentiation is primarily regulated by interacting transcription factors (TFs). Current research suggests that microRNAs (miRNAs) degrade and repress translation of mRNA, but also may target genes involved in differentiation. We focus on getting insights into the transcriptional circuitry regulating miRNA genes expressed during monocytic differentiation. Results: We computationally analysed the transcriptional circuitry of miRNA genes during monocytic differentiation using in vitro time-course expression data for TFs and miRNAs. A set of TF?miRNA associations was derived from predicted TF binding sites in promoter regions of miRNA genes. Time-lagged expression correlation analysis was utilised to evaluate the TF?miRNA associations. Our analysis identified 12 TFs that potentially play a central role in regulating miRNAs throughout the differentiation process. Six of these 12 TFs (ATF2, E2F3, HOXA4, NFE2L1, SP3, and YY1) have not previously been described to be important for monocytic differentiation. The remaining six TFs are CEBPB, CREB1, ELK1, NFE2L2, RUNX1, and USF2. For several miRNAs (miR-21, miR-155, miR-424, and miR-17-92), we show how their inferred transcriptional regulation impacts monocytic differentiation. Conclusions: The study demonstrates that miRNAs and their transcriptional regulatory control are integral molecular mechanisms during differentiation. Furthermore, it is the first study to decipher on a large-scale, how miRNAs are controlled by TFs during human monocytic differentiation. Subsequently, we have identified 12 candidate key controllers of miRNAs during this differentiation process. 2009 Schmeier et al; licensee Bio

  3. Transcriptional regulation of human dual specificity protein phosphatase 1 (DUSP1 gene by glucocorticoids.

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    Lauren E Shipp

    Full Text Available BACKGROUND: Glucocorticoids are potent anti-inflammatory agents commonly used to treat inflammatory diseases. They convey signals through the intracellular glucocorticoid receptor (GR, which upon binding to ligands, associates with genomic glucocorticoid response elements (GREs to regulate transcription of associated genes. One mechanism by which glucocorticoids inhibit inflammation is through induction of the dual specificity phosphatase-1 (DUSP1, a.k.a. mitogen-activated protein kinase phosphatase-1, MKP-1 gene. METHODOLOGY/PRINCIPAL FINDINGS: We found that glucocorticoids rapidly increased transcription of DUSP1 within 10 minutes in A549 human lung adenocarcinoma cells. Using chromatin immunoprecipitation (ChIP scanning, we located a GR binding region between -1421 and -1118 upstream of the DUSP1 transcription start site. This region is active in a reporter system, and mutagenesis analyses identified a functional GRE located between -1337 and -1323. We found that glucocorticoids increased DNase I hypersensitivity, reduced nucleosome density, and increased histone H3 and H4 acetylation within genomic regions surrounding the GRE. ChIP experiments showed that p300 was recruited to the DUSP1 GRE, and RNA interference experiments demonstrated that reduction of p300 decreased glucocorticoid-stimulated DUSP1 gene expression and histone H3 hyperacetylation. Furthermore, overexpression of p300 potentiated glucocorticoid-stimulated activity of a reporter gene containing the DUSP1 GRE, and this coactivation effect was compromised when the histone acetyltransferase domain was mutated. ChIP-reChIP experiments using GR followed by p300 antibodies showed significant enrichment of the DUSP1 GRE upon glucocorticoid treatment, suggesting that GR and p300 are in the same protein complex recruited to the DUSP1 GRE. CONCLUSIONS/SIGNIFICANCE: Our studies identified a functional GRE for the DUSP1 gene. Moreover, the transcriptional activation of DUSP1 by

  4. Expression of SNC73, a transcript of the immunoglobulin α-1 gene, in human epithelial carcinomas

    Institute of Scientific and Technical Information of China (English)

    Li-Yi Geng; Shu Zheng; Zheng-Zhen Shi; Qi Dong; Xin-Han Cai; Yan-Ming Zhang; Wei Cao; Jia-Ping Peng; Yong-Ming Fang; Lei Zheng

    2007-01-01

    AIM: To investigate the expression of SNC73, a transcript of the immunoglobulin α-1 gene (IgA1-H chain), in human epithelia-derived tumor cells.METHODS: Total RNAs and cell lysates were prepared from five different human epithelial cell lines derived from lung, stomach, liver, skin, and breast, respectively. RT-PCR and immunoblot analysis of these five cell lines were done. Both RT-PCR and immunochemistry were used to detect the expression of SNC73 in these cell lines. We also examined the expression of SNC73 in normal epithelial cells of colon mucosa by in situ hybridization. RT-PCR and immunoblot analysis were used to determine whether the recombination activating gene1/2 (RAG1 and RAG2) is present. The expression of three immunoglobulin transcription factors, EBF, E2A and Pax5, and the heavy chain of IgA1 and two types of light chains of immunoglobulin (κ and λ) in the aforementioned cell lines were analyzed by RT-PCR and immunochemistry, respectively. All the RT-PCR products were analyzed by sequencing.RESULTS: The results of RT-PCR and immunochemistry showed that both mRNA and protein of SNC73 were expressed in five human epithelia-derived cancer cell lines. These data were further confirmed in the normal epithelial cells of colon mucosa by in situ hybridization. Also, the heavy chain of IgA1 and κ light chain were detected in these cells, but no λ light chain was observed. Both RAG1 and RAG2 were expressed in these human epithelia-derived cancer cell lines and the sequence was identical to that expressed in pre-B and pre-T cells. In addition to RAG1 and RAG2, the mRNA in one of the immunoglobulin transcription factors, EBF, was also detected in these cell lines, and Pax5 was only expressed in SW480 cells, but no expression of E2A was observed in all the five cell lines.CONCLUSION: Immunoglobulin A1 is originally expressed and V(D)J recombination machine is also present in non-lymphoid cells, suggesting that V(D)J recombination machine mediates the

  5. SIRT1 Suppresses Human T-Cell Leukemia Virus Type 1 Transcription

    Science.gov (United States)

    Tang, Hei-Man Vincent; Gao, Wei-Wei; Chan, Chi-Ping; Cheng, Yun; Deng, Jian-Jun; Yuen, Kit-San; Iha, Hidekatsu

    2015-01-01

    ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1)-associated diseases are poorly treatable, and HTLV-1 vaccines are not available. High proviral load is one major risk factor for disease development. HTLV-1 encodes Tax oncoprotein, which activates transcription from viral long terminal repeats (LTR) and various types of cellular promoters. Counteracting Tax function might have prophylactic and therapeutic benefits. In this work, we report on the suppression of Tax activation of HTLV-1 LTR by SIRT1 deacetylase. The transcriptional activity of Tax on the LTR was largely ablated when SIRT1 was overexpressed, but Tax activation of NF-κB was unaffected. On the contrary, the activation of the LTR by Tax was boosted when SIRT1 was depleted. Treatment of cells with resveratrol shunted Tax activity in a SIRT1-dependent manner. The activation of SIRT1 in HTLV-1-transformed T cells by resveratrol potently inhibited HTLV-1 proviral transcription and Tax expression, whereas compromising SIRT1 by specific inhibitors augmented HTLV-1 mRNA expression. The administration of resveratrol also decreased the production of cell-free HTLV-1 virions from MT2 cells and the transmission of HTLV-1 from MT2 cells to uninfected Jurkat cells in coculture. SIRT1 associated with Tax in HTLV-1-transformed T cells. Treatment with resveratrol prevented the interaction of Tax with CREB and the recruitment of CREB, CRTC1, and p300 to Tax-responsive elements in the LTR. Our work demonstrates the negative regulatory function of SIRT1 in Tax activation of HTLV-1 transcription. Small-molecule activators of SIRT1 such as resveratrol might be considered new prophylactic and therapeutic agents in HTLV-1-associated diseases. IMPORTANCE Human T-cell leukemia virus type 1 (HTLV-1) causes a highly lethal blood cancer or a chronic debilitating disease of the spinal cord. Treatments are unsatisfactory, and vaccines are not available. Disease progression is associated with robust expression of HTLV-1 genes

  6. Discovery of novel human transcript variants by analysis of intronic single-block EST with polyadenylation site

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    Yu Peng

    2009-11-01

    Full Text Available Abstract Background Alternative polyadenylation sites within a gene can lead to alternative transcript variants. Although bioinformatic analysis has been conducted to detect polyadenylation sites using nucleic acid sequences (EST/mRNA in the public databases, one special type, single-block EST is much less emphasized. This bias leaves a large space to discover novel transcript variants. Results In the present study, we identified novel transcript variants in the human genome by detecting intronic polyadenylation sites. Poly(A/T-tailed ESTs were obtained from single-block ESTs and clustered into 10,844 groups standing for 5,670 genes. Most sites were not found in other alternative splicing databases. To verify that these sites are from expressed transcripts, we analyzed the supporting EST number of each site, blasted representative ESTs against known mRNA sequences, traced terminal sequences from cDNA clones, and compared with the data of Affymetrix tiling array. These analyses confirmed about 84% (9,118/10,844 of the novel alternative transcripts, especially, 33% (3,575/10,844 of the transcripts from 2,704 genes were taken as high-reliability. Additionally, RT-PCR confirmed 38% (10/26 of predicted novel transcript variants. Conclusion Our results provide evidence for novel transcript variants with intronic poly(A sites. The expression of these novel variants was confirmed with computational and experimental tools. Our data provide a genome-wide resource for identification of novel human transcript variants with intronic polyadenylation sites, and offer a new view into the mystery of the human transcriptome.

  7. Human genes with a greater number of transcript variants tend to show biological features of housekeeping and essential genes

    DEFF Research Database (Denmark)

    Ryu, Jae Yong; Kim, Hyun Uk; Lee, Sang Yup

    2015-01-01

    64 vertebrate species as orthologs, subjected to regulations by transcription factors and microRNAs, and showed hub node-like properties in the human protein-protein interaction network. These findings were also confirmed by metabolic simulations of 60 cancer metabolic models. All these results......Alternative splicing is a process observed in gene expression that results in a multi-exon gene to produce multiple mRNA variants which might have different functions and activities. Although physiologically important, many aspects of genes with different number of transcript variants (or splice...... variants) still remain to be characterized. In this study, we provide bioinformatic evidence that genes with a greater number of transcript variants are more likely to play functionally important roles in cells, compared with those having fewer transcript variants. Among 21 983 human genes, 3728 genes were...

  8. Novelty and Inductive Generalization in Human Reinforcement Learning.

    Science.gov (United States)

    Gershman, Samuel J; Niv, Yael

    2015-07-01

    In reinforcement learning (RL), a decision maker searching for the most rewarding option is often faced with the question: What is the value of an option that has never been tried before? One way to frame this question is as an inductive problem: How can I generalize my previous experience with one set of options to a novel option? We show how hierarchical Bayesian inference can be used to solve this problem, and we describe an equivalence between the Bayesian model and temporal difference learning algorithms that have been proposed as models of RL in humans and animals. According to our view, the search for the best option is guided by abstract knowledge about the relationships between different options in an environment, resulting in greater search efficiency compared to traditional RL algorithms previously applied to human cognition. In two behavioral experiments, we test several predictions of our model, providing evidence that humans learn and exploit structured inductive knowledge to make predictions about novel options. In light of this model, we suggest a new interpretation of dopaminergic responses to novelty.

  9. Enhancer Turnover Is Associated with a Divergent Transcriptional Response to Glucocorticoid in Mouse and Human Macrophages.

    Science.gov (United States)

    Jubb, Alasdair W; Young, Robert S; Hume, David A; Bickmore, Wendy A

    2016-01-15

    Phenotypic differences between individuals and species are controlled in part through differences in expression of a relatively conserved set of genes. Genes expressed in the immune system are subject to especially powerful selection. We have investigated the evolution of both gene expression and candidate enhancers in human and mouse macrophages exposed to glucocorticoid (GC), a regulator of innate immunity and an important therapeutic agent. Our analyses revealed a very limited overlap in the repertoire of genes responsive to GC in human and mouse macrophages. Peaks of inducible binding of the GC receptor (GR) detected by chromatin immunoprecipitation-Seq correlated with induction, but not repression, of target genes in both species, occurred at distal regulatory sites not promoters, and were strongly enriched for the consensus GR-binding motif. Turnover of GR binding between mice and humans was associated with gain and loss of the motif. There was no detectable signal of positive selection at species-specific GR binding sites, but clear evidence of purifying selection at the small number of conserved sites. We conclude that enhancer divergence underlies the difference in transcriptional activation after GC treatment between mouse and human macrophages. Only the shared inducible loci show evidence of selection, and therefore these loci may be important for the subset of responses to GC that is shared between species.

  10. Functional Domains of Autoimmune Regulator (AIRE) Modulate INS-VNTR Transcription in Human Thymic Epithelial Cells*

    Science.gov (United States)

    Sparks, Avis E.; Chen, Chiachen; Breslin, Mary B.; Lan, Michael S.

    2016-01-01

    INS-VNTR (insulin-variable number of tandem repeats) and AIRE (autoimmune regulator) have been associated with the modulation of insulin gene expression in thymus, which is essential to induce either insulin tolerance or the development of insulin autoimmunity and type 1 diabetes. We sought to analyze whether each functional domain of AIRE is critical for the activation of INS-VNTR in human thymic epithelial cells. Twelve missense or nonsense mutations in AIRE and two chimeric AIRE constructs were generated. A luciferase reporter assay and a pulldown assay using biotinylated INS-class I VNTR probe were performed to examine the transactivation and binding activities of WT, mutant, and chimeric AIREs on the INS-VNTR promoter. Confocal microscopy analysis was performed for WT or mutant AIRE cellular localization. We found that all of the AIRE mutations resulted in loss of transcriptional activation of INS-VNTR except mutant P252L. Using WT/mutant AIRE heterozygous forms to modulate the INS-VNTR target revealed five mutations (R257X, G228W, C311fsX376, L397fsX478, and R433fsX502) that functioned in a dominant negative fashion. The LXXLL-3 motif is identified for the first time to be essential for DNA binding to INS-VNTR, whereas the intact PHD1, PHD2, LXXLL-3, and LXXLL-4 motifs were important for successful transcriptional activation. AIRE nuclear localization in the human thymic epithelial cell line was disrupted by mutations in the homogenously staining region domain and the R257X mutation in the PHD1 domain. This study supports the notion that AIRE mutation could specifically affect human insulin gene expression in thymic epithelial cells through INS-VNTR and subsequently induce either insulin tolerance or autoimmunity. PMID:27048654

  11. SMAD4 regulates cell motility through transcription of N-cadherin in human pancreatic ductal epithelium.

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    Ya'an Kang

    Full Text Available Expression of the cellular adhesion protein N-cadherin is a critical event during epithelial-mesenchymal transition (EMT. The SMAD4 protein has been identified as a mediator of transforming growth factor-β (TGF-β superfamily signaling, which regulates EMT, but the mechanisms linking TGF-β signaling to N-cadherin expression remain unclear. When the TGF-β pathway is activated, SMAD proteins, including the common mediator SMAD4, are subsequently translocated into the nucleus, where they influence gene transcription via SMAD binding elements (SBEs. Here we describe a mechanism for control of CDH2, the gene encoding N-cadherin, through the canonical TGFβ-SMAD4 pathway. We first identified four previously undescribed SBEs within the CDH2 promoter. Using telomerase immortalized human pancreatic ductal epithelium, we found that TGF-β stimulation prompted specific SMAD4 binding to all four SBEs. Luciferase reporter and SMAD4-knockdown experiments demonstrated that specific SMAD4 binding to the SBE located at -3790 bp to -3795 bp within the promoter region of CDH2 was necessary for TGF-β-stimulated transcription. Expression of N-cadherin on the surface of epithelial cells facilitates motility and invasion, and we demonstrated that knockdown of SMAD4 causes decreased N-cadherin expression, which results in diminished migration and invasion of human pancreatic ductal epithelial cells. Similar reduction of cell motility was produced after CDH2 knockdown. Together, these findings suggest that SMAD4 is critical for the TGF-β-driven upregulation of N-cadherin and the resultant invasive phenotype of human pancreatic ductal epithelial cells during EMT.

  12. Identification of transcription factor AML-1 binding site upstream of human cytomegalovirus UL111A gene.

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    Xiaoqun Zheng

    Full Text Available Human cytomegalovirus (HCMV interleukin-10 (hcmvIL-10, encoded by HCMV UL111A gene, is a homolog of human IL-10. It exerts immunomodulatory effects that allow HCMV to evade host defense mechanisms. However, the exact mechanism underlying the regulation of hcmvIL-10 expression is not well understood. The transcription factor acute myeloid leukemia 1 (AML-1 plays an important role in the regulation of various genes involved in the differentiation of hematopoietic lineages. A putative AML-1 binding site is present within the upstream regulatory region (URR of UL111A gene. To provide evidence that AML-1 is involved in regulating UL111A gene expression, we examined the interaction of AML-1 with the URR of UL111A in HCMV-infected human monocytic THP-1 cells using a chromatin immunoprecipitation assay. HcmvIL-10 transcription was detected in differentiated THP-1 cells, but not in undifferentiated ones. Furthermore, the URR of UL111A showed a higher intensity of AML-1 binding, a higher level of histone H3 acetyl-K9, but a lower level of histone H3 dimethyl-K9 in differentiated THP-1 cells than undifferentiated cells. Down-regulation of AML1 by RNA interference decreased the expression of the UL111A gene. Our results suggest that AML-1 may contribute to the epigenetic regulation of UL111A gene via histone modification in HCMV-infected differentiated THP-1 cells. This finding could be useful for the development of new anti-viral therapies.

  13. Functional Domains of Autoimmune Regulator (AIRE) Modulate INS-VNTR Transcription in Human Thymic Epithelial Cells.

    Science.gov (United States)

    Sparks, Avis E; Chen, Chiachen; Breslin, Mary B; Lan, Michael S

    2016-05-20

    INS-VNTR (insulin-variable number of tandem repeats) and AIRE (autoimmune regulator) have been associated with the modulation of insulin gene expression in thymus, which is essential to induce either insulin tolerance or the development of insulin autoimmunity and type 1 diabetes. We sought to analyze whether each functional domain of AIRE is critical for the activation of INS-VNTR in human thymic epithelial cells. Twelve missense or nonsense mutations in AIRE and two chimeric AIRE constructs were generated. A luciferase reporter assay and a pulldown assay using biotinylated INS-class I VNTR probe were performed to examine the transactivation and binding activities of WT, mutant, and chimeric AIREs on the INS-VNTR promoter. Confocal microscopy analysis was performed for WT or mutant AIRE cellular localization. We found that all of the AIRE mutations resulted in loss of transcriptional activation of INS-VNTR except mutant P252L. Using WT/mutant AIRE heterozygous forms to modulate the INS-VNTR target revealed five mutations (R257X, G228W, C311fsX376, L397fsX478, and R433fsX502) that functioned in a dominant negative fashion. The LXXLL-3 motif is identified for the first time to be essential for DNA binding to INS-VNTR, whereas the intact PHD1, PHD2, LXXLL-3, and LXXLL-4 motifs were important for successful transcriptional activation. AIRE nuclear localization in the human thymic epithelial cell line was disrupted by mutations in the homogenously staining region domain and the R257X mutation in the PHD1 domain. This study supports the notion that AIRE mutation could specifically affect human insulin gene expression in thymic epithelial cells through INS-VNTR and subsequently induce either insulin tolerance or autoimmunity.

  14. Transcriptional regulation of human polo-like kinases and early mitotic inhibitors

    Institute of Scientific and Technical Information of China (English)

    Moe Tategu; Hiroki Nakagawa; Kaori Sasaki; Rieko Yamauchi; Sota Sekimachi; Yuka Suita; Naoko Watanabe; Kenichi Yoshida

    2008-01-01

    Human polo-like kinases (PLK1-PLK4) have been implicated in mitotic regulation and carcinogenesis.PLK1 phosphorylates early mitotic inhibitor 1 (Emil) to ensure mitosis entry,whereas Emi2 plays a key role during the meiotic cell cycle.Transcription factor E2F is primarily considered to regulate the G1/S transition of the cell cycle but its involvement in the regulation of mitosis has also been recently suggested.A gap still exists between the molecular basis of E2F and mitotic regulation.The present study was designed to characterize the transcriptional regulation of human PLK and Emi genes.Adenoviral overexpression of E2F1 increased PLK1 and PLK3 mRNA levels in A549 cells.A reporter gene assay revealed that the putative promoter regions of PLK1,PLK3,and PLK4 genes were responsive to ac-tivators E2F,E2F1-E2F3.We further characterized the putative promoter regions of Emil and Emi2 genes,and these could be regulated by activators E2F and E2F1-E2F4,respectively.Finally,PLK1-PLK4,Emil,and Emi2 mRNA expression levels in human adult,fetal tis-sues,and several cell lines indicated that each gene has a unique expression pattern but is uniquely expressed in common tissues and cells such as the testes and thymus.Collectively,these results indicate that E2F can integrate G1/S and G2/Mto oscillate the cell cycle by regu-lating mitotic genes PLK and Emi,leading to determination of the cell fate.

  15. Noncytotoxic Suppression of Human Immunodeficiency Virus Type 1 Transcription by Exosomes Secreted from CD8+ T Cells▿

    OpenAIRE

    2009-01-01

    CD8+ T cells display a noncytotoxic activity that suppresses transcription of human immunodeficiency virus type 1 (HIV-1) in an antigen-independent and major histocompatibility complex-unrestricted manner. To date, the precise cellular and molecular factors mediating this CD8+ T-cell effector function remain unsolved. Despite evidence indicating the dependence of the activity on cell-cell contact, the possibility of a membrane-mediated activity that represses transcription from the viral prom...

  16. EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes

    OpenAIRE

    Sutter, Carrie Hayes; Yin, Hong; Li, Yunbo; Mammen, Jennifer S.; Bodreddigari, Sridevi; Stevens, Gaylene; Cole, Judith A; Sutter, Thomas R.

    2009-01-01

    Dioxin is an extremely potent carcinogen. In highly exposed people, the most commonly observed toxicity is chloracne, a pathological response of the skin. Most of the effects of dioxin are attributed to its activation of the aryl hydrocarbon receptor (AHR), a transcription factor that binds to the Ah receptor nuclear translocator (ARNT) to regulate the transcription of numerous genes, including CYP1A1 and CYP1B1. In cultures of normal human epidermal keratinocytes dioxin accelerates cell diff...

  17. Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor.

    Science.gov (United States)

    Mori, Seiichiro; Takeuchi, Takamasa; Ishii, Yoshiyuki; Yugawa, Takashi; Kiyono, Tohru; Nishina, Hiroshi; Kukimoto, Iwao

    2017-03-15

    The cytidine deaminase APOBEC3B (A3B) underlies the genetic heterogeneity of several human cancers, including cervical cancer, which is caused by human papillomavirus (HPV) infection. We previously identified a region within the A3B promoter that is activated by the viral protein HPV16 E6 in human keratinocytes. Here, we discovered three sites recognized by the TEAD family of transcription factors within this region of the A3B promoter. Reporter assays in HEK293 cells showed that exogenously expressed TEAD4 induced A3B promoter activation through binding to these sites. Normal immortalized human keratinocytes expressing E6 (NIKS-E6) displayed increased levels of TEAD1/4 protein compared to parental NIKS. A series of E6 mutants revealed that E6-mediated degradation of p53 was important for increasing TEAD4 levels. Knockdown of TEADs in NIKS-E6 significantly reduced A3B mRNA levels, whereas ectopic expression of TEAD4 in NIKS increased A3B mRNA levels. Finally, chromatin immunoprecipitation assays demonstrated increased levels of TEAD4 binding to the A3B promoter in NIKS-E6 compared to NIKS. Collectively, these results indicate that E6 induces upregulation of A3B through increased levels of TEADs, highlighting the importance of the TEAD-A3B axis in carcinogenesis.IMPORTANCE The expression of APOBEC3B (A3B), a cellular DNA cytidine deaminase, is upregulated in various human cancers and leaves characteristic, signature mutations in cancer genomes, suggesting that it plays a prominent role in carcinogenesis. Viral oncoproteins encoded by human papillomavirus (HPV) and polyomavirus have been reported to induce A3B expression, implying the involvement of A3B upregulation in virus-associated carcinogenesis. However, the molecular mechanisms causing A3B upregulation remain unclear. Here, we demonstrate that exogenous expression of the cellular transcription factor TEAD activates the A3B promoter. Further, the HPV oncoprotein E6 increases the levels of endogenous TEAD1

  18. HOXB5 cooperates with NKX2-1 in the transcription of human RET.

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    Jiang Zhu

    Full Text Available The enteric nervous system (ENS regulates peristaltic movement of the gut, and abnormal ENS causes Hirschsprung's disease (HSCR in newborns. HSCR is a congenital complex genetic disorder characterised by a lack of enteric ganglia along a variable length of the intestine. The receptor tyrosine kinase gene (RET is the major HSCR gene and its expression is crucial for ENS development. We have previously reported that (i HOXB5 transcription factor mediates RET expression, and (ii mouse with defective HOXB5 activity develop HSCR phenotype. In this study, we (i elucidate the underlying mechanisms that HOXB5 mediate RET expression, and (ii examine the interactions between HOXB5 and other transcription factors implicated in RET expression. We show that human HOXB5 binds to the promoter region 5' upstream of the binding site of NKX2-1 and regulates RET expression. HOXB5 and NKX2-1 form a protein complex and mediate RET expression in a synergistic manner. HSCR associated SNPs at the NKX2-1 binding site (-5G>A rs10900296; -1A>C rs10900297, which reduce NKX2-1 binding, abolish the synergistic trans-activation of RET by HOXB5 and NKX2-1. In contrast to the synergistic activation of RET with NKX2-1, HOXB5 cooperates in an additive manner with SOX10, PAX3 and PHOX2B in trans-activation of RET promoter. Taken together, our data suggests that HOXB5 in coordination with other transcription factors mediates RET expression. Therefore, defects in cis- or trans-regulation of RET by HOXB5 could lead to reduction of RET expression and contribute to the manifestation of the HSCR phenotype.

  19. Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities

    Science.gov (United States)

    Jolma, Arttu; Kivioja, Teemu; Toivonen, Jarkko; Cheng, Lu; Wei, Gonghong; Enge, Martin; Taipale, Mikko; Vaquerizas, Juan M.; Yan, Jian; Sillanpää, Mikko J.; Bonke, Martin; Palin, Kimmo; Talukder, Shaheynoor; Hughes, Timothy R.; Luscombe, Nicholas M.; Ukkonen, Esko; Taipale, Jussi

    2010-01-01

    The genetic code—the binding specificity of all transfer-RNAs—defines how protein primary structure is determined by DNA sequence. DNA also dictates when and where proteins are expressed, and this information is encoded in a pattern of specific sequence motifs that are recognized by transcription factors. However, the DNA-binding specificity is only known for a small fraction of the ∼1400 human transcription factors (TFs). We describe here a high-throughput method for analyzing transcription factor binding specificity that is based on systematic evolution of ligands by exponential enrichment (SELEX) and massively parallel sequencing. The method is optimized for analysis of large numbers of TFs in parallel through the use of affinity-tagged proteins, barcoded selection oligonucleotides, and multiplexed sequencing. Data are analyzed by a new bioinformatic platform that uses the hundreds of thousands of sequencing reads obtained to control the quality of the experiments and to generate binding motifs for the TFs. The described technology allows higher throughput and identification of much longer binding profiles than current microarray-based methods. In addition, as our method is based on proteins expressed in mammalian cells, it can also be used to characterize DNA-binding preferences of full-length proteins or proteins requiring post-translational modifications. We validate the method by determining binding specificities of 14 different classes of TFs and by confirming the specificities for NFATC1 and RFX3 using ChIP-seq. Our results reveal unexpected dimeric modes of binding for several factors that were thought to preferentially bind DNA as monomers. PMID:20378718

  20. Eos negatively regulates human γ-globin gene transcription during erythroid differentiation.

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    Hai-Chuan Yu

    Full Text Available BACKGROUND: Human globin gene expression is precisely regulated by a complicated network of transcription factors and chromatin modifying activities during development and erythropoiesis. Eos (Ikaros family zinc finger 4, IKZF4, a member of the zinc finger transcription factor Ikaros family, plays a pivotal role as a repressor of gene expression. The aim of this study was to examine the role of Eos in globin gene regulation. METHODOLOGY/PRINCIPAL FINDINGS: Western blot and quantitative real-time PCR detected a gradual decrease in Eos expression during erythroid differentiation of hemin-induced K562 cells and Epo-induced CD34+ hematopoietic stem/progenitor cells (HPCs. DNA transfection and lentivirus-mediated gene transfer demonstrated that the enforced expression of Eos significantly represses the expression of γ-globin, but not other globin genes, in K562 cells and CD34+ HPCs. Consistent with a direct role of Eos in globin gene regulation, chromatin immunoprecipitaion and dual-luciferase reporter assays identified three discrete sites located in the DNase I hypersensitivity site 3 (HS3 of the β-globin locus control region (LCR, the promoter regions of the Gγ- and Aγ- globin genes, as functional binding sites of Eos protein. A chromosome conformation capture (3C assay indicated that Eos may repress the interaction between the LCR and the γ-globin gene promoter. In addition, erythroid differentiation was inhibited by enforced expression of Eos in K562 cells and CD34+ HPCs. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that Eos plays an important role in the transcriptional regulation of the γ-globin gene during erythroid differentiation.

