Project Summary The long-term goal of this study is to elucidate the molecular mechanisms of how histone ubiquitylation regulates chromatin activity, which remains largely unknown. To understand the fundamental ways in which histone ubiquitylation influences chromatin-mediated biological processes, this project focuses on revealing (1) how histone ubiquitylation affects the structural dynamics of the nucleosome, the fundamental packing unit of chromatin made of ~147 bp DNA, two histone H2A-H2B dimers and one (H3-H4)2 tetramer, and (2) how it regulates the efficiency of hexasome generation by histone chaperone and transcription elongation by RNA polymerase II (Pol II). These studies take advantage of site-specifically ubiquitylated histones and cutting-edge biochemical and single-molecule approaches. An emerging theme in this project is that histone ubiquitylation alters intra-nucleosomal dynamics and thus physically regulates gene accessibility. The preliminary biochemical and single-molecule studies have shown that histone H2BK34 and H2BK120 ubiquitylation accompanies destabilization of the nucleosome structure, inducing labilization of H2A-H2B dimers. In collaboration with a histone acceptor such as histone chaperone Nap1, H2BK34 and H2BK120 ubiquitylation promote dissociation of one of the two H2A-H2B dimers, producing hexasomes that are emerging as critical sub-nucleosomal intermediates in an active chromatin state. The goal of this proposal is to elucidate how histone ubiquitylation alters the nucleosome dynamics and eventually how the changes are implicated in regulating the efficiencies of hexasome generation and transcription elongation. The specific aims are (1) to define the effects of H2BK34/120 ubiquitylation on the structural dynamics of the nucleosome with a recently developed single-molecule structural flexibility test method and (2) to evaluate the effects of H2BK34/ K120ub on (i) the kinetics of hexasome generation by histone chaperone Nap1 and FACT and (ii) the kinetics of transcription elongation in the context of Nap1- and FACT-mediated nucleosome disassembly and reassembly, both at a single-molecule level based on a recently developed transcription elongation monitoring system. This project will result in fundamental understanding on the direct physical roles of histone ubiquitylation in regulating chromatin dynamics.