Replication-Coupled Dilution of H4K20me2 Guides 53BP1 to Pre-replicative Chromatin

Abstract

SummaryThe bivalent histone modification reader 53BP1 accumulates around DNA double-strand breaks (DSBs), where it dictates repair pathway choice decisions by limiting DNA end resection. How this function is regulated locally and across the cell cycle to channel repair reactions toward non-homologous end joining (NHEJ) in G1 and promote homology-directed repair (HDR) in S/G2 is insufficiently understood. Here, we show that the ability of 53BP1 to accumulate around DSBs declines as cells progress through S phase and reveal that the inverse relationship between 53BP1 recruitment and replicated chromatin is linked to the replication-coupled dilution of 53BP1’s target mark H4K20me2. Consistently, premature maturation of post-replicative chromatin restores H4K20me2 and rescues 53BP1 accumulation on replicated chromatin. The H4K20me2-mediated chromatin association of 53BP1 thus represents an inbuilt mechanism to distinguish DSBs in pre- versus post-replicative chromatin, allowing for localized repair pathway choice decisions based on the availability of replication-generated template strands for HDR.

Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures

“…53BP1 is known to be recruited to damaged chromatin by engaging multiple interactions with H2AK15ub and H4K20me2 but also with γH2AX (for review, see Panier and Boulton, 2014). Our data support this proposed mode of recruitment given that: (1) 53BP1 strongly parallels the pattern observed with γH2AX and the FK2 antibody (although the broad specificity of this antibody toward many ubiquitinated substrates precludes a definitive conclusion regarding the nature of the modified protein) and (2) 53BP1 association with DSB is minimal in G2, in agreement with the proposed dilution of H4K20me2 on post-replicative chromatin (Pellegrino et al., 2017). H1, on the other hand, was recently shown to be displaced from sites of DNA damage (Sellou et al., 2016, Strickfaden et al., 2016).…”

Phase separation of 53 BP 1 determines liquid‐like behavior of DNA repair compartments

“…would react to changes in osmolarity, we monitored the 53BP1 response to ionizing radiation (IR) by quantitative image-based cytometry (QIBC), a high-content microscopy approach that allows for cell cycle resolved profiling of DNA damage responses Toledo et al, 2013;Ochs et al, 2016;Pellegrino et al, 2017;Michelena et al, 2018). As observed previously, we measured a strong IR-induced increase in nuclear 53BP1 foci in G1, which gradually declined in S-phase when increasing amounts of replicated chromatin promote DSB repair by homologous recombination (Chapman et al, 2012;Saredi et al, 2016;Pellegrino et al, 2017), and which rose again in late G2 ( Fig 1A). Consistent with prior work (Giunta et al, 2010;Orthwein et al, 2014), 53BP1 accumulation was blocked when chromosomes condensed in mitosis ( Fig 1B).…”

“…Automated multichannel wide-field microscopy for quantitative image-based cytometry (QIBC) was performed on the Olympus ScanR Screening System described above as done previously (Pellegrino et al, 2017;Michelena et al, 2018). Images were analyzed with the Olympus ScanR Image Analysis Software version 3.0.0, a dynamic background correction was applied, nuclei segmentation was performed using an integrated intensity-based object detection module using the DAPI signal, and foci segmentation was performed using an integrated spot-detection module.…”

DNA end-resection in highly accessible chromatin produces a toxic break

“…Therefore, we induced "HALT" and "GO" breaks and stained the cells for H4K20me2 and H2AX in combination with EdU. In order to rule out previously described cell cycle effects on H4K20me2 levels 48 , we selected G2 phase cells based on the absence of EdU and high DAPI signal 50 . In these cells, we quantified the intensity of H4K20me2 levels at the H2AX focus.…”