  1. Open chromatin mapping identifies transcriptional networks regulating human epididymis epithelial function.

    Science.gov (United States)

    Browne, James A; Yang, Rui; Song, Lingyun; Crawford, Gregory E; Leir, Shih-Hsing; Harris, Ann

    2014-12-01

    The epithelium lining the epididymis in the male reproductive tract maintains a luminal environment that promotes sperm cell maturation. This process is dependent on the coordinated expression of many genes that encode proteins with a role in epithelial transport. We previously generated genome-wide maps of open chromatin in primary human epididymis epithelial (HEE) cells to identify potential regulatory elements controlling coordinated gene expression in the epididymis epithelium. Subsequent in silico analysis identified transcription factor-binding sites (TFBS) that were over-represented in the HEE open chromatin, including the motif for paired box 2 (PAX2). PAX2 is a critical transcriptional regulator of urogenital tract development, which has been well studied in the kidney but is unexplored in the epididymis. Due to the limited lifespan of primary HEE cells in culture, we investigated the role of PAX2 in an immortalized HEE cell line (REP). First, REP cells were evaluated by DNase I digestion followed by high-throughput sequencing and the PAX2-binding motif was again identified as an over-represented TFBS within intergenic open chromatin, though on fewer chromosomes than in the primary HEE cells. To identify PAX2-target genes in REP cells, RNA-seq analysis was performed after siRNA-mediated depletion of PAX2 and compared with that with a non-targeting siRNA. In response to PAX2-repression, 3135 transcripts were differentially expressed (1333 up-regulated and 1802 down-regulated). Novel PAX2 targets included multiple genes encoding proteins with predicted functions in the epididymis epithelium.

  2. Prostate specific membrane antigen (PSM) is expressed in various human tissues: implication for the use of PSM reverse transcription polymerase chain reaction to detect hematogenous prostate cancer spread.

    Science.gov (United States)

    Renneberg, H; Friedetzky, A; Konrad, L; Kurek, R; Weingärtner, K; Wennemuth, G; Tunn, U W; Aumüller, G

    1999-01-01

    Detection of prostate-specific membrane antigen (PSM)-mRNA expression in blood samples using reverse transcription polymerase chain reaction (RT-PCR) is discussed as a new diagnostic marker of circulating micrometastases in prostate cancer patients. We applied the RT-PCR technique to different human tissues and obtained positive signals for PSM transcripts in human genital and multiple extra-genital tissue sites. The cDNAs were prepared from different human tissues and prostatic cell lines. RT-PCR and nested RT-PCR for PSM was performed with primers derived from the published PSM cDNA. The RT-PCR fragments obtained were cloned and showed 100% sequence homology to PSM. Southern blot hybridization with labeled probes was used to confirm the specificity of the amplicons. In addition to the known PSM expression in the human brain, PSM-mRNA was detected in cDNA isolated from human testis, epididymis and seminal vesicles and in the PC-3 prostatic cancer cell line. Furthermore, we found PSM-mRNA in heart, liver, lung, kidney, spleen, and thyroid gland. The results indicate that PSM expression is not restricted to the prostate gland, but represents a more general component of genital and extra-genital human tissues. This must be considered when RT-PCR and nested RT-PCR screening for PSM expression is performed as a diagnostic measure in blood from prostate cancer patients.

  3. Transcriptional regulation of the human acid alpha-glucosidase gene. Identification of a repressor element and its transcription factors Hes-1 and YY1.

    Science.gov (United States)

    Yan, B; Heus, J; Lu, N; Nichols, R C; Raben, N; Plotz, P H

    2001-01-19

    Acid alpha-glucosidase, the product of a housekeeping gene, is a lysosomal enzyme that degrades glycogen. A deficiency of this enzyme is responsible for a recessively inherited myopathy and cardiomyopathy, glycogenesis type II. We have previously demonstrated that the human acid alpha-glucosidase gene expression is regulated by a silencer within intron 1, which is located in the 5'-untranslated region. In this study, we have used deletion analysis, electrophoretic mobility shift assay, and footprint analysis to further localize the silencer to a 25-base pair element. The repressive effect on the TK promoter was about 50% in both orientations in expression plasmid, and two transcriptional factors were identified with antibodies binding specifically to the element. Mutagenesis and functional analyses of the element demonstrated that the mammalian homologue 1 of Drosophila hairy and Enhancer of split (Hes-1) binding to an E box (CACGCG) and global transcription factor-YY1 binding to its core site function as a transcriptional repressor. Furthermore, the overexpression of Hes-1 significantly enhanced the repressive effect of the silencer element. The data should be helpful in understanding the expression and regulation of the human acid alpha-glucosidase gene as well as other lysosomal enzyme genes.

  4. Identification of Tissue-Specific Protein-Coding and Noncoding Transcripts across 14 Human Tissues Using RNA-seq.

    Science.gov (United States)

    Zhu, Jinhang; Chen, Geng; Zhu, Sibo; Li, Suqing; Wen, Zhuo; Bin Li; Zheng, Yuanting; Shi, Leming

    2016-06-22

    Many diseases and adverse drug reactions exhibit tissue specificity. To better understand the tissue-specific expression characteristics of transcripts in different human tissues, we deeply sequenced RNA samples from 14 different human tissues. After filtering many lowly expressed transcripts, 24,729 protein-coding transcripts and 1,653 noncoding transcripts were identified. By analyzing highly expressed tissue-specific protein-coding transcripts (TSCTs) and noncoding transcripts (TSNTs), we found that testis expressed the highest numbers of TSCTs and TSNTs. Brain, monocytes, ovary, and heart expressed more TSCTs than the rest tissues, whereas brain, placenta, heart, and monocytes expressed more TSNTs than other tissues. Co-expression network constructed based on the TSCTs and TSNTs showed that each hub TSNT was co-expressed with several TSCTs, allowing functional annotation of TSNTs. Important biological processes and KEGG pathways highly related to the specific functions or diseases of each tissue were enriched with the corresponding TSCTs. These TSCTs and TSNTs may participate in the tissue-specific physiological or pathological processes. Our study provided a unique data set and systematic analysis of expression characteristics and functions of both TSCTs and TSNTs based on 14 distinct human tissues, and could facilitate future investigation of the mechanisms behind tissue-specific diseases and adverse drug reactions.

  5. Comparative transcriptional analysis of human macrophages exposed to animal and human isolates of Mycobacterium avium subspecies paratuberculosis with diverse genotypes.

    Science.gov (United States)

    Motiwala, Alifiya S; Janagama, Harish K; Paustian, Michael L; Zhu, Xiaochun; Bannantine, John P; Kapur, Vivek; Sreevatsan, Srinand

    2006-11-01

    Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in animals and has been hypothesized to be associated with Crohn's disease in humans. Recently, M. avium subsp. paratuberculosis isolates recovered from Crohn's disease patients were shown to have limited diversity, implying the existence of human disease-associated genotypes and strain sharing with animals (A. H. Ghadiali et al., J. Clin. Microbiol. 42:5345-5348, 2004). To explore whether these genotypic differences or similarities among human and animal isolates translated to functionally significant attributes such as variance in host preference and/or difference in magnitude of infections, we performed a global scale analysis of M. avium subsp. paratuberculosis isolates that were representative of different genotypes and host species using DNA microarrays. Genome-wide characterization of the transcriptional changes was carried out using a human monocytic cell line (THP-1 cells) in response to different genotypes of M. avium subsp. paratuberculosis isolates recovered from various hosts. We identified several differentially expressed genes during early intracellular infection, including those involved in common canonical pathways such as NF-kappaB, interleukin-6 (IL-6), mitogen-activated protein kinase/extracellular signal-regulated kinase, and Jun N-terminal protein kinase signaling, as well as genes involved in T helper type 1 (Th1) responses (such as CCL5 ligand) and those that encode several proinflammatory cytokines and chemokine receptors. The cattle and human isolates of M. avium subsp. paratuberculosis, regardless of their short sequence repeat (SSR) genotype, induced similar global gene expression patterns in THP-1 cells. They differentially regulated genes necessary for cell survival without causing major alterations in proinflammatory genes. In contrast, the sheep isolates representing diverse SSR genotypes closely resembled the global gene expression pattern of an M

  6. Simplified method to predict mutual interactions of human transcription factors based on their primary structure

    KAUST Repository

    Schmeier, Sebastian

    2011-07-05

    Background: Physical interactions between transcription factors (TFs) are necessary for forming regulatory protein complexes and thus play a crucial role in gene regulation. Currently, knowledge about the mechanisms of these TF interactions is incomplete and the number of known TF interactions is limited. Computational prediction of such interactions can help identify potential new TF interactions as well as contribute to better understanding the complex machinery involved in gene regulation. Methodology: We propose here such a method for the prediction of TF interactions. The method uses only the primary sequence information of the interacting TFs, resulting in a much greater simplicity of the prediction algorithm. Through an advanced feature selection process, we determined a subset of 97 model features that constitute the optimized model in the subset we considered. The model, based on quadratic discriminant analysis, achieves a prediction accuracy of 85.39% on a blind set of interactions. This result is achieved despite the selection for the negative data set of only those TF from the same type of proteins, i.e. TFs that function in the same cellular compartment (nucleus) and in the same type of molecular process (transcription initiation). Such selection poses significant challenges for developing models with high specificity, but at the same time better reflects real-world problems. Conclusions: The performance of our predictor compares well to those of much more complex approaches for predicting TF and general protein-protein interactions, particularly when taking the reduced complexity of model utilisation into account. © 2011 Schmeier et al.

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

    KAUST Repository

    Kulakovskiy, Ivan V.

    2012-11-21

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

  8. A comprehensive resource of interacting protein regions for refining human transcription factor networks.

    Directory of Open Access Journals (Sweden)

    Etsuko Miyamoto-Sato

    Full Text Available Large-scale data sets of protein-protein interactions (PPIs are a valuable resource for mapping and analysis of the topological and dynamic features of interactome networks. The currently available large-scale PPI data sets only contain information on interaction partners. The data presented in this study also include the sequences involved in the interactions (i.e., the interacting regions, IRs suggested to correspond to functional and structural domains. Here we present the first large-scale IR data set obtained using mRNA display for 50 human transcription factors (TFs, including 12 transcription-related proteins. The core data set (966 IRs; 943 PPIs displays a verification rate of 70%. Analysis of the IR data set revealed the existence of IRs that interact with multiple partners. Furthermore, these IRs were preferentially associated with intrinsic disorder. This finding supports the hypothesis that intrinsically disordered regions play a major role in the dynamics and diversity of TF networks through their ability to structurally adapt to and bind with multiple partners. Accordingly, this domain-based interaction resource represents an important step in refining protein interactions and networks at the domain level and in associating network analysis with biological structure and function.

  9. Transcriptional analysis of early lineage commitment in human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Wormald Sam

    2007-03-01

    Full Text Available Abstract Background The mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood. In fact, even our knowledge of the phenotype of hESC is limited, because the immunological and molecular criteria presently used to define this phenotype describe the properties of a heterogeneous population of cells. Results We used a novel approach combining immunological and transcriptional analysis (immunotranscriptional profiling to compare gene expression in hESC populations at very early stages of differentiation. Immunotranscriptional profiling enabled us to identify novel markers of stem cells and their differentiated progeny, as well as novel potential regulators of hESC commitment and differentiation. The data show clearly that genes associated with the pluripotent state are downregulated in a coordinated fashion, and that they are co-expressed with lineage specific transcription factors in a continuum during the early stages of stem cell differentiation. Conclusion These findings, that show that maintenance of pluripotency and lineage commitment are dynamic, interactive processes in hESC cultures, have important practical implications for propagation and directed differentiation of these cells, and for the interpretation of mechanistic studies of hESC renewal and commitment. Since embryonic stem cells at defined stages of commitment can be isolated in large numbers by immunological means, they provide a powerful model for studying molecular genetics of stem cell commitment in the embryo.

  10. Transcriptional analysis of early lineage commitment in human embryonic stem cells

    Science.gov (United States)

    Laslett, Andrew L; Grimmond, Sean; Gardiner, Brooke; Stamp, Lincon; Lin, Adelia; Hawes, Susan M; Wormald, Sam; Nikolic-Paterson, David; Haylock, David; Pera, Martin F

    2007-01-01

    Background The mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood. In fact, even our knowledge of the phenotype of hESC is limited, because the immunological and molecular criteria presently used to define this phenotype describe the properties of a heterogeneous population of cells. Results We used a novel approach combining immunological and transcriptional analysis (immunotranscriptional profiling) to compare gene expression in hESC populations at very early stages of differentiation. Immunotranscriptional profiling enabled us to identify novel markers of stem cells and their differentiated progeny, as well as novel potential regulators of hESC commitment and differentiation. The data show clearly that genes associated with the pluripotent state are downregulated in a coordinated fashion, and that they are co-expressed with lineage specific transcription factors in a continuum during the early stages of stem cell differentiation. Conclusion These findings, that show that maintenance of pluripotency and lineage commitment are dynamic, interactive processes in hESC cultures, have important practical implications for propagation and directed differentiation of these cells, and for the interpretation of mechanistic studies of hESC renewal and commitment. Since embryonic stem cells at defined stages of commitment can be isolated in large numbers by immunological means, they provide a powerful model for studying molecular genetics of stem cell commitment in the embryo. PMID:17335568

  11. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors

    Energy Technology Data Exchange (ETDEWEB)

    Yakubov, Eduard [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Rechavi, Gidi [Cancer Research Center, Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rozenblatt, Shmuel [Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Tel-Aviv (Israel); Givol, David, E-mail: david.givol@weizmann.ac.il [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel)

    2010-03-26

    Reprogramming of differentiated cells into induced pluripotent cells (iPS) was accomplished in 2006 by expressing four, or less, embryonic stem cell (ESC)-specific transcription factors. Due to the possible danger of DNA damage and the potential tumorigenicity associated with such DNA damage, attempts were made to minimize DNA integration by the vectors involved in this process without complete success. Here we present a method of using RNA transfection as a tool for reprogramming human fibroblasts to iPS. We used RNA synthesized in vitro from cDNA of the same reprogramming four transcription factors. After transfection of the RNA, we show intracellular expression and nuclear localization of the respective proteins in at least 70% of the cells. We used five consecutive transfections to support continuous protein expression resulting in the formation of iPS colonies that express alkaline phosphatase and several ESC markers and that can be expanded. This method completely avoids DNA integration and may be developed to replace the use of DNA vectors in the formation of iPS.

  12. Circulating Human Eosinophils Share a Similar Transcriptional Profile in Asthma and Other Hypereosinophilic Disorders

    Science.gov (United States)

    Barnig, Cindy; Dembélé, Doulaye; Paul, Nicodème; Poirot, Anh; Uring-Lambert, Béatrice; Georgel, Philippe; de Blay, Fréderic; Bahram, Seiamak

    2015-01-01

    Eosinophils are leukocytes that are released into the peripheral blood in a phenotypically mature state and are capable of being recruited into tissues in response to appropriate stimuli. Eosinophils, traditionally considered cytotoxic effector cells, are leukocytes recruited into the airways of asthma patients where they are believed to contribute to the development of many features of the disease. This perception, however, has been challenged by recent findings suggesting that eosinophils have also immunomodulatory functions and may be involved in tissue homeostasis and wound healing. Here we describe a transcriptome-based approach–in a limited number of patients and controls—to investigate the activation state of circulating human eosinophils isolated by flow cytometry. We provide an overview of the global expression pattern in eosinophils in various relevant conditions, e.g., eosinophilic asthma, hypereosinophilic dermatological diseases, parasitosis and pulmonary aspergillosis. Compared to healthy subjects, circulating eosinophils isolated from asthma patients differed in their gene expression profile which is marked by downregulation of transcripts involved in antigen presentation, pathogen recognition and mucosal innate immunity, whereas up-regulated genes were involved in response to non-specific stimulation, wounding and maintenance of homeostasis. Eosinophils from other hypereosinophilic disorders displayed a very similar transcriptional profile. Taken together, these observations seem to indicate that eosinophils exhibit non-specific immunomodulatory functions important for tissue repair and homeostasis and suggest new roles for these cells in asthma immunobiology. PMID:26524763

  13. Transcripts from a novel human KRAB zinc finger gene contain spliced Alu and endogenous retroviral segments

    Energy Technology Data Exchange (ETDEWEB)

    Baban, S.; Freeman, J.D.; Mager, D.L. [Univ. of British Columbia, Vancouver, British Columbia (Canada)

    1996-05-01

    During the course of an investigation into the potential effects of endogenous retroviruses on adjacent gene expression, we isolated two cDNA clones containing a small sequence segment belonging to the human endogenous retrovirus family, HERV-H. Characterization of the clones revealed that they represent transcripts from a novel KRAB zinc finger gene termed ZNF177. The two cDNA clones differ at their 5{prime} termini and in the presence of a 559-bp internal exon. The clone containing this internal exon has six imperfect zinc finger motifs followed by seven perfect copies of the C{sub 2}H{sub 2} type but has a frame shift between the KRAB domain and the downstream zinc finger region. The smaller clone lacks the six imperfect motifs and has an intact ORF. The 5{prime} putative untranslated regions of both cDNAs contain an 86-bp HERV-H env segment and a segment of an Alu repeat, both in the antisense orientation, that have been incorporated by splicing. RT-PCR experiments show evidence of alternative splicing but the majority of transcripts appear to contain the Alu and env segments. Genomic PCR and hybridization experiments suggest that a partial HERV-H element is integrated within the ZNF177 locus, which Southern analysis has shown to be a single-copy gene. Northern and RT-PCR analyses suggest that ZNF177 is transcribed at a low level in a variety of cell types. 41 refs., 8 figs.

  14. An inducible transcription factor activates expression of human immunodeficiency virus in T cells

    Science.gov (United States)

    Nabel, Gary; Baltimore, David

    1987-04-01

    Human immunodeficiency virus (HIV) production from latently infected T lymphocytes can be induced with compounds that activate the cells to secrete lymphokines1,2. The elements in the HIV genome which control activation are not known but expression might be regulated through a variety of DNA elements. The cis-acting control elements of the viral genome are enhancer and promoter regions. The virus also encodes trans-acting factors specified by the tat-III (refs 3-6) and art genes7. We have examined whether products specific to activated T cells might stimulate viral transcription by binding to regions on viral DNA. Activation of T cells, which increases HIV expression up to 50-fold, correlated with induction of a DNA binding protein indistinguishable from a recognized transcription factor, called NF-κB (ref. 8), with binding sites in the viral enhancer. Mutation of these binding sites abolished inducibility. That NF-κB acts in synergy with the viral tat-III gene product to enhance HIV expression in T cells may have implications for the pathogenesis of AIDS (acquired immune deficiency syndrome).

  15. The human enhancer blocker CTC-binding factor interacts with the transcription factor Kaiso.

    Science.gov (United States)

    Defossez, Pierre-Antoine; Kelly, Kevin F; Filion, Guillaume J P; Pérez-Torrado, Roberto; Magdinier, Frédérique; Menoni, Hervé; Nordgaard, Curtis L; Daniel, Juliet M; Gilson, Eric

    2005-12-30

    CTC-binding factor (CTCF) is a DNA-binding protein of vertebrates that plays essential roles in regulating genome activity through its capacity to act as an enhancer blocker. We performed a yeast two-hybrid screen to identify protein partners of CTCF that could regulate its activity. Using full-length CTCF as bait we recovered Kaiso, a POZ-zinc finger transcription factor, as a specific binding partner. The interaction occurs through a C-terminal region of CTCF and the POZ domain of Kaiso. CTCF and Kaiso are co-expressed in many tissues, and CTCF was specifically co-immunoprecipitated by several Kaiso monoclonal antibodies from nuclear lysates. Kaiso is a bimodal transcription factor that recognizes methylated CpG dinucleotides or a conserved unmethylated sequence (TNGCAGGA, the Kaiso binding site). We identified one consensus unmethylated Kaiso binding site in close proximity to the CTCF binding site in the human 5' beta-globin insulator. We found, in an insulation assay, that the presence of this Kaiso binding site reduced the enhancer-blocking activity of CTCF. These data suggest that the Kaiso-CTCF interaction negatively regulates CTCF insulator activity.

  16. Regulation of Human Cytomegalovirus Transcription in Latency: Beyond the Major Immediate-Early Promoter

    Directory of Open Access Journals (Sweden)

    John Sinclair

    2013-06-01

    Full Text Available Lytic infection of differentiated cell types with human cytomegalovirus (HCMV results in the temporal expression of between 170–200 open reading frames (ORFs. A number of studies have demonstrated the temporal regulation of these ORFs and that this is orchestrated by both viral and cellular mechanisms associated with the co-ordinated recruitment of transcription complexes and, more recently, higher order chromatin structure. Importantly, HCMV, like all herpes viruses, establishes a lifelong latent infection of the host—one major site of latency being the undifferentiated haematopoietic progenitor cells resident in the bone marrow. Crucially, the establishment of latency is concomitant with the recruitment of cellular enzymes that promote extensive methylation of histones bound to the major immediate early promoter. As such, the repressive chromatin structure formed at the major immediate early promoter (MIEP elicits inhibition of IE gene expression and is a major factor involved in maintenance of HCMV latency. However, it is becoming increasingly clear that a distinct subset of viral genes is also expressed during latency. In this review, we will discuss the mechanisms that control the expression of these latency-associated transcripts and illustrate that regulation of these latency-associated promoters is also subject to chromatin mediated regulation and that the instructive observations previously reported regarding the negative regulation of the MIEP during latency are paralleled in the regulation of latent gene expression.

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

    Science.gov (United States)

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

    2013-01-01

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

  18. System-wide analysis of the transcriptional network of human myelomonocytic leukemia cells predicts attractor structure and phorbol-ester-induced differentiation and dedifferentiation transitions

    Science.gov (United States)

    Sakata, Katsumi; Ohyanagi, Hajime; Sato, Shinji; Nobori, Hiroya; Hayashi, Akiko; Ishii, Hideshi; Daub, Carsten O.; Kawai, Jun; Suzuki, Harukazu; Saito, Toshiyuki

    2015-02-01

    We present a system-wide transcriptional network structure that controls cell types in the context of expression pattern transitions that correspond to cell type transitions. Co-expression based analyses uncovered a system-wide, ladder-like transcription factor cluster structure composed of nearly 1,600 transcription factors in a human transcriptional network. Computer simulations based on a transcriptional regulatory model deduced from the system-wide, ladder-like transcription factor cluster structure reproduced expression pattern transitions when human THP-1 myelomonocytic leukaemia cells cease proliferation and differentiate under phorbol myristate acetate stimulation. The behaviour of MYC, a reprogramming Yamanaka factor that was suggested to be essential for induced pluripotent stem cells during dedifferentiation, could be interpreted based on the transcriptional regulation predicted by the system-wide, ladder-like transcription factor cluster structure. This study introduces a novel system-wide structure to transcriptional networks that provides new insights into network topology.

  19. Two P2X1 receptor transcripts able to form functional channels are present in most human monocytes.

    Science.gov (United States)

    López-López, Cintya; Jaramillo-Polanco, Josue; Portales-Pérez, Diana P; Gómez-Coronado, Karen S; Rodríguez-Meléndez, Jessica G; Cortés-García, Juan D; Espinosa-Luna, Rosa; Montaño, Luis M; Barajas-López, Carlos

    2016-12-15

    To characterize the presence and general properties of P2X1 receptors in single human monocytes we used RT-PCR, flow cytometry, and the patch-clamp and the two-electrode voltage-clamp techniques. Most human monocytes expressed the canonical P2X1 (90%) and its splicing variant P2X1del (88%) mRNAs. P2X1 receptor immunoreactivity was also observed in 70% of these cells. Currents mediated by P2X1 (EC50=1.9±0.8µm) and P2X1del (EC50 >1000µm) channels, expressed in Xenopus leavis oocytes, have different ATP sensitivity and kinetics. Both currents mediated by P2X1 and P2X1del channels kept increasing during the continuous presence of high ATP concentrations. Currents mediated by the native P2X1 receptors in human monocytes showed an EC50=6.3±0.2µm. Currents have kinetics that resemble those observed for P2X1 and P2X1del receptors in oocytes. Our study is the first to demonstrate the expression of P2X1 transcript and its splicing variant P2X1del in most human monocytes. We also, for the first time, described functional homomeric P2X1del channels and demonstrated that currents mediated by P2X1 or P2X1del receptors, during heterologous expression, increased in amplitude when activated with high ATP concentrations in a similar fashion to those channels that increase their conductance under similar conditions, such as P2X7, P2X2, and P2X4 channels.

  20. (Some) cellular mechanisms influencing the transcription of human endogenous retrovirus, HERV-Fc1

    DEFF Research Database (Denmark)

    Laska, Magdalena Janina; Nissen, Kari Konstantin; Nexø, Bjørn Andersen

    2013-01-01

    DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. DNA methylation is considered an important mechanism for silencing of retroelements in the mammalian genome. However, the methylation of human endogenous retroviruses (HERVs) is not well...... investigated. The aim of this study was to investigate the transcriptional potential of HERV-Fc1 proviral 5'LTR in more detail, and examined the specific influence of CpG methylation on this LTR in number of cell lines. Specifically, the role of demethylating chemicals e.g. 5-aza-2' deoxycytidine...... and Trichostatin-A, in inducing or reactivating expression of HERV-Fc1 specific sequences and the mechanisms were investigated. In our present study, 5-aza-dC is shown to be a powerful inducer of HERV-Fc1, and at the same time it strongly inhibits methylation of DNA. Treatment with this demethylating agent 5-aza...

  1. Molecular diversity of L-type Ca2+ channel transcripts in human fibroblasts.

    Science.gov (United States)

    Soldatov, N M

    1992-05-15

    The nucleotide sequence of cDNA encoding the human fibroblast Ca2+ channel of L type (HFCC) has been determined. It is highly homologous to L-type channels previously cloned from rabbit lung and heart as well as from rat brain. At least four sites of molecular diversity were identified in the nucleotide sequence of HFCC. Three of these include regions encoding the transmembrane segments IIS6, IIIS2, and IVS3, which are known to be important for channel gating properties. The positions of these sites correlate with RNA splice sites, indicating that the molecular diversity of the transcripts is a result of alternative splicing. The fourth diversity region is located at the C-terminal region and comprises insertions and deletions. It is suggested that these variations may give rise to multiple subforms of HFCC with altered electrophysiological properties.

  2. Alternative spliced CD1d transcripts in human bronchial epithelial cells.

    Directory of Open Access Journals (Sweden)

    Kambez Hajipouran Benam

    Full Text Available CD1d is a MHC I like molecule which presents glycolipid to natural killer T (NKT cells, a group of cells with diverse but critical immune regulatory functions in the immune system. These cells are required for optimal defence against bacterial, viral, protozoan, and fungal infections, and control of immune-pathology and autoimmune diseases. CD1d is expressed on antigen presenting cells but also found on some non-haematopoietic cells. However, it has not been observed on bronchial epithelium, a site of active host defence in the lungs. Here, we identify for the first time, CD1D mRNA variants and CD1d protein expression on human bronchial epithelial cells, describe six alternatively spliced transcripts of this gene in these cells; and show that these variants are specific to epithelial cells. These findings provide the basis for investigations into a role for CD1d in lung mucosal immunity.

  3. Type 2 diabetes and obesity induce similar transcriptional reprogramming in human myocytes

    DEFF Research Database (Denmark)

    Väremo, Leif; Henriksen, Tora Ida; Scheele, Camilla

    2017-01-01

    BACKGROUND: Skeletal muscle is one of the primary tissues involved in the development of type 2 diabetes (T2D). The close association between obesity and T2D makes it difficult to isolate specific effects attributed to the disease alone. Therefore, here we set out to identify and characterize...... intrinsic properties of myocytes, associated independently with T2D or obesity. METHODS: We generated and analyzed RNA-seq data from primary differentiated myotubes from 24 human subjects, using a factorial design (healthy/T2D and non-obese/obese), to determine the influence of each specific factor...... on genome-wide transcription. This setup enabled us to identify intrinsic properties, originating from muscle precursor cells and retained in the corresponding myocytes. Bioinformatic and statistical methods, including differential expression analysis, gene-set analysis, and metabolic network analysis, were...

  4. Identification of transcription factors that promote the differentiation of human pluripotent stem cells into lacrimal gland epithelium-like cells.

