BRCA1/BARD1-Dependent Ubiquitylation of Nucleosomal Histone H2A

Abstract

Breast cancer type-1 susceptibility protein (BRCA1) was the first gene to be linked to heritable breast and ovarian cancer over thirty years ago. Since then, remarkable progress has been made with regards to genetic testing, prophylactic measures, and treatment options available to patients with BRCA1 mutations. However, thousands of BRCA1 variants of unknown significance still exist, leaving doctors and patients with no clear path forward. Understanding the underlying biology of the gene product of BRCA1 (the BRCA1 protein) is an important goal that may improve health outcomes. The BRCA1 protein forms an obligate heterodimeric complex with its binding partner, BARD1 (BRCA1/BARD1). Together, this large complex functions in the nucleus of cells, protecting the integrity of genome by helping to repair double-stranded DNA breaks and regulate the transcription of certain genes. These functions are mediated, in-part, by the sole known enzymatic function of BRCA1/BARD1 as a RING-type E3 ubiquitin (Ub) ligase. Recently, histone H2A in nucleosomes has emerged as a central substrate for BRCA1/BARD1-dependent Ub ligase activity. BRCA1/BARD1 targets specific lysine sites on the extreme C-terminal tail of histone H2A for Ub transfer which serves as a signal for DNA repair and transcriptional regulation. In this thesis, I first review what is known about the E3 ubiquitin ligase function of BRCA1/BARD1 in general (Chapter 1) and specifically at chromatin (Chapter 2). In Chapter 3, I present a cryo-EM structure of the BRCA1/BARD1 RING/RING heterodimer bound to a nucleosome substrate, with additional structural and biochemical data that provide key mechanistic insight into the basis for site-specific ubiquitylation of nucleosomal H2A. In Chapter 4, I reveal interactions between non-RING regions of BARD1 and nucleosome substrates that enhance chromatin binding and H2A ubiquitylation, describing BRCA1/BARD1/chromatin complexes with extensive multivalency. Overall, my work uncovers a critical role for the previously underappreciated partner of BRCA1, BARD1. Together, this body of work sets the stage for additional discoveries into the biological importance of BRCA1/BARD1 H2A-specific E3 Ub ligase activity in DNA repair and transcriptional regulation. These findings may ultimately be used to predict or experimentally measure the impacts of patient mutations of unknown clinical significance in addition to explaining the effects of known pathogenic mutations. Knowledge of the molecular effects of these mutations and their underlying biology may ultimately lead to enhanced prophylactic measures or new avenues of therapeutics for patients.