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Transcriptional elongation requires DNA break-induced signaling

ABSTRACT: We have previously shown that RNA polymerase II (Pol II) pause release and transcriptional elongation involve phosphorylation of the factor TRIM28 by the DNA damage response (DDR) kinases ATM and DNA-PK. Here, we report a significant role for DNA breaks and DDR signaling in the mechanisms of transcriptional elongation in stimulus-inducible genes in humans. Our data show the enrichment of TRIM28 and γH2AX on serum-induced genes and the important function of DNA-PK for Pol II pause release and transcriptional activation-coupled DDR signaling on these genes. γH2AX accumulation decreases when P-TEFb is inhibited, confirming that DDR signaling results from transcriptional elongation. In addition, transcriptional elongation-coupled DDR signaling involves topoisomerase II because inhibiting this enzyme interferes with Pol II pause release and γH2AX accumulation. Our findings propose that DDR signaling is required for effective Pol II pause release and transcriptional elongation through a novel mechanism involving TRIM28, DNA-PK, and topoisomerase II

ORGANISM(S): Homo sapiens

PROVIDER: GSE75170 | GEO | 2015/11/20

SECONDARY ACCESSION(S): PRJNA302670

REPOSITORIES: GEO

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Transcriptional elongation requires DNA break-induced signaling

Project description:We have previously shown that RNA polymerase II (Pol II) pause release and transcriptional elongation involve phosphorylation of the factor TRIM28 by the DNA damage response (DDR) kinases ATM and DNA-PK. Here, we report a significant role for DNA breaks and DDR signaling in the mechanisms of transcriptional elongation in stimulus-inducible genes in humans. Our data show the enrichment of TRIM28 and Î³H2AX on serum-induced genes and the important function of DNA-PK for Pol II pause release and transcriptional activation-coupled DDR signaling on these genes. Î³H2AX accumulation decreases when P-TEFb is inhibited, confirming that DDR signaling results from transcriptional elongation. In addition, transcriptional elongation-coupled DDR signaling involves topoisomerase II because inhibiting this enzyme interferes with Pol II pause release and Î³H2AX accumulation. Our findings propose that DDR signaling is required for effective Pol II pause release and transcriptional elongation through a novel mechanism involving TRIM28, DNA-PK, and topoisomerase II 42 samples in total. IP targets were gammaH2ax, s2-pol-II, pol-II, pTRIM28, DNA-pk, topo-IIB. Experimental conditions included DMSO treatment (control), pTEFb, topoII-i, dnapk-i. Matched non-specific IP samples used for control in peak calling.

2015-11-20 | E-GEOD-75170 | biostudies-arrayexpress

Screening HIF-1 Cofactors in Breast Cancer Cells by RIME

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2022-02-17 | PXD024373 | Pride

HIF-1 Recruits TRIM28 and DNA-PK to Release Paused RNA Polymerase II and Activate Target Gene Transcription in Response to Hypoxia

Project description:Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a master regulator of O2 homeostasis in metazoan species by binding to hypoxia response elements (HREs) and activating the transcription of hundreds of genes in response to reduced O2 availability. RNA polymerase II (Pol II) initiates transcription of many HIF target genes under non-hypoxic conditions, but pauses after 20-100 nucleotides and requires HIF-1 binding for release. Here we report that in hypoxic breast cancer cells, HIF-1 recruits TRIM28 and DNA-dependent protein kinase (DNA-PK) to HREs to release paused Pol II. We show that HIF-1 and TRIM28 assemble the catalytically-active DNA-PK heterotrimer, which phosphorylates TRIM28 at serine-824, enabling recruitment of CDK9, which phosphorylates serine-2 of the Pol II large subunit C-terminal domain and the negative elongation factor to release paused Pol II, thereby stimulating productive transcriptional elongation. Our studies have revealed a critical molecular mechanism by which HIF-1 stimulates gene transcription and reveal that the anticancer effects of drugs targeting DNA-PK in breast cancer may be due in part to their inhibition of HIF-dependent transcription.

