All ETDs from UAB

Advisory Committee Chair

Hengbin Wang

Advisory Committee Members

Tim M Townes

Chenbei Chang

Christopher A Klug

Louise Chow

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Histones are globular proteins which help package and organize DNA in the eukaryotic nucleus. The four core histones, H2A, H2B, H3 and H4, combine with approximately 146 base pairs of DNA to form a nucleosome, the fundamental unit of chromatin. Post-translational modifications to the C-terminal and N-terminal histone tails regulate chromatin structure and function, and play important roles in transcriptional regulation, replication, DNA damage response, and mRNA processing. Histone H2B ubiquitination is an evolutionarily conserved modification with roles in gene expression and DNA damage repair. In addition, H2B ubiquitination may also regulate mRNA splicing. Here, we identify USP49, a cysteine protease which forms a complex with RVB1 and SUG1 to deubiquitinate histone H2B specifically. USP49 mediated H2B deubiquitination regulates gene isoform abundance by influencing the alternative splicing of exons. Histone H2B ubiquitination comprises the formation of an isopeptide bond between the histone H2B lysine 120 residue and the C-terminal glycine of ubiquitin. H2B ubiquitination, catalyzed by the RNF20 ubiquitin ligase in humans, affects nucleosome stability, chromatin compaction, and is a prerequisite for histone H3 lysine 4 and 79 methylation. We show that uH2B level, as modulated by USP49 deubiquitination, also regulates the association of core splicing factors U1A and U2B with chromatin and specific splice sites. Impaired association of the U1A and U2B core splicing factors with chromatin is correlated with altered splicing kinetics in USP49 knockdown cells. Recent studies in yeast and humans suggest that ubiquitinated H2B may be part of the chromatin architecture marking exons and introns. Supporting this idea, we find that USP49 is enriched at exons alternatively spliced in USP49 knockdown cells, and that these exons show a greater fold increase in ubH2B than unaffected exons in USP49 knockdown cells. Our work expands the roles of H2B ubiquitination beyond transcription and DNA damage repair and establishes histone H2B ubiquitination and deubiquitination as important regulators of splicing.

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