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 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 DIPG-03. THERAPEUTIC TARGETING OF TRANSCRIPTIONAL ELONGATION IN DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii287-iii287
Author(s):  
Hiroaki Katagi ◽  
Nozomu Takata ◽  
Yuki Aoi ◽  
Yongzhan Zhang ◽  
Emily J Rendleman ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Xenograft Model ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Transcriptional Elongation ◽  
Pontine Glioma ◽  
Elongation Complex ◽  
Pol Ii ◽  
Repair Genes ◽  
Novel Therapeutic

Abstract Diffuse intrinsic pontine glioma (DIPG) is highly aggressive brain stem tumor and needed to develop novel therapeutic agents for the treatment. The super elongation complex (SEC) is essential for transcription elongation through release of RNA polymerase II (Pol II). We found that AFF4, a scaffold protein of the SEC, is required for the growth of H3K27M-mutant DIPG cells. In addition, the small molecule SEC inhibitor, KL-1, increased promoter-proximal pausing of Pol II, and reduced transcription elongation, resulting in down-regulate cell cycle, transcription and DNA repair genes. KL-1 treatment decreased cell growth and increased apoptosis in H3K27M-mutant DIPG cells, and prolonged animal survival in our human H3K27M-mutant DIPG xenograft model. Our results demonstrate that the SEC disruption by KL-1 is a novel therapeutic strategy for H3K27M-mutant DIPG.

scholarly journals Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

2007 ◽  
Vol 27 (13) ◽  
pp. 4641-4651 ◽  
Author(s):  
Junjiang Fu ◽  
Ho-Geun Yoon ◽  
Jun Qin ◽  
Jiemin Wong
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Elongation Complex ◽  
Complex Activity ◽  
Interacting Protein ◽  
Pol Ii ◽  
Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

scholarly journals A machine learning-based framework for modeling transcription elongation

2021 ◽  
Vol 118 (6) ◽  
pp. e2007450118
Author(s):  
Peiyuan Feng ◽  
An Xiao ◽  
Meng Fang ◽  
Fangping Wan ◽  
Shuya Li ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
High Throughput Sequencing ◽  
Gene Expression Regulation ◽  
Transcription Elongation ◽  
Transcriptional Elongation ◽  
Sequencing Data ◽  
Pol Ii ◽  
High Throughput Sequencing Data ◽  
Pol Ii Pausing ◽  
Elongation Process

RNA polymerase II (Pol II) generally pauses at certain positions along gene bodies, thereby interrupting the transcription elongation process, which is often coupled with various important biological functions, such as precursor mRNA splicing and gene expression regulation. Characterizing the transcriptional elongation dynamics can thus help us understand many essential biological processes in eukaryotic cells. However, experimentally measuring Pol II elongation rates is generally time and resource consuming. We developed PEPMAN (polymerase II elongation pausing modeling through attention-based deep neural network), a deep learning-based model that accurately predicts Pol II pausing sites based on the native elongating transcript sequencing (NET-seq) data. Through fully taking advantage of the attention mechanism, PEPMAN is able to decipher important sequence features underlying Pol II pausing. More importantly, we demonstrated that the analyses of the PEPMAN-predicted results around various types of alternative splicing sites can provide useful clues into understanding the cotranscriptional splicing events. In addition, associating the PEPMAN prediction results with different epigenetic features can help reveal important factors related to the transcription elongation process. All these results demonstrated that PEPMAN can provide a useful and effective tool for modeling transcription elongation and understanding the related biological factors from available high-throughput sequencing data.

scholarly journals Promoter-specific changes in initiation, elongation and homeostasis of histone H3 acetylation during CBP/p300 inhibition

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Emily Hsu ◽  
Nathan R Zemke ◽  
Arnold J Berk
Keyword(s):  
Rna Polymerase Ii ◽  
Control Point ◽  
Histone H3 ◽  
Airway Epithelial Cells ◽  
Proximal Region ◽  
Transferase Activity ◽  
Transcriptional Elongation ◽  
Elongation Complex ◽  
Histone H3 Acetylation ◽  
H3 Acetylation

Regulation of RNA Polymerase II (Pol2) elongation in the promoter proximal region is an important and ubiquitous control point for gene expression in metazoans. We report that transcription of the adenovirus 5 E4 region is regulated during the release of paused Pol2 into productive elongation by recruitment of the super elongation complex (SEC), dependent on promoter H3K18/27 acetylation by CBP/p300. We also establish that this is a general transcriptional regulatory mechanism that applies to ~6% of expressed protein-coding genes in primary human airway epithelial cells. We observed that a homeostatic mechanism maintains promoter, but not enhancer H3K18/27ac in response to extensive inhibition of CBP/p300 acetyl transferase activity by the highly specific small molecule inhibitor A-485. Further, our results suggest a function for BRD4 association at enhancers in regulating paused Pol2 release at nearby promoters. Taken together, our results uncover processes regulating transcriptional elongation by promoter region histone H3 acetylation and homeostatic maintenance of promoter, but not enhancer, H3K18/27ac in response to inhibition of CBP/p300 acetyl transferase activity.

