ORF Sequence: NC_000913 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_000913 gi|16128606 >gi|16128606|ref|NP_415156.1| RNA chaperone, transcription antiterm...inator, affects expression of rpoS and uspA [Escherichia coli K12] MSKIKGNVKWFNESKGFGFITPEDGSKDVFVHFSAIQTNGFKTLAEGQRVEFEITNGAKGPSAANVIAL

Understanding large multiprotein complexes: applying a multiple allosteric networks model to explain the function of the Mediator transcription complex.

Science.gov (United States)

Lewis, Brian A

2010-01-15

The regulation of transcription and of many other cellular processes involves large multi-subunit protein complexes. In the context of transcription, it is known that these complexes serve as regulatory platforms that connect activator DNA-binding proteins to a target promoter. However, there is still a lack of understanding regarding the function of these complexes. Why do multi-subunit complexes exist? What is the molecular basis of the function of their constituent subunits, and how are these subunits organized within a complex? What is the reason for physical connections between certain subunits and not others? In this article, I address these issues through a model of network allostery and its application to the eukaryotic RNA polymerase II Mediator transcription complex. The multiple allosteric networks model (MANM) suggests that protein complexes such as Mediator exist not only as physical but also as functional networks of interconnected proteins through which information is transferred from subunit to subunit by the propagation of an allosteric state known as conformational spread. Additionally, there are multiple distinct sub-networks within the Mediator complex that can be defined by their connections to different subunits; these sub-networks have discrete functions that are activated when specific subunits interact with other activator proteins.

The MYST family histone acetyltransferase complex regulates stress resistance and longevity through transcriptional control of DAF-16/FOXO transcription factors.

Science.gov (United States)

Ikeda, Takako; Uno, Masaharu; Honjoh, Sakiko; Nishida, Eisuke

2017-08-09

The well-known link between longevity and the Sir2 histone deacetylase family suggests that histone deacetylation, a modification associated with repressed chromatin, is beneficial to longevity. However, the molecular links between histone acetylation and longevity remain unclear. Here, we report an unexpected finding that the MYST family histone acetyltransferase complex (MYS-1/TRR-1 complex) promotes rather than inhibits stress resistance and longevity in Caenorhabditis elegans Our results show that these beneficial effects are largely mediated through transcriptional up-regulation of the FOXO transcription factor DAF-16. MYS-1 and TRR-1 are recruited to the promoter regions of the daf-16 gene, where they play a role in histone acetylation, including H4K16 acetylation. Remarkably, we also find that the human MYST family Tip60/TRRAP complex promotes oxidative stress resistance by up-regulating the expression of FOXO transcription factors in human cells. Tip60 is recruited to the promoter regions of the foxo1 gene, where it increases H4K16 acetylation levels. Our results thus identify the evolutionarily conserved role of the MYST family acetyltransferase as a key epigenetic regulator of DAF-16/FOXO transcription factors. © 2017 The Authors.

The Transcription Bubble of the RNA Polymerase-Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics : Implications for Transcription Start-Site Selection

NARCIS (Netherlands)

Robb, Nicole C.; Cordes, Thorben; Hwang, Ling Chin; Gryte, Kristofer; Duchi, Diego; Craggs, Timothy D.; Santoso, Yusdi; Weiss, Shimon; Ebright, Richard H.; Kapanidis, Achillefs N.

2013-01-01

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts similar to 14 bp around the transcription start site and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RPo). There is significant flexibility in the

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

DEFF Research Database (Denmark)

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

2006-01-01

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

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

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NandyMazumdar, Monali; Nedialkov, Yuri; Svetlov, Dmitri; Sevostyanova, Anastasia; Belogurov, Georgiy A; Artsimovitch, Irina

2016-12-27

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

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

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

2016-03-01

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

Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.

Science.gov (United States)

Konermann, Silvana; Brigham, Mark D; Trevino, Alexandro E; Joung, Julia; Abudayyeh, Omar O; Barcena, Clea; Hsu, Patrick D; Habib, Naomi; Gootenberg, Jonathan S; Nishimasu, Hiroshi; Nureki, Osamu; Zhang, Feng

2015-01-29

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

Endoplasmic reticulum stress-responsive transcription factor ATF6α directs recruitment of the Mediator of RNA polymerase II transcription and multiple histone acetyltransferase complexes.

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Sela, Dotan; Chen, Lu; Martin-Brown, Skylar; Washburn, Michael P; Florens, Laurence; Conaway, Joan Weliky; Conaway, Ronald C

2012-06-29

The basic leucine zipper transcription factor ATF6α functions as a master regulator of endoplasmic reticulum (ER) stress response genes. Previous studies have established that, in response to ER stress, ATF6α translocates to the nucleus and activates transcription of ER stress response genes upon binding sequence specifically to ER stress response enhancer elements in their promoters. In this study, we investigate the biochemical mechanism by which ATF6α activates transcription. By exploiting a combination of biochemical and multidimensional protein identification technology-based mass spectrometry approaches, we have obtained evidence that ATF6α functions at least in part by recruiting to the ER stress response enhancer elements of ER stress response genes a collection of RNA polymerase II coregulatory complexes, including the Mediator and multiple histone acetyltransferase complexes, among which are the Spt-Ada-Gcn5 acetyltransferase (SAGA) and Ada-Two-A-containing (ATAC) complexes. Our findings shed new light on the mechanism of action of ATF6α, and they outline a straightforward strategy for applying multidimensional protein identification technology mass spectrometry to determine which RNA polymerase II transcription factors and coregulators are recruited to promoters and other regulatory elements to control transcription.

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.

Isolation and mass spectrometry of transcription factor complexes.

Science.gov (United States)

Sebastiaan Winkler, G; Lacomis, Lynne; Philip, John; Erdjument-Bromage, Hediye; Svejstrup, Jesper Q; Tempst, Paul

2002-03-01

Protocols are described that enable the isolation of novel proteins associated with a known protein and the subsequent identification of these proteins by mass spectrometry. We review the basics of nanosample handling and of two complementary approaches to mass analysis, and provide protocols for the entire process. The protein isolation procedure is rapid and based on two high-affinity chromatography steps. The method does not require previous knowledge of complex composition or activity and permits subsequent biochemical characterization of the isolated factor. As an example, we provide the procedures used to isolate and analyze yeast Elongator, a histone acetyltransferase complex important for transcript elongation, which led to the identification of three novel subunits.

Sequence Classification: 288227 [

Lifescience Database Archive (English)

Full Text Available rone, transcription antiterminator, affects expression of rpoS and uspA || http://www.ncbi.nlm.nih.gov/protein/16128606 ... ...Non-TMB Non-TMH Non-TMB TMB Non-TMB Non-TMB >gi|16128606|ref|NP_415156.1| RNA chape

Roles of mono-ubiquitinated Smad4 in the formation of Smad transcriptional complexes

International Nuclear Information System (INIS)

Wang Bei; Suzuki, Hiroyuki; Kato, Mitsuyasu

2008-01-01

TGF-β activates receptor-regulated Smad (R-Smad) through phosphorylation by type I receptors. Activated R-Smad binds to Smad4 and the complex translocates into the nucleus and stimulates the transcription of target genes through association with co-activators including p300. It is not clear, however, how activated Smad complexes are removed from target genes. In this study, we show that TGF-β enhances the mono-ubiquitination of Smad4. Smad4 mono-ubiquitination was promoted by p300 and suppressed by the c-Ski co-repressor. Smad4 mono-ubiquitination disrupted the interaction with Smad2 in the presence of constitutively active TGF-β type I receptor. Furthermore, mono-ubiquitinated Smad4 was not found in DNA-binding Smad complexes. A Smad4-Ubiquitin fusion protein, which mimics mono-ubiquitinated Smad4, enhanced localization to the cytoplasm. These results suggest that mono-ubiquitination of Smad4 occurs in the transcriptional activator complex and facilitates the turnover of Smad complexes at target genes

Dissection of combinatorial control by the Met4 transcriptional complex.

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

2010-02-01

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

Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements.

Science.gov (United States)

Meier, Daniel; Schindler, Detlev

2011-01-01

The Fanconi anemia (FA) gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M) that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS). In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs), and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements.

Directory of Open Access Journals (Sweden)

Daniel Meier

Full Text Available The Fanconi anemia (FA gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS. In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs, and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

Light-harvesting complex gene expression is controlled by both transcriptional and post-transcriptional mechanisms during photoacclimation in Chlamydomonas reinhardtii

CERN Document Server

Durnford Dion, G; McKim, Sarah M; Sarchfield, Michelle L

2003-01-01

To compensate for increases in photon flux density (PFD), photosynthetic organisms possess mechanisms for reversibly modulating their photosynthetic apparatus to minimize photodamage. The photoacclimation response in Chlamydomonas reinhardtii was assessed following a 10-fold increase in PFD over 24h. In addition to a 50% reduction in the amount of chlorophyll and light-harvesting complexes (LHC) per cell, the expression of genes encoding polypeptides of the light-harvesting antenna were also affected. The abundance of Lhcb (a LHCH gene), Lhcb4 (a CP29-like gene), and Lhca (a LHCI gene) transcripts were reduced by 65 to 80%, within 1-2 h; however, the RNA levels of all three genes recovered to their low-light (LL) concentrations within 6-8 h. To determine the role of transcript turnover in this transient decline in abundance, the stability of all transcripts was measured. Although there was no change in the Lhcb or Lhca transcript turnover time, the Lhcb4 mRNA stability decreased 2.5-fold immediately following...

The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation.

Science.gov (United States)

Malik, Sohail; Roeder, Robert G

2010-11-01

The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action of numerous other co-activators and co-repressors, including those acting at the level of chromatin. These interactions ultimately allow the Mediator to deliver outputs that range from maximal activation of genes to modulation of basal transcription to long-term epigenetic silencing.

Structures of RNA Polymerase Closed and Intermediate Complexes Reveal Mechanisms of DNA Opening and Transcription Initiation.

Science.gov (United States)

Glyde, Robert; Ye, Fuzhou; Darbari, Vidya Chandran; Zhang, Nan; Buck, Martin; Zhang, Xiaodong

2017-07-06

Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ 54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ 54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ 54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Regulation of hepatic lipogenesis by the transcription complex Prep1-Pbx1

OpenAIRE

Cabaro, Serena

2011-01-01

Prep1 is an homeodomain transcription factor belonging to the TALE proteins, including also Pbx1, which plays an essential role in hematopoiesis, organogenesis and development. Prep1 forms transcriptionally active complexes with Pbx1 and regulates the activity of several genes. The Prep1 null mutation leads to embryonic death at a very early stage. Therefore, Prep1 hypomorphic (Prep1i/i) mice have been generated. Prep1 heterozygous (Prep1i/+) mice, which express only 55-57% of protein, have a...

Intracytoplasmic maturation of the human immunodeficiency virus type 1 reverse transcription complexes determines their capacity to integrate into chromatin

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Kashanchi Fatah

2006-01-01

Full Text Available Abstract Background The early events of the HIV-1 life cycle include entry of the viral core into target cell, assembly of the reverse transcription complex (RTCs performing reverse transcription, its transformation into integration-competent complexes called pre-integration complexes (PICs, trafficking of complexes into the nucleus, and finally integration of the viral DNA into chromatin. Molecular details and temporal organization of these processes remain among the least investigated and most controversial problems in the biology of HIV. Results To quantitatively evaluate maturation and nuclear translocation of the HIV-1 RTCs, nucleoprotein complexes isolated from the nucleus (nRTC and cytoplasm (cRTC of HeLa cells infected with MLV Env-pseudotyped HIV-1 were analyzed by real-time PCR. While most complexes completed reverse transcription in the cytoplasm, some got into the nucleus before completing DNA synthesis. The HIV-specific RNA complexes could get into the nucleus when reverse transcription was blocked by reverse transcriptase inhibitor, although nuclear import of RNA complexes was less efficient than of DNA-containing RTCs. Analysis of the RTC nuclear import in synchronized cells infected in the G2/M phase of the cell cycle showed enrichment in the nuclei of RTCs containing incomplete HIV-1 DNA compared to non-synchronized cells, where RTCs with complete reverse transcripts prevailed. Immunoprecipitation assays identified viral proteins IN, Vpr, MA, and cellular Ini1 and PML associated with both cRTCs and nRTCs, whereas CA was detected only in cRTCs and RT was diminished in nRTCs. Cytoplasmic maturation of the complexes was associated with increased immunoreactivity with anti-Vpr and anti-IN antibodies, and decreased reactivity with antibodies to RT. Both cRTCs and nRTCs carried out endogenous reverse transcription reaction in vitro. In contrast to cRTCs, in vitro completion of reverse transcription in nRTCs did not increase their

A pp32-retinoblastoma protein complex modulates androgen receptor-mediated transcription and associates with components of the splicing machinery

International Nuclear Information System (INIS)

Adegbola, Onikepe; Pasternack, Gary R.

2005-01-01

We have previously shown pp32 and the retinoblastoma protein interact. pp32 and the retinoblastoma protein are nuclear receptor transcriptional coregulators: the retinoblastoma protein is a coactivator for androgen receptor, the major regulator of prostate cancer growth, while pp32, which is highly expressed in prostate cancer, is a corepressor of the estrogen receptor. We now show pp32 increases androgen receptor-mediated transcription and the retinoblastoma protein modulates this activity. Using affinity purification and mass spectrometry, we identify members of the pp32-retinoblastoma protein complex as PSF and nonO/p54nrb, proteins implicated in coordinate regulation of nuclear receptor-mediated transcription and splicing. We show that the pp32-retinoblastoma protein complex is modulated during TPA-induced K562 differentiation. Present evidence suggests that nuclear receptors assemble multiprotein complexes to coordinately regulate transcription and mRNA processing. Our results suggest that pp32 and the retinoblastoma protein may be part of a multiprotein complex that coordinately regulates nuclear receptor-mediated transcription and mRNA processing

A real-time view of the TAR:Tat:P-TEFb complex at HIV-1 transcription sites

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Knezevich Anna

2007-05-01

Full Text Available Abstract HIV-1 transcription is tightly regulated: silent in long-term latency and highly active in acutely-infected cells. Transcription is activated by the viral protein Tat, which recruits the elongation factor P-TEFb by binding the TAR sequence present in nascent HIV-1 RNAs. In this study, we analyzed the dynamic of the TAR:Tat:P-TEFb complex in living cells, by performing FRAP experiments at HIV-1 transcription sites. Our results indicate that a large fraction of Tat present at these sites is recruited by Cyclin T1. We found that in the presence of Tat, Cdk9 remained bound to nascent HIV-1 RNAs for 71s. In contrast, when transcription was activated by PMA/ionomycin, in the absence of Tat, Cdk9 turned-over rapidly and resided on the HIV-1 promoter for only 11s. Thus, the mechanism of trans-activation determines the residency time of P-TEFb at the HIV-1 gene, possibly explaining why Tat is such a potent transcriptional activator. In addition, we observed that Tat occupied HIV-1 transcription sites for 55s, suggesting that the TAR:Tat:P-TEFb complex dissociates from the polymerase following transcription initiation, and undergoes subsequent cycles of association/dissociation.

The Mediator complex: a central integrator of transcription

Science.gov (United States)

Allen, Benjamin L.; Taatjes, Dylan J.

2016-01-01

The RNA polymerase II (pol II) enzyme transcribes all protein-coding and most non-coding RNA genes and is globally regulated by Mediator, a large, conformationally flexible protein complex with variable subunit composition (for example, a four-subunit CDK8 module can reversibly associate). These biochemical characteristics are fundamentally important for Mediator's ability to control various processes important for transcription, including organization of chromatin architecture and regulation of pol II pre-initiation, initiation, re-initiation, pausing, and elongation. Although Mediator exists in all eukaryotes, a variety of Mediator functions appear to be specific to metazoans, indicative of more diverse regulatory requirements. PMID:25693131

Transcriptional Analysis of the bgIP Gene from Streptococcus mutans

Science.gov (United States)

2006-04-21

bglP gene from Streptococcus mutans Christopher K Cote1,2 and Allen L Honeyman*1,3 Address: 1Department of Medical Microbiology and Immunology... Streptococcus mutans . A potential ribonucleic antitermination (RAT) site to which the LicT protein would potentially bind has been identified immediately...longisporum [3], Lactobacillus plantarium [4], Bacillus subtilis [5,6], and Streptococcus mutans [7]. All of these organisms rely on the phosphoe

Translational Capacity of a Cell Is Determined during Transcription Elongation via the Ccr4-Not Complex

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Ishaan Gupta

2016-05-01

Full Text Available The current understanding of gene expression considers transcription and translation to be independent processes. Challenging this notion, we found that translation efficiency is determined during transcription elongation through the imprinting of mRNAs with Not1, the central scaffold of the Ccr4-Not complex. We determined that another subunit of the complex, Not5, defines Not1 binding to specific mRNAs, particularly those produced from ribosomal protein genes. This imprinting mechanism specifically regulates ribosomal protein gene expression, which in turn determines the translational capacity of cells. We validate our model by SILAC and polysome profiling experiments. As a proof of concept, we demonstrate that enhanced translation compensates for transcriptional elongation stress. Taken together, our data indicate that in addition to defining mRNA stability, components of the Ccr4-Not imprinting complex regulate RNA translatability, thus ensuring global gene expression homeostasis.

A structural model of the E. coli PhoB Dimer in the transcription initiation complex

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Tung Chang-Shung

2012-03-01

Full Text Available Abstract Background There exist > 78,000 proteins and/or nucleic acids structures that were determined experimentally. Only a small portion of these structures corresponds to those of protein complexes. While homology modeling is able to exploit knowledge-based potentials of side-chain rotomers and backbone motifs to infer structures for new proteins, no such general method exists to extend our understanding of protein interaction motifs to novel protein complexes. Results We use a Motif Binding Geometries (MBG approach, to infer the structure of a protein complex from the database of complexes of homologous proteins taken from other contexts (such as the helix-turn-helix motif binding double stranded DNA, and demonstrate its utility on one of the more important regulatory complexes in biology, that of the RNA polymerase initiating transcription under conditions of phosphate starvation. The modeled PhoB/RNAP/σ-factor/DNA complex is stereo-chemically reasonable, has sufficient interfacial Solvent Excluded Surface Areas (SESAs to provide adequate binding strength, is physically meaningful for transcription regulation, and is consistent with a variety of known experimental constraints. Conclusions Based on a straightforward and easy to comprehend concept, "proteins and protein domains that fold similarly could interact similarly", a structural model of the PhoB dimer in the transcription initiation complex has been developed. This approach could be extended to enable structural modeling and prediction of other bio-molecular complexes. Just as models of individual proteins provide insight into molecular recognition, catalytic mechanism, and substrate specificity, models of protein complexes will provide understanding into the combinatorial rules of cellular regulation and signaling.

Multiple promoters and alternative splicing: Hoxa5 transcriptional complexity in the mouse embryo.

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Yan Coulombe

2010-05-01

Full Text Available The genomic organization of Hox clusters is fundamental for the precise spatio-temporal regulation and the function of each Hox gene, and hence for correct embryo patterning. Multiple overlapping transcriptional units exist at the Hoxa5 locus reflecting the complexity of Hox clustering: a major form of 1.8 kb corresponding to the two characterized exons of the gene and polyadenylated RNA species of 5.0, 9.5 and 11.0 kb. This transcriptional intricacy raises the question of the involvement of the larger transcripts in Hox function and regulation.We have undertaken the molecular characterization of the Hoxa5 larger transcripts. They initiate from two highly conserved distal promoters, one corresponding to the putative Hoxa6 promoter, and a second located nearby Hoxa7. Alternative splicing is also involved in the generation of the different transcripts. No functional polyadenylation sequence was found at the Hoxa6 locus and all larger transcripts use the polyadenylation site of the Hoxa5 gene. Some larger transcripts are potential Hoxa6/Hoxa5 bicistronic units. However, even though all transcripts could produce the genuine 270 a.a. HOXA5 protein, only the 1.8 kb form is translated into the protein, indicative of its essential role in Hoxa5 gene function. The Hoxa6 mutation disrupts the larger transcripts without major phenotypic impact on axial specification in their expression domain. However, Hoxa5-like skeletal anomalies are observed in Hoxa6 mutants and these defects can be explained by the loss of expression of the 1.8 kb transcript. Our data raise the possibility that the larger transcripts may be involved in Hoxa5 gene regulation.Our observation that the Hoxa5 larger transcripts possess a developmentally-regulated expression combined to the increasing sum of data on the role of long noncoding RNAs in transcriptional regulation suggest that the Hoxa5 larger transcripts may participate in the control of Hox gene expression.

Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling

Science.gov (United States)

Honda, Kenya; Yanai, Hideyuki; Mizutani, Tatsuaki; Negishi, Hideo; Shimada, Naoya; Suzuki, Nobutaka; Ohba, Yusuke; Takaoka, Akinori; Yeh, Wen-Chen; Taniguchi, Tadatsugu

2004-01-01

Toll-like receptor (TLR) activation is central to immunity, wherein the activation of the TLR9 subfamily members TLR9 and TLR7 results in the robust induction of type I IFNs (IFN-α/β) by means of the MyD88 adaptor protein. However, it remains unknown how the TLR signal “input” can be processed through MyD88 to “output” the induction of the IFN genes. Here, we demonstrate that the transcription factor IRF-7 interacts with MyD88 to form a complex in the cytoplasm. We provide evidence that this complex also involves IRAK4 and TRAF6 and provides the foundation for the TLR9-dependent activation of the IFN genes. The complex defined in this study represents an example of how the coupling of the signaling adaptor and effector kinase molecules together with the transcription factor regulate the processing of an extracellular signal to evoke its versatile downstream transcriptional events in a cell. Thus, we propose that this molecular complex may function as a cytoplasmic transductional-transcriptional processor. PMID:15492225

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

Science.gov (United States)

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

2015-11-01

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

Bacillus subtilis δ Factor Functions as a Transcriptional Regulator by Facilitating the Open Complex Formation.

Science.gov (United States)

Prajapati, Ranjit Kumar; Sengupta, Shreya; Rudra, Paulami; Mukhopadhyay, Jayanta

2016-01-15

Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2α, β, β', and ω. However, in many Gram-positive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called δ. For over three decades since its identification, it had been thought that δ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that δ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that δ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of δ. Performing biochemical and mutational analysis, we show that Bacillus subtilis δ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, δ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of δ. Using transcription assay, we show that δ-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that δ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the -35 promoter element for transcription activation suggests that δ functions as a transcriptional regulator. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation

OpenAIRE

Malik, Sohail; Roeder, Robert G.

2010-01-01

The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action ...

The herpes viral transcription factor ICP4 forms a novel DNA recognition complex

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Tunnicliffe, Richard B.; Lockhart-Cairns, Michael P.; Levy, Colin; Mould, A. Paul; Jowitt, Thomas A.; Sito, Hilary; Baldock, Clair; Sandri-Goldin, Rozanne M.

2017-01-01

Abstract The transcription factor ICP4 from herpes simplex virus has a central role in regulating the gene expression cascade which controls viral infection. Here we present the crystal structure of the functionally essential ICP4 DNA binding domain in complex with a segment from its own promoter, revealing a novel homo-dimeric fold. We also studied the complex in solution by small angle X-Ray scattering, nuclear magnetic resonance and surface-plasmon resonance which indicated that, in addition to the globular domain, a flanking intrinsically disordered region also recognizes DNA. Together the data provides a rationale for the bi-partite nature of the ICP4 DNA recognition consensus sequence as the globular and disordered regions bind synergistically to adjacent DNA motifs. Therefore in common with its eukaryotic host, the viral transcription factor ICP4 utilizes disordered regions to enhance the affinity and tune the specificity of DNA interactions in tandem with a globular domain. PMID:28505309

The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor.

Science.gov (United States)

Kim, Mi Jung; Jang, In-Cheol; Chua, Nam-Hai

2016-07-01

The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15 However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

Characterisation of major histocompatibility complex class I transcripts in an Australian dragon lizard.

Science.gov (United States)

Hacking, Jessica; Bertozzi, Terry; Moussalli, Adnan; Bradford, Tessa; Gardner, Michael

2018-07-01

Characterisation of squamate major histocompatibility complex (MHC) genes has lagged behind other taxonomic groups. MHC genes encode cell-surface glycoproteins that present self- and pathogen-derived peptides to T cells and play a critical role in pathogen recognition. Here we characterise MHC class I transcripts for an agamid lizard (Ctenophorus decresii) and investigate the evolution of MHC class I in Iguanian lizards. An iterative assembly strategy was used to identify six full-length C. decresii MHC class I transcripts, which were validated as likely to encode classical class I MHC molecules. Evidence for exon shuffling recombination was uncovered for C. decresii transcripts and Bayesian phylogenetic analysis of Iguanian MHC class I sequences revealed a pattern expected under a birth-and-death mode of evolution. This work provides a stepping stone towards further research on the agamid MHC class I region. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Tax oncogene enhances ELL incorporation into p300 and P-TEFb containing protein complexes to activate transcription.

Science.gov (United States)

Fufa, Temesgen D; Byun, Jung S; Wakano, Clay; Fernandez, Alfonso G; Pise-Masison, Cynthia A; Gardner, Kevin

2015-09-11

The eleven-nineteen lysine-rich leukemia protein (ELL) is a key regulator of RNA polymerase II mediated transcription. ELL facilitates RNA polymerase II transcription pause site entry and release by dynamically interacting with p300 and the positive transcription elongation factor b (P-TEFb). In this study, we investigated the role of ELL during the HTLV-1 Tax oncogene induced transactivation. We show that ectopic expression of Tax enhances ELL incorporation into p300 and P-TEFb containing transcriptional complexes and the subsequent recruitment of these complexes to target genes in vivo. Depletion of ELL abrogates Tax induced transactivation of the immediate early genes Fos, Egr2 and NF-kB, suggesting that ELL is an essential cellular cofactor of the Tax oncogene. Thus, our study identifies a novel mechanism of ELL-dependent transactivation of immediate early genes by Tax and provides the rational for further defining the genome-wide targets of Tax and ELL. Published by Elsevier Inc.

A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

KAUST Repository

Law, Julie A.; Ausí n, Israel; Johnson, Lianna M.; Vashisht, Ajay  A Amar; Zhu, Jian-Kang; Wohlschlegel, James  A A.; Jacobsen, Steven E.

2010-01-01

DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

KAUST Repository

Law, Julie A.

2010-05-01

DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo.

Science.gov (United States)

Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

2017-07-12

The Mediator complex has been described as a general transcription factor, but it is unclear if it is essential for Pol II transcription and/or is a required component of the preinitiation complex (PIC) in vivo. Here, we show that depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in transcription. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in transcription. Depletion of head or middle subunits, but not tail subunits, causes a downstream shift in the Pol II occupancy profile, suggesting that Mediator at the core promoter inhibits promoter escape. Interestingly, a functional PIC and Pol II transcription can occur when Mediator is not detected at core promoters. These results provide strong evidence that Mediator is essential for Pol II transcription and stimulates PIC formation, but it is not a required component of the PIC in vivo.

The Complex Transcriptional Response of Acaryochloris marina to Different Oxygen Levels

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Miguel A. Hernández-Prieto

2017-02-01

Full Text Available Ancient oxygenic photosynthetic prokaryotes produced oxygen as a waste product, but existed for a long time under an oxygen-free (anoxic atmosphere, before an oxic atmosphere emerged. The change in oxygen levels in the atmosphere influenced the chemistry and structure of many enzymes that contained prosthetic groups that were inactivated by oxygen. In the genome of Acaryochloris marina, multiple gene copies exist for proteins that are normally encoded by a single gene copy in other cyanobacteria. Using high throughput RNA sequencing to profile transcriptome responses from cells grown under microoxic and hyperoxic conditions, we detected 8446 transcripts out of the 8462 annotated genes in the Cyanobase database. Two-thirds of the 50 most abundant transcripts are key proteins in photosynthesis. Microoxic conditions negatively affected the levels of expression of genes encoding photosynthetic complexes, with the exception of some subunits. In addition to the known regulation of the multiple copies of psbA, we detected a similar transcriptional pattern for psbJ and psbU, which might play a key role in the altered components of photosystem II. Furthermore, regulation of genes encoding proteins important for reactive oxygen species-scavenging is discussed at genome level, including, for the first time, specific small RNAs having possible regulatory roles under varying oxygen levels.

The Complex Transcriptional Response of Acaryochloris marina to Different Oxygen Levels

Science.gov (United States)

Hernández-Prieto, Miguel A.; Lin, Yuankui; Chen, Min

2016-01-01

Ancient oxygenic photosynthetic prokaryotes produced oxygen as a waste product, but existed for a long time under an oxygen-free (anoxic) atmosphere, before an oxic atmosphere emerged. The change in oxygen levels in the atmosphere influenced the chemistry and structure of many enzymes that contained prosthetic groups that were inactivated by oxygen. In the genome of Acaryochloris marina, multiple gene copies exist for proteins that are normally encoded by a single gene copy in other cyanobacteria. Using high throughput RNA sequencing to profile transcriptome responses from cells grown under microoxic and hyperoxic conditions, we detected 8446 transcripts out of the 8462 annotated genes in the Cyanobase database. Two-thirds of the 50 most abundant transcripts are key proteins in photosynthesis. Microoxic conditions negatively affected the levels of expression of genes encoding photosynthetic complexes, with the exception of some subunits. In addition to the known regulation of the multiple copies of psbA, we detected a similar transcriptional pattern for psbJ and psbU, which might play a key role in the altered components of photosystem II. Furthermore, regulation of genes encoding proteins important for reactive oxygen species-scavenging is discussed at genome level, including, for the first time, specific small RNAs having possible regulatory roles under varying oxygen levels. PMID:27974439

Pleiotropy constrains the evolution of protein but not regulatory sequences in a transcription regulatory network influencing complex social behaviours

Directory of Open Access Journals (Sweden)

Daria eMolodtsova

2014-12-01

Full Text Available It is increasingly apparent that genes and networks that influence complex behaviour are evolutionary conserved, which is paradoxical considering that behaviour is labile over evolutionary timescales. How does adaptive change in behaviour arise if behaviour is controlled by conserved, pleiotropic, and likely evolutionary constrained genes? Pleiotropy and connectedness are known to constrain the general rate of protein evolution, prompting some to suggest that the evolution of complex traits, including behaviour, is fuelled by regulatory sequence evolution. However, we seldom have data on the strength of selection on mutations in coding and regulatory sequences, and this hinders our ability to study how pleiotropy influences coding and regulatory sequence evolution. Here we use population genomics to estimate the strength of selection on coding and regulatory mutations for a transcriptional regulatory network that influences complex behaviour of honey bees. We found that replacement mutations in highly connected transcription factors and target genes experience significantly stronger negative selection relative to weakly connected transcription factors and targets. Adaptively evolving proteins were significantly more likely to reside at the periphery of the regulatory network, while proteins with signs of negative selection were near the core of the network. Interestingly, connectedness and network structure had minimal influence on the strength of selection on putative regulatory sequences for both transcription factors and their targets. Our study indicates that adaptive evolution of complex behaviour can arise because of positive selection on protein-coding mutations in peripheral genes, and on regulatory sequence mutations in both transcription factors and their targets throughout the network.

Genome-Wide Phylogenetic Comparative Analysis of Plant Transcriptional Regulation: A Timeline of Loss, Gain, Expansion, and Correlation with Complexity

OpenAIRE

Lang, Daniel; Weiche, Benjamin; Timmerhaus, Gerrit; Richardt, Sandra; Ria?o-Pach?n, Diego M.; Corr?a, Luiz G. G.; Reski, Ralf; Mueller-Roeber, Bernd; Rensing, Stefan A.

2010-01-01

Evolutionary retention of duplicated genes encoding transcription-associated proteins (TAPs, comprising transcription factors and other transcriptional regulators) has been hypothesized to be positively correlated with increasing morphological complexity and paleopolyploidizations, especially within the plant kingdom. Here, we present the most comprehensive set of classification rules for TAPs and its application for genome-wide analyses of plants and algae. Using a dated species tree and phy...

Transcriptional regulation by competing transcription factor modules.

Directory of Open Access Journals (Sweden)

Rutger Hermsen

2006-12-01

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

Mediator, SWI/SNF and SAGA complexes regulate Yap8-dependent transcriptional activation of ACR2 in response to arsenate.

Science.gov (United States)

Menezes, Regina Andrade; Pimentel, Catarina; Silva, Ana Rita Courelas; Amaral, Catarina; Merhej, Jawad; Devaux, Frédéric; Rodrigues-Pousada, Claudina

2017-04-01

Response to arsenic stress in Saccharomyces cerevisiae is orchestrated by the regulatory protein Yap8, which mediates transcriptional activation of ACR2 and ACR3. This study contributes to the state of art knowledge of the molecular mechanisms underlying yeast stress response to arsenate as it provides the genetic and biochemical evidences that Yap8, through cysteine residues 132, 137, and 274, is the sensor of presence of arsenate in the cytosol. Moreover, it is here reported for the first time the essential role of the Mediator complex in the transcriptional activation of ACR2 by Yap8. Based on our data, we propose an order-of-function map to recapitulate the sequence of events taking place in cells injured with arsenate. Modification of the sulfhydryl state of these cysteines converts Yap8 in its activated form, triggering the recruitment of the Mediator complex to the ACR2/ACR3 promoter, through the interaction with the tail subunit Med2. The Mediator complex then transfers the regulatory signals conveyed by Yap8 to the core transcriptional machinery, which culminates with TBP occupancy, ACR2 upregulation and cell adaptation to arsenate stress. Additional co-factors are required for the transcriptional activation of ACR2 by Yap8, particularly the nucleosome remodeling activity of SWI/SNF and SAGA complexes. Copyright © 2017. Published by Elsevier B.V.

Simplified Method for Predicting a Functional Class of Proteins in Transcription Factor Complexes

KAUST Repository

Piatek, Marek J.

2013-07-12

Background:Initiation of transcription is essential for most of the cellular responses to environmental conditions and for cell and tissue specificity. This process is regulated through numerous proteins, their ligands and mutual interactions, as well as interactions with DNA. The key such regulatory proteins are transcription factors (TFs) and transcription co-factors (TcoFs). TcoFs are important since they modulate the transcription initiation process through interaction with TFs. In eukaryotes, transcription requires that TFs form different protein complexes with various nuclear proteins. To better understand transcription regulation, it is important to know the functional class of proteins interacting with TFs during transcription initiation. Such information is not fully available, since not all proteins that act as TFs or TcoFs are yet annotated as such, due to generally partial functional annotation of proteins. In this study we have developed a method to predict, using only sequence composition of the interacting proteins, the functional class of human TF binding partners to be (i) TF, (ii) TcoF, or (iii) other nuclear protein. This allows for complementing the annotation of the currently known pool of nuclear proteins. Since only the knowledge of protein sequences is required in addition to protein interaction, the method should be easily applicable to many species.Results:Based on experimentally validated interactions between human TFs with different TFs, TcoFs and other nuclear proteins, our two classification systems (implemented as a web-based application) achieve high accuracies in distinguishing TFs and TcoFs from other nuclear proteins, and TFs from TcoFs respectively.Conclusion:As demonstrated, given the fact that two proteins are capable of forming direct physical interactions and using only information about their sequence composition, we have developed a completely new method for predicting a functional class of TF interacting protein partners

Modular Organization of the NusA- and NusG-Stimulated RNA Polymerase Pause Signal That Participates in the Bacillus subtilis trp Operon Attenuation Mechanism.

Science.gov (United States)

Mondal, Smarajit; Yakhnin, Alexander V; Babitzke, Paul

2017-07-15

The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to the nascent transcript and blocks the formation of an antiterminator structure such that the formation of an overlapping intrinsic terminator causes termination in the 5' untranslated region (5' UTR). In the absence of bound TRAP, the antiterminator forms and transcription continues into the trp genes. RNA polymerase pauses at positions U107 and U144 in the 5' UTR. The general transcription elongation factors NusA and NusG stimulate pausing at both positions. NusG-stimulated pausing at U144 requires sequence-specific contacts with a T tract in the nontemplate DNA (ntDNA) strand within the paused transcription bubble. Pausing at U144 participates in a trpE translation repression mechanism. Since U107 just precedes the critical overlap between the antiterminator and terminator structures, pausing at this position is thought to participate in attenuation. Here we carried out in vitro pausing and termination experiments to identify components of the U107 pause signal and to determine whether pausing affects the termination efficiency in the 5' UTR. We determined that the U107 and U144 pause signals are organized in a modular fashion containing distinct RNA hairpin, U-tract, and T-tract components. NusA-stimulated pausing was affected by hairpin strength and the U-tract sequence, whereas NusG-stimulated pausing was affected by hairpin strength and the T-tract sequence. We also determined that pausing at U107 results in increased TRAP-dependent termination in the 5' UTR, implying that NusA- and NusG-stimulated pausing participates in the trp operon attenuation mechanism by providing additional time for TRAP binding. IMPORTANCE The expression of several bacterial operons is controlled by regulated termination in the 5' untranslated region (5' UTR). Transcription attenuation is defined as situations in which the binding of a regulatory

Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures

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Hubin, Elizabeth A.; Lilic, Mirjana; Darst, Seth A.; Campbell, Elizabeth A.

2017-07-13

The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2 Å-resolution crystal structure of a Mycobacterium smegmatis (Msm) open promoter complex (RPo), along with structural analysis of the Msm RPo and a previously reported 2.76 Å-resolution crystal structure of an Msm transcription initiation complex with a promoter DNA fragment. We observe the interaction of the Msm RNAP α-subunit C-terminal domain (αCTD) with DNA, and we provide evidence that the αCTD may play a role in Mtb transcription regulation. Our results reveal the structure of an Actinobacteria-unique insert of the RNAP β' subunit. Finally, our analysis reveals the disposition of the N-terminal segment of Msm σA, which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with E. coli.

Core Mediator structure at 3.4 Å extends model of transcription initiation complex.

Science.gov (United States)

Nozawa, Kayo; Schneider, Thomas R; Cramer, Patrick

2017-05-11

Mediator is a multiprotein co-activator that binds the transcription pre-initiation complex (PIC) and regulates RNA polymerase (Pol) II. The Mediator head and middle modules form the essential core Mediator (cMed), whereas the tail and kinase modules play regulatory roles. The architecture of Mediator and its position on the PIC are known, but atomic details are limited to Mediator subcomplexes. Here we report the crystal structure of the 15-subunit cMed from Schizosaccharomyces pombe at 3.4 Å resolution. The structure shows an unaltered head module, and reveals the intricate middle module, which we show is globally required for transcription. Sites of known Mediator mutations cluster at the interface between the head and middle modules, and in terminal regions of the head subunits Med6 (ref. 16) and Med17 (ref. 17) that tether the middle module. The structure led to a model for Saccharomyces cerevisiae cMed that could be combined with the 3.6 Å cryo-electron microscopy structure of the core PIC (cPIC). The resulting atomic model of the cPIC-cMed complex informs on interactions of the submodules forming the middle module, called beam, knob, plank, connector, and hook. The hook is flexibly linked to Mediator by a conserved hinge and contacts the transcription initiation factor IIH (TFIIH) kinase that phosphorylates the carboxy (C)-terminal domain (CTD) of Pol II and was recently positioned on the PIC. The hook also contains residues that crosslink to the CTD and reside in a previously described cradle. These results provide a framework for understanding Mediator function, including its role in stimulating CTD phosphorylation by TFIIH.

Electrostatic study of Alanine mutational effects on transcription: application to GATA-3:DNA interaction complex.

Science.gov (United States)

El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges

2015-01-01

Protein-DNA interaction is of fundamental importance in molecular biology, playing roles in functions as diverse as DNA transcription, DNA structure formation, and DNA repair. Protein-DNA association is also important in medicine; understanding Protein-DNA binding kinetics can assist in identifying disease root causes which can contribute to drug development. In this perspective, this work focuses on the transcription process by the GATA Transcription Factor (TF). GATA TF binds to DNA promoter region represented by `G,A,T,A' nucleotides sequence, and initiates transcription of target genes. When proper regulation fails due to some mutations on the GATA TF protein sequence or on the DNA promoter sequence (weak promoter), deregulation of the target genes might lead to various disorders. In this study, we aim to understand the electrostatic mechanism behind GATA TF and DNA promoter interactions, in order to predict Protein-DNA binding in the presence of mutations, while elaborating on non-covalent binding kinetics. To generate a family of mutants for the GATA:DNA complex, we replaced every charged amino acid, one at a time, with a neutral amino acid like Alanine (Ala). We then applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations, for each mutation. These calculations delineate the contribution to binding from each Ala-replaced amino acid in the GATA:DNA interaction. After analyzing the obtained data in view of a two-step model, we are able to identify potential key amino acids in binding. Finally, we applied the model to GATA-3:DNA (crystal structure with PDB-ID: 3DFV) binding complex and validated it against experimental results from the literature.

Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes

LENUS (Irish Health Repository)

Whan, Vicki

2010-11-23

Abstract Background About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q) tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. Results The current investigation identified 178 bovine poly-Q encoding genes (Q ≥ 5) and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was identified

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

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

2017-04-10

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

Complex Interdependence Regulates Heterotypic Transcription Factor Distribution and Coordinates Cardiogenesis.

Science.gov (United States)

Luna-Zurita, Luis; Stirnimann, Christian U; Glatt, Sebastian; Kaynak, Bogac L; Thomas, Sean; Baudin, Florence; Samee, Md Abul Hassan; He, Daniel; Small, Eric M; Mileikovsky, Maria; Nagy, Andras; Holloway, Alisha K; Pollard, Katherine S; Müller, Christoph W; Bruneau, Benoit G

2016-02-25

Transcription factors (TFs) are thought to function with partners to achieve specificity and precise quantitative outputs. In the developing heart, heterotypic TF interactions, such as between the T-box TF TBX5 and the homeodomain TF NKX2-5, have been proposed as a mechanism for human congenital heart defects. We report extensive and complex interdependent genomic occupancy of TBX5, NKX2-5, and the zinc finger TF GATA4 coordinately controlling cardiac gene expression, differentiation, and morphogenesis. Interdependent binding serves not only to co-regulate gene expression but also to prevent TFs from distributing to ectopic loci and activate lineage-inappropriate genes. We define preferential motif arrangements for TBX5 and NKX2-5 cooperative binding sites, supported at the atomic level by their co-crystal structure bound to DNA, revealing a direct interaction between the two factors and induced DNA bending. Complex interdependent binding mechanisms reveal tightly regulated TF genomic distribution and define a combinatorial logic for heterotypic TF regulation of differentiation. Copyright © 2016 Elsevier Inc. All rights reserved.

RNA-Interference Components Are Dispensable for Transcriptional Silencing of the Drosophila Bithorax-Complex

KAUST Repository

Cernilogar, Filippo M.

2013-06-13

Background:Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated.Principal Findings:Here we show that the endogenous Dicer-2/Argonaute-2 RNAi pathway is dispensable for the PcG mediated silencing of the homeotic Bithorax Complex (BX-C). Although Dicer-2 depletion triggers mild transcriptional activation at Polycomb Response Elements (PREs), this does not induce transcriptional changes at PcG-repressed genes. Moreover, Dicer-2 is not needed to maintain global levels of methylation of lysine 27 of histone H3 and does not affect PRE-mediated higher order chromatin structures within the BX-C. Finally bioinformatic analysis, comparing published data sets of PcG targets with Argonaute-2-bound small RNAs reveals no enrichment of these small RNAs at promoter regions associated with PcG proteins.Conclusions:We conclude that the Dicer-2/Argonaute-2 RNAi pathway, despite its role in pairing sensitive gene silencing of transgenes, does not have a role in PcG dependent silencing of major homeotic gene cluster loci in Drosophila. © 2013 Cernilogar et al.

A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts.

Science.gov (United States)

Fox, Hannah L; Dembowski, Jill A; DeLuca, Neal A

2017-06-13

Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (RNA Pol II). While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22) function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16) was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq). The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq), we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production. IMPORTANCE HSV-1 interacts with many cellular proteins throughout productive infection. Here, we demonstrate the interaction of a viral protein, ICP22, with a subset of cellular proteins known to be involved in transcription elongation. We determined that ICP22 is required to recruit the FACT complex and other transcription

The transcriptional landscape

DEFF Research Database (Denmark)

Nielsen, Henrik

2011-01-01

The application of new and less biased methods to study the transcriptional output from genomes, such as tiling arrays and deep sequencing, has revealed that most of the genome is transcribed and that there is substantial overlap of transcripts derived from the two strands of DNA. In protein coding...... regions, the map of transcripts is very complex due to small transcripts from the flanking ends of the transcription unit, the use of multiple start and stop sites for the main transcript, production of multiple functional RNA molecules from the same primary transcript, and RNA molecules made...... by independent transcription from within the unit. In genomic regions separating those that encode proteins or highly abundant RNA molecules with known function, transcripts are generally of low abundance and short-lived. In most of these cases, it is unclear to what extent a function is related to transcription...

Transcription factor 19 interacts with histone 3 lysine 4 trimethylation and controls gluconeogenesis via the nucleosome-remodeling-deacetylase complex.

Science.gov (United States)

Sen, Sabyasachi; Sanyal, Sulagna; Srivastava, Dushyant Kumar; Dasgupta, Dipak; Roy, Siddhartha; Das, Chandrima

2017-12-15

Transcription factor 19 (TCF19) has been reported as a type 1 diabetes-associated locus involved in maintenance of pancreatic β cells through a fine-tuned regulation of cell proliferation and apoptosis. TCF19 also exhibits genomic association with type 2 diabetes, although the precise molecular mechanism remains unknown. It harbors both a plant homeodomain and a forkhead-associated domain implicated in epigenetic recognition and gene regulation, a phenomenon that has remained unexplored. Here, we show that TCF19 selectively interacts with histone 3 lysine 4 trimethylation through its plant homeodomain finger. Knocking down TCF19 under high-glucose conditions affected many metabolic processes, including gluconeogenesis. We found that TCF19 overexpression represses de novo glucose production in HepG2 cells. The transcriptional repression of key genes, induced by TCF19, coincided with NuRD (nucleosome-remodeling-deacetylase) complex recruitment to the promoters of these genes. TCF19 interacted with CHD4 (chromodomain helicase DNA-binding protein 4), which is a part of the NuRD complex, in a glucose concentration-independent manner. In summary, our results show that TCF19 interacts with an active transcription mark and recruits a co-repressor complex to regulate gluconeogenic gene expression in HepG2 cells. Our study offers critical insights into the molecular mechanisms of transcriptional regulation of gluconeogenesis and into the roles of chromatin readers in metabolic homeostasis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Crystallization and preliminary crystallographic analysis of the transcriptional regulator RfaH from Escherichia coli and its complex with ops DNA

International Nuclear Information System (INIS)

Vassylyeva, Marina N.; Svetlov, Vladimir; Klyuyev, Sergiy; Devedjiev, Yancho D.; Artsimovitch, Irina; Vassylyev, Dmitry G.