    Science.gov (United States)

    Hirayama, Masatoshi; Ko, Shigeru B H; Kawakita, Tetsuya; Akiyama, Tomohiko; Goparaju, Sravan K; Soma, Atsumi; Nakatake, Yuhki; Sakota, Miki; Chikazawa-Nohtomi, Nana; Shimmura, Shigeto; Tsubota, Kazuo; Ko, Minoru S H

    2017-01-01

    Dry eye disease is the most prevalent pathological condition in aging eyes. One potential therapeutic strategy is the transplantation of lacrimal glands, generated in vitro from pluripotent stem cells such as human embryonic stem cells, into patients. One of the preceding requirements is a method to differentiate human embryonic stem cells into lacrimal gland epithelium cells. As the first step for this approach, this study aims to identify a set of transcription factors whose overexpression can promote the differentiation of human embryonic stem cells into lacrimal gland epithelium-like cells. We performed microarray analyses of lacrimal glands and lacrimal glands-related organs obtained from mouse embryos and adults, and identified transcription factors enriched in lacrimal gland epithelium cells. We then transfected synthetic messenger RNAs encoding human orthologues of these transcription factors into human embryonic stem cells and examined whether the human embryonic stem cells differentiate into lacrimal gland epithelium-like cells by assessing cell morphology and marker gene expression. The microarray analysis of lacrimal glands tissues identified 16 transcription factors that were enriched in lacrimal gland epithelium cells. We focused on three of the transcription factors, because they are expressed in other glands such as salivary glands and are also known to be involved in the development of lacrimal glands. We tested the overexpression of various combinations of the three transcription factors and PAX6, which is an indispensable gene for lacrimal glands development, in human embryonic stem cells. Combining PAX6, SIX1, and FOXC1 caused significant changes in morphology, i.e., elongated cell shape and increased expression (both RNAs and proteins) of epithelial markers such as cytokeratin15, branching morphogenesis markers such as BARX2, and lacrimal glands markers such as aquaporin5 and lactoferrin. We identified a set of transcription factors enriched in

  5. Comparative Analysis of Gene Regulation by the Transcription Factor PPARα between Mouse and Human

    Science.gov (United States)

    Rakhshandehroo, Maryam; Hooiveld, Guido; Müller, Michael; Kersten, Sander

    2009-01-01

    Background Studies in mice have shown that PPARα is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARα in human liver. Here we set out to compare the function of PPARα in mouse and human hepatocytes via analysis of target gene regulation. Methodology/Principal Findings Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARα agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARα expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 significantly induced the expression of 362–672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARα in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARα targets, including CPT1A, HMGCS2, FABP1, ACSL1, and ADFP. Several genes were identified that were specifically induced by PPARα in human (MBL2, ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4). Furthermore, several putative novel PPARα targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8. Conclusions/Significance Our results suggest that PPARα activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARα as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARα regulates a mostly divergent set of genes in mouse and human hepatocytes. PMID:19710929

  6. Comparative analysis of gene regulation by the transcription factor PPARalpha between mouse and human.

    Directory of Open Access Journals (Sweden)

    Maryam Rakhshandehroo

    Full Text Available BACKGROUND: Studies in mice have shown that PPARalpha is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARalpha in human liver. Here we set out to compare the function of PPARalpha in mouse and human hepatocytes via analysis of target gene regulation. METHODOLOGY/PRINCIPAL FINDINGS: Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARalpha agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARalpha expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 significantly induced the expression of 362-672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARalpha in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARalpha targets, including CPT1A, HMGCS2, FABP1, ACSL1, and ADFP. Several genes were identified that were specifically induced by PPARalpha in human (MBL2, ALAS1, CYP1A1, TSKU or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4. Furthermore, several putative novel PPARalpha targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PPARalpha activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARalpha as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARalpha regulates a mostly divergent set of genes in mouse and

  7. Methylation and Transcripts Expression at the Imprinted GNAS Locus in Human Embryonic and Induced Pluripotent Stem Cells and Their Derivatives

    Directory of Open Access Journals (Sweden)

    Virginie Grybek

    2014-09-01

    Full Text Available Data from the literature indicate that genomic imprint marks are disturbed in human pluripotent stem cells (PSCs. GNAS is an imprinted locus that produces one biallelic (Gsα and four monoallelic (NESP55, GNAS-AS1, XLsα, and A/B transcripts due to differential methylation of their promoters (DMR. To document imprinting at the GNAS locus in PSCs, we studied GNAS locus DMR methylation and transcript (NESP55, XLsα, and A/B expression in human embryonic stem cells (hESCs and human induced pluripotent stem cells (hiPSCs derived from two human fibroblasts and their progenies. Results showed that (1 methylation at the GNAS locus DMRs is DMR and cell line specific, (2 changes in allelic transcript expression can be independent of a change in allele-specific DNA methylation, and (3 interestingly, methylation at A/B DMR is correlated with A/B transcript expression. These results indicate that these models are valuable to study the mechanisms controlling GNAS methylation, factors involved in transcript expression, and possibly mechanisms involved in the pathophysiology of pseudohypoparathyroidism type 1B.

  8. Generalization of value in reinforcement learning by humans.

    Science.gov (United States)

    Wimmer, G Elliott; Daw, Nathaniel D; Shohamy, Daphna

    2012-04-01

    Research in decision-making has focused on the role of dopamine and its striatal targets in guiding choices via learned stimulus-reward or stimulus-response associations, behavior that is well described by reinforcement learning theories. However, basic reinforcement learning is relatively limited in scope and does not explain how learning about stimulus regularities or relations may guide decision-making. A candidate mechanism for this type of learning comes from the domain of memory, which has highlighted a role for the hippocampus in learning of stimulus-stimulus relations, typically dissociated from the role of the striatum in stimulus-response learning. Here, we used functional magnetic resonance imaging and computational model-based analyses to examine the joint contributions of these mechanisms to reinforcement learning. Humans performed a reinforcement learning task with added relational structure, modeled after tasks used to isolate hippocampal contributions to memory. On each trial participants chose one of four options, but the reward probabilities for pairs of options were correlated across trials. This (uninstructed) relationship between pairs of options potentially enabled an observer to learn about option values based on experience with the other options and to generalize across them. We observed blood oxygen level-dependent (BOLD) activity related to learning in the striatum and also in the hippocampus. By comparing a basic reinforcement learning model to one augmented to allow feedback to generalize between correlated options, we tested whether choice behavior and BOLD activity were influenced by the opportunity to generalize across correlated options. Although such generalization goes beyond standard computational accounts of reinforcement learning and striatal BOLD, both choices and striatal BOLD activity were better explained by the augmented model. Consistent with the hypothesized role for the hippocampus in this generalization, functional

  9. Differential transcriptional responses to Ebola and Marburg virus infection in bat and human cells

    Science.gov (United States)

    Hölzer, Martin; Krähling, Verena; Amman, Fabian; Barth, Emanuel; Bernhart, Stephan H.; Carmelo, Victor A. O.; Collatz, Maximilian; Doose, Gero; Eggenhofer, Florian; Ewald, Jan; Fallmann, Jörg; Feldhahn, Lasse M.; Fricke, Markus; Gebauer, Juliane; Gruber, Andreas J.; Hufsky, Franziska; Indrischek, Henrike; Kanton, Sabina; Linde, Jörg; Mostajo, Nelly; Ochsenreiter, Roman; Riege, Konstantin; Rivarola-Duarte, Lorena; Sahyoun, Abdullah H.; Saunders, Sita J.; Seemann, Stefan E.; Tanzer, Andrea; Vogel, Bertram; Wehner, Stefanie; Wolfinger, Michael T.; Backofen, Rolf; Gorodkin, Jan; Grosse, Ivo; Hofacker, Ivo; Hoffmann, Steve; Kaleta, Christoph; Stadler, Peter F.; Becker, Stephan; Marz, Manja

    2016-01-01

    The unprecedented outbreak of Ebola in West Africa resulted in over 28,000 cases and 11,000 deaths, underlining the need for a better understanding of the biology of this highly pathogenic virus to develop specific counter strategies. Two filoviruses, the Ebola and Marburg viruses, result in a severe and often fatal infection in humans. However, bats are natural hosts and survive filovirus infections without obvious symptoms. The molecular basis of this striking difference in the response to filovirus infections is not well understood. We report a systematic overview of differentially expressed genes, activity motifs and pathways in human and bat cells infected with the Ebola and Marburg viruses, and we demonstrate that the replication of filoviruses is more rapid in human cells than in bat cells. We also found that the most strongly regulated genes upon filovirus infection are chemokine ligands and transcription factors. We observed a strong induction of the JAK/STAT pathway, of several genes encoding inhibitors of MAP kinases (DUSP genes) and of PPP1R15A, which is involved in ER stress-induced cell death. We used comparative transcriptomics to provide a data resource that can be used to identify cellular responses that might allow bats to survive filovirus infections. PMID:27713552

  10. Human-specific duplication and mosaic transcripts: the recent paralogous structure of chromosome 22.

    Science.gov (United States)

    Bailey, Jeffrey A; Yavor, Amy M; Viggiano, Luigi; Misceo, Doriana; Horvath, Juliann E; Archidiacono, Nicoletta; Schwartz, Stuart; Rocchi, Mariano; Eichler, Evan E

    2002-01-01

    In recent decades, comparative chromosomal banding, chromosome painting, and gene-order studies have shown strong conservation of gross chromosome structure and gene order in mammals. However, findings from the human genome sequence suggest an unprecedented degree of recent (homologous duplications (> or = 1 kb and > or = 90%) on chromosome 22. Overall, 10.8% (3.7/33.8 Mb) of chromosome 22 is duplicated, with an average sequence identity of 95.4%. To organize the duplications into tractable units, intron-exon structure and well-defined duplication boundaries were used to define 78 duplicated modules (minimally shared evolutionary segments) with 157 copies on chromosome 22. Analysis of these modules provides evidence for the creation or modification of 11 novel transcripts. Comparative FISH analyses of human, chimpanzee, gorilla, orangutan, and macaque reveal qualitative and quantitative differences in the distribution of these duplications--consistent with their recent origin. Several duplications appear to be human specific, including a approximately 400-kb duplication (99.4%-99.8% sequence identity) that transposed from chromosome 14 to the most proximal pericentromeric region of chromosome 22. Experimental and in silico data further support a pericentromeric gradient of duplications where the most recent duplications transpose adjacent to the centromere. Taken together, these data suggest that segmental duplications have been an ongoing process of primate genome evolution, contributing to recent gene innovation and the dynamic transformation of genome architecture within and among closely related species.

  11. Differential transcriptional responses to Ebola and Marburg virus infection in bat and human cells.

    Science.gov (United States)

    Hölzer, Martin; Krähling, Verena; Amman, Fabian; Barth, Emanuel; Bernhart, Stephan H; Carmelo, Victor A O; Collatz, Maximilian; Doose, Gero; Eggenhofer, Florian; Ewald, Jan; Fallmann, Jörg; Feldhahn, Lasse M; Fricke, Markus; Gebauer, Juliane; Gruber, Andreas J; Hufsky, Franziska; Indrischek, Henrike; Kanton, Sabina; Linde, Jörg; Mostajo, Nelly; Ochsenreiter, Roman; Riege, Konstantin; Rivarola-Duarte, Lorena; Sahyoun, Abdullah H; Saunders, Sita J; Seemann, Stefan E; Tanzer, Andrea; Vogel, Bertram; Wehner, Stefanie; Wolfinger, Michael T; Backofen, Rolf; Gorodkin, Jan; Grosse, Ivo; Hofacker, Ivo; Hoffmann, Steve; Kaleta, Christoph; Stadler, Peter F; Becker, Stephan; Marz, Manja

    2016-10-07

    The unprecedented outbreak of Ebola in West Africa resulted in over 28,000 cases and 11,000 deaths, underlining the need for a better understanding of the biology of this highly pathogenic virus to develop specific counter strategies. Two filoviruses, the Ebola and Marburg viruses, result in a severe and often fatal infection in humans. However, bats are natural hosts and survive filovirus infections without obvious symptoms. The molecular basis of this striking difference in the response to filovirus infections is not well understood. We report a systematic overview of differentially expressed genes, activity motifs and pathways in human and bat cells infected with the Ebola and Marburg viruses, and we demonstrate that the replication of filoviruses is more rapid in human cells than in bat cells. We also found that the most strongly regulated genes upon filovirus infection are chemokine ligands and transcription factors. We observed a strong induction of the JAK/STAT pathway, of several genes encoding inhibitors of MAP kinases (DUSP genes) and of PPP1R15A, which is involved in ER stress-induced cell death. We used comparative transcriptomics to provide a data resource that can be used to identify cellular responses that might allow bats to survive filovirus infections.

  12. Targeting signal transducers and activators of transcription (STAT) in human cancer by dietary polyphenolic antioxidants.

    Science.gov (United States)

    Amani, Hamed; Ajami, Marjan; Maleki, Solmaz Nasseri; Pazoki-Toroudi, Hamidreza; Daglia, Maria; Tsetegho Sokeng, Arold Jorel; Di Lorenzo, Arianna; Nabavi, Seyed Fazel; Devi, Kasi Pandima; Nabavi, Seyed Mohammad

    2017-08-11

    Over the course of the last three decades, a large body of evidence has shown that polyphenols, the secondary metabolites occurring in plant foods and beverages, exert protective effects due to their antioxidant activity mediated through different mechanisms ranging from direct radical scavenging and metal chelating activities, to the capacity to inhibit pro-oxidant enzymes and to target specific cell-signalling pathways. In the last decade, dietary components, and polyphenols in particular have gained considerable attention as chemopreventive agents against different types of cancer. The signal transducers and activators of transcription (STAT) family is a group of cytoplasmic transcription factors which interact with specific sequences of DNA, inducing the expression of specific genes which in turn give rise to adaptive and highly specific biological responses. Growing evidence suggests that, of the seven STAT members identified, STAT3 is over-expressed in many human tumors (i.e. solid tumors and hematological malignancies) promoting the onset and development of cancer in humans by inhibiting apoptosis or by inducing cell proliferation, angiogenesis, invasion, and metastasis. This review article aims to assess the most recent studies on the role of STATs, with focus on STAT3, in oncogenesis, and the promising effects of some polyphenols on STAT expression. Moreover, the mechanisms behind the anti-inflammatory and antioxidant activities of polyphenols which have an influence on STAT expression are discussed, with a focus on their ability to target specific cell-signalling pathways. Copyright © 2017 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

  13. Expression of a novel alternative transcript of the novel retinal pigment epithelial cell gene NORPEG in human testes

    Institute of Scientific and Technical Information of China (English)

    Wa Yuan; Ying Zheng; Ran Huo; Li Lu; Xiao-Yan Huang; Lan-Lan Yin; Jian-Min Li; Zuo-Min Zhou; Jia-Hao Sha

    2005-01-01

    Aim: To identify a novel alternative transcript of the novel retinal pigment epithelial cell gene (NORPEG) expressed in the human testis. Methods: A human testis cDNA microarray was established and hybridized with cDNA probes from human fetal testes, adult testes and human spermatozoa. Differentially expressed clones were sequenced and analyzed. One of these clones was a short transcript of NORPEG which we proceeded to analyze by RT-PCR.Results: The novel short alternative transcript of NORPEG was isolated and named sNORPEG. It was 3486 bp in length and contained a 2952-bp open reading frame, encoding a 110.4-kDa protein of 983 amino acids. Amino acid sequence analysis showed that the sNORPEG protein contains six ankyrin repeats and two coiled-coil domains. It shares a high homology with the NORPEG and ankycorbin proteins in both its sequence and motifs. Blasting the human genome database localized sNORPEG to human chromosome 5p13.2-13.3. Expression profiles showed that sNORPEG was expressed in human fetal testes, adult testes and spermatozoa. Moreover, sNORPEG was found to be ubiquitously expressed in human tissues. Conclusion: sNORPEG is expressed in different developmental stages of the testis and encodes a protein that may have roles in human testis development and spermatogenesis.

  14. 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.

  15. Transcription of human resistin gene involves an interaction of Sp1 with peroxisome proliferator-activating receptor gamma (PPARgamma.

    Directory of Open Access Journals (Sweden)

    Anil K Singh

    Full Text Available BACKGROUND: Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin. METHODOLOGY/PRINCIPAL FINDING: We show that the minimal promoter of human resistin lies within approximately 80 bp sequence upstream of the transcriptional start site (-240 whereas binding sites for cRel, CCAAT enhancer binding protein alpha (C/EBP-alpha, activating transcription factor 2 (ATF-2 and activator protein 1 (AP-1 transcription factors, important for induced expression, are present within sequences up to -619. Specificity Protein 1(Sp1 binding site (-276 to -295 is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPARgamma is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPARgamma in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPARgamma on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPARgamma interaction. Chromatin immunoprecipitation (ChIP assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPARgamma, chromatin modifier histone deacetylase 1 (HDAC1 and the acetylated form of histone H3 but not cRel, C/EBP-alpha and phospho c-Jun during resistin gene transcription. CONCLUSION: Our findings suggest a complex interplay of Sp1 and PPARgamma along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.

  16. Transcription and processing of mitochondrial RNA in the human pathogen Acanthamoeba castellanii.

    Science.gov (United States)

    Accari, Jessica; Barth, Christian

    2015-07-01

    The size, structure, gene content and organisation of mitochondrial genomes can be highly diverse especially amongst the protists. We investigated the transcription and processing of the mitochondrial genome of the opportunistic pathogen Acanthamoeba castellanii and here we present a detailed transcription map of the 41.6 kb genome that encodes 33 proteins, 16 tRNAs and 2 rRNAs. Northern hybridisation studies identified six major polycistronic transcripts, most of which are co-transcriptionally processed into smaller mono-, di- and tricistronic RNAs. The maturation of the polycistronic transcripts is likely to involve endonucleolytic cleavage where tRNAs serve as processing signals. Reverse transcription polymerase chain reactions across the intervening regions between the six major polycistronic transcripts suggest that these transcripts were once part of an even larger transcript. Our findings indicate that the mitochondrial genome of A. castellanii is transcribed from only one or two promoters, very similar to the mode of transcription in the mitochondria of its close relative Dictyostelium discoideum, where transcription is known to occur from only a single transcription initiation site. Transcription initiation from a minimal number of promoters despite a large genome size may be an emerging trend in the mitochondria of protists.

  17. BCL11B is a general transcriptional repressor of the HIV-1 long terminal repeat in T lymphocytes through recruitment of the NuRD complex.

    Science.gov (United States)

    Cismasiu, Valeriu B; Paskaleva, Elena; Suman Daya, Sneha; Canki, Mario; Duus, Karen; Avram, Dorina

    2008-10-25

    In this study we provide evidence that the transcription factor BCL11B represses expression from the HIV-1 long terminal repeat (LTR) in T lymphocytes through direct association with the HIV-1 LTR. We also demonstrate that the NuRD corepressor complex mediates BCL11B transcriptional repression of the HIV-1 LTR. In addition, BCL11B and the NuRD complex repressed TAT-mediated transactivation of the HIV-1 LTR in T lymphocytes, pointing to a potential role in initiation of silencing. In support of all the above results, we demonstrate that BCL11B affects HIV-1 replication and virus production, most likely by blocking LTR transcriptional activity. BCL11B showed specific repression for the HIV-1 LTR sequences isolated from seven different HIV-1 subtypes, demonstrating that it is a general transcriptional repressor for all LTRs.

  18. The SET2-RPB1 interaction domain of human RECQ5 is important for transcription-associated genome stability.

    Science.gov (United States)

    Li, Min; Xu, Xiaohua; Liu, Yilun

    2011-05-01

    The conserved RECQ5 DNA helicase is a tumor suppressor in mammalian cells. Defects in RECQ5 lead to the accumulation of spontaneous DNA double-stranded breaks (DSBs) during replication, despite the fact that these cells are proficient in DSB repair by homologous recombination (HR). The reason for this is unknown. Here, we demonstrate that these DSBs are linked to RNA polymerase II (RNAPII)-dependent transcription. In human RECQ5-depleted cells, active RNAPII accumulates on chromatin, and DNA breaks are associated with an RNAPII-dependent transcribed locus. Hence, transcription inhibition eliminates both active RNAPII and spontaneous DSB formation. In addition, the regulatory effect of RECQ5 on transcription and its interaction with RNAPII are enhanced in S-phase cells, supporting a role for RECQ5 in preventing transcription-associated DSBs during replication. Finally, we show that the SET2-RPB1 interaction (SRI) domain of human RECQ5 is important for suppressing spontaneous DSBs and the p53-dependent transcription stress response caused by the stalling of active RNAPII on DNA. Thus, our studies provide novel insights into a mechanism by which RECQ5 regulates the transcription machinery via its dynamic interaction with RNAPII, thereby preventing genome instability.

  19. Genome-wide analysis of differential transcriptional and epigenetic variability across human immune cell types

    DEFF Research Database (Denmark)

    Ecker, Simone; Chen, Lu; Pancaldi, Vera

    2017-01-01

    Background: A healthy immune system requires immune cells that adapt rapidly to environmental challenges. This phenotypic plasticity can be mediated by transcriptional and epigenetic variability. Results: We apply a novel analytical approach to measure and compare transcriptional and epigenetic v...

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

    OpenAIRE

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

    2015-01-01

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

  1. Expression of a splice variant of the platelet-activating factor receptor transcript 2 in various human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Ibtissam Youlyouz

    2002-01-01

    Full Text Available Platelet-activating factor receptor (PAF-R transcripts were analysed by reverse transcriptase-polymerase chain reaction in five human cancer cell lines derived from the breast (BT20, SKBR3 and T47D cells, the pancreas (Miapaca cells and the bladder (5637 cells in order to confirm the existence of a splice variant of the PAF-R transcript 2. After cloning and sequencing, we confirmed its existence in all cell lines. It consisted of the PAF-R transcript 2 lengthening with 82 nucleotides from the 3' end of exon 1 of the PAF-R gene. The role of this elongated form of the tissue-type PAF-R transcript in cell physiology remains to be elucidated.

  2. Aging affects the transcriptional regulation of human skeletal muscle disuse atrophy.

    Directory of Open Access Journals (Sweden)

    Charlotte Suetta

    Full Text Available Important insights concerning the molecular basis of skeletal muscle disuse-atrophy and aging related muscle loss have been obtained in cell culture and animal models, but these regulatory signaling pathways have not previously been studied in aging human muscle. In the present study, muscle atrophy was induced by immobilization in healthy old and young individuals to study the time-course and transcriptional factors underlying human skeletal muscle atrophy. The results reveal that irrespectively of age, mRNA expression levels of MuRF-1 and Atrogin-1 increased in the very initial phase (2-4 days of human disuse-muscle atrophy along with a marked reduction in PGC-1α and PGC-1β (1-4 days and a ~10% decrease in myofiber size (4 days. Further, an age-specific decrease in Akt and S6 phosphorylation was observed in young muscle within the first days (1-4 days of immobilization. In contrast, Akt phosphorylation was unchanged in old muscle after 2 days and increased after 4 days of immobilization. Further, an age-specific down-regulation of MuRF-1 and Atrogin-1 expression levels was observed following 2 weeks of immobilization, along with a slowing atrophy response in aged skeletal muscle. Neither the immediate loss of muscle mass, nor the subsequent age-differentiated signaling responses could be explained by changes in inflammatory mediators, apoptosis markers or autophagy indicators. Collectively, these findings indicate that the time-course and regulation of human skeletal muscle atrophy is age dependent, leading to an attenuated loss in aging skeletal muscle when exposed to longer periods of immobility-induced disuse.

  3. Expression of a novel dipeptidyl peptidase 8 (DPP8) transcript variant, DPP8-v3, in human testis

    Institute of Scientific and Technical Information of China (English)

    Hui Zhu; Zuo-Min Zhou; Li Lu; Min Xu; Hui Wang; Jian-Min Li; Jia-Hao Sha

    2005-01-01

    Aim: To investigate the role of a novel dipeptidyl peptidase 8 transcript variant (DPP8-v3) gene in testis development and/or spermatogenesis. Methods: A human testis cDNA microarray was hybridized with mRNA of human adult and fetal testes. Differentially expressed clones were sequenced and characterized and their expression was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Southern-blot analysis. Results: A new transcript variant of the human dipeptidyl peptidase (DPP8), exhibiting a 5-fold higher expression level in human adult than that in fetal testes, was cloned and was named DPP8 variant 3 (DPP8-v3). The full-length sequence of DPP8-v3was 3,030 bp, encoding a protein of 898 amino acids. Conclusion: DPP8-v3 is a novel human DPP8 transcript variant highly expressed in the adult testis. Similar to DPPⅣ, DPP8-v3 may play a key role in the immunoregulation of testes and accordingly may influence spermatogenesis and male fertility.

  4. The eukaryotic gene transcription machinery.

    Science.gov (United States)

    Kornberg, R D

    2001-08-01

    Seven purified proteins may be combined to reconstitute regulated, promoter-dependent RNA polymerase II transcription: five general transcription factors, Mediator, and RNA polymerase II. The entire system has been conserved across species from yeast to humans. The structure of RNA polymerase II, consisting of 10 polypeptides with a mass of about 500 kDa, has been determined at atomic resolution. On the basis of this structure, that of an actively transcribing RNA polymerase II complex has been determined as well.

  5. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells.

    Science.gov (United States)

    Katz, Adam J; Tholpady, Ashok; Tholpady, Sunil S; Shang, Hulan; Ogle, Roy C

    2005-03-01

    Adult human subcutaneous adipose tissue contains cells with intriguing multilineage developmental plasticity, much like marrow-derived mesenchymal stem cells. Putative stem or progenitor cells from fat have been given many different names in the literature, reflecting an early and evolving consensus regarding their phenotypic characterization. The study reported here used microarrays to evaluate over 170 genes relating to angiogenesis and extracellular matrix in undifferentiated, early-passage human adipose-derived adherent stromal (hADAS) cells isolated from three separate donors. The hADAS populations unanimously transcribed 66% of the screened genes, and 83% were transcribed by at least two of the three populations. The most highly transcribed genes relate to functional groupings such as cell adhesion, matrix proteins, growth factors and receptors, and proteases. The transcriptome of hADAS cells demonstrated by this work reveals many similarities to published profiles of bone marrow mesenchymal stem cells (MSCs). In addition, flow analysis of over 24 hADAS cell surface proteins (n = 7 donors) both confirms and expands on the existing literature and reveals strong intergroup correlation, despite an inconsistent nomenclature and the lack of standardized protocols for cell isolation and culture. Finally, based on flow analysis and reverse transcription polymerase chain reaction studies, our results suggest that hADAS cells do not express several proteins that are implicated as markers of "stemness" in other stem cell populations, including telomerase, CD133, and the membrane transporter ABCG2.

  6. The human mitochondrial transcription factor A is a versatile G-quadruplex binding protein

    Science.gov (United States)

    Lyonnais, Sébastien; Tarrés-Soler, Aleix; Rubio-Cosials, Anna; Cuppari, Anna; Brito, Reicy; Jaumot, Joaquim; Gargallo, Raimundo; Vilaseca, Marta; Silva, Cristina; Granzhan, Anton; Teulade-Fichou, Marie-Paule; Eritja, Ramon; Solà, Maria

    2017-01-01

    The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a high-mobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4-dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria. PMID:28276514

  7. Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells.

    Science.gov (United States)

    Chen, Lu; Ge, Bing; Casale, Francesco Paolo; Vasquez, Louella; Kwan, Tony; Garrido-Martín, Diego; Watt, Stephen; Yan, Ying; Kundu, Kousik; Ecker, Simone; Datta, Avik; Richardson, David; Burden, Frances; Mead, Daniel; Mann, Alice L; Fernandez, Jose Maria; Rowlston, Sophia; Wilder, Steven P; Farrow, Samantha; Shao, Xiaojian; Lambourne, John J; Redensek, Adriana; Albers, Cornelis A; Amstislavskiy, Vyacheslav; Ashford, Sofie; Berentsen, Kim; Bomba, Lorenzo; Bourque, Guillaume; Bujold, David; Busche, Stephan; Caron, Maxime; Chen, Shu-Huang; Cheung, Warren; Delaneau, Oliver; Dermitzakis, Emmanouil T; Elding, Heather; Colgiu, Irina; Bagger, Frederik O; Flicek, Paul; Habibi, Ehsan; Iotchkova, Valentina; Janssen-Megens, Eva; Kim, Bowon; Lehrach, Hans; Lowy, Ernesto; Mandoli, Amit; Matarese, Filomena; Maurano, Matthew T; Morris, John A; Pancaldi, Vera; Pourfarzad, Farzin; Rehnstrom, Karola; Rendon, Augusto; Risch, Thomas; Sharifi, Nilofar; Simon, Marie-Michelle; Sultan, Marc; Valencia, Alfonso; Walter, Klaudia; Wang, Shuang-Yin; Frontini, Mattia; Antonarakis, Stylianos E; Clarke, Laura; Yaspo, Marie-Laure; Beck, Stephan; Guigo, Roderic; Rico, Daniel; Martens, Joost H A; Ouwehand, Willem H; Kuijpers, Taco W; Paul, Dirk S; Stunnenberg, Hendrik G; Stegle, Oliver; Downes, Kate; Pastinen, Tomi; Soranzo, Nicole

    2016-11-17

    Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14(+) monocytes, CD16(+) neutrophils, and naive CD4(+) T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.

  8. (Some cellular mechanisms influencing the transcription of human endogenous retrovirus, HERV-Fc1.

    Directory of Open Access Journals (Sweden)

    Magdalena Janina Laska

    Full Text Available DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. DNA methylation is considered an important mechanism for silencing of retroelements in the mammalian genome. However, the methylation of human endogenous retroviruses (HERVs is not well investigated. The aim of this study was to investigate the transcriptional potential of HERV-Fc1 proviral 5'LTR in more detail, and examined the specific influence of CpG methylation on this LTR in number of cell lines. Specifically, the role of demethylating chemicals e.g. 5-aza-2' deoxycytidine and Trichostatin-A, in inducing or reactivating expression of HERV-Fc1 specific sequences and the mechanisms were investigated. In our present study, 5-aza-dC is shown to be a powerful inducer of HERV-Fc1, and at the same time it strongly inhibits methylation of DNA. Treatment with this demethylating agent 5-aza-dC, results in significantly increased levels of HERV-Fc1 expression in cells previously not expressing HERV-Fc1, or with a very low expression level. The extent of expression of HERV-Fc1 RNAs precisely correlates with the apparent extent of demethylation of the related DNA sequences. In conclusion, the results suggest that inhibition of DNA methylation/histone deacetylase can interfere with gene silencing mechanisms affecting HERV-Fc1 expression in human cells.

  9. Novel Mutations in the Transcriptional Activator Domain of the Human TBX20 in Patients with Atrial Septal Defect

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    Irma Eloisa Monroy-Muñoz

    2015-01-01

    Full Text Available Background. The relevance of TBX20 gene in heart development has been demonstrated in many animal models, but there are few works that try to elucidate the effect of TBX20 mutations in human congenital heart diseases. In these studies, all missense mutations associated with atrial septal defect (ASD were found in the DNA-binding T-box domain, none in the transcriptional activator domain. Methods. We search for TBX20 mutations in a group of patients with ASD or ventricular septal defect (VSD using the High Resolution Melting (HRM method and DNA sequencing. Results. We report three missense mutations (Y309D, T370O, and M395R within the transcriptional activator domain of human TBX20 that were associated with ASD. Conclusions. This is the first association of TBX20 transcriptional activator domain missense mutations with ASD. These findings could have implications for diagnosis, genetic screening, and patient follow-up.

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

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    Arnoldo J Müller-Molina

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

  11. Inhibition of mitochondrial gene transcription suppresses neurotensin secretion in the human carcinoid cell line BON.

