Characterization of Nucleosomes Containing Specific Forms of the Histone Variant H2A.Z
Ng, Marlee Kam
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H2A.Z is a highly conserved histone variant that replaces canonical histone H2A at specific loci to regulate diverse nuclear processes. Amongst these, the role of H2A.Z in transcriptional regulation is of particular interest due to its enrichment at promoters of most genes in yeast and higher eukaryotes. However, its precise role in regulation is complex, as it has been linked to both repression and activation. One possibility is that H2A.Z activity is regulated by post-translational modifications since H2A.Z can be acetylated or monoubiquitylated in mammals. For example, H2A.Z can be multiply acetylated at several lysine residues at its N-terminus, and such modified form is associated with active promoters. In contrast, our lab has previously shown that a fraction of H2A.Z is monoubiquitylated at its C-terminus, and this form is associated with silent chromatin. One aim of this thesis is to characterize monoubiquitylated H2A.Z-nucleosomes in the context of transcriptional regulation. To this end, we devised a biotinylation-based method to enrich for H2A.ZUb1-mononucleosomes, and further characterized their composition and genomic distribution. In the second chapter, I demonstrate that H2A.ZUb1-enriched mononucleosomes are enriched with the histone post-translational modification H3K27me3, but depleted of H3K4 methylation and other modifications associated with transcriptional activity. H2A.ZUb1-eniched mononucleosomes also preferentially co-purify with proteins typically involved in repression, and with CTCF and cohesin. Consistent with these, ChIP-Seq analysis of H2A.ZUb1-nucleosomes identifies non-expressed genes as sites of H2A.ZUb1 enrichment. In addition to post-translational modification, vertebrate H2A.Z is differentiated into non-allelic isoforms H2A.Z-1 and H2A.Z-2. Previously, we used mass-spectrometry to identify proteins that preferentially associate with H2A.Z-mononucleosomes over H2A-mononucleosomes. In the third chapter, I show that one of these proteins, USP39, is enriched on mononucleosomes containing H2A.Z-1 over those containing H2A.Z-2, and that this selectivity can be mapped to an isoform-specific residue in its C-terminal tail. USP39 is a component of the U4/U6.U5 tri-snRNP, and consistent with a functional link between H2A.Z-1 and USP39, we identify a subset of shared alternative splicing events. Altogether, these data support functional diversification of H2A.Z through monoubiquitylation and isoform-specific amino acid substitution, and collectively, contribute to our understanding of biological pathways converging on H2A.Z.