2006-01-01

The E. coli transcriptional regulator RfaH was cloned, expressed, purified and crystallized and the complex of RfaH with its target DNA oligonucleotide was cocrystallized. Complete diffraction data sets were collected for the apo protein and its nucleic acid complex at 2.4 and at 1.6 Å resolution, respectively. The bacterial transcriptional factor and virulence regulator RfaH binds to rapidly moving transcription elongation complexes through specific interactions with the exposed segment of the non-template DNA strand. To elucidate this unusual mechanism of recruitment, determination of the three-dimensional structure of RfaH and its complex with DNA was initiated. To this end, the Escherichia coli rfaH gene was cloned and expressed. The purified protein was crystallized by the sitting-drop vapor-diffusion technique. The space group was P6 1 22 or P6 5 22, with unit-cell parameters a = b = 45.46, c = 599.93 Å. A complex of RfaH and a nine-nucleotide oligodeoxyribonucleotide was crystallized by the same technique, but under different crystallization conditions, yielding crystals that belonged to space group P1 (unit-cell parameters a = 36.79, b = 44.01, c = 62.37 Å, α = 80.62, β = 75.37, γ = 75.41°). Complete diffraction data sets were collected for RfaH and its complex with DNA at 2.4 and 1.6 Å resolution, respectively. Crystals of selenomethionine-labeled proteins in both crystal forms were obtained by cross-microseeding using the native microcrystals. The structure determination of RfaH and its complex with DNA is in progress

Structure-aided prediction of mammalian transcription factor complexes in conserved non-coding elements

KAUST Repository

Guturu, H.

2013-11-11

Mapping the DNA-binding preferences of transcription factor (TF) complexes is critical for deciphering the functions of cis-regulatory elements. Here, we developed a computational method that compares co-occurring motif spacings in conserved versus unconserved regions of the human genome to detect evolutionarily constrained binding sites of rigid TF complexes. Structural data were used to estimate TF complex physical plausibility, explore overlapping motif arrangements seldom tackled by non-structure-aware methods, and generate and analyse three-dimensional models of the predicted complexes bound to DNA. Using this approach, we predicted 422 physically realistic TF complex motifs at 18% false discovery rate, the majority of which (326, 77%) contain some sequence overlap between binding sites. The set of mostly novel complexes is enriched in known composite motifs, predictive of binding site configurations in TF-TF-DNA crystal structures, and supported by ChIP-seq datasets. Structural modelling revealed three cooperativity mechanisms: direct protein-protein interactions, potentially indirect interactions and \\'through-DNA\\' interactions. Indeed, 38% of the predicted complexes were found to contain four or more bases in which TF pairs appear to synergize through overlapping binding to the same DNA base pairs in opposite grooves or strands. Our TF complex and associated binding site predictions are available as a web resource at http://bejerano.stanford.edu/complex.

Structure-aided prediction of mammalian transcription factor complexes in conserved non-coding elements

KAUST Repository

Guturu, H.; Doxey, A. C.; Wenger, A. M.; Bejerano, G.

2013-01-01

Mapping the DNA-binding preferences of transcription factor (TF) complexes is critical for deciphering the functions of cis-regulatory elements. Here, we developed a computational method that compares co-occurring motif spacings in conserved versus unconserved regions of the human genome to detect evolutionarily constrained binding sites of rigid TF complexes. Structural data were used to estimate TF complex physical plausibility, explore overlapping motif arrangements seldom tackled by non-structure-aware methods, and generate and analyse three-dimensional models of the predicted complexes bound to DNA. Using this approach, we predicted 422 physically realistic TF complex motifs at 18% false discovery rate, the majority of which (326, 77%) contain some sequence overlap between binding sites. The set of mostly novel complexes is enriched in known composite motifs, predictive of binding site configurations in TF-TF-DNA crystal structures, and supported by ChIP-seq datasets. Structural modelling revealed three cooperativity mechanisms: direct protein-protein interactions, potentially indirect interactions and 'through-DNA' interactions. Indeed, 38% of the predicted complexes were found to contain four or more bases in which TF pairs appear to synergize through overlapping binding to the same DNA base pairs in opposite grooves or strands. Our TF complex and associated binding site predictions are available as a web resource at http://bejerano.stanford.edu/complex.

Core Transcriptional Regulatory Circuit Controlled by the TAL1 Complex in Human T Cell Acute Lymphoblastic Leukemia

OpenAIRE

Sanda, Takaomi; Lawton, Lee N.; Barrasa, M. Inmaculada; Fan, Zi Peng; Kohlhammer, Holger; Gutierrez, Alejandro; Ma, Wenxue; Tatarek, Jessica; Ahn, Yebin; Kelliher, Michelle A.; Jamieson, Catriona H.M.; Staudt, Louis M.; Young, Richard A.; Look, A. Thomas

2012-01-01

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T-cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3 and RUNX1. We show that TAL1 forms a positive interconnected auto-regulatory loop with GATA3 and RUNX1, and that the TAL1 complex directly activates the MY...

Engagement of Components of DNA-Break Repair Complex and NFκB in Hsp70A1A Transcription Upregulation by Heat Shock.

Science.gov (United States)

Hazra, Joyita; Mukherjee, Pooja; Ali, Asif; Poddar, Soumita; Pal, Mahadeb

2017-01-01

An involvement of components of DNA-break repair (DBR) complex including DNA-dependent protein kinase (DNA-PK) and poly-ADP-ribose polymerase 1 (PARP-1) in transcription regulation in response to distinct cellular signalling has been revealed by different laboratories. Here, we explored the involvement of DNA-PK and PARP-1 in the heat shock induced transcription of Hsp70A1A. We find that inhibition of both the catalytic subunit of DNA-PK (DNA-PKc), and Ku70, a regulatory subunit of DNA-PK holo-enzyme compromises transcription of Hsp70A1A under heat shock treatment. In immunoprecipitation based experiments we find that Ku70 or DNA-PK holoenzyme associates with NFκB. This NFκB associated complex also carries PARP-1. Downregulation of both NFκB and PARP-1 compromises Hsp70A1A transcription induced by heat shock treatment. Alteration of three bases by site directed mutagenesis within the consensus κB sequence motif identified on the promoter affected inducibility of Hsp70A1A transcription by heat shock treatment. These results suggest that NFκB engaged with the κB motif on the promoter cooperates in Hsp70A1A activation under heat shock in human cells as part of a DBR complex including DNA-PK and PARP-1.

Structure and functional analysis of the RNA- and viral phosphoprotein-binding domain of respiratory syncytial virus M2-1 protein.

Directory of Open Access Journals (Sweden)

Marie-Lise Blondot

Full Text Available Respiratory syncytial virus (RSV protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-1(58-177 core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-1(58-177, as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1.

Polycomb group protein-mediated repression of transcription

DEFF Research Database (Denmark)

Morey, Lluís; Helin, Kristian

2010-01-01

The polycomb group (PcG) proteins are essential for the normal development of multicellular organisms. They form multi-protein complexes that work as transcriptional repressors of several thousand genes controlling differentiation pathways during development. How the PcG proteins work as transcri......The polycomb group (PcG) proteins are essential for the normal development of multicellular organisms. They form multi-protein complexes that work as transcriptional repressors of several thousand genes controlling differentiation pathways during development. How the PcG proteins work...... as transcriptional repressors is incompletely understood, but involves post-translational modifications of histones by two major PcG protein complexes: polycomb repressive complex 1 and polycomb repressive complex 2....

A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions

Science.gov (United States)

Khalil, Ahmad S.; Lu, Timothy K.; Bashor, Caleb J.; Ramirez, Cherie L.; Pyenson, Nora C.; Joung, J. Keith; Collins, James J.

2013-01-01

SUMMARY Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks. PMID:22863014

The Mediator Complex: At the Nexus of RNA Polymerase II Transcription.

Science.gov (United States)

Jeronimo, Célia; Robert, François

2017-10-01

Mediator is an essential, large, multisubunit, transcriptional co-activator highly conserved across eukaryotes. Mediator interacts with gene-specific transcription factors at enhancers as well as with the RNA polymerase II (RNAPII) transcription machinery bound at promoters. It also interacts with several other factors involved in various aspects of transcription, chromatin regulation, and mRNA processing. Hence, Mediator is at the nexus of RNAPII transcription, regulating its many steps and connecting transcription with co-transcriptional events. To achieve this flexible role, Mediator, which is divided into several functional modules, reorganizes its conformation and composition while making transient contacts with other components. Here, we review the mechanisms of action of Mediator and propose a unifying model for its function. Copyright © 2017 Elsevier Ltd. All rights reserved.

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription.

Science.gov (United States)

Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien; Pira, Dorcas; Angrand, Pierre-Olivier

2012-05-01

Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation.

Family values in the age of genomics: comparative analyses of temperate bacteriophage HK022.

Science.gov (United States)

Weisberg, R A; Gottesmann, M E; Hendrix, R W; Little, J W

1999-01-01

HK022 is a temperate coliphage related to phage lambda. Its chromosome has been completely sequenced, and several aspects of its life cycle have been intensively studied. In the overall arrangement, expression, and function of most of its genes, HK022 broadly resembles lambda and other members of the lambda family. Upon closer view, significant differences emerge. The differences reveal alternative strategies used by related phages to cope with similar problems and illuminate previously unknown regulatory and structural motifs. HK022 prophages protect lysogens from superinfection by producing a sequence-specific RNA binding protein that prematurely terminates nascent transcripts of infecting phage. It uses a novel RNA-based mechanism to antiterminate its own early transcription. The HK022 protein shell is strengthened by a complex pattern of covalent subunit interlinking to form a unitary structure that resembles chain-mail armour. Its integrase and repressor proteins are similar to those of lambda, but the differences provide insights into the evolution of biological specificity and the elements needed for construction of a stable genetic switch.

A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts

Directory of Open Access Journals (Sweden)

Hannah L. Fox

2017-06-01

Full Text Available Herpes simplex virus 1 (HSV-1 genes are transcribed by cellular RNA polymerase II (RNA Pol II. While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22 function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16 was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq. The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq, we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production.

Complex and extensive post-transcriptional regulation revealed by integrative proteomic and transcriptomic analysis of metabolite stress response in Clostridium acetobutylicum.

Science.gov (United States)

Venkataramanan, Keerthi P; Min, Lie; Hou, Shuyu; Jones, Shawn W; Ralston, Matthew T; Lee, Kelvin H; Papoutsakis, E Terry

2015-01-01

Clostridium acetobutylicum is a model organism for both clostridial biology and solvent production. The organism is exposed to its own toxic metabolites butyrate and butanol, which trigger an adaptive stress response. Integrative analysis of proteomic and RNAseq data may provide novel insights into post-transcriptional regulation. The identified iTRAQ-based quantitative stress proteome is made up of 616 proteins with a 15 % genome coverage. The differentially expressed proteome correlated poorly with the corresponding differential RNAseq transcriptome. Up to 31 % of the differentially expressed proteins under stress displayed patterns opposite to those of the transcriptome, thus suggesting significant post-transcriptional regulation. The differential proteome of the translation machinery suggests that cells employ a different subset of ribosomal proteins under stress. Several highly upregulated proteins but with low mRNA levels possessed mRNAs with long 5'UTRs and strong RBS scores, thus supporting the argument that regulatory elements on the long 5'UTRs control their translation. For example, the oxidative stress response rubrerythrin was upregulated only at the protein level up to 40-fold without significant mRNA changes. We also identified many leaderless transcripts, several displaying different transcriptional start sites, thus suggesting mRNA-trimming mechanisms under stress. Downregulation of Rho and partner proteins pointed to changes in transcriptional elongation and termination under stress. The integrative proteomic-transcriptomic analysis demonstrated complex expression patterns of a large fraction of the proteome. Such patterns could not have been detected with one or the other omic analyses. Our analysis proposes the involvement of specific molecular mechanisms of post-transcriptional regulation to explain the observed complex stress response.

CRISPR-Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b

Science.gov (United States)

He, Fei; Vestergaard, Gisle; Peng, Wenfang; She, Qunxin

2017-01-01

Abstract CRISPR-Cas (clustered regularly interspaced short palindromic repeats and the associated genes) constitute adaptive immune systems in bacteria and archaea and they provide sequence specific immunity against foreign nucleic acids. CRISPR-Cas systems are activated by viral infection. However, little is known about how CRISPR-Cas systems are activated in response to viral infection or how their expression is controlled in the absence of viral infection. Here, we demonstrate that both the transcriptional regulator Csa3b, and the type I-A interference complex Cascade, are required to transcriptionally repress the interference gene cassette in the archaeon Sulfolobus. Csa3b binds to two palindromic repeat sites in the promoter region of the cassette and facilitates binding of the Cascade to the promoter region. Upon viral infection, loading of Cascade complexes onto crRNA-matching protospacers leads to relief of the transcriptional repression. Our data demonstrate a mechanism coupling CRISPR-Cas surveillance of protospacers to transcriptional regulation of the interference gene cassette thereby allowing a fast response to viral infection. PMID:27980065

Zipper-interacting protein kinase is involved in regulation of ubiquitination of the androgen receptor, thereby contributing to dynamic transcription complex assembly.

Science.gov (United States)

Felten, A; Brinckmann, D; Landsberg, G; Scheidtmann, K H

2013-10-10

We have recently identified apoptosis-antagonizing transcription factor (AATF), tumor-susceptibility gene 101 (TSG101) and zipper-interacting protein kinase (ZIPK) as novel coactivators of the androgen receptor (AR). The mechanisms of coactivation remained obscure, however. Here we investigated the interplay and interdependence between these coactivators and the AR using the endogenous prostate specific antigen (PSA) gene as model for AR-target genes. Chromatin immunoprecipitation in combination with siRNA-mediated knockdown revealed that recruitment of AATF and ZIPK to the PSA enhancer was dependent on AR, whereas recruitment of TSG101 was dependent on AATF. Association of AR and its coactivators with the PSA enhancer or promoter occurred in cycles. Dissociation of AR-transcription complexes was due to degradation because inhibition of the proteasome system by MG132 caused accumulation of AR at enhancer/promoter elements. Moreover, inhibition of degradation strongly reduced transcription, indicating that continued and efficient transcription is based on initiation, degradation and reinitiation cycles. Interestingly, knockdown of ZIPK by siRNA had a similar effect as MG132, leading to reduced transcription but enhanced accumulation of AR at androgen-response elements. In addition, knockdown of ZIPK, as well as overexpression of a dominant-negative ZIPK mutant, diminished polyubiquitination of AR. Furthermore, ZIPK cooperated with the E3 ligase Mdm2 in AR-dependent transactivation, assembled into a single complex on chromatin and phosphorylated Mdm2 in vitro. These results suggest that ZIPK has a crucial role in regulation of ubiquitination and degradation of the AR, and hence promoter clearance and efficient transcription.

Genetic control of nitrate assimilation in Klebsiella oxytoca. Final technical report; FINAL

International Nuclear Information System (INIS)

Stewart, Valley J.

2001-01-01

Some microorganisms can use nitrate as the sole source of nitrogen for biosynthesis. This project focused on the bacterium Klebsiella oxytoca, an enterobacterium found in soil and water. Mutagenesis and molecular cloning identified the nasFEDCBA operon encoding enzymes for the uptake and reduction of nitrate and nitrite to ammonium, and the adjacent nasR regulatory gene. Analysis of nasF operon expression revealed that transcription is activated by the Ntr (general nitrogen regulation ) system in response to nitrogen limitation. Transcription antitermination control in response to nitrate and nitrite is mediated by the NasR protein. Additional work established that the NasR protein is an RNA-binding protein that interacts with nasF operon leader RNA to control transcription readthrough

Functional organization of cytoplasmic inclusion bodies in cells infected by respiratory syncytial virus.

Science.gov (United States)

Rincheval, Vincent; Lelek, Mickael; Gault, Elyanne; Bouillier, Camille; Sitterlin, Delphine; Blouquit-Laye, Sabine; Galloux, Marie; Zimmer, Christophe; Eleouet, Jean-François; Rameix-Welti, Marie-Anne

2017-09-15

Infection of cells by respiratory syncytial virus induces the formation of cytoplasmic inclusion bodies (IBs) where all the components of the viral RNA polymerase complex are concentrated. However, the exact organization and function of these IBs remain unclear. In this study, we use conventional and super-resolution imaging to dissect the internal structure of IBs. We observe that newly synthetized viral mRNA and the viral transcription anti-terminator M2-1 concentrate in IB sub-compartments, which we term "IB-associated granules" (IBAGs). In contrast, viral genomic RNA, the nucleoprotein, the L polymerase and its cofactor P are excluded from IBAGs. Live imaging reveals that IBAGs are highly dynamic structures. Our data show that IBs are the main site of viral RNA synthesis. They further suggest that shortly after synthesis in IBs, viral mRNAs and M2-1 transiently concentrate in IBAGs before reaching the cytosol and suggest a novel post-transcriptional function for M2-1.Respiratory syncytial virus (RSV) induces formation of inclusion bodies (IBs) sheltering viral RNA synthesis. Here, Rincheval et al. identify highly dynamic IB-associated granules (IBAGs) that accumulate newly synthetized viral mRNA and the viral M2-1 protein but exclude viral genomic RNA and RNA polymerase complexes.

CRISPR-Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b.

Science.gov (United States)

He, Fei; Vestergaard, Gisle; Peng, Wenfang; She, Qunxin; Peng, Xu

2017-02-28

CRISPR-Cas (clustered regularly interspaced short palindromic repeats and the associated genes) constitute adaptive immune systems in bacteria and archaea and they provide sequence specific immunity against foreign nucleic acids. CRISPR-Cas systems are activated by viral infection. However, little is known about how CRISPR-Cas systems are activated in response to viral infection or how their expression is controlled in the absence of viral infection. Here, we demonstrate that both the transcriptional regulator Csa3b, and the type I-A interference complex Cascade, are required to transcriptionally repress the interference gene cassette in the archaeon Sulfolobus. Csa3b binds to two palindromic repeat sites in the promoter region of the cassette and facilitates binding of the Cascade to the promoter region. Upon viral infection, loading of Cascade complexes onto crRNA-matching protospacers leads to relief of the transcriptional repression. Our data demonstrate a mechanism coupling CRISPR-Cas surveillance of protospacers to transcriptional regulation of the interference gene cassette thereby allowing a fast response to viral infection. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Transcriptional intermediary factor 1γ binds to the anaphase-promoting complex/cyclosome and promotes mitosis

DEFF Research Database (Denmark)

Sedgwick, G.G.; Townsend, K.; Martin, A.

2013-01-01

The anaphase-promoting complex/cyclosome (APC/C) is an ubiquitin ligase that functions during mitosis. Here we identify the transcriptional regulator, transcriptional intermediary factor 1γ, TIF1γ, as an APC/C-interacting protein that regulates APC/C function. TIF1γ is not a substrate for APC....../C-dependent ubiquitylation but instead, associates specifically with the APC/C holoenzyme and Cdc20 to affect APC/C activity and progression through mitosis. RNA interference studies indicate that TIF1γ knockdown results in a specific reduction in APC/C ubiquitin ligase activity, the stabilization of APC/C substrates......, and an increase in the time taken for cells to progress through mitosis from nuclear envelope breakdown to anaphase. TIF1γ knockdown cells are also characterized by the inappropriate presence of cyclin A at metaphase, and an increase in the number of cells that fail to undergo metaphase-to-anaphase transition...

HYPER RECOMBINATION1 of the THO/TREX complex plays a role in controlling transcription of the REVERSION-TO-ETHYLENE SENSITIVITY1 gene in Arabidopsis.

Directory of Open Access Journals (Sweden)

Congyao Xu

2015-02-01

Full Text Available Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1 represses ethylene hormone responses by promoting ethylene receptor ETHYLENE RESPONSE1 (ETR1 signaling, which negatively regulates ethylene responses. To investigate the regulation of RTE1, we performed a genetic screening for mutations that suppress ethylene insensitivity conferred by RTE1 overexpression in Arabidopsis. We isolated HYPER RECOMBINATION1 (HPR1, which is required for RTE1 overexpressor (RTE1ox ethylene insensitivity at the seedling but not adult stage. HPR1 is a component of the THO complex, which, with other proteins, forms the TRanscription EXport (TREX complex. In yeast, Drosophila, and humans, the THO/TREX complex is involved in transcription elongation and nucleocytoplasmic RNA export, but its role in plants is to be fully determined. We investigated how HPR1 is involved in RTE1ox ethylene insensitivity in Arabidopsis. The hpr1-5 mutation may affect nucleocytoplasmic mRNA export, as revealed by in vivo hybridization of fluorescein-labeled oligo(dT45 with unidentified mRNA in the nucleus. The hpr1-5 mutation reduced the total and nuclear RTE1 transcript levels to a similar extent, and RTE1 transcript reduction rate was not affected by hpr1-5 with cordycepin treatment, which prematurely terminates transcription. The defect in the THO-interacting TEX1 protein of TREX but not the mRNA export factor SAC3B also reduced the total and nuclear RTE1 levels. SERINE-ARGININE-RICH (SR proteins are involved mRNA splicing, and we found that SR protein SR33 co-localized with HPR1 in nuclear speckles, which agreed with the association of human TREX with the splicing machinery. We reveal a role for HPR1 in RTE1 expression during transcription elongation and less likely during export. Gene expression involved in ethylene signaling suppression was not reduced by the hpr1-5 mutation, which indicates selectivity of HPR1 for RTE1 expression affecting the consequent ethylene response. Thus

ATF1 Modulates the Heat Shock Response by Regulating the Stress-Inducible Heat Shock Factor 1 Transcription Complex

Science.gov (United States)

Takii, Ryosuke; Fujimoto, Mitsuaki; Tan, Ke; Takaki, Eiichi; Hayashida, Naoki; Nakato, Ryuichiro; Shirahige, Katsuhiko

2014-01-01

The heat shock response is an evolutionally conserved adaptive response to high temperatures that controls proteostasis capacity and is regulated mainly by an ancient heat shock factor (HSF). However, the regulation of target genes by the stress-inducible HSF1 transcription complex has not yet been examined in detail in mammalian cells. In the present study, we demonstrated that HSF1 interacted with members of the ATF1/CREB family involved in metabolic homeostasis and recruited them on the HSP70 promoter in response to heat shock. The HSF1 transcription complex, including the chromatin-remodeling factor BRG1 and lysine acetyltransferases p300 and CREB-binding protein (CBP), was formed in a manner that was dependent on the phosphorylation of ATF1. ATF1-BRG1 promoted the establishment of an active chromatin state and HSP70 expression during heat shock, whereas ATF1-p300/CBP accelerated the shutdown of HSF1 DNA-binding activity during recovery from acute stress, possibly through the acetylation of HSF1. Furthermore, ATF1 markedly affected the resistance to heat shock. These results revealed the unanticipated complexity of the primitive heat shock response mechanism, which is connected to metabolic adaptation. PMID:25312646

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription.

Science.gov (United States)

Herdman, Chelsea; Mars, Jean-Clement; Stefanovsky, Victor Y; Tremblay, Michel G; Sabourin-Felix, Marianne; Lindsay, Helen; Robinson, Mark D; Moss, Tom

2017-07-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription.

Directory of Open Access Journals (Sweden)

Chelsea Herdman

2017-07-01

Full Text Available Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state

Ménage à trois: the complex relationships between mitogen-activated protein kinases, WRKY transcription factors, and VQ-motif-containing proteins.

Science.gov (United States)

Weyhe, Martin; Eschen-Lippold, Lennart; Pecher, Pascal; Scheel, Dierk; Lee, Justin

2014-01-01

Out of the 34 members of the VQ-motif-containing protein (VQP) family, 10 are phosphorylated by the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6. Most of these MPK3/6-targeted VQPs (MVQs) interacted with specific sub-groups of WRKY transcription factors in a VQ-motif-dependent manner. In some cases, the MAPK appears to phosphorylate either the MVQ or the WRKY, while in other cases, both proteins have been reported to act as MAPK substrates. We propose a network of dynamic interactions between members from the MAPK, MVQ and WRKY families - either as binary or as tripartite interactions. The compositions of the WRKY-MVQ transcriptional protein complexes may change - for instance, through MPK3/6-mediated modulation of protein stability - and therefore control defense gene transcription.

Real-time observation of the initiation of RNA polymerase II transcription.

Science.gov (United States)

Fazal, Furqan M; Meng, Cong A; Murakami, Kenji; Kornberg, Roger D; Block, Steven M

2015-09-10

Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.

Two sides of the same coin: TFIIH complexes in transcription and DNA repair.

Science.gov (United States)

Zhovmer, Alexander; Oksenych, Valentyn; Coin, Frédéric

2010-04-13

TFIIH is organized into a seven-subunit core associated with a three-subunit Cdk-activating kinase (CAK) module. TFIIH has roles in both transcription initiation and DNA repair. During the last 15 years, several studies have been conducted to identify the composition of the TFIIH complex involved in DNA repair. Recently, a new technique combining chromatin immunoprecipitation and western blotting resolved the hidden nature of the TFIIH complex participating in DNA repair. Following the recruitment of TFIIH to the damaged site, the CAK module is released from the core TFIIH, and the core subsequently associates with DNA repair factors. The release of the CAK is specifically driven by the recruitment of the DNA repair factor XPA and is required to promote the incision/excision of the damaged DNA. Once the DNA lesions have been repaired, the CAK module returns to the core TFIIH on the chromatin, together with the release of the repair factors. These data highlight the dynamic composition of a fundamental cellular factor that adapts its subunit composition to the cell needs.

Two Sides of the Same Coin: TFIIH Complexes in Transcription and DNA Repair

Directory of Open Access Journals (Sweden)

Alexander Zhovmer

2010-01-01

Full Text Available TFIIH is organized into a seven-subunit core associated with a three-subunit Cdk-activating kinase (CAK module. TFIIH has roles in both transcription initiation and DNA repair. During the last 15 years, several studies have been conducted to identify the composition of the TFIIH complex involved in DNA repair. Recently, a new technique combining chromatin immunoprecipitation and western blotting resolved the hidden nature of the TFIIH complex participating in DNA repair. Following the recruitment of TFIIH to the damaged site, the CAK module is released from the core TFIIH, and the core subsequently associates with DNA repair factors. The release of the CAK is specifically driven by the recruitment of the DNA repair factor XPA and is required to promote the incision/excision of the damaged DNA. Once the DNA lesions have been repaired, the CAK module returns to the core TFIIH on the chromatin, together with the release of the repair factors. These data highlight the dynamic composition of a fundamental cellular factor that adapts its subunit composition to the cell needs.

Novel Functions for TAF7, a Regulator of TAF1-independent Transcription

OpenAIRE

Devaiah, Ballachanda N.; Lu, Hanxin; Gegonne, Anne; Sercan, Zeynep; Zhang, Hongen; Clifford, Robert J.; Lee, Maxwell P.; Singer, Dinah S.

2010-01-01

The transcription factor TFIID components TAF7 and TAF1 regulate eukaryotic transcription initiation. TAF7 regulates transcription initiation of TAF1-dependent genes by binding to the acetyltransferase (AT) domain of TAF1 and inhibiting the enzymatic activity that is essential for transcription. TAF7 is released from the TAF1-TFIID complex upon completion of preinitiation complex assembly, allowing transcription to initiate. However, not all transcription is TAF1-dependent, and the role of TA...

SAF-A forms a complex with BRG1 and both components are required for RNA polymerase II mediated transcription.

Directory of Open Access Journals (Sweden)

Dzeneta Vizlin-Hodzic

Full Text Available BACKGROUND: Scaffold attachment factor A (SAF-A participates in the regulation of gene expression by organizing chromatin into transcriptionally active domains and by interacting directly with RNA polymerase II. METHODOLOGY: Here we use co-localization, co-immunoprecipitation (co-IP and in situ proximity ligation assay (PLA to identify Brahma Related Gene 1 (BRG1, the ATP-driven motor of the human SWI-SNF chromatin remodeling complex, as another SAF-A interaction partner in mouse embryonic stem (mES cells. We also employ RNA interference to investigate functional aspects of the SAF-A/BRG1 interaction. PRINCIPAL FINDINGS: We find that endogenous SAF-A protein interacts with endogenous BRG1 protein in mES cells, and that the interaction does not solely depend on the presence of mRNA. Moreover the interaction remains intact when cells are induced to differentiate. Functional analyses reveal that dual depletion of SAF-A and BRG1 abolishes global transcription by RNA polymerase II, while the nucleolar RNA polymerase I transcription machinery remains unaffected. CONCLUSIONS: We demonstrate that SAF-A interacts with BRG1 and that both components are required for RNA Polymerase II Mediated Transcription.

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

Science.gov (United States)

Hurst, H C; Masson, N; Jones, N C; Lee, K A

1990-12-01

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.

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

Energy Technology Data Exchange (ETDEWEB)

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

2008-03-01

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

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

International Nuclear Information System (INIS)

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

2008-01-01

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

Downregulation of RND3/RhoE in glioblastoma patients promotes tumorigenesis through augmentation of notch transcriptional complex activity

International Nuclear Information System (INIS)

Liu, Baohui; Lin, Xi; Yang, Xiangsheng; Dong, Huimin; Yue, Xiaojing; Andrade, Kelsey C; Guo, Zhentao; Yang, Jian; Wu, Liquan; Zhu, Xiaonan; Zhang, Shenqi; Tian, Daofeng; Wang, Junmin; Cai, Qiang; Chen, Qizuan; Mao, Shanping; Chen, Qianxue; Chang, Jiang

2015-01-01

Activation of Notch signaling contributes to glioblastoma multiform (GBM) tumorigenesis. However, the molecular mechanism that promotes the Notch signaling augmentation during GBM genesis remains largely unknown. Identification of new factors that regulate Notch signaling is critical for tumor treatment. The expression levels of RND3 and its clinical implication were analyzed in GBM patients. Identification of RND3 as a novel factor in GBM genesis was demonstrated in vitro by cell experiments and in vivo by a GBM xenograft model. We found that RND3 expression was significantly decreased in human glioblastoma. The levels of RND3 expression were inversely correlated with Notch activity, tumor size, and tumor cell proliferation, and positively correlated with patient survival time. We demonstrated that RND3 functioned as an endogenous repressor of the Notch transcriptional complex. RND3 physically interacted with NICD, CSL, and MAML1, the Notch transcriptional complex factors, promoted NICD ubiquitination, and facilitated the degradation of these cofactor proteins. We further revealed that RND3 facilitated the binding of NICD to FBW7, a ubiquitin ligase, and consequently enhanced NICD protein degradation. Therefore, Notch transcriptional activity was inhibited. Forced expression of RND3 repressed Notch signaling, which led to the inhibition of glioblastoma cell proliferation in vitro and tumor growth in the xenograft mice in vivo. Downregulation of RND3, however, enhanced Notch signaling activity, and subsequently promoted glioma cell proliferation. Inhibition of Notch activity abolished RND3 deficiency-mediated GBM cell proliferation. We conclude that downregulation of RND3 is responsible for the enhancement of Notch activity that promotes glioblastoma genesis

Multiple 5' ends of human cytomegalovirus UL57 transcripts identify a complex, cycloheximide-resistant promoter region that activates oriLyt

International Nuclear Information System (INIS)

Kiehl, Anita; Huang, Lili; Franchi, David; Anders, David G.

2003-01-01

The human cytomegalovirus (HCMV) UL57 gene lies adjacent to HCMV oriLyt, from which it is separated by an organizationally conserved, mostly noncoding region that is thought to both regulate UL57 expression and activate oriLyt function. However, the UL57 promoter has not been studied. We determined the 5' ends of UL57 transcripts toward an understanding of the potential relationship between UL57 expression and oriLyt activation. The results presented here identified three distinct 5' ends spread over 800 bp, at nt 90302, 90530, and 91138; use of these sites exhibited differential sensitivity to phosphonoformic acid treatment. Interestingly, a 10-kb UL57 transcript accumulated in cycloheximide-treated infected cells, even though other early transcripts were not detectable. However, the 10-kb transcript did not accumulate in cells treated with the more stringent translation inhibitor anisomycin. Consistent with the notion that the identified 5' ends arise from distinct transcription start sites, the sequences upstream of sites I and II functioned as promoters responsive to HCMV infection in transient assays. However, the origin-proximal promoter region III required downstream sequences for transcriptional activity. Mutation of candidate core promoter elements suggested that promoter III is regulated by an initiator region (Inr) and a downstream promoter element. Finally, a 42-bp sequence containing the candidate Inr activated a minimal oriLyt core construct in transient replication assays. Thus, these studies showed that a large, complex promoter region with novel features controls UL57 expression, and identified a sequence that regulates both UL57 transcription and oriLyt activation

The E2F-DP1 Transcription Factor Complex Regulates Centriole Duplication in Caenorhabditis elegans

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Jacqueline G. Miller

2016-03-01

Full Text Available Centrioles play critical roles in the organization of microtubule-based structures, from the mitotic spindle to cilia and flagella. In order to properly execute their various functions, centrioles are subjected to stringent copy number control. Central to this control mechanism is a precise duplication event that takes place during S phase of the cell cycle and involves the assembly of a single daughter centriole in association with each mother centriole . Recent studies have revealed that posttranslational control of the master regulator Plk4/ZYG-1 kinase and its downstream effector SAS-6 is key to ensuring production of a single daughter centriole. In contrast, relatively little is known about how centriole duplication is regulated at a transcriptional level. Here we show that the transcription factor complex EFL-1-DPL-1 both positively and negatively controls centriole duplication in the Caenorhabditis elegans embryo. Specifically, we find that down regulation of EFL-1-DPL-1 can restore centriole duplication in a zyg-1 hypomorphic mutant and that suppression of the zyg-1 mutant phenotype is accompanied by an increase in SAS-6 protein levels. Further, we find evidence that EFL-1-DPL-1 promotes the transcription of zyg-1 and other centriole duplication genes. Our results provide evidence that in a single tissue type, EFL-1-DPL-1 sets the balance between positive and negative regulators of centriole assembly and thus may be part of a homeostatic mechanism that governs centriole assembly.

Widespread anti-sense transcription in apple is correlated with siRNA production and indicates a large potential for transcriptional and/or post-transcriptional control.

Science.gov (United States)

Celton, Jean-Marc; Gaillard, Sylvain; Bruneau, Maryline; Pelletier, Sandra; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Navarro, Lionel; Laurens, François; Renou, Jean-Pierre

2014-07-01

Characterizing the transcriptome of eukaryotic organisms is essential for studying gene regulation and its impact on phenotype. The realization that anti-sense (AS) and noncoding RNA transcription is pervasive in many genomes has emphasized our limited understanding of gene transcription and post-transcriptional regulation. Numerous mechanisms including convergent transcription, anti-correlated expression of sense and AS transcripts, and RNAi remain ill-defined. Here, we have combined microarray analysis and high-throughput sequencing of small RNAs (sRNAs) to unravel the complexity of transcriptional and potential post-transcriptional regulation in eight organs of apple (Malus × domestica). The percentage of AS transcript expression is higher than that identified in annual plants such as rice and Arabidopsis thaliana. Furthermore, we show that a majority of AS transcripts are transcribed beyond 3'UTR regions, and may cover a significant portion of the predicted sense transcripts. Finally we demonstrate at a genome-wide scale that anti-sense transcript expression is correlated with the presence of both short (21-23 nt) and long (> 30 nt) siRNAs, and that the sRNA coverage depth varies with the level of AS transcript expression. Our study provides a new insight on the functional role of anti-sense transcripts at the genome-wide level, and a new basis for the understanding of sRNA biogenesis in plants. © 2014 INRA. New Phytologist © 2014 New Phytologist Trust.

Nucleic Acid Analogue Induced Transcription of Double Stranded DNA

DEFF Research Database (Denmark)

1998-01-01

RNA is transcribed from a double stranded DNA template by forming a complex by hybridizing to the template at a desired transcription initiation site one or more oligonucleic acid analogues of the PNA type capable of forming a transcription initiation site with the DNA and exposing the complex...... to the action of a DNA dependant RNA polymerase in the presence of nucleoside triphosphates. Equal length transcripts may be obtained by placing a block to transcription downstream from the initiation site or by cutting the template at such a selected location. The initiation site is formed by displacement...... of one strand of the DNA locally by the PNA hybridization....

The Journey of a Transcription Factor

DEFF Research Database (Denmark)

Pireyre, Marie

Plants have developed astonishing networks regulating their metabolism to adapt to their environment. The complexity of these networks is illustrated by the expansion of families of regulators such as transcription factors in the plant kingdom. Transcription factors specifically impact...... transcriptional networks by integrating exogenous and endogenous stimuli and regulating gene expression accordingly. Regulation of transcription factors and their activation is thus highly important to modulate the transcriptional programs and increase fitness of the plant in a given environment. Plant metabolism....... The biosynthetic machinery of GLS is governed by interplay of six MYB and three bHLH transcription factors. MYB28, MYB29 and MYB76 regulate methionine-derived GLS, and MYB51, MYB34 and MYB122 regulate tryptophan-derived GLS. The three bHLH transcription factors MYC2, MYC3 and MYC4 physically interact with all six...

Core transcriptional regulatory circuit controlled by the TAL1 complex in human T cell acute lymphoblastic leukemia.

Science.gov (United States)

Sanda, Takaomi; Lawton, Lee N; Barrasa, M Inmaculada; Fan, Zi Peng; Kohlhammer, Holger; Gutierrez, Alejandro; Ma, Wenxue; Tatarek, Jessica; Ahn, Yebin; Kelliher, Michelle A; Jamieson, Catriona H M; Staudt, Louis M; Young, Richard A; Look, A Thomas

2012-08-14

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Phorbol-ester-induced activation of the NF-κB transcription factor involves dissociation of an apparently cytoplasmic NF-κB/inhibitor complex

International Nuclear Information System (INIS)

Baeuerle, P.A.; Lenardo, M.; Pierce, J.W.; Baltimore, D.

1988-01-01

There is increasing evidence that inducible transcription of genes is mediated through the induction of the activity of trans-acting protein factors. The NF-κB transcription factor provides a model system to study the posttranslational activation of a phorbol-ester-inducible transcription factor. The finding that NF-κB activity is undectable in subcellular fractions from unstimulated cells suggests that NF-κB exists as an inactive precursor. The authors showed that NF-κB is detectable in two different forms. After selective removal of endogenous NF-κB, they demonstrate the existence of a protein inhibitor in cytosolic fractions of unstimulated cells that is able in vitro to convert NF-κB into an inactive desoxycholate-dependent form. The data are consistent with a molecular mechanism of inducible gene expression by which an apparently cytoplasmic transcription factor-inhibitor complex is dissociated by the action of TPA-activated protein kinase C

Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

Energy Technology Data Exchange (ETDEWEB)

Tennyson, C.N.; Worton, R.G. [Univ. of Toronto and the Hospital for Sick Children, Ontario (Canada)

1994-09-01

The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

Complex formation of p65/RelA with nuclear Akt1 for enhanced transcriptional activation of NF-κB

International Nuclear Information System (INIS)

Kwon, Osong; Kim, Kyung A; He, Long; Jung, Mira; Jeong, Sook Jung; Ahn, Jong Seog; Kim, Bo Yeon

2008-01-01

Akt1 was revealed to interact with Ki-Ras in the cytoplasm of Ki-Ras-transformed human prostate epithelial cells, 267B1/K-ras. Moreover, p65/RelA in the nucleus was found to interact with both Ki-Ras and Akt1, suggesting the nuclear translocation of Akt1:Ki-Ras complex for NF- κB activation. In support of this, compared with wild type Akt1, the dominant negative Akt1 mutant was decreased in its nuclear expression, reducing the Ki-Ras-induced NF-κB transcriptional activation. Moreover, inhibitors of Ras (sulindac sulfide and farnesyltransferase inhibitor I) or PI3K/Akt (wortmannin), reduced the amounts of Akt1 and Ki-Ras in the nucleus as well as partial NF-κB activity. The complete inhibition of Ki-Ras-induced NF-κB activation, however, could only be obtained by combined treatment with wortmannin and proteasome inhibitor-1. Accordingly, clonogenic assay showed Akt1 contribution to IκBα-mediated NF-κB activation for oncogenic cell growth by Ki-Ras. Our data suggest a crucial role of Ki-Ras:Akt1 complex in NF-κB transcriptional activation and enhancement of cell survival

The multitalented Mediator complex.

Science.gov (United States)

Carlsten, Jonas O P; Zhu, Xuefeng; Gustafsson, Claes M

2013-11-01

The Mediator complex is needed for regulated transcription of RNA polymerase II (Pol II)-dependent genes. Initially, Mediator was only seen as a protein bridge that conveyed regulatory information from enhancers to the promoter. Later studies have added many other functions to the Mediator repertoire. Indeed, recent findings show that Mediator influences nearly all stages of transcription and coordinates these events with concomitant changes in chromatin organization. We review the multitude of activities associated with Mediator and discuss how this complex coordinates transcription with other cellular events. We also discuss the inherent difficulties associated with in vivo characterization of a coactivator complex that can indirectly affect diverse cellular processes via changes in gene transcription. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Scc2/Scc4 complex acts in sister chromatid cohesion and transcriptional regulation by maintaining nucleosome-free regions

Science.gov (United States)

Lopez-Serra, Lidia; Kelly, Gavin; Patel, Harshil; Stewart, Aengus; Uhlmann, Frank

2014-01-01

The cohesin complex is at the heart of many chromosomal activities, including sister chromatid cohesion and transcriptional regulation1-3. Cohesin loading onto chromosomes depends on the Scc2/Scc4 cohesin loader complex4-6, but the chromatin features that form cohesin loading sites remain poorly understood. Here, we show that the RSC chromatin remodeling complex recruits budding yeast Scc2/Scc4 to broad nucleosome-free regions, that the cohesin loader itself helps to maintain. Consequently, inactivation of the cohesin loader or RSC complex have similar effects on nucleosome positioning, gene expression and sister chromatid cohesion. These results reveal an intimate link between local chromatin structure and higher order chromosome architecture. Our findings pertain to the similarities between two severe human disorders, Cornelia de Lange syndrome, caused by mutations in the human cohesin loader, and Coffin-Siris syndrome, resulting from mutations in human RSC complex components7-9. Both could arise from gene misregulation due to related changes in the nucleosome landscape. PMID:25173104

Contradiction between plastid gene transcription and function due to complex posttranscriptional splicing: an exemplary study of ycf15 function and evolution in angiosperms.

Directory of Open Access Journals (Sweden)

Chao Shi

Full Text Available Plant chloroplast genes are usually co-transcribed while its posttranscriptional splicing is fairly complex and remains largely unsolved. On basis of sequencing the three complete Camellia (Theaceae chloroplast genomes for the first time, we comprehensively analyzed the evolutionary patterns of ycf15, a plastid gene quite paradoxical in terms of its function and evolution, along the inferred angiosperm phylogeny. Although many species in separate lineages including the three species reported here contained an intact ycf15 gene in their chloroplast genomes, the phylogenetic mixture of both intact and obviously disabled ycf15 genes imply that they are all non-functional. Both intracellular gene transfer (IGT and horizontal gene transfer (HGT failed to explain such distributional anomalies. While, transcriptome analyses revealed that ycf15 was transcribed as precursor polycistronic transcript which contained ycf2, ycf15 and antisense trnL-CAA. The transcriptome assembly was surprisingly found to cover near the complete Camellia chloroplast genome. Many non-coding regions including pseudogenes were mapped by multiple transcripts, indicating the generality of pseudogene transcriptions. Our results suggest that plastid DNA posttranscriptional splicing may involve complex cleavage of non-functional genes.

Mechanism of transcription activation at the comG promoter by the competence transcription factor ComK of Bacillus subtilis

NARCIS (Netherlands)

Susanna, KA; van der Werff, AF; den Hengst, CD; Calles, B; Salas, M; Venema, G; Hamoen, LW; Kuipers, OP

The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding

In silico and biological survey of transcription-associated proteins implicated in the transcriptional machinery during the erythrocytic development of Plasmodium falciparum

Directory of Open Access Journals (Sweden)

Bischoff Emmanuel

2010-01-01

Full Text Available Abstract Background Malaria is the most important parasitic disease in the world with approximately two million people dying every year, mostly due to Plasmodium falciparum infection. During its complex life cycle in the Anopheles vector and human host, the parasite requires the coordinated and modulated expression of diverse sets of genes involved in epigenetic, transcriptional and post-transcriptional regulation. However, despite the availability of the complete sequence of the Plasmodium falciparum genome, we are still quite ignorant about Plasmodium mechanisms of transcriptional gene regulation. This is due to the poor prediction of nuclear proteins, cognate DNA motifs and structures involved in transcription. Results A comprehensive directory of proteins reported to be potentially involved in Plasmodium transcriptional machinery was built from all in silico reports and databanks. The transcription-associated proteins were clustered in three main sets of factors: general transcription factors, chromatin-related proteins (structuring, remodelling and histone modifying enzymes, and specific transcription factors. Only a few of these factors have been molecularly analysed. Furthermore, from transcriptome and proteome data we modelled expression patterns of transcripts and corresponding proteins during the intra-erythrocytic cycle. Finally, an interactome of these proteins based either on in silico or on 2-yeast-hybrid experimental approaches is discussed. Conclusion This is the first attempt to build a comprehensive directory of potential transcription-associated proteins in Plasmodium. In addition, all complete transcriptome, proteome and interactome raw data were re-analysed, compared and discussed for a better comprehension of the complex biological processes of Plasmodium falciparum transcriptional regulation during the erythrocytic development.

Post-transcriptional generation of miRNA variants by multiple nucleotidyl transferases contributes to miRNA transcriptome complexity

OpenAIRE

Wyman, Stacia K.; Knouf, Emily C.; Parkin, Rachael K.; Fritz, Brian R.; Lin, Daniel W.; Dennis, Lucas M.; Krouse, Michael A.; Webster, Philippa J.; Tewari, Muneesh

2011-01-01

Modification of microRNA sequences by the 3′ addition of nucleotides to generate so-called “isomiRs” adds to the complexity of miRNA function, with recent reports showing that 3′ modifications can influence miRNA stability and efficiency of target repression. Here, we show that the 3′ modification of miRNAs is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species ranging from C. elegans to human. The modifications resul...

X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila.

Science.gov (United States)

Larschan, Erica; Bishop, Eric P; Kharchenko, Peter V; Core, Leighton J; Lis, John T; Park, Peter J; Kuroda, Mitzi I

2011-03-03

The evolution of sex chromosomes has resulted in numerous species in which females inherit two X chromosomes but males have a single X, thus requiring dosage compensation. MSL (Male-specific lethal) complex increases transcription on the single X chromosome of Drosophila males to equalize expression of X-linked genes between the sexes. The biochemical mechanisms used for dosage compensation must function over a wide dynamic range of transcription levels and differential expression patterns. It has been proposed that the MSL complex regulates transcriptional elongation to control dosage compensation, a model subsequently supported by mapping of the MSL complex and MSL-dependent histone 4 lysine 16 acetylation to the bodies of X-linked genes in males, with a bias towards 3' ends. However, experimental analysis of MSL function at the mechanistic level has been challenging owing to the small magnitude of the chromosome-wide effect and the lack of an in vitro system for biochemical analysis. Here we use global run-on sequencing (GRO-seq) to examine the specific effect of the MSL complex on RNA Polymerase II (RNAP II) on a genome-wide level. Results indicate that the MSL complex enhances transcription by facilitating the progression of RNAP II across the bodies of active X-linked genes. Improving transcriptional output downstream of typical gene-specific controls may explain how dosage compensation can be imposed on the diverse set of genes along an entire chromosome.