    Science.gov (United States)

    Li, Nan; Wang, Qingding; Li, Jing; Wang, Xiaofu; Hellmich, Mark R; Rajaraman, Srinivasan; Greeley, George H; Townsend, Courtney M; Evers, B Mark

    2005-02-01

    Mitochondria, organelles essential for ATP production, play a central role in a number of cellular functions, including the regulation of insulin secretion. Neurotensin (NT), an important regulatory intestinal hormone, has been implicated in fatty acid translocation, gut motility and secretion, and intestinal cell growth; however, mechanisms regulating NT secretion have not been entirely defined. The purpose of this study was to determine the effect of inhibition of mitochondrial gene transcription on NT secretion. BON cells, a novel human carcinoid cell line that produces and secretes NT peptide and expresses the gene encoding NT (designated NT/N), were treated with ethidium bromide (EB; 0.05, 0.1, and 0.4 microg/ml), an inhibitor of DNA and RNA synthesis, or vehicle over a time course (1-4 days). Cells were then stimulated with either ACh (100 microM) or phorbol 12 myristate,13-acetate (PMA, 10 nM) for 30 min. Media and cells were extracted, and NT peptide measured by RIA. Treatment with EB had no effect on BON cell viability or cell cycle distribution over the 4-day course. In contrast, EB treatment produced a dose-dependent reduction of mitochondrial gene expression; however, NT/N gene expression was not altered. Mitochondrial inhibition by EB treatment suppressed NT secretion induced by ACh and PMA, both in a dose-dependent manner. EB-mediated inhibition of NT secretion and mitochondrial gene expression was reversed with removal of EB. Our results demonstrate that inhibition of mitochondrial gene transcription suppresses both ACh- and PMA-stimulated NT release. These findings are the first to demonstrate that mitochondrial function is important for agonist-mediated NT secretion.

  12. Activation of hypoxia inducible factor 1 is a general phenomenon in infections with human pathogens.

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    Nadine Werth

    Full Text Available BACKGROUND: Hypoxia inducible factor (HIF-1 is the key transcriptional factor involved in the adaptation process of cells and organisms to hypoxia. Recent findings suggest that HIF-1 plays also a crucial role in inflammatory and infectious diseases. METHODOLOGY/PRINCIPAL FINDINGS: Using patient skin biopsies, cell culture and murine infection models, HIF-1 activation was determined by immunohistochemistry, immunoblotting and reporter gene assays and was linked to cellular oxygen consumption. The course of a S. aureus peritonitis was determined upon pharmacological HIF-1 inhibition. Activation of HIF-1 was detectable (i in all ex vivo in biopsies of patients suffering from skin infections, (ii in vitro using cell culture infection models and (iii in vivo using murine intravenous and peritoneal S. aureus infection models. HIF-1 activation by human pathogens was induced by oxygen-dependent mechanisms. Small colony variants (SCVs of S. aureus known to cause chronic infections did not result in cellular hypoxia nor in HIF-1 activation. Pharmaceutical inhibition of HIF-1 activation resulted in increased survival rates of mice suffering from a S. aureus peritonitis. CONCLUSIONS/SIGNIFICANCE: Activation of HIF-1 is a general phenomenon in infections with human pathogenic bacteria, viruses, fungi and protozoa. HIF-1-regulated pathways might be an attractive target to modulate the course of life-threatening infections.

  13. Genome-wide profiling of H3K56 acetylation and transcription factor binding sites in human adipocytes.

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    Kinyui Alice Lo

    Full Text Available The growing epidemic of obesity and metabolic diseases calls for a better understanding of adipocyte biology. The regulation of transcription in adipocytes is particularly important, as it is a target for several therapeutic approaches. Transcriptional outcomes are influenced by both histone modifications and transcription factor binding. Although the epigenetic states and binding sites of several important transcription factors have been profiled in the mouse 3T3-L1 cell line, such data are lacking in human adipocytes. In this study, we identified H3K56 acetylation sites in human adipocytes derived from mesenchymal stem cells. H3K56 is acetylated by CBP and p300, and deacetylated by SIRT1, all are proteins with important roles in diabetes and insulin signaling. We found that while almost half of the genome shows signs of H3K56 acetylation, the highest level of H3K56 acetylation is associated with transcription factors and proteins in the adipokine signaling and Type II Diabetes pathways. In order to discover the transcription factors that recruit acetyltransferases and deacetylases to sites of H3K56 acetylation, we analyzed DNA sequences near H3K56 acetylated regions and found that the E2F recognition sequence was enriched. Using chromatin immunoprecipitation followed by high-throughput sequencing, we confirmed that genes bound by E2F4, as well as those by HSF-1 and C/EBPα, have higher than expected levels of H3K56 acetylation, and that the transcription factor binding sites and acetylation sites are often adjacent but rarely overlap. We also discovered a significant difference between bound targets of C/EBPα in 3T3-L1 and human adipocytes, highlighting the need to construct species-specific epigenetic and transcription factor binding site maps. This is the first genome-wide profile of H3K56 acetylation, E2F4, C/EBPα and HSF-1 binding in human adipocytes, and will serve as an important resource for better understanding adipocyte

  14. Noncoding RNA in the Transcriptional Landscape of Human Neural Progenitor Cell Differentiation

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    Patrick eHecht

    2015-10-01

    Full Text Available Increasing evidence suggests that noncoding RNAs play key roles in cellular processes, particularly in the brain. The present study used RNA sequencing to identify the transcriptional landscape of two human neural progenitor cell lines, SK-N-SH and ReNcell CX, as they differentiate into human cortical projection neurons. Protein coding genes were found to account for 54.8% and 57.0% of expressed genes, respectively, and alignment of RNA sequencing reads revealed that only 25.5-28.1% mapped to exonic regions of the genome. Differential expression analysis in the two cell lines identified altered gene expression in both protein coding and noncoding RNAs as they undergo neural differentiation with 222 differentially expressed genes observed in SK-N-SH cells and 19 differentially expressed genes in ReNcell CX. Interestingly, genes showing differential expression in SK-N-SH cells are enriched in genes implicated in autism spectrum disorder, but not in gene sets related to cancer or Alzheimer’s disease. Weighted gene co-expression network analysis (WGCNA was used to detect modules of co-expressed protein coding and noncoding RNAs in SK-N-SH cells and found four modules to be associated with neural differentiation. These modules contain varying levels of noncoding RNAs ranging from 10.7% to 49.7% with gene ontology suggesting roles in numerous cellular processes important for differentiation. These results indicate that noncoding RNAs are highly expressed in human neural progenitor cells and likely hold key regulatory roles in gene networks underlying neural differentiation and neurodevelopmental disorders.

  15. Transcriptional profiling defines the effects of nickel in human epidermal keratinocytes.

    Science.gov (United States)

    Gazel, Alix; Rosdy, Martin; Tornier, Carine; De Fraissinette, Anne De Brugerolle; Blumenberg, Miroslav

    2008-12-01

    Nickel is a ubiquitous and virtually unavoidable environmental pollutant and occupational hazard, but its molecular and cellular effects are not well understood. Human epidermal keratinocytes are the sentinel and the primary target for nickel. We treated with nickel salts skin equivalents containing differentiating epidermal keratinocytes grown on air-liquid interface in standard cell culture conditions. We identified the transcriptional profiles affected by nickel in reconstructed human epidermis (RHE) using DNA microarrays. The Ni-regulated genes were determined at two time points, immediate-early, 30 min after treatment, and late, at 6 h. Using in silico data analysis, we determined that 134 genes are regulated by nickel; of these, 97 are induced and 37 suppressed. Functional categories of regulated genes suggest that Ni inhibits apoptosis, promotes cell cycle and induces synthesis of extracellular matrix proteins and extracellular proteases. Importantly, Ni also regulates a set of secreted signaling proteins, inducing VEGF, amphiregulin, PGF, GDF15, and BST2, while suppressing IL-18, galectin-3, and LITAF. These secreted proteins may be important in Ni-caused allergic reactions. Ni induced inhibitors of the NFkappaB signaling pathway, and suppressed its activators. Correspondingly, NFkappaB binding sites were found to be overrepresented in the Ni-suppressed genes, whereas cFOS/AP1 binding sites were common in the Ni-induced genes. Significant parallels were found between the Ni-regulated genes and the genes regulated by TGFbeta, EGF, glucocorticoids, or Oncostatin-M. The comprehensive identification of Ni-regulated genes in human epidermal equivalents significantly advances our understanding of the molecular effects of nickel in skin.

  16. Benzo (a) pyrene induced tumorigenesity of human immortalized oral epithelial cells: transcription profiling

    Institute of Scientific and Technical Information of China (English)

    LI Jin-zhong; PAN Hong-ya; ZHENG Jia-wei; ZHOU Xiao-jian; ZHANG Ping; CHEN Wan-tao; ZHANG Zhi-yuan

    2008-01-01

    Background The present study was designed to examine and analyze the global gene expression changes during the tumorigenesis of a human immortalized oral epithelial cell line, and search for the possible genes that may play a role in the carcinogenesis of oral cancer associated with benzo (a) pyrene.Methods The human immortalized oral epithelial cells, which have been established through transfection of E6/E7 genasof human papillomavirus type 16 and proved to be non-tumorigenic in nude mice, were treated with benzo (a) pyrene.Tumorigenesity of the treated cells were examined through nude mice subcutaneous injection. The global gene expression profiles of immortalized cells and the tumorigenic cells were acquired through hybridization of a microarray of Affymetrix U133 plus 2.0. The data were analyzed using Spring 7.0 software and treated statistically using one-way analysis of variance (ANOVA). The differentially expressed genes were classified using a Venn diagram and annotated with gene ontology. Several highlighted genes were validated in cells using a real-time polymerase chain reaction.Results There were 883 differentially expressed genes during the tumorigenesis and most of them changed expression in the early stage of tumorigenesis. These genes mainly involved in macromolecule metabolism and signal transduction,possessed the molecular function of transition metal ion binding, nucleotide binding and kinase activity; their protein products were mainly integral to membranes or localized in the nucleus and cytoskeleton. The expression patterns of IGFBP3, S100A8, MAP2K, KRT6B, GDF15, MET were validated in cells using a real-time polymerase chain reaction; the expression of IGFBP3 was further validated in clinical oral cancer specimens.Concluslona This study provides the global transcription profiling associated with the tumorigenesis of oral epithelial cells exposed to benzo (a) pyrene; IGFBP3 may play a potential role in the initiation of oral cancer related to

  17. Functional analysis of human mutations in homeodomain transcription factor PITX3

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    Sorokina Elena

    2007-09-01

    Full Text Available Abstract Background The homeodomain-containing transcription factor PITX3 was shown to be essential for normal eye development in vertebrates. Human patients with point mutations in PITX3 demonstrate congenital cataracts along with anterior segment defects in some cases when one allele is affected and microphthalmia with brain malformations when both copies are mutated. The functional consequences of these human mutations remain unknown. Results We studied the PITX3 mutant proteins S13N and G219fs to determine the type and severity of functional defects. Our results demonstrate alterations in DNA-binding profiles and/or transactivation activities and suggest a partial loss-of-function in both mutants with the G219fs form being more severely affected. No anomalies in cellular distribution and no dominant-negative effects were discovered for these mutants. Interestingly, the impairment of the G219fs activity varied between different ocular cell lines. Conclusion The G219fs mutation was found in multiple families affected with congenital cataracts along with anterior segment malformations in many members. Our data suggest that the presence/severity of anterior segment defects in families affected with G219fs may be determined by secondary factors that are expressed in the developing anterior segment structures and may modify the effect(s of this mutation. The S13N mutant showed only minor alteration of transactivation ability and DNA binding pattern and may represent a rare polymorphism in the PITX3 gene. A possible contribution of this mutation to human disease needs to be further investigated.

  18. Functional Characterization of Single-Nucleotide Polymorphisms in the Human Undifferentiated Embryonic-Cell Transcription Factor 1 Gene

    NARCIS (Netherlands)

    Thummer, Rajkumar P.; Drenth-Diephuis, Loes J.; Carney, Karen E.; Eggen, Bart J. L.

    2010-01-01

    Single-nucleotide polymorphisms (SNPs) are single-nucleotide sequence variations between individuals. Two missense SNPs are present in the human undifferentiated embryonic-cell transcription factor 1 (UTF1) gene and their consequences for UTF1 function are investigated in this study. Expression of t

  19. Two type XVII collagen (BP180) mRNA transcripts in human keratinocytes : a long and a short form

    NARCIS (Netherlands)

    Molnar, K; van der Steege, G; Jonkman, MF; Nijenhuis, Albertine; Husz, S; van der Meer, JB; Pas, HH

    We have analysed BP180 mRNA expression in normal human keratinocytes. Here we report the presence in normal keratinocytes of two COL17A1 transcripts which differ by 0.6 kb in length. Both mRNAs hybridized on Northern blot with probes directed to sequences encoding intracellular and extracellular

  20. PAI-1 transcriptional regulation during the G0 --> G1 transition in human epidermal keratinocytes.

    Science.gov (United States)

    Qi, Li; Allen, Rosalie R; Lu, Qi; Higgins, Craig E; Garone, Rosemarie; Staiano-Coico, Lisa; Higgins, Paul J

    2006-10-01

    Plasminogen activator inhibitor type-1 (PAI-1) is the major negative regulator of the plasmin-dependent pericellular proteolytic cascade. PAI-1 gene expression is normally growth state regulated but frequently elevated in chronic fibroproliferative and neoplastic diseases affecting both stromal restructuring and cellular migratory activities. Kinetic modeling of cell cycle transit in synchronized human keratinocytes (HaCaT cells) indicated that PAI-1 transcription occurred early after serum stimulation of quiescent (G0) cells and prior to entry into a cycling G1 condition. PAI-1 repression (in G0) was associated with upstream stimulatory factor-1 (USF-1) occupancy of two consensus E box motifs (5'-CACGTG-3') at the PE1 and PE2 domains in the PF1 region (nucleotides -794 to -532) of the PAI-1 promoter. Chromatin immunoprecipitation (ChIP) analysis established that the PE1 and PE2 site E boxes were occupied by USF-1 in quiescent cells and by USF-2 in serum-activated, PAI-1-expressing keratinocytes. This reciprocal and growth state-dependent residence of USF family members (USF-1 vs. USF-2) at PE1/PE2 region chromatin characterized the G0 --> G1 transition period and the transcriptional status of the PAI-1 gene. A consensus E box motif was required for USF/E box interactions, as a CG --> AT substitution at the two central nucleotides inhibited formation of USF/probe complexes. The 5' flanking sites (AAT or AGAC) in the PE2 segment were not necessary for USF binding. USF recognition of the PE1/PE2 region E box sites required phosphorylation with several potential involved residues, including T153, maping to the USF-specific region (USR). A T153A substitution in USF-1 did not repress serum-induced PAI-1 expression whereas the T153D mutant was an effective suppressor. As anticipated from the ChIP results, transfection of wild-type USF-2 failed to inhibit PAI-1 induction. Collectively, these data suggest that USF family members are important regulators of PAI-1 gene

  1. Transcriptional Regulatory Networks Activated by PI3K and ERK Transduced Growth Signals in Human Glioblastoma Cells

    Institute of Scientific and Technical Information of China (English)

    Peter M. Haverty; Zhi-Ping Weng; Ulla Hansen

    2005-01-01

    Determining how cells regulate their transcriptional response to extracellular signals is key to the understanding of complex eukaryotic systems. This study was initiated with the goals of furthering the study of mammalian transcriptional regulation and analyzing the relative benefits of related computational methodologies. One dataset available for such an analysis involved gene expression profiling of the early growth factor response to platelet derived growth factor (PDGF)in a human glioblastoma cell line; this study differentiated genes whose expression was regulated by signaling through the phosphoinositide-3-kinase (PI3K) versus the extracellular-signal regulated kinase (ERK) pathways. We have compared the inferred transcription factors from this previous study with additional predictions of regulatory transcription factors using two alternative promoter sequence analysis techniques. This comparative analysis, in which the algorithms predict overlapping,although not identical, sets of factors, argues for meticulous benchmarking of promoter sequence analysis methods to determine the positive and negative attributes that contribute to their varying results. Finally, we inferred transcriptional regulatory networks deriving from various signaling pathways using the CARRIE program suite. These networks not only included previously described transcriptional features of the response to growth signals, but also predicted new regulatory features for the propagation and modulation of the growth signal.

  2. Inhibiting cell migration and cell invasion by silencing the transcription factor ETS-1 in human bladder cancer.

    Science.gov (United States)

    Liu, Li; Liu, Yuchen; Zhang, Xintao; Chen, Mingwei; Wu, Hanwei; Lin, Muqi; Zhan, Yonghao; Zhuang, Chengle; Lin, Junhao; Li, Jianfa; Xu, Wen; Fu, Xing; Zhang, Qiaoxia; Sun, Xiaojuan; Zhao, Guoping; Huang, Weiren

    2016-05-03

    As one of the members of the ETS gene family, the transcription factor v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS-1) plays key role in the regulation of physiological processes in normal cells and tumors. In this study, we aimed to investigate the relationship between the transcription factor ETS-1 and malignant phenotypes of bladder cancer. We demonstrated that ETS-1 was up-regulated in human bladder cancer tissue compared to paired normal bladder tissue. In order to evaluate the functional role of ETS-1 in human bladder cancer, vectors expressing ETS-1 shRNA and ETS-1 protein were constructed in vitro and transfected into the human bladder cancer T24 and 5637 cells. Our results showed that the transcription factor ETS-1 could promote cell migration and cell invasion in human bladder cancer, without affecting cell proliferation and apoptosis. In conclusion, ETS-1 plays oncogenic roles through inducing cell migration and invasion in human bladder cancer, and it can be used as a therapeutic target for treating human bladder cancer.

  3. Sequences in the intergenic spacer influence RNA Pol I transcription from the human rRNA promoter

    Energy Technology Data Exchange (ETDEWEB)

    Li, W.M.; Sylvester, J.E. [Hahnemann Univ., Philadelphia, PA (United States)

    1994-09-01

    In most eucaryotic species, ribosomal genes are tandemly repeated about 100-5000 times per haploid genome. The 43 Kb human rDNA repeat consists of a 13 Kb coding region for the 18S, 5.8S, 28S ribosomal RNAs (rRNAs) and transcribed spacers separated by a 30 Kb intergenic spacer. For species such as frog, mouse and rat, sequences in the intergenic spacer other than the gene promoter have been shown to modulate transcription of the ribosomal gene. These sequences are spacer promoters, enhancers and the terminator for spacer transcription. We are addressing whether the human ribosomal gene promoter is similarly influenced. In-vitro transcription run-off assays have revealed that the 4.5 kb region (CBE), directly upstream of the gene promoter, has cis-stimulation and trans-competition properties. This suggests that the CBE fragment contains an enhancer(s) for ribosomal gene transcription. Further experiments have shown that a fragment ({approximately}1.6 kb) within the CBE fragment also has trans-competition function. Deletion subclones of this region are being tested to delineate the exact sequences responsible for these modulating activities. Previous sequence analysis and functional studies have revealed that CBE contains regions of DNA capable of adopting alternative structures such as bent DNA, Z-DNA, and triple-stranded DNA. Whether these structures are required for modulating transcription remains to be determined as does the specific DNA-protein interaction involved.

  4. Human erythrocytes analyzed by generalized 2D Raman correlation spectroscopy

    Science.gov (United States)

    Wesełucha-Birczyńska, Aleksandra; Kozicki, Mateusz; Czepiel, Jacek; Łabanowska, Maria; Nowak, Piotr; Kowalczyk, Grzegorz; Kurdziel, Magdalena; Birczyńska, Malwina; Biesiada, Grażyna; Mach, Tomasz; Garlicki, Aleksander

    2014-07-01

    The most numerous elements of the blood cells, erythrocytes, consist mainly of two components: homogeneous interior filled with hemoglobin and closure which is the cell membrane. To gain insight into their specific properties we studied the process of disintegration, considering these two constituents, and comparing the natural aging process of human healthy blood cells. MicroRaman spectra of hemoglobin within the single RBC were recorded using 514.5, and 785 nm laser lines. The generalized 2D correlation method was applied to analyze the collected spectra. The time passed from blood donation was regarded as an external perturbation. The time was no more than 40 days according to the current storage limit of blood banks, although, the average RBC life span is 120 days. An analysis of the prominent synchronous and asynchronous cross peaks allow us to get insight into the mechanism of hemoglobin decomposition. Appearing asynchronous cross-peaks point towards globin and heme separation from each other, while synchronous shows already broken globin into individual amino acids. Raman scattering analysis of hemoglobin "wrapping", i.e. healthy erythrocyte ghosts, allows for the following peculiarity of their behavior. The increasing power of the excitation laser induced alterations in the assemblage of membrane lipids. 2D correlation maps, obtained with increasing laser power recognized as an external perturbation, allows for the consideration of alterations in the erythrocyte membrane structure and composition, which occurs first in the proteins. Cross-peaks were observed indicating an asynchronous correlation between the senescent-cell antigen (SCA) and heme or proteins vibrations. The EPR spectra of the whole blood was analyzed regarding time as an external stimulus. The 2D correlation spectra points towards participation of the selected metal ion centers in the disintegration process.

  5. Dissecting the transcriptional regulatory properties of human chromosome 16 highly conserved non-coding regions.

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    José Luis Royo

    Full Text Available Non-coding DNA conservation across species has been often used as a predictor for transcriptional enhancer activity. However, only a few systematic analyses of the function of these highly conserved non-coding regions (HCNRs have been performed. Here we use zebrafish transgenic assays to perform a systematic study of 113 HCNRs from human chromosome 16. By comparing transient and stable transgenesis, we show that the first method is highly inefficient, leading to 40% of false positives and 20% of false negatives. When analyzed in stable transgenic lines, a great majority of HCNRs were active in the central nervous system, although some of them drove expression in other organs such as the eye and the excretory system. Finally, by testing a fraction of the HCNRs lacking enhancer activity for in vivo insulator activity, we find that 20% of them may contain enhancer-blocking function. Altogether our data indicate that HCNRs may contain different types of cis-regulatory activity, including enhancer, insulators as well as other not yet discovered functions.

  6. Human TFIIH Kinase CDK7 Regulates Transcription-Associated Chromatin Modifications

    Directory of Open Access Journals (Sweden)

    Christopher C. Ebmeier

    2017-08-01

    Full Text Available CDK7 phosphorylates the RNA polymerase II (pol II C-terminal domain CTD and activates the P-TEFb-associated kinase CDK9, but its regulatory roles remain obscure. Here, using human CDK7 analog-sensitive (CDK7as cells, we observed reduced capping enzyme recruitment, increased pol II promoter-proximal pausing, and defective termination at gene 3′ ends upon CDK7 inhibition. We also noted that CDK7 regulates chromatin modifications downstream of transcription start sites. H3K4me3 spreading was restricted at gene 5′ ends and H3K36me3 was displaced toward gene 3′ ends in CDK7as cells. Mass spectrometry identified factors that bound TFIIH-phosphorylated versus P-TEFb-phosphorylated CTD (versus unmodified; capping enzymes and H3K4 methyltransferase complexes, SETD1A/B, selectively bound phosphorylated CTD, and the H3K36 methyltransferase SETD2 specifically bound P-TEFb-phosphorylated CTD. Moreover, TFIIH-phosphorylated CTD stimulated SETD1A/B activity toward nucleosomes, revealing a mechanistic basis for CDK7 regulation of H3K4me3 spreading. Collectively, these results implicate a CDK7-dependent “CTD code” that regulates chromatin marks in addition to RNA processing and pol II pausing.

  7. Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene

    Energy Technology Data Exchange (ETDEWEB)

    Kouzarides, T.; Bankier, A.T.; Satchwell, S.C.; Weston, K.; Tomlinson, P.; Barrell, B.G.

    1987-01-01

    DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. The authors present here a 5280 base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, they were able to analyze the extent of homology between the DNA polymerases of three distantly related herpes viruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpes viruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type I and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barr virus.

  8. Quantifiable mRNA transcripts for tamoxifen-metabolising enzymes in human endometrium.

    Science.gov (United States)

    Singh, Maneesh N; Stringfellow, Helen F; Walsh, Michael J; Ashton, Kate M; Paraskevaidis, Evangelos; Abdo, Khalil R; Martin-Hirsch, Pierre L; Phillips, David H; Martin, Francis L

    2008-07-10

    Tamoxifen has been used in the management of receptor-positive breast cancer for >20 years. Usage confers an elevated risk of developing endometrial carcinoma. Its mechanism of carcinogenicity remains unresolved with controversy as to whether or not this is mediated through a genotoxic mechanism. Usage is not only associated with an elevated occurrence of endometrioid endometrial carcinoma, but also type 2 and mixed epithelial-stromal tumours (MESTs) that have a poorer prognosis. Following hysterectomy, endometrial tissues (n=18) classified as benign (n=6), non-tamoxifen-associated carcinoma (n=6) and tamoxifen-associated carcinoma (n=6) were obtained; quantitative gene expression was performed. Employing real-time RT-PCR, the relative gene expressions of phase I/II metabolic enzymes CYP1A2, CYP1B1 and CYP3A4, cathechol-O-methyltransferase (COMT) and SULT2A1 were ascertained. Measurable mRNA transcripts, especially for those genes associated with tamoxifen bioactivation, were quantifiable in all the tissues examined. Whether this is evidence that generation of genotoxic tamoxifen metabolites may occur in human endometrial tissue remains to be ascertained.

  9. Post-transcriptional regulation of neurofibromin level in cultured human melanocytes in response to growth factors.

    Science.gov (United States)

    Griesser, J; Kaufmann, D; Maier, B; Mailhammer, R; Kuehl, P; Krone, W

    1997-03-01

    Among the symptoms that characterize neurofibromatosis type 1 (NF1) are pigmentation anomalies such as cafe au lait spots. It has been suggested that the reduction of the neurofibromin level in the epidermis of NF1 patients is responsible for the observed signs such as altered melanogenesis and altered density of melanocytes. Our studies show that in cultured normal human melanocytes, the neurofibromin level can be varied in vitro over a wide range by using different culture conditions. The influence of factors that control differentiation and proliferation of melanocytes on neurofibromin levels was studied. Immunoprecipitation followed by western blotting showed a 3- to 4-fold increase of neurofibromin after stimulation by PMA or bFGF, respectively, and a 1.5-fold increase in cells stimulated with steel factor. The increase of neurofibromin was not paralleled by a higher NF1 mRNA level as proved by northern blotting. Pulse-chase experiments with 35S-labeled melanocytes revealed an approximately 3-fold increase in the half-life of neurofibromin in bFGF- or PMA-stimulated cells compared to controls. These results indicate that the neurofibromin level of cultured melanocytes can be regulated by a mechanism independent of NF1 gene transcription and translation, which might influence the degradation rate of the protein.

  10. Transcriptional regulation of the human Liver X Receptor α gene by Hepatocyte Nuclear Factor 4α

    Energy Technology Data Exchange (ETDEWEB)

    Theofilatos, Dimitris; Anestis, Aristomenis [University of Crete Medical School and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, Heraklion, 71003, Crete (Greece); Hashimoto, Koshi [Department of Preemptive Medicine and Metabolism, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-city, Tokyo, 113-8510 (Japan); Kardassis, Dimitris, E-mail: kardasis@imbb.forth.gr [University of Crete Medical School and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, Heraklion, 71003, Crete (Greece)

    2016-01-15

    Liver X Receptors (LXRs) are sterol-activated transcription factors that play major roles in cellular cholesterol homeostasis, HDL biogenesis and reverse cholesterol transport. The aim of the present study was to investigate the mechanisms that control the expression of the human LXRα gene in hepatic cells. A series of reporter plasmids containing consecutive 5′ deletions of the hLXRα promoter upstream of the luciferase gene were constructed and the activity of each construct was measured in HepG2 cells. This analysis showed that the activity of the human LXRα promoter was significantly reduced by deleting the −111 to −42 region suggesting the presence of positive regulatory elements in this short proximal fragment. Bioinformatics data including motif search and ChIP-Seq revealed the presence of a potential binding motif for Hepatocyte Nuclear Factor 4 α (HNF-4α) in this area. Overexpression of HNF-4α in HEK 293T cells increased the expression of all LXRα promoter constructs except −42/+384. In line, silencing the expression of endogenous HNF-4α in HepG2 cells was associated with reduced LXRα protein levels and reduced activity of the −111/+384 LXRα promoter but not of the −42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4α specific binding motif (H4-SBM) in the −50 to −40 region of the human LXRα promoter. A triple mutation in this H4-SBM abolished HNF-4α binding and reduced the activity of the promoter to 65% relative to the wild type. Furthermore, the mutant promoter could not be transactivated by HNF-4α. In conclusion, our data indicate that HNF-4α may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXRα in hepatic cells. - Highlights: • The human LXRα promoter contains a HNF-4α specific binding motif in the proximal −50/−40 region. • Mutations in this motif abolished HNF4α binding and transactivation of the h

  11. The yeast and human FACT chromatin-reorganizing complexes solve R-loop-mediated transcription-replication conflicts.

    Science.gov (United States)

    Herrera-Moyano, Emilia; Mergui, Xénia; García-Rubio, María L; Barroso, Sonia; Aguilera, Andrés

    2014-04-01

    FACT (facilitates chromatin transcription) is a chromatin-reorganizing complex that swaps nucleosomes around the RNA polymerase during transcription elongation and has a role in replication that is not fully understood yet. Here we show that recombination factors are required for the survival of yeast FACT mutants, consistent with an accumulation of DNA breaks that we detected by Rad52 foci and transcription-dependent hyperrecombination. Breaks also accumulate in FACT-depleted human cells, as shown by γH2AX foci and single-cell electrophoresis. Furthermore, FACT-deficient yeast and human cells show replication impairment, which in yeast we demonstrate by ChIP-chip (chromatin immunoprecipitation [ChIP] coupled with microarray analysis) of Rrm3 to occur genome-wide but preferentially at highly transcribed regions. Strikingly, in yeast FACT mutants, high levels of Rad52 foci are suppressed by RNH1 overexpression; R loops accumulate at high levels, and replication becomes normal when global RNA synthesis is inhibited in FACT-depleted human cells. The results demonstrate a key function of FACT in the resolution of R-loop-mediated transcription-replication conflicts, likely associated with a specific chromatin organization.

