Role of 53BP1 During the DNA Damage Response

Carpenter, Phillip

Abstract

The failure to repair DMA double-stranded breaks (DSBs) impacts several important cellular processes and can lead to deleterious consequences. DSBs are created during the development of lymphoid T and B cells, in response to stalled replication forks, or through exposure to ionizing radiation (IR). Our lab has played a key role in defining 53BP1 as a critical component in DDR pathways, particularly in B cells activated for class switch recombination (CSR). Importantly, we demonstrated in a seminal paper that 53BP1 operates in the """"""""joining phase"""""""" of CSR, a NHEJ-like mechanism specific to the immunoglobulin heavy chain (IgH) locus of B cells. We have proposed that such a joining-like function operates in response to DSBs and tethers free, broken DNA ends and chromatin together by """"""""anchoring"""""""" them through novel repair mechanisms that promote CSR. The failure to perform 53BP1-mediated anchoring perturbs the DSB repair response and contributes to the generation of cells with abnormal ploidy and/or translocations. How 53BP1 functions in this novel aspect of DNA repair is unknown, but as detailed within, we hypothesize that 53BP1 cooperates with various factors that bind modified histone tails (i.e. the Nsd1 histone methyltransferase [HMTase] and Phf20) in a manner that facilitates DSB repair through the modulation of chromatin structure near breaks. Our goal is to elucidate the mechanisms of how 53BP1 coordinates DSB repair (i.e., anchoring) with the regulation of chromatin structure.
The specific aims of this proposal are: I. To determine the nature of histone lysine methylation near sites of DSBs in 53BP1+/+ and 53BPT1' B cells activated for DNA repair (switching) through chromatin immunoprecipitation at the IgH locus. Through the use of a B cell model system, we will determine the levels and types of specific chromatin modifications near sites of DSBs at the IgH locus as they likely contribute to DSB repair. II. To examine the relationship between 53BP1 function and histone lysine methylation through both a structure/function analysis of 53BP1-Nsd1 complex formation and a determination of its effect on HMTase activity, histone deacetylase activity, and nucleosome binding. As we have shown that 53BP1 binds the chromatin modulators Nsd1 (and Phf20), they may have a common function in regulating DSB repair and chromatin structure. III. To examine the contribution of Nsd1 in histone lysine methylation and DSB repair. Surprisingly, very little is known about Nsd1 function, but its relationship to 53BP1 suggests a role for the HMTases in DSB repair. As NsdT1' mice display embryonic lethality, we will use RNA interference to determine the role of Nds1 in lysine methylation and DSB repair, including CSR at the IgH locus.