Characterizing highly dynamic conformational states: The transcription bubble in RNAP-promoter open complex as an example

Science.gov (United States)

Lerner, Eitan; Ingargiola, Antonino; Weiss, Shimon

2018-03-01

Bio-macromolecules carry out complicated functions through structural changes. To understand their mechanism of action, the structure of each step has to be characterized. While classical structural biology techniques allow the characterization of a few "structural snapshots" along the enzymatic cycle (usually of stable conformations), they do not cover all (and often fast interconverting) structures in the ensemble, where each may play an important functional role. Recently, several groups have demonstrated that structures of different conformations in solution could be solved by measuring multiple distances between different pairs of residues using single-molecule Förster resonance energy transfer (smFRET) and using them as constrains for hybrid/integrative structural modeling. However, this approach is limited in cases where the conformational dynamics is faster than the technique's temporal resolution. In this study, we combine existing tools that elucidate sub-millisecond conformational dynamics together with hybrid/integrative structural modeling to study the conformational states of the transcription bubble in the bacterial RNA polymerase-promoter open complex (RPo). We measured microsecond alternating laser excitation-smFRET of differently labeled lacCONS promoter dsDNA constructs. We used a combination of burst variance analysis, photon-by-photon hidden Markov modeling, and the FRET-restrained positioning and screening approach to identify two conformational states for RPo. The experimentally derived distances of one conformational state match the known crystal structure of bacterial RPo. The experimentally derived distances of the other conformational state have characteristics of a scrunched RPo. These findings support the hypothesis that sub-millisecond dynamics in the transcription bubble are responsible for transcription start site selection.

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription

DEFF Research Database (Denmark)

Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien

2012-01-01

site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial...... diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites...... and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain...

Subunit architecture and functional modular rearrangements of the transcriptional mediator complex.

Science.gov (United States)

Tsai, Kuang-Lei; Tomomori-Sato, Chieri; Sato, Shigeo; Conaway, Ronald C; Conaway, Joan W; Asturias, Francisco J

2014-06-05

The multisubunit Mediator, comprising ∼30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA-binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.

A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure.

Science.gov (United States)

Zhang, Jinwei; Landick, Robert

2016-04-01

The vectorial (5'-to-3' at varying velocity) synthesis of RNA by cellular RNA polymerases (RNAPs) creates a rugged kinetic landscape, demarcated by frequent, sometimes long-lived, pauses. In addition to myriad gene-regulatory roles, these pauses temporally and spatially program the co-transcriptional, hierarchical folding of biologically active RNAs. Conversely, these RNA structures, which form inside or near the RNA exit channel, interact with the polymerase and adjacent protein factors to influence RNA synthesis by modulating pausing, termination, antitermination, and slippage. Here, we review the evolutionary origin, mechanistic underpinnings, and regulatory consequences of this interplay between RNAP and nascent RNA structure. We categorize and rationalize the extensive linkage between the transcriptional machinery and its product, and provide a framework for future studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mediator and p300/CBP-Steroid Receptor Coactivator Complexes Have Distinct Roles, but Function Synergistically, during Estrogen Receptor α-Dependent Transcription with Chromatin Templates

OpenAIRE

Acevedo, Mari Luz; Kraus, W. Lee

2003-01-01

Ligand-dependent transcriptional activation by nuclear receptors involves the recruitment of various coactivators to the promoters of hormone-regulated genes assembled into chromatin. Nuclear receptor coactivators include histone acetyltransferase complexes, such as p300/CBP-steroid receptor coactivator (SRC), as well as the multisubunit mediator complexes (“Mediator”), which may help recruit RNA polymerase II to the promoter. We have used a biochemical approach, including an in vitro chromat...

Probing the interaction between the histone methyltransferase/deacetylase subunit RBBP4/7 and the transcription factor BCL11A in epigenetic complexes.

Science.gov (United States)

Moody, Rebecca Reed; Lo, Miao-Chia; Meagher, Jennifer L; Lin, Chang-Ching; Stevers, Nicholas O; Tinsley, Samantha L; Jung, Inkyung; Matvekas, Aleksas; Stuckey, Jeanne A; Sun, Duxin

2018-02-09

The transcription factor BCL11A has recently been reported to be a driving force in triple-negative breast cancer (TNBC), contributing to the maintenance of a chemoresistant breast cancer stem cell (BCSC) population. Although BCL11A was shown to suppress γ-globin and p21 and to induce MDM2 expression in the hematopoietic system, its downstream targets in TNBC are still unclear. For its role in transcriptional repression, BCL11A was found to interact with several corepressor complexes; however, the mechanisms underlying these interactions remain unknown. Here, we reveal that BCL11A interacts with histone methyltransferase (PRC2) and histone deacetylase (NuRD and SIN3A) complexes through their common subunit, RBBP4/7. In fluorescence polarization assays, we show that BCL11A competes with histone H3 for binding to the negatively charged top face of RBBP4. To define that interaction, we solved the crystal structure of RBBP4 in complex with an N-terminal peptide of BCL11A (residues 2-16, BCL11A(2-16)). The crystal structure identifies novel interactions between BCL11A and the side of the β-propeller of RBBP4 that are not seen with histone H3. We next show that BCL11A(2-16) pulls down RBBP4, RBBP7, and other components of PRC2, NuRD, and SIN3A from the cell lysate of the TNBC cell line SUM149. Furthermore, we demonstrate the therapeutic potential of targeting the RBBP4-BCL11A binding by showing that a BCL11A peptide can decrease aldehyde dehydrogenase-positive BCSCs and mammosphere formation capacity in SUM149. Together, our findings have uncovered a previously unidentified mechanism that BCL11A may use to recruit epigenetic complexes to regulate transcription and promote tumorigenesis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Extracellular Matrix-Regulated Gene Expression RequiresCooperation of SWI/SNF and Transcription Factors

Energy Technology Data Exchange (ETDEWEB)

Xu, Ren; Spencer, Virginia A.; Bissell, Mina J.

2006-05-25

Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation (ChIP) assays and mammary-specific genes as models, we show here that extracellular matrix (ECM) molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the {beta}- and ?-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both {beta}- and ?-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. ChIP analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Coimmunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, C/EBP{beta}, and glucocorticoid receptor (GR). Thus, ECM- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors.

Structures of BmrR-Drug Complexes Reveal a Rigid Multidrug Binding Pocket And Transcription Activation Through Tyrosine Expulsion

Energy Technology Data Exchange (ETDEWEB)

Newberry, K.J.; Huffman, J.L.; Miller, M.C.; Vazquez-Laslop, N.; Neyfakh, A.A.; Brennan, R.G.

2009-05-22

BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets, that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.

The Mediator complex of Caenorhabditis elegans: insights into the developmental and physiological roles of a conserved transcriptional coregulator.

Science.gov (United States)

Grants, Jennifer M; Goh, Grace Y S; Taubert, Stefan

2015-02-27

The Mediator multiprotein complex ('Mediator') is an important transcriptional coregulator that is evolutionarily conserved throughout eukaryotes. Although some Mediator subunits are essential for the transcription of all protein-coding genes, others influence the expression of only subsets of genes and participate selectively in cellular signaling pathways. Here, we review the current knowledge of Mediator subunit function in the nematode Caenorhabditis elegans, a metazoan in which established and emerging genetic technologies facilitate the study of developmental and physiological regulation in vivo. In this nematode, unbiased genetic screens have revealed critical roles for Mediator components in core developmental pathways such as epidermal growth factor (EGF) and Wnt/β-catenin signaling. More recently, important roles for C. elegans Mediator subunits have emerged in the regulation of lipid metabolism and of systemic stress responses, engaging conserved transcription factors such as nuclear hormone receptors (NHRs). We emphasize instances where similar functions for individual Mediator subunits exist in mammals, highlighting parallels between Mediator subunit action in nematode development and in human cancer biology. We also discuss a parallel between the association of the Mediator subunit MED12 with several human disorders and the role of its C. elegans ortholog mdt-12 as a regulatory hub that interacts with numerous signaling pathways. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Transcription-based model for the induction of chromosomal exchange events by ionising radiation

International Nuclear Information System (INIS)

Radford, I.A.

2003-01-01

The mechanistic basis for chromosomal aberration formation, following exposure of mammalian cells to ionising radiation, has long been debated. Although chromosomal aberrations are probably initiated by DNA double-strand breaks (DSB), little is understood about the mechanisms that generate and modulate DNA rearrangement. Based on results from our laboratory and data from the literature, a novel model of chromosomal aberration formation has been suggested (Radford 2002). The basic postulates of this model are that: (1) DSB, primarily those involving multiple individual damage sites (i.e. complex DSB), are the critical initiating lesion; (2) only those DSB occurring in transcription units that are associated with transcription 'factories' (complexes containing multiple transcription units) induce chromosomal exchange events; (3) such DSB are brought into contact with a DNA topoisomerase I molecule through RNA polymerase II catalysed transcription and give rise to trapped DNA-topo I cleavage complexes; and (4) trapped complexes interact with another topo I molecule on a temporarily inactive transcription unit at the same transcription factory leading to DNA cleavage and subsequent strand exchange between the cleavage complexes. We have developed a method using inverse PCR that allows the detection and sequencing of putative ionising radiation-induced DNA rearrangements involving different regions of the human genome (Forrester and Radford 1998). The sequences detected by inverse PCR can provide a test of the prediction of the transcription-based model that ionising radiation-induced DNA rearrangements occur between sequences in active transcription units. Accordingly, reverse transcriptase PCR was used to determine if sequences involved in rearrangements were transcribed in the test cells. Consistent with the transcription-based model, nearly all of the sequences examined gave a positive result to reverse transcriptase PCR (Forrester and Radford unpublished)

Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters

DEFF Research Database (Denmark)

Chen, Yun; Pai, Athma A; Herudek, Jan

2016-01-01

Mammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation s...... suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provide a framework with which evolution shapes transcriptomes.......Mammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation...

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

Science.gov (United States)

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

2017-01-01

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

Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

Science.gov (United States)

Ducrot, Cécile; Lefebvre, Olivier; Landrieux, Emilie; Guirouilh-Barbat, Josée; Sentenac, André; Acker, Joel

2006-04-28

Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

Human GW182 Paralogs Are the Central Organizers for RNA-Mediated Control of Transcription.

Science.gov (United States)

Hicks, Jessica A; Li, Liande; Matsui, Masayuki; Chu, Yongjun; Volkov, Oleg; Johnson, Krystal C; Corey, David R

2017-08-15

In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

A transcript cleavage factor of Mycobacterium tuberculosis important for its survival.

Directory of Open Access Journals (Sweden)

Arnab China

Full Text Available After initiation of transcription, a number of proteins participate during elongation and termination modifying the properties of the RNA polymerase (RNAP. Gre factors are one such group conserved across bacteria. They regulate transcription by projecting their N-terminal coiled-coil domain into the active center of RNAP through the secondary channel and stimulating hydrolysis of the newly synthesized RNA in backtracked elongation complexes. Rv1080c is a putative gre factor (MtbGre in the genome of Mycobacterium tuberculosis. The protein enhanced the efficiency of promoter clearance by lowering abortive transcription and also rescued arrested and paused elongation complexes on the GC rich mycobacterial template. Although MtbGre is similar in domain organization and shares key residues for catalysis and RNAP interaction with the Gre factors of Escherichia coli, it could not complement an E. coli gre deficient strain. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein-protein interactions for transcript cleavage. Decrease in the level of MtbGre reduced the bacterial survival by several fold indicating its essential role in mycobacteria. Another Gre homolog, Rv3788 was not functional in transcript cleavage activity indicating that a single Gre is sufficient for efficient transcription of the M. tuberculosis genome.

The electrostatic role of the Zn-Cys2His2 complex in binding of operator DNA with transcription factors: mouse EGR-1 from the Cys2His2 family.

Science.gov (United States)

Chirgadze, Y N; Boshkova, E A; Polozov, R V; Sivozhelezov, V S; Dzyabchenko, A V; Kuzminsky, M B; Stepanenko, V A; Ivanov, V V

2018-01-07

The mouse factor Zif268, known also as early growth response protein EGR-1, is a classical representative for the Cys2His2 transcription factor family. It is required for binding the RNA polymerase with operator dsDNA to initialize the transcription process. We have shown that only in this family of total six Zn-finger protein families the Zn complex plays a significant role in the protein-DNA binding. Electrostatic feature of this complex in the binding of factor Zif268 from Mus musculus with operator DNA has been considered. The factor consists of three similar Zn-finger units which bind with triplets of coding DNA. Essential contacts of the factor with the DNA phosphates are formed by three conservative His residues, one in each finger. We describe here the results of calculations of the electrostatic potentials for the Zn-Cys2His2 complex, Zn-finger unit 1, and the whole transcription factor. The potential of Zif268 has a positive area on the factor surface, and it corresponds exactly to the binding sites of each of Zn-finger units. The main part of these areas is determined by conservative His residues, which form contacts with the DNA phosphate groups. Our result shows that the electrostatic positive potential of this histidine residue is enhanced due to the Zn complex. The other contacts of the Zn-finger with DNA are related to nucleotide bases, and they are responsible for the sequence-specific binding with DNA. This result may be extended to all other members of the Cys2His2 transcription factor family.

Interference of transcription across H-NS binding sites and repression by H-NS.

Science.gov (United States)

Rangarajan, Aathmaja Anandhi; Schnetz, Karin

2018-05-01

Nucleoid-associated protein H-NS represses transcription by forming extended DNA-H-NS complexes. Repression by H-NS operates mostly at the level of transcription initiation. Less is known about how DNA-H-NS complexes interfere with transcription elongation. In vitro H-NS has been shown to enhance RNA polymerase pausing and to promote Rho-dependent termination, while in vivo inhibition of Rho resulted in a decrease of the genome occupancy by H-NS. Here we show that transcription directed across H-NS binding regions relieves H-NS (and H-NS/StpA) mediated repression of promoters in these regions. Further, we observed a correlation of transcription across the H-NS-bound region and de-repression. The data suggest that the transcribing RNA polymerase is able to remodel the H-NS complex and/or dislodge H-NS from the DNA and thus relieve repression. Such an interference of transcription and H-NS mediated repression may imply that poorly transcribed AT-rich loci are prone to be repressed by H-NS, while efficiently transcribed loci escape repression. © 2018 John Wiley & Sons Ltd.

Factor requirements for transcription in the Archaeon Sulfolobus shibatae.

Science.gov (United States)

Qureshi, S A; Bell, S D; Jackson, S P

1997-05-15

Archaea (archaebacteria) constitute a domain of life that is distinct from Bacteria (eubacteria) and Eucarya (eukaryotes). Although archaeal cells share many morphological features with eubacteria, their transcriptional apparatus is more akin to eukaryotic RNA polymerases I, II and III than it is to eubacterial transcription systems. Thus, in addition to possessing a 10 subunit RNA polymerase and a homologue of the TATA-binding protein (TBP), Archaea possess a polypeptide termed TFB that is homologous to eukaryotic TFIIB. Here, we investigate the factor requirements for transcription of several promoters of the archaeon Sulfolobus shibatae and its associated virus SSV. Through in vitro transcription and immunodepletion, we demonstrate that S. shibatae TBP, TFB and RNA polymerase are not complexed tightly with one another and that each is required for efficient transcription of all promoters tested. Furthermore, full transcription is restored by supplementing respective depleted extracts with recombinant TBP or TFB, indicating that TBP-associated factors or TFB-associated factors are not required. Indeed, gel-filtration suggests that Sulfolobus TBP and TFB are not associated stably with other proteins. Finally, all promoters analysed are transcribed accurately and efficiently in an in vitro system comprising recombinant TBP and TFB, together with essentially homogeneous preparation of RNA polymerase. Transcription in Archaea is therefore fundamentally homologous to that in eukaryotes, although factor requirements appear to be much less complex.

Transcription factor interplay in T helper cell differentiation

Science.gov (United States)

Evans, Catherine M.

2013-01-01

The differentiation of CD4 helper T cells into specialized effector lineages has provided a powerful model for understanding immune cell differentiation. Distinct lineages have been defined by differential expression of signature cytokines and the lineage-specifying transcription factors necessary and sufficient for their production. The traditional paradigm of differentiation towards Th1 and Th2 subtypes driven by T-bet and GATA3, respectively, has been extended to incorporate additional T cell lineages and transcriptional regulators. Technological advances have expanded our view of these lineage-specifying transcription factors to the whole genome and revealed unexpected interplay between them. From these data, it is becoming clear that lineage specification is more complex and plastic than previous models might have suggested. Here, we present an overview of the different forms of transcription factor interplay that have been identified and how T cell phenotypes arise as a product of this interplay within complex regulatory networks. We also suggest experimental strategies that will provide further insight into the mechanisms that underlie T cell lineage specification and plasticity. PMID:23878131

Transcription factor interplay in T helper cell differentiation.

Science.gov (United States)

Evans, Catherine M; Jenner, Richard G

2013-11-01

The differentiation of CD4 helper T cells into specialized effector lineages has provided a powerful model for understanding immune cell differentiation. Distinct lineages have been defined by differential expression of signature cytokines and the lineage-specifying transcription factors necessary and sufficient for their production. The traditional paradigm of differentiation towards Th1 and Th2 subtypes driven by T-bet and GATA3, respectively, has been extended to incorporate additional T cell lineages and transcriptional regulators. Technological advances have expanded our view of these lineage-specifying transcription factors to the whole genome and revealed unexpected interplay between them. From these data, it is becoming clear that lineage specification is more complex and plastic than previous models might have suggested. Here, we present an overview of the different forms of transcription factor interplay that have been identified and how T cell phenotypes arise as a product of this interplay within complex regulatory networks. We also suggest experimental strategies that will provide further insight into the mechanisms that underlie T cell lineage specification and plasticity.

Messenger RNA Interferase RelE Controls relBE Transcription by Conditional Cooperativity

DEFF Research Database (Denmark)

Overgaard, Martin; Borch, Jonas; Jørgensen, Mikkel G

2008-01-01

Prokaryotic toxin-antitoxin (TA) loci consist of two genes in an operon that encodes a metabolically stable toxin and an unstable antitoxin. The antitoxin neutralises its cognate toxin by forming a tight complex with it. In all cases known, the antitoxin autoregulates TA operon transcription by b...... operator DNA. A mutational analysis of the operator-sites showed that RelE in excess counteracted cooperative binding of the RelB(2)*RelE complexes to the operator-sites. Thus, RelE controls relBE transcription by conditional cooperativity.......Prokaryotic toxin-antitoxin (TA) loci consist of two genes in an operon that encodes a metabolically stable toxin and an unstable antitoxin. The antitoxin neutralises its cognate toxin by forming a tight complex with it. In all cases known, the antitoxin autoregulates TA operon transcription...... by binding to one or more operators in the promoter region while the toxin functions as a co-repressor of transcription. Interestingly, the toxin can also stimulate TA operon transcription. Here we analyse mechanistic aspects of how RelE of Escherichia coli can function both as a co-repressor and derepressor...

Therapeutic potential of Mediator complex subunits in metabolic diseases.

Science.gov (United States)

Ranjan, Amol; Ansari, Suraiya A

2018-01-01

The multisubunit Mediator is an evolutionary conserved transcriptional coregulatory complex in eukaryotes. It is needed for the transcriptional regulation of gene expression in general as well as in a gene specific manner. Mediator complex subunits interact with different transcription factors as well as components of RNA Pol II transcription initiation complex and in doing so act as a bridge between gene specific transcription factors and general Pol II transcription machinery. Specific interaction of various Mediator subunits with nuclear receptors (NRs) and other transcription factors involved in metabolism has been reported in different studies. Evidences indicate that ligand-activated NRs recruit Mediator complex for RNA Pol II-dependent gene transcription. These NRs have been explored as therapeutic targets in different metabolic diseases; however, they show side-effects as targets due to their overlapping involvement in different signaling pathways. Here we discuss the interaction of various Mediator subunits with transcription factors involved in metabolism and whether specific interaction of these transcription factors with Mediator subunits could be potentially utilized as therapeutic strategy in a variety of metabolic diseases. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B

Energy Technology Data Exchange (ETDEWEB)

Reimers, Kerstin [Klinik fuer Plastische, Hand-und Wiederherstellungschirurgie, Podbielskistrasse 380, D-30659 Hannover (Germany); Buchholz, Katja [Institut fuer Medizinische Mikrobiologie, Otto-von-Guericke-Universitaet Magdeburg, Leipzigerstrasse 44, D-39120 Magdeburg (Germany); Werchau, Hermann [Institut fuer Medizinische Mikrobiologie, Otto-von-Guericke-Universitaet Magdeburg, Leipzigerstrasse 44, D-39120 Magdeburg (Germany)

2005-01-20

Respiratory syncytial virus (RSV) induces the production of a number of cytokines and chemokines by activation of nuclear factor kappa B (NF-{kappa}B). The activation of NF-{kappa}B has been shown to depend on viral replication in the infected cells. In this study, we demonstrate that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-{kappa}B in A549 cells. Electromobility shift assays show increased NF-{kappa}B complexes in the nuclei of M2-1-expressing cells. M2-1 protein is found in nuclei of M2-1-expressing cells and in RSV-infected cells. Co-immunoprecipitations of nuclear extracts of M2-1-expressing cells and of RSV-infected cells revealed an association of M2-1 with Rel A protein. Furthermore, the activation of NF-{kappa}B depends on the C-terminus of the RSV M2-1 protein, as shown by NF-{kappa}B-induced gene expression of a reporter gene construct.

Protein-protein interactions in the regulation of WRKY transcription factors.

Science.gov (United States)

Chi, Yingjun; Yang, Yan; Zhou, Yuan; Zhou, Jie; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

2013-03-01

It has been almost 20 years since the first report of a WRKY transcription factor, SPF1, from sweet potato. Great progress has been made since then in establishing the diverse biological roles of WRKY transcription factors in plant growth, development, and responses to biotic and abiotic stress. Despite the functional diversity, almost all analyzed WRKY proteins recognize the TTGACC/T W-box sequences and, therefore, mechanisms other than mere recognition of the core W-box promoter elements are necessary to achieve the regulatory specificity of WRKY transcription factors. Research over the past several years has revealed that WRKY transcription factors physically interact with a wide range of proteins with roles in signaling, transcription, and chromatin remodeling. Studies of WRKY-interacting proteins have provided important insights into the regulation and mode of action of members of the important family of transcription factors. It has also emerged that the slightly varied WRKY domains and other protein motifs conserved within each of the seven WRKY subfamilies participate in protein-protein interactions and mediate complex functional interactions between WRKY proteins and between WRKY and other regulatory proteins in the modulation of important biological processes. In this review, we summarize studies of protein-protein interactions for WRKY transcription factors and discuss how the interacting partners contribute, at different levels, to the establishment of the complex regulatory and functional network of WRKY transcription factors.

Ubiquitin fusion constructs allow the expression and purification of multi-KOW domain complexes of the Saccharomyces cerevisiae transcription elongation factor Spt4/5.

Science.gov (United States)

Blythe, Amanda; Gunasekara, Sanjika; Walshe, James; Mackay, Joel P; Hartzog, Grant A; Vrielink, Alice

2014-08-01

Spt4/5 is a hetero-dimeric transcription elongation factor that can both inhibit and promote transcription elongation by RNA polymerase II (RNAPII). However, Spt4/5's mechanism of action remains elusive. Spt5 is an essential protein and the only universally-conserved RNAP-associated transcription elongation factor. The protein contains multiple Kyrpides, Ouzounis and Woese (KOW) domains. These domains, in other proteins, are thought to bind RNA although there is little direct evidence in the literature to support such a function in Spt5. This could be due, at least in part, to difficulties in expressing and purifying recombinant Spt5. When expressed in Escherichia coli (E. coli), Spt5 is innately insoluble. Here we report a new approach for the successful expression and purification of milligram quantities of three different multi-KOW domain complexes of Saccharomyces cerevisiae Spt4/5 for use in future functional studies. Using the E. coli strain Rosetta2 (DE3) we have developed strategies for co-expression of Spt4 and multi-KOW domain Spt5 complexes from the bi-cistronic pET-Duet vector. In a second strategy, Spt4/5 was expressed via co-transformation of Spt4 in the vector pET-M11 with Spt5 ubiquitin fusion constructs in the vector pHUE. We characterized the multi-KOW domain Spt4/5 complexes by Western blot, limited proteolysis, circular dichroism, SDS-PAGE and size exclusion chromatography-multiangle light scattering and found that the proteins are folded with a Spt4:Spt5 hetero-dimeric stoichiometry of 1:1. These expression constructs encompass a larger region of Spt5 than has previously been reported, and will provide the opportunity to elucidate the biological function of the multi-KOW containing Spt5. Copyright © 2014 Elsevier Inc. All rights reserved.

Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings.

Science.gov (United States)

Gonzalez, Antonio; Zhao, Mingzhe; Leavitt, John M; Lloyd, Alan M

2008-03-01

In all higher plants studied to date, the anthocyanin pigment pathway is regulated by a suite of transcription factors that include Myb, bHLH and WD-repeat proteins. However, in Arabidopsis thaliana, the Myb regulators remain to be conclusively identified, and little is known about anthocyanin pathway regulation by TTG1-dependent transcriptional complexes. Previous overexpression of the PAP1 Myb suggested that genes from the entire phenylpropanoid pathway are targets of regulation by Myb/bHLH/WD-repeat complexes in Arabidopsis, in contrast to other plants. Here we demonstrate that overexpression of Myb113 or Myb114 results in substantial increases in pigment production similar to those previously seen as a result of over-expression of PAP1, and pigment production in these overexpressors remains TTG1- and bHLH-dependent. Also, plants harboring an RNAi construct targeting PAP1 and three Myb candidates (PAP2, Myb113 and Myb114) showed downregulated Myb gene expression and obvious anthocyanin deficiencies. Correlated with these anthocyanin deficiencies is downregulation of the same late anthocyanin structural genes that are downregulated in ttg1 and bHLH anthocyanin mutants. Expression studies using GL3:GR and TTG1:GR fusions revealed direct regulation of the late biosynthetic genes only. Functional diversification between GL3 and EGL3 with regard to activation of gene targets was revealed by GL3:GR studies in single and double bHLH mutant seedlings. Expression profiles for Myb and bHLH regulators are also presented in the context of pigment production in young seedlings.

Transcriptional regulation by Polycomb group proteins

DEFF Research Database (Denmark)

Di Croce, Luciano; Helin, Kristian

2013-01-01

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

Hydrogen peroxide sensing, signaling and regulation of transcription factors

Directory of Open Access Journals (Sweden)

H. Susana Marinho

2014-01-01

Full Text Available The regulatory mechanisms by which hydrogen peroxide (H2O2 modulates the activity of transcription factors in bacteria (OxyR and PerR, lower eukaryotes (Yap1, Maf1, Hsf1 and Msn2/4 and mammalian cells (AP-1, NRF2, CREB, HSF1, HIF-1, TP53, NF-κB, NOTCH, SP1 and SCREB-1 are reviewed. The complexity of regulatory networks increases throughout the phylogenetic tree, reaching a high level of complexity in mammalians. Multiple H2O2 sensors and pathways are triggered converging in the regulation of transcription factors at several levels: (1 synthesis of the transcription factor by upregulating transcription or increasing both mRNA stability and translation; (ii stability of the transcription factor by decreasing its association with the ubiquitin E3 ligase complex or by inhibiting this complex; (iii cytoplasm–nuclear traffic by exposing/masking nuclear localization signals, or by releasing the transcription factor from partners or from membrane anchors; and (iv DNA binding and nuclear transactivation by modulating transcription factor affinity towards DNA, co-activators or repressors, and by targeting specific regions of chromatin to activate individual genes. We also discuss how H2O2 biological specificity results from diverse thiol protein sensors, with different reactivity of their sulfhydryl groups towards H2O2, being activated by different concentrations and times of exposure to H2O2. The specific regulation of local H2O2 concentrations is also crucial and results from H2O2 localized production and removal controlled by signals. Finally, we formulate equations to extract from typical experiments quantitative data concerning H2O2 reactivity with sensor molecules. Rate constants of 140 M−1 s−1 and ≥1.3 × 103 M−1 s−1 were estimated, respectively, for the reaction of H2O2 with KEAP1 and with an unknown target that mediates NRF2 protein synthesis. In conclusion, the multitude of H2O2 targets and mechanisms provides an opportunity for

Frequency Modulation of Transcriptional Bursting Enables Sensitive and Rapid Gene Regulation.

Science.gov (United States)

Li, Congxin; Cesbron, François; Oehler, Michael; Brunner, Michael; Höfer, Thomas

2018-04-25

Gene regulation is a complex non-equilibrium process. Here, we show that quantitating the temporal regulation of key gene states (transcriptionally inactive, active, and refractory) provides a parsimonious framework for analyzing gene regulation. Our theory makes two non-intuitive predictions. First, for transcription factors (TFs) that regulate transcription burst frequency, as opposed to amplitude or duration, weak TF binding is sufficient to elicit strong transcriptional responses. Second, refractoriness of a gene after a transcription burst enables rapid responses to stimuli. We validate both predictions experimentally by exploiting the natural, optogenetic-like responsiveness of the Neurospora GATA-type TF White Collar Complex (WCC) to blue light. Further, we demonstrate that differential regulation of WCC target genes is caused by different gene activation rates, not different TF occupancy, and that these rates are tuned by both the core promoter and the distance between TF-binding site and core promoter. In total, our work demonstrates the relevance of a kinetic, non-equilibrium framework for understanding transcriptional regulation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Modelling reveals kinetic advantages of co-transcriptional splicing.

Directory of Open Access Journals (Sweden)

Stuart Aitken

2011-10-01

Full Text Available Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3' end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3' end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter.

Modelling reveals kinetic advantages of co-transcriptional splicing.

Science.gov (United States)

Aitken, Stuart; Alexander, Ross D; Beggs, Jean D

2011-10-01

Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3' end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3' end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter.

SUMOylation of the ING1b tumor suppressor regulates gene transcription

DEFF Research Database (Denmark)

Satpathy, Shankha; Guérillon, Claire; Kim, Tae-Sun

2014-01-01

members of histone deacetylase complexes, whereas ING3-5 are stoichiometric components of different histone acetyltransferase complexes. The INGs target these complexes to histone marks, thus acting as epigenetic regulators. ING proteins affect angiogenesis, apoptosis, DNA repair, metastasis......1b E195A), we further demonstrate that ING1b SUMOylation regulates the binding of ING1b to the ISG15 and DGCR8 promoters, consequently regulating ISG15 and DGCR8 transcription. These results suggest a role for ING1b SUMOylation in the regulation of gene transcription....

Structural Basis of Mitochondrial Transcription Initiation.

Science.gov (United States)

Hillen, Hauke S; Morozov, Yaroslav I; Sarfallah, Azadeh; Temiakov, Dmitry; Cramer, Patrick

2017-11-16

Transcription in human mitochondria is driven by a single-subunit, factor-dependent RNA polymerase (mtRNAP). Despite its critical role in both expression and replication of the mitochondrial genome, transcription initiation by mtRNAP remains poorly understood. Here, we report crystal structures of human mitochondrial transcription initiation complexes assembled on both light and heavy strand promoters. The structures reveal how transcription factors TFAM and TFB2M assist mtRNAP to achieve promoter-dependent initiation. TFAM tethers the N-terminal region of mtRNAP to recruit the polymerase to the promoter whereas TFB2M induces structural changes in mtRNAP to enable promoter opening and trapping of the DNA non-template strand. Structural comparisons demonstrate that the initiation mechanism in mitochondria is distinct from that in the well-studied nuclear, bacterial, or bacteriophage transcription systems but that similarities are found on the topological and conceptual level. These results provide a framework for studying the regulation of gene expression and DNA replication in mitochondria. Copyright © 2017 Elsevier Inc. All rights reserved.

Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

Science.gov (United States)

Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

2015-04-28

A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

How salicylic acid takes transcriptional control over jasmonic acid signaling

Directory of Open Access Journals (Sweden)

Lotte eCaarls

2015-03-01

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

Human RNA polymerase II associated factor 1 complex promotes tumorigenesis by activating c-MYC transcription in non-small cell lung cancer

International Nuclear Information System (INIS)

Zhi, Xiuyi; Giroux-Leprieur, Etienne; Wislez, Marie; Hu, Mu; Zhang, Yi; Shi, Huaiyin; Du, Kaiqi; Wang, Lei

2015-01-01

Human RNA polymerase II (RNAPII)-associated factor 1 complex (hPAF1C) plays a crucial role in protein-coding gene transcription. Overexpression of hPAF1C has been implicated in the initiation and progression of various human cancers. However, the molecular pathways involved in tumorigenesis through hPAF1C remain to be elucidated. The current study suggested hPAF1C expression as a prognostic biomarker for early stage non-small cell lung cancer (NSCLC) and patients with low hPAF1C expression levels had significantly better overall survival. Furthermore, the expression of hPAF1C was found to be positively correlated with c-MYC expression in patient tumor samples and in cancer cell lines. Mechanistic studies indicated that hPAF1C could promote lung cancer cell proliferation through regulating c-MYC transcription. These results demonstrated the prognostic value of hPAF1C in early-stage NSCLC and the role of hPAF1C in the transcriptional regulation of c-MYC oncogene during NSCLC tumorigenesis. - Highlights: • hPAF1C expression is a prognostic biomarker for early stage non-small cell lung cancer. • The expression of hPAF1C was positively correlated with c-MYC in tumor samples of patients and in several NSCLC cell lines. • hPAF1C could promote lung cancer cell proliferation through regulating c-MYC transcription.

Genomic binding profiles of functionally distinct RNA polymerase III transcription complexes in human cells.

Science.gov (United States)

Moqtaderi, Zarmik; Wang, Jie; Raha, Debasish; White, Robert J; Snyder, Michael; Weng, Zhiping; Struhl, Kevin

2010-05-01

Genome-wide occupancy profiles of five components of the RNA polymerase III (Pol III) machinery in human cells identified the expected tRNA and noncoding RNA targets and revealed many additional Pol III-associated loci, mostly near short interspersed elements (SINEs). Several genes are targets of an alternative transcription factor IIIB (TFIIIB) containing Brf2 instead of Brf1 and have extremely low levels of TFIIIC. Strikingly, expressed Pol III genes, unlike nonexpressed Pol III genes, are situated in regions with a pattern of histone modifications associated with functional Pol II promoters. TFIIIC alone associates with numerous ETC loci, via the B box or a novel motif. ETCs are often near CTCF binding sites, suggesting a potential role in chromosome organization. Our results suggest that human Pol III complexes associate preferentially with regions near functional Pol II promoters and that TFIIIC-mediated recruitment of TFIIIB is regulated in a locus-specific manner.

An X11alpha/FSBP complex represses transcription of the GSK3beta gene promoter.

LENUS (Irish Health Repository)

Lau, Kwok-Fai

2010-08-04

X11alpha is a neuronal adaptor protein that interacts with the amyloid precursor protein (APP) through a centrally located phosphotyrosine binding domain to inhibit the production of Abeta peptide that is deposited in Alzheimer\\'s disease brains. X11alpha also contains two C-terminal postsynaptic density-95, large discs, zona occludens 1 (PDZ) domains, and we show here that through its PDZ domains, X11alpha interacts with a novel transcription factor, fibrinogen silencer binding protein. Moreover, we show that an X11alpha\\/fibrinogen silencer binding protein complex signals to the nucleus to repress glycogen synthase kinase-3beta promoter activity. Glycogen synthase kinase-3beta is a favoured candidate kinase for phosphorylating tau in Alzheimer\\'s disease. Our findings show a new function for X11alpha that may impact on Alzheimer\\'s disease pathogenesis.

Colon cancer associated transcripts in human cancers.

Science.gov (United States)

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

2017-10-01

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

Transcriptional Waves in the Yeast Cell Cycle

OpenAIRE

Oliva, Anna; Rosebrock, Adam; Ferrezuelo, Francisco; Pyne, Saumyadipta; Chen, Haiying; Skiena, Steve; Futcher, Bruce; Leatherwood, Janet

2005-01-01

Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillat...

The Mediator Complex and Lipid Metabolism

OpenAIRE

Zhang, Yi; Xiaoli,; Zhao, Xiaoping; Yang, Fajun

2013-01-01

The precise control of gene expression is essential for all biological processes. In addition to DNA-binding transcription factors, numerous transcription cofactors contribute another layer of regulation of gene transcription in eukaryotic cells. One of such transcription cofactors is the highly conserved Mediator complex, which has multiple subunits and is involved in various biological processes through directly interacting with relevant transcription factors. Although the current understan...

Overexpression of the PAP1 transcription factor reveals a complex regulation of flavonoid and phenylpropanoid metabolism in Nicotiana tabacum plants attacked by Spodoptera litura.

Science.gov (United States)

Mitsunami, Tomoko; Nishihara, Masahiro; Galis, Ivan; Alamgir, Kabir Md; Hojo, Yuko; Fujita, Kohei; Sasaki, Nobuhiro; Nemoto, Keichiro; Sawasaki, Tatsuya; Arimura, Gen-ichiro

2014-01-01

Anthocyanin pigments and associated flavonoids have demonstrated antioxidant properties and benefits for human health. Consequently, current plant bioengineers have focused on how to modify flavonoid metabolism in plants. Most of that research, however, does not consider the role of natural biotic stresses (e.g., herbivore attack). To understand the influence of herbivore attack on the metabolic engineering of flavonoids, we examined tobacco plants overexpressing the Arabidopsis PAP1 gene (encoding an MYB transcription factor), which accumulated anthocyanin pigments and other flavonoids/phenylpropanoids. In comparison to wild-type and control plants, transgenic plants exhibited greater resistance to Spodoptera litura. Moreover, herbivory suppressed the PAP1-induced increase of transcripts of flavonoid/phenylpropanoid biosynthetic genes (e.g., F3H) and the subsequent accumulation of these genes' metabolites, despite the unaltered PAP1 mRNA levels after herbivory. The instances of down-regulation were independent of the signaling pathways mediated by defense-related jasmonates but were relevant to the levels of PAP1-induced and herbivory-suppressed transcription factors, An1a and An1b. Although initially F3H transcripts were suppressed by herbivory, after the S. litura feeding was interrupted, F3H transcripts increased. We hypothesize that in transgenic plants responding to herbivory, there is a complex mechanism regulating enriched flavonoid/phenylpropanoid compounds, via biotic stress signals.

Overexpression of the PAP1 transcription factor reveals a complex regulation of flavonoid and phenylpropanoid metabolism in Nicotiana tabacum plants attacked by Spodoptera litura.

Directory of Open Access Journals (Sweden)

Tomoko Mitsunami

Full Text Available Anthocyanin pigments and associated flavonoids have demonstrated antioxidant properties and benefits for human health. Consequently, current plant bioengineers have focused on how to modify flavonoid metabolism in plants. Most of that research, however, does not consider the role of natural biotic stresses (e.g., herbivore attack. To understand the influence of herbivore attack on the metabolic engineering of flavonoids, we examined tobacco plants overexpressing the Arabidopsis PAP1 gene (encoding an MYB transcription factor, which accumulated anthocyanin pigments and other flavonoids/phenylpropanoids. In comparison to wild-type and control plants, transgenic plants exhibited greater resistance to Spodoptera litura. Moreover, herbivory suppressed the PAP1-induced increase of transcripts of flavonoid/phenylpropanoid biosynthetic genes (e.g., F3H and the subsequent accumulation of these genes' metabolites, despite the unaltered PAP1 mRNA levels after herbivory. The instances of down-regulation were independent of the signaling pathways mediated by defense-related jasmonates but were relevant to the levels of PAP1-induced and herbivory-suppressed transcription factors, An1a and An1b. Although initially F3H transcripts were suppressed by herbivory, after the S. litura feeding was interrupted, F3H transcripts increased. We hypothesize that in transgenic plants responding to herbivory, there is a complex mechanism regulating enriched flavonoid/phenylpropanoid compounds, via biotic stress signals.

Overexpression of the PAP1 Transcription Factor Reveals a Complex Regulation of Flavonoid and Phenylpropanoid Metabolism in Nicotiana tabacum Plants Attacked by Spodoptera litura

Science.gov (United States)

Mitsunami, Tomoko; Nishihara, Masahiro; Galis, Ivan; Alamgir, Kabir Md; Hojo, Yuko; Fujita, Kohei; Sasaki, Nobuhiro; Nemoto, Keichiro; Sawasaki, Tatsuya; Arimura, Gen-ichiro

2014-01-01

Anthocyanin pigments and associated flavonoids have demonstrated antioxidant properties and benefits for human health. Consequently, current plant bioengineers have focused on how to modify flavonoid metabolism in plants. Most of that research, however, does not consider the role of natural biotic stresses (e.g., herbivore attack). To understand the influence of herbivore attack on the metabolic engineering of flavonoids, we examined tobacco plants overexpressing the Arabidopsis PAP1 gene (encoding an MYB transcription factor), which accumulated anthocyanin pigments and other flavonoids/phenylpropanoids. In comparison to wild-type and control plants, transgenic plants exhibited greater resistance to Spodoptera litura. Moreover, herbivory suppressed the PAP1-induced increase of transcripts of flavonoid/phenylpropanoid biosynthetic genes (e.g., F3H) and the subsequent accumulation of these genes' metabolites, despite the unaltered PAP1 mRNA levels after herbivory. The instances of down-regulation were independent of the signaling pathways mediated by defense-related jasmonates but were relevant to the levels of PAP1-induced and herbivory-suppressed transcription factors, An1a and An1b. Although initially F3H transcripts were suppressed by herbivory, after the S. litura feeding was interrupted, F3H transcripts increased. We hypothesize that in transgenic plants responding to herbivory, there is a complex mechanism regulating enriched flavonoid/phenylpropanoid compounds, via biotic stress signals. PMID:25268129

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

KAUST Repository

Schaefer, Ulf; Schmeier, Sebastian; Bajic, Vladimir B.

2010-01-01

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.

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.

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

Science.gov (United States)

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

2016-06-07

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

RelB and RelE of Escherichia coli Form a Tight Complex That Represses Transcription via The Ribbon-Helix-Helix Motif in RelB

DEFF Research Database (Denmark)

Overgaard, Martin; Borch, Jonas; Gerdes, Kenn

2009-01-01

RelB, the Ribbon-Helix-Helix (RHH) repressor encoded by the relBE toxin-antitoxin locus of Escherichia coli, forms a tight complex with RelE and thereby counteracts the mRNA cleavage activity of RelE. In addition, RelB dimers repress the strong relBE promoter and this repression by RelB is enhanced...... by RelE - that is - RelE functions as a transcriptional co-repressor. RelB is a Lon protease substrate and Lon is required both for activation of relBE transcription and for activation of the mRNA cleavage activity of RelE. Here we characterize the molecular interactions important for transcriptional...... motif recognizes four 6 bp repeats within the bipartite binding site. The spacing between each half-site was found to be essential for cooperative interactions between adjacently bound RelB dimers stabilized by the co-repressor RelE. Kinetic and stoichiometric measurements of the interaction between Rel...

5' diversity of human hepatic PXR (NR1I2) transcripts and identification of the major transcription initiation site.

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Kurose, Kouichi; Koyano, Satoru; Ikeda, Shinobu; Tohkin, Masahiro; Hasegawa, Ryuichi; Sawada, Jun-Ichi

2005-05-01

The human pregnane X receptor (PXR) is a crucial regulator of the genes encoding several major cytochrome P450 enzymes and transporters, such as CYP3A4 and MDR1, but its own transcriptional regulation remains unclear. To elucidate the transcriptional mechanisms of human PXR gene, we first endeavored to identify the transcription initiation site of human PXR using 5'-RACE. Five types of 5'-variable transcripts (a, b, c, d, and e) with common exon 2 sequence were found, and comparison of these sequences with the genomic sequence suggested that their 5' diversity is derived from initiation by alternative promoters and alternative splicing. None of the exons found in our study contain any new in-frame coding regions. Newly identified introns IVS-a and IVS-b were found to have CT-AC splice sites that do not follow the GT-AG rule of conventional donor and acceptor splice sites. Of the five types of 5' variable transcripts identified, RT-PCR showed that type-a was the major transcript type. Four transcription initiation sites (A-D) for type-a transcript were identified by 5'-RACE using GeneRacer RACE Ready cDNA (human liver) constructed by the oligo-capping method. Putative TATA boxes were located approximately 30 bp upstream from the transcriptional start sites of the major transcript (C) and the longest minor transcript (A) expressed in the human liver. These results indicate that the initiation of transcription of human PXR is more complex than previously reported.

Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation.

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Fishburn, James; Tomko, Eric; Galburt, Eric; Hahn, Steven

2015-03-31

Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5' → 3' direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

Transcription profiling suggests that mitochondrial topoisomerase IB acts as a topological barrier and regulator of mitochondrial DNA transcription.

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Dalla Rosa, Ilaria; Zhang, Hongliang; Khiati, Salim; Wu, Xiaolin; Pommier, Yves

2017-12-08

Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that Top1mt KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the Top1mt KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation.

A comprehensive analysis of microProteins reveals their potentially widespread mechanism of transcriptional regulation

NARCIS (Netherlands)

Magnani, Enrico; de Klein, Niek; Nam, Hye-In; Kim, Jung-Gun; Pham, Kimberly; Fiume, Elisa; Mudgett, Mary Beth; Rhee, Seung Yon

2014-01-01

Truncated transcription factor-like proteins called microProteins (miPs) can modulate transcription factor activities, thereby increasing transcriptional regulatory complexity. To understand their prevalence, evolution, and function, we predicted over 400 genes that encode putative miPs from

DNA Topoisomerases in Transcription

DEFF Research Database (Denmark)

Rødgaard, Morten Terpager

2015-01-01

This Ph.D. thesis summarizes the main results of my studies on the interplay between DNA topoisomerases and transcription. The work was performed from 2011 to 2015 at Aarhus University in the Laboratory of Genome Research, and was supervised by associate professor Anni H. Andersen. Most of the ex......This Ph.D. thesis summarizes the main results of my studies on the interplay between DNA topoisomerases and transcription. The work was performed from 2011 to 2015 at Aarhus University in the Laboratory of Genome Research, and was supervised by associate professor Anni H. Andersen. Most...... topoisomerase-DNA cleavage complex. The second study is an investigation of how topoisomerases influence gene regulation by keeping the genome in an optimal topological state....

THRAP3 interacts with and inhibits the transcriptional activity of SOX9 during chondrogenesis.

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Sono, Takashi; Akiyama, Haruhiko; Miura, Shigenori; Deng, Jian Min; Shukunami, Chisa; Hiraki, Yuji; Tsushima, Yu; Azuma, Yoshiaki; Behringer, Richard R; Matsuda, Shuichi

2018-07-01

Sex-determining region Y (Sry)-box (Sox)9 is required for chondrogenesis as a transcriptional activator of genes related to chondrocyte proliferation, differentiation, and cartilage-specific extracellular matrix. Although there have been studies investigating the Sox9-dependent transcriptional complexes, not all their components have been identified. In the present study, we demonstrated that thyroid hormone receptor-associated protein (THRAP)3 is a component of a SOX9 transcriptional complex by liquid chromatography mass spectrometric analysis of FLAG-tagged Sox9-binding proteins purified from FLAG-HA-tagged Sox9 knock-in mice. Thrap3 knockdown in ATDC5 chondrogenic cells increased the expression of Collagen type II alpha 1 chain (Col2a1) without affecting Sox9 expression. THRAP3 and SOX9 overexpression reduced Col2a1 levels to a greater degree than overexpression of SOX9 alone. The negative regulation of SOX9 transcriptional activity by THRAP3 was mediated by interaction between the proline-, glutamine-, and serine-rich domain of SOX9 and the innominate domain of THRAP3. These results indicate that THRAP3 negatively regulates SOX9 transcriptional activity as a cofactor of a SOX9 transcriptional complex during chondrogenesis.

Dynamics of Fos-Jun-NFAT1 complexes.

