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The DNA damage response is essential to safeguard genome integrity. Although the contribution of chromatin in DNA repair has been investigated1,2, the contribution of chromosome folding to these processes remains unclear3. Here we report that, after the production of double-stranded breaks (DSBs) in mammalian cells, ATM drives the formation of a new chromatin compartment (D compartment) through the clustering of damaged topologically associating domains, decorated with γH2AX and 53BP1. This compartment forms by a mechanism that is consistent with polymer-polymer phase separation rather than liquid-liquid phase separation. The D compartment arises mostly in G1 phase, is independent of cohesin and is enhanced after pharmacological inhibition of DNA-dependent protein kinase (DNA-PK) or R-loop accumulation. Importantly, R-loop-enriched DNA-damage-responsive genes physically localize to the D compartment, and this contributes to their optimal activation, providing a function for DSB clustering in the DNA damage response. However, DSB-induced chromosome reorganization comes at the expense of an increased rate of translocations, also observed in cancer genomes. Overall, we characterize how DSB-induced compartmentalization orchestrates the DNA damage response and highlight the critical impact of chromosome architecture in genomic instability.

The repair of DNA double-strand breaks (DSBs) is essential for safeguarding genome integrity. When a DSB forms, the PI3K-related ATM kinase rapidly triggers the establishment of megabase-sized, chromatin domains decorated with phosphorylated histone H2AX ([gamma]H2AX), which act as seeds for the formation of DNA-damage response foci.sup.1. It is unclear how these foci are rapidly assembled to establish a 'repair-prone' environment within the nucleus. Topologically associating domains are a key feature of 3D genome organization that compartmentalize transcription and replication, but little is known about their contribution to DNA repair processes.sup.2,3. Here we show that topologically associating domains are functional units of the DNA damage response, and are instrumental for the correct establishment of [gamma]H2AX-53BP1 chromatin domains in a manner that involves one-sided cohesin-mediated loop extrusion on both sides of the DSB. We propose a model in which H2AX-containing nucleosomes are rapidly phosphorylated as they actively pass by DSB-anchored cohesin. Our work highlights the importance of chromosome conformation in the maintenance of genome integrity and demonstrates the establishment of a chromatin modification by loop extrusion. During the repair of double-stranded DNA breaks, cohesin mediates the extrusion of loops of DNA along which phosphorylated H2AX spreads to establish a repair zone., Author(s): Coline Arnould [sup.1] , Vincent Rocher [sup.1] , Anne-Laure Finoux [sup.1] , Thomas Clouaire [sup.1] , Kevin Li [sup.2] , Felix Zhou [sup.2] , Pierre Caron [sup.1] , Philippe. [...]

CpG islands (CGIs) are prominent in the mammalian genome owing to their GC-rich base composition and high density of CpG dinucleotides (1,2). Most human gene promoters are embedded within CGIs that lack DNA methylation and coincide with sites of histone H3 lysine 4 trimethylation (H3K4me3), irrespective of transcriptional activity (3,4). In spite of these intriguing correlations, the functional significance of non-methylated CGI sequences with respect to chromatin structure and transcription is unknown. By performing a search for proteins that are common to all CGIs, here we show high enrichment for Cfp1, which selectivelybinds to non-methylated CpGs in vitro (5,6). Chromatin immunoprecipitation of a mono-allelically methylated CGI confirmed that Cfp1 specifically associates with non-methylated CpG sites in vivo. High throughput sequencing of Cfp1-bound chromatin identified a notable concordance with non-methylated CGIs and sites of H3K4me3 in the mouse brain. Levels of H3K4me3 at CGIs were markedly reduced in Cfp1-depleted cells, consistent with the finding that Cfp1 associates with the H3K4 methyltransferase Setd1 (refs 7, 8). To test whether non-methylated CpG-dense sequences are sufficient to establish domains of H3K4me3, we analysed artificial CpG clusters that were integrated into the mouse genome. Despite the absence of promoters, the insertions recruited Cfp1 and created new peaks of H3K4me3. The data indicate that a primary function of non-methylated CGIs is to genetically influence the local chromatin modification state by interaction with Cfp1 and perhaps other CpG-binding proteins., To characterize the chromatin modifications typical of CGIs, we used the methyl-CpG-sensitive restriction endonuclease HinPI (cleavage site GCGC) to release small chromatin fragments from purified brain nuclei, as described previously [...]

We have identified a novel evolutionarily conserved protein motif--designated the THAP domain--that defines a new family of cellular factors. We have found that the THAP domain presents striking similarities with the site-specific DNA-binding domain (DBD) of Drosophila P element transposase, including a similar size, N-terminal location, and conservation of the residues that define the THAP motif, such as the C2CH signature (Cys-[Xaa.sub.2-4]-Cys-[Xaa.sub.35-50]-Cys-[Xaa.sub.2]-His). Our results suggest that the THAP domain is a novel example of a DBD that is shared between cellular proteins and transposases from mobile genomic parasites.

Upon DNA damage, histone modifications are dynamically reshaped to accommodate DNA damage signaling and repair within chromatin. In this study, we report the identification of the histone demethylase KDM5A as a key regulator of the bromodomain protein ZMY ND8 and NuRD (nucleosome remodeling and histone deacetylation) complex in the DNA damage response. We observe KDM5A-dependent H3K4me3 demethylation within chromatin near DNA double-strand break (DSB) sites. Mechanistically, demethylation of H3K4me3 is required for ZMY ND8-NuRD binding to chromatin and recruitment to DNA damage. Functionally, KDM5A deficiency results in impaired transcriptional silencing and repair of DSBs by homologous recombination. Thus, this study identifies a crucial function for KDM5A in demethylating H3K4 to allow ZMY ND8-NuRD to operate within damaged chromatin to repair DSBs. [ABSTRACT FROM AUTHOR]

How chromatin shapes pathways that promote genome-epigenome integrity in response to DNA damage is an issue of crucial importance. We report that human bromodomain (BRD)-containing proteins, the primary "readers" of acetylated chromatin, are vital for the DNA damage response (DDR). We discovered that more than one-third of all human BRD proteins change localization in response to DNA damage. We identified ZMYND8 (zinc finger and MYND [myeloid, Nervy, and DEAF-1] domain containing 8) as a novel DDR factor that recruits the nucleosome remodeling and histone deacetylation (NuRD) complex to damaged chromatin. Our data define a transcription-associated DDR pathway mediated by ZMYND8 and the NuRD complex that targets DNA damage, including when it occurs within transcriptionally active chromatin, to repress transcription and promote repair by homologous recombination. Thus, our data identify human BRD proteins as key chromatin modulators of the DDR and provide novel insights into how DNA damage within actively transcribed regions requires chromatin-binding proteins to orchestrate the appropriate response in concordance with the damage-associated chromatin context. [ABSTRACT FROM AUTHOR]