Elucidating the function of the histone H4 basic patch in SAGA-mediated histone H2B deubiquitination and histone acetylation

The eukaryotic genome is packaged into the nucleus by the wrapping of DNA around histones to form the nucleosome—the fundamental repeating unit of chromatin. In addition to its structural role, chromatin regulates DNA accessibility and thereby DNA-templated processes such as DNA replication and transcription. Chromatin structure and function is mediated by the covalent modification of the histone proteins with a vast array of post-translational modifications (PTMs) including acetylation, methylation, phosphorylation, and monoubiquitylation. Histone PTMs can affect chromatin structure directly or serve as binding sites for effector proteins and complexes that mediate a specific function. Proper regulation of histone PTMs is critical for many cellular and developmental processes and their aberrant placement or removal is a hallmark of many diseases including cancer and neurodegenerative disorders. Histone H2B monoubiquitylation (H2Bub1) has central functions in multiple DNA-templated processes including gene transcription, DNA repair, and replication. In addition, H2Bub1 is required for the trans-histone regulation of H3K4 and H3K79 methylation. Although previous studies have elucidated the basic mechanisms that establish and remove H2Bub1, we have only an incomplete understanding of how H2Bub1 is regulated. We report a novel regulator of H2Bub1 – the histone H4 basic patch. The H4 basic patch is a hub for chromatin modifiers, yet the H4 basic patch regulates H2Bub1 levels independently of interactions with chromatin remodelers and separately from its regulation of H3K79 methylation. To measure H2B ubiquitylation and deubiquitination kinetics in vivo, we used a rapid and reversible optogenetic tool, LINX (light-inducible nuclear exporter), to control the subcellular location of the H2Bub1 E3-ligase, Bre1. The ability of Bre1 to ubiquitylate H2B was unaffected by a H4 basic patch mutant. In contrast, H2Bub1 deubiquitination by SAGA-associated Ubp8, but not by Ubp10, increased in the mutant. Consistent with a function for the H4 basic patch in regulating SAGA deubiquitinase activity, we also detected increased histone acetylation by SAGA in H4 basic patch mutants. The work in this dissertation discusses the discovery a new regulatory mechanism of the H4 basic patch in SAGA-mediated functionsDoctor of Philosoph