ATM pathway activation limits R-loop-associated genomic instability in Werner syndrome cells

Abstract

Werner syndrome (WS) is a cancer-prone disease caused by deficiency of Werner protein (WRN). WRN maintains genome integrity by promoting replication-fork stability after various forms of replication stress. Under mild replication stress, WS cells show impaired ATR-mediated CHK1 activation. However, it remains unclear if WS cells elicit other repair pathway. We demonstrate that loss of WRN leads to enhanced ATM phosphorylation upon prolonged exposure to aphidicolin, a specific inhibitor of DNA polymerases, resulting in CHK1 activation. Moreover, we find that loss of WRN sensitises cells to replication-transcription collisions and promotes accumulation of R-loops, which undergo XPG-dependent cleavage responsible for ATM signalling activation. Importantly, we observe that ATM pathway limits chromosomal instability in WS cells. Finally, we prove that, in WS cells, genomic instability enhanced upon chemical inhibition of ATM kinase activity is counteracted by direct or indirect suppression of R-loop formation or by XPG abrogation. Together, these findings suggest a potential role of WRN as regulator of R-loop-associated genomic instability, strengthening the notion that conflicts between replication and transcription can affect DNA replication, leading to human disease and cancer.

Generation of inducible SMARCAL1 knock-down iPSC to model severe Schimke immune-osseous dysplasia reveals a link between replication stress and altered expression of master differentiation genes

“…The recent observation of a non-canonical ATM activation that is dependent on R-loop accumulation and alternative processing 35 , and increased upon defective replication or mild replication stress 19 , is consistent with our data. Notably, although Smarcal1 KO MEFs do not have any significant proliferation defect compared with wild-type cells, they show a slow-growth phenotype if treated with α -amanitin 9 .…”

“…immunofluorescence. DRB is a transcription inhibitor and is widely used to prevent replicationtranscription conflicts without affecting, in the short-term, proliferation 18 , thus any reduction of γ -H2AX levels in cells treated with DRB is likely correlated with faulty resolution of accumulation of these events as demonstrated also un our group 19 . Interestingly, while DRB treatment did not affect significantly the presence of Increased number of replication-transcription conflicts can be associated to enhanced accumulation of R-loops 20,21 .…”

Checkpoint Defects Elicit a WRNIP1-Mediated Response to Counteract R-Loop-Associated Genomic Instability

“…Consistently with a role of WRNIP1 in activating an ATM signaling, combined loss of WRNIP1 and ATM did not have any additive effect on CHK1 phosphorylation ( Figure 2D). Absence of WRN differently affects CHK1 activation upon MRS: it reduces the ATR-dependent CHK1 activation early after Aph treatment but stimulates that dependent on ATM at late time-points [18]. The two phenotypes are interlinked and expression of a phospho-mimic form of CHK1, CHK1 317D/345D [33], prevents the late phenotype [18].…”

Checkpoint defects require WRNIP1 to counteract R-loop-associated genomic instability

“…Also, we find that chromatin-bound WRNIP1 is related to the late ATM-dependent CHK1 phosphorylation. Supporting this, WRNIP1 recruitment is counteracted by overexpression of a constitutively active CHK1 that, compensating for defective ATR pathway, abolishes the need to activate ATM as well as in WRN helicase dead cells that efficiently phosphorylate CHK1 (Basile et al, 2014;Marabitti et al, 2019). Interestingly, degradation of Rloops weakens the association of WRNIP1 with chromatin.…”