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Regulation of the RNAPII Pool Is Integral to the DNA Damage Response [TT-seq CSBKO]

ABSTRACT: In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNAPII and genome-wide transcription shutdown. How these responses are connected has remained unclear. Here we show that damage-induced ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA damage response coordination. K1268-ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. It is also crucial for transcriptional shutdown, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery – indeed, depletion of the RNAPII pool underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA damage response, and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programmes, in genome instability disorders and even normal cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE143529 | GEO | 2020/01/14

REPOSITORIES: GEO

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Regulation of the RNAPII Pool Is Integral to the DNA Damage Response [TT-seq]

Project description:In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNAPII and genome-wide transcription shutdown. How these responses are connected has remained unclear. Here we show that damage-induced ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA damage response coordination. K1268-ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. It is also crucial for transcriptional shutdown, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery – indeed, depletion of the RNAPII pool underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA damage response, and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programmes, in genome instability disorders and even normal cells.

2020-01-14 | GSE143518 | GEO

Regulation of the RNAPII Pool Is Integral to the DNA Damage Response [RNA-seq]

2020-01-14 | GSE143537 | GEO

ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation

Project description:Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation at a single lysine (K1268) by the CRL4CSA ubiquitin ligase. How CRL4CSA is specifically directed toward the K1268 site is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to the K1268 site, revealing ELOF1 as a specificity factor that interacts with and positions CRL4CSA for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also reveals a CSB-independent compensatory pathway in which ELOF1 protects cells against DNA replication stress by preventing DNA damage-induced R-loops. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between the transcriptional stress response and DNA replication.

2021-04-21 | PXD024051 | Pride

ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation

Project description:Removal of transcription-blocking DNA lesions requires the binding of transcription-coupled repair (TCR) factors to DNA damage-stalled RNA polymerase II (RNAPII). The full repertoire of factors required for TCR remains to be elucidated. Through genome-wide CRISPR screens and strand-specific ChIP-seq, we identify the RNAPII-associated factor ELOF1 as a new core TCR component. Mechanistically, ELOF1 does not affect the association of TCR components CSB and the CRL4CSA ubiquitin ligase, but instead regulates RNAPII ubiquitylation, and subsequent ubiquitin-dependent recruitment of repair factors. This function requires its constitutive interaction with RNAPII close to the K1268 site, revealing ELOF1 as a specificity factor that positions CRL4CSA for optimal RNAPII ubiquitylation to orchestrate transcription-coupled repair. Finally, ELOF1 operates in a second transcription stress-response pathway, regulated by the deubiquitylating enzyme OTUD5.

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Ubiquitin ligase WWP2 targets RNA polymerase II in a DNA-PK-dependent transcription shutoff circuitry for DNA repair

Project description:DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII)-transcribed genes lead to inhibition of transcription. DNA protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remained unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII-transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of non-ubiquitylatable RPB1 abrogates the loading of Non-Homologous End-Joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shut-off circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.

2019-03-29 | PXD012606 | Pride

A CSB-PAF1 axis activates RNA polymerase II from promoter-proximal sites and restores efficient transcription elongation after DNA damage repair

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2021-02-28 | PXD016198 | Pride

Tyrosine 1 phosphorylated RNA polymerase II transcribes PROMPTs to promote proximal promoter pausing and induce global transcriptional repression in response to DNA damage

Project description:DNA faces continuous threats from various endogenous and exogenous stresses. When damaged, DNA requires repair to enable proper cell functioning. Cells have evolved multiple repair pathways to maintain genome stability. Transcription mediated by RNA polymerase II (RNA Pol II), which is conventionally linked with gene expression, plays an integral role in DNA repair. Transcription occurring at sites of DNA damage facilitates repair by generating functional transcripts, recruiting DNA repair factors, or modulating chromatin architecture. Conversely, the shutdown of transcription in nearby genes is crucial to ensure effective DNA repair. UV-induced DNA lesions have been shown to lead to a genome-wide suppression of transcription through RNA Pol II ubiquitylation and subsequent degradation. However, the global transcriptional response to double-strand breaks remains unclear.In this study, we employed mNET-Seq analysis to examine nascent transcripts bound to the CTD Y1P, S2P and S5P of RNA Pol II. Our data reveal a global transcriptional shutdown, as evidenced by a greater number of downregulated protein coding genes in response to irradiation. Interestingly, we observed a significant increase in Y1P antisense transcripts near promoters, known as PROMPTs, which are associated with the downregulated protein coding genes upon irradiation. Furthermore, an increased promoter pausing index correlated with elevated expression of PROMPTs in response to double strand breaks induction. Motif enrichment analysis uncovered binding sites for the Polycomb repressive complex 2 (PRC2) in upregulated PROMPTs. Using proximity ligation assays, we demonstrated an increased proximity between Y1P RNA Pol II and PRC2 upon irradiation. Finally, we also report that inhibition of Y1P or depletion of PRC2 results in the loss of transcriptional repression of protein coding genes, associated with PROMPTs.

