Release of promoter-proximal paused Pol II in response to histone deactylatase inhibition
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE141871 | GEO | 2020/03/30
REPOSITORIES: GEO
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Project description:Transcription by RNA Polymerase II (Pol II) in metazoan is regulated in several steps, including preinitiation complex (PIC) formation, initiation, Pol II escape, productive elongation, cotranscriptional RNA-processing and termination. Genome-wide studies have demonstrated that the phenomenon of promoter-bound Pol II pausing is widespread, especially for genes involved in developmental and stimulus-responsive pathways. However, a mechanistic understanding of the paused Pol II states at promoters is limited. For example, at a global level, it’s unclear to what extent the engaged paused Pol II is stably tethered to the promoter or undergoes rapid cycles of initiation and termination. Here we used the small molecule Triptolide (TPL), an XPB/TFIIH inhibitor, to block transcriptional initiation followed by measuring Pol II occupancy by ChIP-seq. This inhibition of initiation enables us to investigate different states of paused Pol II. Specifically, our global analysis reveals that most genes with paused Pol II, as defined by pausing index, show significant clearance of Pol II during the period of TPL treatment. Our study further identifies a group of genes with unexpectedly stably-paused Pol II, with unchanged Pol II occupancy even after one hour of inhibition of initiation. This group of genes constitutes a small portion of all paused genes defined by the conventional criterion of pausing index. These findings could pave the way for evaluating the contribution of different elongation/pausing factors on different states of Pol II pausing in developmental and other stimulus-responsive pathways. ChIP-Seq of total/Ser5P Pol II in HCT116 cells treated with DMSO or TPL in serum starvation/activation or normal conditions. Nascent RNA-seq in HCT116 cells treated with DMSO or TPL in starved condition.
2015-01-05 | E-GEOD-63314 | biostudies-arrayexpress
Project description:The S. cerevisiae Rpd3 large (Rpd3L) and small (Rpd3S) histone deacetylase (HDAC) complexes are prototypes for understanding transcriptional repression in eukaryotes [1]. The current view is that they function by deacetylating chromatin, thereby limiting accessibility of transcriptional factors to the underlying DNA. However, an Rpd3 catalytic mutant retains substantial repression capability when targeted to a promoter as a LexA fusion protein [2]. We investigated the HDAC-independent properties of the Rpd3 complexes biochemically and discovered a chaperone function, which promotes histone deposition onto DNA, and a novel activity, which prevents nucleosome eviction but not remodeling mediated by the ATP-dependent RSC complex. These HDAC-independent activities inhibit Pol II transcription on a nucleosomal template. The functions of the endogenous Rpd3 complexes can be recapitulated with recombinant Rpd3 core complex comprising Sin3, Rpd3, and Ume1. To test the hypothesis that Rpd3 contributes to chromatin stabilization in vivo, we measured histone H3 density genomewide and found that it was reduced at promoters in an Rpd3 deletion mutant but partially restored in a catalytic mutant. Importantly, the effects on H3 density are most apparent on RSC-enriched genes [3]. Our data suggest that the Rpd3 core complex could contribute to repression via a novel nucleosome stabilization function. H3 were ChIP'd from yeast strains and normalized to input.
2012-01-01 | E-GEOD-33829 | biostudies-arrayexpress
Project description:Histone acetylation is a dynamic modification regulated by the opposing actions of histone acetyltransferases and histone deacetylases (HDACs). Deacetylation of histone tails results in chromatin tightening and therefore HDACs are generally regarded as transcriptional repressors. Counterintuitively, simultaneous deletion of HDAC1 and HDAC2 in embryonic stem cells (ESC) reduced expression of pluripotent transcription factors, Oct4, Sox2 and Nanog (OSN). By shaping global histone acetylation HDACs indirectly regulate the activity of acetyl-lysine readers, such as BRD4. To examine the direct effects of HDAC (LBH589) and BRD4 (JQ1) inhibition on the ESC transcriptome, we performed precision nuclear run-on and sequencing (PRO-seq) analysis. Both LBH589 and JQ1 caused a marked reduction in the pluripotent network. However, while JQ1 treatment induced widespread transcriptional pausing of genes, HDAC inhibition caused a reduction in both paused and elongating polymerase, suggesting an overall reduction in recruitment of RNAPII. Using enhancer RNA (eRNA) expression to measure enhancer activity, we found that while HDAC activity regulates fewer enhancers than JQ1, LBH589 sensitive eRNAs were preferentially associated with super-enhancers and OSN binding sites. These findings suggest that HDAC activity is required for maintenance of pluripotency by regulating the OSN enhancer network via optimal recruitment of RNA polymerase II.