  12. Comparative transcriptional analysis of three human ligaments with distinct biomechanical properties.

    Science.gov (United States)

    Lorda-Diez, Carlos I; Canga-Villegas, Ana; Cerezal, Luis; Plaza, Santiago; Hurlé, Juan M; García-Porrero, Juan A; Montero, Juan A

    2013-12-01

    One major aim of regenerative medicine targeting the musculoskeletal system is to provide complementary and/or alternative therapeutic approaches to current surgical therapies, often involving the removal and prosthetic substitution of damaged tissues such as ligaments. For these approaches to be successful, detailed information regarding the cellular and molecular composition of different musculoskeletal tissues is required. Ligaments have often been considered homogeneous tissues with common biomechanical properties. However, advances in tissue engineering research have highlighted the functional relevance of the organisational and compositional differences between ligament types, especially in those with higher risks of injury. The aim of this study was to provide information concerning the relative expression levels of a subset of key genes (including extracellular matrix components, transcription factors and growth factors) that confer functional identity to ligaments. We compared the transcriptomes of three representative human ligaments subjected to different biomechanical demands: the anterior cruciate ligament (ACL); the ligamentum teres of the hip (LT); and the iliofemoral ligament (IL). We revealed significant differences in the expression of type I collagen, elastin, fibromodulin, biglycan, transforming growth factor β1, transforming growth interacting factor 1, hypoxia-inducible factor 1-alpha and transforming growth factor β-induced gene between the IL and the other two ligaments. Thus, considerable molecular heterogeneity can exist between anatomically distinct ligaments with differing biomechanical demands. However, the LT and ACL were found to show remarkable molecular homology, suggesting common functional properties. This finding provides experimental support for the proposed role of the LT as a hip joint stabiliser in humans.

  13. Comparative transcriptional analysis of three human ligaments with distinct biomechanical properties

    Science.gov (United States)

    Lorda-Diez, Carlos I; Canga-Villegas, Ana; Cerezal, Luis; Plaza, Santiago; Hurlé, Juan M; García-Porrero, Juan A; Montero, Juan A

    2013-01-01

    One major aim of regenerative medicine targeting the musculoskeletal system is to provide complementary and/or alternative therapeutic approaches to current surgical therapies, often involving the removal and prosthetic substitution of damaged tissues such as ligaments. For these approaches to be successful, detailed information regarding the cellular and molecular composition of different musculoskeletal tissues is required. Ligaments have often been considered homogeneous tissues with common biomechanical properties. However, advances in tissue engineering research have highlighted the functional relevance of the organisational and compositional differences between ligament types, especially in those with higher risks of injury. The aim of this study was to provide information concerning the relative expression levels of a subset of key genes (including extracellular matrix components, transcription factors and growth factors) that confer functional identity to ligaments. We compared the transcriptomes of three representative human ligaments subjected to different biomechanical demands: the anterior cruciate ligament (ACL); the ligamentum teres of the hip (LT); and the iliofemoral ligament (IL). We revealed significant differences in the expression of type I collagen, elastin, fibromodulin, biglycan, transforming growth factor β1, transforming growth interacting factor 1, hypoxia-inducible factor 1-alpha and transforming growth factor β-induced gene between the IL and the other two ligaments. Thus, considerable molecular heterogeneity can exist between anatomically distinct ligaments with differing biomechanical demands. However, the LT and ACL were found to show remarkable molecular homology, suggesting common functional properties. This finding provides experimental support for the proposed role of the LT as a hip joint stabiliser in humans. PMID:24128114

  14. Transcriptional analysis reveals gender-specific changes in the aging of the human immune system.

    Science.gov (United States)

    Marttila, Saara; Jylhävä, Juulia; Nevalainen, Tapio; Nykter, Matti; Jylhä, Marja; Hervonen, Antti; Tserel, Liina; Peterson, Pärt; Hurme, Mikko

    2013-01-01

    Aging and gender have a strong influence on the functional capacity of the immune system. In general, the immune response in females is stronger than that in males, but there is scant information about the effect of aging on the gender difference in the immune response. To address this question, we performed a transcriptomic analysis of peripheral blood mononuclear cells derived from elderly individuals (nonagenarians, n = 146) and young controls (aged 19-30 years, n = 30). When compared to young controls, we found 339 and 248 genes that were differentially expressed (p1.5 or human immune system are significantly different in males and females.

  15. H-InvDB in 2009: extended database and data mining resources for human genes and transcripts.

    Science.gov (United States)

    Yamasaki, Chisato; Murakami, Katsuhiko; Takeda, Jun-ichi; Sato, Yoshiharu; Noda, Akiko; Sakate, Ryuichi; Habara, Takuya; Nakaoka, Hajime; Todokoro, Fusano; Matsuya, Akihiro; Imanishi, Tadashi; Gojobori, Takashi

    2010-01-01

    We report the extended database and data mining resources newly released in the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). H-InvDB is a comprehensive annotation resource of human genes and transcripts, and consists of two main views and six sub-databases. The latest release of H-InvDB (release 6.2) provides the annotation for 219,765 human transcripts in 43,159 human gene clusters based on human full-length cDNAs and mRNAs. H-InvDB now provides several new annotation features, such as mapping of microarray probes, new gene models, relation to known ncRNAs and information from the Glycogene database. H-InvDB also provides useful data mining resources-'Navigation search', 'H-InvDB Enrichment Analysis Tool (HEAT)' and web service APIs. 'Navigation search' is an extended search system that enables complicated searches by combining 16 different search options. HEAT is a data mining tool for automatically identifying features specific to a given human gene set. HEAT searches for H-InvDB annotations that are significantly enriched in a user-defined gene set, as compared with the entire H-InvDB representative transcripts. H-InvDB now has web service APIs of SOAP and REST to allow the use of H-InvDB data in programs, providing the users extended data accessibility.

  16. Post-transcriptional modifications modulate conformational dynamics in human U2-U6 snRNA complex.

    Science.gov (United States)

    Karunatilaka, Krishanthi S; Rueda, David

    2014-01-01

    The spliceosome catalyzes precursor-mRNA splicing in all eukaryotes. It consists of over 100 proteins and five small nuclear RNAs (snRNAs), including U2 and U6 snRNAs, which are essential for catalysis. Human and yeast snRNAs share structural similarities despite the fact that human snRNAs contain numerous post-transcriptional modifications. Although functions for these modifications have been proposed, their exact roles are still not well understood. To help elucidate these roles in pre-mRNA splicing, we have used single-molecule fluorescence to characterize the effect of several post-transcriptional modifications in U2 snRNA on the conformation and dynamics of the U2-U6 complex in vitro. Consistent with yeast, the human U2-U6 complex reveals the presence of a magnesium-dependent dynamic equilibrium among three conformations. Interestingly, our data show that modifications in human U2 stem I modulate the dynamic equilibrium of the U2-U6 complex by stabilizing the four-helix structure. However, the small magnitude of this effect suggests that post-transcriptional modifications in human snRNAs may have a primary role in the mediation of specific RNA-protein interactions in vivo.

  17. Post-transcriptional modifications modulate conformational dynamics in human U2–U6 snRNA complex

    Science.gov (United States)

    Karunatilaka, Krishanthi S.; Rueda, David

    2014-01-01

    The spliceosome catalyzes precursor-mRNA splicing in all eukaryotes. It consists of over 100 proteins and five small nuclear RNAs (snRNAs), including U2 and U6 snRNAs, which are essential for catalysis. Human and yeast snRNAs share structural similarities despite the fact that human snRNAs contain numerous post-transcriptional modifications. Although functions for these modifications have been proposed, their exact roles are still not well understood. To help elucidate these roles in pre-mRNA splicing, we have used single-molecule fluorescence to characterize the effect of several post-transcriptional modifications in U2 snRNA on the conformation and dynamics of the U2–U6 complex in vitro. Consistent with yeast, the human U2–U6 complex reveals the presence of a magnesium-dependent dynamic equilibrium among three conformations. Interestingly, our data show that modifications in human U2 stem I modulate the dynamic equilibrium of the U2–U6 complex by stabilizing the four-helix structure. However, the small magnitude of this effect suggests that post-transcriptional modifications in human snRNAs may have a primary role in the mediation of specific RNA–protein interactions in vivo. PMID:24243115

  18. Human Sex Determination at the Edge of Ambiguity: INHERITED XY SEX REVERSAL DUE TO ENHANCED UBIQUITINATION AND PROTEASOMAL DEGRADATION OF A MASTER TRANSCRIPTION FACTOR.

    Science.gov (United States)

    Racca, Joseph D; Chen, Yen-Shan; Yang, Yanwu; Phillips, Nelson B; Weiss, Michael A

    2016-10-14

    A general problem is posed by analysis of transcriptional thresholds governing cell fate decisions in metazoan development. A model is provided by testis determination in therian mammals. Its key step, Sertoli cell differentiation in the embryonic gonadal ridge, is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to XY female development (Swyer syndrome). Here, we have characterized an inherited mutation compatible with either male or female somatic phenotypes as observed in an XY father and XY daughter, respectively. The mutation (a crevice-forming substitution at a conserved back surface of the SRY high mobility group box) markedly destabilizes the domain but preserves specific DNA affinity and induced DNA bend angle. On transient transfection of diverse human and rodent cell lines, the variant SRY exhibited accelerated proteasomal degradation (relative to wild type) associated with increased ubiquitination; in vitro susceptibility to ubiquitin-independent ("default") cleavage by the 20S core proteasome was unchanged. The variant's gene regulatory activity (as assessed in a cellular model of the rat embryonic XY gonadal ridge) was reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. Chemical proteasome inhibition restored native-like SRY expression and transcriptional activity in association with restored occupancy of a sex-specific enhancer element in principal downstream gene Sox9, demonstrating that the variant SRY exhibits essentially native activity on a per molecule basis. Our findings define a novel mechanism of impaired organogenesis, accelerated ubiquitin-directed proteasomal degradation of a master transcription factor leading to a developmental decision poised at the edge of ambiguity.

  19. Human teneurin-1 is a direct target of the homeobox transcription factor EMX2 at a novel alternate promoter

    Directory of Open Access Journals (Sweden)

    Brož Daniela

    2011-06-01

    Full Text Available Abstract Background Teneurin-1 is a member of a family of type II transmembrane proteins conserved from C.elegans to vertebrates. Teneurin expression in vertebrates is best studied in mouse and chicken, where the four members teneurin-1 to -4 are predominantly expressed in the developing nervous system in area specific patterns. Based on their distinct, complementary expression a possible function in the establishment of proper connectivity in the brain was postulated. However, the transcription factors contributing to these distinctive expression patterns are largely unknown. Emx2 is a homeobox transcription factor, known to be important for area specification in the developing cortex. A study of Emx2 knock-out mice suggested a role of Emx2 in regulating patterned teneurin expression. Results 5'RACE of human teneurin-1 revealed new alternative untranslated exons that are conserved in mouse and chicken. Closer analysis of the conserved region around the newly identified transcription start revealed promoter activity that was induced by EMX2. Mutation of a predicted homeobox binding site decreased the promoter activity in different reporter assays in vitro and in vivo in electroporated chick embryos. We show direct in vivo binding of EMX2 to the newly identified promoter element and finally confirm that the endogenous alternate transcript is specifically upregulated by EMX2. Conclusions We found that human teneurin-1 is directly regulated by EMX2 at a newly identified and conserved promoter region upstream of the published transcription start site, establishing teneurin-1 as the first human EMX2 target gene. We identify and characterize the EMX2 dependent promoter element of human teneurin-1.

  20. TEFM (c17orf42) is necessary for transcription of human mtDNA

    NARCIS (Netherlands)

    Minczuk, M.; He, J.; Duch, A.M.; Ettema, T.J.G.; Chlebowski, A.; Dzionek, K.; Nijtmans, L.G.J.; Huynen, M.A.; Holt, I.J.

    2011-01-01

    Here we show that c17orf42, hereafter TEFM (transcription elongation factor of mitochondria), makes a critical contribution to mitochondrial transcription. Inactivation of TEFM in cells by RNA interference results in respiratory incompetence owing to decreased levels of H- and L-strand promoter-dist

  1. Regulation of human papillomavirus transcription by the differentiation-dependent epithelial factor Epoc-1/skn-1a.

    Science.gov (United States)

    Yukawa, K; Butz, K; Yasui, T; Kikutani, H; Hoppe-Seyler, F

    1996-01-01

    Human papillomavirus (HPV) early gene expression is closely linked to the differentiation status of infected epithelial cells. Typically, HPV type 16 (HPV16) or HPV18 E6 and E7 transcripts are only barely detectable within the undifferentiated basal cell layer, but their levels increase concomitantly with higher degrees of epithelial cell differentiation in suprabasal cells. A similar differentiation-dependent distribution of expression has been reported for the recently cloned epithelial cell specific transcription factor Epoc-1/skn-1a. We therefore examined whether Epoc-1/skn-1a may be directly involved in the activation of HPV E6/E7 transcription. Transient transfection studies showed that Epoc-1/skn-1a specifically stimulated the HPV16 and HPV18 E6/E7 promoters. Moreover, ectopically expressed Epoc-1/skn-1a was sufficient to stimulate HPV transcription also in nonepithelial cells. By deletion analyses, the Epoc-1/skn-1a-responsive element was mapped to the promoter-proximal portion of the HPV18 transcriptional control region. Footprint analyses and gel retardation assays demonstrated direct binding of Epoc-1/skn-1a to a hitherto uncharacterized site within this region. Mutation of the Epoc-1/skn-1a recognition site within the context of the complete HPV18 upstream regulatory region inhibited Epoc-1/skn-1a-mediated transactivation. These results show that Epoc-1/skn-1a can directly activate the E6/E7 promoter by binding to the viral transcriptional control region. Thus, Epoc-1/skn-1a may be involved in the differentiation-dependent regulation of HPV transcription.

  2. Mapping of HPV transcripts in four human cervical lesions using RNAseq suggests quantitative rearrangements during carcinogenic progression.

    Science.gov (United States)

    Chen, Jinmiao; Xue, Yuezhen; Poidinger, Michael; Lim, Thimothy; Chew, Sung Hock; Pang, Chai Ling; Abastado, Jean-Pierre; Thierry, Françoise

    2014-08-01

    Two classes of Human papillomaviruses (HPV) infect the anogenital track: high risk viruses that are associated with risk of cervical cancer and low risk types that drive development of benign lesions, such as condylomas. In the present study, we established quantitative transcriptional maps of the viral genome in clinical lesions associated with high risk HPV16 or low risk HPV6b. Marked qualitative and quantitative changes in the HPV16 transcriptome were associated with progression from low to high grade lesions. Specific transcripts encoding essential regulatory proteins such as E7, E2, E1^E4 and E5 were identified. We also identified intrinsic differences between the HPV6b-associated condyloma transcript map and that of the HPV16-associated low grade CIN specifically regarding promoter usage. Characterization and quantification of HPV transcripts in patient samples thus establish the impact of viral transcriptional regulation on the status of HPV-associated lesions and may therefore help in defining new biologically-relevant prognosis markers. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Diesel exhaust particulate extracts inhibit transcription of nuclear respiratory factor-1 and cell viability in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mattingly, Kathleen A.; Klinge, Carolyn M. [University of Louisville School of Medicine, Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, Louisville, KY (United States)

    2012-04-15

    Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1-regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17{beta}-estradiol (E{sub 2}), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription, and this suppression was not ablated by concomitant treatment with E{sub 2}, 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E{sub 2} increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects. (orig.)

  4. Activin/Nodal signaling controls divergent transcriptional networks in human embryonic stem cells and in endoderm progenitors.

    Science.gov (United States)

    Brown, Stephanie; Teo, Adrian; Pauklin, Siim; Hannan, Nicholas; Cho, Candy H-H; Lim, Bing; Vardy, Leah; Dunn, N Ray; Trotter, Matthew; Pedersen, Roger; Vallier, Ludovic

    2011-08-01

    Activin/Nodal signaling is necessary to maintain pluripotency of human embryonic stem cells (hESCs) and to induce their differentiation toward endoderm. However, the mechanisms by which Activin/Nodal signaling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3, which represent the direct effectors of Activin/Nodal signaling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterizing pluripotency, which includes Oct-4, Nanog, FoxD3, Dppa4, Tert, Myc, and UTF1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signaling appears to control divergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs; whereas, functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signaling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signaling pathways can orchestrate divergent cell fate decisions.

  5. Revised genomic structure of the human ghrelin gene and identification of novel exons, alternative splice variants and natural antisense transcripts

    Directory of Open Access Journals (Sweden)

    Herington Adrian C

    2007-08-01

    Full Text Available Abstract Background Ghrelin is a multifunctional peptide hormone expressed in a range of normal tissues and pathologies. It has been reported that the human ghrelin gene consists of five exons which span 5 kb of genomic DNA on chromosome 3 and includes a 20 bp non-coding first exon (20 bp exon 0. The availability of bioinformatic tools enabling comparative analysis and the finalisation of the human genome prompted us to re-examine the genomic structure of the ghrelin locus. Results We have demonstrated the presence of an additional novel exon (exon -1 and 5' extensions to exon 0 and 1 using comparative in silico analysis and have demonstrated their existence experimentally using RT-PCR and 5' RACE. A revised exon-intron structure demonstrates that the human ghrelin gene spans 7.2 kb and consists of six rather than five exons. Several ghrelin gene-derived splice forms were detected in a range of human tissues and cell lines. We have demonstrated ghrelin gene-derived mRNA transcripts that do not code for ghrelin, but instead may encode the C-terminal region of full-length preproghrelin (C-ghrelin, which contains the coding region for obestatin and a transcript encoding obestatin-only. Splice variants that differed in their 5' untranslated regions were also found, suggesting a role of these regions in the post-transcriptional regulation of preproghrelin translation. Finally, several natural antisense transcripts, termed ghrelinOS (ghrelin opposite strand transcripts, were demonstrated via orientation-specific RT-PCR, 5' RACE and in silico analysis of ESTs and cloned amplicons. Conclusion The sense and antisense alternative transcripts demonstrated in this study may function as non-coding regulatory RNA, or code for novel protein isoforms. This is the first demonstration of putative obestatin and C-ghrelin specific transcripts and these findings suggest that these ghrelin gene-derived peptides may also be produced independently of preproghrelin

  6. Transforming growth factor-beta inhibits aromatase gene transcription in human trophoblast cells via the Smad2 signaling pathway

    Directory of Open Access Journals (Sweden)

    Fu Guodong

    2009-12-01

    Full Text Available Abstract Background Transforming growth factor-beta (TGF-beta is known to exert multiple regulatory functions in the human placenta, including inhibition of estrodial production. We have previously reported that TGF-beta1 decreased aromatase mRNA levels in human trophoblast cells. The objective of this study was to investigate the molecular mechanisms underlying the regulatory effect of TGF-beta1 on aromatase expression. Methods To determine if TGF-beta regulates aromatase gene transcription, several reporter constructs containing different lengths of the placental specific promoter of the human aromatase gene were generated. JEG-3 cells were transiently transfected with a promoter construct and treated with or without TGF-beta1. The promoter activity was measured by luciferase assays. To examine the downstream signaling molecule mediating the effect of TGF-beta on aromatase transcription, cells were transiently transfected with dominant negative mutants of TGF-beta type II (TbetaRII and type I receptor (ALK5 receptors before TGF-beta treatment. Smad2 activation was assessed by measuring phophorylated Smad2 protein levels in cytosolic and nuclear fractions. Smad2 expression was silenced using a siRNA expression construct. Finally, aromatase mRNA half-life was determined by treating cells with actinomycin D together with TGF-beta1 and measuring aromatase mRNA levels at various time points after treatment. Results and Discussion TGF-beta1 inhibited the aromatase promoter activity in a time- and dose-dependent manner. Deletion analysis suggests that the TGF-β1 response element resides between -422 and -117 nucleotides upstream from the transcription start site where a Smad binding element was found. The inhibitory effect of TGF-beta1 was blocked by dominant negative mutants of TbetaRII and ALK5. TGF-beta1 treatment induced Smad2 phosphorylation and translocation into the nucleus. On the other hand, knockdown of Smad2 expression reversed the

  7. Fear generalization in humans: impact of verbal instructions

    NARCIS (Netherlands)

    Vervliet, B.; Kindt, M.; Vansteenwegen, D.; Hermans, D.

    2010-01-01

    Fear generalization lies at the heart of many anxiety problems, but little is known about the factors that can influence this phenomenon. The present study investigated whether verbal instructions about specific stimulus features can influence conditioned fear generalization. All participants were f

  8. Data in support of transcriptional regulation and function of Fas-antisense long noncoding RNA during human erythropoiesis

    Directory of Open Access Journals (Sweden)

    Olga Villamizar

    2016-06-01

    Full Text Available This paper describes data related to a research article titled, “Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death” [1]. Long noncoding RNAs (lncRNAs are increasingly appreciated for their capacity to regulate many steps of gene expression. While recent studies suggest that many lncRNAs are functional, the scope of their actions throughout human biology is largely undefined including human red blood cell development (erythropoiesis. Here we include expression data for 82 lncRNAs during early, intermediate and late stages of human erythropoiesis using a commercial qPCR Array. From these data, we identified lncRNA Fas-antisense 1 (Fas-AS1 or Saf described in the research article. Also included are 5′ untranslated sequences (UTR for lncRNA Saf with transcription factor target sequences identified. Quantitative RT-PCR data demonstrate relative levels of critical erythroid transcription factors, GATA-1 and KLF1, in K562 human erythroleukemia cells and maturing erythroblasts derived from human CD34+ cells. End point and quantitative RT-PCR data for cDNA prepared using random hexamers versus oligo(dT18 revealed that lncRNA Saf is not effectively polyadenylated. Finally, we include flow cytometry histograms demonstrating Fas levels on maturing erythroblasts derived from human CD34+ cells transduced using mock conditions or with lentivirus particles encoding for Saf.

  9. Transcriptional regulation of the differentiation-linked human K4 promoter is dependent upon esophageal-specific nuclear factors.

    Science.gov (United States)

    Opitz, O G; Jenkins, T D; Rustgi, A K

    1998-09-11

    The stratified squamous epithelium comprises actively proliferating basal cells that undergo a program of differentiation accompanied by morphological, biochemical, and genetic changes. The transcriptional regulatory signals and the genes that orchestrate this switch from proliferation to differentiation can be studied through the keratin gene family. Given the localization of keratin 4 (K4) to the early differentiated suprabasal compartment and having previously demonstrated that targeted disruption of this gene in murine embryonic stem cells results in impairment of the normal differentiation program in esophageal and corneal epithelial cells, we studied the transcriptional regulation of the human K4 promoter. A panel of K4 promoter deletions were found in transient transfection assays to be predominantly active in esophageal and corneal cell lines. A critical cis-regulatory element resides between -163 and -140 bp and contains an inverted CACACCT motif. A site-directed mutated version of this motif within the K4 promoter renders it inactive, whereas the wild-type version is active in a heterologous promoter system. It specifically binds esophageal-specific zinc-dependent transcriptional factors. Our studies demonstrate that regulation of the human K4 promoter is in part mediated through tissue-specific transcriptional factors.

  10. Definition of the transcriptional activation domains of three human HOX proteins depends on the DNA-binding context.

    Science.gov (United States)

    Viganò, M A; Di Rocco, G; Zappavigna, V; Mavilio, F

    1998-11-01

    Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions which ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analysis. We report the identification of the transcriptional activation domains of three human Hox proteins, HOXB1, HOXB3, and HOXD9, which interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery depending on the structure of their target regulatory elements.

  11. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

    Science.gov (United States)

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms.

  12. Genome-wide transcriptional comparison of MPP+ treated human neuroblastoma cells with the state space model

    Directory of Open Access Journals (Sweden)

    Jin Hwan Do

    2015-10-01

    Full Text Available This study compared a parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+ response in two distinct phenotypes of human neuroblastoma cell lines: neuronal N-type SH-SY5Y cells and flat substrate-adherent S-type SH-EP cells. SH-SY5Y and SH-EP cells shared only 14% of their own MPP+ response genes, and their gene ontology (GO analysis revealed significant endoplasmic reticulum (ER stress by misfolded proteins. Gene modules, which are groups of transcriptionally co-expressed genes with similar biological functions, were identified for SH-SY5Y and SH-EP cells by using time-series microarray data with the state space model (SSM. All modules of SH-SY5Y and SH-EP cells showed strong positive auto-regulation that was often mediated via signal molecules and may cause bi-stability. Interactions in gene levels were calculated by using SSM parameters obtained in the process of module identification. Gene networks that were constructed from the gene interaction matrix showed different hub genes with high node degrees between SH-SY5Y and SH-EP cells. That is, key hub genes of SH-SY5Y cells were DCN, HIST1H2BK, and C5orf40, whereas those of SH-EP cells were MSH6, RBCK1, MTHFD2, ZNF26, CTH, and CARS. These results suggest that inhibition of the mitochondrial complex I by MPP+ might induce different downstream processes that are cell type dependent.

  13. Alternative splicing and extensive RNA editing of human TPH2 transcripts.

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    Maik Grohmann

    Full Text Available Brain serotonin (5-HT neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2 in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1 is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre-mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour.

  14. Clone-specific expression, transcriptional regulation, and action of interleukin-6 in human colon carcinoma cells

    Directory of Open Access Journals (Sweden)

    Fabjani Gerhild

    2008-01-01

    Full Text Available Abstract Background Many cancer cells produce interleukin-6 (IL-6, a cytokine that plays a role in growth stimulation, metastasis, and angiogenesis of secondary tumours in a variety of malignancies, including colorectal cancer. Effectiveness of IL-6 in this respect may depend on the quantity of basal and inducible IL-6 expressed as the tumour progresses through stages of malignancy. We therefore have evaluated the effect of IL-6 modulators, i.e. IL-1β, prostaglandin E2, 17β-estradiol, and 1,25-dihydroxyvitamin D3, on expression and synthesis of the cytokine at different stages of tumour progression. Methods We utilized cultures of the human colon carcinoma cell clones Caco-2/AQ, COGA-1A and COGA-13, all of which expressed differentiation and proliferation markers typical of distinct stages of tumour progression. IL-6 mRNA and protein levels were assayed by RT-PCR and ELISA, respectively. DNA sequencing was utilized to detect polymorphisms in the IL-6 gene promoter. Results IL-6 mRNA and protein concentrations were low in well and moderately differentiated Caco-2/AQ and COGA-1A cells, but were high in poorly differentiated COGA-13 cells. Addition of IL-1β (5 ng/ml to a COGA-13 culture raised IL-6 production approximately thousandfold via a prostaglandin-independent mechanism. Addition of 17β-estradiol (10-7 M reduced basal IL-6 production by one-third, but IL-1β-inducible IL-6 was unaffected. Search for polymorphisms in the IL-6 promoter revealed the presence of a single haplotype, i.e., -597A/-572G/-174C, in COGA-13 cells, which is associated with a high degree of transcriptional activity of the IL-6 gene. IL-6 blocked differentiation only in Caco-2/AQ cells and stimulated mitosis through up-regulation of c-myc proto-oncogene expression. These effects were inhibited by 10-8 M 1,25-dihydroxyvitamin D3. Conclusion In human colon carcinoma cells derived from well and moderately differentiated tumours, IL-6 expression is low and only marginally

  15. hELP3 Subunit of the Elongator Complex Regulates the Transcription of HSP70 Gene in Human Cells

    Institute of Scientific and Technical Information of China (English)

    Qiuju HAN; Xiaozhe HOU; Dongmei SU; Lina PAN; Jizhou DUAN; Liguo CUI; Baiqu HUANG; Jun LU

    2007-01-01

    The human Elongator complex is remarkably similar to its yeast counterpart in several aspects.In a previous study, we analyzed the functions of the human elongation protein 3 (hELP3) subunit of the human Elongator by using an in vivo yeast complementation system. However, direct evidence for hELP3 functions in regulating gene expression in human cells was not obtained. In this study, we used hELP3 antisense oligonucleotide inhibitors to knock down hELP3 gene expression to investigate its function in human 293T cells. The results showed that specific reduction of hELP3 mRNA and protein caused a significant suppression of HSP70-2 gene expression, and this was accompanied by histone H3 hypoacetylation and decreased RNA polymerase Ⅱ density at the HSP70-2 gene. Moreover, the data also showed that hELP3 exerted the transcriptional regulatory function directly through its presence on the HSP70-2 gene. Data presented in this report provide further insight and direct evidence of the functions of hELP3 in HSP70-2 gene transcriptional elongation in human cells.

