scholarly journals Pdx-1 Links Histone H3-Lys-4 Methylation to RNA Polymerase II Elongation during Activation of Insulin Transcription

2005 ◽  
Vol 280 (43) ◽  
pp. 36244-36253 ◽  
Author(s):  
Joshua Francis ◽  
Swarup K. Chakrabarti ◽  
James C. Garmey ◽  
Raghavendra G. Mirmira
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Structure ◽  
Small Interfering Rna ◽  
Histone H3 ◽  
Insulin Gene ◽  
Transcriptional Elongation ◽  
Pol Ii ◽  
Insulin Promoter ◽  
Interfering Rna

Expression of the insulin gene is nearly exclusive to the β cells of the pancreatic islets. Although the sequence-specific transcription factors that regulate insulin expression have been well studied, the interrelationship between these factors, chromatin structure, and transcriptional elongation by RNA polymerase II (pol II) has remained undefined. In this regard, recent studies have begun to establish a role for the methylation of histone H3 in the initiation or elongation of transcription by pol II. To determine a role for the transcriptional activator Pdx-1 in the maintenance of chromatin structure and pol II recruitment at the insulin gene, we performed small interfering RNA-mediated knockdown of Pdx-1 in βTC3 cells and subsequently studied histone modifications and pol II recruitment by chromatin immunoprecipitation. We demonstrated here that the 50% fall in insulin transcription following knockdown of Pdx-1 is accompanied by a 60% fall in dimethylated histone H3-Lys-4 at the insulin promoter. H3-Lys-4 methylation at the insulin promoter may be mediated, at least partially, by the methyltransferase Set9. Immunohistochemical analysis revealed that Set9 is expressed in an islet-enriched pattern in the pancreas, similar to the pattern of Pdx-1 expression. The recruitment of Set9 to the insulin gene appears to be a consequence of its direct interaction with Pdx-1, and small interfering RNA-mediated knockdown of Set9 attenuates insulin transcription. Pdx-1 knockdown was also associated with an overall shift in the recruitment of pol II isoforms to the insulin gene, from an elongation isoform (Ser(P)-2) to an initiation isoform (Ser(P)-5). Our findings therefore suggest a model whereby Pdx-1 plays a novel role in linking H3-Lys-4 dimethylation and pol II elongation to insulin transcription.

scholarly journals Drosophila UTX Is a Histone H3 Lys27 Demethylase That Colocalizes with the Elongating Form of RNA Polymerase II

2007 ◽  
Vol 28 (3) ◽  
pp. 1041-1046 ◽  
Author(s):  
Edwin R. Smith ◽  
Min Gyu Lee ◽  
Benjamin Winter ◽  
Nathan M. Droz ◽  
Joel C. Eissenberg ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Histone H3 ◽  
Silent Chromatin ◽  
H3k4 Methylation ◽  
Active Chromatin ◽  
Pol Ii ◽  
H3k27 Methylation ◽  
Histone H3 Methylation ◽  
Heat Shock Induction

ABSTRACT Histone H3 methylation at Lys27 (H3K27 methylation) is a hallmark of silent chromatin, while H3K4 methylation is associated with active chromatin regions. Here we report that a Drosophila JmjC family member, dUTX, specifically demethylates di- and trimethylated but not monomethylated H3K27. dUTX localization on chromatin correlates with the elongating form of RNA polymerase II (Pol II), and dUTX can associate with Pol II. Furthermore, heat shock induction results in the recruitment of dUTX to the hsp70 gene, like that of several other Pol II elongation factors. Our data indicate that dUTX is intimately associated with actively transcribed genes and may provide a paradigm for how H3K27 demethylation is required for the activation of preinitiated Pol II on transcriptionally poised genes.

scholarly journals The Spt4p Subunit of Yeast DSIF Stimulates Association of the Paf1 Complex with Elongating RNA Polymerase II

2006 ◽  
Vol 26 (8) ◽  
pp. 3135-3148 ◽  
Author(s):  
Hongfang Qiu ◽  
Cuihua Hu ◽  
Chi-Ming Wong ◽  
Alan G. Hinnebusch
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Histone Methylation ◽  
Histone H3 ◽  
Coding Sequences ◽  
Pol Ii ◽  
Cell Extracts ◽  
Carboxy Terminal Domain ◽  
Paf1 Complex ◽  
Carboxy Terminal

