C8orf33 dictates DNA double-strand break repair choice by modulating KAT8-mediated H4K16 acetylation.

Homologous recombination (HR) and non-homologous end joining (NHEJ) are two main repair pathways of DNA double-strand breaks (DSBs). The regulation of repair pathway choice is crucial for maintaining genomic stability and preventing carcinogenesis. Consequently, there is increasing interest in elucidating the molecular mechanisms that govern DSB repair pathway selection. Here, we identify chromosome 8 open reading frame 33 (C8orf33) as a novel regulator of DSB repair choice. We show that C8orf33 is a nuclear protein localized predominantly to the nucleolus and is recruited to DSB sites within both nuclear and nucleolar regions. We demonstrate that C8orf33 promotes the recruitment of 53BP1, thus channeling DSB repair toward NHEJ. Consequently, C8orf33 inhibits DNA end resection and counteracts the recruitment of HR factors, BRCA1 and RAD51, to DSB sites. Mechanistically, chromatin profiling analysis reveals that C8orf33 antagonizes the chromatin association of KAT8 acetyltransferase at DSB sites leading to reduced histone 4 lysine 16 acetylation (H4K16ac) levels. Accordingly, the loss of C8orf33 enhances KAT8 chromatin binding that increases H4K16ac levels. This promotes the recruitment of HR factors while suppressing the accumulation of NHEJ factors at DSB sites, thereby favoring HR over NHEJ. Additionally, we demonstrate that the elevated HR activity in C8orf33-deficient cells causes genomic instability, as evidenced by accelerated loss of ribosomal DNA repeats and increases cell death. Collectively, our findings establish C8orf33 as a critical regulator of DSB repair pathway choice, safeguarding genomic integrity.