Stabilization of chromatin topology safeguards genome integrity

Abstract

To safeguard genome integrity in response to DNA double-strand breaks (DSB), mammalian cells mobilize the neighboring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes 1. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin-CST-Polα complex 2. How this pathway reflects and impacts on the three-dimensional (3D) nuclear architecture is not known. Here, we applied super-resolution microscopy to show that 53BP1 and RIF1 form an autonomous functional module that stabilizes 3D chromatin topology at sites of DNA breakage. This is initiated by 53BP1 accrual at compact chromatin regions colocalizing with topology-associated domain (TAD) sequences and followed by RIF1 recruitment to boundaries between such domains. The alternating 53BP1 and RIF1 distribution stabilizes several neighboring TAD-sized structures at a single DBS site to an ordered, circular arrangement. Depletion of 53BP1 or RIF1 (but not shieldin) disrupts this arrangement, leading to decompaction of DSBflanking chromatin, reduction of interchromatin space, aberrant spreading of DNA repair proteins, and DNA-end hyper-resection. Similar topological distortions are triggered by depletion of cohesin, suggesting that maintenance of chromatin structure after DNA breakage involves basic mechanisms that shape 3D nuclear organization. Since topological stabilization of DSB-flanking chromatin is independent of DNA repair, we propose that besides providing a structural scaffold to protect DNA ends against aberrant processing, 53BP1 and RIF1 safeguard epigenetic integrity at loci disrupted by DNA breakage. quantitative nanoscopy texture (QUANTEX) analysis tool, which indeed revealed a significant increase in 53BP1-MD Mean breadth and Principal axis length (Fig. 1e, f; Extended Data Fig. 3a-c). It was further reproduced by silencing RIF1 with multiple siRNAs, by replacing endogenous 53BP1 with a mutant unable to promote RIF1 recruitment 10 , and in several cancer-derived as well as non-cancerous cells (Extended Data Fig. 4a-e). Together, these data indicate that 53BP1 and RIF1 form an autonomous module where RIF1 is required to stabilize 53BP1-NDs into ordered, circular chromatin architecture (Extended Data Fig. 4f). In support of this, knockdown of 53BP1 or RIF1 phenocopied each other by disrupting γH2AX-marked chromatin into disordered and elongated shapes (Extended Data Fig. 4g-i). To study how 53BP1 and RIF1 cooperate to stabilize chromatin topology, we set out to determine RIF localization with respect to 53BP1. While conventional microscopy only generally indicates 53BP1 and RIF1 proximity at DSB-sites, 3D-SIM and STED revealed that RIF1 localized to the chromatin boundaries between neighboring 53BP1-NDs (Fig. 2a). To understand the purpose of this alternating localization, we tracked 53BP1 dynamics from pre-to post-damaged state using live-cell 3D-SIM (live-SIM; Extended Data Fig. 5a). The fir...

Topologically Dependent Abundance of Spontaneous DNA Damage in Single Human Cells

“…Recently, Ochs et al has shown that genome topology plays important roles in safeguarding the genome integrity in response to the double strand breaks (33). In this report, we showed that the genome topology also influences the degree of spontaneous DNA damage.…”

Loop extrusion as a mechanism for DNA Double-Strand Breaks repair foci formation

“…Divergent one-sided loop extrusion hence takes place at the DSB, which in turn allows the locally-recruited ATM to phosphorylate H2AX containing nucleosomes as the chromatin fiber is pulled by the cohesin ring. Given that current estimates of cohesin-mediated loop extrusion suggest a rate of 0.5-2 kb per second in vitro 24,25 , such a mechanism would allow a rapid way to assemble DDR foci, with the entire megabase-sized chromatin domain being modified in about 10-30 min, which 9 fits with the observed rate of H2AX foci assembly 9 . This model would be in agreement with the recent finding that in yeast, the ATM ortholog Tel1 mediates H2A phosphorylation in a manner that agrees with a 1D sliding model rather than a 3D diffusion model 35 .…”

PALB2 chromatin recruitment restores homologous recombination in BRCA1-deficient cells depleted of 53BP1

“…As 53BP1 works together with the rest of its downstream cofactors to block over-resection of DNA DSBs, loss of any of these proteins partially restores RAD51 filament formation, functional HR and PARP-inhibitor resistance in BRCA1-deficient cells. Notably, whereas recruitment of RIF1, REV7 and SHLD1/2/3 depends on intact 53BP1, losing the downstream proteins does not prevent 53BP1 from forming IRIF [44][45][46][47] . Thus, in the absence of its co-factors, 53BP1 is still bound to DSB-proximal chromatin and therefore might interfere with efficient PALB2 recruitment, especially in BRCA1-deficient settings.…”

“…Thus, in the absence of its co-factors, 53BP1 is still bound to DSB-proximal chromatin and therefore might interfere with efficient PALB2 recruitment, especially in BRCA1-deficient settings. Importantly, according to a recent study that employed super-resolution microscopy to characterise chromatin conformations orchestrated by 53BP1 and its co-factors, 53BP1 accumulates near DSB sites and, together with RIF1, forms topologically associating domains (TADs) of compacted chromatin 47 . Whereas the integrity of these TADs is fully dependent on 53BP1 and is partially compromised by RIF1 depletion, loss of Shieldin components does not affect 53BP1/ RIF1-mediated chromatin compaction 47 .…”

“…Importantly, according to a recent study that employed super-resolution microscopy to characterise chromatin conformations orchestrated by 53BP1 and its co-factors, 53BP1 accumulates near DSB sites and, together with RIF1, forms topologically associating domains (TADs) of compacted chromatin 47 . Whereas the integrity of these TADs is fully dependent on 53BP1 and is partially compromised by RIF1 depletion, loss of Shieldin components does not affect 53BP1/ RIF1-mediated chromatin compaction 47 . In line with these observations and our model that loss of 53BP1 in BRCA1deficient cells creates permissive conditions for PALB2 accumulation on chromatin, we see that only co-depletion of 53BP1 fully rescues Venus-PALB2 and RAD51 foci in BRCA1-depleted RPE1 cells, while RIF1 depletion has a weaker effect and the loss of REV7 or Shieldin components does not allow detectable recruitment of PALB2 to DSB-proximal chromatin (Supplementary Fig.…”