Science.gov (United States)

Ramirez-Carrozzi, V R; Kerppola, T K

2001-04-24

Transcription initiation in eukaryotes is controlled by nucleoprotein complexes formed through cooperative interactions among multiple transcription regulatory proteins. These complexes may be assembled via stochastic collisions or defined pathways. We investigated the dynamics of Fos-Jun-NFAT1 complexes by using a multicolor fluorescence resonance energy transfer assay. Fos-Jun heterodimers can bind to AP-1 sites in two opposite orientations, only one of which is populated in mature Fos-Jun-NFAT1 complexes. We studied the reversal of Fos-Jun binding orientation in response to NFAT1 by measuring the efficiencies of energy transfer from donor fluorophores linked to opposite ends of an oligonucleotide to an acceptor fluorophore linked to one subunit of the heterodimer. The reorientation of Fos-Jun by NFAT1 was not inhibited by competitor oligonucleotides or heterodimers. The rate of Fos-Jun reorientation was faster than the rate of heterodimer dissociation at some binding sites. The facilitated reorientation of Fos-Jun heterodimers therefore can enhance the efficiency of Fos-Jun-NFAT1 complex formation. We also examined the influence of the preferred orientation of Fos-Jun binding on the stability and transcriptional activity of Fos-Jun-NFAT1 complexes. Complexes formed at sites where Fos-Jun favored the same binding orientation in the presence and absence of NFAT1 exhibited an 8-fold slower dissociation rate than complexes formed at sites where Fos-Jun favored the opposite binding orientation. Fos-Jun-NFAT1 complexes also exhibited greater transcription activation at promoter elements that favored the same orientation of Fos-Jun binding in the presence and absence of NFAT1. Thus, the orientation of heterodimer binding can influence both the dynamics and promoter selectivity of multiprotein transcription regulatory complexes.

In Situ Tagged nsp15 Reveals Interactions with Coronavirus Replication/Transcription Complex-Associated Proteins

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Jeremiah Athmer

2017-01-01

Full Text Available Coronavirus (CoV replication and transcription are carried out in close proximity to restructured endoplasmic reticulum (ER membranes in replication/transcription complexes (RTC. Many of the CoV nonstructural proteins (nsps are required for RTC function; however, not all of their functions are known. nsp15 contains an endoribonuclease domain that is conserved in the CoV family. While the enzymatic activity and crystal structure of nsp15 are well defined, its role in replication remains elusive. nsp15 localizes to sites of RNA replication, but whether it acts independently or requires additional interactions for its function remains unknown. To begin to address these questions, we created an in situ tagged form of nsp15 using the prototypic CoV, mouse hepatitis virus (MHV. In MHV, nsp15 contains the genomic RNA packaging signal (P/S, a 95-bp RNA stem-loop structure that is not required for viral replication or nsp15 function. Utilizing this knowledge, we constructed an internal hemagglutinin (HA tag that replaced the P/S. We found that nsp15-HA was localized to discrete perinuclear puncta and strongly colocalized with nsp8 and nsp12, both well-defined members of the RTC, but not the membrane (M protein, involved in virus assembly. Finally, we found that nsp15 interacted with RTC-associated proteins nsp8 and nsp12 during infection, and this interaction was RNA independent. From this, we conclude that nsp15 localizes and interacts with CoV proteins in the RTC, suggesting it plays a direct or indirect role in virus replication. Furthermore, the use of in situ epitope tags could be used to determine novel nsp-nsp interactions in coronaviruses.

The crystal and solution structure of a putative transcriptional antiterminator from Mycobacterium tuberculosis

DEFF Research Database (Denmark)

Morth, J.P.; Feng, V.; Perry, L.J.

2004-01-01

We describe the crystal structure of Rv1626 from Mycobacterium tuberculosis at 1.48 A resolution and the corresponding solution structure determined from small angle X-ray scattering. The N-terminal domain shows structural homology to the receiver domains found in bacterial two-component systems....... regulators, so far only found in bacteria, and includes NasT, a protein from the assimilatory nitrate/nitrite reductase operon of Azetobacter vinelandii....

Regulation of metabolism by the Mediator complex.

Science.gov (United States)

Youn, Dou Yeon; Xiaoli, Alus M; Pessin, Jeffrey E; Yang, Fajun

2016-01-01

The Mediator complex was originally discovered in yeast, but it is conserved in all eukaryotes. Its best-known function is to regulate RNA polymerase II-dependent gene transcription. Although the mechanisms by which the Mediator complex regulates transcription are often complicated by the context-dependent regulation, this transcription cofactor complex plays a pivotal role in numerous biological pathways. Biochemical, molecular, and physiological studies using cancer cell lines or model organisms have established the current paradigm of the Mediator functions. However, the physiological roles of the mammalian Mediator complex remain poorly defined, but have attracted a great interest in recent years. In this short review, we will summarize some of the reported functions of selective Mediator subunits in the regulation of metabolism. These intriguing findings suggest that the Mediator complex may be an important player in nutrient sensing and energy balance in mammals.

Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

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Carlo Cattani

2011-01-01

Full Text Available This paper deals with the sequence analysis of acute myeloid leukemia mRNA. Six transcript variants of mlf1 mRNA, with more than 2000 bps, are analyzed by focusing on the autocorrelation of each distribution. Through the correlation matrix, some patches and similarities are singled out and commented, with respect to similar distributions. The comparison of Kolmogorov fractal dimension will be also given in order to classify the six variants. The existence of a fractal shape, patterns, and symmetries are discussed as well.

DNA Binding by the Ribosomal DNA Transcription Factor Rrn3 Is Essential for Ribosomal DNA Transcription*

Science.gov (United States)

Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H.; Rothblum, Katrina; Schneider, David A.; Rothblum, Lawrence I.

2013-01-01

The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382–400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I. PMID:23393135

DNA binding by the ribosomal DNA transcription factor rrn3 is essential for ribosomal DNA transcription.

Science.gov (United States)

Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H; Rothblum, Katrina; Schneider, David A; Rothblum, Lawrence I

2013-03-29

The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382-400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I.

Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning.

Science.gov (United States)

Albert, Nick W; Lewis, David H; Zhang, Huaibi; Schwinn, Kathy E; Jameson, Paula E; Davies, Kevin M

2011-03-01

We present an investigation of anthocyanin regulation over the entire petunia plant, determining the mechanisms governing complex floral pigmentation patterning and environmentally induced vegetative anthocyanin synthesis. DEEP PURPLE (DPL) and PURPLE HAZE (PHZ) encode members of the R2R3-MYB transcription factor family that regulate anthocyanin synthesis in petunia, and control anthocyanin production in vegetative tissues and contribute to floral pigmentation. In addition to these two MYB factors, the basic helix-loop-helix (bHLH) factor ANTHOCYANIN1 (AN1) and WD-repeat protein AN11, are also essential for vegetative pigmentation. The induction of anthocyanins in vegetative tissues by high light was tightly correlated to the induction of transcripts for PHZ and AN1. Interestingly, transcripts for PhMYB27, a putative R2R3-MYB active repressor, were highly expressed during non-inductive shade conditions and repressed during high light. The competitive inhibitor PhMYBx (R3-MYB) was expressed under high light, which may provide feedback repression. In floral tissues DPL regulates vein-associated anthocyanin pigmentation in the flower tube, while PHZ determines light-induced anthocyanin accumulation on exposed petal surfaces (bud-blush). A model is presented suggesting how complex floral and vegetative pigmentation patterns are derived in petunia in terms of MYB, bHLH and WDR co-regulators. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Proteins mediating DNA loops effectively block transcription.

Science.gov (United States)

Vörös, Zsuzsanna; Yan, Yan; Kovari, Daniel T; Finzi, Laura; Dunlap, David

2017-07-01

Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

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

NARCIS (Netherlands)

Abbink, Truus E. M.; Berkhout, Ben

2008-01-01

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

Extensive polycistronism and antisense transcription in the mammalian Hox clusters.

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Gaëll Mainguy

Full Text Available The Hox clusters play a crucial role in body patterning during animal development. They encode both Hox transcription factor and micro-RNA genes that are activated in a precise temporal and spatial sequence that follows their chromosomal order. These remarkable collinear properties confer functional unit status for Hox clusters. We developed the TranscriptView platform to establish high resolution transcriptional profiling and report here that transcription in the Hox clusters is far more complex than previously described in both human and mouse. Unannotated transcripts can represent up to 60% of the total transcriptional output of a cluster. In particular, we identified 14 non-coding Transcriptional Units antisense to Hox genes, 10 of which (70% have a detectable mouse homolog. Most of these Transcriptional Units in both human and mouse present conserved sizeable sequences (>40 bp overlapping Hox transcripts, suggesting that these Hox antisense transcripts are functional. Hox clusters also display at least seven polycistronic clusters, i.e., different genes being co-transcribed on long isoforms (up to 30 kb. This work provides a reevaluated framework for understanding Hox gene function and dys-function. Such extensive transcriptions may provide a structural explanation for Hox clustering.

Chick Hairy1 protein interacts with Sap18, a component of the Sin3/HDAC transcriptional repressor complex

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Andrade Raquel P

2007-07-01

Full Text Available Abstract Background The vertebrate adult axial skeleton, trunk and limb skeletal muscles and dermis of the back all arise from early embryonic structures called somites. Somites are symmetrically positioned flanking the embryo axial structures (neural tube and notochord and are periodically formed in a anterior-posterior direction from the presomitic mesoderm. The time required to form a somite pair is constant and species-specific. This extraordinary periodicity is proposed to depend on an underlying somitogenesis molecular clock, firstly evidenced by the cyclic expression of the chick hairy1 gene in the unsegmented presomitic mesoderm with a 90 min periodicity, corresponding to the time required to form a somite pair in the chick embryo. The number of hairy1 oscillations at any given moment is proposed to provide the cell with both temporal and positional information along the embryo's anterior-posterior axis. Nevertheless, how this is accomplished and what biological processes are involved is still unknown. Aiming at understanding the molecular events triggered by the somitogenesis clock Hairy1 protein, we have employed the yeast two-hybrid system to identify Hairy1 interaction partners. Results Sap18, an adaptor molecule of the Sin3/HDAC transcriptional repressor complex, was found to interact with the C-terminal portion of the Hairy1 protein in a yeast two-hybrid assay and the Hairy1/Sap18 interaction was independently confirmed by co-immunoprecipitation experiments. We have characterized the expression patterns of both sap18 and sin3a genes during chick embryo development, using in situ hybridization experiments. We found that both sap18 and sin3a expression patterns co-localize in vivo with hairy1 expression domains in chick rostral presomitic mesoderm and caudal region of somites. Conclusion Hairy1 belongs to the hairy-enhancer-of-split family of transcriptional repressor proteins. Our results indicate that during chick somitogenesis

Fungal mediator tail subunits contain classical transcriptional activation domains.

Science.gov (United States)

Liu, Zhongle; Myers, Lawrence C

2015-04-01

Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Transcription regulatory networks analysis using CAGE

KAUST Repository

Tegnér, Jesper N.

2009-10-01

Mapping out cellular networks in general and transcriptional networks in particular has proved to be a bottle-neck hampering our understanding of biological processes. Integrative approaches fusing computational and experimental technologies for decoding transcriptional networks at a high level of resolution is therefore of uttermost importance. Yet, this is challenging since the control of gene expression in eukaryotes is a complex multi-level process influenced by several epigenetic factors and the fine interplay between regulatory proteins and the promoter structure governing the combinatorial regulation of gene expression. In this chapter we review how the CAGE data can be integrated with other measurements such as expression, physical interactions and computational prediction of regulatory motifs, which together can provide a genome-wide picture of eukaryotic transcriptional regulatory networks at a new level of resolution. © 2010 by Pan Stanford Publishing Pte. Ltd. All rights reserved.

Nuclear stability and transcriptional directionality separate functionally distinct RNA species

DEFF Research Database (Denmark)

Andersson, Robin; Refsing Andersen, Peter; Valen, Eivind

2014-01-01

Mammalian genomes are pervasively transcribed, yielding a complex transcriptome with high variability in composition and cellular abundance. Although recent efforts have identified thousands of new long non-coding (lnc) RNAs and demonstrated a complex transcriptional repertoire produced by protei...

Bayesian error analysis model for reconstructing transcriptional regulatory networks

OpenAIRE

Sun, Ning; Carroll, Raymond J.; Zhao, Hongyu

2006-01-01

Transcription regulation is a fundamental biological process, and extensive efforts have been made to dissect its mechanisms through direct biological experiments and regulation modeling based on physical–chemical principles and mathematical formulations. Despite these efforts, transcription regulation is yet not well understood because of its complexity and limitations in biological experiments. Recent advances in high throughput technologies have provided substantial amounts and diverse typ...

FACT facilitates chromatin transcription by RNA polymerases I and III

DEFF Research Database (Denmark)

Birch, Joanna L; Tan, Bertrand C-M; Panov, Kostya I

2009-01-01

Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle....... The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results...... in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III...

Novel isoforms of the TFIID subunit TAF4 modulate nuclear receptor-mediated transcriptional activity

International Nuclear Information System (INIS)

Brunkhorst, Adrian; Neuman, Toomas; Hall, Anita; Arenas, Ernest; Bartfai, Tamas; Hermanson, Ola; Metsis, Madis

2004-01-01

The transcription factor TFIID consists of TATA-binding protein (TBP) and TBP-associated factors (TAFs). TAFs are essential for modulation of transcriptional activity but the regulation of TAFs is complex and many important aspects remain unclear. In this study, we have identified and characterized five novel truncated forms of the TFIID subunit TAF4 (TAF II 135). Analysis of the mouse gene structure revealed that all truncations were the results of alternative splicing and resulted in the loss of domains or parts of domains implicated in TAF4 functional interactions. Results from transcriptional assays showed that several of the TAF4 isoforms exerted dominant negative effects on TAF4 activity in nuclear receptor-mediated transcriptional activation. In addition, alternative TAF4 isoforms could be detected in specific cell types. Our results indicate an additional level of complexity in TAF4-mediated regulation of transcription and suggest context-specific roles for these new TAF4 isoforms in transcriptional regulation in vivo

Deciphering Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli

DEFF Research Database (Denmark)

Seo, Sang Woo; Kim, Donghyuk; Latif, Haythem

2014-01-01

The ferric uptake regulator (Fur) plays a critical role in the transcriptional regulation of iron metabolism. However, the full regulatory potential of Fur remains undefined. Here we comprehensively reconstruct the Fur transcriptional regulatory network in Escherichia coli K-12 MG1655 in response...

Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

DEFF Research Database (Denmark)

Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook

2015-01-01

A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co...

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

KAUST Repository

Kulakovskiy, Ivan V.

2011-08-18

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

Conflict Resolution in the Genome: How Transcription and Replication Make It Work.

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Hamperl, Stephan; Cimprich, Karlene A

2016-12-01

The complex machineries involved in replication and transcription translocate along the same DNA template, often in opposing directions and at different rates. These processes routinely interfere with each other in prokaryotes, and mounting evidence now suggests that RNA polymerase complexes also encounter replication forks in higher eukaryotes. Indeed, cells rely on numerous mechanisms to avoid, tolerate, and resolve such transcription-replication conflicts, and the absence of these mechanisms can lead to catastrophic effects on genome stability and cell viability. In this article, we review the cellular responses to transcription-replication conflicts and highlight how these inevitable encounters shape the genome and impact diverse cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

Murine Leukemia Virus Uses TREX Components for Efficient Nuclear Export of Unspliced Viral Transcripts

Directory of Open Access Journals (Sweden)

Toshie Sakuma

2014-03-01

Full Text Available Previously we reported that nuclear export of both unspliced and spliced murine leukemia virus (MLV transcripts depends on the nuclear export factor (NXF1 pathway. Although the mRNA export complex TREX, which contains Aly/REF, UAP56, and the THO complex, is involved in the NXF1-mediated nuclear export of cellular mRNAs, its contribution to the export of MLV mRNA transcripts remains poorly understood. Here, we studied the involvement of TREX components in the export of MLV transcripts. Depletion of UAP56, but not Aly/REF, reduced the level of both unspliced and spliced viral transcripts in the cytoplasm. Interestingly, depletion of THO components, including THOC5 and THOC7, affected only unspliced viral transcripts in the cytoplasm. Moreover, the RNA immunoprecipitation assay showed that only the unspliced viral transcript interacted with THOC5. These results imply that MLV requires UAP56, THOC5 and THOC7, in addition to NXF1, for nuclear export of viral transcripts. Given that naturally intronless mRNAs, but not bulk mRNAs, require THOC5 for nuclear export, it is plausible that THOC5 plays a key role in the export of unspliced MLV transcripts.

Deciphering Transcriptional Regulation

DEFF Research Database (Denmark)

Valen, Eivind

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

The Not5 subunit of the ccr4-not complex connects transcription and translation.

Directory of Open Access Journals (Sweden)

Zoltan Villanyi

2014-10-01

Full Text Available Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription.

Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway.

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Rajput, Pallavi; Pandey, Vijaya; Kumar, Vijay

2016-08-01

The well-studied Pol II transcription factor Sp1 has not been investigated for its regulatory role in rDNA transcription. Here, we show that Sp1 bound to specific sites on rDNA and localized into the nucleoli during the G1 phase of cell cycle to activate rDNA transcription. It facilitated the recruitment of Pol I pre-initiation complex and impeded the binding of nucleolar remodeling complex (NoRC) to rDNA resulting in the formation of euchromatin active state. More importantly, Sp1 also orchestrated the site-specific binding of Gadd45a-nucleotide excision repair (NER) complex resulting in active demethylation and transcriptional activation of rDNA. Interestingly, knockdown of Sp1 impaired rDNA transcription due to reduced engagement of the Gadd45a-NER complex and hypermethylation of rDNA. Thus, the present study unveils a novel role of Sp1 in rDNA transcription involving promoter demethylation. Copyright © 2016 Elsevier B.V. All rights reserved.

Transcriptional and phylogenetic analysis of five complete ambystomatid salamander mitochondrial genomes.

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Samuels, Amy K; Weisrock, David W; Smith, Jeramiah J; France, Katherine J; Walker, John A; Putta, Srikrishna; Voss, S Randal

2005-04-11

We report on a study that extended mitochondrial transcript information from a recent EST project to obtain complete mitochondrial genome sequence for 5 tiger salamander complex species (Ambystoma mexicanum, A. t. tigrinum, A. andersoni, A. californiense, and A. dumerilii). We describe, for the first time, aspects of mitochondrial transcription in a representative amphibian, and then use complete mitochondrial sequence data to examine salamander phylogeny at both deep and shallow levels of evolutionary divergence. The available mitochondrial ESTs for A. mexicanum (N=2481) and A. t. tigrinum (N=1205) provided 92% and 87% coverage of the mitochondrial genome, respectively. Complete mitochondrial sequences for all species were rapidly obtained by using long distance PCR and DNA sequencing. A number of genome structural characteristics (base pair length, base composition, gene number, gene boundaries, codon usage) were highly similar among all species and to other distantly related salamanders. Overall, mitochondrial transcription in Ambystoma approximated the pattern observed in other vertebrates. We inferred from the mapping of ESTs onto mtDNA that transcription occurs from both heavy and light strand promoters and continues around the entire length of the mtDNA, followed by post-transcriptional processing. However, the observation of many short transcripts corresponding to rRNA genes indicates that transcription may often terminate prematurely to bias transcription of rRNA genes; indeed an rRNA transcription termination signal sequence was observed immediately following the 16S rRNA gene. Phylogenetic analyses of salamander family relationships consistently grouped Ambystomatidae in a clade containing Cryptobranchidae and Hynobiidae, to the exclusion of Salamandridae. This robust result suggests a novel alternative hypothesis because previous studies have consistently identified Ambystomatidae and Salamandridae as closely related taxa. Phylogenetic analyses of tiger

Methodology for the analysis of transcription and translation in transcription-coupled-to-translation systems in vitro.

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Castro-Roa, Daniel; Zenkin, Nikolay

2015-09-15

The various properties of RNA polymerase (RNAP) complexes with nucleic acids during different stages of transcription involve various types of regulation and different cross-talk with other cellular entities and with fellow RNAP molecules. The interactions of transcriptional apparatus with the translational machinery have been focused mainly in terms of outcomes of gene expression, whereas the study of the physical interaction of the ribosome and the RNAP remains obscure partly due to the lack of a system that allows such observations. In this article we will describe the methodology needed to set up a pure, transcription-coupled-to-translation system in which the translocation of the ribosome can be performed in a step-wise manner towards RNAP allowing investigation of the interactions between the two machineries at colliding and non-colliding distances. In the same time RNAP can be put in various types of states, such as paused, roadblocked, backtracked, etc. The experimental system thus allows studying the effects of the ribosome on different aspects of transcription elongation and the effects by RNAP on translation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Proteomic Analysis of the Mediator Complex Interactome in Saccharomyces cerevisiae.

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Uthe, Henriette; Vanselow, Jens T; Schlosser, Andreas

2017-02-27

Here we present the most comprehensive analysis of the yeast Mediator complex interactome to date. Particularly gentle cell lysis and co-immunopurification conditions allowed us to preserve even transient protein-protein interactions and to comprehensively probe the molecular environment of the Mediator complex in the cell. Metabolic 15 N-labeling thereby enabled stringent discrimination between bona fide interaction partners and nonspecifically captured proteins. Our data indicates a functional role for Mediator beyond transcription initiation. We identified a large number of Mediator-interacting proteins and protein complexes, such as RNA polymerase II, general transcription factors, a large number of transcriptional activators, the SAGA complex, chromatin remodeling complexes, histone chaperones, highly acetylated histones, as well as proteins playing a role in co-transcriptional processes, such as splicing, mRNA decapping and mRNA decay. Moreover, our data provides clear evidence, that the Mediator complex interacts not only with RNA polymerase II, but also with RNA polymerases I and III, and indicates a functional role of the Mediator complex in rRNA processing and ribosome biogenesis.

eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci

DEFF Research Database (Denmark)

Mousavi, Kambiz; Zare, Hossein; Dell'orso, Stefania

2013-01-01

)RNA acted to activate the downstream myogenic genes. The deployment of transcriptional machinery to appropriate loci is contingent on chromatin accessibility, a rate-limiting step preceding Pol II assembly. By nuclease sensitivity assay, we found that eRNAs regulate genomic access of the transcriptional...... complex to defined regulatory regions. In conclusion, our data suggest that eRNAs contribute to establishing a cell-type-specific transcriptional circuitry by directing chromatin-remodeling events....

Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

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Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

2013-08-01

Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

Structural and functional aspects of winged-helix domains at the core of transcription initiation complexes.

Science.gov (United States)

Teichmann, Martin; Dumay-Odelot, Hélène; Fribourg, Sébastien

2012-01-01

The winged helix (WH) domain is found in core components of transcription systems in eukaryotes and prokaryotes. It represents a sub-class of the helix-turn-helix motif. The WH domain participates in establishing protein-DNA and protein-protein-interactions. Here, we discuss possible explanations for the enrichment of this motif in transcription systems.

p53 Maintains Genomic Stability by Preventing Interference between Transcription and Replication

Directory of Open Access Journals (Sweden)

Constance Qiao Xin Yeo

2016-04-01

Full Text Available p53 tumor suppressor maintains genomic stability, typically acting through cell-cycle arrest, senescence, and apoptosis. We discovered a function of p53 in preventing conflicts between transcription and replication, independent of its canonical roles. p53 deficiency sensitizes cells to Topoisomerase (Topo II inhibitors, resulting in DNA damage arising spontaneously during replication. Topoisomerase IIα (TOP2A-DNA complexes preferentially accumulate in isogenic p53 mutant or knockout cells, reflecting an increased recruitment of TOP2A to regulate DNA topology. We propose that p53 acts to prevent DNA topological stress originating from transcription during the S phase and, therefore, promotes normal replication fork progression. Consequently, replication fork progression is impaired in the absence of p53, which is reversed by transcription inhibition. Pharmacologic inhibition of transcription also attenuates DNA damage and decreases Topo-II-DNA complexes, restoring cell viability in p53-deficient cells. Together, our results demonstrate a function of p53 that may underlie its role in tumor suppression.

Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes*

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Hindman, Ryan; Gollnick, Paul

2016-01-01

Vaccinia virus early genes are transcribed immediately upon infection. Nucleoside triphosphate phosphohydrolase I (NPH I) is an essential component of the early gene transcription complex. NPH I hydrolyzes ATP to release transcripts during transcription termination. The ATPase activity of NPH I requires single-stranded (ss) DNA as a cofactor; however, the source of this cofactor within the transcription complex is not known. Based on available structures of transcription complexes it has been hypothesized that the ssDNA cofactor is obtained from the unpaired non-template strand within the transcription bubble. In vitro transcription on templates that lack portions of the non-template strand within the transcription bubble showed that the upstream portion of the transcription bubble is required for efficient NPH I-mediated transcript release. Complementarity between the template and non-template strands in this region is also required for NPH I-mediated transcript release. This observation complicates locating the source of the ssDNA cofactor within the transcription complex because removal of the non-template strand also disrupts transcription bubble reannealing. Prior studies have shown that ssRNA binds to NPH I, but it does not activate ATPase activity. Chimeric transcription templates with RNA in the non-template strand confirm that the source of the ssDNA cofactor for NPH I is the upstream portion of the non-template strand in the transcription bubble. Consistent with this conclusion we also show that isolated NPH I acts as a 5′ to 3′ translocase on single-stranded DNA. PMID:27189950

Archaeal RNA polymerase arrests transcription at DNA lesions.

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Gehring, Alexandra M; Santangelo, Thomas J

2017-01-01

Transcription elongation is not uniform and transcription is often hindered by protein-bound factors or DNA lesions that limit translocation and impair catalysis. Despite the high degree of sequence and structural homology of the multi-subunit RNA polymerases (RNAP), substantial differences in response to DNA lesions have been reported. Archaea encode only a single RNAP with striking structural conservation with eukaryotic RNAP II (Pol II). Here, we demonstrate that the archaeal RNAP from Thermococcus kodakarensis is sensitive to a variety of DNA lesions that pause and arrest RNAP at or adjacent to the site of DNA damage. DNA damage only halts elongation when present in the template strand, and the damage often results in RNAP arresting such that the lesion would be encapsulated with the transcription elongation complex. The strand-specific halt to archaeal transcription elongation on modified templates is supportive of RNAP recognizing DNA damage and potentially initiating DNA repair through a process akin to the well-described transcription-coupled DNA repair (TCR) pathways in Bacteria and Eukarya.

Deciphering Transcriptome and Complex Alternative Splicing Transcripts in Mammary Gland Tissues from Cows Naturally Infected with Staphylococcus aureus Mastitis

Science.gov (United States)

Jiang, Qiang; Yang, Chun Hong; Zhang, Yan; Sun, Yan; Li, Rong Ling; Wang, Chang Fa; Zhong, Ji Feng; Huang, Jin Ming

2016-01-01

Alternative splicing (AS) contributes to the complexity of the mammalian proteome and plays an important role in diseases, including infectious diseases. The differential AS patterns of these transcript sequences between the healthy (HS3A) and mastitic (HS8A) cows naturally infected by Staphylococcus aureus were compared to understand the molecular mechanisms underlying mastitis resistance and susceptibility. In this study, using the Illumina paired-end RNA sequencing method, 1352 differentially expressed genes (DEGs) with higher than twofold changes were found in the HS3A and HS8A mammary gland tissues. Gene ontology and KEGG pathway analyses revealed that the cytokine–cytokine receptor interaction pathway is the most significantly enriched pathway. Approximately 16k annotated unigenes were respectively identified in two libraries, based on the bovine Bos taurus UMD3.1 sequence assembly and search. A total of 52.62% and 51.24% annotated unigenes were alternatively spliced in term of exon skipping, intron retention, alternative 5′ splicing and alternative 3ʹ splicing. Additionally, 1,317 AS unigenes were HS3A-specific, whereas 1,093 AS unigenes were HS8A-specific. Some immune-related genes, such as ITGB6, MYD88, ADA, ACKR1, and TNFRSF1B, and their potential relationships with mastitis were highlighted. From Chromosome 2, 4, 6, 7, 10, 13, 14, 17, and 20, 3.66% (HS3A) and 5.4% (HS8A) novel transcripts, which harbor known quantitative trait locus associated with clinical mastitis, were identified. Many DEGs in the healthy and mastitic mammary glands are involved in immune, defense, and inflammation responses. These DEGs, which exhibit diverse and specific splicing patterns and events, can endow dairy cattle with the potential complex genetic resistance against mastitis. PMID:27459697

Elk3 from hamster-a ternary complex factor with strong transcriptional repressor activity

DEFF Research Database (Denmark)

Hjortoe, G.M.; Weilguny, D.; Willumsen, Berthe Marie

2005-01-01

the transcription of genes that are activated during entry into G1. We have isolated the Cricetulus griseus Elk3 gene from the Chinese hamster ovary (CHO) cell line and investigated the transcriptional potential of this factor. Transient transfections revealed that, in addition to its regulation of the c......-fos promoter, Elk3 from CHO cells seems to inhibit other promoters controlling expression of proteins involved in G1/S phase progression; Cyclin D1 and DHFR. As has been described for the Elk3 homologs Net (Mouse) and Sap-2 (Human), the results of the present study further indicate that hamster Elk3...

Metabolic Network Topology Reveals Transcriptional Regulatory Signatures of Type 2 Diabetes

DEFF Research Database (Denmark)

Zelezniak, Aleksej; Pers, Tune Hannes; Pinho Soares, Simao Pedro

2010-01-01

mechanisms underlying these transcriptional changes and their impact on the cellular metabolic phenotype is a challenging task due to the complexity of transcriptional regulation and the highly interconnected nature of the metabolic network. In this study we integrate skeletal muscle gene expression datasets...... with human metabolic network reconstructions to identify key metabolic regulatory features of T2DM. These features include reporter metabolites—metabolites with significant collective transcriptional response in the associated enzyme-coding genes, and transcription factors with significant enrichment...... factor regulatory network connecting several parts of metabolism. The identified transcription factors include members of the CREB, NRF1 and PPAR family, among others, and represent regulatory targets for further experimental analysis. Overall, our results provide a holistic picture of key metabolic...

Prevalence of transcription promoters within archaeal operons and coding sequences.

Science.gov (United States)

Koide, Tie; Reiss, David J; Bare, J Christopher; Pang, Wyming Lee; Facciotti, Marc T; Schmid, Amy K; Pan, Min; Marzolf, Bruz; Van, Phu T; Lo, Fang-Yin; Pratap, Abhishek; Deutsch, Eric W; Peterson, Amelia; Martin, Dan; Baliga, Nitin S

2009-01-01

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of approximately 64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein-DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3' ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes-events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements.

Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells.

Science.gov (United States)

Li, LiQi; Jothi, Raja; Cui, Kairong; Lee, Jan Y; Cohen, Tsadok; Gorivodsky, Marat; Tzchori, Itai; Zhao, Yangu; Hayes, Sandra M; Bresnick, Emery H; Zhao, Keji; Westphal, Heiner; Love, Paul E

2011-02-01

The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non-DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex-binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs.

Genome-wide identification and characterization of Notch transcription complex-binding sequence paired sites in leukemia cells

Science.gov (United States)

Severson, Eric; Arnett, Kelly L.; Wang, Hongfang; Zang, Chongzhi; Taing, Len; Liu, Hudan; Pear, Warren S.; Liu, X. Shirley; Blacklow, Stephen C.; Aster, Jon C.

2018-01-01

Notch transcription complexes (NTCs) drive target gene expression by binding to two distinct types of genomic response elements, NTC monomer-binding sites and sequence-paired sites (SPSs) that bind NTC dimers. SPSs are conserved and are linked to the Notch-responsiveness of a few genes, but their overall contribution to Notch-dependent gene regulation is unknown. To address this issue, we determined the DNA sequence requirements for NTC dimerization using a fluorescence resonance energy transfer (FRET) assay, and applied insights from these in vitro studies to Notch-“addicted” leukemia cells. We find that SPSs contribute to the regulation of approximately a third of direct Notch target genes. While originally described in promoters, SPSs are present mainly in long-range enhancers, including an enhancer containing a newly described SPS that regulates HES5. Our work provides a general method for identifying sequence-paired sites in genome-wide data sets and highlights the widespread role of NTC dimerization in Notch-transformed leukemia cells. PMID:28465412

Coordinating repair of oxidative DNA damage with transcription and replication

International Nuclear Information System (INIS)

Cooper, P.K.

2003-01-01

Transcription-coupled repair (TCR) preferentially removes DNA lesions from template strands of active genes. Defects in TCR, which acts both on lesions removed by nucleotide excision repair (NER) and on oxidative lesions removed by base excision repair (BER), underlie the fatal developmental disorder Cockayne syndrome. Although its detailed mechanism remains unknown, TCR involves recognition of a stalled RNA polymerase (RNAP), removal or remodeling of RNAP to allow access to the lesion, and recruitment of repair enzymes. At a minimum, these early steps require a non-enzymatic function of the multifunctional repair protein XPG, the CSB protein with ATP-dependent chromatin remodeling activity, and the TFIIH complex (including the XPB and XPD helicases) that is also required for basal transcription initiation and NER. XPG exists in the cell in a complex with TFIIH, and in vitro evidence has suggested that it interacts with CSB. To address the mechanism of TCR, we are characterizing protein-DNA and protein-protein interactions of XPG. We show that XPG preferentially binds to double-stranded DNA containing bubbles resembling in size the unpaired regions associated with transcription. Two distinct domains of XPG are required for the observed strong binding specificity and stability. XPG both interacts directly with CSB and synergistically binds with it to bubble DNA, and it strongly stimulates the bubble DNA-dependent ATPase activity of CSB. Significantly for TCR, XPG also interacts directly with RNAP II, binds both the protein and nucleic acid components (the R-loop) of a stalled RNA polymerase, and forms a ternary complex with CSB and the stalled RNAP. These results are consistent with the model that XPG and CSB jointly interact with the DNA/chromatin structure in the vicinity of the stalled transcriptional apparatus and with the transcriptional machinery itself to remodel the chromatin and either move or remodel the blocked RNA polymerase to expose the lesion

Protein Phosphatase 1-Dependent Transcriptional Programs for Long-Term Memory and Plasticity

Science.gov (United States)

Graff, Johannes; Koshibu, Kyoko; Jouvenceau, Anne; Dutar, Patrick; Mansuy, Isabelle M.

2010-01-01

Gene transcription is essential for the establishment and the maintenance of long-term memory (LTM) and for long-lasting forms of synaptic plasticity. The molecular mechanisms that control gene transcription in neuronal cells are complex and recruit multiple signaling pathways in the cytoplasm and the nucleus. Protein kinases (PKs) and…

Post-transcriptional trafficking and regulation of neuronal gene expression.

Science.gov (United States)

Goldie, Belinda J; Cairns, Murray J

2012-02-01

Intracellular messenger RNA (mRNA) traffic and translation must be highly regulated, both temporally and spatially, within eukaryotic cells to support the complex functional partitioning. This capacity is essential in neurons because it provides a mechanism for rapid input-restricted activity-dependent protein synthesis in individual dendritic spines. While this feature is thought to be important for synaptic plasticity, the structures and mechanisms that support this capability are largely unknown. Certainly specialized RNA binding proteins and binding elements in the 3' untranslated region (UTR) of translationally regulated mRNA are important, but the subtlety and complexity of this system suggests that an intermediate "specificity" component is also involved. Small non-coding microRNA (miRNA) are essential for CNS development and may fulfill this role by acting as the guide strand for mediating complex patterns of post-transcriptional regulation. In this review we examine post-synaptic gene regulation, mRNA trafficking and the emerging role of post-transcriptional gene silencing in synaptic plasticity.

The Wnt Transcriptional Switch: TLE Removal or Inactivation?

Science.gov (United States)

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

2018-02-01

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

Step out of the groove : epigenetic gene control systems and engineered transcription factors

NARCIS (Netherlands)

Verschure, P.J.; Visser, A.E.; Rots, M.G.

2006-01-01

At the linear DNA level, gene activity is believed to be driven by binding of transcription factors, which subsequently recruit the RNA polymerase to the gene promoter region. However, it has become clear that transcriptional activation involves large complexes of many different proteins, which not

Dynamic usage of transcription start sites within core promoters

DEFF Research Database (Denmark)

Kawaji, Hideya; Frith, Martin C; Katayama, Shintaro

2006-01-01

BACKGROUND: Mammalian promoters do not initiate transcription at single, well defined base pairs, but rather at multiple, alternative start sites spread across a region. We previously characterized the static structures of transcription start site usage within promoters at the base pair level......, based on large-scale sequencing of transcript 5' ends. RESULTS: In the present study we begin to explore the internal dynamics of mammalian promoters, and demonstrate that start site selection within many mouse core promoters varies among tissues. We also show that this dynamic usage of start sites...... is associated with CpG islands, broad and multimodal promoter structures, and imprinting. CONCLUSION: Our results reveal a new level of biologic complexity within promoters--fine-scale regulation of transcription starting events at the base pair level. These events are likely to be related to epigenetic...

A human transcription factor in search mode.

Science.gov (United States)

Hauser, Kevin; Essuman, Bernard; He, Yiqing; Coutsias, Evangelos; Garcia-Diaz, Miguel; Simmerling, Carlos

2016-01-08

Transcription factors (TF) can change shape to bind and recognize DNA, shifting the energy landscape from a weak binding, rapid search mode to a higher affinity recognition mode. However, the mechanism(s) driving this conformational change remains unresolved and in most cases high-resolution structures of the non-specific complexes are unavailable. Here, we investigate the conformational switch of the human mitochondrial transcription termination factor MTERF1, which has a modular, superhelical topology complementary to DNA. Our goal was to characterize the details of the non-specific search mode to complement the crystal structure of the specific binding complex, providing a basis for understanding the recognition mechanism. In the specific complex, MTERF1 binds a significantly distorted and unwound DNA structure, exhibiting a protein conformation incompatible with binding to B-form DNA. In contrast, our simulations of apo MTERF1 revealed significant flexibility, sampling structures with superhelical pitch and radius complementary to the major groove of B-DNA. Docking these structures to B-DNA followed by unrestrained MD simulations led to a stable complex in which MTERF1 was observed to undergo spontaneous diffusion on the DNA. Overall, the data support an MTERF1-DNA binding and recognition mechanism driven by intrinsic dynamics of the MTERF1 superhelical topology. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

CREB and FoxO1: two transcription factors for the regulation of hepatic gluconeogenesis

Science.gov (United States)

Oh, Kyoung-Jin; Han, Hye-Sook; Kim, Min-Jung; Koo, Seung-Hoi

2013-01-01

Liver plays a major role in maintaining glucose homeostasis in mammals. Under fasting conditions, hepatic glucose production is critical as a source of fuel to maintain the basic functions in other tissues, including skeletal muscle, red blood cells, and the brain. Fasting hormones glucagon and cortisol play major roles during the process, in part by activating the transcription of key enzyme genes in the gluconeogenesis such as phosphoenol pyruvate carboxykinase (PEPCK) and glucose 6 phosphatase catalytic subunit (G6Pase). Conversely, gluconeogenic transcription is repressed by pancreatic insulin under feeding conditions, which effectively inhibits transcriptional activator complexes by either promoting post-translational modifications or activating transcriptional inhibitors in the liver, resulting in the reduction of hepatic glucose output. The transcriptional regulatory machineries have been highlighted as targets for type 2 diabetes drugs to control glycemia, so understanding of the complex regulatory mechanisms for transcription circuits for hepatic gluconeogenesis is critical in the potential development of therapeutic tools for the treatment of this disease. In this review, the current understanding regarding the roles of two key transcriptional activators, CREB and FoxO1, in the regulation of hepatic gluconeogenic program is discussed. [BMB Reports 2013; 46(12): 567-574] PMID:24238363

The Drosophila Helicase MLE Targets Hairpin Structures in Genomic Transcripts.

Directory of Open Access Journals (Sweden)

Simona Cugusi

2016-01-01

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

The logic of communication: roles for mobile transcription factors in plants.

Science.gov (United States)

Long, Yuchen; Scheres, Ben; Blilou, Ikram

2015-02-01

Mobile transcription factors play many roles in plant development. Here, we compare the use of mobile transcription factors as signals with some canonical signal transduction processes in prokaryotes and eukaryotes. After an initial survey, we focus on the SHORT-ROOT pathway in Arabidopsis roots to show that, despite the simplicity of the concept of mobile transcription factor signalling, many lines of evidence reveal a surprising complexity in control mechanisms linked to this process. We argue that these controls bestow precision, robustness, and versatility on mobile transcription factor signalling. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Distributed biotin-streptavidin transcription roadblocks for mapping cotranscriptional RNA folding.

Science.gov (United States)

Strobel, Eric J; Watters, Kyle E; Nedialkov, Yuri; Artsimovitch, Irina; Lucks, Julius B

2017-07-07

RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2΄-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks. While effective, the distribution of elongation complexes using EcoRIE111Q requires laborious PCR using many different oligonucleotides for each sequence analyzed. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent biotin-streptavidin (SAv) roadblocking strategy that simplifies the preparation of roadblocking DNA templates. We first determine the properties of biotin-SAv roadblocks. We then show that randomly distributed biotin-SAv roadblocks can be used in cotranscriptional SHAPE-Seq experiments to identify the same RNA structural transitions related to a riboswitch decision-making process that we previously identified using EcoRIE111Q. Lastly, we find that EcoRIE111Q maps nascent RNA structure to specific transcript lengths more precisely than biotin-SAv and propose guidelines to leverage the complementary strengths of each transcription roadblock in cotranscriptional SHAPE-Seq. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Transcription of minute virus of mice, an autonomous parvovirus, may be regulated by attenuation

International Nuclear Information System (INIS)

Ben-Asher, E.; Aloni, Y.

1984-01-01

To characterize the transcriptional organization and regulation of minute virus of mice, an autonomous parvovirus, viral transcriptional complexes were isolated and cleaved with restriction enzymes. The in vivo preinitiated nascent RNA was elongated in vitro in the presence of [alpha- 32 P]UTP to generate runoff transcripts. The lengths of the runoff transcripts were analyzed by gel electrophoresis under denaturing conditions. On the basis of the map locations of the restriction sites and the lengths of the runoff transcripts, the in vivo initiation sites were determined. Two major initiation sites having similar activities were thus identified at residues 201 +/- 5 and 2005 +/- 5; both of them were preceded by a TATAA sequence. When uncleaved viral transcriptional complexes or isolated nuclei were incubated in vitro in the presence of [alpha- 32 P]UTP or [alpha- 32 P]CTP, they synthesized labeled RNA that, as determined by polyacrylamide gel electrophoresis, contained a major band of 142 nucleotides. The RNA of the major band was mapped between the initiation site at residue 201 +/- 5 and residue 342. We noticed the potential of forming two mutually exclusive stem-and-loop structures in the 142-nucleotide RNA; one of them is followed by a string of uridylic acid residues typical of a procaryotic transcription termination signal. We propose that, as in the transcription of simian virus 40, RNA transcription in minute virus of mice may be regulated by attenuation and may involve eucaryotic polymerase B, which can respond to a transcription termination signal similar to that of the procaryotic polymerase

Transcriptional regulation of Caenorhabditis elegans FOXO/DAF-16 modulates lifespan.

Science.gov (United States)

Bansal, Ankita; Kwon, Eun-Soo; Conte, Darryl; Liu, Haibo; Gilchrist, Michael J; MacNeil, Lesley T; Tissenbaum, Heidi A

2014-01-01

Insulin/IGF-1 signaling plays a central role in longevity across phylogeny. In C. elegans, the forkhead box O (FOXO) transcription factor, DAF-16, is the primary target of insulin/IGF-1 signaling, and multiple isoforms of DAF-16 (a, b, and d/f) modulate lifespan, metabolism, dauer formation, and stress resistance. Thus far, across phylogeny modulation of mammalian FOXOs and DAF-16 have focused on post-translational regulation with little focus on transcriptional regulation. In C. elegans, we have previously shown that DAF-16d/f cooperates with DAF-16a to promote longevity. In this study, we generated transgenic strains expressing near-endogenous levels of either daf-16a or daf-16d/f, and examined temporal expression of the isoforms to further define how these isoforms contribute to lifespan regulation. Here, we show that DAF-16a is sensitive both to changes in gene dosage and to alterations in the level of insulin/IGF-1 signaling. Interestingly, we find that as worms age, the intestinal expression of daf-16d/f but not daf-16a is dramatically upregulated at the level of transcription. Preventing this transcriptional upregulation shortens lifespan, indicating that transcriptional regulation of daf-16d/f promotes longevity. In an RNAi screen of transcriptional regulators, we identify elt-2 (GATA transcription factor) and swsn-1 (core subunit of SWI/SNF complex) as key modulators of daf-16d/f gene expression. ELT-2 and another GATA factor, ELT-4, promote longevity via both DAF-16a and DAF-16d/f while the components of SWI/SNF complex promote longevity specifically via DAF-16d/f. Our findings indicate that transcriptional control of C. elegans FOXO/daf-16 is an essential regulatory event. Considering the conservation of FOXO across species, our findings identify a new layer of FOXO regulation as a potential determinant of mammalian longevity and age-related diseases such as cancer and diabetes.

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

KAUST Repository

Schmeier, Sebastian; Alam, Tanvir; Essack, Magbubah; Bajic, Vladimir B.

2016-01-01

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/.

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/.

Molding a peptide into an RNA site by in vivo peptide evolution

OpenAIRE

Harada, Kazuo; Martin, Shelley S.; Tan, Ruoying; Frankel, Alan D.

1997-01-01

Short peptides corresponding to the arginine-rich domains of several RNA-binding proteins are able to bind to their specific RNA sites with high affinities and specificities. In the case of the HIV-1 Rev-Rev response element (RRE) complex, the peptide forms a single α-helix that binds deeply in a widened, distorted RNA major groove and makes a substantial set of base-specific and backbone contacts. Using a reporter system based on antitermination by the bacteriophage λ N protein, it has been ...

An Optogenetic Platform for Real-Time, Single-Cell Interrogation of Stochastic Transcriptional Regulation.

Science.gov (United States)

Rullan, Marc; Benzinger, Dirk; Schmidt, Gregor W; Milias-Argeitis, Andreas; Khammash, Mustafa

2018-05-17

Transcription is a highly regulated and inherently stochastic process. The complexity of signal transduction and gene regulation makes it challenging to analyze how the dynamic activity of transcriptional regulators affects stochastic transcription. By combining a fast-acting, photo-regulatable transcription factor with nascent RNA quantification in live cells and an experimental setup for precise spatiotemporal delivery of light inputs, we constructed a platform for the real-time, single-cell interrogation of transcription in Saccharomyces cerevisiae. We show that transcriptional activation and deactivation are fast and memoryless. By analyzing the temporal activity of individual cells, we found that transcription occurs in bursts, whose duration and timing are modulated by transcription factor activity. Using our platform, we regulated transcription via light-driven feedback loops at the single-cell level. Feedback markedly reduced cell-to-cell variability and led to qualitative differences in cellular transcriptional dynamics. Our platform establishes a flexible method for studying transcriptional dynamics in single cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Repression of class I transcription by cadmium is mediated by the protein phosphatase 2A

Science.gov (United States)

Zhou, Lei; Le Roux, Gwenaëlle; Ducrot, Cécile; Chédin, Stéphane; Labarre, Jean; Riva, Michel; Carles, Christophe

2013-01-01

Toxic metals are part of our environment, and undue exposure to them leads to a variety of pathologies. In response, most organisms adapt their metabolism and have evolved systems to limit this toxicity and to acquire tolerance. Ribosome biosynthesis being central for protein synthesis, we analyzed in yeast the effects of a moderate concentration of cadmium (Cd2+) on Pol I transcription that represents >60% of the transcriptional activity of the cells. We show that Cd2+ rapidly and drastically shuts down the expression of the 35S rRNA. Repression does not result from a poisoning of any of the components of the class I transcriptional machinery by Cd2+, but rather involves a protein phosphatase 2A (PP2A)-dependent cellular signaling pathway that targets the formation/dissociation of the Pol I–Rrn3 complex. We also show that Pol I transcription is repressed by other toxic metals, such as Ag+ and Hg2+, which likewise perturb the Pol I–Rrn3 complex, but through PP2A-independent mechanisms. Taken together, our results point to a central role for the Pol I–Rrn3 complex as molecular switch for regulating Pol I transcription in response to toxic metals. PMID:23640330

Gene expression patterns of oxidative phosphorylation complex I subunits are organized in clusters.

