Abstract 6096: BRCA1-BARD1 ubiquitylates histones for genome maintenance

Abstract

Abstract Background: Breast cancer type 1 susceptibility protein (BRCA1) is a tumor suppressor gene involved in DNA double strand break repair with well-known cancer implications. BRCA1 heterodimerizes with BRCA1 associated Ring domain 1 (BARD1) to form a complex with DNA binding and ubiquitin E3 ligase function capable of interacting with proteins of diverse biological processes, most notably homology-directed DNA repair. During DNA repair, BRCA1-BARD1 directly interfaces with nucleosomes and transfers mono ubiquitin (Ub) to lysine residues on the C-terminal tail of histone H2A. Although truncation of the enzymatic BRCA1-BARD1 RING-RING domain retains H2A ubiquitylating activity, full-length BRCA1-BARD1 binds more tightly with nucleosomes and displays higher H2A-Ub activity. However, the molecular basis and biological significance for this enhanced nucleosome binding and H2A-Ub activity is uncharacterized. Methods: Full length BRCA1-BARD1 or truncated mutants and histones were purified from E. coli. or insect cells. Nucleosomes were assembled for in vitro ubiquitylation reaction and binding assays. To determine the biological significance, mammalian cell lines that stably express wild type or mutant forms of BARD1 were established for cellular fractionation, foci analysis, and clonogenic survival studies alongside various DNA damage agents. Results: Our results show multiple interaction sites exist between BRCA1-BARD1 and nucleosomes which allow high-affinity chromatin binding and promote increased histone H2A ubiquitylation activity. Multivalent BARD1-nucleosome interactions, namely those using strong binding motifs located in the intrinsically disordered region (IDR) of BARD1, and the weak “kiss” interaction mediated by the RING domains of both BRCA1 and BARD1, are essential for H2A ubiquitylation by BRCA1-BARD1. Further, we isolated two types of specific histone binding and/or ubiquitylation-defective mutants of BARD1: a BARD1-IDR mutant with disrupted nucleosome binding withe retained H2A ubiquitylation ability, and a RING mutant that solely impairs H2A ubiquitylation. In both cases, we demonstrate that these mutants are hypersensitive to DNA damage agents, including polyADP-ribose polymerase (PARP) inhibitors, and demonstrate reduced capacity of BARD1 to associate with chromatin and foci formation owing to attenuated repair capacity. Conclusion: Our studies provide convincing evidence BRCA1-BARD1 interacts with nucleosomes and ubiquitylates histones via its E3 ligase activity. Further, it plays a critical role in DNA damage response and repair that contributes to genome stability, which when disrupted sensitizes them to DNA damage agents. Our results open new avenues towards understanding whether and how these mutations in BRCA1-BARD1 affect its tumor suppression functions and their implications clinically, ultimately with the goal to translate these findings for the benefit of cancer patients. Citation Format: Wenjing Li, Samuel R. Witus, Meiling Wang, Peter S. Brzovic, Rachel E. Klevit, Weixing Zhao. BRCA1-BARD1 ubiquitylates histones for genome maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6096.