Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks.

Recruitment of 53BP1 to chromatin flanking double strand breaks
(DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of
chromatin and interaction of 53BP1 with histone H4 methylated on
lysine 20 (H4K20me). Several histone methyltransferases have been
implicated in 53BP1 recruitment, but their quantitative
contributions to the 53BP1 response are unclear. We have developed
a multi-photon laser (MPL) system to target DSBs to subfemtoliter
nuclear volumes and used this to mathematically model DSB response
kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed
first order kinetics, the 53BP1 MPL-DSB response is best fitted by
a Gompertz growth function. The 53BP1 MPL response shows the
expected dependency on MDC1 and RNF8. We determined the impact of
altered H4K20 methylation on 53BP1 MPL response kinetics in mouse
embryonic fibroblasts (MEFs) lacking key H4K20 histone
methyltransferases. This revealed no major requirement for the
known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1
recruitment or DSB repair function, but a key role for the H4K20
monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1
has recently been implicated in 53BP1 DSB recruitment. We found
that WHSC1 homozygous mutant MEFs reveal an alteration in balance
of H4K20 methylation patterns; however, 53BP1 DSB responses in
these cells appear normal.