2020-08-01 | GSE136860 | GEO
Project description:Histone deacetylase (HDAC) inhibitors are part of a new generation of epigenetic drugs for cancer treatment. It is known that histone acetylation plays a key role in controlling essential chromosome functions, including gene regulation, and this process has been linked with cancer development and progression. Better understanding of molecular mechanisms involving HDAC inhibitors is needed for the design of new targeted drugs, and also to evaluate the effectiveness of current treatments. In this study, an untargeted metabolomics approach was used to identify intracellular metabolite deregulation after treating cancer cell lines with the HDAC inhibitor HC-Toxin. Metabolomics analysis was performed using high resolution mass spectrometry, in combination with univariate and multivariate statistics and pathway analysis. HDAC inhibition showed highly specific metabolic changes in cancer cell lines compared to non-cancerous cells. In particular, N-acetyl-L-cysteine, N-acetylmethionine, and N-acetyl-L-carnitine showed a dose dependent change. Moreover, pathways controlling protein biosynthesis, as well as tryptophan, cysteine and methionine metabolism were significantly altered by HDAC inhibition. This study illustrates that HDAC inhibition has multiple effects on different metabolic pathways and our results can be extrapolated to inform on the molecular transitions in human cells.
2022-05-04 | MTBLS682 | MetaboLights
Project description:Transcription by RNA Polymerase II (Pol II) in metazoan is regulated in several steps, including preinitiation complex (PIC) formation, initiation, Pol II escape, productive elongation, cotranscriptional RNA-processing and termination. Genome-wide studies have demonstrated that the phenomenon of promoter-bound Pol II pausing is widespread, especially for genes involved in developmental and stimulus-responsive pathways. However, a mechanistic understanding of the paused Pol II states at promoters is limited. For example, at a global level, it’s unclear to what extent the engaged paused Pol II is stably tethered to the promoter or undergoes rapid cycles of initiation and termination. Here we used the small molecule Triptolide (TPL), an XPB/TFIIH inhibitor, to block transcriptional initiation followed by measuring Pol II occupancy by ChIP-seq. This inhibition of initiation enables us to investigate different states of paused Pol II. Specifically, our global analysis reveals that most genes with paused Pol II, as defined by pausing index, show significant clearance of Pol II during the period of TPL treatment. Our study further identifies a group of genes with unexpectedly stably-paused Pol II, with unchanged Pol II occupancy even after one hour of inhibition of initiation. This group of genes constitutes a small portion of all paused genes defined by the conventional criterion of pausing index. These findings could pave the way for evaluating the contribution of different elongation/pausing factors on different states of Pol II pausing in developmental and other stimulus-responsive pathways.
2015-01-05 | GSE63314 | GEO
Project description:The S. cerevisiae Rpd3 large (Rpd3L) and small (Rpd3S) histone deacetylase (HDAC) complexes are prototypes for understanding transcriptional repression in eukaryotes [1]. The current view is that they function by deacetylating chromatin, thereby limiting accessibility of transcriptional factors to the underlying DNA. However, an Rpd3 catalytic mutant retains substantial repression capability when targeted to a promoter as a LexA fusion protein [2]. We investigated the HDAC-independent properties of the Rpd3 complexes biochemically and discovered a chaperone function, which promotes histone deposition onto DNA, and a novel activity, which prevents nucleosome eviction but not remodeling mediated by the ATP-dependent RSC complex. These HDAC-independent activities inhibit Pol II transcription on a nucleosomal template. The functions of the endogenous Rpd3 complexes can be recapitulated with recombinant Rpd3 core complex comprising Sin3, Rpd3, and Ume1. To test the hypothesis that Rpd3 contributes to chromatin stabilization in vivo, we measured histone H3 density genomewide and found that it was reduced at promoters in an Rpd3 deletion mutant but partially restored in a catalytic mutant. Importantly, the effects on H3 density are most apparent on RSC-enriched genes [3]. Our data suggest that the Rpd3 core complex could contribute to repression via a novel nucleosome stabilization function.