Directory of Open Access Journals (Sweden)

Yael Garbian

Full Text Available After the radiation of eukaryotes, the NUO operon, controlling the transcription of the NADH dehydrogenase complex of the oxidative phosphorylation system (OXPHOS complex I, was broken down and genes encoding this protein complex were dispersed across the nuclear genome. Seven genes, however, were retained in the genome of the mitochondrion, the ancient symbiote of eukaryotes. This division, in combination with the three-fold increase in subunit number from bacteria (N = approximately 14 to man (N = 45, renders the transcription regulation of OXPHOS complex I a challenge. Recently bioinformatics analysis of the promoter regions of all OXPHOS genes in mammals supported patterns of co-regulation, suggesting that natural selection favored a mechanism facilitating the transcriptional regulatory control of genes encoding subunits of these large protein complexes. Here, using real time PCR of mitochondrial (mtDNA- and nuclear DNA (nDNA-encoded transcripts in a panel of 13 different human tissues, we show that the expression pattern of OXPHOS complex I genes is regulated in several clusters. Firstly, all mtDNA-encoded complex I subunits (N = 7 share a similar expression pattern, distinct from all tested nDNA-encoded subunits (N = 10. Secondly, two sub-clusters of nDNA-encoded transcripts with significantly different expression patterns were observed. Thirdly, the expression patterns of two nDNA-encoded genes, NDUFA4 and NDUFA5, notably diverged from the rest of the nDNA-encoded subunits, suggesting a certain degree of tissue specificity. Finally, the expression pattern of the mtDNA-encoded ND4L gene diverged from the rest of the tested mtDNA-encoded transcripts that are regulated by the same promoter, consistent with post-transcriptional regulation. These findings suggest, for the first time, that the regulation of complex I subunits expression in humans is complex rather than reflecting global co-regulation.

Post-translational regulation of Oct4 transcriptional activity.

Directory of Open Access Journals (Sweden)

Jonathan P Saxe

Full Text Available Oct4 is a key component of the molecular circuitry which regulates embryonic stem cell proliferation and differentiation. It is essential for maintenance of undifferentiated, pluripotent cell populations, and accomplishes these tasks by binding DNA in multiple heterodimer and homodimer configurations. Very little is known about how formation of these complexes is regulated, or the mechanisms through which Oct4 proteins respond to complex extracellular stimuli which regulate pluripotency. Here, we provide evidence for a phosphorylation-based mechanism which regulates specific Oct4 homodimer conformations. Point mutations of a putative phosphorylation site can specifically abrogate transcriptional activity of a specific homodimer assembly, with little effect on other configurations. Moreover, we performed bioinformatic predictions to identify a subset of Oct4 target genes which may be regulated by this specific assembly, and show that altering Oct4 protein levels affects transcription of Oct4 target genes which are regulated by this assembly but not others. Finally, we identified several signaling pathways which may mediate this phosphorylation and act in combination to regulate Oct4 transcriptional activity and protein stability. These results provide a mechanism for rapid and reversible alteration of Oct4 transactivation potential in response to extracellular signals.

Susceptibility to bystander DNA damage is influenced by replication and transcriptional activity

Science.gov (United States)

Dickey, Jennifer S.; Baird, Brandon J.; Redon, Christophe E.; Avdoshina, Valeriya; Palchik, Guillermo; Wu, Junfang; Kondratyev, Alexei; Bonner, William M.; Martin, Olga A.

2012-01-01

Direct cellular DNA damage may lead to genome destabilization in unexposed, bystander, cells sharing the same milieu with directly damaged cells by means of the bystander effect. One proposed mechanism involves double strand break (DSB) formation in S phase cells at sites of single strand lesions in the DNA of replication complexes, which has a more open structure compared with neighboring DNA. The DNA in transcription complexes also has a more open structure, and hence may be susceptible to bystander DSB formation from single strand lesions. To examine whether transcription predisposes non-replicating cells to bystander effect-induced DNA DSBs, we examined two types of primary cells that exhibit high levels of transcription in the absence of replication, rat neurons and human lymphocytes. We found that non-replicating bystander cells with high transcription rates exhibited substantial levels of DNA DSBs, as monitored by γ-H2AX foci formation. Additionally, as reported in proliferating cells, TGF-β and NO were found to mimic bystander effects in cell populations lacking DNA synthesis. These results indicate that cell vulnerability to bystander DSB damage may result from transcription as well as replication. The findings offer insights into which tissues may be vulnerable to bystander genomic destabilization in vivo. PMID:22941641

Binding of transcription termination protein nun to nascent RNA and template DNA.

Science.gov (United States)

Watnick, R S; Gottesman, M E

1999-12-17

The amino-terminal arginine-rich motif of coliphage HK022 Nun binds phage lambda nascent transcript, whereas the carboxyl-terminal domain interacts with RNA polymerase (RNAP) and blocks transcription elongation. RNA binding is inhibited by zinc (Zn2+) and stimulated by Escherichia coli NusA. To study these interactions, the Nun carboxyl terminus was extended by a cysteine residue conjugated to a photochemical cross-linker. The carboxyl terminus contacted NusA and made Zn2+-dependent intramolecular contacts. When Nun was added to a paused transcription elongation complex, it cross-linked to the DNA template. Nun may arrest transcription by anchoring RNAP to DNA.

Cryptic Transcription and Early Termination in the Control of Gene Expression

Directory of Open Access Journals (Sweden)

Jessie Colin

2011-01-01

Full Text Available Recent studies on yeast transcriptome have revealed the presence of a large set of RNA polymerase II transcripts mapping to intergenic and antisense regions or overlapping canonical genes. Most of these ncRNAs (ncRNAs are subject to termination by the Nrd1-dependent pathway and rapid degradation by the nuclear exosome and have been dubbed cryptic unstable transcripts (CUTs. CUTs are often considered as by-products of transcriptional noise, but in an increasing number of cases they play a central role in the control of gene expression. Regulatory mechanisms involving expression of a CUT are diverse and include attenuation, transcriptional interference, and alternative transcription start site choice. This review focuses on the impact of cryptic transcription on gene expression, describes the role of the Nrd1-complex as the main actor in preventing nonfunctional and potentially harmful transcription, and details a few systems where expression of a CUT has an essential regulatory function. We also summarize the most recent studies concerning other types of ncRNAs and their possible role in regulation.

Polyphenol Compound as a Transcription Factor Inhibitor

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Seyeon Park

2015-10-01

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

Polyphenol Compound as a Transcription Factor Inhibitor.

Science.gov (United States)

Park, Seyeon

2015-10-30

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

Characterization of Mediator Complex and its Associated Proteins from Rice.

Science.gov (United States)

Samanta, Subhasis; Thakur, Jitendra Kumar

2017-01-01

The Mediator complex is a multi-protein complex that acts as a molecular bridge conveying transcriptional messages from the cis element-bound transcription factor to the RNA Polymerase II machinery. It is found in all eukaryotes including members of the plant kingdom. Increasing number of reports from plants regarding different Mediator subunits involved in a multitude of processes spanning from plant development to environmental interactions have firmly established it as a central hub of plant regulatory networks. Routine isolation of Mediator complex in a particular species is a necessity because of many reasons. First, composition of the Mediator complex varies from species to species. Second, the composition of the Mediator complex in a particular species is not static under all developmental and environmental conditions. Besides this, at times, Mediator complex is used in in vitro transcription systems. Rice, a staple food crop of the world, is used as a model monocot crop. Realizing the need of a reliable protocol for the isolation of Mediator complex from plants, we describe here the isolation of Mediator complex from rice.

A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo

DEFF Research Database (Denmark)

Damgaard, Christian Kroun; Kahns, Søren; Lykke-Andersen, Søren

2008-01-01

RNAs, harboring wild-type or various 5′ splice site mutations, we demonstrate a strong positive correlation between splicing efficiency and transcription activity. Interestingly, a 5′ splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced...... a promoter-proximal 5′ splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing preinitiation complex assembly....

Enhanceosomes as integrators of hypoxia inducible factor (HIF) and other transcription factors in the hypoxic transcriptional response.

Science.gov (United States)

Pawlus, Matthew R; Hu, Cheng-Jun

2013-09-01

Hypoxia is a prevalent attribute of the solid tumor microenvironment that promotes the expression of genes through posttranslational modifications and stabilization of alpha subunits (HIF1α and HIF2α) of hypoxia-inducible factors (HIFs). Despite significant similarities, HIF1 (HIF1α/ARNT) and HIF2 (HIF2α/ARNT) activate common as well as unique target genes and exhibit different functions in cancer biology. More surprisingly, accumulating data indicates that the HIF1- and/or HIF2-mediated hypoxia responses can be oncogenic as well as tumor suppressive. While the role of HIF in the hypoxia response is well established, recent data support the concept that HIF is necessary, but not sufficient for the hypoxic response. Other transcription factors that are activated by hypoxia are also required for the HIF-mediated hypoxia response. HIFs, other transcription factors, co-factors and RNA poll II recruited by HIF and other transcription factors form multifactorial enhanceosome complexes on the promoters of HIF target genes to activate hypoxia inducible genes. Importantly, HIF1 or HIF2 requires distinct partners in activating HIF1 or HIF2 target genes. Because HIF enhanceosome formation is required for the gene activation and distinct functions of HIF1 and HIF2 in tumor biology, disruption of the HIF1 or HIF2 specific enhanceosome complex may prove to be a beneficial strategy in tumor treatment in which tumor growth is specifically dependent upon HIF1 or HIF2 activity. Copyright © 2013 Elsevier Inc. All rights reserved.

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity.

Science.gov (United States)

Traverse, Charles C; Ochman, Howard

2017-08-29

Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola , which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location. IMPORTANCE The high level of mistakes generated during transcription can result in the accumulation of malfunctioning and misfolded proteins which can alter global gene regulation and in the expenditure of energy to degrade these nonfunctional proteins. The transcriptome-wide occurrence of base substitutions has been elucidated in bacteria, but information on transcription insertions and deletions-errors that potentially have more dire effects on protein function-is limited to reporter gene constructs. Here, we capture the transcriptome-wide spectrum of insertions and deletions in Escherichia coli and Buchnera aphidicola and show that they occur at rates approaching those of base substitutions

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

OpenAIRE

Hurst, H C; Masson, N; Jones, N C; Lee, K A

1990-01-01

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We...

Mediator Undergoes a Compositional Change during Transcriptional Activation.

Science.gov (United States)

Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

2016-11-03

Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter. Copyright © 2016 Elsevier Inc. All rights reserved.

TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity

DEFF Research Database (Denmark)

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

2011-01-01

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

Alkane Biosynthesis Genes in Cyanobacteria and Their Transcriptional Organization

International Nuclear Information System (INIS)

Klähn, Stephan; Baumgartner, Desirée; Pfreundt, Ulrike; Voigt, Karsten; Schön, Verena; Steglich, Claudia; Hess, Wolfgang R.

2014-01-01

In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl–acyl carrier protein reductase and aldehyde deformylating oxygenase. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short-chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado) and sll0209 (aar), which give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313, and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in cyanobacteria.

Alkane biosynthesis genes in cyanobacteria and their transcriptional organization

Directory of Open Access Journals (Sweden)

Stephan eKlähn

2014-07-01

Full Text Available In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl-acyl carrier protein reductase (AAR and aldehyde deformylating oxygenase (ADO. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado and sll0209 (aar, that give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313 and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in

Alkane Biosynthesis Genes in Cyanobacteria and Their Transcriptional Organization

Energy Technology Data Exchange (ETDEWEB)

Klähn, Stephan; Baumgartner, Desirée; Pfreundt, Ulrike; Voigt, Karsten; Schön, Verena; Steglich, Claudia; Hess, Wolfgang R., E-mail: wolfgang.hess@biologie.uni-freiburg.de [Genetics and Experimental Bioinformatics, Institute of Biology 3, Faculty of Biology, University of Freiburg, Freiburg (Germany)

2014-07-14

In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl–acyl carrier protein reductase and aldehyde deformylating oxygenase. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short-chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado) and sll0209 (aar), which give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313, and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in cyanobacteria.

Circadian Enhancers Coordinate Multiple Phases of Rhythmic Gene Transcription In Vivo

Science.gov (United States)

Fang, Bin; Everett, Logan J.; Jager, Jennifer; Briggs, Erika; Armour, Sean M.; Feng, Dan; Roy, Ankur; Gerhart-Hines, Zachary; Sun, Zheng; Lazar, Mitchell A.

2014-01-01

SUMMARY Mammalian transcriptomes display complex circadian rhythms with multiple phases of gene expression that cannot be accounted for by current models of the molecular clock. We have determined the underlying mechanisms by measuring nascent RNA transcription around the clock in mouse liver. Unbiased examination of eRNAs that cluster in specific circadian phases identified functional enhancers driven by distinct transcription factors (TFs). We further identify on a global scale the components of the TF cistromes that function to orchestrate circadian gene expression. Integrated genomic analyses also revealed novel mechanisms by which a single circadian factor controls opposing transcriptional phases. These findings shed new light on the diversity and specificity of TF function in the generation of multiple phases of circadian gene transcription in a mammalian organ. PMID:25416951

Insulated transcriptional elements enable precise design of genetic circuits.

Science.gov (United States)

Zong, Yeqing; Zhang, Haoqian M; Lyu, Cheng; Ji, Xiangyu; Hou, Junran; Guo, Xian; Ouyang, Qi; Lou, Chunbo

2017-07-03

Rational engineering of biological systems is often complicated by the complex but unwanted interactions between cellular components at multiple levels. Here we address this issue at the level of prokaryotic transcription by insulating minimal promoters and operators to prevent their interaction and enable the biophysical modeling of synthetic transcription without free parameters. This approach allows genetic circuit design with extraordinary precision and diversity, and consequently simplifies the design-build-test-learn cycle of circuit engineering to a mix-and-match workflow. As a demonstration, combinatorial promoters encoding NOT-gate functions were designed from scratch with mean errors of 96% using our insulated transcription elements. Furthermore, four-node transcriptional networks with incoherent feed-forward loops that execute stripe-forming functions were obtained without any trial-and-error work. This insulation-based engineering strategy improves the resolution of genetic circuit technology and provides a simple approach for designing genetic circuits for systems and synthetic biology.Unwanted interactions between cellular components can complicate rational engineering of biological systems. Here the authors design insulated minimal promoters and operators that enable biophysical modeling of bacterial transcription without free parameters for precise circuit design.

Function and regulation of the Mediator complex.

Science.gov (United States)

Conaway, Ronald C; Conaway, Joan Weliky

2011-04-01

Over the past few years, advances in biochemical and genetic studies of the structure and function of the Mediator complex have shed new light on its subunit architecture and its mechanism of action in transcription by RNA polymerase II (pol II). The development of improved methods for reconstitution of recombinant Mediator subassemblies is enabling more in-depth analyses of basic features of the mechanisms by which Mediator interacts with and controls the activity of pol II and the general initiation factors. The discovery and characterization of multiple, functionally distinct forms of Mediator characterized by the presence or absence of the Cdk8 kinase module have led to new insights into how Mediator functions in both Pol II transcription activation and repression. Finally, progress in studies of the mechanisms by which the transcriptional activation domains (ADs) of DNA binding transcription factors target Mediator have brought to light unexpected complexities in the way Mediator participates in signal transduction. Copyright © 2011 Elsevier Ltd. All rights reserved.

TRE5-A retrotransposition profiling reveals putative RNA polymerase III transcription complex binding sites on the Dictyostelium extrachromosomal rDNA element.

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Thomas Spaller

Full Text Available The amoeba Dictyostelium discoideum has a haploid genome in which two thirds of the DNA encodes proteins. Consequently, the space available for selfish mobile elements to expand without excess damage to the host genome is limited. The non-long terminal repeat retrotransposon TRE5-A maintains an active population in the D. discoideum genome and apparently adapted to this gene-dense environment by targeting positions ~47 bp upstream of tRNA genes that are devoid of protein-coding regions. Because only ~24% of tRNA genes are associated with a TRE5-A element in the reference genome, we evaluated whether TRE5-A retrotransposition is limited to this subset of tRNA genes. We determined that a tagged TRE5-A element (TRE5-Absr integrated at 384 of 405 tRNA genes, suggesting that expansion of the current natural TRE5-A population is not limited by the availability of targets. We further observed that TRE5-Absr targets the ribosomal 5S gene on the multicopy extrachromosomal DNA element that carries the ribosomal RNA genes, indicating that TRE5-A integration may extend to the entire RNA polymerase III (Pol III transcriptome. We determined that both natural TRE5-A and cloned TRE5-Absr retrotranspose to locations on the extrachromosomal rDNA element that contain tRNA gene-typical A/B box promoter motifs without displaying any other tRNA gene context. Based on previous data suggesting that TRE5-A targets tRNA genes by locating Pol III transcription complexes, we propose that A/B box loci reflect Pol III transcription complex assembly sites that possess a function in the biology of the extrachromosomal rDNA element.

Transcriptional landscape estimation from tiling array data using a model of signal shift and drift

DEFF Research Database (Denmark)

Rasmussen, Simon; Jarmer, Hanne Østergaard; Nicolas, P

2009-01-01

MOTIVATION: High-density oligonucleotide tiling array technology holds the promise of a better description of the complexity and the dynamics of transcriptional landscapes. In organisms such as bacteria and yeasts, transcription can be measured on a genome-wide scale with a resolution >25 bp...

“Jump Start and Gain” Model for Dosage Compensation in Drosophila Based on Direct Sequencing of Nascent Transcripts

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Francesco Ferrari

2013-11-01

Full Text Available Dosage compensation in Drosophila is mediated by the MSL complex, which increases male X-linked gene expression approximately 2-fold. The MSL complex preferentially binds the bodies of active genes on the male X, depositing H4K16ac with a 3′ bias. Two models have been proposed for the influence of the MSL complex on transcription: one based on promoter recruitment of RNA polymerase II (Pol II, and a second featuring enhanced transcriptional elongation. Here, we utilize nascent RNA sequencing to document dosage compensation during transcriptional elongation. We also compare X and autosomes from published data on paused and elongating polymerase in order to assess the role of Pol II recruitment. Our results support a model for differentially regulated elongation, starting with release from 5′ pausing and increasing through X-linked gene bodies. Our results highlight facilitated transcriptional elongation as a key mechanism for the coordinated regulation of a diverse set of genes.

Functional characterization of tobacco transcription factor TGA2.1

DEFF Research Database (Denmark)

Kegler, C.; Lenk, I.; Krawczyk, S.

2004-01-01

Activation sequence-1 (as-1)-like regulatory cis elements mediate transcriptional activation in response to increased levels of plant signalling molecules auxin and salicylic acid (SA). Our earlier work has shown that tobacco cellular as-1-binding complex SARP (salicylic acid responsive protein...

Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment.

Science.gov (United States)

Eyboulet, Fanny; Cibot, Camille; Eychenne, Thomas; Neil, Helen; Alibert, Olivier; Werner, Michel; Soutourina, Julie

2013-12-01

Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3' endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes.

Gibberellic acid and cGMP-dependent transcriptional regulation in arabidopsis thaliana

KAUST Repository

Bastian, René

2010-03-01

An ever increasing amount of transcriptomic data and analysis tools provide novel insight into complex responses of biological systems. Given these resources we have undertaken to review aspects of transcriptional regulation in response to the plant hormone gibberellic acid (GA) and its second messenger guanosine 3\\',5\\'-cyclic monophosphate (cGMP) in Arabidopsis thaliana, both wild type and selected mutants. Evidence suggests enrichment of GA-responsive (GARE) elements in promoters of genes that are transcriptionally upregulated in response to cGMP but downregulated in a GA insensitive mutant (ga1-3). In contrast, in the genes upregulated in the mutant, no enrichment in the GARE is observed suggesting that GARE motifs are diagnostic for GA-induced and cGMP-dependent transcriptional upregulation. Further, we review how expression studies of GA-dependent transcription factors and transcriptional networks based on common promoter signatures derived from ab initio analyses can contribute to our understanding of plant responses at the systems level. © 2010 Landes Bioscience.

In vitro fluorescence studies of transcription factor IIB-DNA interaction.

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Górecki, Andrzej; Figiel, Małgorzata; Dziedzicka-Wasylewska, Marta

2015-01-01

General transcription factor TFIIB is one of the basal constituents of the preinitiation complex of eukaryotic RNA polymerase II, acting as a bridge between the preinitiation complex and the polymerase, and binding promoter DNA in an asymmetric manner, thereby defining the direction of the transcription. Methods of fluorescence spectroscopy together with circular dichroism spectroscopy were used to observe conformational changes in the structure of recombinant human TFIIB after binding to specific DNA sequence. To facilitate the exploration of the structural changes, several site-directed mutations have been introduced altering the fluorescence properties of the protein. Our observations showed that binding of specific DNA sequences changed the protein structure and dynamics, and TFIIB may exist in two conformational states, which can be described by a different microenvironment of W52. Fluorescence studies using both intrinsic and exogenous fluorophores showed that these changes significantly depended on the recognition sequence and concerned various regions of the protein, including those interacting with other transcription factors and RNA polymerase II. DNA binding can cause rearrangements in regions of proteins interacting with the polymerase in a manner dependent on the recognized sequences, and therefore, influence the gene expression.

Protein intrinsic disorder in Arabidopsis NAC transcription factors

DEFF Research Database (Denmark)

O'Shea, Charlotte; Jensen, Mikael Kryger; Stender, Emil G.P.

2015-01-01

of differences in binding mechanisms. Although substitution of both hydrophobic and acidic residues of the ANAC046 MoRF region abolished binding, substitution of other residues, even with α-helix-breaking proline, was less disruptive. Together, the biophysical analyses suggest that RCD1-ANAC046 complex formation......Protein ID (intrinsic disorder) plays a significant, yet relatively unexplored role in transcription factors (TFs). In the present paper, analysis of the transcription regulatory domains (TRDs) of six phylogenetically representative, plant-specific NAC [no apical meristem, ATAF (Arabidopsis...

Transcriptional Regulatory Network Analysis of MYB Transcription Factor Family Genes in Rice

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Shuchi eSmita

2015-12-01

Full Text Available MYB transcription factor (TF is one of the largest TF families and regulates defense responses to various stresses, hormone signaling as well as many metabolic and developmental processes in plants. Understanding these regulatory hierarchies of gene expression networks in response to developmental and environmental cues is a major challenge due to the complex interactions between the genetic elements. Correlation analyses are useful to unravel co-regulated gene pairs governing biological process as well as identification of new candidate hub genes in response to these complex processes. High throughput expression profiling data are highly useful for construction of co-expression networks. In the present study, we utilized transcriptome data for comprehensive regulatory network studies of MYB TFs by top down and guide gene approaches. More than 50% of OsMYBs were strongly correlated under fifty experimental conditions with 51 hub genes via top down approach. Further, clusters were identified using Markov Clustering (MCL. To maximize the clustering performance, parameter evaluation of the MCL inflation score (I was performed in terms of enriched GO categories by measuring F-score. Comparison of co-expressed cluster and clads analyzed from phylogenetic analysis signifies their evolutionarily conserved co-regulatory role. We utilized compendium of known interaction and biological role with Gene Ontology enrichment analysis to hypothesize function of coexpressed OsMYBs. In the other part, the transcriptional regulatory network analysis by guide gene approach revealed 40 putative targets of 26 OsMYB TF hubs with high correlation value utilizing 815 microarray data. The putative targets with MYB-binding cis-elements enrichment in their promoter region, functional co-occurrence as well as nuclear localization supports our finding. Specially, enrichment of MYB binding regions involved in drought-inducibility implying their regulatory role in drought

Transcriptional and chromatin regulation during fasting – The genomic era

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Goldstein, Ido; Hager, Gordon L.

2015-01-01

An elaborate metabolic response to fasting is orchestrated by the liver and is heavily reliant upon transcriptional regulation. In response to hormones (glucagon, glucocorticoids) many transcription factors (TFs) are activated and regulate various genes involved in metabolic pathways aimed at restoring homeostasis: gluconeogenesis, fatty acid oxidation, ketogenesis and amino acid shuttling. We summarize the recent discoveries regarding fasting-related TFs with an emphasis on genome-wide binding patterns. Collectively, the summarized findings reveal a large degree of co-operation between TFs during fasting which occurs at motif-rich DNA sites bound by a combination of TFs. These new findings implicate transcriptional and chromatin regulation as major determinants of the response to fasting and unravels the complex, multi-TF nature of this response. PMID:26520657

Engineering yeast transcription machinery for improved ethanol tolerance and production.

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Alper, Hal; Moxley, Joel; Nevoigt, Elke; Fink, Gerald R; Stephanopoulos, Gregory

2006-12-08

Global transcription machinery engineering (gTME) is an approach for reprogramming gene transcription to elicit cellular phenotypes important for technological applications. Here we show the application of gTME to Saccharomyces cerevisiae for improved glucose/ethanol tolerance, a key trait for many biofuels programs. Mutagenesis of the transcription factor Spt15p and selection led to dominant mutations that conferred increased tolerance and more efficient glucose conversion to ethanol. The desired phenotype results from the combined effect of three separate mutations in the SPT15 gene [serine substituted for phenylalanine (Phe(177)Ser) and, similarly, Tyr(195)His, and Lys(218)Arg]. Thus, gTME can provide a route to complex phenotypes that are not readily accessible by traditional methods.

Pokemon (FBI-1) interacts with Smad4 to repress TGF-β-induced transcriptional responses.

Science.gov (United States)

Yang, Yutao; Cui, Jiajun; Xue, Feng; Zhang, Chuanfu; Mei, Zhu; Wang, Yue; Bi, Mingjun; Shan, Dapeng; Meredith, Alex; Li, Hui; Xu, Zhi-Qing David

2015-03-01

Pokemon, an important proto-oncoprotein, is a transcriptional repressor that belongs to the POK (POZ and Krüppel) family. Smad4, a key component of TGF-β pathway, plays an essential role in TGF-β-induced transcriptional responses. In this study, we show that Pokemon can interact directly with Smad4 both in vitro and in vivo. Overexpression of Pokemon decreases TGF-β-induced transcriptional activities, whereas knockdown of Pokemon increases these activities. Interestingly, Pokemon does not affect activation of Smad2/3, formation of Smads complex, or DNA binding activity of Smad4. TGF-β1 treatment increases the interaction between Pokemon and Smad4, and also enhances the recruitment of Pokemon to Smad4-DNA complex. In addition, we also find that Pokemon recruits HDAC1 to Smad4 complex but decreases the interaction between Smad4 and p300/CBP. Taken together, all these data suggest that Pokemon is a new partner of Smad4 and plays a negative role in TGF-β pathway. Copyright © 2014. Published by Elsevier B.V.

Myocardin-related transcription factors are required for cardiac development and function

OpenAIRE

Mokalled, Mayssa H.; Carroll, Kelli J.; Cenik, Bercin K.; Chen, Beibei; Liu, Ning; Olson, Eric N.; Bassel-Duby, Rhonda

2015-01-01

Myocardin-Related Transcription Factors A and B (MRTF-A and MRTF-B) are highly homologous proteins that function as powerful coactivators of serum response factor (SRF), a ubiquitously expressed transcription factor essential for cardiac development. The SRF/MRTF complex binds to CArG boxes found in the control regions of genes that regulate cytoskeletal dynamics and muscle contraction, among other processes. While SRF is required for heart development and function, the role of MRTFs in the d...

Insights into the post-transcriptional regulation of the mitochondrial electron transport chain.

Science.gov (United States)

Sirey, Tamara M; Ponting, Chris P

2016-10-15

The regulation of the mitochondrial electron transport chain is central to the control of cellular homeostasis. There are significant gaps in our understanding of how the expression of the mitochondrial and nuclear genome-encoded components of the electron transport chain are co-ordinated, and how the assembly of the protein complexes that constitute the electron transport chain are regulated. Furthermore, the role post-transcriptional gene regulation may play in modulating these processes needs to be clarified. This review summarizes the current knowledge regarding the post-transcriptional gene regulation of the electron transport chain and highlights how noncoding RNAs may contribute significantly both to complex electron transport chain regulatory networks and to mitochondrial dysfunction. © 2016 The Author(s).

CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex.

Science.gov (United States)

Bae, Brian; Chen, James; Davis, Elizabeth; Leon, Katherine; Darst, Seth A; Campbell, Elizabeth A

2015-09-08

A key point to regulate gene expression is at transcription initiation, and activators play a major role. CarD, an essential activator in Mycobacterium tuberculosis, is found in many bacteria, including Thermus species, but absent in Escherichia coli. To delineate the molecular mechanism of CarD, we determined crystal structures of Thermus transcription initiation complexes containing CarD. The structures show CarD interacts with the unique DNA topology presented by the upstream double-stranded/single-stranded DNA junction of the transcription bubble. We confirm that our structures correspond to functional activation complexes, and extend our understanding of the role of a conserved CarD Trp residue that serves as a minor groove wedge, preventing collapse of the transcription bubble to stabilize the transcription initiation complex. Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter complexes, explaining the need for CarD.

Role of the σ⁵⁴ Activator Interacting Domain in Bacterial Transcription Initiation

Energy Technology Data Exchange (ETDEWEB)

Siegel, Alexander R. [Univ. of California, Berkeley, CA (United States); Wemmer, David E. [Univ. of California, Berkeley, CA (United States)

2016-10-11

Bacterial sigma factors are subunits of RNA polymerase that direct the holoenzyme to specific sets of promoters in the genome and are a central element of regulating transcription. Most polymerase holoenzymes open the promoter and initiate transcription rapidly after binding. However, polymerase containing the members of the σ⁵⁴ family must be acted on by a transcriptional activator before DNA opening and initiation occur. A key domain in these transcriptional activators forms a hexameric AAA + ATPase that acts through conformational changes brought on by ATP hydrolysis. Contacts between the transcriptional activator and σ⁵⁴ are primarily made through an N-terminal σ⁵⁴ activator interacting domain (AID). To better understand this mechanism of bacterial transcription initiation, we characterized the σ⁵⁴ AID by NMR spectroscopy and other biophysical methods and show that it is an intrinsically disordered domain in σ⁵⁴ alone. In this paper, we identified a minimal construct of the Aquifex aeolicus σ⁵⁴ AID that consists of two predicted helices and retains native-like binding affinity for the transcriptional activator NtrC1. Using the NtrC1 ATPase domain, bound with the non-hydrolyzable ATP analog ADP-beryllium fluoride, we studied the NtrC1–σ⁵⁴ AID complex using NMR spectroscopy. We show that the σ⁵⁴ AID becomes structured after associating with the core loops of the transcriptional activators in their ATP state and that the primary site of the interaction is the first predicted helix. Finally, understanding this complex, formed as the first step toward initiation, will help unravel the mechanism of σ⁵⁴ bacterial transcription initiation.

TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter

OpenAIRE

Wright, Kevin J.; Marr, Michael T.; Tjian, Robert

2006-01-01

Activator-dependent recruitment of TFIID initiates formation of the transcriptional preinitiation complex. TFIID binds core promoter DNA elements and directs the assembly of other general transcription factors, leading to binding of RNA polymerase II and activation of RNA synthesis. How TATA box-binding protein (TBP) and the TBP-associated factors (TAFs) are assembled into a functional TFIID complex with promoter recognition and coactivator activities in vivo remains unknown. Here, we use RNA...

A broad but restricted requirement for TAF-5 (human TAFII100) for embryonic transcription in Caenorhabditis elegans.

Science.gov (United States)

Walker, Amy K; Blackwell, T Keith

2003-02-21

As conserved components of the transcription factor (TF) IID- and TFTC/SAGA-related complexes, TATA-binding protein-associated factors (TAF(II)s) are important for eukaryotic mRNA transcription. In yeast, genetic analyses suggest that, although some individual TAF(II)s are required for transcription of most genes, others have highly specialized functions. Much less is known about the functions of TAF(II)s in metazoans, which have more complex genomes that include many tissue-specific genes. TAF-5 (human (h) TAF(II)100) is of particular interest because it is predicted to have an important structural role. Here we describe the first genetics-based analysis of TAF-5 in a metazoan. By performing RNA interference in Caenorhabditis elegans embryos, which can survive for several cell generations without transcription, we found that taf-5 is important for a significant fraction of transcription. However, TAF-5 is apparently not essential for the expression of multiple developmental and other metazoan-specific genes. This phenotype remarkably resembles the previously described effects of similarly depleting two C. elegans histone fold TAF(II)s, TAF-9 (hTAF(II)31/32) and TAF-10 (hTAF(II)30), but is distinct from the widespread transcription block caused by TAF-4 (hTAF(II)130) depletion. Our findings suggest that TAF-5, TAF-9, and TAF-10 are part of a functional module of TFIID- and TFTC/SAGA-related complexes that can be bypassed in many metazoan-specific genes.

Microphthalmia-associated transcription factor (MITF – from Waardenburg syndrome genetics to melanoma therapy

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Ivan Šamija

2010-11-01

Full Text Available Microphthalmia-associated transcription factor (MITF was first discovered as protein coded by gene whose mutations are associated with Waardenburg syndrome. Later, MITF was shown to be key transcription factor regulating melanogenesis. Further studies have shown that in addition to regulating melanogenesis MITF also plays central role in regulation of melanocyte development and survival. MITF gene is amplified in a proportion of melanomas and ectopic MITF expression can transform melanocytes so MITF can function as melanoma “lineage survival” oncogene. Different studies have further revealed MITF’s important but complex role in tumorigenesis and progression of melanoma. As expected from its important role in melanocytes and melanoma MITF is intricately regulated on all the levels from transcription to post-translational modifications. Although complex mechanisms of MITF functioning are still being revealed, MITF already has a valuable role in managing melanoma patients. Immunohistochemical analysis of MITF has shown both diagnostic and prognostic value in patients with melanoma. MITF is also a valuable specific marker for detection of circulating melanoma cells by reverse-transcription – polymerase chain reaction. MITF has recently been investigated as a potential target for melanoma therapy.

25-hydroxycholesterol promotes RANKL-induced osteoclastogenesis through coordinating NFATc1 and Sp1 complex in the transcription of miR-139-5p

International Nuclear Information System (INIS)

Zhang, Lishan; Lv, Yinping; Xian, Guozhe; Lin, Yanliang

2017-01-01

25-hydroxycholesterol (25-HC) is implicated in many processes, including lipid metabolism and the immune response. However, the role of 25-HC in RANKL-induced osteoclastogenesis remains largely unknown. Our results showed that 25-HC inhibited miR-139-5p expression in mouse bone marrow macrophages (BMMs) cultured in receptor activator of NF-κB ligand (RANKL) and monocyte macrophage colony-stimulating factor (M-CSF). Further investigation suggested that 25-HC promoted the expression of nuclear factor of activated T cell cytoplasmic 1 (NFATc1) and Sp1, especially in the presence of RANKL and M-CSF. Meanwhile, 25-HC induced nuclear translocation of NFATc1, resulting in the interaction between NFATc1 and Sp1 that was confirmed by co-immunoprecipitation. Chromatin immunoprecipitation assay indicated that Sp1 could bind to miR-139-5p promoter, but NFATc1 had no binding capacity. Although forming NFATc1/Sp1 complex increased its binding to miR-139-5p promoter, the complex inhibited the transcriptional activity of Sp1. Inhibition of NFATc1 increase the expression of miR-139-5p, which might be due to the release of free Sp1 that could bind to the promoter of miR-139-5p. Enforced expression of miR-139-5p impaired osteoclastogenesis induced by co-treatment with 25-HC and RANKL. These results suggested that 25-HC induced the interaction between NFATc1 and Sp1, reducing the level of free Sp1 to inhibit miR-139-5p expression and promote osteoclastogenesis. - Highlights: • 25-hydroxycholesterol inhibited miR-139-5p expression in bone marrow macrophages. • 25-hydroxycholesterol promoted the expression of NFATc1 and Sp1. • 25-hydroxycholesterol induced the interaction between NFATc1 and Sp1. • NFATc1/Sp1 complex inhibited the transcription of miR-139-5p. • MiR-139-5p impaired osteoclastogenesis induced by 25-hydroxycholesterol and RANKL.

Transcription elongation factor GreA has functional chaperone activity.

Science.gov (United States)

Li, Kun; Jiang, Tianyi; Yu, Bo; Wang, Limin; Gao, Chao; Ma, Cuiqing; Xu, Ping; Ma, Yanhe

2012-01-01

Bacterial GreA is an indispensable factor in the RNA polymerase elongation complex. It plays multiple roles in transcriptional elongation, and may be implicated in resistance to various stresses. In this study, we show that Escherichia coli GreA inhibits aggregation of several substrate proteins under heat shock condition. GreA can also effectively promote the refolding of denatured proteins. These facts reveal that GreA has chaperone activity. Distinct from many molecular chaperones, GreA does not form stable complexes with unfolded substrates. GreA overexpression confers the host cells with enhanced resistance to heat shock and oxidative stress. Moreover, GreA expression in the greA/greB double mutant could suppress the temperature-sensitive phenotype, and dramatically alleviate the in vivo protein aggregation. The results suggest that bacterial GreA may act as chaperone in vivo. These results suggest that GreA, in addition to its function as a transcription factor, is involved in protection of cellular proteins against aggregation.

Purification, crystallization and preliminary X-ray analysis of OsAREB8 from rice, a member of the AREB/ABF family of bZIP transcription factors, in complex with its cognate DNA

International Nuclear Information System (INIS)

Miyazono, Ken-ichi; Koura, Tsubasa; Kubota, Keiko; Yoshida, Takuya; Fujita, Yasunari; Yamaguchi-Shinozaki, Kazuko; Tanokura, Masaru

2012-01-01

OsAREB8 from rice (O. sativa), a member of the AREB/ABF family of bZIP transcription factors, was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. A crystal of OsAREB8 in complex with its cognate DNA diffracted X-rays to 3.65 Å resolution. The AREB/ABF family of bZIP transcription factors play a key role in drought stress response and tolerance during the vegetative stage in plants. To reveal the DNA-recognition mechanism of the AREB/ABF family of proteins, the bZIP domain of OsAREB8, an AREB/ABF-family protein from Oryza sativa, was expressed in Escherichia coli, purified and crystallized with its cognate DNA. Crystals of the OsAREB8–DNA complex were obtained by the sitting-drop vapour-diffusion method at 277 K with a reservoir solution consisting of 50 mM MES pH 6.4, 29% MPD, 2 mM spermidine, 20 mM magnesium acetate and 100 mM sodium chloride. A crystal diffracted X-rays to 3.65 Å resolution and belonged to space group C222, with unit-cell parameters a = 155.1, b = 206.7, c = 38.5 Å. The crystal contained one OsAREB8–DNA complex in the asymmetric unit

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

Science.gov (United States)

Lebar, Tina; Jerala, Roman

2018-01-01

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

New PAH gene promoter KLF1 and 3'-region C/EBPalpha motifs influence transcription in vitro.

Science.gov (United States)

Klaassen, Kristel; Stankovic, Biljana; Kotur, Nikola; Djordjevic, Maja; Zukic, Branka; Nikcevic, Gordana; Ugrin, Milena; Spasovski, Vesna; Srzentic, Sanja; Pavlovic, Sonja; Stojiljkovic, Maja

2017-02-01

Phenylketonuria (PKU) is a metabolic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. Although the PAH genotype remains the main determinant of PKU phenotype severity, genotype-phenotype inconsistencies have been reported. In this study, we focused on unanalysed sequences in non-coding PAH gene regions to assess their possible influence on the PKU phenotype. We transiently transfected HepG2 cells with various chloramphenicol acetyl transferase (CAT) reporter constructs which included PAH gene non-coding regions. Selected non-coding regions were indicated by in silico prediction to contain transcription factor binding sites. Furthermore, electrophoretic mobility shift assay (EMSA) and supershift assays were performed to identify which transcriptional factors were engaged in the interaction. We found novel KLF1 motif in the PAH promoter, which decreases CAT activity by 50 % in comparison to basal transcription in vitro. The cytosine at the c.-170 promoter position creates an additional binding site for the protein complex involving KLF1 transcription factor. Moreover, we assessed for the first time the role of a multivariant variable number tandem repeat (VNTR) region located in the 3'-region of the PAH gene. We found that the VNTR3, VNTR7 and VNTR8 constructs had approximately 60 % of CAT activity. The regulation is mediated by the C/EBPalpha transcription factor, present in protein complex binding to VNTR3. Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the PAH gene which decrease transcription in vitro and, thus, could be considered as PAH expression modifiers. New transcription motifs in non-coding regions will contribute to better understanding of the PKU phenotype complexity and may become important for the optimisation of PKU treatment.

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

Science.gov (United States)

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

2016-07-01

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

A Genome-Scale Resource for the Functional Characterization of Arabidopsis Transcription Factors

Directory of Open Access Journals (Sweden)

Jose L. Pruneda-Paz

2014-07-01

Full Text Available Extensive transcriptional networks play major roles in cellular and organismal functions. Transcript levels are in part determined by the combinatorial and overlapping functions of multiple transcription factors (TFs bound to gene promoters. Thus, TF-promoter interactions provide the basic molecular wiring of transcriptional regulatory networks. In plants, discovery of the functional roles of TFs is limited by an increased complexity of network circuitry due to a significant expansion of TF families. Here, we present the construction of a comprehensive collection of Arabidopsis TFs clones created to provide a versatile resource for uncovering TF biological functions. We leveraged this collection by implementing a high-throughput DNA binding assay and identified direct regulators of a key clock gene (CCA1 that provide molecular links between different signaling modules and the circadian clock. The resources introduced in this work will significantly contribute to a better understanding of the transcriptional regulatory landscape of plant genomes.

Quick change: post-transcriptional regulation in Pseudomonas.

Science.gov (United States)

Grenga, Lucia; Little, Richard H; Malone, Jacob G

2017-08-01

Pseudomonas species have evolved dynamic and intricate regulatory networks to fine-tune gene expression, with complex regulation occurring at every stage in the processing of genetic information. This approach enables Pseudomonas to generate precise individual responses to the environment in order to improve their fitness and resource economy. The weak correlations we observe between RNA and protein abundance highlight the significant regulatory contribution of a series of intersecting post-transcriptional pathways, influencing mRNA stability, translational activity and ribosome function, to Pseudomonas environmental responses. This review examines our current understanding of three major post-transcriptional regulatory systems in Pseudomonas spp.; Gac/Rsm, Hfq and RimK, and presents an overview of new research frontiers, emerging genome-wide methodologies, and their potential for the study of global regulatory responses in Pseudomonas. © FEMS 2017.

The Mediator Complex and Lipid Metabolism.

Science.gov (United States)

Zhang, Yi; Xiaoli; Zhao, Xiaoping; Yang, Fajun

2013-03-01

The precise control of gene expression is essential for all biological processes. In addition to DNA-binding transcription factors, numerous transcription cofactors contribute another layer of regulation of gene transcription in eukaryotic cells. One of such transcription cofactors is the highly conserved Mediator complex, which has multiple subunits and is involved in various biological processes through directly interacting with relevant transcription factors. Although the current understanding on the biological functions of Mediator remains incomplete, research in the past decade has revealed an important role of Mediator in regulating lipid metabolism. Such function of Mediator is dependent on specific transcription factors, including peroxisome proliferator-activated receptor-gamma (PPARγ) and sterol regulatory element-binding proteins (SREBPs), which represent the master regulators of lipid metabolism. The medical significance of these findings is apparent, as aberrant lipid metabolism is intimately linked to major human diseases, such as type 2 diabetes and cardiovascular disease. Here, we briefly review the functions and molecular mechanisms of Mediator in regulation of lipid metabolism.

Circular chromatin complexes in human lymphocytes high-resolution autoradiography

International Nuclear Information System (INIS)

Becak, M.L.; Fukuda-Pizzocaro, K.; Santos, R. de C.S. dos; Brunner, O.

1985-01-01

Transcriptionally active chromatin fibers were observed in chromosomes presenting the loops/scaffold configuration. The active fibers showed altered nucleosomes and presented multiforked aspects which led to the formation of ring complexes. The ribonucleoprotein transcripts (RNP) appeared as networks of 0.1 μm or multiples tandemly disposed along the fiber. It is suggested that the ring complexes belong to the human genome. The possibility that these circular structures come from a prokaryote is also considered. (author) [pt

Dissecting specific and global transcriptional regulation of bacterial gene expression

NARCIS (Netherlands)

Gerosa, Luca; Kochanowski, Karl; Heinemann, Matthias; Sauer, Uwe

Gene expression is regulated by specific transcriptional circuits but also by the global expression machinery as a function of growth. Simultaneous specific and global regulation thus constitutes an additional-but often neglected-layer of complexity in gene expression. Here, we develop an

ORF Alignment: NC_004431 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available DDQQREKVVMGRGIGFQKRPGERINSSGIEKEYAL 60 ... MQITKIL ... IDDQQREKVVMGRGIGFQKRPGERINSS...GIEKEYAL Sbjct: 1 ... MQITKILNNNVVVVIDDQQREKVVMGRGIGFQKRPGERINSSGIEKEYAL 50 ... ... operon Antiterminator [Escherichia coli CFT073] ... Length = 50 ... Query: 11 MQITKILXXXXXXXI

Set1/COMPASS and Mediator are repurposed to promote epigenetic transcriptional memory.

Science.gov (United States)

D'Urso, Agustina; Takahashi, Yoh-Hei; Xiong, Bin; Marone, Jessica; Coukos, Robert; Randise-Hinchliff, Carlo; Wang, Ji-Ping; Shilatifard, Ali; Brickner, Jason H

2016-06-23

In yeast and humans, previous experiences can lead to epigenetic transcriptional memory: repressed genes that exhibit mitotically heritable changes in chromatin structure and promoter recruitment of poised RNA polymerase II preinitiation complex (RNAPII PIC), which enhances future reactivation. Here, we show that INO1 memory in yeast is initiated by binding of the Sfl1 transcription factor to the cis-acting Memory Recruitment Sequence, targeting INO1 to the nuclear periphery. Memory requires a remodeled form of the Set1/COMPASS methyltransferase lacking Spp1, which dimethylates histone H3 lysine 4 (H3K4me2). H3K4me2 recruits the SET3C complex, which plays an essential role in maintaining this mark. Finally, while active INO1 is associated with Cdk8(-) Mediator, during memory, Cdk8(+) Mediator recruits poised RNAPII PIC lacking the Kin28 CTD kinase. Aspects of this mechanism are generalizable to yeast and conserved in human cells. Thus, COMPASS and Mediator are repurposed to promote epigenetic transcriptional poising by a highly conserved mechanism.

[Participation of the piRNA pathway in recruiting a component of RNA polymerase I transcription initiation complex to germline cell nucleoli].

