Abstract 1097 Determining the functions of BRCA1 that are mediated through nucleosome ubiquitylation

BRCA1 and BARD1 proteins heterodimerize in human cells and ubiquitylate nucleosomes to aid in DNA damage repair and gene repression. While this is hypothesized to contribute to tumor-suppression functions of the two proteins, the heterodimer also acts as a protein-protein interaction scaffold in many cellular processes leaving ambiguity about the molecular mechanisms that drive various BRCA1 functions. To address some of these remaining questions we are utilizing the model organism C. elegans to connect how the biochemical functions of the heterodimer affect phenotypes associated with BRCA1 in the context of the organism. We used in vitro biochemistry and ex vivo transcript quantification to establish that nucleosome ubiquitylation and gene repression are conserved functions in the C. elegans homologs, BRC-1 and BRD-1. We found that worm strains with mutated brc-1 or brd-1 overexpressed members of the cytochrome P450 (cyp) gene family that is most similar in sequence to cyp genes repressed by the human homologs. We also used purified proteins and reconstituted nucleosomes to show that while BRD-1 and BARD1 use different interfaces to interact with the nucleosome substrate, BRC-1 and BRCA1 have a conserved nucleosome binding interface that is vital for nucleosome ubiquitylation. In order to investigate the specific role that nucleosome ubiquitylation plays in the many functions of BRC-1, we generated a C. elegans strain containing a mutation of two conserved amino acids essential for binding nucleosomes. Unlike other brc-1 mutant worm strains, all other BRC-1-protein interfaces remain intact. We have utilized this strain to determine BRC-1 nucleosome ubiquitylation plays a role in meiotic X chromosome nondisjunction as measured by the number of male self-progeny and in managing reactive oxygen species using an in vivo fluorescence assay. However, we find that the phenotypes are intermediate, characterized by effect sizes falling between wild-type and complete loss of the brc-1 gene. This implies other BRC-1 functions, besides nucleosome ubiquitylation, also play a role in these phenotypes. Ongoing experiments with this strain will also be presented to demonstrate the role of nucleosome ubiquitylation in repression of genes and microsatellite DNA as assayed by RT-PCR. We will share our findings regarding the conserved features of BRC-1 and BRD-1 nucleosome ubiquitylation and data regarding its contribution to the loss-of-BRC-1 phenotypes. Thank you to TCU Biology Department and NIH for funding (R15GM135900).