Timely stalling and resumption of RNA polymerases at damaged chromatin are actively regulated processes. Prior work showed an importance of FACT histone chaperone in such process. Here we provide a new role of OTUD5 deubiquitinase in the FACT-dependent process. Through a DUB RNAi screen, we found OTUD5 as a specific stabilizer of the UBR5 E3 ligase. OTUD5 localizes to DNA double strand breaks (DSBs), interacts with UBR5 and represses the RNA Pol II elongation and RNA synthesis. OTUD5 co-localizes and interacts with the FACT component SPT16 and antagonizes the histone H2A deposition at DSB lesions. OTUD5 interacts with UBR5 and SPT16 independently through two distinct regions, and both interactions are necessary for arresting the Pol II elongation at lesions. These analyses suggested that the catalytic (through UBR5 stabilization) as well as scaffolding (through FACT binding) activities of OTUD5 are involved in the FACT-dependent transcription. We found that a cancer-associated missense mutation within the OTUD5 Ubiquitin Interacting Motif (UIM) abrogates the FACT association and the Pol II arrest, providing a possible link between the transcriptional regulation and tumor suppression. Our work establishes OTUD5 as a new regulator of the DNA damage response, and provides an insight into the FACT-dependent transcription at damaged chromatin.

BMI1 is a component of the Polycomb Repressive Complex 1 (PRC1), which plays a key role in maintaining epigenetic silencing during development. BMI1 also participates in gene silencing during DNA damage response, but the precise downstream function of BMI1 in gene silencing is unclear. Here we identified the UBR5 E3 ligase as a downstream factor of BMI1. We found that UBR5 forms damage-inducible nuclear foci in a manner dependent on the PRC1 components BMI1, RNF1 (RING1a), and RNF2 (RING1b). Whereas transcription is repressed at UV-induced lesions on chromatin, depletion of the PRC1 members or UBR5 alone derepressed transcription elongation at these sites, suggesting that UBR5 functions in a linear pathway with PRC1 in inducing gene silencing at lesions. Mass spectrometry (MS) analysis revealed that UBR5 associates with BMI1 as well as FACT components SPT16 and SSRP1. We found that UBR5 localizes to the UV-induced lesions along with SPT16. We show that UBR5 ubiquitinates SPT16, and depletion of UBR5 or BMI1 leads to an enlargement of SPT16 foci size at UV lesions, suggesting that UBR5 and BMI1 repress SPT16 enrichment at the damaged sites. Consistently, depletion of the FACT components effectively reversed the transcriptional derepression incurred in the UBR5 and BMI1 KO cells. Finally, UBR5 and BMI1 KO cells are hypersensitive to UV, which supports the notion that faulty RNA synthesis at damaged sites is harmful to the cell fitness. Altogether, these results suggest that BMI1 and UBR5 repress the polymerase II (Pol II)-mediated transcription at damaged sites, by negatively regulating the FACT-dependent Pol II elongation.erturbation of chromatin structures can cause inappropriate gene expression and loss of genome integrity. Polycomb proteins are recognized in all metazoans for their conserved transcriptional repressive function. The canonical Polycomb Repressive Complex 1 (PRC1) contains BMI1, RNF1 (RING1a), RNF2 (RING1b), core components (PC), Polyhomeotic (PH), and CBX proteins (1, 2). BMI1 serves as a key regulatory component of the PRC1 complex, which is required to maintain the transcriptionally repressed state of many genes throughout development via chromatin remodeling and histone modification (1). The only known enzymatic activity of the BMI1-containing PRC1 complex is to monoubiquitinate histone H2A at Lys-119 (K119) residue, which is associated with transcriptional repression (1, 3, 4). However, the E3 ubiquitin ligase activity of RNF2 or the H2AK119-Ub is dispensable for repression of canonical PRC1 target genes during mouse or Drosophila embryonic development, respectively (5, 6), suggesting that the PRC1 complex may also induce gene silencing through other mechanisms (7). A series of studies has suggested that multiple distinct forms of the PRC1 complex with varying components could exist, and each of these may have distinct modes of regulation and functions (reviewed in ref. 2).In addition to its well-known role as an oncogene, recent evidence suggests that BMI1 participates in the DNA damage res...

Common fragile sites (CFSs) are breakage-prone genomic loci, and are considered to be hotspots for genomic rearrangements frequently observed in cancers. Understanding the underlying mechanisms for CFS instability will lead to better insight on cancer etiology. Here we show that Polycomb group proteins BMI1 and RNF2 are suppressors of transcriptionreplication conflicts (TRCs) and CFS instability. Cells depleted of BMI1 or RNF2 showed slower replication forks and elevated fork stalling. These phenotypes are associated with increase occupancy of RNA Pol II (RNAPII) at CFSs, suggesting that the BMI1-RNF2 complex regulate RNAPII elongation at these fragile regions. Using proximity ligase assays, we showed that depleting BMI1 or RNF2 causes increased associations between RNAPII with EdU-labeled nascent forks and replisomes, suggesting increased TRC incidences. Increased occupancy of a fork protective factor FANCD2 and R-loop resolvase RNH1 at CFSs are observed in RNF2 CRISPR-KO cells, which are consistent with increased transcription-associated replication stress in RNF2-deficient cells. Depleting FANCD2 or FANCI proteins further increased genomic instability and cell death of the RNF2-deficient cells, suggesting that in the absence of RNF2, cells depend on these fork-protective factors for survival. These data suggest that the Polycomb proteins have non-canonical roles in suppressing TRC and preserving genomic integrity.

Common fragile sites (CFSs) are breakage-prone genomic loci, and are considered to be hotspots for genomic rearrangements frequently observed in cancers. Understanding the underlying mechanisms for CFS instability will lead to better insight on cancer etiology. Here we show that Polycomb group proteins BMI1 and RNF2 are suppressors of transcription-replication conflicts (TRCs) and CFS instability. Cells depleted of BMI1 or RNF2 showed slower replication forks and elevated fork stalling. These phenotypes are associated with increase occupancy of RNA Pol II (RNAPII) at CFSs, suggesting that the BMI1-RNF2 complex regulate RNAPII elongation at these fragile regions. Using proximity ligase assays, we showed that depleting BMI1 or RNF2 causes increased associations between RNAPII with EdU-labeled nascent forks and replisomes, suggesting increased TRC incidences. Increased occupancy of a fork protective factor FANCD2 and R-loop resolvase RNH1 at CFSs are observed in RNF2 CRISPR-KO cells, which are consistent with increased transcription-associated replication stress in RNF2-deficient cells. Depleting FANCD2 or FANCI proteins further increased genomic instability and cell death of the RNF2-deficient cells, suggesting that in the absence of RNF2, cells depend on these fork-protective factors for survival. These data suggest that the Polycomb proteins have non-canonical roles in suppressing TRC and preserving genomic integrity.Author summaryIncreasing evidence suggest that instabilities at common fragile sites (CFSs), breakage-prone genomic loci, may be source of genomic aberration seen in cancer cells. Among the proposed mechanisms that can cause CFSs instabilities is the conflict between transcription and replication, and the mechanisms or factors that resolve the possible conflicts are only beginning to be understood. Here we found that deficiency in the Polycomb group proteins BMI1 or RNF2 leads to the CFS instability, and is associated with transcription-associated replication fork stresses. We further found that in the absence of RNF2, cells depend on the Fanconi Anemia fork-protective proteins for genome maintenance and survival. These results underscore that the Polycomb proteins are important for genome maintenance.