Missense mutations that disrupt the RING domain of the tumor suppressor gene lead to increased risk of breast and ovarian cancer. The BRCA1 RING domain is a ubiquitin ligase, whose structure and function rely critically on forming a heterodimer with BARD1, which also harbors a RING domain. The function of the BARD1 RING domain is unknown. In families severely affected with breast cancer, we identified inherited BARD1 missense mutations Cys53Trp, Cys71Tyr, and Cys83Arg that alter three zinc-binding residues of the BARD1 RING domain. Each of these mutant BARD1 proteins retained the ability to form heterodimeric complexes with BRCA1 to make an active ubiquitin ligase, but the mutant BRCA1/BARD1 complexes were deficient in binding to nucleosomes and in ubiquitylating histone H2A. The BARD1 mutations also caused loss of transcriptional repression of BRCA1-regulated estrogen metabolism genes and ; breast epithelial cells edited to create heterozygous loss of showed significantly higher expression of and Reintroduction of wild-type into these cells restored and transcription to normal levels, but introduction of the cancer-predisposing RING mutants failed to do so. These results indicate that an intact BARD1 RING domain is critical to BRCA1/BARD1 binding to nucleosomes and hence to ubiquitylation of histone H2A and also critical to transcriptional repression of BRCA1-regulated genes active in estrogen metabolism.

Reversible post-translational modifications of histone proteins in eukaryotic chromatin are closely tied to gene function and cellular development. Specific combinations of histone modifications, or marks, are implicated in distinct DNA-templated processes mediated by a range of chromatin-associated enzymes that install, erase and interpret the histone code. Mechanistic studies of the precise biochemical relationship between sets of marks and their effects on chromatin function are significantly complicated by the dynamic nature and heterogeneity of marks in cellular chromatin. Protein semisynthesis is a chemical technique that enables the piecewise assembly of uniformly and site-specifically modified histones in quantities sufficient for biophysical and biochemical analyses. Recent pioneering efforts in semisynthesis have yielded access to histones site-specifically modified by entire proteins, such as ubiquitin (Ub) and the small ubiquitin-like modifier (SUMO). Herein, we highlight key studies of biochemical crosstalk involving Ub and SUMO in chromatin that were enabled by histone semisynthesis.