SummaryDouble-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.

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H2A.Z histone variant is an important regulator of gene transcription, which is enriched at regulatory regions but is also found within gene bodies. Recent evidence suggests that active recruitment of H2A.Z within gene bodies is required to induce gene repression. In contrast to this view, we show that global inhibition of transcription results in H2A.Z accumulation at gene transcription start sites, as well as within gene bodies. Our results indicate that accumulation of H2A.Z within repressed genes can also be a consequence of the repression of gene transcription rather than an active mechanism required to establish the repression.

Regulation of the chromatin state is crucial for biological processes such as the regulation of transcription, DNA replication, and DNA damage repair. Here we show that knockdown of the BRD8 bromodomain protein - a subunit of the p400/Tip60 complex - leads to p21 induction, and concomitant cell cycle arrest in G1/S. We further demonstrate that the p53 transcriptional pathway is activated in BRD8-depleted cells, and this accounts for upregulation of not only p21 but also of pro-apoptotic genes, leading to subsequent apoptosis. Importantly, the DNA damage response (DDR) is induced upon BRD8 depletion, and DNA damage foci are detectable in BRD8-depleted cells under normal growth conditions. Consistently with an activated DDR, we find that in BRD8-depleted cells, the ATM-CHK2 DDR pathway is turned on but, CHK1 proteins levels are severely reduced and replication stress is detectable as enhanced replication protein A (RPA32) phosphorylation levels. Notably, acetylation of histone H4 at K16 (H4K16ac) is reduced in BRD8-depleted cells, suggesting that BRD8 may have a role in the recruitment and/or stabilization of the p400/Tip60 complex within chromatin, thereby facilitating DNA repair. Taken together, our results suggest that BRD8 is involved not only in p53-dependent gene suppression, but also in the maintenance of genome stability.

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