Program Director/Principal Investigator (Last, First, Middle): Peterson, Craig, Lewis The overall objective of our research is to determine how chromosome structure influences gene transcription, DNA replication and repair, with special emphasis on identifying and characterizing the chromatin remodeling machines that control chromosome dynamics. Notably, genetic experiments have revealed ATP- dependent chromatin remodeling enzymes as essential regulators of virtually every chromosomal process, and their dysregulation leads to a variety of diseases, including cancer. Our research efforts can be organized into three inter-related areas: (1) Mechanistic studies of ATP-dependent chromatin remodeling enzymes;; (2) Role of chromatin dynamics in genome stability pathways;; and (3) Assembly/function of chromatin higher order structures. A major focus of our mechanistic studies is to continue to dissect the structure and biochemical mechanisms of the INO80C and SWR1C enzymes. These remodeling enzymes catalyze novel, ATP-dependent histone exchange events that control the deposition and distribution of the H2A.Z histone variant within nucleosomes that flank promoters of genes transcribed by RNA polymerase II, as well as nucleosomes that flank chromatin boundary elements, centromeres, and replication origins. Mammalian homologs of SWR1C and INO80C, including the p400/Tip60 and hINO80 complexes, are key for proper stem cell function, genome stability, development, and gene expression. During the past budget period, we identified a novel regulatory interaction between SWR1C and the acetylation of lysine 56 of histone H3 (H3-K56Ac) that regulates nucleosome dynamics, noncoding RNA expression, and assembly of large-scale, chromosome interaction domains (CIDs) that are related to mammalian topologically-associated domains (TADs). These mechanistic studies will include quantitative, fluorescence-based assays to define steps of the histone dimer exchange reaction, as well as the reconstitution of these multi-subunit enzymes with recombinant subunits. Studies from us and others over the past 10 years have demonstrated that chromatin dynamics play a large role in stabilizing the replisome and in controlling various steps in DNA double strand break repair. Our recent data suggests that changes in chromatin dynamics can also lead to dysregulation of transcription which impacts genome stability pathways. Our research will address several key unanswered questions focused on genome stability pathways: (1) Do Remodelers regulate the homology search step of homologous recombination and do they function in concert with histone acetylases? (2) How does INO80C stabilize the replisome and is this role due to the regulation of ncRNA expression? (3) How does INO80C prevent ncRNA expression from intergenic regions? (4) Does the hyperacetylation of H3-K56Ac lead to formation of R-loops that disrupt replisome function? (5) Does hypoacet...