Dicer promotes genome stability via the bromodomain transcriptional co-activator BRD4
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ABSTRACT:
PROVIDER: PRJNA722747 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14267900_1.fastq.gz | Fastqsanger.gz | |||
| SRR14267900_2.fastq.gz | Fastqsanger.gz | |||
| SRR14267901_1.fastq.gz | Fastqsanger.gz | |||
| SRR14267901_2.fastq.gz | Fastqsanger.gz | |||
| SRR14267902_1.fastq.gz | Fastqsanger.gz |
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Project description:RNA interference is required for post-transcriptional silencing, but also has additional roles in transcriptional silencing of centromeres and genome stability. However, these roles have been difficult to detect in mammals. Strikingly, we found that Dicer-deficient embryonic stem cells have strong proliferation and chromosome segregation defects as well as increased transcription of centromeric satellite repeats, which triggers the interferon response. We conducted a CRISPR-Cas9 genetic screen to restore viability and identified transcriptional activators, histone H3K9 methyltransferases, and chromosome segregation factors as suppressors, resembling Dicer suppressors identified in independent screens in fission yeast. The strongest suppressors were mutations in the transcriptional co-activator Brd4, which reversed the strand-specific transcription of major satellite repeats suppressing the interferon response, and in the histone acetyltransferase Elp3. We show that identical mutations in the second bromodomain of Brd4 rescue Dicer-dependent silencing and chromosome segregation defects in both mammalian cells and fission yeast. This remarkable conservation demonstrates that RNA interference has an ancient role in transcriptional silencing of satellite repeats, which is essential for cell cycle progression and proper chromosome segregation. Our results have pharmacological implications for cancer and autoimmune diseases characterized by unregulated transcription of satellite repeats.
2021-11-13 | GSE172282 | GEO
Project description:RNA interference is required for post-transcriptional silencing, but also has additional roles in transcriptional silencing of centromeres and genome stability. However, these roles have been controversial in mammals. Strikingly, we found that Dicer-deficient embryonic stem cells have strong proliferation and chromosome segregation defects as well as increased transcription of centromeric satellite repeats, which triggers the interferon response. We conducted a CRISPR-Cas9 genetic screen to restore viability and identified transcriptional activators, histone H3K9 methyltransferases, and chromosome segregation factors as suppressors, resembling Dicer suppressors identified in independent screens in fission yeast. The strongest suppressors were mutations in the transcriptional co-activator Brd4, which reversed the strand-specific transcription of major satellite repeats suppressing the interferon response, and in the histone acetyltransferase Elp3. We show that identical mutations in the second bromodomain of Brd4 rescue Dicer-dependent silencing and chromosome segregation defects in both mammalian cells and fission yeast. This remarkable conservation demonstrates that RNA interference has an ancient role in transcriptional silencing and in particular of satellite repeats, which is essential for cell cycle progression and proper chromosome segregation. Our results have pharmacological implications for cancer and autoimmune diseases characterized by unregulated transcription of satellite repeats.
| S-EPMC8863982 | biostudies-literature
Project description:During heat stress cyto-protective genes including heat shock proteins are transcriptionally up-regulated and post-transcriptional splicing is inhibited. In contrast, co-transcriptional mRNA-splicing is maintained. These factors closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response. Furthermore, there is evidence for BRD4's role in splicing regulation; Using RNA-Seq analyses we indeed found a significant increase in splicing inhibition, in particular intron retentions, during heat treatment in BRD4-deficient cells, but not under normal conditions. Subsequent experiments revealed that heat stress leads to the recruitment of BRD4 to nuclear stress bodies, to the interaction with the heat shock factor 1 (HSF1) and to the transcriptional up-regulation of non-coding Sat III RNA transcripts. These findings implicate BRD4 as a central regulator of splicing during heat stress. Since BRD4 is a potent target for anti-cancer therapies, our data linking BRD4 to the splicing machinery and the heat stress response - give additional insight into the mode of action of BRD4 inhibitors. WI38 cells have been treated by heatshock and anti BRD4 siRNA and combination.