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

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    Sakaki Yoshiyuki

    2004-02-01

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

  17. GATA3 is a master regulator of the transcriptional response to low-dose ionizing radiation in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bonin, F.; Molina, M.; Berthier-Vergnes, O.; Lamartine, J. [Universite de Lyon, Lyon, F-69003 (France); Universite Lyon 1, Lyon, F-69003 (France); CNRS, UMR5534, Centre de Genetique Moleculaire et Cellulaire, Villeurbanne, F-69622 (France); Malet, C.; Ginestet, C. [Centre Leon Berard, Service de Radiotherapie, Lyon F-69008 (France); Martin, M.T. [Laboratoire de Genomique et Radiobiologie de la Keratinopoiese, CEA, IRCM, Evry F-91000 (France)

    2009-07-01

    Background: The general population is constantly exposed to low levels of radiation through natural, occupational or medical irradiation. Even if the biological effects of low-level radiation have been intensely debated and investigated, the molecular mechanisms underlying the cellular response to low doses remain largely unknown. Results: The present study investigated the role of GATA3 protein in the control of the cellular and molecular response of human keratinocytes exposed to a 1 cGy dose of X-rays. Chromatin immunoprecipitation showed GATA3 to be able to bind the promoter of 4 genes responding to a 1 cGy exposure. To go further into the role of GATA3 after ionizing radiation exposure, we studied the cellular and molecular consequences of radiation in GATA3 knock-down cells. Knockdown was obtained by lentiviral-mediated expression of an shRNA targeting the GATA3 transcript in differentiated keratinocytes. First, radiosensitivity was assessed: the toxicity, in terms of immediate survival (with XTT test), associated with 1 cGy radiation was found to be increased in GATA3 knock-down cells. The impact of GATA3 knock-down on the transcriptome of X-ray irradiated cells was also investigated, using oligonucleotide micro-arrays to assess changes between 3 h and 72 h post-irradiation in normal vs GATA3 knock-down backgrounds; transcriptome response was found to be completely altered in GATA3 knock-down cells, with a strong induction/repression peak 48 h after irradiation. Functional annotation revealed enrichment in genes known to be involved in chaperone activity, TGF{beta} signalling and stress response. Conclusion: Taken together, these data indicate that GATA3 is an important regulator of the cellular and molecular response of epidermal cells to very low doses of radiation. (authors)

  18. Transcription Elongation Factor NusA Is a General Antagonist of Rho-dependent Termination in Escherichia coli.

    Science.gov (United States)

    Qayyum, M Zuhaib; Dey, Debashish; Sen, Ranjan

    2016-04-08

    NusA is an essential protein that binds to RNA polymerase and also to the nascent RNA and influences transcription by inducing pausing and facilitating the process of transcription termination/antitermination. Its participation in Rho-dependent transcription termination has been perceived, but the molecular nature of this involvement is not known. We hypothesized that, because both Rho and NusA are RNA-binding proteins and have the potential to target the same RNA, the latter is likely to influence the global pattern of the Rho-dependent termination. Analyses of the nascent RNA binding properties and consequent effects on the Rho-dependent termination functions of specific NusA-RNA binding domain mutants revealed an existence of Rho-NusA direct competition for the overlappingnut(NusA-binding site) andrut(Rho-binding site) sites on the RNA. This leads to delayed entry of Rho at therutsite that inhibits the latter's RNA release process. High density tiling microarray profiles of these NusA mutants revealed that a significant number of genes, together with transcripts from intergenic regions, are up-regulated. Interestingly, the majority of these genes were also up-regulated when the Rho function was compromised. These results provide strong evidence for the existence of NusA-binding sites in different operons that are also the targets of Rho-dependent terminations. Our data strongly argue in favor of a direct competition between NusA and Rho for the access of specific sites on the nascent transcripts in different parts of the genome. We propose that this competition enables NusA to function as a global antagonist of the Rho function, which is unlike its role as a facilitator of hairpin-dependent termination.

  19. Transcription-factor occupancy at HOT regions quantitatively predicts RNA polymerase recruitment in five human cell lines.

    KAUST Repository

    Foley, Joseph W

    2013-10-20

    BACKGROUND: High-occupancy target (HOT) regions are compact genome loci occupied by many different transcription factors (TFs). HOT regions were initially defined in invertebrate model organisms, and we here show that they are a ubiquitous feature of the human gene-regulation landscape. RESULTS: We identified HOT regions by a comprehensive analysis of ChIP-seq data from 96 DNA-associated proteins in 5 human cell lines. Most HOT regions co-localize with RNA polymerase II binding sites, but many are not near the promoters of annotated genes. At HOT promoters, TF occupancy is strongly predictive of transcription preinitiation complex recruitment and moderately predictive of initiating Pol II recruitment, but only weakly predictive of elongating Pol II and RNA transcript abundance. TF occupancy varies quantitatively within human HOT regions; we used this variation to discover novel associations between TFs. The sequence motif associated with any given TF\\'s direct DNA binding is somewhat predictive of its empirical occupancy, but a great deal of occupancy occurs at sites without the TF\\'s motif, implying indirect recruitment by another TF whose motif is present. CONCLUSIONS: Mammalian HOT regions are regulatory hubs that integrate the signals from diverse regulatory pathways to quantitatively tune the promoter for RNA polymerase II recruitment.

  20. Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans

    Directory of Open Access Journals (Sweden)

    Stahl Stefanie

    2002-02-01

    Full Text Available Abstract Background Mucolipidosis type IV (MLIV is an autosomal recessive lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. Recently the MLIV gene, MCOLN1, has been identified as a new member of the transient receptor potential (TRP cation channel superfamily. Here we report the cloning and characterization of the mouse homologue, Mcoln1, and report a novel splice variant that is not seen in humans. Results The human and mouse genes display a high degree of synteny. Mcoln1 shows 91% amino acid and 86% nucleotide identity to MCOLN1. Also, Mcoln1 maps to chromosome 8 and contains an open reading frame of 580 amino acids, with a transcript length of approximately 2 kb encoded by 14 exons, similar to its human counterpart. The transcript that results from murine specific alternative splicing encodes a 611 amino acid protein that differs at the c-terminus. Conclusions Mcoln1 is highly similar to MCOLN1, especially in the transmembrane domains and ion pore region. Also, the late endosomal/lysosomal targeting signal is conserved, supporting the hypothesis that the protein is localized to these vesicle membranes. To date, there are very few reports describing species-specific splice variants. While identification of Mcoln1 is crucial to the development of mouse models for MLIV, the fact that there are two transcripts in mice suggests an additional or alternate function of the gene that may complicate phenotypic assessment.

  1. Transcriptional regulation of human FE65, a ligand of Alzheimer's disease amyloid precursor protein, by Sp1.

    LENUS (Irish Health Repository)

    Yu, Hoi-Tin

    2010-03-01

    FE65 is a neuronal-enriched adaptor protein that binds to the Alzheimer\\'s disease amyloid precursor protein (APP). FE65 forms a transcriptionally active complex with the APP intracellular domain (AICD). The precise gene targets for this complex are unclear but several Alzheimer\\'s disease-linked genes have been proposed. Additionally, evidence suggests that FE65 influences APP metabolism. The mechanism by which FE65 expression is regulated is as yet unknown. To gain insight into the regulatory mechanism, we cloned a 1.6 kb fragment upstream of the human FE65 gene and found that it possesses particularly strong promoter activity in neurones. To delineate essential regions in the human FE65 promoter, a series of deletion mutants were generated. The minimal FE65 promoter was located between -100 and +5, which contains a functional Sp1 site. Overexpression of the transcription factor Sp1 potentiates the FE65 promoter activity. Conversely, suppression of the FE65 promoter was observed in cells either treated with an Sp1 inhibitor or in which Sp1 was knocked down. Furthermore, reduced levels of Sp1 resulted in downregulation of endogenous FE65 mRNA and protein. These findings reveal that Sp1 plays a crucial role in transcriptional control of the human FE65 gene.

  2. Transcription factor 7-like 1 is involved in hypothalamo–pituitary axis development in mice and humans

    Science.gov (United States)

    Gaston-Massuet, Carles; McCabe, Mark J.; Scagliotti, Valeria; Young, Rodrigo M.; Carreno, Gabriela; Gregory, Louise C.; Jayakody, Sujatha A.; Pozzi, Sara; Gualtieri, Angelica; Basu, Basudha; Koniordou, Markela; Wu, Chun-I; Bancalari, Rodrigo E.; Rahikkala, Elisa; Veijola, Riitta; Lopponen, Tuija; Graziola, Federica; Turton, James; Signore, Massimo; Mousavy Gharavy, Seyedeh Neda; Charolidi, Nicoletta; Sokol, Sergei Y.; Merrill, Bradley J.; Dattani, Mehul T.; Martinez-Barbera, Juan Pedro

    2016-01-01

    Aberrant embryonic development of the hypothalamus and/or pituitary gland in humans results in congenital hypopituitarism (CH). Transcription factor 7-like 1 (TCF7L1), an important regulator of the WNT/β-catenin signaling pathway, is expressed in the developing forebrain and pituitary gland, but its role during hypothalamo–pituitary (HP) axis formation or involvement in human CH remains elusive. Using a conditional genetic approach in the mouse, we first demonstrate that TCF7L1 is required in the prospective hypothalamus to maintain normal expression of the hypothalamic signals involved in the induction and subsequent expansion of Rathke’s pouch progenitors. Next, we reveal that the function of TCF7L1 during HP axis development depends exclusively on the repressing activity of TCF7L1 and does not require its interaction with β-catenin. Finally, we report the identification of two independent missense variants in human TCF7L1, p.R92P and p.R400Q, in a cohort of patients with forebrain and/or pituitary defects. We demonstrate that these variants exhibit reduced repressing activity in vitro and in vivo relative to wild-type TCF7L1. Together, our data provide support for a conserved molecular function of TCF7L1 as a transcriptional repressor during HP axis development in mammals and identify variants in this transcription factor that are likely to contribute to the etiology of CH. PMID:26764381

  3. In Vitro Anticancer Activity of Phlorofucofuroeckol A via Upregulation of Activating Transcription Factor 3 against Human Colorectal Cancer Cells

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    Hyun Ji Eo

    2016-03-01

    Full Text Available Phlorofucofuroeckol A (PFF-A, one of the phlorotannins found in brown algae, has been reported to exert anti-cancer property. However, the molecular mechanism for the anti-cancer effect of PFF-A has not been known. Activating transcription factor 3 (ATF3 has been reported to be associated with apoptosis in colorectal cancer. The present study was performed to investigate the molecular mechanism by which PFF-A stimulates ATF3 expression and apoptosis in human colorectal cancer cells. PFF-A decreased cell viability through apoptosis of human colorectal cancer cells. PFF-A increased ATF3 expression through regulating transcriptional activity. The responsible cis-element for ATF3 transcriptional activation by PFF-A was cAMP response element binding protein (CREB, located between positions −147 and −85 of the ATF3 promoter. Inhibition of p38, c-Jun N-terminal kinases (JNK, glycogen synthase kinase (GSK 3β, and IκB kinase (IKK-α blocked PFF-A-mediated ATF3 expression. ATF3 knockdown by ATF3 siRNA attenuated the cleavage of poly (ADP-ribose polymerase (PARP by PFF-A, while ATF3 overexpression increased PFF-A-mediated cleaved PARP. These results suggest that PFF-A may exert anti-cancer property through inducing apoptosis via the ATF3-mediated pathway in human colorectal cancer cells.

  4. Effect of short-term fasting and refeeding on transcriptional regulation of metabolic genes in human skeletal muscle.

    Science.gov (United States)

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P Darrell

    2003-03-01

    During short-term fasting, substrate utilization in skeletal muscle shifts from predominantly carbohydrate to fat as a means of conserving glucose. To examine the potential influence of short-term fasting and refeeding on transcriptional regulation in skeletal muscle, muscle biopsies were obtained from nine male subjects at rest, after 20 h of fasting, and 1 h after consuming either a high-carbohydrate (CHO trial) or a low-carbohydrate (FAT trial) meal. Fasting induced an increase in transcription of the pyruvate dehydrogenase kinase 4 (PDK4) (10-fold), lipoprotein lipase (LPL) ( approximately 2-fold), uncoupling protein 3 (UCP3) ( approximately 5-fold), and carnitine palmitoyltransferase I (CPT I) ( approximately 2.5-fold) genes. Surprisingly, transcription of PDK4 and LPL increased further in response to refeeding (both trials) to more than 50-fold and 6- to 10-fold, respectively, over prefasting levels. However, responses varied among subjects with two subjects in particular displaying far greater activation of PDK4 (>100-fold) and LPL (>20-fold) than the other subjects (mean approximately 8-fold and approximately 2-fold, respectively). Transcription of UCP3 decreased to basal levels after the CHO meal but remained elevated after the FAT meal, whereas CPT I remained elevated after both refeeding meals. The present findings demonstrate that short-term fasting/refeeding in humans alters the transcription of several genes in skeletal muscle related to lipid metabolism. Marked heterogeneity in the transcriptional response to the fasting/refeeding protocol suggests that individual differences in genetic profile may play an important role in adaptive molecular responses to metabolic challenges.

  5. Early Transcriptional Signatures of the Immune Response to a Live Attenuated Tetravalent Dengue Vaccine Candidate in Non-human Primates

    Science.gov (United States)

    Strouts, Fiona R.; Popper, Stephen J.; Partidos, Charalambos D.; Stinchcomb, Dan T.; Osorio, Jorge E.; Relman, David A.

    2016-01-01

    Background The development of a vaccine against dengue faces unique challenges, including the complexity of the immune responses to the four antigenically distinct serotypes. Genome-wide transcriptional profiling provides insight into the pathways and molecular features that underlie responses to immune system stimulation, and may facilitate predictions of immune protection. Methodology/Principal Findings In this study, we measured early transcriptional responses in the peripheral blood of cynomolgus macaques following vaccination with a live, attenuated tetravalent dengue vaccine candidate, TDV, which is based on a DENV-2 backbone. Different doses and routes of vaccine administration were used, and viral load and neutralizing antibody titers were measured at different time-points following vaccination. All 30 vaccinated animals developed a neutralizing antibody response to each of the four dengue serotypes, and only 3 of these animals had detectable serum viral RNA after challenge with wild-type dengue virus (DENV), suggesting protection of vaccinated animals to DENV infection. The vaccine induced statistically significant changes in 595 gene transcripts on days 1, 3, 5 and 7 as compared with baseline and placebo-treated animals. Genes involved in the type I interferon (IFN) response, including IFI44, DDX58, MX1 and OASL, exhibited the highest fold-change in transcript abundance, and this response was strongest following double dose and subcutaneous (versus intradermal) vaccine administration. In addition, modules of genes involved in antigen presentation, dendritic cell activation, and T cell activation and signaling were enriched following vaccination. Increased abundance of gene transcripts related to T cell activation on day 5, and the type I IFN response on day 7, were significantly correlated with the development of high neutralizing antibody titers on day 30. Conclusions/Significance These results suggest that early transcriptional responses may be

  6. Early Transcriptional Signatures of the Immune Response to a Live Attenuated Tetravalent Dengue Vaccine Candidate in Non-human Primates.

    Directory of Open Access Journals (Sweden)

    Fiona R Strouts

    2016-05-01

    Full Text Available The development of a vaccine against dengue faces unique challenges, including the complexity of the immune responses to the four antigenically distinct serotypes. Genome-wide transcriptional profiling provides insight into the pathways and molecular features that underlie responses to immune system stimulation, and may facilitate predictions of immune protection.In this study, we measured early transcriptional responses in the peripheral blood of cynomolgus macaques following vaccination with a live, attenuated tetravalent dengue vaccine candidate, TDV, which is based on a DENV-2 backbone. Different doses and routes of vaccine administration were used, and viral load and neutralizing antibody titers were measured at different time-points following vaccination. All 30 vaccinated animals developed a neutralizing antibody response to each of the four dengue serotypes, and only 3 of these animals had detectable serum viral RNA after challenge with wild-type dengue virus (DENV, suggesting protection of vaccinated animals to DENV infection. The vaccine induced statistically significant changes in 595 gene transcripts on days 1, 3, 5 and 7 as compared with baseline and placebo-treated animals. Genes involved in the type I interferon (IFN response, including IFI44, DDX58, MX1 and OASL, exhibited the highest fold-change in transcript abundance, and this response was strongest following double dose and subcutaneous (versus intradermal vaccine administration. In addition, modules of genes involved in antigen presentation, dendritic cell activation, and T cell activation and signaling were enriched following vaccination. Increased abundance of gene transcripts related to T cell activation on day 5, and the type I IFN response on day 7, were significantly correlated with the development of high neutralizing antibody titers on day 30.These results suggest that early transcriptional responses may be predictive of development of adaptive immunity to TDV

  7. HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells

    Directory of Open Access Journals (Sweden)

    Ennifar Eric

    2008-06-01

    Full Text Available Abstract Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1 is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT, is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex – the reverse transcription complex (RTC – consisting of viral genomic RNA associated with viral proteins (including RT and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein – the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF. We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT.

  8. Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

    Science.gov (United States)

    Stump, Craig S.; Short, Kevin R.; Bigelow, Maureen L.; Schimke, Jill M.; Sreekumaran Nair, K.

    2003-06-01

    Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels. cytochrome c oxidase | NADH dehydrogenase subunit IV | amino acids | citrate synthase

  9. The Transcription Factor GATA-6 is Overexpressed in Vivo and Contributes to Silencing 15-LOX-1 in Vitro in Human Colon Cancer

    OpenAIRE

    Shureiqi, Imad; Zuo, Xiangsheng; Broaddus, Russell; Wu, Yuanqing; Guan, Baoxiang; Morris, Jeffrey S.; Lippman, Scott M.

    2006-01-01

    Transcriptional suppression of 15-lipoxygenase-1 (15-LOX-1) helps enable human colorectal cancer cells escape apoptosis, a critical mechanism for colonic tumorigenesis. GATA-6 is strongly expressed in vitro in cancer cells; its downregulation by pharmaceuticals is associated with reversal of 15-LOX-1 transcriptional suppression. The mechanistic contribution of GATA-6 overexpression to colonic tumorigenesis, especially concerning 15-LOX-1 transcriptional suppression, remains unknown. We tested...

  10. Generalization Gradients in Human Predictive Learning: Effects of Discrimination Training and within-Subjects Testing

    Science.gov (United States)

    Vervliet, Bram; Iberico, Carlos; Vervoort, Ellen; Baeyens, Frank

    2011-01-01

    Generalization gradients have been investigated widely in animal conditioning experiments, but much less so in human predictive learning tasks. Here, we apply the experimental design of a recent study on conditioned fear generalization in humans (Lissek et al., 2008) to a predictive learning task, and examine the effects of a number of relevant…

  11. Noncytotoxic suppression of human immunodeficiency virus type 1 transcription by exosomes secreted from CD8+ T cells.

    Science.gov (United States)

    Tumne, Ashwin; Prasad, Varsha Shridhar; Chen, Yue; Stolz, Donna B; Saha, Kunal; Ratner, Deena M; Ding, Ming; Watkins, Simon C; Gupta, Phalguni

    2009-05-01

    CD8(+) T cells display a noncytotoxic activity that suppresses transcription of human immunodeficiency virus type 1 (HIV-1) in an antigen-independent and major histocompatibility complex-unrestricted manner. To date, the precise cellular and molecular factors mediating this CD8(+) T-cell effector function remain unsolved. Despite evidence indicating the dependence of the activity on cell-cell contact, the possibility of a membrane-mediated activity that represses transcription from the viral promoter remains unexplored. We therefore investigated whether this inhibition of HIV-1 transcription might be elicited by a membrane-bound determinant. Using a CD8(+) T-cell line displaying potent noncytotoxic HIV-1 suppression activity, we have identified a membrane-localized HIV-1-suppressing activity that is concomitantly secreted as 30- to 100-nm endosome-derived tetraspanin-rich vesicles known as exosomes. Purified exosomes from CD8(+) T-cell culture supernatant noncytotoxically suppressed CCR5-tropic (R5) and CXCR4-tropic (X4) replication of HIV-1 in vitro through a protein moiety. Similar antiviral activity was also found in exosomes isolated from two HIV-1-infected subjects. The antiviral exosomes specifically inhibited HIV-1 transcription in both acute and chronic models of infection. Our results, for the first time, indicate the existence of an antiviral membrane-bound factor consistent with the hallmarks defining noncytotoxic CD8(+) T-cell suppression of HIV-1.

  12. Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair.

    Science.gov (United States)

    Morales, Julio C; Richard, Patricia; Rommel, Amy; Fattah, Farjana J; Motea, Edward A; Patidar, Praveen L; Xiao, Ling; Leskov, Konstantin; Wu, Shwu-Yuan; Hittelman, Walter N; Chiang, Cheng-Ming; Manley, James L; Boothman, David A

    2014-04-01

    Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.

  13. Cryptic DNA-binding domain in the C terminus of RNA polymerase II general transcription factor RAP30.

    Science.gov (United States)

    Tan, S; Garrett, K P; Conaway, R C; Conaway, J W

    1994-10-11

    The C terminus of mammalian transcription factor RAP30 has been found to be a cryptic DNA-binding domain strikingly similar to the C-terminal DNA-binding domain present in conserved region 4 of members of the sigma 70 family of bacterial sigma factors. This RAP30 domain shares strongest sequence similarity with the DNA-binding domain present in region 4 of Bacillus subtilis sporulation-specific sigma K. Like the region 4 DNA-binding activity of Escherichia coli sigma 70, the RAP30 C-terminal DNA binding activity is masked in intact RAP30 but is readily detectable when the RAP30 C terminus is expressed as a fusion protein. Consistent with a role for RAP30 DNA-binding activity in transcription, mutations that abolish DNA binding also abolish transcription. Therefore, RAP30 may function at least in part through the action of an evolutionarily ancient DNA-binding domain that first appeared prior to the divergence of bacteria and eukaryotes.

  14. Changes in transcriptional output of human peripheral blood mononuclear cells following resistance exercise.

    Science.gov (United States)

    Carlson, Lara A; Tighe, S W; Kenefick, R W; Dragon, J; Westcott, N W; Leclair, R J

    2011-12-01

    Various types of exercise alter the population of circulating peripheral blood mononuclear cells (PBMCs) and change their transcriptional output. This work examines changes in PBMC populations and transcription in response to resistance exercise training (RET), and identify key transcriptional changes in PBMCs that may play a role in altering peripheral tissues in response to RET. Ten resistance-trained men (20-24 years), performed an acute bout of RET for ~30 min following a 12 h fast. Venous blood was sampled at rest, immediately following exercise, and at 2 h post-exercise and analyzed for total and differential leukocytes and global gene expression using Affymetrix Genechips. Results showed elevated leukocytes, monocytes, lymphocytes, and lactate values immediately post-exercise (P transcriptional changes in pathways related to immune response, inflammation, and cellular communication. The change in PBMC population (2 h time point) correlated with a dramatic decrease in the expression of CD160, and XCL1, markers of lymphocyte populations. At the 2 h recovery time point upregulation of matrix metalloproteinase 9, orosomucoid 1, dishevelled-associated activator of morphogenesis 2, and arginase 1 suggest an induction in muscle damage and repair during this time frame. These results demonstrate that an acute bout of RET disrupts cellular homeostasis, induces a transient redistribution of certain leukocytes, and results in transcriptional changes in PBMCs translating into systemic changes in response to RET.

  15. Some theoretical aspects of human categorization behavior: similarity and generalization

    OpenAIRE

    Jäkel, Frank

    2007-01-01

    Explanations of human categorization behavior often invoke similarity. Stimuli that are similar to each other are grouped together whereas stimuli that are very different are kept separate. Despite serious problems in defining similarity, both conceptually and experimentally, this is the prevailing view of categorization in prototype models (Posner & Keele, 1968; Reed, 1972) and exemplar models (Medin & Schaffer, 1978; Nosofsky, 1986). This is also the prevailing approach in machine learning ...

  16. Human and mouse ZFY genes produce a conserved testis-specific transcript encoding a zinc finger protein with a short acidic domain and modified transactivation potential.

    Science.gov (United States)

    Decarpentrie, Fanny; Vernet, Nadège; Mahadevaiah, Shantha K; Longepied, Guy; Streichemberger, Eric; Aknin-Seifer, Isabelle; Ojarikre, Obah A; Burgoyne, Paul S; Metzler-Guillemain, Catherine; Mitchell, Michael J

    2012-06-15

    Mammalian ZFY genes are located on the Y chromosome, and code putative transcription factors with 12-13 zinc fingers preceded by a large acidic (activating) domain. In mice, there are two genes, Zfy1 and Zfy2, which are expressed mainly in the testis. Their transcription increases in germ cells as they enter meiosis, both are silenced by meiotic sex chromosome inactivation (MSCI) during pachytene, and Zfy2 is strongly reactivated later in spermatids. Recently, we have shown that mouse Zfy2, but not Zfy1, is involved in triggering the apoptotic elimination of specific types of sex chromosomally aberrant spermatocytes. In humans, there is a single widely transcribed ZFY gene, and there is no evidence for a specific role in the testis. Here, we characterize ZFY transcription during spermatogenesis in mice and humans. In mice, we define a variety of Zfy transcripts, among which is a Zfy2 transcript that predominates in spermatids, and a Zfy1 transcript, lacking an exon encoding approximately half of the acidic domain, which predominates prior to MSCI. In humans, we have identified a major testis-specific ZFY transcript that encodes a protein with the same short acidic domain. This represents the first evidence that ZFY has a conserved function during human spermatogenesis. We further show that, in contrast to the full acidic domain, the short domain does not activate transcription in yeast, and we hypothesize that this explains the functional difference observed between Zfy1 and Zfy2 during mouse meiosis.

  17. Mapping the transcription repressive domain in the highly conserved human gene hnulp1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    HNULP1,a new member of the basic helixloop-helix transcription factors,contains a DUF654 domain in its C-terminus and is highly conserved from Drosophilae,yeast,zebrafish to mouse.The function of this motif,however,is currently unknown.In this research,we fused five deletion fragments of the DUF654 domain to the GAL4 DNA-binding domain and then co-transfected with plasmids L8G5-Luc and VP-16.The analysis of the GAL4 luciferase reporter gene indicated that fragments from 228 to 407 amino acids in the DUF654 domain had a strong transcription repression activity.Therefore,this study lays a solid foundation for research on the mechanism of hnulp1 transcriptional regulation and the function of the DUF654 domain.

  18. Transcriptional organization of a 450-kb region of the human X chromosome in Xq28

    Energy Technology Data Exchange (ETDEWEB)

    Bione, S.; Tamanini, F.; Maestrini, E.; Tribioli, C.; Rivella, S.; Toniolo, D. (Instituto di Genetica Biochimica ed Evoluzionistica, Pavia (Italy)); Poustka, A. (German Cancer Research Center, Heidelberg (Germany))

    1993-11-15

    In this paper, the authors report the transcriptional organization of a 450-kb gene cluster in Xq28, flanked by the glucose-6-phosphate dehydrogenase and the color vision genes. CpG islands previously identified and mapped to distal Xq28 have helped in construction of a continuous contig of cosmids and in identification of cDNAs corresponding to eight transcripts. Thirteen to 16 small genes with CpG islands are clustered in a region of 250-300 kb. Many are highly expressed in muscle or brain and may be the genes responsible for muscle or neurological disorders mapped to distal Xq28. The analysis indicates that, in this region of the genome, genes not related in sequence are organized in transcriptional domains of 100 kb and that this organization may be important for establishing and regulating gene expression in relation to tissue distribution and X chromosome inactivation.

  19. Transcriptional organization of a 450-kb region of the human X chromosome in Xq28.

    Science.gov (United States)

    Bione, S; Tamanini, F; Maestrini, E; Tribioli, C; Poustka, A; Torri, G; Rivella, S; Toniolo, D

    1993-12-01

    In this paper, we report the transcriptional organization of a 450-kb gene cluster in Xq28, flanked by the glucose-6-phosphate dehydrogenase and the color vision genes. CpG islands previously identified and mapped to distal Xq28 have helped in construction of a continuous contig of cosmids and in identification of cDNAs corresponding to eight transcripts. Thirteen to 16 small genes with CpG islands are clustered in a region of 250-300 kb. Many are highly expressed in muscle or brain and may be the genes responsible for muscle or neurological disorders mapped to distal Xq28. Our analysis indicates that, in this region of the genome, genes not related in sequence are organized in transcriptional domains of 100 kb and that this organization may be important for establishing and regulating gene expression in relation to tissue distribution and X chromosome inactivation.

  20. The transcription factor SOX18 regulates the expression of matrix metalloproteinase 7 and guidance molecules in human endothelial cells.

    Directory of Open Access Journals (Sweden)

    Martina Hoeth

    Full Text Available Mutations in the transcription factor SOX18 are responsible for specific cardiovascular defects in humans and mice. In order to gain insight into the molecular basis of its action, we identified target genes of SOX18 and analyzed one, MMP7, in detail.SOX18 was expressed in HUVEC using a recombinant adenoviral vector and the altered gene expression profile was analyzed using microarrays. Expression of several regulated candidate SOX18 target genes was verified by real-time PCR. Knock-down of SOX18 using RNA interference was then used to confirm the effect of the transcription factor on selected genes that included the guidance molecules ephrin B2 and semaphorin 3G. One gene, MMP7, was chosen for further analysis, including detailed promoter studies using reporter gene assays, electrophoretic mobility shift analysis and chromatin-immunoprecipitation, revealing that it responds directly to SOX18. Immunohistochemical analysis demonstrated the co-expression of SOX18 and MMP7 in blood vessels of human skin.The identification of MMP7 as a direct SOX18 target gene as well as other potential candidates including guidance molecules provides a molecular basis for the proposed function of this transcription factor in the regulation of vessel formation.

  1. Chemically Modified Oligonucleotides Modulate an Epigenetically Varied and Transient Form of Transcription Silencing of HIV-1 in Human Cells

    Directory of Open Access Journals (Sweden)

    Stuart Knowling

    2012-01-01

    Full Text Available Small noncoding RNAs (ncRNAs have been shown to guide epigenetic silencing complexes to target loci in human cells. When targeted to gene promoters, these small RNAs can lead to long-term stable epigenetic silencing of gene transcription. To date, small RNAs have been shown to modulate transcriptional gene silencing (TGS of human immunodeficiency virus type 1 (HIV-1 as well as several other disease-related genes, but it has remained unknown as to what extent particular chemistries can be used to generate single-stranded backbone-modified oligonucleotides that are amenable to this form of gene targeting and regulation. Here, we present data indicating that specific combinations of backbone modifications can be used to generate single-stranded antisense oligonucleotides that can functionally direct TGS of HIV-1 in a manner that is however, independent of epigenetic changes at the target loci. Furthermore, this functionality appears contingent on the absence of a 5′ phosphate in the oligonucleotide. These data suggest that chemically modified oligonucleotide based approaches could be implemented as a means to regulate gene transcription in an epigenetically independent manner.

