Structural and biochemical studies on AMC and PR-DUB chromatin modifying complexes

Abstract

Nucleosome is a basic unit of chromatin in Eukaryotes, which is made up of DNA and histone octamer. A variety of chromatin modifiers put covalent modifications on histones, which activate or repress the gene expression depending on the types and locations of the modifications. In this research, I investigated the structure and function of Ash1-Mrg15-Caf1 (AMC) and Polycomb Repressive DeUBiquitinase (PR-DUB) complexes, which are H3K36 dimethyltransferase and H2A K119 deubiquitinase respectively. AMC is a complex consist of Ash1, Mrg15 and Caf1 (also known as Nurf55 or p55 in fly, and RbAp48 in human). Among these subunits, the binding mode and biological function of Caf1 remains unclear. In this research, I dissected the binding mode of Caf1 via biochemical and structural approaches. I found that Caf1 interacts to the proximal region of histone binding module cluster of Ash1 using its H4 binding pocket. Furthermore, I revealed the novel function of Caf1 in AMC complex as a sensor to discriminate the unmodified or H3K4me3 modified histone H3. This finding suggest the interplay between TrxG histone marks on active genes. PR-DUB is a complex composed of BAP1 and ASXL1. In PR-DUB, ASXL1 provides substrate specificity to BAP1. However, the mechanism of PR-DUB recruitment to its substrate nucleosome has not been clearly identified. In this study, I investigated the cryo-EM structures of PR-DUB in complexes with unmodified/H2A K119Ub nucleosome. By comparing these two distinct structures, I proposed a binding model that PR-DUB initially binds to nucleosome by interacting on the acidic patch of nucleosome, then tightly positioned by interacting with H2A K119Ub. This model explains how PR-DUB is recruited and recognizes its target nucleosome on chromatin. These results provide thorough understanding of the molecular mechanism by which chromatin modifiers recognize their substrate in the context of distinct histone modifications.