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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE149760 | GEO | 2021/04/20

REPOSITORIES: GEO

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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 anchors CRL4CSA to RNA pol II to activate transcription-coupled repair

Project description:Transcription-coupled DNA repair (TCR) removes transcription-blocking DNA lesions through the coordinated assembly of repair factors on arrested RNA polymerase II (Pol II). This process requires the site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase. Pol II ubiquitylation is facilitated by ELOF1, a recently identified TCR factor. Using cryogenic electron microscopy, biochemical assays, and cell biology, we reveal that ELOF1 functions as an adaptor to correctly dock CRL4CSA onto Pol II and facilitate the recruitment of UVSSA. The ELOF1-CSA-UVSSA interaction positions CRL4CSA on arrested Pol II, leading to its ubiquitylation upon ligase activation by neddylation. ELOF1-mediated repositioning enables a TFIIS-like element in UVSSA to reach the Pol II active site and the pore region to prevent Pol II re-activation. These findings provide the structural and molecular basis underlying the transition of Pol II from a transcriptionally active to an arrested state, primed for DNA repair.

2023-11-24 | PXD042388 | Pride

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

Project description:The coordinated transcription of genes involves RNA polymerase II enzymes (RNAPII), which pull DNA through their active sites. DNA lesions in transcribed strands block RNAPII elongation and induce a strong transcriptional arrest. The transcription-coupled repair (TCR) pathway ensures the efficient removal of transcription-blocking DNA lesions, but this is not sufficient to overcome this arrest and resume transcription. Through proteomics screens, we find that the TCR-specific CSB protein loads the evolutionary conserved PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions specifically in response to DNA damage. PAF1C is dispensable for TCR-mediated repair,but is essential to resume RNA synthesis after UV irradiation, suggesting an unexpected uncoupling between DNA repair and transcription restart. Moreover, we find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation waves throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.

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

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 | GSE143529 | GEO

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

2020-01-14 | GSE143518 | GEO

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

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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-PAF1C axis restores processive transcription elongation after DNA damage repair

Project description:The coordinated transcription of genes involves the regulated release of RNA polymerase II (RNAPII) from promoter-proximal sites into active elongation. DNA lesions in transcribed strands block elongation and induce a strong transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but this is not sufficient to resume transcription. Through proteomics screens, we find that the TCR-specific CSB protein loads the evolutionary conserved PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions in response to DNA damage. PAF1C is dispensable for TCR-mediated repair, but is essential for recovery of RNA synthesis after UV irradiation, suggesting an uncoupling between DNA repair and transcription recovery. Moreover, we find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.

2021-01-15 | GSE140930 | GEO

Interactome analysis of elongating RNAPII after induction of DNA damage by IP-MS/MS

Project description:Endogenous aldehydes induce inter-strand crosslinks (ICL) and DNA-protein crosslinks (DPC). While DNA-repair and aldehyde-clearance systems cope with cellular toxicity, deficiencies in these mechanisms cause genome-instability disorders. The FA-pathway, defective in Fanconi anemia (FA), specifically removes ICL. In contrast, SPRTN, compromised in Ruijs-Aalfs syndrome, eliminates DPC during replication. However, AMeDS patients lacking aldehyde-detoxification display combined features of FA and Cockayne syndrome, associated with transcription-coupled repair (TCR) deficiency, suggesting a novel repair mechanism for aldehyde-induced DNA lesions in active genes. In this report, we demonstrate efficient resolution of aldehyde-induced transcription-blocking lesions by TCR. Mass-spectrometry and DPC-seq identify the TCR complex and additional factors involved in DPC removal and formaldehyde-induced damage tolerance. Notably, TFIIS-dependent cleavage of stalled-RNAPII transcripts exclusively protects against formaldehyde-induced damage. A mouse-model lacking both aldehyde-clearance and TCR pathways confirms endogenous DPC accumulation in transcribed regions. These findings highlight the importance of DPC removal in preventing transcription-roadblocks and contribute to understanding disorders related to aldehyde clearance and TCR deficiencies.

2024-02-12 | PXD044310 | Pride

Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6

Project description:Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The mono-ubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae and K120 in humans) occurs co-transcriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its Histone Modification Domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that specifically target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro crosslinking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic and biochemical experiments, we identified separation-of-function mutations in RAD6 that greatly impair H2BK123 ubiquitylation but not other Rad6 functions. Finally, by employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.

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