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RBM7 activates P-TEFb for a pro-survival transcriptional response to DNA damage

Project description:DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain obscure. Here we show that following DNA damage, the RNA-binding motif protein 7 (RBM7) stimulates RNA polymerase II (Pol II) elongation and increases cell viability by activating the positive transcription elongation factor b (P-TEFb). This is mediated by DNA damage-enhanced binding of RBM7 to 7SK, the scaffold of the 7SK small nuclear ribonucleoprotein (snRNP) that inhibits P-TEFb. As a result, P-TEFb relocates from 7SK snRNP to chromatin to activate transcription of key DDR genes and multiple classes of non-coding RNAs. By alleviating the inhibition of P-TEFb, RBM7 thus promotes Pol II elongation to activate a transcriptional response that is crucial for cell fate upon genotoxic insult. Our work highlights a novel paradigm in stress-dependent control of Pol II pause release, and offers the promise of combating cancer by RBM7 and P-TEFb antagonists in combination with established DNA damage-inducing chemotherapeutics.

2018-06-01 | E-MTAB-6475 | biostudies-arrayexpress

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2022-08-09 | PXD033771 | Pride

The PNUTS phosphatase complex controls transcription pause release

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TOX4 Facilitates Promoter-Proximal Pausing and C-Terminal Domain Dephosphorylation of RNA Polymerase II in Human Cells

Project description:TOX4 is one of the regulatory proteins of PP1 phosphatases with poorly understood functions. Here we show that chromatin occupancy pattern of TOX4 resembles that of RNA polymerase II (Pol II), and its loss increases cellular level of C-terminal domain (CTD) phosphorylated Pol II but mainly decreases Pol II occupancy on promoters. In addition, elongation rate analyses by 4sUDRB-seq suggest that TOX4 restricts pause release and early elongation but promotes late elongation. Moreover, TT-seq analyses indicate that TOX4 loss mainly decreases transcriptional output. Mechanistically, TOX4 may restrict pause release through facilitating CTD serine 2 and DSIF dephosphorylation, and promote Pol II recycling and reinitiation through facilitating CTD serines 2 and 5 dephosphorylation. Furthermore, among the PP1 phosphatases, TOX4 preferentially binds PP1α and is capable of facilitating Pol II CTD dephosphorylation in vitro. These results lay the foundation for a better understanding of the role of TOX4 in transcriptional regulation.

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SPT6 functions in transcriptional pause-release via PAF1C recruitment [RNA-seq]

Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in release of paused RNA Pol II. Short genes demonstrate a prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Unexpectedly, the recruitment of PAF1 complex (PAF1C) to RNA Pol II fails upon SPT6 depletion, leading to the release of NELF-bound RNA Pol II into the gene bodies. Furthermore, SPT6 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause-release through PAF1C recruitment and NELF removal during the early elongation.

2022-08-09 | GSE202189 | GEO

SPT6 functions in transcriptional pause-release via PAF1C recruitment [PRO-seq]

Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in release of paused RNA Pol II. Short genes demonstrate a prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Unexpectedly, the recruitment of PAF1 complex (PAF1C) to RNA Pol II fails upon SPT6 depletion, leading to the release of NELF-bound RNA Pol II into the gene bodies. Furthermore, SPT6 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause-release through PAF1C recruitment and NELF removal during the early elongation.

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SPT6 functions in transcriptional pause-release via PAF1C recruitment [ChIP-seq]

Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in release of paused RNA Pol II. Short genes demonstrate a prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Unexpectedly, the recruitment of PAF1 complex (PAF1C) to RNA Pol II fails upon SPT6 depletion, leading to the release of NELF-bound RNA Pol II into the gene bodies. Furthermore, SPT6 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause-release through PAF1C recruitment and NELF removal during the early elongation.

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