ABSTRACT The Paf1 complex (Paf1C) interacts with RNA polymerase II (Pol II) and promotes histone methylation of transcribed coding sequences, but the mechanism of Paf1C recruitment is unknown. We show that Paf1C is not recruited directly by the activator Gcn4p but is dependent on preinitiation complex assembly and Ser5 carboxy-terminal domain phosphorylation for optimal association with ARG1 coding sequences. Importantly, Spt4p is required for Paf1C occupancy at ARG1 (and other genes) and for Paf1C association with Ser5-phosphorylated Pol II in cell extracts, whereas Spt4p-Pol II association is independent of Paf1C. Since spt4Δ does not reduce levels of Pol II at ARG1, Ser5 phosphorylation, or Paf1C expression, it appears that Spt4p (or its partner in DSIF, Spt5p) provides a platform on Pol II for recruiting Paf1C following Ser5 phosphorylation and promoter clearance. spt4Δ reduces trimethylation of Lys4 on histone H3, demonstrating a new role for yeast DSIF in promoting a Paf1C-dependent function in elongation.

The Transcriptional Intermediary Factor TIF1γ Controls Erythroid Cell Fate by Specifically Regulating Transcriptional Elongation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 254-254
Author(s):  
Xiaoying Bai ◽  
Joseph Lee ◽  
Jocelyn LeBlanc ◽  
Anna Sessa ◽  
Zhongan Yang ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Fate ◽  
Erythroid Cell ◽  
Transcriptional Elongation ◽  
Pol Ii ◽  
Physical Interactions ◽  
Paf1 Complex

Abstract Abstract 254 Vertebrate erythropoiesis is regulated by cell-specific transcription factors, RNA polymerase-associated basal machinery and chromatin remodeling factors. One critical chromatin factor is the transcriptional intermediary factor TIF1γ. Loss of TIF1γfunction in zebrafish mutant moonshine causes a profound anemia during embryogenesis, associated with a progressive decrease in expression of most erythroid mRNAs such as GATA1 and globin. TIF1γdeficiency has also been linked to TGF-βsignaling, although the in vivo mechanism for the anemia remains unclear. In an effort to find genes that interact with TIF1γ, we undertook a genetic suppressor screen in which we sought mutations in another gene that would restore blood to normal levels in the background of moonshine deficiency. Few suppressor screens have been done in vertebrate genetic models, and the haploid genetics of zebrafish was a great advantage for this screen. After screening 800 families of fish, two suppressor mutants, “eclipse” and “sunrise”, were found that could greatly rescue the erythroid defects in moonshine. The deficient gene in sunrise has been mapped to the locus of cdc73 (also known as parafibromin/HRPT2), a subunit of the PAF1 complex known to regulate RNA polymerase II (Pol II) elongation and chromatin modification. Furthermore, we have found that knocking down other subunits in the PAF1 complex also rescued the blood defect in moonshine, suggesting that PAF1 as a complex antagonizes TIF1γfunction during erythropoiesis. In yeast, PAF1 has been shown to physically or genetically interact with other elongation factors including DSIF, FACT and p-TEFb. We have found that knocking down DSIF, which is known to induce Pol II pausing during early elongation, also rescues moonshine. FACT and p-TEFb are both known to counteract DSIF to release Pol II from pausing, and knocking down FACT and p-TEFb caused the zebrafish to develop anemia. This strongly suggests that the erythroid defects in TIF1γdeficiency is caused by attenuated Pol II elongation. In an effort to understand the cell-specific phenotype of TIF1γdeficiency, we introduced a FLAG tagged TIF1γinto K562 erythroleukemia cells to pull down interacting proteins. Physical interactions were found among TIF1γ, FACT, p-TEFb and surprisingly the SCL hematopoietic transcription complex. The interaction with the SCL complex provides a cell-specific control over transcriptional elongation. The physical interactions, taken together with the genetic data, suggest a novel mechanism regulating erythropoiesis. TIF1γphysically and functionally links blood-specific transcription factors like SCL to Pol II-associated elongation machinery to regulate blood cell fate. In light of the recent discoveries of widespread Pol II stalling in the promoter proximal region in metazoan genomes, we speculate that similar mechanisms will regulate cell fates in other blood lineages as well as non-blood tissues. Disclosures: Zon: FATE Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Stemgent: Consultancy.

