Chromatin remodelling enzymes, such as BRG1, alter local chromatin structure by modulating nucleosome positions. BRG1 has a well-established canonical role in transcription; however, emerging data indicates a non-canonical role in DNA replication through a yet undefined mechanism. We recently identified BRG1 as an essential gene for response to replication stress in a whole genome CRISPR screen. Replication stress is any process that impedes replication fork progression, moreover endogenous replication stress is the primary driver of genome instability in cancer. Therefore, we hypothesised that BRG1 plays a central role in genome stability by regulating DNA replication and replication stress.
Using ChIP-seq data we found a strong co-localization between BRG1 and factors involved in replication initiation, suggesting a role for BRG1 at replication origins. Therefore, we performed molecular combing assays to probe the replication dynamics with BRG1 depletion. We determined that BRG1 depletion reduces origin firing but does not affect the rate of replication. As we have previously shown BRG1 regulates the expression of genes involved in replication initiation, we repeated our molecular combing assays in cells positive for HPV E6E7 to determine if reduced origin firing was due to gene expression changes. In these cells the BRG1-Rb interaction is disrupted, and depletion of BRG1 fails to down regulate replication initiation genes. However, we found reduced origin firing occurred in the HPV E6E7 positive cells implying that the BRG1 has a role here independent of its role in transcription. We next investigated how BRG1 functions in the replication stress response. We found that after a replication stress challenged with either aphidicolin or hydroxyurea, there is reduced phosphorylation of CHK1, a marker of the replication stress response, with depletion of BRG1.
Together, our data indicates BRG1 has a role in DNA replication at replication origins, and regulates the chromatin response to replication stress.