SUMMARY Appropriate DNA double-strand break (DSB) repair factor choice is essential for ensuring accurate repair outcome and genomic integrity. The factors that regulate this process remain poorly understood. Here, we identify two repressive chromatin components, the macrohistone variant macroH2A1 and the H3K9 methyltransferase and tumor suppressor PRDM2, which together direct the choice between the antagonistic DSB repair mediators BRCA1 and 53BP1. The macroH2A1/PRDM2 module mediates an unexpected shift from accessible to condensed chromatin that requires the ataxia telangiectasia mutated (ATM)-dependent accumulation of both proteins at DSBs in order to promote DSB-flanking H3K9 dimethylation. Remarkably, loss of macroH2A1 or PRDM2, as well as experimentally induced chromatin decondensation, impairs the retention of BRCA1, but not 53BP1, at DSBs. As a result, mac-roH2A1 and/or PRDM2 depletion causes epistatic defects in DSB end resection, homology-directed repair, and the resistance to poly(ADP-ribose) polymerase (PARP) inhibition—all hallmarks of BRCA1-deficient tumors. Together, these findings identify dynamic, DSB-associated chromatin reorganization as a critical modulator of BRCA1-dependent genome maintenance.

Genetic or pharmacological alteration of the activity of the histone deacetylase 6 (HDAC6) induces a parallel alteration in cell migration. Using tubacin to block deacetylation of α-tubulin, and not other HDAC6 substrates, yielded a motility reduction equivalent to agents that block all NAD-independent HDACs. Accordingly, we investigated how the failure to deacetylate tubulin contributes to decreased motility in HDAC6-inhibited cells. Testing the hypothesis that motility is reduced because cellular adhesion is altered, we found that inhibiting HDAC6 activity towards tubulin rapidly increased total adhesion area. Next, we investigated the mechanism of the adhesion area increase. Formation of adhesions proceeded normally and cell spreading was more rapid in the absence of active HDAC6; however, photobleaching assays and adhesion breakdown showed that adhesion turnover was slower. To test the role of hyperacetylated tubulin in altering adhesion turnover, we measured microtubule dynamics in HDAC6-inhibited cells because dynamic microtubules are required to target adhesions for turnover. HDAC6 inhibition yielded a decrease in microtubule dynamics that was sufficient to decrease focal adhesion turnover. Thus, our results suggest a scenario in which the decreased dynamics of hyperacetylated microtubules in HDAC6-inhibited cells compromises their capacity to mediate the focal adhesion dynamics required for rapid cell migration.

Recent integrative epigenome analyses highlight the importance of functionally distinct chromatin states for accurate cell function. How these states are established and maintained is a matter of intense investigation. Here, we present evidence for DNA damage as an unexpected means to shape a protective chromatin environment at regions of recurrent replication stress (RS). Upon aberrant fork stalling, DNA damage signaling and concomitant H2AX phosphorylation coordinate the FACT-dependent deposition of macroH2A1.2, a histone variant that promotes DNA repair by homologous recombination (HR). MacroH2A1.2, in turn, facilitates the accumulation of the tumor suppressor and HR effector BRCA1 at replication forks to protect from RS-induced DNA damage. Consequently, replicating primary cells steadily accrue macroH2A1.2 at fragile regions, whereas macroH2A1.2 loss in these cells triggers DNA damage signaling-dependent senescence, a hallmark of RS. Altogether, our findings demonstrate that recurrent DNA damage contributes to the chromatin landscape to ensure the epigenomic integrity of dividing cells.

Tumor growth relies on efficient DNA repair to mitigate the detrimental impact of DNA damage associated with excessive cell division. Modulating repair factor function, thus, provides a promising strategy to manipulate malignant growth. Here, we identify the ubiquitin-specific protease USP21 as a positive regulator of BRCA2, a key mediator of DNA repair by homologous recombination. USP21 interacts with, deubiquitinates and stabilizes BRCA2 to promote efficient RAD51 loading at DNA double-strand breaks. As a result, depletion of USP21 decreases homologous recombination efficiency, causes an increase in DNA damage load and impairs tumor cell survival. Importantly, BRCA2 overexpression partially restores the USP21-associated survival defect. Moreover, we show that USP21 is overexpressed in hepatocellular carcinoma, where it promotes BRCA2 stability and inversely correlates with patient survival. Together, our findings identify deubiquitination as a means to regulate BRCA2 function and point to USP21 as a potential therapeutic target in BRCA2-proficient tumors.