scholarly journals Apoptosis and Autophagy Induction in Mammalian Cells by Small Interfering RNA Knockdown of mRNA Capping Enzymes

2008 ◽  
Vol 28 (19) ◽  
pp. 5829-5836 ◽  
Author(s):  
Chun Chu ◽  
Aaron J. Shatkin
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Fluorescent Protein ◽  
Mrna Translation ◽  
Proteolytic Processing ◽  
Tunel Staining ◽  
Interfering Rna ◽  
Capping Enzymes

ABSTRACT Addition of a 5′ cap to RNA polymerase II transcripts, the first step of pre-mRNA processing in eukaryotes from yeasts to mammals, is catalyzed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine-N-7)methyltransferase. The effects of knockdown of these capping enzymes in mammalian cells were investigated using T7 RNA polymerase-synthesized small interfering RNA and also a lentivirus-based inducible, short hairpin RNA system. Decreasing either guanylyltransferase or methyltransferase resulted in caspase-3 activation and elevated terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining characteristic of apoptosis. Induction of apoptosis was independent of p53 tumor suppressor but dependent on BAK or BAX. In addition, levels of the BH3 family member Bim increased, while Mcl-1 and Bik levels remained unchanged during apoptosis. In contrast to capping enzyme knockdown, apoptosis induced by cycloheximide inhibition of protein synthesis required BAK but not BAX. Both Bim and Mcl-1 levels decreased in cycloheximide-induced apoptosis while Bik levels were unchanged, suggesting that apoptosis in siRNA-treated cells is not a direct consequence of loss of mRNA translation. siRNA-treated BAK−/− BAX−/− double-knockout mouse embryonic fibroblasts failed to activate capase-3 or increase TUNEL staining but instead exhibited autophagy, as demonstrated by proteolytic processing of microtubule-associated protein 1 light chain 3 (LC3) and translocation of transfected green fluorescent protein-LC3 from the nucleus to punctate cytoplasmic structures.

scholarly journals Rhythmic E-Box Binding by CLK-CYC Controls Daily Cycles in per and tim Transcription and Chromatin Modifications

2008 ◽  
Vol 28 (14) ◽  
pp. 4642-4652 ◽  
Author(s):  
Pete Taylor ◽  
Paul E. Hardin
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Specific Binding ◽  
Histone H3 ◽  
Time Of Day ◽  
Transcriptional Elongation ◽  
Chromatin Modifications ◽  
Pol Ii ◽  
E Box ◽  
E Boxes

ABSTRACT The Drosophila melanogaster circadian oscillator comprises interlocked per/tim and Clk transcriptional feedback loops. In the per/tim loop, CLK-CYC-dependent transcriptional activation is rhythmically repressed by PER or PER-TIM to control circadian gene expression that peaks around dusk. Here we show that rhythmic transcription of per and tim involves time-of-day-specific binding of CLK-CYC and associated cycles in chromatin modifications. Activation of per and tim transcription occurs in concert with CLK-CYC binding to upstream and/or intronic E-boxes, acetylation of histone H3-K9, and trimethylation of histone H3-K4. These events are associated with RNA polymerase II (Pol II) binding to the tim promoter and transcriptional elongation by Pol II that is constitutively bound to the per promoter. Repression of per and tim transcription is associated with PER-dependent reversal of these events. Rhythms in H3-K9 acetylation and H3-K4 trimethylation are also associated with CLOCK-BMAL1-dependent transcription in mammals, indicating that the mechanism that controls rhythmic transcription is a conserved feature of the circadian clock even though feedback repression is mediated by different proteins.

scholarly journals Evidence for Eviction and Rapid Deposition of Histones upon Transcriptional Elongation by RNA Polymerase II

2004 ◽  
Vol 24 (23) ◽  
pp. 10111-10117 ◽  
Author(s):  
Marc A. Schwabish ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dynamic Equilibrium ◽  
Yeast Genome ◽  
Transcriptional Elongation ◽  
Coding Region ◽  
Pol Ii ◽  
Core Histones ◽  
Histone Exchange