Science.gov (United States)

Fefelova, E A; Stolyarenko, A D; Yakushev, E Y; Gvozdev, V A; Klenov, M S

2017-01-01

Proteins of the Piwi family and short Piwi-interacting RNAs (piRNAs) ensure the protection of the genome from transposable elements. We have previously shown that nuclear Piwi protein tends to concentrate in the nucleoli of the cells of Drosophila melanogaster ovaries. It could be hypothesized that the function of Piwi in the nucleolus is associated with the repression of R1 and R2 retrotransposons inserted into the rDNA cluster. Here, we show that Piwi participates in recruiting Udd protein to nucleoli. Udd is a component of the conserved Selectivity Factor I-like (SL1-like) complex, which is required for transcription initiation by RNA polymerase I. We found that Udd localization depends on Piwi in germline cells, but not in somatic cells of the ovaries. In contrast, knockdowns of the SL1-like components (Udd or TAF1b) do not disrupt Piwi localization. We also observed that the absence of Udd or TAF1b in germline cells, as well as the impairment of Piwi nuclear localization lead to the accumulation of late stage egg chambers in the ovaries, which could be explained by reduced rRNA transcription. These results allow us to propose for the first time a role for Piwi in the nucleolus that is not directly associated with transposable element repression.

Temporal regulation of Drosophila salivary gland degeneration by the Broad-Complex transcription factors

Czech Academy of Sciences Publication Activity Database

Kuchárová-Mahmood, S.; Raška, Ivan; Mechler, B. M.; Farkaš, R.

2002-01-01

Roč. 140, - (2002), s. 67-78 ISSN 1047-8477 R&D Projects: GA ČR GA304/02/0342 Grant - others:GA-(SK) VEGA:2/7194/20 Institutional research plan: CEZ:AV0Z5039906; CEZ:MSM 111100003 Keywords : programmed cell death * BR-C transcription factors * drosophila Subject RIV: EA - Cell Biology Impact factor: 4.194, year: 2002

Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters.

Directory of Open Access Journals (Sweden)

Christopher A Lavender

2016-08-01

Full Text Available Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment.

Identification of a Dynamic Core Transcriptional Network in t(8;21 AML that Regulates Differentiation Block and Self-Renewal

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Anetta Ptasinska

2014-09-01

Full Text Available Oncogenic transcription factors such as RUNX1/ETO, which is generated by the chromosomal translocation t(8;21, subvert normal blood cell development by impairing differentiation and driving malignant self-renewal. Here, we use digital footprinting and chromatin immunoprecipitation sequencing (ChIP-seq to identify the core RUNX1/ETO-responsive transcriptional network of t(8;21 cells. We show that the transcriptional program underlying leukemic propagation is regulated by a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes, which bind to identical DNA sites in a mutually exclusive fashion. Perturbation of this equilibrium in t(8;21 cells by RUNX1/ETO depletion leads to a global redistribution of transcription factor complexes within preexisting open chromatin, resulting in the formation of a transcriptional network that drives myeloid differentiation. Our work demonstrates on a genome-wide level that the extent of impaired myeloid differentiation in t(8;21 is controlled by the dynamic balance between RUNX1/ETO and RUNX1 activities through the repression of transcription factors that drive differentiation.

Unexpected complexity of the reef-building coral Acropora millepora transcription factor network.

KAUST Repository

Ryu, Tae Woo

2011-04-28

Coral reefs are disturbed on a global scale by environmental changes including rising sea surface temperatures and ocean acidification. Little is known about how corals respond or adapt to these environmental changes especially at the molecular level. This is mostly because of the paucity of genome-wide studies on corals and the application of systems approaches that incorporate the latter. Like in any other organism, the response of corals to stress is tightly controlled by the coordinated interplay of many transcription factors.

Unexpected complexity of the reef-building coral Acropora millepora transcription factor network.

KAUST Repository

Ryu, Tae Woo; Mavromatis, Charalampos Harris; Bayer, Till; Voolstra, Christian R.; Ravasi, Timothy

2011-01-01

Coral reefs are disturbed on a global scale by environmental changes including rising sea surface temperatures and ocean acidification. Little is known about how corals respond or adapt to these environmental changes especially at the molecular level. This is mostly because of the paucity of genome-wide studies on corals and the application of systems approaches that incorporate the latter. Like in any other organism, the response of corals to stress is tightly controlled by the coordinated interplay of many transcription factors.

Cocaine- and amphetamine-regulated transcript peptide increases mitochondrial respiratory chain complex II activity and protects against oxygen-glucose deprivation in neurons.

Science.gov (United States)

Sha, Dujuan; Wang, Luna; Zhang, Jun; Qian, Lai; Li, Qiming; Li, Jin; Qian, Jian; Gu, Shuangshuang; Han, Ling; Xu, Peng; Xu, Yun

2014-09-25

The mechanisms of ischemic stroke, a main cause of disability and death, are complicated. Ischemic stroke results from the interaction of various factors including oxidative stress, a key pathological mechanism that plays an important role during the acute stage of ischemic brain injury. This study demonstrated that cocaine- and amphetamine-regulated transcript (CART) peptide, specifically CART55-102, increased the survival rate, but decreased the mortality of neurons exposed to oxygen-glucose deprivation (OGD), in a dose-dependent manner. The above-mentioned effects of CART55-102 were most significant at 0.4nM. These results indicated that CART55-102 suppressed neurotoxicity and enhanced neuronal survival after oxygen-glucose deprivation. CART55-102 (0.4nM) significantly diminished reactive oxygen species levels and markedly increased the activity of mitochondrial respiratory chain complex II in oxygen-glucose deprived neurons. In summary, CART55-102 suppressed oxidative stress in oxygen-glucose deprived neurons, possibly through elevating the activity of mitochondrial respiratory chain complex II. This result provides evidence for the development of CART55-102 as an antioxidant drug. Copyright © 2014 Elsevier B.V. All rights reserved.

The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line

DEFF Research Database (Denmark)

Suzuki, Harukazu; Forrest, Alistair R R; van Nimwegen, Erik

2009-01-01

, we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks......Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites...... involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process....

In vitro-reconstituted nucleoids can block mitochondrial DNA replication and transcription.

Science.gov (United States)

Farge, Géraldine; Mehmedovic, Majda; Baclayon, Marian; van den Wildenberg, Siet M J L; Roos, Wouter H; Gustafsson, Claes M; Wuite, Gijs J L; Falkenberg, Maria

2014-07-10

The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Emerging functions of multi-protein complex Mediator with special emphasis on plants.

Science.gov (United States)

Malik, Naveen; Agarwal, Pinky; Tyagi, Akhilesh

2017-10-01

Mediator is a multi-subunit protein complex which is involved in transcriptional regulation in yeast and other eukaryotes. As a co-activator, it connects information from transcriptional activators/repressors to transcriptional machinery including RNA polymerase II and general transcription factors. It is not only involved in transcription initiation but also has important roles to play in transcription elongation and termination. Functional attributes of different Mediator subunits have been largely defined in yeast and mammalian systems earlier, while such studies in plants have gained momentum recently. Mediator regulates various processes related to plant development and is also involved in biotic and abiotic stress response. Thus, plant Mediator, like yeast and mammalian Mediator complex, is indispensable for plant growth and survival. Interaction of its multiple subunits with other regulatory proteins and their ectopic expression or knockdown in model plant like Arabidopsis and certain crop plants are paving the way to biochemical analysis and unravel molecular mechanisms of action of Mediator in plants.

Basic leucine zipper protein Cnc-C is a substrate and transcriptional regulator of the Drosophila 26S proteasome.

Science.gov (United States)

Grimberg, Kristian Björk; Beskow, Anne; Lundin, Daniel; Davis, Monica M; Young, Patrick

2011-02-01

While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

Was that Infinity or Affinity? Applying Insights from Translation Studies to Qualitative Research Transcription

Directory of Open Access Journals (Sweden)

Jen Ross

2010-02-01

Full Text Available Despite a small but compelling body of literature arguing that transcription represents a key moment of choice and the exercise of power in the research process, many qualitative researchers appear to believe (or at least proceed as if they believe that transcription is relatively unproblematic. Translation studies and its engagement with visibility, power, authenticity and fidelity has a lot to offer to qualitative researchers working critically with transcription theory and practice. This paper explores the translation studies theories of equivalence, overt and covert translation, foreignisation and domestication, and the remainder, and demonstrates some fertile connections between transcription and translation. These connections help us to think about some broader political and cultural issues in relation to transcription and academic discourse, the complexity of equivalence and the central role of the situated transcriber. URN: urn:nbn:de:0114-fqs100223

Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes

International Nuclear Information System (INIS)

Zhang, H.; Wang, J.C.; Liu, L.F.

1988-01-01

Treatment of HeLa cells with a DNA topoisomerase I-specific inhibitor, camptothecin, results in rapid cessation of the synthesis of the 45S rRNA precursor. The inhibition of rRNA synthesis is reversible following drug removal and correlates with the presence of camptothecin-trapped topoisomerase I-DNA abortive complexes, which can be detected as topoisomerase I-linked DNA breaks upon lysis with sodium dodecyl sulfate. These breaks were found to be concentrated within the transcribed region of human rRNA genes. No such sites can be detected in the inactive human rRNA genes in mouse-human hybrid cells, suggesting a preferential association of topoisomerase I with actively transcribed genes. The distribution of RNA polymerase molecules along the transcription unit of human rRNA genes in camptothecin-treated HeLa cells, as assayed by nuclear run-on transcription, shows a graded decrease of the RNA polymerase density toward the 3' end of the transcription unit; the density is minimally affected near the 5' start of the transcription unit. These results suggest that DNA topoisomerase I is normally involved in the elongation step of transcription, especially when the transcripts are long, and that camptothecin interferes with this role

TACO: a general-purpose tool for predicting cell-type-specific transcription factor dimers.

Science.gov (United States)

Jankowski, Aleksander; Prabhakar, Shyam; Tiuryn, Jerzy

2014-03-19

Cooperative binding of transcription factor (TF) dimers to DNA is increasingly recognized as a major contributor to binding specificity. However, it is likely that the set of known TF dimers is highly incomplete, given that they were discovered using ad hoc approaches, or through computational analyses of limited datasets. Here, we present TACO (Transcription factor Association from Complex Overrepresentation), a general-purpose standalone software tool that takes as input any genome-wide set of regulatory elements and predicts cell-type-specific TF dimers based on enrichment of motif complexes. TACO is the first tool that can accommodate motif complexes composed of overlapping motifs, a characteristic feature of many known TF dimers. Our method comprehensively outperforms existing tools when benchmarked on a reference set of 29 known dimers. We demonstrate the utility and consistency of TACO by applying it to 152 DNase-seq datasets and 94 ChIP-seq datasets. Based on these results, we uncover a general principle governing the structure of TF-TF-DNA ternary complexes, namely that the flexibility of the complex is correlated with, and most likely a consequence of, inter-motif spacing.

Estradiol-Induced Transcriptional Regulation of Long Non-Coding RNA, HOTAIR.

Science.gov (United States)

Bhan, Arunoday; Mandal, Subhrangsu S

2016-01-01

HOTAIR (HOX antisense intergenic RNA) is a 2.2 kb long non-coding RNA (lncRNA), transcribed from the antisense strand of homeobox C (HOXC) gene locus in chromosome 12. HOTAIR acts as a scaffolding lncRNA. It interacts and guides various chromatin-modifying complexes such as PRC2 (polycomb-repressive complex 2) and LSD1 (lysine-specific demethylase 1) to the target gene promoters leading to their gene silencing. Various studies have demonstrated that HOTAIR overexpression is associated with breast cancer. Recent studies from our laboratory demonstrate that HOTAIR is required for viability of breast cancer cells and is transcriptionally regulated by estradiol (E2) in vitro and in vivo. This chapter describes protocols for analysis of the HOTAIR promoter, cloning, transfection and dual luciferase assays, knockdown of protein synthesis by antisense oligonucleotides, and chromatin immunoprecipitation (ChIP) assay. These protocols are useful for studying the estrogen-mediated transcriptional regulation of lncRNA HOTAIR, as well as other protein coding genes and non-coding RNAs.

Transcriptional regulation by histone modifications: towards a theory of chromatin re-organization during stem cell differentiation

International Nuclear Information System (INIS)

Binder, Hans; Steiner, Lydia; Przybilla, Jens; Rohlf, Thimo; Prohaska, Sonja; Galle, Jörg

2013-01-01

Chromatin-related mechanisms, as e.g. histone modifications, are known to be involved in regulatory switches within the transcriptome. Only recently, mathematical models of these mechanisms have been established. So far they have not been applied to genome-wide data. We here introduce a mathematical model of transcriptional regulation by histone modifications and apply it to data of trimethylation of histone 3 at lysine 4 (H3K4me3) and 27 (H3K27me3) in mouse pluripotent and lineage-committed cells. The model describes binding of protein complexes to chromatin which are capable of reading and writing histone marks. Molecular interactions of the complexes with DNA and modified histones create a regulatory switch of transcriptional activity. The regulatory states of the switch depend on the activity of histone (de-) methylases, the strength of complex-DNA-binding and the number of nucleosomes capable of cooperatively contributing to complex-binding. Our model explains experimentally measured length distributions of modified chromatin regions. It suggests (i) that high CpG-density facilitates recruitment of the modifying complexes in embryonic stem cells and (ii) that re-organization of extended chromatin regions during lineage specification into neuronal progenitor cells requires targeted de-modification. Our approach represents a basic step towards multi-scale models of transcriptional control during development and lineage specification. (paper)

Transcriptional regulators of legume-rhizobia symbiosis: nuclear factors Ys and GRAS are two for tango.

Science.gov (United States)

Rípodas, Carolina; Clúa, Joaquín; Battaglia, Marina; Baudin, Maël; Niebel, Andreas; Zanetti, María Eugenia; Blanco, Flavio

2014-01-01

Transcription factors are DNA binding proteins that regulate gene expression. The nitrogen fixing symbiosis established between legume plants and soil bacteria is a complex interaction, in which plants need to integrate signals derived from the symbiont and the surrounding environment to initiate the developmental program of nodule organogenesis and the infection process. Several transcription factors that play critical roles in these processes have been reported in the past decade, including proteins of the GRAS and NF-Y families. Recently, we reported the characterization of a new GRAS domain containing-protein that interacts with a member of the C subunit of the NF-Y family, which plays an important role in nodule development and the progression of bacterial infection during the symbiotic interaction. The connection between transcription factors of these families highlights the significance of multimeric complexes in the fabulous capacity of plants to integrate and respond to multiple environmental stimuli.

Directing traffic on DNA-How transcription factors relieve or induce transcriptional interference.

Science.gov (United States)

Hao, Nan; Palmer, Adam C; Dodd, Ian B; Shearwin, Keith E

2017-03-15

Transcriptional interference (TI) is increasingly recognized as a widespread mechanism of gene control, particularly given the pervasive nature of transcription, both sense and antisense, across all kingdoms of life. Here, we discuss how transcription factor binding kinetics strongly influence the ability of a transcription factor to relieve or induce TI.

Transcriptional plant responses critical for resistance towards necrotrophic pathogens

Directory of Open Access Journals (Sweden)

Rainer P. Birkenbihl

2011-11-01

Full Text Available Plant defenses aimed at necrotrophic pathogens appear to be genetically complex. Despite the apparent lack of a specific recognition of such necrotrophs by products of major R genes, biochemical, molecular, and genetic studies, in particular using the model plant Arabidopsis, have uncovered numerous host components critical for the outcome of such interactions. Although the JA signaling pathway plays a central role in plant defense towards necrotrophs additional signaling pathways contribute to the plant response network. Transcriptional reprogramming is a vital part of the host defense machinery and several key regulators have recently been identified. Some of these transcription factors positively affect plant resistance whereas others play a role in enhancing host susceptibility towards these phytopathogens.

Interaction between C/EBPβ and Tax down-regulates human T-cell leukemia virus type I transcription

International Nuclear Information System (INIS)

Hivin, P.; Gaudray, G.; Devaux, C.; Mesnard, J.-M.

2004-01-01

The human T-cell leukemia virus type I (HTLV-I) Tax protein trans-activates viral transcription through three imperfect tandem repeats of a 21-bp sequence called Tax-responsive element (TxRE). Tax regulates transcription via direct interaction with some members of the activating transcription factor/CRE-binding protein (ATF/CREB) family including CREM, CREB, and CREB-2. By interacting with their ZIP domain, Tax stimulates the binding of these cellular factors to the CRE-like sequence present in the TxREs. Recent observations have shown that CCAAT/enhancer binding protein β (C/EBPβ) forms stable complexes on the CRE site in the presence of CREB-2. Given that C/EBPβ has also been found to interact with Tax, we analyzed the effects of C/EBPβ on viral Tax-dependent transcription. We show here that C/EBPβ represses viral transcription and that Tax is no more able to form a stable complex with CREB-2 on the TxRE site in the presence of C/EBPβ. We also analyzed the physical interactions between Tax and C/EBPβ and found that the central region of C/EBPβ, excluding its ZIP domain, is required for direct interaction with Tax. It is the first time that Tax is described to interact with a basic leucine-zipper (bZIP) factor without recognizing its ZIP domain. Although unexpected, this result explains why C/EBPβ would be unable to form a stable complex with Tax on the TxRE site and could then down-regulate viral transcription. Lastly, we found that C/EBPβ was able to inhibit Tax expression in vivo from an infectious HTLV-I molecular clone. In conclusion, we propose that during cell activation events, which stimulate the Tax synthesis, C/EBPβ may down-regulate the level of HTLV-I expression to escape the cytotoxic-T-lymphocyte response

FACT, the Bur kinase pathway, and the histone co-repressor HirC have overlapping nucleosome-related roles in yeast transcription elongation.

Directory of Open Access Journals (Sweden)

Jennifer R Stevens

Full Text Available Gene transcription is constrained by the nucleosomal nature of chromosomal DNA. This nucleosomal barrier is modulated by FACT, a conserved histone-binding heterodimer. FACT mediates transcription-linked nucleosome disassembly and also nucleosome reassembly in the wake of the RNA polymerase II transcription complex, and in this way maintains the repression of 'cryptic' promoters found within some genes. Here we focus on a novel mutant version of the yeast FACT subunit Spt16 that supplies essential Spt16 activities but impairs transcription-linked nucleosome reassembly in dominant fashion. This Spt16 mutant protein also has genetic effects that are recessive, which we used to show that certain Spt16 activities collaborate with histone acetylation and the activities of a Bur-kinase/Spt4-Spt5/Paf1C pathway that facilitate transcription elongation. These collaborating activities were opposed by the actions of Rpd3S, a histone deacetylase that restores a repressive chromatin environment in a transcription-linked manner. Spt16 activity paralleling that of HirC, a co-repressor of histone gene expression, was also found to be opposed by Rpd3S. Our findings suggest that Spt16, the Bur/Spt4-Spt5/Paf1C pathway, and normal histone abundance and/or stoichiometry, in mutually cooperative fashion, facilitate nucleosome disassembly during transcription elongation. The recessive nature of these effects of the mutant Spt16 protein on transcription-linked nucleosome disassembly, contrasted to its dominant negative effect on transcription-linked nucleosome reassembly, indicate that mutant FACT harbouring the mutant Spt16 protein competes poorly with normal FACT at the stage of transcription-linked nucleosome disassembly, but effectively with normal FACT for transcription-linked nucleosome reassembly. This functional difference is consistent with the idea that FACT association with the transcription elongation complex depends on nucleosome disassembly, and that the

Experimental Incubations Elicit Profound Changes in Community Transcription in OMZ Bacterioplankton

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Stewart, Frank J.; Dalsgaard, Tage; Young, Curtis R.; Thamdrup, Bo; Revsbech, Niels Peter; Ulloa, Osvaldo; Canfield, Don E.; DeLong, Edward F.

2012-01-01

metabolic linkages between community members. These data highlight the impressive capacity for transcriptional changes within complex microbial communities, underscoring the need for caution when inferring in situ metabolism based on transcript abundances in experimental incubations. PMID:22615914

Two genes in Balbiani ring 2 with metabolically different 75S transcripts

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Galler, R.; Saiga, H.; Widmer, R. M.; Lezzi, M.; Edström, J.-E.

1985-01-01

Balbiani ring 2 (BR2) in salivary glands of Chironomus pallidivittatus and C. tentans (two sibling species of the subgenus Camptochironomus) is a favoured model system for studies of gene organization and transcript formation. Here we show that BR2 is more complex than hitherto believed, containing two 75S RNA-producing genes, BR2a and BR2b, present in different 35–40 kb blocks of DNA. The transcripts hybridizing to two different repeat units originating in BR2 differ in size. Further support...

Menin and RNF20 recruitment is associated with dynamic histone modifications that regulate signal transducer and activator of transcription 1 (STAT1-activated transcription of the interferon regulatory factor 1 gene (IRF1

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Buro Lauren J

2010-09-01

Full Text Available Abstract Background Signal transducer and activator of transcription (STAT activation of gene expression is both rapid and transient, and when properly executed it affects growth, differentiation, homeostasis and the immune response, but when dysregulated it contributes to human disease. Transcriptional activation is regulated by alterations to the chromatin template. However, the role of histone modification at gene loci that are activated for transcription in response to STAT signaling is poorly defined. Results Using chromatin immunoprecipitation, we profiled several histone modifications during STAT1 activation of the interferon regulatory factor 1 gene (IRF1. Methylated lysine histone proteins H3K4me2, H3K4me3, H3K79me3, H3K36me3 and monoubiquitinated histone ubH2B are dynamic and correlate with interferon (IFNγ induction of STAT1 activity. Chemical inhibition of H3K4 methylation downregulates IRF1 transcription and decreases RNA polymerase II (Pol II occupancy at the IRF1 promoter. MEN1, a component of a complex proteins associated with Set1 (COMPASS-like complex and the hBRE1 component, RNF20, are localized to IRF1 in the uninduced state and are further recruited when IRF1 is activated. RNAi-mediated depletion of RNF20 lowers both ubH2B and H3K4me3, but surprisingly, upregulates IFNγ induced IRF1 transcription. The dynamics of phosphorylation in the C-terminal domain (CTD of Pol II are disrupted during gene activation as well. Conclusions H2B monoubiquitination promotes H3K4 methylation, but the E3 ubiquitin ligase, RNF20, is repressive of inducible transcription at the IRF1 gene locus, suggesting that ubH2B can, directly or indirectly, affect Pol II CTD phosphorylation cycling to exert control on ongoing transcription.

DNA damage mediated transcription arrest: Step back to go forward.

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Mullenders, Leon

2015-12-01

The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

Alteration of light-dependent gene regulation by the absence of the RCO-1/RCM-1 repressor complex in the fungus Neurospora crassa.

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Carmen Ruger-Herreros

Full Text Available The activation of transcription by light in the fungus Neurospora crassa requires the White Collar Complex (WCC, a photoreceptor and transcription factor complex. After light reception two WCCs interact and bind the promoters of light-regulated genes to activate transcription. This process is regulated by VVD, a small photoreceptor that disrupts the interaction between WCCs and leads to a reduction in transcription after long exposures to light. The N. crassa RCO-1/RCM-1 repressor complex is the homolog of the Tup1-Ssn6 repressor complex in yeast, and its absence modifies photoadaptation. We show that the absence of the RCO-1/RCM-1 repressor complex leads to several alterations in transcription that are gene-specific: an increase in the accumulation of mRNAs in the dark, a repression of transcription, and a derepression of transcription after long exposures to light. The absence of the RCO-1/RCM-1 repressor complex leads to lower VVD levels that are available for the regulation of the activity of the WCC. The reduction in the amount of VVD results in increased WCC binding to the promoters of light-regulated genes in the dark and after long exposures to light, leading to the modification of photoadaptation that has been observed in rco-1 and rcm-1 mutants. Our results show that the photoadaptation phenotype of mutants in the RCO-1/RCM-1 repressor complex is, at least in part, an indirect consequence of the reduction of vvd transcription, and the resulting modification in the regulation of transcription by the WCC.

SUMO-, MAPK- and resistance protein-signaling converge at transcription complexes that regulate plant innate immunity

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Burg, van den H.A.; Takken, F.L.W.

2010-01-01

Upon pathogen perception plant innate immune receptors activate various signaling pathways that trigger host defenses. PAMP-triggered defense signaling requires mitogen-activated protein kinase (MAPK) pathways, which modulate the activity of transcription factors through phosphorylation. Here, we

SUMO-, MAPK-, and resistance protein-signaling converge at transcription complexes that regulate plant innate immunity

NARCIS (Netherlands)

van den Burg, H.A.; Takken, F.L.W.

2010-01-01

Upon pathogen perception plant innate immune receptors activate various signaling pathways that trigger host defenses. PAMP-triggered defense signaling requires mitogen-activated protein kinase (MAPK) pathways, which modulate the activity of transcription factors through phosphorylation. Here, we

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

NARCIS (Netherlands)

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

2015-01-01

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

MITIE: Simultaneous RNA-Seq-based transcript identification and quantification in multiple samples.

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Behr, Jonas; Kahles, André; Zhong, Yi; Sreedharan, Vipin T; Drewe, Philipp; Rätsch, Gunnar

2013-10-15

High-throughput sequencing of mRNA (RNA-Seq) has led to tremendous improvements in the detection of expressed genes and reconstruction of RNA transcripts. However, the extensive dynamic range of gene expression, technical limitations and biases, as well as the observed complexity of the transcriptional landscape, pose profound computational challenges for transcriptome reconstruction. We present the novel framework MITIE (Mixed Integer Transcript IdEntification) for simultaneous transcript reconstruction and quantification. We define a likelihood function based on the negative binomial distribution, use a regularization approach to select a few transcripts collectively explaining the observed read data and show how to find the optimal solution using Mixed Integer Programming. MITIE can (i) take advantage of known transcripts, (ii) reconstruct and quantify transcripts simultaneously in multiple samples, and (iii) resolve the location of multi-mapping reads. It is designed for genome- and assembly-based transcriptome reconstruction. We present an extensive study based on realistic simulated RNA-Seq data. When compared with state-of-the-art approaches, MITIE proves to be significantly more sensitive and overall more accurate. Moreover, MITIE yields substantial performance gains when used with multiple samples. We applied our system to 38 Drosophila melanogaster modENCODE RNA-Seq libraries and estimated the sensitivity of reconstructing omitted transcript annotations and the specificity with respect to annotated transcripts. Our results corroborate that a well-motivated objective paired with appropriate optimization techniques lead to significant improvements over the state-of-the-art in transcriptome reconstruction. MITIE is implemented in C++ and is available from http://bioweb.me/mitie under the GPL license.

The transcriptional regulatory network mediated by banana (Musa acuminata) dehydration-responsive element binding (MaDREB) transcription factors in fruit ripening.

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Kuang, Jian-Fei; Chen, Jian-Ye; Liu, Xun-Cheng; Han, Yan-Chao; Xiao, Yun-Yi; Shan, Wei; Tang, Yang; Wu, Ke-Qiang; He, Jun-Xian; Lu, Wang-Jin

2017-04-01

Fruit ripening is a complex, genetically programmed process involving the action of critical transcription factors (TFs). Despite the established significance of dehydration-responsive element binding (DREB) TFs in plant abiotic stress responses, the involvement of DREBs in fruit ripening is yet to be determined. Here, we identified four genes encoding ripening-regulated DREB TFs in banana (Musa acuminata), MaDREB1, MaDREB2, MaDREB3, and MaDREB4, and demonstrated that they play regulatory roles in fruit ripening. We showed that MaDREB1-MaDREB4 are nucleus-localized, induced by ethylene and encompass transcriptional activation activities. We performed a genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) experiment for MaDREB2 and identified 697 genomic regions as potential targets of MaDREB2. MaDREB2 binds to hundreds of loci with diverse functions and its binding sites are distributed in the promoter regions proximal to the transcriptional start site (TSS). Most of the MaDREB2-binding targets contain the conserved (A/G)CC(G/C)AC motif and MaDREB2 appears to directly regulate the expression of a number of genes involved in fruit ripening. In combination with transcriptome profiling (RNA sequencing) data, our results indicate that MaDREB2 may serve as both transcriptional activator and repressor during banana fruit ripening. In conclusion, our study suggests a hierarchical regulatory model of fruit ripening in banana and that the MaDREB TFs may act as transcriptional regulators in the regulatory network. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Network analysis of inflammatory genes and their transcriptional regulators in coronary artery disease.

Directory of Open Access Journals (Sweden)

Jiny Nair

Full Text Available Network analysis is a novel method to understand the complex pathogenesis of inflammation-driven atherosclerosis. Using this approach, we attempted to identify key inflammatory genes and their core transcriptional regulators in coronary artery disease (CAD. Initially, we obtained 124 candidate genes associated with inflammation and CAD using Polysearch and CADgene database for which protein-protein interaction network was generated using STRING 9.0 (Search Tool for the Retrieval of Interacting Genes and visualized using Cytoscape v 2.8.3. Based on betweenness centrality (BC and node degree as key topological parameters, we identified interleukin-6 (IL-6, vascular endothelial growth factor A (VEGFA, interleukin-1 beta (IL-1B, tumor necrosis factor (TNF and prostaglandin-endoperoxide synthase 2 (PTGS2 as hub nodes. The backbone network constructed with these five hub genes showed 111 nodes connected via 348 edges, with IL-6 having the largest degree and highest BC. Nuclear factor kappa B1 (NFKB1, signal transducer and activator of transcription 3 (STAT3 and JUN were identified as the three core transcription factors from the regulatory network derived using MatInspector. For the purpose of validation of the hub genes, 97 test networks were constructed, which revealed the accuracy of the backbone network to be 0.7763 while the frequency of the hub nodes remained largely unaltered. Pathway enrichment analysis with ClueGO, KEGG and REACTOME showed significant enrichment of six validated CAD pathways - smooth muscle cell proliferation, acute-phase response, calcidiol 1-monooxygenase activity, toll-like receptor signaling, NOD-like receptor signaling and adipocytokine signaling pathways. Experimental verification of the above findings in 64 cases and 64 controls showed increased expression of the five candidate genes and the three transcription factors in the cases relative to the controls (p<0.05. Thus, analysis of complex networks aid in the

Regulation of TCF ETS-domain transcription factors by helix-loop-helix motifs.

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Stinson, Julie; Inoue, Toshiaki; Yates, Paula; Clancy, Anne; Norton, John D; Sharrocks, Andrew D

2003-08-15

DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecular interactions. The helix-loop-helix (HLH)-containing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2/Net/ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also contains an HLH-like motif. The NID also acts as a transcriptional repression domain. Here, we have studied the role of HLH motifs in regulating DNA binding and transcription by the TCF protein SAP-1 and how Cdk-mediated phosphorylation affects the inhibitory activity of the Id proteins towards the TCFs. We demonstrate that the NID region of SAP-1 is an autoinhibitory motif that acts to inhibit DNA binding and also functions as a transcription repression domain. This region can be functionally replaced by fusion of Id proteins to SAP-1, whereby the Id moiety then acts to repress DNA binding in cis. Phosphorylation of the Ids by cyclin-Cdk complexes results in reduction in protein-protein interactions between the Ids and TCFs and relief of their DNA-binding inhibitory activity. In revealing distinct mechanisms through which HLH motifs modulate the activity of TCFs, our results therefore provide further insight into the role of HLH motifs in regulating TCF function and how the inhibitory properties of the trans-acting Id HLH proteins are themselves regulated by phosphorylation.

Decoding transcriptional enhancers: Evolving from annotation to functional interpretation.

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Engel, Krysta L; Mackiewicz, Mark; Hardigan, Andrew A; Myers, Richard M; Savic, Daniel

2016-09-01

Deciphering the intricate molecular processes that orchestrate the spatial and temporal regulation of genes has become an increasingly major focus of biological research. The differential expression of genes by diverse cell types with a common genome is a hallmark of complex cellular functions, as well as the basis for multicellular life. Importantly, a more coherent understanding of gene regulation is critical for defining developmental processes, evolutionary principles and disease etiologies. Here we present our current understanding of gene regulation by focusing on the role of enhancer elements in these complex processes. Although functional genomic methods have provided considerable advances to our understanding of gene regulation, these assays, which are usually performed on a genome-wide scale, typically provide correlative observations that lack functional interpretation. Recent innovations in genome editing technologies have placed gene regulatory studies at an exciting crossroads, as systematic, functional evaluation of enhancers and other transcriptional regulatory elements can now be performed in a coordinated, high-throughput manner across the entire genome. This review provides insights on transcriptional enhancer function, their role in development and disease, and catalogues experimental tools commonly used to study these elements. Additionally, we discuss the crucial role of novel techniques in deciphering the complex gene regulatory landscape and how these studies will shape future research. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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

2012-01-01

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

RNA polymerase III transcription - regulated by chromatin structure and regulator of nuclear chromatin organization.

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Pascali, Chiara; Teichmann, Martin

2013-01-01

RNA polymerase III (Pol III) transcription is regulated by modifications of the chromatin. DNA methylation and post-translational modifications of histones, such as acetylation, phosphorylation and methylation have been linked to Pol III transcriptional activity. In addition to being regulated by modifications of DNA and histones, Pol III genes and its transcription factors have been implicated in the organization of nuclear chromatin in several organisms. In yeast, the ability of the Pol III transcription system to contribute to nuclear organization seems to be dependent on direct interactions of Pol III genes and/or its transcription factors TFIIIC and TFIIIB with the structural maintenance of chromatin (SMC) protein-containing complexes cohesin and condensin. In human cells, Pol III genes and transcription factors have also been shown to colocalize with cohesin and the transcription regulator and genome organizer CCCTC-binding factor (CTCF). Furthermore, chromosomal sites have been identified in yeast and humans that are bound by partial Pol III machineries (extra TFIIIC sites - ETC; chromosome organizing clamps - COC). These ETCs/COC as well as Pol III genes possess the ability to act as boundary elements that restrict spreading of heterochromatin.

The histone chaperone TAF-I/SET/INHAT is required for transcription in vitro of chromatin templates.

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Gamble, Matthew J; Erdjument-Bromage, Hediye; Tempst, Paul; Freedman, Leonard P; Fisher, Robert P

2005-01-01

To uncover factors required for transcription by RNA polymerase II on chromatin, we fractionated a mammalian cell nuclear extract. We identified the histone chaperone TAF-I (also known as INHAT [inhibitor of histone acetyltransferase]), which was previously proposed to repress transcription, as a potent activator of chromatin transcription responsive to the vitamin D3 receptor or to Gal4-VP16. TAF-I associates with chromatin in vitro and can substitute for the related protein NAP-1 in assembling chromatin onto cloned DNA templates in cooperation with the remodeling enzyme ATP-dependent chromatin assembly factor (ACF). The chromatin assembly and transcriptional activation functions are distinct, however, and can be dissociated temporally. Efficient transcription of chromatin assembled with TAF-I still requires the presence of TAF-I during the polymerization reaction. Conversely, TAF-I cannot stimulate transcript elongation when added after the other factors necessary for assembly of a preinitiation complex on naked DNA. Thus, TAF-I is required to facilitate transcription at a step after chromatin assembly but before transcript elongation.

Mutually Exclusive CBC-Containing Complexes Contribute to RNA Fate

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Simone Giacometti

2017-03-01

Full Text Available The nuclear cap-binding complex (CBC stimulates processing reactions of capped RNAs, including their splicing, 3′-end formation, degradation, and transport. CBC effects are particular for individual RNA families, but how such selectivity is achieved remains elusive. Here, we analyze three main CBC partners known to impact different RNA species. ARS2 stimulates 3′-end formation/transcription termination of several transcript types, ZC3H18 stimulates degradation of a diverse set of RNAs, and PHAX functions in pre-small nuclear RNA/small nucleolar RNA (pre-snRNA/snoRNA transport. Surprisingly, these proteins all bind capped RNAs without strong preferences for given transcripts, and their steady-state binding correlates poorly with their function. Despite this, PHAX and ZC3H18 compete for CBC binding and we demonstrate that this competitive binding is functionally relevant. We further show that CBC-containing complexes are short lived in vivo, and we therefore suggest that RNA fate involves the transient formation of mutually exclusive CBC complexes, which may only be consequential at particular checkpoints during RNA biogenesis.

Changes in signal transducer and activator of transcription 3 (STAT3) dynamics induced by complexation with pharmacological inhibitors of Src homology 2 (SH2) domain dimerization.

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Resetca, Diana; Haftchenary, Sina; Gunning, Patrick T; Wilson, Derek J

2014-11-21

The activity of the transcription factor signal transducer and activator of transcription 3 (STAT3) is dysregulated in a number of hematological and solid malignancies. Development of pharmacological STAT3 Src homology 2 (SH2) domain interaction inhibitors holds great promise for cancer therapy, and a novel class of salicylic acid-based STAT3 dimerization inhibitors that includes orally bioavailable drug candidates has been recently developed. The compounds SF-1-066 and BP-1-102 are predicted to bind to the STAT3 SH2 domain. However, given the highly unstructured and dynamic nature of the SH2 domain, experimental confirmation of this prediction was elusive. We have interrogated the protein-ligand interaction of STAT3 with these small molecule inhibitors by means of time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry. Analysis of site-specific evolution of deuterium uptake induced by the complexation of STAT3 with SF-1-066 or BP-1-102 under physiological conditions enabled the mapping of the in silico predicted inhibitor binding site to the STAT3 SH2 domain. The binding of both inhibitors to the SH2 domain resulted in significant local decreases in dynamics, consistent with solvent exclusion at the inhibitor binding site and increased rigidity of the inhibitor-complexed SH2 domain. Interestingly, inhibitor binding induced hot spots of allosteric perturbations outside of the SH2 domain, manifesting mainly as increased deuterium uptake, in regions of STAT3 important for DNA binding and nuclear localization. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The Mediator subunit MED23 couples H2B mono-ubiquitination to transcriptional control and cell fate determination.

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Yao, Xiao; Tang, Zhanyun; Fu, Xing; Yin, Jingwen; Liang, Yan; Li, Chonghui; Li, Huayun; Tian, Qing; Roeder, Robert G; Wang, Gang

2015-12-02

The Mediator complex orchestrates multiple transcription factors with the Pol II apparatus for precise transcriptional control. However, its interplay with the surrounding chromatin remains poorly understood. Here, we analyze differential histone modifications between WT and MED23(-/-) (KO) cells and identify H2B mono-ubiquitination at lysine 120 (H2Bub) as a MED23-dependent histone modification. Using tandem affinity purification and mass spectrometry, we find that MED23 associates with the RNF20/40 complex, the enzyme for H2Bub, and show that this association is critical for the recruitment of RNF20/40 to chromatin. In a cell-free system, Mediator directly and substantially increases H2Bub on recombinant chromatin through its cooperation with RNF20/40 and the PAF complex. Integrative genome-wide analyses show that MED23 depletion specifically reduces H2Bub on a subset of MED23-controlled genes. Importantly, MED23-coupled H2Bub levels are oppositely regulated during myogenesis and lung carcinogenesis. In sum, these results establish a mechanistic link between the Mediator complex and a critical chromatin modification in coordinating transcription with cell growth and differentiation. © 2015 The Authors.

In vitro-reconstituted nucleoids can block mitochondrial DNA replication and transcription

NARCIS (Netherlands)

Farge, Géraldine; Mehmedovic, Majda; Baclayon, Marian; van den Wildenberg, Siet M J L; Roos, Wouter H; Gustafsson, Claes M; Wuite, Gijs J L; Falkenberg, Maria

2014-01-01

The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A

Mutant Forms of the Azotobacter vinelandii Transcriptional Activator NifA Resistant to Inhibition by the NifL Regulatory Protein

OpenAIRE

Reyes-Ramirez, Francisca; Little, Richard; Dixon, Ray

2002-01-01

The Azotobacter vinelandii σ54-dependent transcriptional activator protein NifA is regulated by the NifL protein in response to redox, carbon, and nitrogen status. Under conditions inappropriate for nitrogen fixation, NifL inhibits transcription activation by NifA through the formation of the NifL-NifA protein complex. NifL inhibits the ATPase activity of the central AAA+ domain of NifA required to drive open complex formation by σ54-RNA polymerase and may also inhibit the activator-polymeras...

Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

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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.

WRKY transcription factors

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Bakshi, Madhunita; Oelmüller, Ralf

2014-01-01

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

Nuclear Matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling

International Nuclear Information System (INIS)

Sreenath, Kadreppa; Pavithra, Lakshminarasimhan; Singh, Sandeep; Sinha, Surajit; Dash, Prasanta K.; Siddappa, Nagadenahalli B.; Ranga, Udaykumar; Mitra, Debashis; Chattopadhyay, Samit

2010-01-01

Nuclear Matrix and MARs have been implicated in the transcriptional regulation of host as well as viral genes but their precise role in HIV-1 transcription remains unclear. Here, we show that > 98% of HIV sequences contain consensus MAR element in their promoter. We show that SMAR1 binds to the LTR MAR and reinforces transcriptional silencing by tethering the LTR MAR to nuclear matrix. SMAR1 associated HDAC1-mSin3 corepressor complex is dislodged from the LTR upon cellular activation by PMA/TNFα leading to an increase in the acetylation and a reduction in the trimethylation of histones, associated with the recruitment of RNA Polymerase II on the LTR. Overexpression of SMAR1 lead to reduction in LTR mediated transcription, both in a Tat dependent and independent manner, resulting in a decreased virion production. These results demonstrate the role of SMAR1 in regulating viral transcription by alternative compartmentalization of LTR between the nuclear matrix and chromatin.

Non-circadian expression masking clock-driven weak transcription rhythms in U2OS cells.

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Julia Hoffmann

Full Text Available U2OS cells harbor a circadian clock but express only a few rhythmic genes in constant conditions. We identified 3040 binding sites of the circadian regulators BMAL1, CLOCK and CRY1 in the U2OS genome. Most binding sites even in promoters do not correlate with detectable rhythmic transcript levels. Luciferase fusions reveal that the circadian clock supports robust but low amplitude transcription rhythms of representative promoters. However, rhythmic transcription of these potentially clock-controlled genes is masked by non-circadian transcription that overwrites the weaker contribution of the clock in constant conditions. Our data suggest that U2OS cells harbor an intrinsically rather weak circadian oscillator. The oscillator has the potential to regulate a large number of genes. The contribution of circadian versus non-circadian transcription is dependent on the metabolic state of the cell and may determine the apparent complexity of the circadian transcriptome.

bZIPs and WRKYs: two large transcription factor families executing two different functional strategies

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Carles eMarco Llorca

2014-04-01

Full Text Available bZIPs and WRKYs are two important plant transcription factor families regulating diverse developmental and stress-related processes. Since a partial overlap in these biological processes is obvious, it can be speculated that they fulfill non-redundant functions in a complex regulatory network. Here, we focus on the regulatory mechanisms that are so far described for bZIPs and WRKYs. bZIP factors need to heterodimerize for DNA-binding and regulation of transcription, and based on a bioinformatics approach, bZIPs can build up more than the double of protein interactions than WRKYs. In contrast, an enrichment of the WRKY DNA-binding motifs can be found in WRKY promoters, a phenomenon which is not observed for the bZIP family. Thus, the two transcription factor families follow two different functional strategies in which WRKYs regulate each other’s transcription in a transcriptional network whereas bZIP action relies on intensive heterodimerization.

Factor C*, the specific initiation component of the mouse RNA polymerase I holoenzyme, is inactivated early in the transcription process.

OpenAIRE

Brun, R P; Ryan, K; Sollner-Webb, B

1994-01-01

Factor C* is the component of the RNA polymerase I holoenzyme (factor C) that allows specific transcriptional initiation on a factor D (SL1)- and UBF-activated rRNA gene promoter. The in vitro transcriptional capacity of a preincubated rDNA promoter complex becomes exhausted very rapidly upon initiation of transcription. This is due to the rapid depletion of C* activity. In contrast, C* activity is not unstable in the absence of transcription, even in the presence of nucleoside triphosphates ...

Dendritic Cytoskeletal Architecture Is Modulated by Combinatorial Transcriptional Regulation in Drosophila melanogaster.

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Das, Ravi; Bhattacharjee, Shatabdi; Patel, Atit A; Harris, Jenna M; Bhattacharya, Surajit; Letcher, Jamin M; Clark, Sarah G; Nanda, Sumit; Iyer, Eswar Prasad R; Ascoli, Giorgio A; Cox, Daniel N

2017-12-01

Transcription factors (TFs) have emerged as essential cell autonomous mediators of subtype specific dendritogenesis; however, the downstream effectors of these TFs remain largely unknown, as are the cellular events that TFs control to direct morphological change. As dendritic morphology is largely dictated by the organization of the actin and microtubule (MT) cytoskeletons, elucidating TF-mediated cytoskeletal regulatory programs is key to understanding molecular control of diverse dendritic morphologies. Previous studies in Drosophila melanogaster have demonstrated that the conserved TFs Cut and Knot exert combinatorial control over aspects of dendritic cytoskeleton development, promoting actin and MT-based arbor morphology, respectively. To investigate transcriptional targets of Cut and/or Knot regulation, we conducted systematic neurogenomic studies, coupled with in vivo genetic screens utilizing multi-fluor cytoskeletal and membrane marker reporters. These analyses identified a host of putative Cut and/or Knot effector molecules, and a subset of these putative TF targets converge on modulating dendritic cytoskeletal architecture, which are grouped into three major phenotypic categories, based upon neuromorphometric analyses: complexity enhancer, complexity shifter, and complexity suppressor. Complexity enhancer genes normally function to promote higher order dendritic growth and branching with variable effects on MT stabilization and F-actin organization, whereas complexity shifter and complexity suppressor genes normally function in regulating proximal-distal branching distribution or in restricting higher order branching complexity, respectively, with spatially restricted impacts on the dendritic cytoskeleton. Collectively, we implicate novel genes and cellular programs by which TFs distinctly and combinatorially govern dendritogenesis via cytoskeletal modulation. Copyright © 2017 by the Genetics Society of America.

Dual Regulation of Bacillus subtilis kinB Gene Encoding a Sporulation Trigger by SinR through Transcription Repression and Positive Stringent Transcription Control.

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Fujita, Yasutaro; Ogura, Mitsuo; Nii, Satomi; Hirooka, Kazutake

2017-01-01

It is known that transcription of kinB encoding a trigger for Bacillus subtilis sporulation is under repression by SinR, a master repressor of biofilm formation, and under positive stringent transcription control depending on the adenine species at the transcription initiation nucleotide (nt). Deletion and base substitution analyses of the kinB promoter (P kinB ) region using lacZ fusions indicated that either a 5-nt deletion (Δ5, nt -61/-57, +1 is the transcription initiation nt) or the substitution of G at nt -45 with A (G-45A) relieved kinB repression. Thus, we found a pair of SinR-binding consensus sequences (GTTCTYT; Y is T or C) in an inverted orientation (SinR-1) between nt -57/-42, which is most likely a SinR-binding site for kinB repression. This relief from SinR repression likely requires SinI, an antagonist of SinR. Surprisingly, we found that SinR is essential for positive stringent transcription control of P kinB . Electrophoretic mobility shift assay (EMSA) analysis indicated that SinR bound not only to SinR-1 but also to SinR-2 (nt -29/-8) consisting of another pair of SinR consensus sequences in a tandem repeat arrangement; the two sequences partially overlap the '-35' and '-10' regions of P kinB . Introduction of base substitutions (T-27C C-26T) in the upstream consensus sequence of SinR-2 affected positive stringent transcription control of P kinB , suggesting that SinR binding to SinR-2 likely causes this positive control. EMSA also implied that RNA polymerase and SinR are possibly bound together to SinR-2 to form a transcription initiation complex for kinB transcription. Thus, it was suggested in this work that derepression of kinB from SinR repression by SinI induced by Spo0A∼P and occurrence of SinR-dependent positive stringent transcription control of kinB might induce effective sporulation cooperatively, implying an intimate interplay by stringent response, sporulation, and biofilm formation.