2016-09-07 | E-GEOD-73471 | biostudies-arrayexpress
Project description:Transcriptional elongation by RNA polymerase II (Pol II) is regulated by positive transcription elongation factor b (P-TEFb) in association with Bromodomain-containing protein 4 (BRD4). We used genome-wide chromatin immunoprecipitation sequencing in primary human CD4+ T cells to reveal that BRD4 co-localizes with Ser2-phosphorylated Pol II (Pol II Ser2) at both enhancers and promoters of active genes. Disruption of bromodomain:histone acetylation interactions by JQ1, a small-molecule bromodomain inhibitor, resulted in decreased BRD4 binding, reduced Pol II Ser2, and reduced expression of lineage-specific genes in primary human CD4+ T cells. A large number of JQ1-disrupted BRD4 binding regions exhibited di-acetylated H4 (lysine-5 and -8) and H3K27 acetylation (H3K27ac), which correlated with the presence of histone acetyltransferases and deacetylases. Genes associated with BRD4/H3K27ac co-occupancy exhibited significantly higher activity than those associated with H3K27ac or BRD4 binding alone. Comparison of BRD4 binding in T cells and in human embryonic stem cells revealed that enhancer BRD4 binding sites were predominantly lineage-specific. Our findings suggest that BRD4-driven Pol II phosphorylation at serine 2 plays an important role in regulating lineage-specific gene transcription in human CD4+ T cells. Examination of BRD4, total Pol II, serine 2 phosphorylated Pol II and serine 5 phosphorylated Pol II binding in CD4+ T cells (with and without JQ1 treatment) and BRD4 binding in human embryonic stems cell; PolyA RNA expression in CD4+ T cells( with and without JQ1 treatment) using RNA-seq
2012-11-14 | E-GEOD-33281 | biostudies-arrayexpress
Project description:DNA damage activates a complex signaling network in cells that blocks cell cycle progression, recruits factors involved in DNA repair, and/or triggers programs that control senescence or programmed cell death. Alterations in chromatin structure are known to be important for the initiation and propagation of the DNA damage response, although the molecular details are unclear. We investigated the role of chromatin structure in the DNA damage response by monitoring multiple timedependent checkpoint signaling and response events with a high-content multiplex image-based RNAi screen of chromatin modifying and interacting genes. We discovered that Brd4, a double bromodomain-containing protein, functions as an endogenous inhibitor of DNA damage signaling by binding to acetylated histones at sites of open chromatin and altering chromatin accessibility. Loss of Brd4 or disruption of acetyl-lysine binding results in an increase in both the number and size of radiation-induced !H2AX nuclear foci while overexpression of a Brd4 splice isoform completely suppresses !H2AX formation, despite equivalent double strand break formation. Brd4 knock-down cells displayed altered chromatin structure, prolonged cell cycle checkpoint arrest and enhanced survival after irradiation, while overexpression of Brd4 isoform B results in enhanced radiationinduced lethality. Brd4 is the target of the t(15;19) chromosomal translocation in a rare form of cancer, NUT Midline Carcinoma. Acetyl lysine-bromodomain interactions of the Brd4-NUT fusion protein suppresses !H2AX foci in discrete nuclear compartments, rendering cells more radiosensitive, mimicking overexpression of Brd4 isoform B. NUT Midline Carcinoma is sensitive to radiotherapy, however tumor material from this rare cancer is scarce. We therefore investigated Brd4 expression in another human cancer commonly treated with radiotherapy, glioblastoma multiforme, and found that expression of Brd4 isoform B correlated specifically with treatment response to radiotherapy. These data implicate Brd4 as an endogenous insulator of DNA damage signaling through recognition of epigenetic modifications in chromatin and suggest that expression of the Brd4 in human cancer can modulate the clinical response to DNA-damaging cancer therapy. Two replicates each of U2OS cells expressing either Brd4 shRNA or a control shRNA
2013-07-14 | E-GEOD-30700 | biostudies-arrayexpress
Project description:The BET family protein BRD4, which forms the CDK9-containing BRD4-PTEFb complex, is considered to be a master regulator of RNA polymerase II (Pol II) pause release. Because its tandem bromodomains interact with acetylated histone lysine residues, it has long been thought that BRD4 requires these bromodomains for its recruitment to chromatin and transcriptional regulatory function. Here, using rapid depletion and genetic complementation with domain deletion mutants, we demonstrate that BRD4 bromodomains are dispensable for Pol II pause release. A minimal, bromodomain-less C-terminal BRD4 fragment containing the PTEFb-interacting C-terminal motif (CTM) is instead both necessary and sufficient to mediate Pol II pause release in the absence of full-length BRD4. Although BRD4-PTEFb can associate with chromatin through acetyl recognition, our results indicate that a distinct, active BRD4-PTEFb population functions to regulate transcription independently of bromodomain-mediated chromatin association. These findings may enable more effective pharmaceutical modulation of BRD4-PTEFb activity.