  2. An Algorithm for Generating Small RNAs Capable of Epigenetically Modulating Transcriptional Gene Silencing and Activation in Human Cells

    Directory of Open Access Journals (Sweden)

    Amanda Ackley

    2013-01-01

    Full Text Available Small noncoding antisense RNAs (sasRNAs guide epigenetic silencing complexes to target loci in human cells and modulate gene transcription. When these targeted loci are situated within a promoter, long-term, stable epigenetic silencing of transcription can occur. Recent studies suggest that there exists an endogenous form of such epigenetic regulation in human cells involving long noncoding RNAs. In this article, we present and validate an algorithm for the generation of highly effective sasRNAs that can mimic the endogenous noncoding RNAs involved in the epigenetic regulation of gene expression. We validate this algorithm by targeting several oncogenes including AKT-1, c-MYC, K-RAS, and H-RAS. We also target a long antisense RNA that mediates the epigenetic repression of the tumor suppressor gene DUSP6, silenced in pancreatic cancer. An algorithm that can efficiently design small noncoding RNAs for the epigenetic transcriptional silencing or activation of specific genes has potential therapeutic and experimental applications.

  3. Deregulation of the Pit-1 transcription factor in human breast cancer cells promotes tumor growth and metastasis

    Science.gov (United States)

    Ben-Batalla, Isabel; Seoane, Samuel; Garcia-Caballero, Tomas; Gallego, Rosalia; Macia, Manuel; Gonzalez, Luis O.; Vizoso, Francisco; Perez-Fernandez, Roman

    2010-01-01

    The Pit-1 transcription factor (also know as POU1F1) plays a critical role in cell differentiation during organogenesis of the anterior pituitary in mammals and is a transcriptional activator for pituitary gene transcription. Increased expression of Pit-1 has been reported in human tumorigenic breast cells. Here, we found that Pit-1 overexpression or knockdown in human breast cancer cell lines induced profound phenotypic changes in the expression of proteins involved in cell proliferation, apoptosis, and invasion. Some of these protumorigenic effects of Pit-1 were mediated by upregulation of Snai1, an inductor of the epithelial-mesenchymal transition. In immunodeficient mice, Pit-1 overexpression induced tumoral growth and promoted metastasis in lung. In patients with invasive ductal carcinoma of the breast and node-positive tumor, high expression of Pit-1 was significantly correlated with Snai1 positivity. Notably, in these patients elevated expression of Pit-1 was significantly and independently associated with the occurrence of distant metastasis. These findings suggest that Pit-1 could help to make a more accurate prognosis in patients with node-positive breast cancer and may represent a new therapeutic target. PMID:21060149

  4. Gene transcription profiles associated with inter-modular hubs and connection distance in human functional magnetic resonance imaging networks

    Science.gov (United States)

    Vértes, Petra E.; Rittman, Timothy; Whitaker, Kirstie J.; Romero-Garcia, Rafael; Váša, František; Wagstyl, Konrad; Fonagy, Peter; Dolan, Raymond J.; Jones, Peter B.; Goodyer, Ian M.

    2016-01-01

    Human functional magnetic resonance imaging (fMRI) brain networks have a complex topology comprising integrative components, e.g. long-distance inter-modular edges, that are theoretically associated with higher biological cost. Here, we estimated intra-modular degree, inter-modular degree and connection distance for each of 285 cortical nodes in multi-echo fMRI data from 38 healthy adults. We used the multivariate technique of partial least squares (PLS) to reduce the dimensionality of the relationships between these three nodal network parameters and prior microarray data on regional expression of 20 737 genes. The first PLS component defined a transcriptional profile associated with high intra-modular degree and short connection distance, whereas the second PLS component was associated with high inter-modular degree and long connection distance. Nodes in superior and lateral cortex with high inter-modular degree and long connection distance had local transcriptional profiles enriched for oxidative metabolism and mitochondria, and for genes specific to supragranular layers of human cortex. In contrast, primary and secondary sensory cortical nodes in posterior cortex with high intra-modular degree and short connection distance had transcriptional profiles enriched for RNA translation and nuclear components. We conclude that, as predicted, topologically integrative hubs, mediating long-distance connections between modules, are more costly in terms of mitochondrial glucose metabolism. This article is part of the themed issue ‘Interpreting BOLD: a dialogue between cognitive and cellular neuroscience’. PMID:27574314

  5. Human corneal epithelial subpopulations: oxygen dependent ex vivo expansion and transcriptional profiling.

    Science.gov (United States)

    Bath, Chris

    2013-06-01

    differentiation pathway in human corneal epithelium according to an optimized protocol for maintenance of expression profiles. Isolated total RNA from basal limbal crypts (BLCs), superficial limbal crypts (SLCs), paracentral/central cornea and limbal stroma was amplified and converted to fragmented cDNA libraries for use in deep paired-end next-generation sequencing. Global transcriptional profiling was carried out using bioinformatics. The location of primitive cells in BLCs, migratory and activated cells in SLCs and differentiated cells in paracentral/central cornea was evident from mapping of significantly upregulated genes in each compartment to the gene ontology (GO). Interestingly, many GO terms in BLCs were also involved in neurogenic processes, whereas many GO terms in SLCs were related to vasculature. Mapping upregulated genes in BLCs to pathway annotations in Kyoto Encyclopedia of Genes and Genomes described many active pathways as signalling and cancer-associated pathways. We supply extensive information on possible novel biomarkers, reveal insight into both active pathways and novel regulators of LESCs such as Lrig1 and SOX9 and provide an immense amount of data for future exploration (Bath et al. 2013b). Selective ex vivo expansion of LESCs in hypoxia and the comprehensive molecular characterization of corneal epithelial subpopulations in situ are expected to be beneficial for the future treatment of LSCD by cultured limbal epithelial transplantation. © 2013 Acta Ophthalmologica Scandinavica Foundation.

  6. The amino-terminal domain of human signal transducers and activators of transcription 1: Overexpression, purification and characterization

    Indian Academy of Sciences (India)

    Arati Prabhu; Evans Coutinho; Sudha Srivastava

    2005-12-01

    The dual functional signal transducers and activators of transcription (STAT) proteins are latent cytoplasmic transcription factors that play crucial roles in host defense. Animals that lack these proteins are highly susceptible to microbial and viral infections and chemically induced primary tumours. We have over expressed the aminoterminal domain of human STAT1 (hSTAT1) in Escherichia coli and purified it by affinity chromatography and gel filtration chromatography. The entire process has been monitored by gel electrophoresis. The pure protein has been characterized by mass spectrometry and 2-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy. Our results indicate that the N-terminus of hSTAT1 exists as a dimer in solution.

  7. The forkhead transcription factor Foxl2 is sumoylated in both human and mouse: sumoylation affects its stability, localization, and activity.

    Directory of Open Access Journals (Sweden)

    Mara Marongiu

    Full Text Available The FOXL2 forkhead transcription factor is expressed in ovarian granulosa cells, and mutated FOXL2 causes the blepharophimosis, ptosis and epicanthus inversus syndrome (BPES and predisposes to premature ovarian failure. Inactivation of Foxl2 in mice demonstrated its indispensability for female gonadal sex determination and ovary development and revealed its antagonism of Sox9, the effector of male testis development. To help to define the regulatory activities of FOXL2, we looked for interacting proteins. Based on yeast two-hybrid screening, we found that FOXL2 interacts with PIAS1 and UBC9, both parts of the sumoylation machinery. We showed that human FOXL2 is sumoylated in transfected cell lines, and that endogenous mouse Foxl2 is comparably sumoylated. This modification changes its cellular localization, stability and transcriptional activity. It is intriguing that similar sumoylation and regulatory consequences have also been reported for SOX9, the male counterpart of FOXL2 in somatic gonadal tissues.

  8. Multifunctional human transcriptional coactivator protein PC4 is a substrate of Aurora kinases and activates the Aurora enzymes.

    Science.gov (United States)

    Dhanasekaran, Karthigeyan; Kumari, Sujata; Boopathi, Ramachandran; Shima, Hiroki; Swaminathan, Amrutha; Bachu, Mahesh; Ranga, Udaykumar; Igarashi, Kazuhiko; Kundu, Tapas K

    2016-03-01

    Positive coactivator 4 (PC4), a human transcriptional coactivator, is involved in diverse processes like chromatin organization and transcription regulation. It is hyperphosphorylated during mitosis, with unknown significance. For the first time, we demonstrate the function of PC4 outside the nucleus upon nuclear envelope breakdown. A fraction of PC4 associates with Aurora A and Aurora B and undergoes phosphorylation, following which PC4 activates both Aurora A and B to sustain optimal kinase activity to maintain the phosphorylation gradient for the proper functioning of the mitotic machinery. This mitotic role is evident in PC4 knockdown cells where the defects are rescued only by the catalytically active Aurora kinases, but not the kinase-dead mutants. Similarly, the PC4 phosphodeficient mutant failed to rescue such defects. Hence, our observations establish a novel mitotic function of PC4 that might be dependent on Aurora kinase-mediated phosphorylation.

  9. N-Acetyl-L-Cystein downregulates beta-amyloid precursor protein gene transcription in human neuroblastoma cells.

    Science.gov (United States)

    Studer, R; Baysang, G; Brack, C

    2001-01-01

    The causes for the sporadic form of Alzheimer's disease (AD) are still poorly understood, except from the fact that age is an important risk factor. The main component of the characteristic amyloid plaques in brains of AD patients are Abeta peptides, derivatives of the amyloid precursor protein APP. Oxidative stress may contribute to the aetiology of AD by dysregulation of APP metabolism. Overexpression of the APP gene could result in an increased secretion of neurotoxic Abeta peptides, while preventing the overexpression might be protective. We here report that the antioxidant N-Acetyl-L-Cystein (NAC) downregulates APP gene transcription in human neuroblastoma cells. The effect is reversible when cells are returned to NAC free medium. These results open up new possibilities for the development of therapeutic agents that intervene at the transcriptional level.

  10. Transcriptional Activation of a Constitutive Heterochromatic Domain of the Human Genome in Response to Heat ShockD⃞

    Science.gov (United States)

    Rizzi, Nicoletta; Denegri, Marco; Chiodi, Ilaria; Corioni, Margherita; Valgardsdottir, Rut; Cobianchi, Fabio; Riva, Silvano; Biamonti, Giuseppe

    2004-01-01

    Heat shock triggers the assembly of nuclear stress bodies that contain heat shock factor 1 and a subset of RNA processing factors. These structures are formed on the pericentromeric heterochromatic regions of specific human chromosomes, among which chromosome 9. In this article we show that these heterochromatic domains are characterized by an epigenetic status typical of euchromatic regions. Similarly to transcriptionally competent portions of the genome, stress bodies are, in fact, enriched in acetylated histone H4. Acetylation peaks at 6 h of recovery from heat shock. Moreover, heterochromatin markers, such as HP1 and histone H3 methylated on lysine 9, are excluded from these nuclear districts. In addition, heat shock triggers the transient accumulation of RNA molecules, heterogeneous in size, containing the subclass of satellite III sequences found in the pericentromeric heterochromatin of chromosome 9. This is the first report of a transcriptional activation of a constitutive heterochromatic portion of the genome in response to stress stimuli. PMID:14617804

  11. Nanoparticle-mediated transcriptional modification enhances neuronal differentiation of human neural stem cells following transplantation in rat brain.

    Science.gov (United States)

    Li, Xiaowei; Tzeng, Stephany Y; Liu, Xiaoyan; Tammia, Markus; Cheng, Yu-Hao; Rolfe, Andrew; Sun, Dong; Zhang, Ning; Green, Jordan J; Wen, Xuejun; Mao, Hai-Quan

    2016-04-01

    Strategies to enhance survival and direct the differentiation of stem cells in vivo following transplantation in tissue repair site are critical to realizing the potential of stem cell-based therapies. Here we demonstrated an effective approach to promote neuronal differentiation and maturation of human fetal tissue-derived neural stem cells (hNSCs) in a brain lesion site of a rat traumatic brain injury model using biodegradable nanoparticle-mediated transfection method to deliver key transcriptional factor neurogenin-2 to hNSCs when transplanted with a tailored hyaluronic acid (HA) hydrogel, generating larger number of more mature neurons engrafted to the host brain tissue than non-transfected cells. The nanoparticle-mediated transcription activation method together with an HA hydrogel delivery matrix provides a translatable approach for stem cell-based regenerative therapy.

  12. Transcriptional activation of immediate-early gene ETR101 by human T-cell leukaemia virus type I Tax

    DEFF Research Database (Denmark)

    Chen, Li; Ma, Shiliang; Li, Bo

    2003-01-01

    Human T-cell leukaemia virus type I (HTLV-I) Tax regulates viral and cellular gene expression through interactions with multiple cellular transcription pathways. This study describes the finding of immediate-early gene ETR101 expression in HTLV-I-infected cells and its regulation by Tax. ETR101...... was persistently expressed in HTLV-I-infected cells but not in HTLV-I uninfected cells. Expression of ETR101 was dependent upon Tax expression in the inducible Tax-expressing cell line JPX-9 and also in Jurkat cells transiently transfected with Tax-expressing vectors. Tax transactivated the ETR101 gene promoter......-DNA complex in HTLV-I-infected cell lines. EMSA with specific antibodies confirmed that the CREB transcription factor was responsible for formation of this specific protein-DNA complex. These results suggested that Tax directly transactivated ETR101 gene expression, mainly through a CRE sequence via the CREB...

  13. Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ren, He, E-mail: herenrh@yahoo.com.cn [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China); Zhao, Tiansuo; Wang, Xiuchao; Gao, Chuntao; Wang, Jian; Yu, Ming [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China); Hao, Jihui, E-mail: jihuihao@yahoo.com [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China)

    2010-03-26

    The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breast cancer.

  14. Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

    Energy Technology Data Exchange (ETDEWEB)

    Kamada, Katsuhiko; De Angelis, Jacqueline; Roeder, Robert G.; Burley, Stephen K. (Rockefeller)

    2012-12-13

    The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-{angstrom} resolution. The {alpha}/{beta} structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3{gamma} (HNF-3{gamma}), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3{gamma} and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the {beta} subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

  15. Activation of the Long Terminal Repeat of Human Endogenous Retrovirus K by Melanoma-Specific Transcription Factor MITF-M

    Directory of Open Access Journals (Sweden)

    Iyoko Katoh

    2011-11-01

    Full Text Available The human and Old World primate genomes possess conserved endogenous retrovirus sequences that have been implicated in evolution, reproduction, and carcinogenesis. Human endogenous retrovirus (HERV-K with 5′LTR-gag-pro-pol-env-rec/np9-3′LTR sequences represents the newest retrovirus family that integrated into the human genome 1 to 5 million years ago. Although a high-level expression of HERV-K in melanomas, breast cancers, and terato-carcinomas has been demonstrated, the mechanism of the lineage-specific activation of the long terminal repeat (LTR remains obscure. We studied chromosomal HERV-K expression in MeWo melanoma cells in comparison with the basal expression in human embryonic kidney 293 (HEK293 cells. Cloned LTR of HERV-K (HML-2.HOM was also characterized by mutation and transactivation experiments. We detected multiple transcriptional initiator (Inr sites in the LTR by rapid amplification of complementary DNA ends (5′ RACE. HEK293 and MeWo showed different Inr usage. The most potent Inr was associated with a TATA box and three binding motifs of microphthalmia-associated transcription factor (MITF. Both chromosomal HERV-K expression and the cloned LTR function were strongly activated in HEK293 by transfection with MITF-M, a melanocyte/melanoma–specific isoform of MITF. Coexpression of MITF and the HERV-K core antigen was detected in retinal pigmented epithelium by an immunofluorescence analysis. Although malignant melanoma lines MeWo, G361, and SK-MEL-28 showed enhanced HERV-K transcription compared with normal melanocytes, the level of MITF-M messenger RNA persisted from normal to transformed melanocytes. Thus, MITF-M may be a prerequisite for the pigmented cell lineage–specific function of HERV-K LTR, leading to the high-level expression in malignant melanomas.

  16. Transcriptional and functional studies of Human Endogenous Retrovirus envelope EnvP(b) and EnvV genes in human trophoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Vargas, Amandine, E-mail: amandine.vargas@voila.fr; Thiery, Maxime, E-mail: thiery.maxime@courrier.uqam.ca; Lafond, Julie, E-mail: lafond.julie@uqam.ca; Barbeau, Benoit, E-mail: barbeau.benoit@uqam.ca

    2012-03-30

    HERV (Human Endogenous Retrovirus)-encoded envelope proteins are implicated in the development of the placenta. Indeed, Syncytin-1 and -2 play a crucial role in the fusion of human trophoblasts, a key step in placentation. Other studies have identified two other HERV env proteins, namely EnvP(b) and EnvV, both expressed in the placenta. In this study, we have fully characterized both env transcripts and their expression pattern and have assessed their implication in trophoblast fusion. Through RACE analyses, standard spliced transcripts were detected, while EnvV transcripts demonstrated alternative splicing at its 3 Prime end. Promoter activity and expression of both genes were induced in forskolin-stimulated BeWo cells and in primary trophoblasts. Although we have confirmed the fusogenic activity of EnvP(b), overexpression or silencing experiments revealed no impact of this protein on trophoblast fusion. Our results demonstrate that both env genes are expressed in human trophoblasts but are not required for syncytialization.

  17. Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood.

    NARCIS (Netherlands)

    Fradin, C.; de Groot, P.W.J.; Maccallum, D.; Schaller, M.; Klis, F.M.; Odds, F.C.; Hube, B.

    2005-01-01

    Survival in blood and escape from blood vessels into tissues are essential steps for the yeast Candida albicans to cause systemic infections. To elucidate the influence of blood components on fungal growth, morphology and transcript profile during bloodstream infections, we exposed C. albicans to bl

  18. Ensemble approach combining multiple methods improves human transcription start site prediction.

    LENUS (Irish Health Repository)

    Dineen, David G

    2010-01-01

    The computational prediction of transcription start sites is an important unsolved problem. Some recent progress has been made, but many promoters, particularly those not associated with CpG islands, are still difficult to locate using current methods. These methods use different features and training sets, along with a variety of machine learning techniques and result in different prediction sets.

  19. The forkhead transcription factor FOXP1 represses human plasma cell differentiation

    NARCIS (Netherlands)

    M. Van Keimpema (Martine); L.J. Grüneberg (Leonie J.); M. Mokry (Michal); R. Van Boxtel (Ruben); M.C. van Zelm (Menno); P.J. Coffer (Paul); S. Pals; M. Spaargaren

    2015-01-01

    textabstractExpression of the for khead transcription factor FOXP1 is essential for early B-cell development, whereas down regulation ofFOXP1at the germinal center (GC) stage is required for GC B-cell function. Aberrantly high FOXP1 expression is frequently observed in diffuse large B-cell lymphoma

  20. Human endothelin subtype A receptor enhancement during tissue culture via de novo transcription

    DEFF Research Database (Denmark)

    Hansen-Schwartz, Jacob; Nordström, Carl-Henrik; Edvinsson, Lars

    2002-01-01

    was observed; the response was preserved in some segments and abolished in others, and potentiation of the precontraction was observed in yet other segments. No isolated contractile response to sarafotoxin 6c was observed, however. In reverse transcription-polymerase chain reaction assays, both ET(A) and ET...

  1. Signal transducer and activator of transcription 3 and 5 regulate system Xc- and redox balance in human breast cancer cells.

    Science.gov (United States)

    Linher-Melville, Katja; Haftchenary, Sina; Gunning, Patrick; Singh, Gurmit

    2015-07-01

    System Xc- is a cystine/glutamate antiporter that contributes to the maintenance of cellular redox balance. The human xCT (SLC7A11) gene encodes the functional subunit of system Xc-. Transcription factors regulating antioxidant defense mechanisms including system Xc- are of therapeutic interest, especially given that aggressive breast cancer cells exhibit increased system Xc- function. This investigation provides evidence that xCT expression is regulated by STAT3 and/or STAT5A, functionally affecting the antiporter in human breast cancer cells. Computationally analyzing two kilobase pairs of the xCT promoter/5' flanking region identified a distal gamma-activated site (GAS) motif, with truncations significantly increasing luciferase reporter activity. Similar transcriptional increases were obtained after treating cells transiently transfected with the full-length xCT promoter construct with STAT3/5 pharmacological inhibitors. Knock-down of STAT3 or STAT5A with siRNAs produced similar results. However, GAS site mutation significantly reduced xCT transcriptional activity, suggesting that STATs may interact with other transcription factors at more proximal promoter sites. STAT3 and STAT5A were bound to the xCT promoter in MDA-MB-231 cells, and binding was disrupted by pre-treatment with STAT inhibitors. Pharmacologically suppressing STAT3/5 activation significantly increased xCT mRNA and protein levels, as well as cystine uptake, glutamate release, and total levels of intracellular glutathione. Our data suggest that STAT proteins negatively regulate basal xCT expression. Blocking STAT3/5-mediated signaling induces an adaptive, compensatory mechanism to protect breast cancer cells from stress, including reactive oxygen species, by up-regulating xCT expression and the function of system Xc-. We propose that targeting system Xc- together with STAT3/5 inhibitors may heighten therapeutic anti-cancer effects.

  2. HUMAN BABESIOSIS: A GENERAL REVIEW WITH SPECIAL REFERENCE TO EGYPT.

    Science.gov (United States)

    Saleh, Ahmad Megahed Ahmad; Adam, Samia Mohammad Adam; Abdel-Motagaly, Amr M El-Sayed; Mohammad, Abeer; Ibrahim, Abdallah; Morsy, Tosson Aly

    2015-12-01

    Babesiosis is a tick-borne malaria-like illness caused by species of the intra-erythrocytic protozoan Babesia. Humans are opportunistic hosts for Babesia when bitten by nymph or adult ticks. Currently, Babesia infection is transmitted by various tick vectors in Europe, Asia, Africa and the northwestern and northeastern United States. Human babesiosis was first described in 1957 but is now known to have worldwide distribution. The increase in reported cases is likely due to increases in actual incidence as well as increased awareness of the disease. Despite the diagnostic and preventive advances resulting from extensive research and a greater understanding of the disease, babesiosis continues to have significant medical impact as a confounding variable in the diagnosis and treatment of Lyme disease and as a potential threat to the blood supply, especially in the United States. Diagnostic advances, like the development of PCR assays, have resulted in increased sensitivity for detection as well as the discovery and characterization of new babesial species. Further studies using the molecular tools now available and those to be developed will lead to a better understanding of the natural history of these organisms, including the transmission cycle and the, potential role of Babesia parasites themselves as immunomodulAtor. Human babesiosis is usually an asymptomatic infection in healthy individuals. Several patients become symptomatic, and, within these subpopulations, significant morbidity and mortality occur, especially in elderly, immunocompromised, or asplenic patients. It is difficult to diagnose. Although the index of suspicion should be high in rural Babesia endemic areas, patients with babesiosis have few, if any, localizing signs to suggest the disease. Diagnosis confirmation depends on the parasitemia degree, the expertise and well trained laboratory personnel. Most patients infected by B microti who are otherwise healthy appear to have a mild illness and

  3. General Base-General Acid Catalysis in Human Histone Deacetylase 8.

    Science.gov (United States)

    Gantt, Sister M Lucy; Decroos, Christophe; Lee, Matthew S; Gullett, Laura E; Bowman, Christine M; Christianson, David W; Fierke, Carol A

    2016-02-09

    Histone deacetylases (HDACs) regulate cellular processes such as differentiation and apoptosis and are targeted by anticancer therapeutics in development and in the clinic. HDAC8 is a metal-dependent class I HDAC and is proposed to use a general acid-base catalytic pair in the mechanism of amide bond hydrolysis. Here, we report site-directed mutagenesis and enzymological measurements to elucidate the catalytic mechanism of HDAC8. Specifically, we focus on the catalytic function of Y306 and the histidine-aspartate dyads H142-D176 and H143-D183. Additionally, we report X-ray crystal structures of four representative HDAC8 mutants: D176N, D176N/Y306F, D176A/Y306F, and H142A/Y306F. These structures provide a useful framework for understanding enzymological measurements. The pH dependence of kcat/KM for wild-type Co(II)-HDAC8 is bell-shaped with two pKa values of 7.4 and 10.0. The upper pKa reflects the ionization of the metal-bound water molecule and shifts to 9.1 in Zn(II)-HDAC8. The H142A mutant has activity 230-fold lower than that of wild-type HDAC8, but the pKa1 value is not altered. Y306F HDAC8 is 150-fold less active than the wild-type enzyme; crystal structures show that Y306 hydrogen bonds with the zinc-bound substrate carbonyl, poised for transition state stabilization. The H143A and H142A/H143A mutants exhibit activity that is >80000-fold lower than that of wild-type HDAC8; the buried D176N and D176A mutants have significant catalytic effects, with more subtle effects caused by D183N and D183A. These enzymological and structural studies strongly suggest that H143 functions as a single general base-general acid catalyst, while H142 remains positively charged and serves as an electrostatic catalyst for transition state stabilization.

  4. Application technology on human general function as a group

    Energy Technology Data Exchange (ETDEWEB)

    Numano, Masayoshi; Matsuoka, Takeshi; Tanaka, Kunihiko; Fukuto, Junji; Mitomo, Nobuo; Miyazaki; Keiko; Matsukura, Hiroshi; Niwa, Yasuyuki; Ando, Hirotomo [Ship Research Inst., Tokyo (Japan)

    2000-02-01

    An operation assistant system for two operators as an object of plant model simulated on PWR was made experimentally, effectiveness on applying 3D-VR spatial indication and repulsive feedback input apparatus to plant operation assistance was investigated. By adopting a transmission type HMD, combination use with the conventional type operation monitoring system was made possible, and then it could be thought to become possible that human error was found by oneself by adding intuitive indication and feedback to judgement and operation used by a system assisting with logic understanding. And, by sharing these informations in a group, it was also found that correction of not only selfish but also other operator's error was made possible. (G.K.)

  5. Human hydatidosis granulosus in greater Cairo, Egypt: with general review.

    Science.gov (United States)

    Ibrahim, Badawyia B; Haridy, Fouad M; Hegazi, Mamdoud M; Morsy, Tosson A

    2007-08-01

    In this study, human cases (41) of proved hydatidosis granulosus were obtained from Al Kasr Al Aini University Hospitals from 2000-2006. They were 22 females with ages from 8 to 70 years and 19 males with ages from 5 to 75 years. The highly infected organ was the liver. Infection in other organs as lung, spleen, brain, eye, pelvic and bones of upper and lower limbs were reported. Most of the hydatidosis patients were from governorates of Giza (24 cases), Qalyobia (7 cases), Cairo (5 cases), Bani-Swef (3 cases), Sharkia (1 case) and Demiatta (1 case). Biopsies of infected liver and lung were processed for the histopathological studies and photographed. The results were discussed on the light of work done before Egypt.

  6. Generalization of category knowledge and dimensional categorization in humans (Homo sapiens) and nonhuman primates (Macaca mulatta).

    Science.gov (United States)

    Smith, J David; Zakrzewski, Alexandria C; Johnston, Jennifer J R; Roeder, Jessica L; Boomer, Joseph; Ashby, F Gregory; Church, Barbara A

    2015-10-01

    A theoretical framework within neuroscience distinguishes humans' implicit and explicit systems for category learning. We used a perceptual-categorization paradigm to ask whether nonhumans share elements of these systems. Participants learned categories that foster implicit or explicit categorization in humans, because they had a multidimensional, information-integration (II) solution or a unidimensional, rule-based (RB) solution. Then humans and macaques generalized their category knowledge to new, untested regions of the stimulus space. II generalization was impaired, suggesting that II category learning is conditioned and constrained by stimulus generalization to its original, trained stimulus contexts. RB generalization was nearly seamless, suggesting that RB category knowledge in humans and monkeys has properties that grant it some independence from the original, trained stimulus contexts. These findings raise the questions of (a) how closely macaques' dimensional categorization verges on humans' explicit/declarative categorization, and (b) how far macaques' dimensional categorization has advanced beyond that in other vertebrate species.

  7. A novel activating role of SRC and STAT3 on HGF transcription in human breast cancer cells

    Directory of Open Access Journals (Sweden)

    Elliott Bruce E

    2007-10-01

    Full Text Available Abstract We have previously determined that the HGF promoter can be transactivated by a combination of activated Src and wild-type Stat3 in the mouse breast cell lines HC11 and SP1. To determine if this pathway is of relevance for the human disease, a series of human breast and other human cells lines were examined, and the status of key proteins in these cells determined. All of the human breast cell lines exhibited strong transactivation by a combination of activated Src and Stat3. This activation was dependent on a Stat3 recognition element present at nt-95. The exception was the ErbB2 over-expressing cell line SK-BR-3 where Stat3 alone could transactivate HGF though Src augmented this effect. Increased phosphorylation of Stat3 tyrosine 705 was also observed in this line. Analysis of three ovarian cell lines revealed that Src/Stat3 expression was not able to activate the HGF promoter in two of these lines (SKOV3 and IOSE-80PC. Src/Stat3 expression did activate HGF transcription in OVCAR3 cells, but this effect was not mediated by the Stat3 site at nt-95. Stat3 phosphorylation at tyrosine 705 was observed in IOSE-80PC cells, but was insufficient to allow for activation of the HGF promoter. Human kidney (HEK293 and cervical carcinoma (HeLa cells were also not Src/Stat3 permissive, despite high levels of Stat3 phospho-Y705. These results suggest that human breast cells are a uniquely permissive environment for HGF transactivation by Src/Stat3 which may allow for the inappropriate activation of HGF transcription during the early stages of breast transformation. This could lead to paracrine or autocrine activation of the Met receptor in breast carcinoma cells.

  8. Hsp90 inhibition suppresses NF-κB transcriptional activation via Sirt-2 in human lung microvascular endothelial cells.

