Characterization of KAP1 acetylation and its roles in DNA damage repair

Abstract

DNA damage repair is a complex process governing genomic integrity, cell cycle progression and the overall survival of all kinds of species. It involves the orchestrating of various protein machineries for detection of DNA damage and subsequent repair of the damage sites. KAP1, also known as TRIM28, is a heterochromatin-associated protein that plays essential roles in DNA damage sensing and repair especially in response to hazardous DNA double strand breaks (DSBs). According to previous reports, its serine phosphorylation levels at S473 and S824 will be upregulated upon DSBs, mediating its mobilization, chromatin relaxation and dynamics of DNA damage repair proteins like 53BP1 and BRCA1. However, the functional relevance of KAP1 acetylation/ deacetylation has been rarely studied, especially in the context of DSB repair. Moreover, the deacetylase(s) and acetyltransferase(s) modifying KAP1 have remained unknown. I hereby identify the deacetylase(s) and acetyltransferase(s) of KAP1. KAP1 acetylation can be enhanced by MOF, with HP1 box being the predominant acetylated region. Moreover, KAP1 can be deacetylated predominantly by SIRT6 in vitro and in vivo. By means of mass spectrometry, the deacetylation sites of KAP1 by SIRT6 and SIRT1 were also identified. In the biological context, KAP1 will be deacetylated in response to irradiation-induced DSBs in a SIRT6-dependent manner. SIRT6 deacetylates KAP1 upon DSBs, in turns upregulating phosphorylation at S473 as early as 0.5 h after irradiation. Depletion of SIRT6 compromises phosphorylation of KAP1 at S473 from 0 to 6 h after irradiaiton. At the early stage of DSB repair, I show that SIRT6 facilitates dissociation of KAP1 from chromatin 0.5 h after irradiation-induced DSBs, parallel to its deacetylase activity on KAP1. Consistently, KAP1 hypoacetylation at K304 exhibited diminished tethering to H3. This suggests a possible link between SIRT6-induced deacetylation of KAP1 and dissociation of KAP1 from chromatin in response to DSBs. Towards the end of DSB repair, SIRT6 knockout cells displayed a defect in clearance of 53BP1 foci from the repaired damage sites 24 h after irradiation. SIRT6-mediated KAP1 deacetylation at K289 and K304 promotes such resolution of 53BP1 foci. Notably, hypoacetylation of KAP1 alleviates 53BP1 foci resolution defects in SIRT6 knockout cells. To conclude, my findings show that SIRT6-mediated KAP1 deacetylation plays a significant role in both early and late stage of DNA repair in terms of mobilization of KAP1, accessbility of chromatin, clearnace of 53BP1 foci and negative regulation of DNA damage signalling. Notably, my study is the first to suggest the crosstalks between SIRT6 and ATM-KAP1 signalling in DSB repair processes. It also provides additional insights on SIRT6-mediated DNA damage repair signalling.