MMSET regulates histone H4K20 methylation and 53BP1 accumulation at DNA damage sites

Abstract

p53-binding protein 1 (53BP1) is known to be an important mediator of the DNA damage response, with dimethylation of histone H4 lysine 20 (H4K20me2) critical to the recruitment of 53BP1 to double-strand breaks (DSBs). However, it is not clear how 53BP1 is specifically targeted to the sites of DNA damage, as the overall level of H4K20me2 does not seem to increase following DNA damage. It has been proposed that DNA breaks may cause exposure of methylated H4K20 previously buried within the chromosome; however, experimental evidence for such a model is lacking. Here we found that H4K20 methylation actually increases locally upon the induction of DSBs and that methylation of H4K20 at DSBs is mediated by the histone methyltransferase MMSET (also known as NSD2 or WHSC1) in mammals. Downregulation of MMSET significantly decreases H4K20 methylation at DSBs and the subsequent accumulation of 53BP1. Furthermore, we found that the recruitment of MMSET to DSBs requires the γH2AX-MDC1 pathway; specifically, the interaction between the MDC1 BRCT domain and phosphorylated Ser 102 of MMSET. Thus, we propose that a pathway involving γH2AX-MDC1-MMSET regulates the induction of H4K20 methylation on histones around DSBs, which, in turn, facilitates 53BP1 recruitment.

Figure 1. Induction of H4K20 methylation and recruitment of MMSET at DSBs

a and d, Examples of ChIP analysis by PCR of indicated proteins on a DSB induced by I-SceI, where input demonstrates equal amount of DNA for ChIP. b and e Quantitative PCR (Q-PCR) of indicated ChIP samples, where the Y axis represents the relative enrichment of the indicated proteins compared to the IgG control (after normalized with a PCR internal control to a locus other than the DSB; ± s.e.m., n=3). c and f immunofluorescence (IF) staining of 293T cells after indicated treatments, then stained with indicated antibodies.

Figure 2. MMSET is required for H4K20me2/3 and 53BP1 accumulation at DSBs

a, Quantitative PCR analysis of ChIP samples from HeLa-DR-GFP cells transfected with control or MMSET shRNA, where the Y axis represents the relative enrichment of the indicated proteins compared to the IgG control (± s.e.m., n=3). b, EtBr staining of ChIP samples from 2a analyzed by PCR, where input demonstrates equal loading of DNA for PCR. c–d, IF of HCT116 cells transfected with the indicated siRNA or shRNA, irradiated (5Gy), and stained with indicated antibodies. e, Phosphorylation of CHK1/2 in the cell lysates from 2c analyzed by immunoblotting.

Figure 3. Recruitment of MMSET to DSBs requires the ATM-H2AX-MDC1 pathway

a, ChIP analysis by PCR of indicated proteins at DSBs in HeLa-DR-GFP cells transfected with the indicated siRNA. Right panels: Western blots of H2AX and MDC1. b, Coimmunoprecipitation of MMSET and MDC1 in HeLa cells before or after IR. c, GST pull down assay of MMSET using indicated GST-fusion proteins. d, 293T cells treated and immunoprecipitated as indicated, then analyzed with anti-pSQ/TQ antibody. e, 293T cells transfected with the indicated constructs were treated as indicated, then immunoprecipitated and immunoblotted with indicated antibodies. f, the interaction between GST-MDC1-BRCT and indicated peptides were measured by BIAcore 3000.

Figure 4. Phosphorylation of MMSET is important for H4K20 methylation, 53BP1 recruitment and DNA damage response

a, HeLa DR-GFP cells were transfected with the indicated constructs, H4K20 methylation and MMSET recruitment was analyzed by PCR of ChIP samples. b–c, HCT116 cells transfected with indicated constructs were irradiated, and 10 min later, stained with indicated antibodies. d, Radiation sensitivity of cells from Figure 4c was determined by colony formation (±s.e.m., n=3). E, Model demonstrating how the MDC1-MMSET pathway regulates DNA damage-induced histone H4 Lysine 20 methylation and 53BP1 foci formation.