2023-07-14 | GSE232869 | GEO
Project description:To study the role of the bromodomain (BD) in BRD4-dependent gene expression, we compared the function of wild type BRD4 and a mutant BRD4-mBD, which carries a point mutation in each of the two BDs. We first knocked down endogenous BRD4 by shRNA (shBRD4) and then stably reconstituted the cells with shBRD4-resistant YFP-BRD4 (wild type or mBD mutant). Following BRD4 reconstitution, the degree of recovery in gene expression was analyzed by Affymetrix Mouse Exon 1.0 ST array. Wild-type BRD4 restored gene expression significantly and far more efficiently than the mBD mutant.
2014-11-10 | GSE56370 | GEO
Project description:Transcriptional elongation by RNA polymerase II (Pol II) is regulated by positive transcription elongation factor b (P-TEFb) in association with Bromodomain-containing protein 4 (BRD4). We used genome-wide chromatin immunoprecipitation sequencing in primary human CD4+ T cells to reveal that BRD4 co-localizes with Ser2-phosphorylated Pol II (Pol II Ser2) at both enhancers and promoters of active genes. Disruption of bromodomain:histone acetylation interactions by JQ1, a small-molecule bromodomain inhibitor, resulted in decreased BRD4 binding, reduced Pol II Ser2, and reduced expression of lineage-specific genes in primary human CD4+ T cells. A large number of JQ1-disrupted BRD4 binding regions exhibited di-acetylated H4 (lysine-5 and -8) and H3K27 acetylation (H3K27ac), which correlated with the presence of histone acetyltransferases and deacetylases. Genes associated with BRD4/H3K27ac co-occupancy exhibited significantly higher activity than those associated with H3K27ac or BRD4 binding alone. Comparison of BRD4 binding in T cells and in human embryonic stem cells revealed that enhancer BRD4 binding sites were predominantly lineage-specific. Our findings suggest that BRD4-driven Pol II phosphorylation at serine 2 plays an important role in regulating lineage-specific gene transcription in human CD4+ T cells.
2012-11-14 | GSE33281 | GEO
Project description:During heat stress cyto-protective genes including heat shock proteins are transcriptionally up-regulated and post-transcriptional splicing is inhibited. In contrast, co-transcriptional mRNA-splicing is maintained. These factors closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response. Furthermore, there is evidence for BRD4's role in splicing regulation; Using RNA-Seq analyses we indeed found a significant increase in splicing inhibition, in particular intron retentions, during heat treatment in BRD4-deficient cells, but not under normal conditions. Subsequent experiments revealed that heat stress leads to the recruitment of BRD4 to nuclear stress bodies, to the interaction with the heat shock factor 1 (HSF1) and to the transcriptional up-regulation of non-coding Sat III RNA transcripts. These findings implicate BRD4 as a central regulator of splicing during heat stress. Since BRD4 is a potent target for anti-cancer therapies, our data linking BRD4 to the splicing machinery and the heat stress response - give additional insight into the mode of action of BRD4 inhibitors.
2016-09-07 | GSE73471 | GEO
Project description:NUT midline carcinoma (NMC) is a rare, aggressive subtype of squamous carcinoma that is driven by the BRD4-NUT fusion oncoprotein. BRD4, a BET protein, binds to chromatin through its two bromodomains, and NUT recruits the p300 histone acetyltransferse (HAT) to activate transcription of oncogenic target genes. BET selective bromodomain inhibitors have demonstrated on-target activity in NMC patients, but with limited efficacy. P300, like BRD4, contains a bromodomain. We show that combining selective p300/CBP and BET bromodomain inhibitors, GNE-781 and OTX015, respectively, induces synergistic inhibition of NMC growth. Treatment of NMC cells with the novel dual p300/CBP and BET bromodomain selective inhibitor, NEO2734, potently inhibits growth and induces differentiation of NMC cells in vitro; findings that correspond with potentiated transcriptional effects from combined BET and p300 bromodomain inhibition. In three disseminated NMC xenograft models, NEO2734 provided greater growth inhibition, with tumor regression and significant survival benefit seen in two of three models, compared with a lead clinical BET inhibitor or 'standard' chemotherapy.
2020-06-19 | GSE142481 | GEO