Final Report Summary - TELOMERES (Genome-wide identification of factors controlling the telomere damage response and telomere-driven genomic instability.)
Our genome is continuously challenged with different kinds of DNA damage. To handle this and maintain genome integrity, cells have complex mechanisms to detect and repair different DNA lesions. Despite being essential, these mechanisms are a potential threat to the natural ends of our chromosomes, which should not be handled as broken DNA, as this would prevent cell proliferation or lead to chromosomal instability. To prevent inappropriate activation of DNA damage responses and repair activities at natural chromosome ends, these ends are shielded by specialized nucleoprotein structures, known as telomeres. Chromosome ends lose telomere protection as a consequence of incomplete telomere replication during cell division or upon telomeric protein malfunctioning. It has become clear that dysfunctional telomeres play important roles in aging and cancer development by inducing DNA damage responses and repair activities that result in genomic instability. However, we have relatively poor understanding of the mechanisms underlying these processes. In this project we aimed to significantly increase our understanding of the mechanisms underlying the telomere damage response and telomere-driven genomic instability. Hereto we performed functional genetic screens to identify factors with critical roles in telomere damage responses and telomere-driven genomic instability, followed by mechanistic studies on the roles of these factors in the cellular responses to telomere uncapping and upon DNA damage. We identified MAD2L2 (a.k.a. MAD2B or REV7) as a novel factor controlling DNA repair both at mammalian telomeres and at DNA double-strand breaks (DSBs). We found that MAD2L2 contributes to the DNA repair pathway choice between the NHEJ and HDR DNA repair pathways, by inhibiting 5’ DNA end-resection downstream of the factors 53BP1 and RIF1. End-resection blocks NHEJ while committing to HDR. By inhibiting end-resection MAD2L2 promotes NHEJ and thereby promotes fusion of deprotected telomeres and end-joining of DNA DSBs in several settings, including during immunoglobulin class switch recombination. Absence of MAD2L2 on the other hand allows end-resection, and thereby initiation of HDR at the cost of NHEJ. In additional follow-up work we addressed how DNA repair pathway choice decisions are made while cells replicate their DNA in the S-phase of the cell cycle. In S-phase both NHEJ (activated by 53BP1) and HDR (activated by BRCA1) are functional and non-replicated and replicated DNA regions co-exist, each requiring a different DNA repair mechanism to maintain genome integrity. We found that MAD2L2 is recruited to DSBs in H4K20 dimethylated (H4K20me2) chromatin by forming a complex with 53BP1 and RIF1. In addition, MAD2L2 suppresses DSB accumulation of BRCA1 in S/G2, suggesting that MAD2L2 could be a limiting factor in DNA repair pathway choice. Moreover, we found that the differential H4K20 methylation status between pre-replicative and post-replicative DNA represents an intrinsic mechanism that locally ensures appropriate recruitment of 53BP1-RIF1-MAD2L2 at DSBs to engage the correct repair pathway. In addition, in a collaborative study we found that efficient NHEJ at uncapped telomeres requires the chromatin remodeler CHD2 and deposition of the histone variant H3.3. Altogether these findings have significantly advanced our understanding of the mechanisms underlying the telomere damage response and telomere-driven genomic instability. Furthermore, our work also yielded critical new insights beyond telomeres, in the control of DNA repair pathway choice at DNA double-strand breaks. As appropriate repair of DNA lesions and the inhibition of DNA repair activities at telomeres are crucial to prevent genomic instability, cancer development and aging-related disease, we anticipate that these findings will eventually contribute to the development of novel strategies for the prevention, diagnosis or treatment of cancer and aging-related pathologies.