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 Pausing sites of RNA polymerase II on actively transcribed genes are enriched in DNA double-stranded breaks

2020 ◽  
Vol 295 (12) ◽  
pp. 3990-4000 ◽  
Author(s):  
Sandeep Singh ◽  
Karol Szlachta ◽  
Arkadi Manukyan ◽  
Heather M. Raimer ◽  
Manikarna Dinda ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Topoisomerase I ◽  
Cell Types ◽  
Dna Breaks ◽  
Transcription Start ◽  
Pol Ii ◽  
Transcription Start Sites

DNA double-stranded breaks (DSBs) are strongly associated with active transcription, and promoter-proximal pausing of RNA polymerase II (Pol II) is a critical step in transcriptional regulation. Mapping the distribution of DSBs along actively expressed genes and identifying the location of DSBs relative to pausing sites can provide mechanistic insights into transcriptional regulation. Using genome-wide DNA break mapping/sequencing techniques at single-nucleotide resolution in human cells, we found that DSBs are preferentially located around transcription start sites of highly transcribed and paused genes and that Pol II promoter-proximal pausing sites are enriched in DSBs. We observed that DSB frequency at pausing sites increases as the strength of pausing increases, regardless of whether the pausing sites are near or far from annotated transcription start sites. Inhibition of topoisomerase I and II by camptothecin and etoposide treatment, respectively, increased DSBs at the pausing sites as the concentrations of drugs increased, demonstrating the involvement of topoisomerases in DSB generation at the pausing sites. DNA breaks generated by topoisomerases are short-lived because of the religation activity of these enzymes, which these drugs inhibit; therefore, the observation of increased DSBs with increasing drug doses at pausing sites indicated active recruitment of topoisomerases to these sites. Furthermore, the enrichment and locations of DSBs at pausing sites were shared among different cell types, suggesting that Pol II promoter-proximal pausing is a common regulatory mechanism. Our findings support a model in which topoisomerases participate in Pol II promoter-proximal pausing and indicated that DSBs at pausing sites contribute to transcriptional activation.

scholarly journals TATA-Binding Protein Mutants That Are Lethal in the Absence of the Nhp6 High-Mobility-Group Protein

2004 ◽  
Vol 24 (14) ◽  
pp. 6419-6429 ◽  
Author(s):  
Peter Eriksson ◽  
Debabrata Biswas ◽  
Yaxin Yu ◽  
James M. Stewart ◽  
David J. Stillman
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Synthetic Lethality ◽  
Binding Protein ◽  
High Mobility ◽  
Tata Binding Protein ◽  
High Mobility Group ◽  
Multicopy Plasmid ◽  
Pol Ii ◽  
Pol Iii

ABSTRACT The Saccharomyces cerevisiae Nhp6 protein is related to the high-mobility-group B family of architectural DNA-binding proteins that bind DNA nonspecifically but bend DNA sharply. Nhp6 is involved in transcriptional activation by both RNA polymerase II (Pol II) and Pol III. Our previous genetic studies have implicated Nhp6 in facilitating TATA-binding protein (TBP) binding to some Pol II promoters in vivo, and we have used a novel genetic screen to isolate 32 new mutations in TBP that are viable in wild-type cells but lethal in the absence of Nhp6. The TBP mutations that are lethal in the absence of Nhp6 cluster in three regions: on the upper surface of TBP that may have a regulatory role, near residues that contact Spt3, or near residues known to contact either TFIIA or Brf1 (in TFIIIB). The latter set of mutations suggests that Nhp6 becomes essential when a TBP mutant compromises its ability to interact with either TFIIA or Brf1. Importantly, the synthetic lethality for some of the TBP mutations is suppressed by a multicopy plasmid with SNR6 or by an spt3 mutation. It has been previously shown that nhp6ab mutants are defective in expressing SNR6, a Pol III-transcribed gene encoding the U6 splicing RNA. Chromatin immunoprecipitation experiments show that TBP binding to SNR6 is reduced in an nhp6ab mutant. Nhp6 interacts with Spt16/Pob3, the yeast equivalent of the FACT elongation complex, consistent with nhp6ab cells being extremely sensitive to 6-azauracil (6-AU). However, this 6-AU sensitivity can be suppressed by multicopy SNR6 or BRF1. Additionally, strains with SNR6 promoter mutations are sensitive to 6-AU, suggesting that decreased SNR6 RNA levels contribute to 6-AU sensitivity. These results challenge the widely held belief that 6-AU sensitivity results from a defect in transcriptional elongation.

scholarly journals Yeast NC2 Associates with the RNA Polymerase II Preinitiation Complex and Selectively Affects Transcription In Vivo

2001 ◽  
Vol 21 (8) ◽  
pp. 2736-2742 ◽  
Author(s):  
Joseph V. Geisberg ◽  
Frank C. Holstege ◽  
Richard A. Young ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Negative Regulator ◽  
Preinitiation Complex ◽  
Positive Role ◽  
Pol Ii ◽  
Basal Transcription Factors

ABSTRACT NC2 (Dr1-Drap1 or Bur6-Ydr1) has been characterized in vitro as a general negative regulator of RNA polymerase II (Pol II) transcription that interacts with TATA-binding protein (TBP) and inhibits its function. Here, we show that NC2 associates with promoters in vivo in a manner that correlates with transcriptional activity and with occupancy by basal transcription factors. NC2 rapidly associates with promoters in response to transcriptional activation, and it remains associated under conditions in which transcription is blocked after assembly of the Pol II preinitiation complex. NC2 positively and negatively affects approximately 17% of Saccharomyces cerevisiaegenes in a pattern that resembles the response to general environmental stress. Relative to TBP, NC2 occupancy is high at promoters where NC2 is positively required for normal levels of transcription. Thus, NC2 is associated with the Pol II preinitiation complex, and it can play a direct and positive role at certain promoters in vivo.

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.

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