    Science.gov (United States)

    Thangjam, Gagan S; Birmpas, Charalampos; Barabutis, Nektarios; Gregory, Betsy W; Clemens, Mary Ann; Newton, Joseph R; Fulton, David; Catravas, John D

    2016-05-15

    The ability of anti-heat shock protein 90 (Hsp90) drugs to attenuate NF-κB-mediated transcription is the major basis for their anti-inflammatory properties. While the molecular mechanisms underlying this effect are not clear, they appear to be distinct in human endothelial cells. We now show for the first time that type 2 sirtuin (Sirt-2) histone deacetylase binds human NF-κB target gene promoter and prevents the recruitment of NF-κB proteins and subsequent assembly of RNA polymerase II complex in human lung microvascular endothelial cells. Hsp90 inhibitors stabilize the Sirt-2/promoter interaction and impose a "transcriptional block," which is reversed by either inhibition or downregulation of Sirt-2 protein expression. Furthermore, this process is independent of NF-κB (p65) Lysine 310 deacetylation, suggesting that it is distinct from known Sirt-2-dependent mechanisms. We demonstrate that Sirt-2 is recruited to NF-κB target gene promoter via interaction with core histones. Upon inflammatory challenge, chromatin remodeling and core histone H3 displacement from the promoter region removes Sirt-2 and allows NF-κB/coactivator recruitment essential for RNA Pol II-dependent mRNA induction. This novel mechanism may have important implications in pulmonary inflammation.

  9. Proneural Transcription Factor Atoh1 Drives Highly Efficient Differentiation of Human Pluripotent Stem Cells Into Dopaminergic Neurons

    Science.gov (United States)

    Sagal, Jonathan; Zhan, Xiping; Xu, Jinchong; Tilghman, Jessica; Karuppagounder, Senthilkumar S.; Chen, Li; Dawson, Valina L.; Dawson, Ted M.; Laterra, John

    2014-01-01

    Human pluripotent stem cells (PSCs) are a promising cell resource for various applications in regenerative medicine. Highly efficient approaches that differentiate human PSCs into functional lineage-specific neurons are critical for modeling neurological disorders and testing potential therapies. Proneural transcription factors are crucial drivers of neuron development and hold promise for driving highly efficient neuronal conversion in PSCs. Here, we study the functions of proneural transcription factor Atoh1 in the neuronal differentiation of PSCs. We show that Atoh1 is induced during the neuronal conversion of PSCs and that ectopic Atoh1 expression is sufficient to drive PSCs into neurons with high efficiency. Atoh1 induction, in combination with cell extrinsic factors, differentiates PSCs into functional dopaminergic (DA) neurons with >80% purity. Atoh1-induced DA neurons recapitulate key biochemical and electrophysiological features of midbrain DA neurons, the degeneration of which is responsible for clinical symptoms in Parkinson’s disease (PD). Atoh1-induced DA neurons provide a reliable disease model for studying PD pathogenesis, such as neurotoxin-induced neurodegeneration in PD. Overall, our results determine the role of Atoh1 in regulating neuronal differentiation and neuron subtype specification of human PSCs. Our Atoh1-mediated differentiation approach will enable large-scale applications of PD patient-derived midbrain DA neurons in mechanistic studies and drug screening for both familial and sporadic PD. PMID:24904172

  10. A synthetic interaction screen identifies factors selectively required for proliferation and TERT transcription in p53-deficient human cancer cells.

    Directory of Open Access Journals (Sweden)

    Li Xie

    Full Text Available Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi-based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53- human cancer cells. We find that compared to p53-competent (p53+ human cancer cell lines, diverse p53- human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53- cells, RNAi-mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53- but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53- cancer cells.

  11. Characterization of CRTAM gene promoter: AP-1 transcription factor control its expression in human T CD8 lymphocytes.

    Science.gov (United States)

    Valle-Rios, Ricardo; Patiño-Lopez, Genaro; Medina-Contreras, Oscar; Canche-Pool, Elsy; Recillas-Targa, Felix; Lopez-Bayghen, Esther; Zlotnik, Albert; Ortiz-Navarrete, Vianney

    2009-10-01

    Class-I MHC-restricted T-cell associated molecule (CRTAM) is a member of the Nectin-like adhesion molecule family. It is rapidly induced in NK, NKT and CD8(+) T cells. Interaction with its ligand Nectin-like 2 results in increased secretion of IFN-gamma by activated CD8(+) T lymphocytes. Through sequential bioinformatic analyses of the upstream region of the human CRTAM gene, we detected cis-elements potentially important for CRTAM gene transcription. Analyzing 2kb upstream from the ATG translation codon by mutation analysis in conjunction with luciferase reporter assays, electrophoretic mobility shify assay (EMSA) and supershift assays, we identified an AP-1 binding site, located at 1.4kb from the ATG translation codon of CRTAM gene as an essential element for CRTAM expression in activated but not resting human CD8(+) T cells. CRTAM expression was reduced in activated CD8(+) T cells treated with the JNK inhibitor SP600125, indicating that CRTAM expression is driven by the JNK-AP-1 signaling pathway. This study represents the first CRTAM gene promoter analysis in human T cells and indicates that AP-1 is a positive transcriptional regulator of this gene, a likely important finding because CRTAM has recently been shown to play a role in IFN-gamma and IL-17 production and T cell proliferation.

  12. Phenobarbital induces cell cycle transcriptional responses in mouse liver humanized for constitutive androstane and pregnane x receptors.

    Science.gov (United States)

    Luisier, Raphaëlle; Lempiäinen, Harri; Scherbichler, Nina; Braeuning, Albert; Geissler, Miriam; Dubost, Valerie; Müller, Arne; Scheer, Nico; Chibout, Salah-Dine; Hara, Hisanori; Picard, Frank; Theil, Diethilde; Couttet, Philippe; Vitobello, Antonio; Grenet, Olivier; Grasl-Kraupp, Bettina; Ellinger-Ziegelbauer, Heidrun; Thomson, John P; Meehan, Richard R; Elcombe, Clifford R; Henderson, Colin J; Wolf, C Roland; Schwarz, Michael; Moulin, Pierre; Terranova, Rémi; Moggs, Jonathan G

    2014-06-01

    The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CAR(KO)-PXR(KO)), double humanized CAR and PXR (CAR(h)-PXR(h)), and wild-type C57BL/6 mice. Wild-type and CAR(h)-PXR(h) mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CAR(KO)-PXR(KO) mouse livers and largely reversible in wild-type and CAR(h)-PXR(h) mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CAR(h)-PXR(h) mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.

  13. In Vivo Imaging of Human MDR1 Transcription in the Brain and Spine of MDR1-Luciferase Reporter Mice.

    Science.gov (United States)

    Yasuda, Kazuto; Cline, Cynthia; Lin, Yvonne S; Scheib, Rachel; Ganguly, Samit; Thirumaran, Ranjit K; Chaudhry, Amarjit; Kim, Richard B; Schuetz, Erin G

    2015-11-01

    P-glycoprotein (Pgp) [the product of the MDR1 (ABCB1) gene] at the blood-brain barrier (BBB) limits central nervous system (CNS) entry of many prescribed drugs, contributing to the poor success rate of CNS drug candidates. Modulating Pgp expression could improve drug delivery into the brain; however, assays to predict regulation of human BBB Pgp are lacking. We developed a transgenic mouse model to monitor human MDR1 transcription in the brain and spinal cord in vivo. A reporter construct consisting of ∼10 kb of the human MDR1 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line (MDR1-luc). Fluorescence in situ hybridization localized the MDR1-luciferase transgene on chromosome 3. Reporter gene expression was monitored with an in vivo imaging system following D-luciferin injection. Basal expression was detectable in the brain, and treatment with activators of the constitutive androstane, pregnane X, and glucocorticoid receptors induced brain and spinal MDR1-luc transcription. Since D-luciferin is a substrate of ABCG2, the feasibility of improving D-luciferin brain accumulation (and luciferase signal) was tested by coadministering the dual ABCB1/ABCG2 inhibitor elacridar. The brain and spine MDR1-luc signal intensity was increased by elacridar treatment, suggesting enhanced D-luciferin brain bioavailability. There was regional heterogeneity in MDR1 transcription (cortex > cerebellum) that coincided with higher mouse Pgp protein expression. We confirmed luciferase expression in brain vessel endothelial cells by ex vivo analysis of tissue luciferase protein expression. We conclude that the MDR1-luc mouse provides a unique in vivo system to visualize MDR1 CNS expression and regulation.

  14. A Premature Termination of Human Epidermal Growth Factor Receptor Transcription in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jihene Elloumi-Mseddi

    2014-01-01

    Full Text Available Our success in producing an active epidermal growth factor receptor (EGFR tyrosine kinase in Escherichia coli encouraged us to express the full-length receptor in the same host. Despite its large size, we were successful at producing the full-length EGFR protein fused to glutathione S-transferase (GST that was detected by Western blot analysis. Moreover, we obtained a majoritarian truncated GST-EGFR form detectable by gel electrophoresis and Western blot. This truncated protein was purified and confirmed by MALDI-TOF/TOF analysis to belong to the N-terminal extracellular region of the EGFR fused to GST. Northern blot analysis showed two transcripts suggesting the occurrence of a transcriptional arrest.

  15. Genome-wide transcriptional reorganization associated with senescence-to-immortality switch during human hepatocellular carcinogenesis.

    Science.gov (United States)

    Yildiz, Gokhan; Arslan-Ergul, Ayca; Bagislar, Sevgi; Konu, Ozlen; Yuzugullu, Haluk; Gursoy-Yuzugullu, Ozge; Ozturk, Nuri; Ozen, Cigdem; Ozdag, Hilal; Erdal, Esra; Karademir, Sedat; Sagol, Ozgul; Mizrak, Dilsa; Bozkaya, Hakan; Ilk, Hakki Gokhan; Ilk, Ozlem; Bilen, Biter; Cetin-Atalay, Rengul; Akar, Nejat; Ozturk, Mehmet

    2013-01-01

    Senescence is a permanent proliferation arrest in response to cell stress such as DNA damage. It contributes strongly to tissue aging and serves as a major barrier against tumor development. Most tumor cells are believed to bypass the senescence barrier (become "immortal") by inactivating growth control genes such as TP53 and CDKN2A. They also reactivate telomerase reverse transcriptase. Senescence-to-immortality transition is accompanied by major phenotypic and biochemical changes mediated by genome-wide transcriptional modifications. This appears to happen during hepatocellular carcinoma (HCC) development in patients with liver cirrhosis, however, the accompanying transcriptional changes are virtually unknown. We investigated genome-wide transcriptional changes related to the senescence-to-immortality switch during hepatocellular carcinogenesis. Initially, we performed transcriptome analysis of senescent and immortal clones of Huh7 HCC cell line, and identified genes with significant differential expression to establish a senescence-related gene list. Through the analysis of senescence-related gene expression in different liver tissues we showed that cirrhosis and HCC display expression patterns compatible with senescent and immortal phenotypes, respectively; dysplasia being a transitional state. Gene set enrichment analysis revealed that cirrhosis/senescence-associated genes were preferentially expressed in non-tumor tissues, less malignant tumors, and differentiated or senescent cells. In contrast, HCC/immortality genes were up-regulated in tumor tissues, or more malignant tumors and progenitor cells. In HCC tumors and immortal cells genes involved in DNA repair, cell cycle, telomere extension and branched chain amino acid metabolism were up-regulated, whereas genes involved in cell signaling, as well as in drug, lipid, retinoid and glycolytic metabolism were down-regulated. Based on these distinctive gene expression features we developed a 15-gene

  16. Human immunodeficiency virus trans-activator of transcription peptide detection via ribonucleic acid aptamer on aminated diamond biosensor

    Science.gov (United States)

    Rahim Ruslinda, A.; Wang, Xianfen; Ishii, Yoko; Ishiyama, Yuichiro; Tanabe, Kyosuke; Kawarada, Hiroshi

    2011-09-01

    The potential of ribonucleic acid (RNA) as both informational and ligand binding molecule have opened a scenario in the development of biosensors. An aminated diamond-based RNA aptasensor is presented for human immunodeficiency virus (HIV) trans-activator of transcription (Tat) peptide protein detection that not only gives a labeled or label-free detection method but also provides a reusable platform for a simple, sensitive, and selective detection of proteins. The immobilized procedure was based on the binding interaction between positively charged amine terminated diamond and the RNA aptamer probe molecules with the negatively charged surface carboxylic compound linker molecule such as terephthalic acid.

  17. Identification of NR4A2 as a transcriptional activator of IL-8 expression in human inflammatory arthritis.

    LENUS (Irish Health Repository)

    Aherne, Carol M

    2009-10-01

    Expression of the orphan nuclear receptor NR4A2 is controlled by pro-inflammatory mediators, suggesting that NR4A2 may contribute to pathological processes in the inflammatory lesion. This study identifies the chemoattractant protein, interleukin 8 (IL-8\\/CXCL8), as a molecular target of NR4A2 in human inflammatory arthritis and examines the mechanism through which NR4A2 modulates IL-8 expression. In TNF-alpha-activated human synoviocyte cells, enhanced expression of IL-8 mRNA and protein correspond to temporal changes in NR4A2 transcription and nuclear distribution. Ectopic expression of NR4A2 leads to robust changes in endogenous IL-8 mRNA levels and co-treatment with TNF-alpha results in significant (p<0.001) secretion of IL-8 protein. Transcriptional effects of NR4A2 on the human IL-8 promoter are enhanced in the presence of TNF-alpha, suggesting molecular crosstalk between TNF-alpha signalling and NR4A2. A dominant negative IkappaB kinase antagonizes the combined effects of NR4A2 and TNF-alpha on IL-8 promoter activity. Co-expression of NR4A2 and the p65 subunit of NF-kappaB enhances IL-8 transcription and functional studies indicate that transactivation occurs independently of NR4A2 binding to DNA or heterodimerization with additional nuclear receptors. The IL-8 minimal promoter region is sufficient to support NR4A2 and NF-kappaB\\/p65 co-operative activity and NR4A2 can interact with NF-kappaB\\/p65 on a 39bp sequence within this region. In patients treated with methotrexate for active inflammatory arthritis, a reduction in NR4A2 synovial tissue levels correlate significantly (n=10, r=0.73, p=0.002) with changes in IL-8 expression. Collectively, these data delineate an important role for NR4A2 in modulating IL-8 expression and reveal novel transcriptional responses to TNF-alpha in human inflammatory joint disease.

  18. Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

    Energy Technology Data Exchange (ETDEWEB)

    Grierson, Patrick M. [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Acharya, Samir, E-mail: samir.acharya@osumc.edu [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Groden, Joanna [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States)

    2013-03-15

    Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription.

  19. Argonaute-1 binds transcriptional enhancers and controls constitutive and alternative splicing in human cells

    Science.gov (United States)

    Alló, Mariano; Agirre, Eneritz; Bessonov, Sergey; Bertucci, Paola; Gómez Acuña, Luciana; Buggiano, Valeria; Bellora, Nicolás; Singh, Babita; Petrillo, Ezequiel; Blaustein, Matías; Miñana, Belén; Dujardin, Gwendal; Pozzi, Berta; Pelisch, Federico; Bechara, Elías; Agafonov, Dmitry E.; Srebrow, Anabella; Lührmann, Reinhard; Valcárcel, Juan; Eyras, Eduardo; Kornblihtt, Alberto R.

    2014-01-01

    The roles of Argonaute proteins in cytoplasmic microRNA and RNAi pathways are well established. However, their implication in small RNA-mediated transcriptional gene silencing in the mammalian cell nucleus is less understood. We have recently shown that intronic siRNAs cause chromatin modifications that inhibit RNA polymerase II elongation and modulate alternative splicing in an Argonaute-1 (AGO1)-dependent manner. Here we used chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) to investigate the genome-wide distribution of AGO1 nuclear targets. Unexpectedly, we found that about 80% of AGO1 clusters are associated with cell-type-specific transcriptional enhancers, most of them (73%) overlapping active enhancers. This association seems to be mediated by long, rather than short, enhancer RNAs and to be more prominent in intragenic, rather than intergenic, enhancers. Paradoxically, crossing ChIP-seq with RNA-seq data upon AGO1 depletion revealed that enhancer-bound AGO1 is not linked to the global regulation of gene transcription but to the control of constitutive and alternative splicing, which was confirmed by an individual gene analysis explaining how AGO1 controls inclusion levels of the cassette exon 107 in the SYNE2 gene. PMID:25313066

  20. Differential expression of ETS family transcription factors in NCCIT human embryonic carcinoma cells upon retinoic acid-induced differentiation.

    Science.gov (United States)

    Park, Sung-Won; Do, Hyun-Jin; Ha, Woo Tae; Han, Mi-Hee; Song, Hyuk; Uhm, Sang-Jun; Chung, Hak-Jae; Kim, Jae-Hwan

    2014-01-01

    E26 transformation-specific (ETS) transcription factors play important roles in normal and tumorigenic processes during development, differentiation, homeostasis, proliferation, and apoptosis. To identify critical ETS factor(s) in germ cell-derived cancer cells, we examined the expression patterns of the 27 ETS transcription factors in naive and differentiated NCCIT human embryonic carcinoma cells, which exhibit both pluripotent and tumorigenic characteristics. Overall, expression of ETS factors was relatively low in NCCIT cells. Among the 27 ETS factors, polyomavirus enhancer activator 3 (PEA3) and epithelium-specific ETS transcription factor-1 (ESE-1) exhibited the most significant changes in their expression levels. Western blot analysis confirmed these patterns, revealing reduced levels of PEA3 protein and elevated levels of ESE-1 protein in differentiated cells. PEA3 increased the proportion of cells in S-phase and promoted cell growth, whereas ESE-1 reduced proliferation potential. These data suggest that PEA3 and ESE-1 may play important roles in pluripotent and tumorigenic embryonic carcinoma cells. These findings contribute to our understanding of the functions of oncogenic ETS factors in germ cell-derived stem cells during processes related to tumorigenesis and pluripotency.

  1. [Colorimetric detection of human influenza A H1N1 virus by reverse transcription loop mediated isothermal amplification].

    Science.gov (United States)

    Nie, Kai; Wang, Da-Yan; Qin, Meng; Gao, Rong-Bao; Wang, Miao; Zou, Shu-Mei; Han, Feng; Zhao, Xiang; Li, Xi-Yan; Shu, Yue-Long; Ma, Xue-Jun

    2010-03-01

    A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.

  2. EGF stimulates Pit-1 independent transcription of the human prolactin pituitary promoter in human breast cancer SK-BR-3 cells through its proximal AP-1 response element.

    Science.gov (United States)

    Manfroid, Isabelle; Van de Weerdt, Cécile; Baudhuin, Ariane; Martial, Joseph A; Muller, Marc

    2005-01-14

    Normal and neoplastic human mammary gland cells are targets for the proliferative action of prolactin. These cells also synthesize prolactin, thereby inducing an autocrine/paracrine proliferative loop. We present the first extensive analysis of the transcriptional regulation of the human prolactin gene (hPRL) in human mammary tumor cells, SK-BR-3. We show that the pituitary promoter is functional in these cells in the absence of the pituitary-specific factor Pit-1. Expression of exogenous Pit-1 or epidermal growth factor (EGF) treatment stimulates the transfected hPRL pituitary promoter and the endogenous hPRL expression. EGF stimulation is mediated by increased synthesis of c-fos and c-jun, resulting in AP-1 binding to the proximal hPRL pituitary promoter. This regulation involves the EGF receptor, possibly ErbB2 that is highly expressed in SK-BR-3 cells, and a PI3K/JNK pathway. The stimulation of hPRL gene transcription by EGF in mammary cells may include hPRL in a complex regulatory network controlling growth of human mammary cells.

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

    Directory of Open Access Journals (Sweden)

    Surleen Kaur

    2016-03-01

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

  4. Transcription Factor SomA Is Required for Adhesion, Development and Virulence of the Human Pathogen Aspergillus fumigatus.

    Directory of Open Access Journals (Sweden)

    Chi-Jan Lin

    Full Text Available The transcription factor Flo8/Som1 controls filamentous growth in Saccharomyces cerevisiae and virulence in the plant pathogen Magnaporthe oryzae. Flo8/Som1 includes a characteristic N-terminal LUG/LUH-Flo8-single-stranded DNA binding (LUFS domain and is activated by the cAMP dependent protein kinase A signaling pathway. Heterologous SomA from Aspergillus fumigatus rescued in yeast flo8 mutant strains several phenotypes including adhesion or flocculation in haploids and pseudohyphal growth in diploids, respectively. A. fumigatus SomA acts similarly to yeast Flo8 on the promoter of FLO11 fused with reporter gene (LacZ in S. cerevisiae. FLO11 expression in yeast requires an activator complex including Flo8 and Mfg1. Furthermore, SomA physically interacts with PtaB, which is related to yeast Mfg1. Loss of the somA gene in A. fumigatus resulted in a slow growth phenotype and a block in asexual development. Only aerial hyphae without further differentiation could be formed. The deletion phenotype was verified by a conditional expression of somA using the inducible Tet-on system. A adherence assay with the conditional somA expression strain indicated that SomA is required for biofilm formation. A ptaB deletion strain showed a similar phenotype supporting that the SomA/PtaB complex controls A. fumigatus biofilm formation. Transcriptional analysis showed that SomA regulates expression of genes for several transcription factors which control conidiation or adhesion of A. fumigatus. Infection assays with fertilized chicken eggs as well as with mice revealed that SomA is required for pathogenicity. These data corroborate a complex control function of SomA acting as a central factor of the transcriptional network, which connects adhesion, spore formation and virulence in the opportunistic human pathogen A. fumigatus.

  5. Purinergic P2Y2 Receptor Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: NEW AP-1 TRANSCRIPTION FACTOR SITE AND NEGATIVE REGULATOR.

    Science.gov (United States)

    Liu, Yiwei; Zhang, Lingxin; Wang, Chuan; Roy, Shama; Shen, Jianzhong

    2016-01-22

    We recently reported that the P2Y2 receptor (P2Y2R) is the predominant nucleotide receptor expressed in human coronary artery endothelial cells (HCAEC) and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), a key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R-TF axis remains unclear. Here, we report the role of a newly identified AP-1 consensus sequence in the TF gene promoter and its original binding components in P2Y2R regulation of TF transcription. Using bioinformatics tools, we found that a novel AP-1 site at -1363 bp of the human TF promoter region is highly conserved across multiple species. Activation of P2Y2R increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion, and mutation of this distal AP-1 site all significantly suppressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c-Jun, ATF-2, and Fra-1, but not the typical c-Fos, bound to the new AP-1 site. In addition, loss-of-function studies using siRNAs confirmed a positive transactivation role of c-Jun and ATF-2 but unexpectedly revealed a strong negative role of Fra-1 in P2Y2R-induced TF up-regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation through Src, leading to Fra-1 activation, whereas Rho/JNK mediated P2Y2R-induced activation of c-Jun and ATF-2. These findings reveal the molecular basis for P2Y G protein-coupled receptor control of endothelial TF expression and indicate that targeting the P2Y2R-Fra-1-TF pathway may be an attractive new strategy for controlling vascular inflammation and thrombogenicity associated with endothelial dysfunction.

  6. Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Mizutani, Naoki [Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya (Japan); College of Life and Health Sciences, Chubu University, Kasugai (Japan); Omori, Yukari [Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya (Japan); Kawamoto, Yoshiyuki; Sobue, Sayaka; Ichihara, Masatoshi [College of Life and Health Sciences, Chubu University, Kasugai (Japan); Suzuki, Motoshi [Division of Molecular Carcinogenesis, Nagoya University Graduate School of Medicine, Nagoya (Japan); Kyogashima, Mamoru [Department of Microbiology and Molecular Biology, Nihon Pharmaceutical University, Saitama (Japan); Nakamura, Mitsuhiro [Department of Drug Information, Gifu Pharmaceutical University, Gifu (Japan); Tamiya-Koizumi, Keiko [College of Life and Health Sciences, Chubu University, Kasugai (Japan); Nozawa, Yoshinori [Tokai Gakuin University, Kakamigahara (Japan); Murate, Takashi, E-mail: murate@isc.chubu.ac.jp [College of Life and Health Sciences, Chubu University, Kasugai (Japan)

    2016-02-19

    Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5′-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment. - Highlights: • Resveratrol inhibited cell proliferation of K562 and HCT116 cells. • Resveratrol increased cellular ceramide and decreased sphingomyelin and S1P. • ASMase mRNA and activity were increased with resveratrol. • ASMase inhibition suppressed RSV-induced ceramide accumulation. • Increased ASMase transcription was at least partially due to EGR family proteins.

  7. Imprinting of IGF2 P0 transcript and novel alternatively spliced INS-IGF2 isoforms show differences between mouse and human.

    Science.gov (United States)

    Monk, D; Sanches, R; Arnaud, P; Apostolidou, S; Hills, F A; Abu-Amero, S; Murrell, A; Friess, H; Reik, W; Stanier, P; Constância, M; Moore, G E

    2006-04-15

    Genomic imprinting is limited to a subset of genes that play critical roles in fetal growth, development and behaviour. One of the most studied imprinted genes encodes insulin-like growth factor 2, and aberrant imprinting and DNA methylation of this gene is associated with the growth disorders Beckwith-Wiedemann and Silver-Russell syndromes and many human cancers. Specific isoforms of this gene have been shown to be essential for normal placental function, as mice carrying paternal null alleles for the Igf2-P0 transcript are growth restricted at birth. We report here the identification of three novel human transcripts from the IGF2 locus. One is equivalent to the mouse Igf2-P0 transcript, whereas the two others (INSIGF long and short) originate from the upstream INS gene that alternatively splices to downstream IGF2 exons. In order to elucidate the molecular mechanisms involved in the complex imprinting of these novel IGF2 transcripts, both the allele-specific expression and methylation for all the IGF2 promoters including P0 and the INSIGF transcripts were analysed in human tissues. Similar to the mouse, the human IGF2-P0 transcript is paternally expressed; however, its expression is not limited to placenta. This expression correlates with tissue-specific promoter methylation on the maternal allele. The two novel INSIGF transcripts reported here use the INS promoter and show highly restricted tissue expression profiles including the pancreas. As previously reported for INS in the yolk sac, we demonstrate complex, tissue-specific imprinting of these transcripts. The finding of additional transcripts within this locus will have important implications for IGF2 regulation in both cancer and metabolism.

  8. Human mitochondrial transcription factor A functions in both nuclei and mitochondria and regulates cancer cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Han, Bin [Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Department of Urology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Department of Urology, Shengjing Hospital of China Medical University, Shenyang (China); Izumi, Hiroto; Yasuniwa, Yoshihiro; Akiyama, Masaki; Yamaguchi, Takahiro [Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Fujimoto, Naohiro; Matsumoto, Tetsuro [Department of Urology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Wu, Bin [Department of Urology, Shengjing Hospital of China Medical University, Shenyang (China); Tanimoto, Akihide [Department of Pathology, Kagoshima University Graduate School of Medical and Dental Science, Kagoshima (Japan); Sasaguri, Yasuyuki [Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Kohno, Kimitoshi, E-mail: k-kohno@med.uoeh-u.ac.jp [Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan)

    2011-04-29

    Highlights: {yields} Mitochondrial transcription factor A (mtTFA) localizes in nuclei and binds tightly to the nuclear chromatin. {yields} mtTFA contains two putative nuclear localization signals (NLS) in the HMG-boxes. {yields} Overexpression of mtTFA enhances the growth of cancer cells, whereas downregulation of mtTFA inhibits their growth by regulating mtTFA target genes, such as baculoviral IAP repeat-containing 5 (BIRC5; also known as survivin). {yields} Knockdown of mtTFA expression induces p21-dependent G1 cell cycle arrest. -- Abstract: Mitochondrial transcription factor A (mtTFA) is one of the high mobility group protein family and is required for both transcription from and maintenance of mitochondrial genomes. However, the roles of mtTFA have not been extensively studied in cancer cells. Here, we firstly reported the nuclear localization of mtTFA. The proportion of nuclear-localized mtTFA varied among different cancer cells. Some mtTFA binds tightly to the nuclear chromatin. DNA microarray and chromatin immunoprecipitation assays showed that mtTFA can regulate the expression of nuclear genes. Overexpression of mtTFA enhanced the growth of cancer cell lines, whereas downregulation of mtTFA inhibited their growth by regulating mtTFA target genes, such as baculoviral IAP repeat-containing 5 (BIRC5; also known as survivin). Knockdown of mtTFA expression induced p21-dependent G1 cell cycle arrest. These results imply that mtTFA functions in both nuclei and mitochondria to promote cell growth.

  9. Dynamic transcriptional signatures and network responses for clinical symptoms in influenza-infected human subjects using systems biology approaches.

    Science.gov (United States)

    Linel, Patrice; Wu, Shuang; Deng, Nan; Wu, Hulin

    2014-10-01

    Recent studies demonstrate that human blood transcriptional signatures may be used to support diagnosis and clinical decisions for acute respiratory viral infections such as influenza. In this article, we propose to use a newly developed systems biology approach for time course gene expression data to identify significant dynamically response genes and dynamic gene network responses to viral infection. We illustrate the methodological pipeline by reanalyzing the time course gene expression data from a study with healthy human subjects challenged by live influenza virus. We observed clear differences in the number of significant dynamic response genes (DRGs) between the symptomatic and asymptomatic subjects and also identified DRG signatures for symptomatic subjects with influenza infection. The 505 common DRGs shared by the symptomatic subjects have high consistency with the signature genes for predicting viral infection identified in previous works. The temporal response patterns and network response features were carefully analyzed and investigated.

  10. NF-κB Mediated Transcription in Human Monocytic Cells and Endothelial Cells.

    Science.gov (United States)

    Parry, G C; Mackman, N

    1998-04-01

    Monocytes and endothelial cells become activated at sites of inflammation and contribute to the pathology