2012-01-01 | GSE33829 | GEO
Project description:Histone acetylation is a dynamic modification regulated by the opposing actions of histone acetyltransferases and histone deacetylases (HDACs). Deacetylation of histone tails results in chromatin tightening and therefore HDACs are generally regarded as transcriptional repressors. Counterintuitively, simultaneous deletion of HDAC1 and HDAC2 in embryonic stem cells (ESC) reduced expression of pluripotent transcription factors, Oct4, Sox2 and Nanog (OSN). By shaping global histone acetylation HDACs indirectly regulate the activity of acetyl-lysine readers, such as BRD4. To examine the direct effects of HDAC (LBH589) and BRD4 (JQ1) inhibition on the ESC transcriptome, we performed precision nuclear run-on and sequencing (PRO-seq) analysis. Both LBH589 and JQ1 caused a marked reduction in the pluripotent network. However, while JQ1 treatment induced widespread transcriptional pausing of genes, HDAC inhibition caused a reduction in both paused and elongating polymerase, suggesting an overall reduction in recruitment of RNAPII. Using enhancer RNA (eRNA) expression to measure enhancer activity, we found that while HDAC activity regulates fewer enhancers than JQ1, LBH589 sensitive eRNAs were preferentially associated with super-enhancers and OSN binding sites. These findings suggest that HDAC activity is required for maintenance of pluripotency by regulating the OSN enhancer network via optimal recruitment of RNA polymerase II. Comparative gene expression using the Illumina mouseWG-6 v2 expression BeadChip platform. The effects of Panobinostat/LBH589 (50nM)
2020-08-01 | GSE136618 | GEO
Project description:Histone acetylation is a dynamic modification regulated by the opposing actions of histone acetyltransferases and histone deacetylases (HDACs). Deacetylation of histone tails results in chromatin tightening and therefore HDACs are generally regarded as transcriptional repressors. Counterintuitively, simultaneous deletion of HDAC1 and HDAC2 in embryonic stem cells (ESC) reduced expression of pluripotent transcription factors, Oct4, Sox2 and Nanog (OSN). By shaping global histone acetylation HDACs indirectly regulate the activity of acetyl-lysine readers, such as BRD4. To examine the direct effects of HDAC (LBH589) and BRD4 (JQ1) inhibition on the ESC transcriptome, we performed precision nuclear run-on and sequencing (PRO-seq) analysis. Both LBH589 and JQ1 caused a marked reduction in the pluripotent network. However, while JQ1 treatment induced widespread transcriptional pausing of genes, HDAC inhibition caused a reduction in both paused and elongating polymerase, suggesting an overall reduction in recruitment of RNAPII. Using enhancer RNA (eRNA) expression to measure enhancer activity, we found that while HDAC activity regulates fewer enhancers than JQ1, LBH589 sensitive eRNAs were preferentially associated with super-enhancers and OSN binding sites. These findings suggest that HDAC activity is required for maintenance of pluripotency by regulating the OSN enhancer network via optimal recruitment of RNA polymerase II. Comparative gene expression using the Illumina mouseWG-6 v2 expression BeadChip platform. Wild type (Day 0) ESC compared to HDAC1-/-; HDAC2-/wt; CreER ESCs on day 4 following for OHT treatment in presence (+LIF) or absence (-LIF) of Leukemia inhibitory factor (LIF).
2020-08-01 | GSE136620 | GEO
Project description:Histone acetyltransferases (HATs) and deacetylases (HDACs) function antagonistically to control histone acetylation. As acetylation is a histone mark for active transcription, HATs have been associated with active and HDACs with inactive genes. We describe here genome-wide mapping of HATs and HDACs binding on chromatin and find that both are found at active genes with acetylated histones. Our data provide evidence that HATs and HDACs are both targeted to transcribed regions of active genes by phosphorylated RNA Pol II. Furthermore, the majority of HDACs in the human genome function to reset chromatin by removing acetylation at active genes. Inactive genes that are primed by MLL-mediated histone H3K4 methylation are subject to a dynamic cycle of acetylation and deacetylation by transient HAT/HDAC binding, preventing Pol II from binding to these genes but poising them for future activation. Silent genes without any H3K4 methylation signal show no evidence of being bound by HDACs. high throughput sequencing: genome-wide analysis of 5 HATs, 4 HDACs in human CD4+ cells, Tip60 and HDAC6 in activated CD4 cells, genome-wide analysis of 2 histone acetylations and RNA Polymerase II after HDAC inhibitor treatment in CD4, histone modification with WDR5 knock-down and HDAC inhibitor treatment in HeLa cells expression profiling: Global change in gene expression in human CD4+ T cells after HDAC inhibitor treatment for 2hours and 12 hours. (9 samples in total)
2009-08-20 | E-GEOD-15735 | biostudies-arrayexpress
Project description:Release of promoter-proximal paused Pol II in response to histone deactylatase inhibition
| PRJNA594989 | ENA