ABSTRACT Biochemical experiments indicate that transcriptional elongation by RNA polymerase II (Pol II) is inhibited by nucleosomes and hence requires chromatin-modifying activities. Here, we examine the fate of histones upon passage of elongating Pol II in vivo. Histone density throughout the entire Saccharomyces cerevisiae GAL10 coding region is inversely correlated with Pol II association and transcriptional activity, suggesting that the elongating Pol II machinery efficiently evicts core histones from the DNA. Furthermore, new histones appear to be deposited onto DNA less than 1 min after passage of Pol II. Transcription-dependent deposition of histones requires the FACT complex that travels with elongating Pol II. Our results suggest that Pol II transcription generates a highly dynamic equilibrium of histone eviction and histone deposition and that there is significant histone exchange throughout most of the yeast genome within a single cell cycle.

scholarly journals HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription

2002 ◽  
Vol 364 (3) ◽  
pp. 649-657 ◽  
Author(s):  
Sergei NEKHAI ◽  
Meisheng ZHOU ◽  
Anne FERNANDEZ ◽  
William S. LANE ◽  
Ned J.C. LAMB ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Viral Gene ◽  
Transcriptional Elongation ◽  
Rna Pol Ii ◽  
Hela Nuclear Extract ◽  
Pol Ii ◽  
Terminal Domain ◽  
Hiv 1

HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246–256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation.

Negotiating the nucleosome: factors that allow RNA polymerase II to elongate through chromatinThis paper is one of a selection of papers published in this Special Issue, entitled 28th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

2007 ◽  
Vol 85 (4) ◽  
pp. 426-434 ◽  
Author(s):  
Jennifer A. Armstrong
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Remodeling ◽  
Peer Review Process ◽  
Histone Variants ◽  
Transcriptional Elongation ◽  
Histone Chaperones ◽  
Nucleosome Assembly ◽  
Pol Ii ◽  
Assembly Factors

Initiation by RNA polymerase II (Pol II) involves a host of enzymes, and the process of elongation appears similarly complex. Transcriptional elongation through chromatin requires the coordinated efforts of Pol II and its associated transcription factors: C-terminal domain kinases, elongation complexes, chromatin-modifying enzymes, chromatin remodeling factors, histone chaperones (nucleosome assembly factors), and histone variants. This review examines the following: (i) the consequences of the encounter between elongating Pol II and a nucleosome, and (ii) chromatin remodeling factors and nucleosome assembly factors that have recently been identified as important for the elongation stage of transcription.

scholarly journals Control of RNA polymerase II promoter-proximal pausing by DNA supercoiling

2020 ◽  
Author(s):  
A. Herrero-Ruiz ◽  
P. Martínez-García ◽  
J. Terrón-Bautista ◽  
J.A. Lieberman ◽  
S. Jimeno-González ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Regulation Of Gene Expression ◽  
Dna Supercoiling ◽  
General Assumption ◽  
Transcriptional Elongation ◽  
Gene Promoters ◽  
Pol Ii ◽  
Topoisomerase Iia ◽  
Promoter Dna

SummaryThe accumulation of topological stress in the form of DNA supercoiling is inherent to the advance of RNA polymerase II (Pol II) complexes, and needs to be resolved by DNA topoisomerases to sustain productive transcriptional elongation. Topoisomerases are therefore considered general positive facilitators of transcription. Here we show that, in contrast to this general assumption, human topoisomerase IIa accumulates at gene promoters, where it removes transcription-associated negative DNA supercoiling and represses transcription by enforcing promoter-proximal pausing of Pol II. We demonstrate that this topological balance is essential to maintain Immediate Early Genes under basal repression conditions, and that its disruption creates a positive feedback loop that explains their typical bursting behavior in response to stimulus. We therefore describe the control of promoter DNA supercoiling by topoisomerases as a novel layer for the regulation of gene expression, which can act as a molecular switch to rapidly activate transcription.

scholarly journals Drosophila Paf1 Modulates Chromatin Structure at Actively Transcribed Genes

2006 ◽  
Vol 26 (1) ◽  
pp. 250-260 ◽  
Author(s):  
Karen Adelman ◽  
Wenxiang Wei ◽  
M. Behfar Ardehali ◽  
Janis Werner ◽  
Bing Zhu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Chromatin Structure ◽  
Chromatin Immunoprecipitation ◽  
Promoter Region ◽  
Associated Factors ◽  
Histone H3 ◽  
Pol Ii ◽  
Stable Part ◽  
Paf1 Complex

ABSTRACT The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. We have identified and characterized the Drosophila homologs of three subunits of the yeast Paf1 complex and found striking differences between the yeast and Drosophila Paf1 complexes. We demonstrate that although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure.

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