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, focusing primarily on the structure and function of the 14-subunit, SWR1C remodeler; (2) Investigating roles for the INO80C remodeler in DNA replication and the maintenance of genome stability; and (3) Probing how the expression of newly replicated genes is repressed following replication fork passage, a process termed transcriptional buffering. The SWR1C remodeling enzyme catalyzes a novel, ATP-dependent histone exchange event that controls the deposition of the H2A.Z histone variant within nucleosomes that flank promoters of genes transcribed by RNA polymerase II, as well as nucleosomes that flank 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. How SWR1C catalyzes ATP-dependent deposition of H2A.Z remains largely unknown, and our proposed mechanistic studies will include ensemble and single molecule fluorescence-based assays to define steps of the histone dimer exchange reaction, as well as a combination of mass spectrometry and cryoEM methods to probe how SWR1C distinguishes different nucleosomal substrates. Studies from us and others have demonstrated that chromatin dynamics play a large role in regulating transcription of both coding and noncoding RNAs, and disruption of this balance can impact genomic stability. In particular, our work on INO80C has found that it prevents pervasive noncoding transcription from impinging on replisome function in both yeast and mammalian cells. We propose a variety of genomic methods to probe key unanswered questions: How does INO80C block noncoding transcription? How does transcription impact fork structure? Does INO80C collaborate with the conserved forkhead transcription factors to organize origins into a nuclear compartment? Our in vivo studies will extend to transcriptional regulation during S phase. We have used Nascent transcript sequencing to confirm that newly replicated genes are transiently repressed 2- fold until the subsequent G2 phase. Termed “transcriptional buffering” this process is conserved in mammals and is believed to prevent transient aneuploid states during S phase. How buffering is established and removed is not known, an...