2024-02-13 | GSE244353 | GEO

Ubiquitin-dependent transfer of PAF1c from MYC onto RNA Polymerase controls double-strand break repair at active promoters

Project description:MYC family proteins are oncogenic transcription factors that can globally affect the function of RNA Polymerase II (RNAPII). The ability of MYC proteins to promote transcription elongation depends on their ubiquitination, but the underlying mechanism and its biological relevance are unknown. Here we show that MYC and the Polymerase II associated factor, PAF1c, interact directly and their function is mutually dependent, since the specific binding of MYC to active promoters depends on PAF1c and, conversely, PAF1c is required for MYC-dependent pause release. Upon binding, PAF1c is rapidly transferred from MYC onto RNAPII and this transfer is driven by the HUWE1 ubiquitin ligase. Both MYC and HUWE1 globally control histone H2B ubiquitylation, which promotes transcriptional elongation and alters chromatin structure for double-strand break repair. Consistently, MYC suppresses the accumulation of double-strand breaks at promoters in response to topoisomerase II inhibition. While depletion of PAF1c has only minor effects on MYC-dependent gene expression, MYC induces rampant transcription-dependent DNA damage in PAF1c-depleted cells. We propose that the HUWE1-dependent transfer of PAF1c from MYC onto RNAPII is critical for absorbing the topological stress accompanied with deregulated and oncogenic transcription.

2021-02-15 | PXD023242 | Pride

A role for the Trim33 ubiquitin ligase in cellular response to replicative stress

Project description:Deregulation of RNA polymerase II (RNAPII) by oncoproteins, such as transcription factor Myc, interferes with DNA replication and is a major source of DNA damage and genomic instability. Ubiquitination is a key pathway controlling RNAPII activity via modification of RNAPII subunits or associated regulatory proteins. We uncover a mechanism for genome maintenance by ubiquitin ligase Trim33 and transcription factor E2f4. We show that Trim33 promotes E2f4 protein turnover, restricting interactions of E2f4 with chromatin and with the Recql DNA helicase. Replicative stress blunts Trim33-dependent regulation, which stimulates E2f4 and Recql recruitment to chromatin and facilitates recovery of DNA replication. Deletion of Trim33 triggers chronic recruitment of Recql to chromatin and accelerates DNA replication under stress, accompanied by compromised DDR signaling and DNA repair. Depletion of Trim33 in Myc-overexpressing cells leads to accumulation of replication-associated DNA damage and delays Myc-driven tumorigenesis. We propose that the Trim33-E2f4-Recql axis provides a mechanism to control DNA replication at transcriptionally active chromatin to maintain genome integrity.

2023-07-24 | PXD037854 | Pride

Expression data from Saccharomyces cerevisiae strains carrying the rna14-1 or the rna15-1 allele

Project description:In the yeast Saccharomyces cerevisiae, cleavage factor I (CFI) and cleavage and polyadenylation factor (CPF) build the core of the transcription termination machinery. CFI comprises the Rna14, Rna15, Pcf11, and Clp1 proteins, as well as the associated Hrp5 RNA-binding protein. We found that CFI participates in the DNA damage response and that rna14-1 shows synthetic growth defects with mutants of different repair pathways, including homologous recombination, non-homologous end joining, post replicative repair, mismatch repair, and nucleotide excision repair, implicating that impaired RNAPII termination and 3â-end processing decreases the cellular tolerance for DNA damage. Beyond replication progression defects, we found that bypass of the G1/S checkpoint in rna14-1 cells leads to synthetic sickness, accumulation of phosphorylated H2A, as well as increase in Rad52-foci and in recombination. Our data provide evidence that CFI dysfunction impairs RNAPII turnover, leading to replication hindrance and lower tolerance to exogenous DNA damage. These findings underscore the importance of coordination between transcription termination, DNA repair and replication in the maintenance of genomic stability. S. cerevisiae strains were grown in YPAD liquid culture at 30Â°C, total RNA was isolated and hybridized on Affymetrix microarrays.

2014-03-31 | E-GEOD-50947 | biostudies-arrayexpress

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