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

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

2017-01-01

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

Nitrogen fixation and molecular oxygen: comparative genomic reconstruction of transcription regulation in Alphaproteobacteria

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Olga V Tsoy

2016-08-01

Full Text Available Biological nitrogen fixation plays a crucial role in the nitrogen cycle. An ability to fix atmospheric nitrogen, reducing it to ammonium, was described for multiple species of Bacteria and Archaea. Being a complex and sensitive process, nitrogen fixation requires a complicated regulatory system, also, on the level of transcription. The transcriptional regulatory network for nitrogen fixation was extensively studied in several representatives of the class Alphaproteobacteria. This regulatory network includes the activator of nitrogen fixation NifA, working in tandem with the alternative sigma-factor RpoN as well as oxygen-responsive regulatory systems, one-component regulators FnrN/FixK and two-component system FixLJ. Here we used a comparative genomics analysis for in silico study of the transcriptional regulatory network in 50 genomes of Alphaproteobacteria. We extended the known regulons and proposed the scenario for the evolution of the nitrogen fixation transcriptional network. The reconstructed network substantially expands the existing knowledge of transcriptional regulation in nitrogen-fixing microorganisms and can be used for genetic experiments, metabolic reconstruction, and evolutionary analysis.

Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation

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Raj Kumar

2008-08-01

Full Text Available Raj Kumar1, William J Calhoun21Division of Gastroenterology; 2Division of Allergy, Pulmonary, Immunology, Critical Care, and Sleep (APICS, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USAAbstract: Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade

Waveband specific transcriptional control of select genetic pathways in vertebrate skin (Xiphophorus maculatus).

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Walter, Ronald B; Boswell, Mikki; Chang, Jordan; Boswell, William T; Lu, Yuan; Navarro, Kaela; Walter, Sean M; Walter, Dylan J; Salinas, Raquel; Savage, Markita

2018-05-10

Evolution occurred exclusively under the full spectrum of sunlight. Conscription of narrow regions of the solar spectrum by specific photoreceptors suggests a common strategy for regulation of genetic pathways. Fluorescent light (FL) does not possess the complexity of the solar spectrum and has only been in service for about 60 years. If vertebrates evolved specific genetic responses regulated by light wavelengths representing the entire solar spectrum, there may be genetic consequences to reducing the spectral complexity of light. We utilized RNA-Seq to assess changes in the transcriptional profiles of Xiphophorus maculatus skin after exposure to FL ("cool white"), or narrow wavelength regions of light between 350 and 600 nm (i.e., 50 nm or 10 nm regions, herein termed "wavebands"). Exposure to each 50 nm waveband identified sets of genes representing discrete pathways that showed waveband specific transcriptional modulation. For example, 350-400 or 450-500 nm waveband exposures resulted in opposite regulation of gene sets marking necrosis and apoptosis (i.e., 350-400 nm; necrosis suppression, apoptosis activation, while 450-500 nm; apoptosis suppression, necrosis activation). Further investigation of specific transcriptional modulation employing successive 10 nm waveband exposures between 500 and 550 nm showed; (a) greater numbers of genes may be transcriptionally modulated after 10 nm exposures, than observed for 50 nm or FL exposures, (b) the 10 nm wavebands induced gene sets showing greater functional specificity than 50 nm or FL exposures, and (c) the genetic effects of FL are primarily due to 30 nm between 500 and 530 nm. Interestingly, many genetic pathways exhibited completely opposite transcriptional effects after different waveband exposures. For example, the epidermal growth factor (EGF) pathway exhibits transcriptional suppression after FL exposure, becomes highly active after 450-500 nm waveband exposure, and again, exhibits strong

Arabidopsis MAP Kinase 4 regulates gene expression via transcription factor release in the nucleus

DEFF Research Database (Denmark)

Qiu, Jin-Long; Fiil, Berthe Katrine; Petersen, Klaus

2008-01-01

kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from...... MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further...... supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation....

NMR assignments of SPOC domain of the human transcriptional corepressor SHARP in complex with a C-terminal SMRT peptide.

Science.gov (United States)

Mikami, Suzuka; Kanaba, Teppei; Ito, Yutaka; Mishima, Masaki

2013-10-01

The transcriptional corepressor SMRT/HDAC1-associated repressor protein (SHARP) recruits histone deacetylases. Human SHARP protein is thought to function in processes involving steroid hormone responses and the Notch signaling pathway. SHARP consists of RNA recognition motifs (RRMs) in the N-terminal region and the spen paralog and ortholog C-terminal (SPOC) domain in the C-terminal region. It is known that the SPOC domain binds the LSD motif in the C-terminal tail of corepressors silencing mediator for retinoid and thyroid receptor (SMRT)/nuclear receptor corepressor (NcoR). We are interested in delineating the mechanism by which the SPOC domain recognizes the LSD motif of the C-terminal tail of SMRT/NcoR. To this end, we are investigating the tertiary structure of the SPOC/SMRT peptide using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of the SPOC domain in complex with a SMRT peptide, which contributes towards a structural understanding of the SPOC/SMRT peptide and its molecular recognition.

Activating RNAs associate with Mediator to enhance chromatin architecture and transcription.

Science.gov (United States)

Lai, Fan; Orom, Ulf A; Cesaroni, Matteo; Beringer, Malte; Taatjes, Dylan J; Blobel, Gerd A; Shiekhattar, Ramin

2013-02-28

Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions. We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism. To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease- displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

TGF-β Suppression of HBV RNA through AID-Dependent Recruitment of an RNA Exosome Complex

Science.gov (United States)

Kitamura, Kouichi; Wang, Zhe; Chowdhury, Sajeda; Monjurul, Ahasan Md; Wakae, Kousho; Koura, Miki; Shimadu, Miyuki; Kinoshita, Kazuo; Muramatsu, Masamichi

2015-01-01

Transforming growth factor (TGF)-β inhibits hepatitis B virus (HBV) replication although the intracellular effectors involved are not determined. Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication. Immunoprecipitation reveals that AID physically associates with viral P protein that binds to specific virus RNA sequence called epsilon. AID also binds to an RNA degradation complex (RNA exosome proteins), indicating that AID, RNA exosome, and P protein form an RNP complex. Suppression of HBV transcripts by TGF-β was abrogated by depletion of either AID or RNA exosome components, suggesting that AID and the RNA exosome involve in TGF-β mediated suppression of HBV RNA. Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited. These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner. PMID:25836330

hCLE/C14orf166 associates with DDX1-HSPC117-FAM98B in a novel transcription-dependent shuttling RNA-transporting complex.

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Alicia Pérez-González

Full Text Available hCLE/C14orf166 is a nuclear and cytoplasmic protein that interacts with the RNAP II, modulates nuclear RNA metabolism and is present in cytoplasmic RNA granules involved in localized translation. Here we have studied whether hCLE shares common interactors in the nucleus and the cytosol, which could shed light on its participation in the sequential phases of RNA metabolism. Nuclear and cytoplasmic purified hCLE-associated factors were identified and proteins involved in mRNA metabolism, motor-related proteins, cytoskeletal and translation-related factors were found. Purified hCLE complexes also contain RNAs and as expected some hCLE-interacting proteins (DDX1, HSPC117, FAM98B were found both in the nucleus and the cytoplasm. Moreover, endogenous hCLE fractionates in protein complexes together with DDX1, HSPC117 and FAM98B and silencing of hCLE down-regulates their nuclear and cytosolic accumulation levels. Using a photoactivatable hCLE-GFP protein, nuclear import and export of hCLE was observed indicating that hCLE is a shuttling protein. Interestingly, hCLE nuclear import required active transcription, as did the import of DDX1, HSPC117 and FAM98B proteins. The data indicate that hCLE probably as a complex with DDX1, HSPC117 and FAM98B shuttles between the nucleus and the cytoplasm transporting RNAs suggesting that this complex has a prominent role on nuclear and cytoplasmic RNA fate.

Mediator can regulate mitotic entry and direct periodic transcription in fission yeast.

Science.gov (United States)

Banyai, Gabor; Lopez, Marcela Davila; Szilagyi, Zsolt; Gustafsson, Claes M

2014-11-01

Cdk8 is required for correct timing of mitotic progression in fission yeast. How the activity of Cdk8 is regulated is unclear, since the kinase is not activated by T-loop phosphorylation and its partner, CycC, does not oscillate. Cdk8 is, however, a component of the multiprotein Mediator complex, a conserved coregulator of eukaryotic transcription that is connected to a number of intracellular signaling pathways. We demonstrate here that other Mediator components regulate the activity of Cdk8 in vivo and thereby direct the timing of mitotic entry. Deletion of Mediator components Med12 and Med13 leads to higher cellular Cdk8 protein levels, premature phosphorylation of the Cdk8 target Fkh2, and earlier entry into mitosis. We also demonstrate that Mediator is recruited to clusters of mitotic genes in a periodic fashion and that the complex is required for the transcription of these genes. We suggest that Mediator functions as a hub for coordinated regulation of mitotic progression and cell cycle-dependent transcription. The many signaling pathways and activator proteins shown to function via Mediator may influence the timing of these cell cycle events. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

Science.gov (United States)

Bykowski, Tomasz; Babb, Kelly; von Lackum, Kate; Riley, Sean P; Norris, Steven J; Stevenson, Brian

2006-07-01

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.

Two Gene Clusters Coordinate Galactose and Lactose Metabolism in Streptococcus gordonii

Science.gov (United States)

Zeng, Lin; Martino, Nicole C.

2012-01-01

Streptococcus gordonii is an early colonizer of the human oral cavity and an abundant constituent of oral biofilms. Two tandemly arranged gene clusters, designated lac and gal, were identified in the S. gordonii DL1 genome, which encode genes of the tagatose pathway (lacABCD) and sugar phosphotransferase system (PTS) enzyme II permeases. Genes encoding a predicted phospho-β-galactosidase (LacG), a DeoR family transcriptional regulator (LacR), and a transcriptional antiterminator (LacT) were also present in the clusters. Growth and PTS assays supported that the permease designated EIILac transports lactose and galactose, whereas EIIGal transports galactose. The expression of the gene for EIIGal was markedly upregulated in cells growing on galactose. Using promoter-cat fusions, a role for LacR in the regulation of the expressions of both gene clusters was demonstrated, and the gal cluster was also shown to be sensitive to repression by CcpA. The deletion of lacT caused an inability to grow on lactose, apparently because of its role in the regulation of the expression of the genes for EIILac, but had little effect on galactose utilization. S. gordonii maintained a selective advantage over Streptococcus mutans in a mixed-species competition assay, associated with its possession of a high-affinity galactose PTS, although S. mutans could persist better at low pHs. Collectively, these results support the concept that the galactose and lactose systems of S. gordonii are subject to complex regulation and that a high-affinity galactose PTS may be advantageous when S. gordonii is competing against the caries pathogen S. mutans in oral biofilms. PMID:22660715

Ctr9, a Protein in the Transcription Complex Paf1, Regulates Dopamine Transporter Activity at the Plasma Membrane.

Science.gov (United States)

De Gois, Stéphanie; Slama, Patrick; Pietrancosta, Nicolas; Erdozain, Amaia M; Louis, Franck; Bouvrais-Veret, Caroline; Daviet, Laurent; Giros, Bruno

2015-07-17

Dopamine (DA) is a major regulator of sensorimotor and cognitive functions. The DA transporter (DAT) is the key protein that regulates the spatial and temporal activity of DA release into the synaptic cleft via the rapid reuptake of DA into presynaptic termini. Several lines of evidence have suggested that transporter-interacting proteins may play a role in DAT function and regulation. Here, we identified the tetratricopeptide repeat domain-containing protein Ctr9 as a novel DAT binding partner using a yeast two-hybrid system. We showed that Ctr9 is expressed in dopaminergic neurons and forms a stable complex with DAT in vivo via GST pulldown and co-immunoprecipitation assays. In mammalian cells co-expressing both proteins, Ctr9 partially colocalizes with DAT at the plasma membrane. This interaction between DAT and Ctr9 results in a dramatic enhancement of DAT-mediated DA uptake due to an increased number of DAT transporters at the plasma membrane. We determined that the binding of Ctr9 to DAT requires residues YKF in the first half of the DAT C terminus. In addition, we characterized Ctr9, providing new insight into this protein. Using three-dimensional modeling, we identified three novel tetratricopeptide repeat domains in the Ctr9 sequence, and based on deletion mutation experiments, we demonstrated the role of the SH2 domain of Ctr9 in nuclear localization. Our results demonstrate that Ctr9 localization is not restricted to the nucleus, as previously described for the transcription complex Paf1. Taken together, our data provide evidence that Ctr9 modulates DAT function by regulating its trafficking. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional analysis of limb transcriptional enhancers in the mouse.

Science.gov (United States)

Nolte, Mark J; Wang, Ying; Deng, Jian Min; Swinton, Paul G; Wei, Caimiao; Guindani, Michele; Schwartz, Robert J; Behringer, Richard R

2014-01-01

Transcriptional enhancers are genomic sequences bound by transcription factors that act together with basal transcriptional machinery to regulate gene transcription. Several high-throughput methods have generated large datasets of tissue-specific enhancer sequences with putative roles in developmental processes. However, few enhancers have been deleted from the genome to determine their roles in development. To understand the roles of two enhancers active in the mouse embryonic limb bud we deleted them from the genome. Although the genes regulated by these enhancers are unknown, they were selected because they were identified in a screen for putative limb bud-specific enhancers associated with p300, an acetyltransferase that participates in protein complexes that promote active transcription, and because the orthologous human enhancers (H1442 and H280) drive distinct lacZ expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. We show that the orthologous mouse sequences, M1442 and M280, regulate dynamic expression in the developing limb. Although significant transcriptional differences in enhancer-proximal genes in embryonic limb buds accompany the deletion of M1442 and M280 no gross limb malformations during embryonic development were observed, demonstrating that M1442 and M280 are not required for mouse limb development. However, M280 is required for the development and/or maintenance of body size; M280 mice are significantly smaller than controls. M280 also harbors an "ultraconserved" sequence that is identical between human, rat, and mouse. This is the first report of a phenotype resulting from the deletion of an ultraconserved element. These studies highlight the importance of determining enhancer regulatory function by experiments that manipulate them in situ and suggest that some of an enhancer's regulatory capacities may be developmentally tolerated rather than developmentally required. © 2014 Wiley Periodicals, Inc.

Tissue-specific 5' heterogeneity of PPARα transcripts and their differential regulation by leptin.

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Emma S Garratt

Full Text Available The genes encoding nuclear receptors comprise multiple 5'untranslated exons, which give rise to several transcripts encoding the same protein, allowing tissue-specific regulation of expression. Both human and mouse peroxisome proliferator activated receptor (PPAR α genes have multiple promoters, although their function is unknown. Here we have characterised the rat PPARα promoter region and have identified three alternative PPARα transcripts, which have different transcription start sites owing to the utilisation of distinct first exons. Moreover these alternative PPARα transcripts were differentially expressed between adipose tissue and liver. We show that while the major adipose (P1 and liver (P2 transcripts were both induced by dexamethasone, they were differentially regulated by the PPARα agonist, clofibric acid, and leptin. Leptin had no effect on the adipose-specific P1 transcript, but induced liver-specific P2 promoter activity via a STAT3/Sp1 mechanism. Moreover in Wistar rats, leptin treatment between postnatal day 3-13 led to an increase in P2 but not P1 transcription in adipose tissue which was sustained into adulthood. This suggests that the expression of the alternative PPARα transcripts are in part programmed by early life exposure to leptin leading to persistent change in adipose tissue fatty acid metabolism through specific activation of a quiescent PPARα promoter. Such complexity in the regulation of PPARα may allow the expression of PPARα to be finely regulated in response to environmental factors.

Fractional dynamics of globally slow transcription and its impact on deterministic genetic oscillation.

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Kun Wei

Full Text Available In dynamical systems theory, a system which can be described by differential equations is called a continuous dynamical system. In studies on genetic oscillation, most deterministic models at early stage are usually built on ordinary differential equations (ODE. Therefore, gene transcription which is a vital part in genetic oscillation is presupposed to be a continuous dynamical system by default. However, recent studies argued that discontinuous transcription might be more common than continuous transcription. In this paper, by appending the inserted silent interval lying between two neighboring transcriptional events to the end of the preceding event, we established that the running time for an intact transcriptional event increases and gene transcription thus shows slow dynamics. By globally replacing the original time increment for each state increment by a larger one, we introduced fractional differential equations (FDE to describe such globally slow transcription. The impact of fractionization on genetic oscillation was then studied in two early stage models--the Goodwin oscillator and the Rössler oscillator. By constructing a "dual memory" oscillator--the fractional delay Goodwin oscillator, we suggested that four general requirements for generating genetic oscillation should be revised to be negative feedback, sufficient nonlinearity, sufficient memory and proper balancing of timescale. The numerical study of the fractional Rössler oscillator implied that the globally slow transcription tends to lower the chance of a coupled or more complex nonlinear genetic oscillatory system behaving chaotically.

Fractional dynamics of globally slow transcription and its impact on deterministic genetic oscillation.

Science.gov (United States)

Wei, Kun; Gao, Shilong; Zhong, Suchuan; Ma, Hong

2012-01-01

In dynamical systems theory, a system which can be described by differential equations is called a continuous dynamical system. In studies on genetic oscillation, most deterministic models at early stage are usually built on ordinary differential equations (ODE). Therefore, gene transcription which is a vital part in genetic oscillation is presupposed to be a continuous dynamical system by default. However, recent studies argued that discontinuous transcription might be more common than continuous transcription. In this paper, by appending the inserted silent interval lying between two neighboring transcriptional events to the end of the preceding event, we established that the running time for an intact transcriptional event increases and gene transcription thus shows slow dynamics. By globally replacing the original time increment for each state increment by a larger one, we introduced fractional differential equations (FDE) to describe such globally slow transcription. The impact of fractionization on genetic oscillation was then studied in two early stage models--the Goodwin oscillator and the Rössler oscillator. By constructing a "dual memory" oscillator--the fractional delay Goodwin oscillator, we suggested that four general requirements for generating genetic oscillation should be revised to be negative feedback, sufficient nonlinearity, sufficient memory and proper balancing of timescale. The numerical study of the fractional Rössler oscillator implied that the globally slow transcription tends to lower the chance of a coupled or more complex nonlinear genetic oscillatory system behaving chaotically.

Mediator MED23 regulates basal transcription in vivo via an interaction with P-TEFb.

Science.gov (United States)

Wang, Wei; Yao, Xiao; Huang, Yan; Hu, Xiangming; Liu, Runzhong; Hou, Dongming; Chen, Ruichuan; Wang, Gang

2013-01-01

The Mediator is a multi-subunit complex that transduces regulatory information from transcription regulators to the RNA polymerase II apparatus. Growing evidence suggests that Mediator plays roles in multiple stages of eukaryotic transcription, including elongation. However, the detailed mechanism by which Mediator regulates elongation remains elusive. In this study, we demonstrate that Mediator MED23 subunit controls a basal level of transcription by recruiting elongation factor P-TEFb, via an interaction with its CDK9 subunit. The mRNA level of Egr1, a MED23-controlled model gene, is reduced 4-5 fold in Med23 (-/-) ES cells under an unstimulated condition, but Med23-deficiency does not alter the occupancies of RNAP II, GTFs, Mediator complex, or activator ELK1 at the Egr1 promoter. Instead, Med23 depletion results in a significant decrease in P-TEFb and RNAP II (Ser2P) binding at the coding region, but no changes for several other elongation regulators, such as DSIF and NELF. ChIP-seq revealed that Med23-deficiency partially reduced the P-TEFb occupancy at a set of MED23-regulated gene promoters. Further, we demonstrate that MED23 interacts with CDK9 in vivo and in vitro. Collectively, these results provide the mechanistic insight into how Mediator promotes RNAP II into transcription elongation.

Circuit-wide Transcriptional Profiling Reveals Brain Region-Specific Gene Networks Regulating Depression Susceptibility.

Science.gov (United States)

Bagot, Rosemary C; Cates, Hannah M; Purushothaman, Immanuel; Lorsch, Zachary S; Walker, Deena M; Wang, Junshi; Huang, Xiaojie; Schlüter, Oliver M; Maze, Ian; Peña, Catherine J; Heller, Elizabeth A; Issler, Orna; Wang, Minghui; Song, Won-Min; Stein, Jason L; Liu, Xiaochuan; Doyle, Marie A; Scobie, Kimberly N; Sun, Hao Sheng; Neve, Rachael L; Geschwind, Daniel; Dong, Yan; Shen, Li; Zhang, Bin; Nestler, Eric J

2016-06-01

Depression is a complex, heterogeneous disorder and a leading contributor to the global burden of disease. Most previous research has focused on individual brain regions and genes contributing to depression. However, emerging evidence in humans and animal models suggests that dysregulated circuit function and gene expression across multiple brain regions drive depressive phenotypes. Here, we performed RNA sequencing on four brain regions from control animals and those susceptible or resilient to chronic social defeat stress at multiple time points. We employed an integrative network biology approach to identify transcriptional networks and key driver genes that regulate susceptibility to depressive-like symptoms. Further, we validated in vivo several key drivers and their associated transcriptional networks that regulate depression susceptibility and confirmed their functional significance at the levels of gene transcription, synaptic regulation, and behavior. Our study reveals novel transcriptional networks that control stress susceptibility and offers fundamentally new leads for antidepressant drug discovery. Copyright © 2016 Elsevier Inc. All rights reserved.

Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair.

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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.

The G-quadruplex DNA stabilizing drug pyridostatin promotes DNA damage and downregulates transcription of Brca1 in neurons.

Science.gov (United States)

Moruno-Manchon, Jose F; Koellhoffer, Edward C; Gopakumar, Jayakrishnan; Hambarde, Shashank; Kim, Nayun; McCullough, Louise D; Tsvetkov, Andrey S

2017-09-12

The G-quadruplex is a non-canonical DNA secondary structure formed by four DNA strands containing multiple runs of guanines. G-quadruplexes play important roles in DNA recombination, replication, telomere maintenance, and regulation of transcription. Small molecules that stabilize the G-quadruplexes alter gene expression in cancer cells. Here, we hypothesized that the G-quadruplexes regulate transcription in neurons. We discovered that pyridostatin, a small molecule that specifically stabilizes G-quadruplex DNA complexes, induced neurotoxicity and promoted the formation of DNA double-strand breaks (DSBs) in cultured neurons. We also found that pyridostatin downregulated transcription of the Brca1 gene, a gene that is critical for DSB repair. Importantly, in an in vitro gel shift assay, we discovered that an antibody specific to the G-quadruplex structure binds to a synthetic oligonucleotide, which corresponds to the first putative G-quadruplex in the Brca1 gene promoter. Our results suggest that the G-quadruplex complexes regulate transcription in neurons. Studying the G-quadruplexes could represent a new avenue for neurodegeneration and brain aging research.

Transcription control and neuronal differentiation by agents that activate the LXR nuclear receptor family.

Science.gov (United States)

Schmidt, A; Vogel, R; Holloway, M K; Rutledge, S J; Friedman, O; Yang, Z; Rodan, G A; Friedman, E

1999-09-10

LXR and PPAR receptors belong to the nuclear receptor superfamily of transcriptional activating factors. Using ligand-dependent transcription assays, we found that 5-tetradecyloxy-2-furancarboxylic acid (TOFA) transactivates chimeric receptors composed of the glucocorticoid receptor DNA binding domain and the ligand binding regions of PPARalpha, PPARbeta (NUC-1) and LXRbeta (NER) receptors. In the same assays, ligands for PPARs (oleic acid, WY-14643 and L-631,033) and LXRs (hydroxycholesterols) maintain their respective receptor selectivity. TOFA and hydroxycholesterols also stimulate transcription from a minimal fibrinogen promoter that is under the control of AP-1 or NF-kappaB transcription factor binding sites. In addition to their effects on transcription, these LXRbeta activators induce neuronal differentiation in rat pheochromocytoma cells. TOFA and the natural LXR agonist, 22 (R)-hydroxycholesterol, stimulate neurite outgrowth in 55 and 28% of cells, respectively. No neurite outgrowth was induced by the related 22(S)-hydroxycholesterol, which does not activate the LXR family. These results suggest that the hydroxycholesterol signaling pathway has a complex effect on transcription that mediates the activity of TOFA and hydroxycholesterol on neuronal differentiation in pheochromocytoma cells.

Amplified in Breast Cancer Regulates Transcription and Translation in Breast Cancer Cells.

Science.gov (United States)

Ochnik, Aleksandra M; Peterson, Mark S; Avdulov, Svetlana V; Oh, Annabell S; Bitterman, Peter B; Yee, Douglas

2016-02-01

Control of mRNA translation is fundamentally altered in cancer. Insulin-like growth factor-I (IGF-I) signaling regulates key translation mediators to modulate protein synthesis (e.g. eIF4E, 4E-BP1, mTOR, and S6K1). Importantly the Amplified in Breast Cancer (AIB1) oncogene regulates transcription and is also a downstream mediator of IGF-I signaling. To determine if AIB1 also affects mRNA translation, we conducted gain and loss of AIB1 function experiments in estrogen receptor alpha (ERα)(+) (MCF-7L) and ERα(-) (MDA-MB-231, MDA-MB-435 and LCC6) breast cancer cells. AIB1 positively regulated IGF-I-induced mRNA translation in both ERα(+) and ERα(-) cells. Formation of the eIF4E-4E-BP1 translational complex was altered in the AIB1 ERα(+) and ERα(-) knockdown cells, leading to a reduction in the eIF4E/4E-BP1 and eIF4G/4E-BP1 ratios. In basal and IGF-I stimulated MCF-7 and LCC6 cells, knockdown of AIB1 decreased the integrity of the cap-binding complex, reduced global IGF-I stimulated polyribosomal mRNA recruitment with a concomitant decrease in ten of the thirteen genes tested in polysome-bound mRNAs mapping to proliferation, cell cycle, survival, transcription, translation and ribosome biogenesis ontologies. Specifically, knockdown of AIB1 decreased ribosome-bound mRNA and steady-state protein levels of the transcription factors ERα and E2F1 in addition to reduced ribosome-bound mRNA of the ribosome biogenesis factor BYSL in a cell-line specific manner to regulate mRNA translation. The oncogenic transcription factor AIB1 has a novel role in the regulation of polyribosome recruitment and formation of the translational complex. Combinatorial therapies targeting IGF signaling and mRNA translation in AIB1 expressing breast cancers may have clinical benefit and warrants further investigation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Cooperative binding of transcription factors promotes bimodal gene expression response.

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Pablo S Gutierrez

Full Text Available In the present work we extend and analyze the scope of our recently proposed stochastic model for transcriptional regulation, which considers an arbitrarily complex cis-regulatory system using only elementary reactions. Previously, we determined the role of cooperativity on the intrinsic fluctuations of gene expression for activating transcriptional switches, by means of master equation formalism and computer simulation. This model allowed us to distinguish between two cooperative binding mechanisms and, even though the mean expression levels were not affected differently by the acting mechanism, we showed that the associated fluctuations were different. In the present generalized model we include other regulatory functions in addition to those associated to an activator switch. Namely, we introduce repressive regulatory functions and two theoretical mechanisms that account for the biphasic response that some cis-regulatory systems show to the transcription factor concentration. We have also extended our previous master equation formalism in order to include protein production by stochastic translation of mRNA. Furthermore, we examine the graded/binary scenarios in the context of the interaction energy between transcription factors. In this sense, this is the first report to show that the cooperative binding of transcription factors to DNA promotes the "all-or-none" phenomenon observed in eukaryotic systems. In addition, we confirm that gene expression fluctuation levels associated with one of two cooperative binding mechanism never exceed the fluctuation levels of the other.

Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

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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.

Modifiers of notch transcriptional activity identified by genome-wide RNAi

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Firnhaber Christopher B

2010-10-01

Full Text Available Abstract Background The Notch signaling pathway regulates a diverse array of developmental processes, and aberrant Notch signaling can lead to diseases, including cancer. To obtain a more comprehensive understanding of the genetic network that integrates into Notch signaling, we performed a genome-wide RNAi screen in Drosophila cell culture to identify genes that modify Notch-dependent transcription. Results Employing complementary data analyses, we found 399 putative modifiers: 189 promoting and 210 antagonizing Notch activated transcription. These modifiers included several known Notch interactors, validating the robustness of the assay. Many novel modifiers were also identified, covering a range of cellular localizations from the extracellular matrix to the nucleus, as well as a large number of proteins with unknown function. Chromatin-modifying proteins represent a major class of genes identified, including histone deacetylase and demethylase complex components and other chromatin modifying, remodeling and replacement factors. A protein-protein interaction map of the Notch-dependent transcription modifiers revealed that a large number of the identified proteins interact physically with these core chromatin components. Conclusions The genome-wide RNAi screen identified many genes that can modulate Notch transcriptional output. A protein interaction map of the identified genes highlighted a network of chromatin-modifying enzymes and remodelers that regulate Notch transcription. Our results open new avenues to explore the mechanisms of Notch signal regulation and the integration of this pathway into diverse cellular processes.

Zinc binding activity of human metapneumovirus M2-1 protein is indispensable for viral replication and pathogenesis in vivo.

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Cai, Hui; Zhang, Yu; Ma, Yuanmei; Sun, Jing; Liang, Xueya; Li, Jianrong

2015-06-01

Human metapneumovirus (hMPV) is a member of the Pneumovirinae subfamily in the Paramyxoviridae family that causes respiratory tract infections in humans. Unlike members of the Paramyxovirinae subfamily, the polymerase complex of pneumoviruses requires an additional cofactor, the M2-1 protein, which functions as a transcriptional antitermination factor. The M2-1 protein was found to incorporate zinc ions, although the specific role(s) of the zinc binding activity in viral replication and pathogenesis remains unknown. In this study, we found that the third cysteine (C21) and the last histidine (H25) in the zinc binding motif (CCCH) of hMPV M2-1 were essential for zinc binding activity, whereas the first two cysteines (C7 and C15) play only minor or redundant roles in zinc binding. In addition, the zinc binding motif is essential for the oligomerization of M2-1. Subsequently, recombinant hMPVs (rhMPVs) carrying mutations in the zinc binding motif were recovered. Interestingly, rhMPV-C21S and -H25L mutants, which lacked zinc binding activity, had delayed replication in cell culture and were highly attenuated in cotton rats. In contrast, rhMPV-C7S and -C15S strains, which retained 60% of the zinc binding activity, replicated as efficiently as rhMPV in cotton rats. Importantly, rhMPVs that lacked zinc binding activity triggered high levels of neutralizing antibody and provided complete protection against challenge with rhMPV. Taken together, these results demonstrate that zinc binding activity is indispensable for viral replication and pathogenesis in vivo. These results also suggest that inhibition of zinc binding activity may serve as a novel approach to rationally attenuate hMPV and perhaps other pneumoviruses for vaccine purposes. The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute

Identification and Classification of New Transcripts in Dorper and Small-Tailed Han Sheep Skeletal Muscle Transcriptomes.

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Tianle Chao

Full Text Available High-throughput mRNA sequencing enables the discovery of new transcripts and additional parts of incompletely annotated transcripts. Compared with the human and cow genomes, the reference annotation level of the sheep genome is still low. An investigation of new transcripts in sheep skeletal muscle will improve our understanding of muscle development. Therefore, applying high-throughput sequencing, two cDNA libraries from the biceps brachii of small-tailed Han sheep and Dorper sheep were constructed, and whole-transcriptome analysis was performed to determine the unknown transcript catalogue of this tissue. In this study, 40,129 transcripts were finally mapped to the sheep genome. Among them, 3,467 transcripts were determined to be unannotated in the current reference sheep genome and were defined as new transcripts. Based on protein-coding capacity prediction and comparative analysis of sequence similarity, 246 transcripts were classified as portions of unannotated genes or incompletely annotated genes. Another 1,520 transcripts were predicted with high confidence to be long non-coding RNAs. Our analysis also revealed 334 new transcripts that displayed specific expression in ruminants and uncovered a number of new transcripts without intergenus homology but with specific expression in sheep skeletal muscle. The results confirmed a complex transcript pattern of coding and non-coding RNA in sheep skeletal muscle. This study provided important information concerning the sheep genome and transcriptome annotation, which could provide a basis for further study.

Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

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Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

2015-11-05

Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division. Copyright © 2015 Elsevier Inc. All rights reserved.

Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis

DEFF Research Database (Denmark)

Bartholdy, Christina; Olszewska, Wieslawa; Stryhn, Anette

2004-01-01

elicited with recombinant vaccinia virus expressing the complete RSV M2 protein, but stronger than those induced by a similar DNA construct without the beta2m gene. DNA vaccination led to enhanced pulmonary disease after RSV challenge, with increased weight loss and cell recruitment to the lung. Depletion......A CD8+ T-cell memory response to respiratory syncytial virus (RSV) was generated by using a DNA vaccine construct encoding the dominant Kd-restricted epitope from the viral transcription anti-terminator protein M2 (M2(82-90)), linked covalently to human beta2-microglobulin (beta2m). Cutaneous gene...... of CD8+ T cells reduced, but did not abolish, enhancement of disease. Mice vaccinated with a construct encoding a class I-restricted lymphocytic choriomeningitis virus epitope and beta2m suffered more severe weight loss after RSV infection than unvaccinated RSV-infected mice, although RSV-specific CD8...

Global transcriptional repression in C. elegans germline precursors by regulated sequestration of TAF-4.

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Guven-Ozkan, Tugba; Nishi, Yuichi; Robertson, Scott M; Lin, Rueyling

2008-10-03

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1-P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

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.

A transcription activator-like effector (TALE) induction system mediated by proteolysis.

Science.gov (United States)

Copeland, Matthew F; Politz, Mark C; Johnson, Charles B; Markley, Andrew L; Pfleger, Brian F

2016-04-01

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic.

Axon Regeneration Is Regulated by Ets-C/EBP Transcription Complexes Generated by Activation of the cAMP/Ca2+ Signaling Pathways.

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Chun Li

2015-10-01

Full Text Available The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. In Caenorhabditis elegans, the JNK and p38 MAPK pathways are important for axon regeneration. Axonal injury induces expression of the svh-2 gene encoding a receptor tyrosine kinase, stimulation of which by the SVH-1 growth factor leads to activation of the JNK pathway. Here, we identify ETS-4 and CEBP-1, related to mammalian Ets and C/EBP, respectively, as transcriptional activators of svh-2 expression following axon injury. ETS-4 and CEBP-1 function downstream of the cAMP and Ca2+-p38 MAPK pathways, respectively. We show that PKA-dependent phosphorylation of ETS-4 promotes its complex formation with CEBP-1. Furthermore, activation of both cAMP and Ca2+ signaling is required for activation of svh-2 expression. Thus, the cAMP/Ca2+ signaling pathways cooperatively activate the JNK pathway, which then promotes axon regeneration.

DNA topology and transcription

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Kouzine, Fedor; Levens, David; Baranello, Laura

2014-01-01

Chromatin is a complex assembly that compacts DNA inside the nucleus while providing the necessary level of accessibility to regulatory factors conscripted by cellular signaling systems. In this superstructure, DNA is the subject of mechanical forces applied by variety of molecular motors. Rather than being a rigid stick, DNA possesses dynamic structural variability that could be harnessed during critical steps of genome functioning. The strong relationship between DNA structure and key genomic processes necessitates the study of physical constrains acting on the double helix. Here we provide insight into the source, dynamics, and biology of DNA topological domains in the eukaryotic cells and summarize their possible involvement in gene transcription. We emphasize recent studies that might inspire and impact future experiments on the involvement of DNA topology in cellular functions. PMID:24755522

Transcriptional regulation of respiration in yeast metabolizing differently repressive carbon substrates

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Fendt Sarah-Maria

2010-02-01

Full Text Available Abstract Background Depending on the carbon source, Saccharomyces cerevisiae displays various degrees of respiration. These range from complete respiration as in the case of ethanol, to almost complete fermentation, and thus very low degrees of respiration on glucose. While many key regulators are known for these extreme cases, we focus here on regulators that are relevant at intermediate levels of respiration. Results We address this question by linking the functional degree of respiration to transcriptional regulation via enzyme abundances. Specifically, we investigated aerobic batch cultures with the differently repressive carbon sources glucose, mannose, galactose and pyruvate. Based on 13C flux analysis, we found that the respiratory contribution to cellular energy production was largely absent on glucose and mannose, intermediate on galactose and highest on pyruvate. In vivo abundances of 40 respiratory enzymes were quantified by GFP-fusions under each condition. During growth on the partly and fully respired substrates galactose and pyruvate, several TCA cycle and respiratory chain enzymes were significantly up-regulated. From these enzyme levels and the known regulatory network structure, we determined the probability for a given transcription factor to cause the coordinated expression changes. The most probable transcription factors to regulate the different degrees of respiration were Gcr1p, Cat8p, the Rtg-proteins and the Hap-complex. For the latter three ones we confirmed their importance for respiration by quantifying the degree of respiration and biomass yields in the corresponding deletion strains. Conclusions Cat8p is required for wild-type like respiration, independent of its known activation of gluconeogenic genes. The Rtg-proteins and the Hap-complex are essential for wild-type like respiration under partially respiratory conditions. Under fully respiratory conditions, the Hap-complex, but not the Rtg-proteins are essential

Transcriptional regulation of respiration in yeast metabolizing differently repressive carbon substrates.

Science.gov (United States)

Fendt, Sarah-Maria; Sauer, Uwe

2010-02-18

Depending on the carbon source, Saccharomyces cerevisiae displays various degrees of respiration. These range from complete respiration as in the case of ethanol, to almost complete fermentation, and thus very low degrees of respiration on glucose. While many key regulators are known for these extreme cases, we focus here on regulators that are relevant at intermediate levels of respiration. We address this question by linking the functional degree of respiration to transcriptional regulation via enzyme abundances. Specifically, we investigated aerobic batch cultures with the differently repressive carbon sources glucose, mannose, galactose and pyruvate. Based on 13C flux analysis, we found that the respiratory contribution to cellular energy production was largely absent on glucose and mannose, intermediate on galactose and highest on pyruvate. In vivo abundances of 40 respiratory enzymes were quantified by GFP-fusions under each condition. During growth on the partly and fully respired substrates galactose and pyruvate, several TCA cycle and respiratory chain enzymes were significantly up-regulated. From these enzyme levels and the known regulatory network structure, we determined the probability for a given transcription factor to cause the coordinated expression changes. The most probable transcription factors to regulate the different degrees of respiration were Gcr1p, Cat8p, the Rtg-proteins and the Hap-complex. For the latter three ones we confirmed their importance for respiration by quantifying the degree of respiration and biomass yields in the corresponding deletion strains. Cat8p is required for wild-type like respiration, independent of its known activation of gluconeogenic genes. The Rtg-proteins and the Hap-complex are essential for wild-type like respiration under partially respiratory conditions. Under fully respiratory conditions, the Hap-complex, but not the Rtg-proteins are essential for respiration.

Combinatorial analysis of lupulin gland transcription factors from R2R3Myb, bHLH and WDR families indicates a complex regulation of chs_H1 genes essential for prenylflavonoid biosynthesis in hop (Humulus lupulus L.)

Czech Academy of Sciences Publication Activity Database

Matoušek, Jaroslav; Kocábek, Tomáš; Patzak, J.; Füssy, Zoltán; Procházková, Jitka; Heyerick, A.

2012-01-01

Roč. 12, č. 27 (2012), s. 1471-2229 ISSN 1471-2229 R&D Projects: GA ČR GA521/08/0740; GA MZe QH81052 Institutional research plan: CEZ:AV0Z50510513 Keywords : transcription factor * protein complexes * transient expression assay Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.354, year: 2012

FoxA1 binding to the MMTV LTR modulates chromatin structure and transcription

International Nuclear Information System (INIS)

Holmqvist, Per-Henrik; Belikov, Sergey; Zaret, Kenneth S.; Wrange, Oerjan

2005-01-01

Novel binding sites for the forkhead transcription factor family member Forkhead box A (FoxA), previously referred to as Hepatocyte Nuclear Factor 3 (HNF3), were found within the mouse mammary tumor virus long terminal repeat (MMTV LTR). The effect of FoxA1 on MMTV LTR chromatin structure, and expression was evaluated in Xenopus laevis oocytes. Mutagenesis of either of the two main FoxA binding sites showed that the distal site, -232/-221, conferred FoxA1-dependent partial inhibition of glucocorticoid receptor (GR) driven MMTV transcription. The proximal FoxA binding segment consisted of two individual FoxA sites at -57/-46 and -45/-34, respectively, that mediated an increased basal MMTV transcription. FoxA1 binding altered the chromatin structure of both the inactive- and the hormone-activated MMTV LTR. Hydroxyl radical foot printing revealed FoxA1-mediated changes in the nucleosome arrangement. Micrococcal nuclease digestion showed the hormone-dependent sub-nucleosome complex, containing ∼120 bp of DNA, to be expanded by FoxA1 binding to the proximal segment into a larger complex containing ∼200 bp. The potential function of the FoxA1-mediated expression of the MMTV provirus for maintenance of expression in different tissues is discussed

Functional Interaction Map of Lyssavirus Phosphoprotein: Identification of the Minimal Transcription Domains

Science.gov (United States)

Jacob, Yves; Real, Eléonore; Tordo, Noël

2001-01-01

Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. The phylogenetically distant rabies virus (PV strain, genotype 1) and Mokola virus (genotype 3) were used to develop a strategy to identify functional homologous interactive domains from two proteins (P and N) which participate in the viral ribonucleoprotein (RNP) transcription-replication complex. This strategy combined two-hybrid and green fluorescent protein–reverse two-hybrid assays in Saccharomyces cerevisiae to analyze protein-protein interactions and a reverse genetic assay in mammalian cells to study the transcriptional activity of the reconstituted RNP complex. Lyssavirus P proteins contain two N-binding domains (N-BDs), a strong one encompassing amino acid (aa) 176 to the C terminus and a weak one in the 189 N-terminal aa. The N-terminal portion of P (aa 52 to 189) also contains a homomultimerization site. Here we demonstrate that N-P interactions, although weaker, are maintained between proteins of the different genotypes. A minimal transcriptional module of the P protein was obtained by fusing the first 60 N-terminal aa containing the L protein binding site to the C-terminal strong N-BD. Random mutation of the strong N-BD on P protein identified three highly conserved K residues crucial for N-P interaction. Their mutagenesis in full-length P induced a transcriptionally defective RNP. The analysis of homologous interactive domains presented here and previously reported dissections of the P protein allowed us to propose a model of the functional interaction network of the lyssavirus P protein. This model underscores the central role of P at the interface between L protein and N-RNA template. PMID:11559793

Tissue-Specific 5′ Heterogeneity of PPARα Transcripts and Their Differential Regulation by Leptin

Science.gov (United States)

Garratt, Emma S.; Vickers, Mark H.; Gluckman, Peter D.; Hanson, Mark A.

2013-01-01

The genes encoding nuclear receptors comprise multiple 5′untranslated exons, which give rise to several transcripts encoding the same protein, allowing tissue-specific regulation of expression. Both human and mouse peroxisome proliferator activated receptor (PPAR) α genes have multiple promoters, although their function is unknown. Here we have characterised the rat PPARα promoter region and have identified three alternative PPARα transcripts, which have different transcription start sites owing to the utilisation of distinct first exons. Moreover these alternative PPARα transcripts were differentially expressed between adipose tissue and liver. We show that while the major adipose (P1) and liver (P2) transcripts were both induced by dexamethasone, they were differentially regulated by the PPARα agonist, clofibric acid, and leptin. Leptin had no effect on the adipose-specific P1 transcript, but induced liver-specific P2 promoter activity via a STAT3/Sp1 mechanism. Moreover in Wistar rats, leptin treatment between postnatal day 3–13 led to an increase in P2 but not P1 transcription in adipose tissue which was sustained into adulthood. This suggests that the expression of the alternative PPARα transcripts are in part programmed by early life exposure to leptin leading to persistent change in adipose tissue fatty acid metabolism through specific activation of a quiescent PPARα promoter. Such complexity in the regulation of PPARα may allow the expression of PPARα to be finely regulated in response to environmental factors. PMID:23825665

Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing.

Science.gov (United States)

Anvar, Seyed Yahya; Allard, Guy; Tseng, Elizabeth; Sheynkman, Gloria M; de Klerk, Eleonora; Vermaat, Martijn; Yin, Raymund H; Johansson, Hans E; Ariyurek, Yavuz; den Dunnen, Johan T; Turner, Stephen W; 't Hoen, Peter A C

2018-03-29

The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells. Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

GCR1, a transcriptional activator in Saccharomyces cerevisiae, complexes with RAP1 and can function without its DNA binding domain.

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Tornow, J; Zeng, X; Gao, W; Santangelo, G M

1993-01-01

In Saccharomyces cerevisiae, efficient expression of glycolytic and translational component genes requires two DNA binding proteins, RAP1 (which binds to UASRPG) and GCR1 (which binds to the CT box). We generated deletions in GCR1 to test the validity of several different models for GCR1 function. We report here that the C-terminal half of GCR1, which includes the domain required for DNA binding to the CT box in vitro, can be removed without affecting GCR1-dependent transcription of either the glycolytic gene ADH1 or the translational component genes TEF1 and TEF2. We have also identified an activation domain within a segment of the GCR1 protein (the N-terminal third) that is essential for in vivo function. RAP1 and GCR1 can be co-immunoprecipitated from whole cell extracts, suggesting that they form a complex in vivo. The data are most consistent with a model in which GCR1 is attracted to DNA through contact with RAP1. Images PMID:8508768

The upstream regulatory sequence of the light harvesting complex Lhcf2 gene of the marine diatom Phaeodactylum tricornutum enhances transcription in an orientation- and distance-independent fashion.

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Russo, Monia Teresa; Annunziata, Rossella; Sanges, Remo; Ferrante, Maria Immacolata; Falciatore, Angela

2015-12-01

Diatoms are a key phytoplankton group in the contemporary ocean, showing extraordinary adaptation capacities to rapidly changing environments. The recent availability of whole genome sequences from representative species has revealed distinct features in their genomes, like novel combinations of genes encoding distinct metabolisms and a significant number of diatom-specific genes. However, the regulatory mechanisms driving diatom gene expression are still largely uncharacterized. Considering the wide variety of fields of study orbiting diatoms, ranging from ecology, evolutionary biology to biotechnology, it is thus essential to increase our understanding of fundamental gene regulatory processes such as transcriptional regulation. To this aim, we explored the functional properties of the 5'-flanking region of the Phaeodatylum tricornutum Lhcf2 gene, encoding a member of the Light Harvesting Complex superfamily and we showed that this region enhances transcription of a GUS reporter gene in an orientation- and distance-independent fashion. This represents the first example of a cis-regulatory sequence with enhancer-like features discovered in diatoms and it is instrumental for the generation of novel genetic tools and diatom exploitation in different areas of study. Copyright © 2015 Elsevier B.V. All rights reserved.

Epigenetic Transcriptional Memory of GAL Genes Depends on Growth in Glucose and the Tup1 Transcription Factor in Saccharomyces cerevisiae.

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Sood, Varun; Cajigas, Ivelisse; D'Urso, Agustina; Light, William H; Brickner, Jason H

2017-08-01

Previously expressed inducible genes can remain poised for faster reactivation for multiple cell divisions, a conserved phenomenon called epigenetic transcriptional memory. The GAL genes in Saccharomyces cerevisiae show faster reactivation for up to seven generations after being repressed. During memory, previously produced Gal1 protein enhances the rate of reactivation of GAL1 , GAL10 , GAL2 , and GAL7 These genes also interact with the nuclear pore complex (NPC) and localize to the nuclear periphery both when active and during memory. Peripheral localization of GAL1 during memory requires the Gal1 protein, a memory-specific cis -acting element in the promoter, and the NPC protein Nup100 However, unlike other examples of transcriptional memory, the interaction with NPC is not required for faster GAL gene reactivation. Rather, downstream of Gal1, the Tup1 transcription factor and growth in glucose promote GAL transcriptional memory. Cells only show signs of memory and only benefit from memory when growing in glucose. Tup1 promotes memory-specific chromatin changes at the GAL1 promoter: incorporation of histone variant H2A.Z and dimethylation of histone H3, lysine 4. Tup1 and H2A.Z function downstream of Gal1 to promote binding of a preinitiation form of RNA Polymerase II at the GAL1 promoter, poising the gene for faster reactivation. This mechanism allows cells to integrate a previous experience (growth in galactose, reflected by Gal1 levels) with current conditions (growth in glucose, potentially through Tup1 function) to overcome repression and to poise critical GAL genes for future reactivation. Copyright © 2017 by the Genetics Society of America.

