H4K20me2 distinguishes pre-replicative from post-replicative chromatin to appropriately direct DNA repair pathway choice by 53BP1-RIF1-MAD2L2

Abstract

The main pathways for the repair of DNA double strand breaks (DSBs) are non-homologous end-joining (NHEJ) and homologous recombination directed repair (HDR). These operate mutually exclusive and are activated by 53BP1 and BRCA1, respectively. As HDR can only succeed in the presence of an intact copy of replicated DNA, cells employ several mechanisms to inactivate HDR in the G1 phase of cell cycle. As cells enter S-phase, these inhibitory mechanisms are released and HDR becomes active. However, during DNA replication, NHEJ and HDR pathways are both functional and non-replicated and replicated DNA regions co-exist, with the risk of aberrant HDR activity at DSBs in non-replicated DNA. It has become clear that DNA repair pathway choice depends on inhibition of DNA end-resection by 53BP1 and its downstream factors RIF1 and MAD2L2. However, it is unknown how MAD2L2 accumulates at DSBs to participate in DNA repair pathway control and how the NHEJ and HDR repair pathways are appropriately activated at DSBs with respect to the replication status of the DNA, such that NHEJ acts at DSBs in pre-replicative DNA and HDR acts on DSBs in post-replicative DNA. Here we show that MAD2L2 is recruited to DSBs in H4K20 dimethylated chromatin by forming a protein complex with 53BP1 and RIF1 and that MAD2L2, similar to 53BP1 and RIF1, suppresses DSB accumulation of BRCA1. Furthermore, we show that the replication status of the DNA locally ensures the engagement of the correct DNA repair pathway, through epigenetics. In non-replicated DNA, saturating levels of the 53BP1 binding site, di-methylated lysine 20 of histone 4 (H4K20me2), lead to robust 53BP1-RIF1-MAD2L2 recruitment at DSBs, with consequent exclusion of BRCA1. Conversely, replication-associated 2-fold dilution of H4K20me2 promotes the release of the 53BP1-RIF1-MAD2L2 complex and favours the access of BRCA1. Thus, the differential H4K20 methylation status between pre-replicative and post-replicative DNA represents an intrinsic mechanism that locally ensures appropriate recruitment of the 53BP1-RIF1-MAD2L2 complex at DNA DSBs, to engage the correct DNA repair pathway.

Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and -deficient cells

“…, consistent with other studies9,10 . IR-induced DNA damage scales with the amount of DNA present in a cell's nucleus and doubles as cells replicate their genome(Figure 2a).…”

“…Of particular interest for the antagonism between the 53BP1 and BRCA1 protein complexes is the H4K20 dimethylation mark (H4K20me2), which is abundant in unreplicated chromatin on parental histones and absent from newly incorporated histones. Hence H4K20me2 is diluted in nascent replicated chromatin and only restored in mature chromatin in late G2/M [10][11][12][13][14] . 53BP1 binds H4K20me2 via its tandem tudor domain (TTD), and both H4K20me2 and the TTD are required for 53BP1 recruitment to DSBs 15,16 .…”

“…The choice between NHEJ and HR is closely linked to the cell cycle and to whether or not an undamaged homologous stretch of DNA is available as a template for HR [4][5][6] . While 53BP1-RIF1-Shieldin restrains DNA end resection of DNA double-strand breaks (DSBs) in the absence of a homologous template strand, after DNA replication BRCA1-BARD1 counteracts 53BP1 binding to damaged chromatin and promotes HR reactions [7][8][9][10][11][12] . The antagonism between 53BP1-RIF1-Shieldin and BRCA1-BARD1 has important implications for cancer therapy, in particular for targeting BRCA-deficient tumors with PARP inhibitors, and for improving CRISPR/Cas9-mediated gene editing.…”

“…Accumulating evidence suggests that this discrimination is linked to the different chromatin makeup ahead of and behind replication forks [10][11][12][13][14] . Of particular interest for the antagonism between the 53BP1 and BRCA1 protein complexes is the H4K20 dimethylation mark (H4K20me2), which is abundant in unreplicated chromatin on parental histones and absent from newly incorporated histones.…”

REV7 confers radioresistance of esophagus squamous cell carcinoma by recruiting PRDX2

“…REV7 also maps to an additional REV7‐binding domain in the C‐terminal region of Rev1 . In addition, REV7 participates in the DNA repair pathway choice through modulation of DNA end‐resection to promote NHEJ repair or cooperating with REV1 or REV3 to promote HR repair . Upregulation of REV7 has been reported in many human cancers, including glioblastoma, and nasopharyngeal, breast and colon cancer, and is related to poor prognoses in late‐stage ovarian, colon cancer and B‐cell lymphoma treated with rituximab .…”