Transcription-replication conflicts as a source of common fragile site instability caused by BMI1-RNF2 deficiency

Abstract

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.

ARID1A regulates R-loop associated DNA replication stress

“…Given the role of the BAF complex in regulating transcriptional programs, we hypothesized that transcription-replication conflicts (TRCs) might be responsible for some of the observed replication stress. We analyzed the proximity between PCNA and RNA polymerase II as a reporter of TRCs [22][23][24] using a proximity ligation assay and found that ARID1A loss significantly increased such conflicts (Fig 1C). These signals were transcription dependent since pretreatment with flavopiridol abolished differences between the WT and ARID1A-knockout cells (Fig 1C).…”

“…PLA experiments were performed using the Duolink PLA kit (Millipore Sigma, DUO92101-1KT) and have been used previously to identify TRCs [22][23][24]. For all PLA experiments, cells were grown on coverslips overnight before fixing.…”

Transcription-Replication Collisions—A Series of Unfortunate Events

“…Although ERFSs have different genetic and epigenetic characteristics from CFSs, they may share similar properties conferring fragility. Transcription has been implicated in DNA breakage at both ERFSs and CFSs, potentially explaining the tissue dependence of fragile site breakage [95,100,101]. CFS damage is hypothesized to arise from under-replicated DNA persisting into mitosis, forming DNA bridges in anaphase [102][103][104][105].…”

Making Connections: Integrative Signaling Mechanisms Coordinate DNA Break Repair in Chromatin

“…The formation of stable R-loops in the genome can give rise to a unique situation where transcription and replication complexes are competing for occupancy of the same DNA template. A growing body of evidence supports a model where a significant source of R-Loop associated DNA damage results from transcription-replication conflicts (TRC), which ultimately can also lead to DSBs ( Helmrich et al, 2011 ; Garcia-Muse and Aguilera, 2019 ; Puget et al, 2019 ; Sanchez et al, 2020 ). The use of novel reporter systems has demonstrated that in both bacterial and human systems, TRCs are most detrimental to cells when they occur in a “head-on” orientation, meaning that the replication and transcription complexes are moving toward each other on the DNA ( Hamperl et al, 2017 ; Lang et al, 2017 ).…”

“…We note that PRC1 has been shown to be positioned on chromatin in proximity to areas of active replication, which raises the possibility of additional interactions between PRC1 and the replisome or aberrant nucleic acid structures (e.g., R-loops), which warrants further investigations. While this localization could be attributed to known PRC1 interactions, recent reports have identified PRC1 as essential for the progression of the replication fork, processing of R-Loop structures, and the integrity of common fragile sites which may indicate a more direct role in these processes (Klusmann et al, 2018;Sanchez et al, 2020). In addition to the PRC1 complex, several other pathways that regulate H2AK119ub at break sites have been identified including PRC2, PBAF, ENL, and FRRUC complexes, for which we refer readers to recent in-depth reviews that have covered the extensive involvement of multiple complexes in repressing transcription at DNA breaks, including through the regulation of H2A ubiquitination (Caron et al, 2019;Puget et al, 2019;Tan and Huen, 2020).…”