Post-transcriptional regulation of ribosome biogenesis in yeast

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Isabelle C. Kos-Braun

2017-05-01

Full Text Available Most microorganisms are exposed to the constantly and often rapidly changing environment. As such they evolved mechanisms to balance their metabolism and energy expenditure with the resources available to them. When resources become scarce or conditions turn out to be unfavourable for growth, cells reduce their metabolism and energy usage to survive. One of the major energy consuming processes in the cell is ribosome biogenesis. Unsurprisingly, cells encountering adverse conditions immediately shut down production of new ribosomes. It is well established that nutrient depletion leads to a rapid repression of transcription of the genes encoding ribosomal proteins, ribosome biogenesis factors as well as ribosomal RNA (rRNA. However, if pre-rRNA processing and ribosome assembly are regulated post-transcriptionally remains largely unclear. We have recently uncovered that the yeast Saccharomyces cerevisiae rapidly switches between two alternative pre-rRNA processing pathways depending on the environmental conditions. Our findings reveal a new level of complexity in the regulation of ribosome biogenesis.

Dissecting interferon-induced transcriptional programs in human peripheral blood cells.

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Simon J Waddell

2010-03-01

Full Text Available 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 alpha, beta, omega and gamma, IL12 and TNFalpha; and (2 characterize the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes to IFNgamma stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNgamma and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFalpha stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNgamma, 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.

The plausible reason why the length of 5' untranslated region is unrelated to organismal complexity

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Chen Feng-Chi

2011-08-01

Full Text Available Abstract Background Organismal complexity is suggested to increase with the complexity of transcriptional and translational regulations. Supporting this notion is a recent study that demonstrated a higher level of tissue-specific gene expression in human than in mouse. However, whether this correlation can be extended beyond mammals remains unclear. In addition, 5' untranslated regions (5'UTRs, which have undergone stochastic elongation during evolution and potentially included an increased number of regulatory elements, may have played an important role in the emergence of organismal complexity. Although the lack of correlation between 5'UTR length and organismal complexity has been proposed, the underlying mechanisms remain unexplored. Results In this study, we select the number of cell types as the measurement of organismal complexity and examine the correlation between (1 organismal complexity and transcriptional regulatory complexity; and (2 organismal complexity and 5'UTR length by comparing the 5'UTRs and multiple-tissue expression profiles of human (Homo sapiens, mouse (Mus musculus, and fruit fly (Drosophila melanogaster. The transcriptional regulatory complexity is measured by using the tissue specificity of gene expression and the ratio of non-constitutively expressed to constitutively expressed genes. We demonstrate that, whereas correlation (1 holds well in the three-way comparison, correlation (2 is not true. Results from a larger dataset that includes more than 15 species, ranging from yeast to human, also reject correlation (2. The reason for the failure of correlation (2 may be ascribed to: Firstly, longer 5'UTRs do not contribute to increased tissue specificity of gene expression. Secondly, the increased numbers of common translational regulatory elements in longer 5'UTRs do not lead to increased organismal complexity. Conclusions Our study has extended the evidence base for the correlation between organismal complexity and

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

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

2014-09-01

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

The POU homeodomain transcription factor POUM2 and broad complex isoform 2 transcription factor induced by 20-hydroxyecdysone collaboratively regulate vitellogenin gene expression and egg formation in the silkworm Bombyx mori.

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Lin, Y; Liu, H; Yang, C; Gu, J; Shen, G; Zhang, H; Chen, E; Han, C; Zhang, Y; Xu, Y; Wu, J; Xia, Q

2017-10-01

Vitellogenin (Vg) is a source of nutrition for embryo development. Our previous study showed that the silkworm (Bombyx mori) transcription factor broad complex isoform 2 (BmBrC-Z2) regulates gene expression of the Vg gene (BmVg) by induction with 20-hydroxyecdysone (20E). However, the mechanism by which 20E regulates BmVg expression was not clarified. In this study, cell transfection experiments showed that the BmVg promoter containing the POU homeodomain transcription factor POUM2 (POUM2) and BrC-Z2 cis-response elements (CREs) showed a more significant response to 20E than that harbouring only the BrC-Z2 or POUM2 CRE. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed that BmPOUM2 could bind to the POUM2 CRE of the BmVg promoter. Over-expression of BmPOUM2 and BmBrC-Z2 in B. mori embryo-derived cell line (BmE) could enhance the activity of the BmVg promoter carrying both the POUM2 and BrC-Z2 CREs following 20E induction. Quantitative PCR and immunofluorescence histochemistry showed that the expression pattern and tissue localization of BmPOUM2 correspond to those of BmVg. Glutathione S-transferase pull-down and co-immunoprecipitation assays confirmed that BmPOUM2 interacts only with BmBrC-Z2 to regulate BmVg expression. Down-regulation of BmPOUM2 in female silkworm by RNA interference significantly reduced BmVg expression, leading to abnormal egg formation. In summary, these results indicate that BmPOUM2 binds only to BmBrC-Z2 to collaboratively regulate BmVg expression by 20E induction to control vitellogenesis and egg formation in the silkworm. Moreover, these findings suggest that homeodomain protein POUM2 plays a novel role in regulating insect vitellogenesis. © 2017 The Royal Entomological Society.

Adaptive evolution of transcription factor binding sites

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Berg Johannes

2004-10-01

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

Transcriptional blockages in a cell-free system by sequence-selective DNA alkylating agents.

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Ferguson, L R; Liu, A P; Denny, W A; Cullinane, C; Talarico, T; Phillips, D R

2000-04-14

There is considerable interest in DNA sequence-selective DNA-binding drugs as potential inhibitors of gene expression. Five compounds with distinctly different base pair specificities were compared in their effects on the formation and elongation of the transcription complex from the lac UV5 promoter in a cell-free system. All were tested at drug levels which killed 90% of cells in a clonogenic survival assay. Cisplatin, a selective alkylator at purine residues, inhibited transcription, decreasing the full-length transcript, and causing blockage at a number of GG or AG sequences, making it probable that intrastrand crosslinks are the blocking lesions. A cyclopropylindoline known to be an A-specific alkylator also inhibited transcription, with blocks at adenines. The aniline mustard chlorambucil, that targets primarily G but also A sequences, was also effective in blocking the formation of full-length transcripts. It produced transcription blocks either at, or one base prior to, AA or GG sequences, suggesting that intrastrand crosslinks could again be involved. The non-alkylating DNA minor groove binder Hoechst 33342 (a bisbenzimidazole) blocked formation of the full-length transcript, but without creating specific blockage sites. A bisbenzimidazole-linked aniline mustard analogue was a more effective transcription inhibitor than either chlorambucil or Hoechst 33342, with different blockage sites occurring immediately as compared with 2 h after incubation. The blockages were either immediately prior to AA or GG residues, or four to five base pairs prior to such sites, a pattern not predicted from in vitro DNA-binding studies. Minor groove DNA-binding ligands are of particular interest as inhibitors of gene expression, since they have the potential ability to bind selectively to long sequences of DNA. The results suggest that the bisbenzimidazole-linked mustard does cause alkylation and transcription blockage at novel DNA sites. in addition to sites characteristic of

Biological data warehousing system for identifying transcriptional regulatory sites from gene expressions of microarray data.

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Tsou, Ann-Ping; Sun, Yi-Ming; Liu, Chia-Lin; Huang, Hsien-Da; Horng, Jorng-Tzong; Tsai, Meng-Feng; Liu, Baw-Juine

2006-07-01

Identification of transcriptional regulatory sites plays an important role in the investigation of gene regulation. For this propose, we designed and implemented a data warehouse to integrate multiple heterogeneous biological data sources with data types such as text-file, XML, image, MySQL database model, and Oracle database model. The utility of the biological data warehouse in predicting transcriptional regulatory sites of coregulated genes was explored using a synexpression group derived from a microarray study. Both of the binding sites of known transcription factors and predicted over-represented (OR) oligonucleotides were demonstrated for the gene group. The potential biological roles of both known nucleotides and one OR nucleotide were demonstrated using bioassays. Therefore, the results from the wet-lab experiments reinforce the power and utility of the data warehouse as an approach to the genome-wide search for important transcription regulatory elements that are the key to many complex biological systems.

An Atlas of Combinatorial Transcriptional Regulation in Mouse and Man

KAUST Repository

Ravasi, Timothy; Suzuki, Harukazu; Cannistraci, Carlo; Katayama, Shintaro; Bajic, Vladimir B.; Tan, Kai; Akalin, Altuna; Schmeier, Sebastian; Kanamori-Katayama, Mutsumi; Bertin, Nicolas; Carninci, Piero; Daub, Carsten O.; Forrest, Alistair R.R.; Gough, Julian; Grimmond, Sean; Han, Jung-Hoon; Hashimoto, Takehiro; Hide, Winston; Hofmann, Oliver; Kamburov, Atanas; Kaur, Mandeep; Kawaji, Hideya; Kubosaki, Atsutaka; Lassmann, Timo; van Nimwegen, Erik; MacPherson, Cameron Ross; Ogawa, Chihiro; Radovanovic, Aleksandar; Schwartz, Ariel; Teasdale, Rohan D.; Tegné r, Jesper; Lenhard, Boris; Teichmann, Sarah A.; Arakawa, Takahiro; Ninomiya, Noriko; Murakami, Kayoko; Tagami, Michihira; Fukuda, Shiro; Imamura, Kengo; Kai, Chikatoshi; Ishihara, Ryoko; Kitazume, Yayoi; Kawai, Jun; Hume, David A.; Ideker, Trey; Hayashizaki, Yoshihide

2010-01-01

Combinatorial interactions among transcription factors are critical to directing tissue-specific gene expression. To build a global atlas of these combinations, we have screened for physical interactions among the majority of human and mouse DNA-binding transcription factors (TFs). The complete networks contain 762 human and 877 mouse interactions. Analysis of the networks reveals that highly connected TFs are broadly expressed across tissues, and that roughly half of the measured interactions are conserved between mouse and human. The data highlight the importance of TF combinations for determining cell fate, and they lead to the identification of a SMAD3/FLI1 complex expressed during development of immunity. The availability of large TF combinatorial networks in both human and mouse will provide many opportunities to study gene regulation, tissue differentiation, and mammalian evolution.

An Atlas of Combinatorial Transcriptional Regulation in Mouse and Man

KAUST Repository

Ravasi, Timothy

2010-03-01

Combinatorial interactions among transcription factors are critical to directing tissue-specific gene expression. To build a global atlas of these combinations, we have screened for physical interactions among the majority of human and mouse DNA-binding transcription factors (TFs). The complete networks contain 762 human and 877 mouse interactions. Analysis of the networks reveals that highly connected TFs are broadly expressed across tissues, and that roughly half of the measured interactions are conserved between mouse and human. The data highlight the importance of TF combinations for determining cell fate, and they lead to the identification of a SMAD3/FLI1 complex expressed during development of immunity. The availability of large TF combinatorial networks in both human and mouse will provide many opportunities to study gene regulation, tissue differentiation, and mammalian evolution.

Stable assembly of HIV-1 export complexes occurs cotranscriptionally

DEFF Research Database (Denmark)

Nawroth, Isabel; Mueller, Florian; Basyuk, Eugenia

2014-01-01

The HIV-1 Rev protein mediates export of unspliced and singly spliced viral transcripts by binding to the Rev response element (RRE) and recruiting the cellular export factor CRM1. Here, we investigated the recruitment of Rev to the transcription sites of HIV-1 reporters that splice either post......- or cotranscriptionally. In both cases, we observed that Rev localized to the transcription sites of the reporters and recruited CRM1. Rev and CRM1 remained at the reporter transcription sites when cells were treated with the splicing inhibitor Spliceostatin A (SSA), showing that the proteins associate with RNA prior...... to or during early spliceosome assembly. Fluorescence recovery after photobleaching (FRAP) revealed that Rev and CRM1 have similar kinetics as the HIV-1 RNA, indicating that Rev, CRM1, and RRE-containing RNAs are released from the site of transcription in one single export complex. These results suggest...

Developmental and transcriptional consequences of mutations in Drosophila TAF(II)60.

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Aoyagi, N; Wassarman, D A

2001-10-01

In vitro, the TAF(II)60 component of the TFIID complex contributes to RNA polymerase II transcription initiation by serving as a coactivator that interacts with specific activator proteins and possibly as a promoter selectivity factor that interacts with the downstream promoter element. In vivo roles for TAF(II)60 in metazoan transcription are not as clear. Here we have investigated the developmental and transcriptional requirements for TAF(II)60 by analyzing four independent Drosophila melanogaster TAF(II)60 mutants. Loss-of-function mutations in Drosophila TAF(II)60 result in lethality, indicating that TAF(II)60 provides a nonredundant function in vivo. Molecular analysis of TAF(II)60 alleles revealed that essential TAF(II)60 functions are provided by two evolutionarily conserved regions located in the N-terminal half of the protein. TAF(II)60 is required at all stages of Drosophila development, in both germ cells and somatic cells. Expression of TAF(II)60 from a transgene rescued the lethality of TAF(II)60 mutants and exposed requirements for TAF(II)60 during imaginal development, spermatogenesis, and oogenesis. Phenotypes of rescued TAF(II)60 mutant flies implicate TAF(II)60 in transcriptional mechanisms that regulate cell growth and cell fate specification and suggest that TAF(II)60 is a limiting component of the machinery that regulates the transcription of dosage-sensitive genes. Finally, TAF(II)60 plays roles in developmental regulation of gene expression that are distinct from those of other TAF(II) proteins.

Enhancer SINEs Link Pol III to Pol II Transcription in Neurons

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Cristina Policarpi

2017-12-01

Full Text Available Summary: Spatiotemporal regulation of gene expression depends on the cooperation of multiple mechanisms, including the functional interaction of promoters with distally located enhancers. Here, we show that, in cortical neurons, a subset of short interspersed nuclear elements (SINEs located in the proximity of activity-regulated genes bears features of enhancers. Enhancer SINEs (eSINEs recruit the Pol III cofactor complex TFIIIC in a stimulus-dependent manner and are transcribed by Pol III in response to neuronal depolarization. Characterization of an eSINE located in proximity to the Fos gene (FosRSINE1 indicated that the FosRSINE1-encoded transcript interacts with Pol II at the Fos promoter and mediates Fos relocation to Pol II factories, providing an unprecedented molecular link between Pol III and Pol II transcription. Strikingly, knockdown of the FosRSINE1 transcript induces defects of both cortical radial migration in vivo and activity-dependent dendritogenesis in vitro, demonstrating that FosRSINE1 acts as a strong enhancer of Fos expression in diverse physiological contexts. : Spatiotemporal regulation of gene expression requires the interaction between promoters and distally located enhancers. Policarpi et al. identify a subset of SINEs that functions as enhancers for activity-dependent neuronal genes. The enhancer SINE FosRSINE1 regulates Fos transcription and is necessary for both activity-dependent dendritogenesis and proper brain development. Keywords: neuroscience, epigenetics, transcription, enhancers, SINEs, neuronal activity, neuronal development

Nucleocytoplasmic shuttling of transcription factors

DEFF Research Database (Denmark)

Cartwright, P; Helin, K

2000-01-01

To elicit the transcriptional response following intra- or extracellular stimuli, the signals need to be transmitted to their site of action within the nucleus. The nucleocytoplasmic shuttling of transcription factors is a mechanism mediating this process. The activation and inactivation...... of the transcriptional response is essential for cells to progress through the cell cycle in a normal manner. The involvement of cytoplasmic and nuclear accessory molecules, and the general nuclear membrane transport components, are essential for this process. Although nuclear import and export for different...... transcription factor families are regulated by similar mechanisms, there are several differences that allow for the specific activation of each transcription factor. This review discusses the general import and export pathways found to be common amongst many different transcription factors, and highlights...

Human more complex than mouse at cellular level.

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Alexander E Vinogradov

Full Text Available The family of transcription factors with the C2H2 zinc finger domain is expanding in the evolution of vertebrates, reaching its highest numbers in the mammals. The question arises: whether an increased amount of these transcription factors is related to embryogenesis, nervous system, pathology or more of them are expressed in individual cells? Among mammals, the primates have a more complex anatomical structure than the rodents (e.g., brain. In this work, I show that a greater number of C2H2-ZF genes are expressed in the human cells than in the mouse cells. The effect is especially pronounced for C2H2-ZF genes accompanied with the KRAB domain. The relative difference between the numbers of C2H2-ZF(-KRAB genes in the human and mouse cellular transcriptomes even exceeds their difference in the genomes (i.e. a greater subset of existing in the genome genes is expressed in the human cellular transcriptomes compared to the mouse transcriptomes. The evolutionary turnover of C2H2-ZF(-KRAB genes acts in the direction of the revealed phenomenon, i.e. gene duplication and loss enhances the difference in the relative number of C2H2-ZF(-KRAB genes between human and mouse cellular transcriptomes. A higher amount of these genes is expressed in the brain and embryonic cells (compared with other tissues, whereas a lower amount--in the cancer cells. It is specifically the C2H2-ZF transcription factors whose repertoire is poorer in the cancer and richer in the brain (other transcription factors taken together do not show this trend. These facts suggest that increase of anatomical complexity is accompanied by a more complex intracellular regulation involving these transcription factors. Malignization is associated with simplification of this regulation. These results agree with the known fact that human cells are more resistant to oncogenic transformation than mouse cells. The list of C2H2-ZF genes whose suppression might be involved in malignization is provided.

Heat shock factor-1 modulates p53 activity in the transcriptional response to DNA damage

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Logan, Ian R.; McNeill, Hesta V.; Cook, Susan; Lu, Xiaohong; Meek, David W.; Fuller-Pace, Frances V.; Lunec, John; Robson, Craig N.

2009-01-01

Here we define an important role for heat shock factor 1 (HSF1) in the cellular response to genotoxic agents. We demonstrate for the first time that HSF1 can complex with nuclear p53 and that both proteins are co-operatively recruited to p53-responsive genes such as p21. Analysis of natural and synthetic cis elements demonstrates that HSF1 can enhance p53-mediated transcription, whilst depletion of HSF1 reduces the expression of p53-responsive transcripts. We find that HSF1 is required for optimal p21 expression and p53-mediated cell-cycle arrest in response to genotoxins while loss of HSF1 attenuates apoptosis in response to these agents. To explain these novel properties of HSF1 we show that HSF1 can complex with DNA damage kinases ATR and Chk1 to effect p53 phosphorylation in response to DNA damage. Our data reveal HSF1 as a key transcriptional regulator in response to genotoxic compounds widely used in the clinical setting, and suggest that HSF1 will contribute to the efficacy of these agents. PMID:19295133

DREAM Controls the On/Off Switch of Specific Activity-Dependent Transcription Pathways

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Mellström, Britt; Sahún, Ignasi; Ruiz-Nuño, Ana; Murtra, Patricia; Gomez-Villafuertes, Rosa; Savignac, Magali; Oliveros, Juan C.; Gonzalez, Paz; Kastanauskaite, Asta; Knafo, Shira; Zhuo, Min; Higuera-Matas, Alejandro; Errington, Michael L.; Maldonado, Rafael; DeFelipe, Javier; Jefferys, John G. R.; Bliss, Tim V. P.; Dierssen, Mara

2014-01-01

Changes in nuclear Ca2+ homeostasis activate specific gene expression programs and are central to the acquisition and storage of information in the brain. DREAM (downstream regulatory element antagonist modulator), also known as calsenilin/KChIP-3 (K+ channel interacting protein 3), is a Ca2+-binding protein that binds DNA and represses transcription in a Ca2+-dependent manner. To study the function of DREAM in the brain, we used transgenic mice expressing a Ca2+-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Using genome-wide analysis, we show that DREAM regulates the expression of specific activity-dependent transcription factors in the hippocampus, including Npas4, Nr4a1, Mef2c, JunB, and c-Fos. Furthermore, DREAM regulates its own expression, establishing an autoinhibitory feedback loop to terminate activity-dependent transcription. Ablation of DREAM does not modify activity-dependent transcription because of gene compensation by the other KChIP family members. The expression of daDREAM in the forebrain resulted in a complex phenotype characterized by loss of recurrent inhibition and enhanced long-term potentiation (LTP) in the dentate gyrus and impaired learning and memory. Our results indicate that DREAM is a major master switch transcription factor that regulates the on/off status of specific activity-dependent gene expression programs that control synaptic plasticity, learning, and memory. PMID:24366545

Whi7 is an unstable cell-cycle repressor of the Start transcriptional program.

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Gomar-Alba, Mercè; Méndez, Ester; Quilis, Inma; Bañó, M Carmen; Igual, J Carlos

2017-08-24

Start is the main decision point in eukaryotic cell cycle in which cells commit to a new round of cell division. It involves the irreversible activation of a transcriptional program by G1 CDK-cyclin complexes through the inactivation of Start transcriptional repressors, Whi5 in yeast or Rb in mammals. Here we provide novel keys of how Whi7, a protein related at sequence level to Whi5, represses Start. Whi7 is an unstable protein, degraded by the SCF Grr1 ubiquitin-ligase, whose stability is cell cycle regulated by CDK1 phosphorylation. Importantly, Whi7 associates to G1/S gene promoters in late G1 acting as a repressor of SBF-dependent transcription. Our results demonstrate that Whi7 is a genuine paralog of Whi5. In fact, both proteins collaborate in Start repression bringing to light that yeast cells, as occurs in mammalian cells, rely on the combined action of multiple transcriptional repressors to block Start transition.The commitment of cells to a new cycle of division involves inactivation of the Start transcriptional repressor Whi5. Here the authors show that the sequence related protein Whi7 associates to G1/S gene promoters in late G1 and collaborates with Whi5 in Start repression.

Nuclear exclusion of transcription factors associated with apoptosis in developing nervous tissue

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R. Linden

1999-07-01

Full Text Available Programmed cell death in the form of apoptosis involves a network of metabolic events and may be triggered by a variety of stimuli in distinct cells. The nervous system contains several neuron and glial cell types, and developmental events are strongly dependent on selective cell interactions. Retinal explants have been used as a model to investigate apoptosis in nervous tissue. This preparation maintains the structural complexity and cell interactions similar to the retina in situ, and contains cells in all stages of development. We review the finding of nuclear exclusion of several transcription factors during apoptosis in retinal cells. The data reviewed in this paper suggest a link between apoptosis and a failure in the nucleo-cytoplasmic partition of transcription factors. It is argued that the nuclear exclusion of transcription factors may be an integral component of apoptosis both in the nervous system and in other types of cells and tissues.

Transcriptional landscape of Mycobacterium tuberculosis infection in macrophages

KAUST Repository

Roy, Sugata

2018-04-24

Mycobacterium tuberculosis (Mtb) infection reveals complex and dynamic host-pathogen interactions, leading to host protection or pathogenesis. Using a unique transcriptome technology (CAGE), we investigated the promoter-based transcriptional landscape of IFNγ (M1) or IL-4/IL-13 (M2) stimulated macrophages during Mtb infection in a time-kinetic manner. Mtb infection widely and drastically altered macrophage-specific gene expression, which is far larger than that of M1 or M2 activations. Gene Ontology enrichment analysis for Mtb-induced differentially expressed genes revealed various terms, related to host-protection and inflammation, enriched in up-regulated genes. On the other hand, terms related to dis-regulation of cellular functions were enriched in down-regulated genes. Differential expression analysis revealed known as well as novel transcription factor genes in Mtb infection, many of them significantly down-regulated. IFNγ or IL-4/IL-13 pre-stimulation induce additional differentially expressed genes in Mtb-infected macrophages. Cluster analysis uncovered significant numbers, prolonging their expressional changes. Furthermore, Mtb infection augmented cytokine-mediated M1 and M2 pre-activations. In addition, we identified unique transcriptional features of Mtb-mediated differentially expressed lncRNAs. In summary we provide a comprehensive in depth gene expression/regulation profile in Mtb-infected macrophages, an important step forward for a better understanding of host-pathogen interaction dynamics in Mtb infection.

Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe

OpenAIRE

Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.

2012-01-01

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription ...

Pnrc2 regulates 3'UTR-mediated decay of segmentation clock-associated transcripts during zebrafish segmentation.

Science.gov (United States)

Gallagher, Thomas L; Tietz, Kiel T; Morrow, Zachary T; McCammon, Jasmine M; Goldrich, Michael L; Derr, Nicolas L; Amacher, Sharon L

2017-09-01

Vertebrate segmentation is controlled by the segmentation clock, a molecular oscillator that regulates gene expression and cycles rapidly. The expression of many genes oscillates during segmentation, including hairy/Enhancer of split-related (her or Hes) genes, which encode transcriptional repressors that auto-inhibit their own expression, and deltaC (dlc), which encodes a Notch ligand. We previously identified the tortuga (tor) locus in a zebrafish forward genetic screen for genes involved in cyclic transcript regulation and showed that cyclic transcripts accumulate post-splicing in tor mutants. Here we show that cyclic mRNA accumulation in tor mutants is due to loss of pnrc2, which encodes a proline-rich nuclear receptor co-activator implicated in mRNA decay. Using an inducible in vivo reporter system to analyze transcript stability, we find that the her1 3'UTR confers Pnrc2-dependent instability to a heterologous transcript. her1 mRNA decay is Dicer-independent and likely employs a Pnrc2-Upf1-containing mRNA decay complex. Surprisingly, despite accumulation of cyclic transcripts in pnrc2-deficient embryos, we find that cyclic protein is expressed normally. Overall, we show that Pnrc2 promotes 3'UTR-mediated decay of developmentally-regulated segmentation clock transcripts and we uncover an additional post-transcriptional regulatory layer that ensures oscillatory protein expression in the absence of cyclic mRNA decay. Copyright © 2017 Elsevier Inc. All rights reserved.

Insights into mRNP biogenesis provided by new genetic interactions among export and transcription factors

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Estruch Francisco

2012-09-01

Full Text Available Abstract Background The various steps of mRNP biogenesis (transcription, processing and export are interconnected. It has been shown that the transcription machinery plays a pivotal role in mRNP assembly, since several mRNA export factors are recruited during transcription and physically interact with components of the transcription machinery. Although the shuttling DEAD-box protein Dbp5p is concentrated on the cytoplasmic fibrils of the NPC, previous studies demonstrated that it interacts physically and genetically with factors involved in transcription initiation. Results We investigated the effect of mutations affecting various components of the transcription initiation apparatus on the phenotypes of mRNA export mutant strains. Our results show that growth and mRNA export defects of dbp5 and mex67 mutant strains can be suppressed by mutation of specific transcription initiation components, but suppression was not observed for mutants acting in the very first steps of the pre-initiation complex (PIC formation. Conclusions Our results indicate that mere reduction in the amount of mRNP produced is not sufficient to suppress the defects caused by a defective mRNA export factor. Suppression occurs only with mutants affecting events within a narrow window of the mRNP biogenesis process. We propose that reducing the speed with which transcription converts from initiation and promoter clearance to elongation may have a positive effect on mRNP formation by permitting more effective recruitment of partially-functional mRNP proteins to the nascent mRNP.

Transcriptional profiling of Medicago truncatula under salt stress identified a novel CBF transcription factor MtCBF4 that plays an important role in abiotic stress responses

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Su Zhen

2011-07-01

Full Text Available Abstract Background Salt stress hinders the growth of plants and reduces crop production worldwide. However, different plant species might possess different adaptive mechanisms to mitigate salt stress. We conducted a detailed pathway analysis of transcriptional dynamics in the roots of Medicago truncatula seedlings under salt stress and selected a transcription factor gene, MtCBF4, for experimental validation. Results A microarray experiment was conducted using root samples collected 6, 24, and 48 h after application of 180 mM NaCl. Analysis of 11 statistically significant expression profiles revealed different behaviors between primary and secondary metabolism pathways in response to external stress. Secondary metabolism that helps to maintain osmotic balance was induced. One of the highly induced transcription factor genes was successfully cloned, and was named MtCBF4. Phylogenetic analysis revealed that MtCBF4, which belongs to the AP2-EREBP transcription factor family, is a novel member of the CBF transcription factor in M. truncatula. MtCBF4 is shown to be a nuclear-localized protein. Expression of MtCBF4 in M. truncatula was induced by most of the abiotic stresses, including salt, drought, cold, and abscisic acid, suggesting crosstalk between these abiotic stresses. Transgenic Arabidopsis over-expressing MtCBF4 enhanced tolerance to drought and salt stress, and activated expression of downstream genes that contain DRE elements. Over-expression of MtCBF4 in M. truncatula also enhanced salt tolerance and induced expression level of corresponding downstream genes. Conclusion Comprehensive transcriptomic analysis revealed complex mechanisms exist in plants in response to salt stress. The novel transcription factor gene MtCBF4 identified here played an important role in response to abiotic stresses, indicating that it might be a good candidate gene for genetic improvement to produce stress-tolerant plants.

Transcriptional profiling of Medicago truncatula under salt stress identified a novel CBF transcription factor MtCBF4 that plays an important role in abiotic stress responses

Science.gov (United States)

2011-01-01

Background Salt stress hinders the growth of plants and reduces crop production worldwide. However, different plant species might possess different adaptive mechanisms to mitigate salt stress. We conducted a detailed pathway analysis of transcriptional dynamics in the roots of Medicago truncatula seedlings under salt stress and selected a transcription factor gene, MtCBF4, for experimental validation. Results A microarray experiment was conducted using root samples collected 6, 24, and 48 h after application of 180 mM NaCl. Analysis of 11 statistically significant expression profiles revealed different behaviors between primary and secondary metabolism pathways in response to external stress. Secondary metabolism that helps to maintain osmotic balance was induced. One of the highly induced transcription factor genes was successfully cloned, and was named MtCBF4. Phylogenetic analysis revealed that MtCBF4, which belongs to the AP2-EREBP transcription factor family, is a novel member of the CBF transcription factor in M. truncatula. MtCBF4 is shown to be a nuclear-localized protein. Expression of MtCBF4 in M. truncatula was induced by most of the abiotic stresses, including salt, drought, cold, and abscisic acid, suggesting crosstalk between these abiotic stresses. Transgenic Arabidopsis over-expressing MtCBF4 enhanced tolerance to drought and salt stress, and activated expression of downstream genes that contain DRE elements. Over-expression of MtCBF4 in M. truncatula also enhanced salt tolerance and induced expression level of corresponding downstream genes. Conclusion Comprehensive transcriptomic analysis revealed complex mechanisms exist in plants in response to salt stress. The novel transcription factor gene MtCBF4 identified here played an important role in response to abiotic stresses, indicating that it might be a good candidate gene for genetic improvement to produce stress-tolerant plants. PMID:21718548

RNA-Seq for enrichment and analysis of IRF5 transcript expression in SLE.

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Rivka C Stone

Full Text Available Polymorphisms in the interferon regulatory factor 5 (IRF5 gene have been consistently replicated and shown to confer risk for or protection from the development of systemic lupus erythematosus (SLE. IRF5 expression is significantly upregulated in SLE patients and upregulation associates with IRF5-SLE risk haplotypes. IRF5 alternative splicing has also been shown to be elevated in SLE patients. Given that human IRF5 exists as multiple alternatively spliced transcripts with distinct function(s, it is important to determine whether the IRF5 transcript profile expressed in healthy donor immune cells is different from that expressed in SLE patients. Moreover, it is not currently known whether an IRF5-SLE risk haplotype defines the profile of IRF5 transcripts expressed. Using standard molecular cloning techniques, we identified and isolated 14 new differentially spliced IRF5 transcript variants from purified monocytes of healthy donors and SLE patients to generate an IRF5 variant transcriptome. Next-generation sequencing was then used to perform in-depth and quantitative analysis of full-length IRF5 transcript expression in primary immune cells of SLE patients and healthy donors by next-generation sequencing. Evidence for additional alternatively spliced transcripts was obtained from de novo junction discovery. Data from these studies support the overall complexity of IRF5 alternative splicing in SLE. Results from next-generation sequencing correlated with cloning and gave similar abundance rankings in SLE patients thus supporting the use of this new technology for in-depth single gene transcript profiling. Results from this study provide the first proof that 1 SLE patients express an IRF5 transcript signature that is distinct from healthy donors, 2 an IRF5-SLE risk haplotype defines the top four most abundant IRF5 transcripts expressed in SLE patients, and 3 an IRF5 transcript signature enables clustering of SLE patients with the H2 risk haplotype.

Transcriptional Silencing of Retroviral Vectors

DEFF Research Database (Denmark)

Lund, Anders Henrik; Duch, M.; Pedersen, F.S.

1996-01-01

. Extinction of long-term vector expression has been observed after implantation of transduced hematopoietic cells as well as fibroblasts, myoblasts and hepatocytes. Here we review the influence of vector structure, integration site and cell type on transcriptional silencing. While down-regulation of proviral...... transcription is known from a number of cellular and animal models, major insight has been gained from studies in the germ line and embryonal cells of the mouse. Key elements for the transfer and expression of retroviral vectors, such as the viral transcriptional enhancer and the binding site for the t......RNA primer for reverse transcription may have a major influence on transcriptional silencing. Alterations of these elements of the vector backbone as well as the use of internal promoter elements from housekeeping genes may contribute to reduce transcriptional silencing. The use of cell culture and animal...

Transcriptional networks controlling adipocyte differentiation

DEFF Research Database (Denmark)

Siersbæk, R; Mandrup, Susanne

2011-01-01

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

Theory on the mechanism of distal action of transcription factors: looping of DNA versus tracking along DNA

International Nuclear Information System (INIS)

Murugan, R

2010-01-01

In this paper, we develop a theory on the mechanism of distal action of the transcription factors, which are bound at their respective cis-regulatory enhancer modules on the promoter-RNA polymerase II (PR) complexes to initiate the transcription event in eukaryotes. We consider both the looping and tracking modes of their distal communication and calculate the mean first passage time that is required for the distal interactions of the complex of enhancer and transcription factor with the PR via both these modes. We further investigate how this mean first passage time is dependent on the length of the DNA segment (L, base-pairs) that connects the cis-regulatory binding site and the respective promoter. When the radius of curvature of this connecting segment of DNA is R that was induced upon binding of the transcription factor at the cis-acting element and RNAPII at the promoter in cis-positions, our calculations indicate that the looping mode of distal action will dominate when L is such that L > 2πR and the tracking mode of distal action will be favored when L 2 bps. It seems that the free energy associated with the binding of the transcription factor with its cis-acting element and the distance of this cis-acting element from the corresponding promoter of the gene of interest is negatively correlated. Our results suggest that the looping and tracking modes of distal action are concurrently operating on the transcription activation and the physics that determines the timescales associated with the looping/tracking in the mechanism of action of these transcription factors on the initiation of the transcription event must put a selection pressure on the distribution of the distances of cis-regulatory modules from their respective promoters of the genes. The computational analysis of the upstream sequences of promoters of various genes in the human and mouse genomes for the presence of putative cis-regulatory elements for a set of known transcription factors using

Regulation of host-pathogen interactions via the post-transcriptional Csr/Rsm system.

Science.gov (United States)

Kusmierek, Maria; Dersch, Petra

2018-02-01

A successful colonization of specific hosts requires a rapid and efficient adaptation of the virulence-relevant gene expression program by bacterial pathogens. An important element in this endeavor is the Csr/Rsm system. This multi-component, post-transcriptional control system forms a central hub within complex regulatory networks and coordinately adjusts virulence properties with metabolic and physiological attributes of the pathogen. A key function is elicited by the RNA-binding protein CsrA/RsmA. CsrA/RsmA interacts with numerous target mRNAs, many of which encode crucial virulence factors, and alters their translation, stability or elongation of transcription. Recent studies highlighted that important colonization factors, toxins, and bacterial secretion systems are under CsrA/RsmA control. CsrA/RsmA deficiency impairs host colonization and attenuates virulence, making this post-transcriptional regulator a suitable drug target. The CsrA/RsmA protein can be inactivated through sequestration by non-coding RNAs, or via binding to specific highly abundant mRNAs and interacting proteins. The wide range of interaction partners and RNA targets, as well as the overarching, interlinked genetic control circuits illustrate the complexity of this regulatory system in the different pathogens. Future work addressing spatio-temporal changes of Csr/Rsm-mediated control during the course of an infection will help us to understand how bacteria reprogram their expression profile to cope with continuous changes experienced in colonized niches. Copyright © 2017 Elsevier Ltd. All rights reserved.

Repression of meiotic genes by antisense transcription and by Fkh2 transcription factor in Schizosaccharomyces pombe.

Science.gov (United States)

Chen, Huei-Mei; Rosebrock, Adam P; Khan, Sohail R; Futcher, Bruce; Leatherwood, Janet K

2012-01-01

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.

Plasticity first: molecular signatures of a complex morphological trait in filamentous cyanobacteria.

Science.gov (United States)

Koch, Robin; Kupczok, Anne; Stucken, Karina; Ilhan, Judith; Hammerschmidt, Katrin; Dagan, Tal

2017-08-31

Filamentous cyanobacteria that differentiate multiple cell types are considered the peak of prokaryotic complexity and their evolution has been studied in the context of multicellularity origins. Species that form true-branching filaments exemplify the most complex cyanobacteria. However, the mechanisms underlying the true-branching morphology remain poorly understood despite of several investigations that focused on the identification of novel genes or pathways. An alternative route for the evolution of novel traits is based on existing phenotypic plasticity. According to that scenario - termed genetic assimilation - the fixation of a novel phenotype precedes the fixation of the genotype. Here we show that the evolution of transcriptional regulatory elements constitutes a major mechanism for the evolution of new traits. We found that supplementation with sucrose reconstitutes the ancestral branchless phenotype of two true-branching Fischerella species and compared the transcription start sites (TSSs) between the two phenotypic states. Our analysis uncovers several orthologous TSSs whose transcription level is correlated with the true-branching phenotype. These TSSs are found in genes that encode components of the septosome and elongasome (e.g., fraC and mreB). The concept of genetic assimilation supplies a tenable explanation for the evolution of novel traits but testing its feasibility is hindered by the inability to recreate and study the evolution of present-day traits. We present a novel approach to examine transcription data for the plasticity first route and provide evidence for its occurrence during the evolution of complex colony morphology in true-branching cyanobacteria. Our results reveal a route for evolution of the true-branching phenotype in cyanobacteria via modification of the transcription level of pre-existing genes. Our study supplies evidence for the 'plasticity-first' hypothesis and highlights the importance of transcriptional regulation in

Effect of ARA9 on dioxin receptor mediated transcription

International Nuclear Information System (INIS)

Lees, M.J.; Whitelaw, M.L.

2002-01-01

The dioxin (Aryl hydrocarbon) receptor (DR) is a unique bHLH transcription factor which is activated by binding of planar aromatic hydrocarbons typified by dioxin (TCDD). The active receptor is key to metabolism of aryl hydrocarbon xenobiotics by being a potent inducer of CYP1A1 gene activity. Chlorinated dioxins are inert to metabolism and initiate multifarious toxicities, including potent tumour promotion. These ill-effects are mediated by the activated DR and we are studying the mechanisms by which the ligand binding domain of the DR controls activity of the protein. The DR ligand binding domain resides within a PAS (Per/Arnt/Sim homology) region which is contiguous with the bHLH. The latent bHLH/PAS dioxin receptor (DR) is found in the cytoplasm of most mammalian cell types in a complex with heat shock protein 90, a novel immunophilin like protein termed ARA9/XAP2/AIP, and the co-chaperone p23. Here we use antisense ARA9 constructs to reveal that in the absence of ARA9, the DR is unable to form a transcriptionally active complex. Co-expression of antisense ARA9 with a form of the DR which is constitutively targeted to the nucleus leads to dramatically decreased levels of the nuclear DR protein, implying that ARA9 may function beyond its currently proposed role in cytoplasmic retention of the latent DR

Reduced Neuronal Transcription of Escargot, the Drosophila Gene Encoding a Snail-Type Transcription Factor, Promotes Longevity

Science.gov (United States)

Symonenko, Alexander V.; Roshina, Natalia V.; Krementsova, Anna V.; Pasyukova, Elena G.

2018-01-01

In recent years, several genes involved in complex neuron specification networks have been shown to control life span. However, information on these genes is scattered, and studies to discover new neuronal genes and gene cascades contributing to life span control are needed, especially because of the recognized role of the nervous system in governing homeostasis, aging, and longevity. Previously, we demonstrated that several genes that encode RNA polymerase II transcription factors and that are involved in the development of the nervous system affect life span in Drosophila melanogaster. Among other genes, escargot (esg) was demonstrated to be causally associated with an increase in the life span of male flies. Here, we present new data on the role of esg in life span control. We show that esg affects the life spans of both mated and unmated males and females to varying degrees. By analyzing the survival and locomotion of the esg mutants, we demonstrate that esg is involved in the control of aging. We show that increased longevity is caused by decreased esg transcription. In particular, we demonstrate that esg knockdown in the nervous system increased life span, directly establishing the involvement of the neuronal esg function in life span control. Our data invite attention to the mechanisms regulating the esg transcription rate, which is changed by insertions of DNA fragments of different sizes downstream of the structural part of the gene, indicating the direction of further research. Our data agree with the previously made suggestion that alterations in gene expression during development might affect adult lifespan, due to epigenetic patterns inherited in cell lineages or predetermined during the development of the structural and functional properties of the nervous system. PMID:29760717

The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage

DEFF Research Database (Denmark)

Larsen, Dorthe H; Hari, Flurina; Clapperton, Julie A

2014-01-01

Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient...... recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1...

Prospects of engineering thermotolerance in crops through modulation of heat stress transcription factor and heat shock protein networks.

Science.gov (United States)

Fragkostefanakis, Sotirios; Röth, Sascha; Schleiff, Enrico; Scharf, Klaus-Dieter

2015-09-01

Cell survival under high temperature conditions involves the activation of heat stress response (HSR), which in principle is highly conserved among different organisms, but shows remarkable complexity and unique features in plant systems. The transcriptional reprogramming at higher temperatures is controlled by the activity of the heat stress transcription factors (Hsfs). Hsfs allow the transcriptional activation of HSR genes, among which heat shock proteins (Hsps) are best characterized. Hsps belong to multigene families encoding for molecular chaperones involved in various processes including maintenance of protein homeostasis as a requisite for optimal development and survival under stress conditions. Hsfs form complex networks to activate downstream responses, but are concomitantly subjected to cell-type-dependent feedback regulation through factor-specific physical and functional interactions with chaperones belonging to Hsp90, Hsp70 and small Hsp families. There is increasing evidence that the originally assumed specialized function of Hsf/chaperone networks in the HSR turns out to be a complex central stress response system that is involved in the regulation of a broad variety of other stress responses and may also have substantial impact on various developmental processes. Understanding in detail the function of such regulatory networks is prerequisite for sustained improvement of thermotolerance in important agricultural crops. © 2014 John Wiley & Sons Ltd.

TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair

NARCIS (Netherlands)

G.S. Winkler (Sebastiaan); U. Fiedler; W. Vermeulen (Wim); F. Coin (Frédéric); R.D. Wood (Richard); H.T.M. Timmers (Marc); G. Weeda (Geert); J.H.J. Hoeijmakers (Jan); S.J. Araú jo; J-M. Egly (Jean-Marc)

2000-01-01

textabstractTFIIH is a multisubunit protein complex involved in RNA polymerase II transcription and nucleotide excision repair, which removes a wide variety of DNA lesions including UV-induced photoproducts. Mutations in the DNA-dependent ATPase/helicase subunits of TFIIH, XPB and

Mechanistic Insight into the Host Transcription Inhibition Function of Rift Valley Fever Virus NSs and Its Importance in Virulence.

Science.gov (United States)

Terasaki, Kaori; Ramirez, Sydney I; Makino, Shinji

2016-10-01

Rift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, causes periodic outbreaks in livestocks and humans in countries of the African continent and Middle East. RVFV NSs protein, a nonstructural protein, is a major virulence factor that exhibits several important biological properties. These include suppression of general transcription, inhibition of IFN-β promoter induction and degradation of double-stranded RNA-dependent protein kinase R. Although each of these biological functions of NSs are considered important for countering the antiviral response in the host, the individual contributions of these functions towards RVFV virulence remains unclear. To examine this, we generated two RVFV MP-12 strain-derived mutant viruses. Each carried mutations in NSs that specifically targeted its general transcription inhibition function without affecting its ability to degrade PKR and inhibit IFN-β promoter induction, through its interaction with Sin3-associated protein 30, a part of the repressor complex at the IFN-β promoter. Using these mutant viruses, we have dissected the transcription inhibition function of NSs and examined its importance in RVFV virulence. Both NSs mutant viruses exhibited a differentially impaired ability to inhibit host transcription when compared with MP-12. It has been reported that NSs suppresses general transcription by interfering with the formation of the transcription factor IIH complex, through the degradation of the p62 subunit and sequestration of the p44 subunit. Our study results lead us to suggest that the ability of NSs to induce p62 degradation is the major contributor to its general transcription inhibition property, whereas its interaction with p44 may not play a significant role in this function. Importantly, RVFV MP-12-NSs mutant viruses with an impaired general transcription inhibition function showed a reduced cytotoxicity in cell culture and attenuated virulence in young mice

Global transcriptional repression in C. elegans germline precursors by regulated sequestration of TFIID component TAF-4

Science.gov (United States)

Guven-Ozkan, Tugba; Nishi, Yuichi; Robertson, Scott M.; Lin, Rueyling

2008-01-01

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1–P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres, but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II following fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wildtype OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators. PMID:18854162

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.

The transcription factor Rbf1 is the master regulator for b-mating type controlled pathogenic development in Ustilago maydis.

Directory of Open Access Journals (Sweden)

Kai Heimel

Full Text Available In the phytopathogenic basidiomycete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled by the heterodimeric bE/bW transcription factor complex encoded by the b-mating type locus. The formation of the active bE/bW heterodimer leads to the formation of filaments, induces a G2 cell cycle arrest, and triggers pathogenicity. Here, we identify a set of 345 bE/bW responsive genes which show altered expression during these developmental changes; several of these genes are associated with cell cycle coordination, morphogenesis and pathogenicity. 90% of the genes that show altered expression upon bE/bW-activation require the zinc finger transcription factor Rbf1, one of the few factors directly regulated by the bE/bW heterodimer. Rbf1 is a novel master regulator in a multilayered network of transcription factors that facilitates the complex regulatory traits of sexual and pathogenic development.

Regulation of Nitrogen Metabolism by GATA Zinc Finger Transcription Factors in Yarrowia lipolytica

Energy Technology Data Exchange (ETDEWEB)

Pomraning, Kyle R.; Bredeweg, Erin L.; Baker, Scott E.

2017-02-15

Fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeastYarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism inY. lipolytica. Deletion of the GATA transcription factor genesgzf3andgzf2resulted in nitrogen source-specific growth defects and greater accumulation of lipids when the cells were growing on a simple nitrogen source. Deletion ofgzf1, which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion ofgzf3results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, whilegzf2is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressormig1, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism.