Replication Stress Shapes a Protective Chromatin Environment across Fragile Genomic Regions.

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.

Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks.

DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to mutagenesis and genomic loss. DSB-repair pathway cross-talk and compensation is incompletely understood, despite its importance for genomic stability, oncogenesis, and genome editing using CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple DNA repair pathways at a DSB. DSB-Spectrum reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibiting the c-NHEJ factor DNA-PKcs increases repair by HR, but also substantially increases mutagenic SSA. Our data indicate that SSA-mediated DSB-repair also occurs at endogenous genomic loci, driven by Alu elements or homologous gene regions. Finally, we demonstrate that long-range end-resection factors DNA2 and Exo1 promote SSA and reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.

Ubiquitin-specific protease 21 stabilizes BRCA2 to control DNA repair and tumor growth.

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.BRCA2 is essential for the repair of DNA damage; therefore, defects in BRCA2 are associated with tumorigenesis but also with increased susceptibility to genotoxic stress. Here the authors show that USP21 regulates the ability of tumor cells to repair damaged DNA by regulating BRCA2 stability.