Characterizing vertebrate histone H2A.Z: acetylation, isoforms and function




Dryhurst, Deanna

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Histone H2A.Z is a highly conserved replication-independent histone variant that is essential for survival in diverse organisms including Tetrahymena thermophila, Drosophila melanogaster, Xenopus laevis, and Mus musculus. H2A.Z has been shown to play a role in many cellular processes including, but not limited to, gene expression, chromosome segregation, cell cycle progression, heterochromatin maintenance and epigenetic transcriptional memory. However, the mechanism by which H2A.Z and its post-translationally modified forms participate in these diverse cellular events and their subsequent effects on chromatin structure and function are not entirely clear. A thorough review of H2A.Z is provided in Chapter 1. We have isolated native non-acetylated and acetylated forms of H2A.Z and characterized nucleosome core particles (NCPs) reconstituted with these proteins using the analytical ultracentrifuge (Chapter 2). We report that NCPs reconstituted with native non-acetylated H2A.Z exhibit a slightly more compact conformation compared to those reconstituted with H2A. Furthermore, we show that acetylation of H2A.Z in conjunction with acetylation of the histone complement, results in NCPs that are less compact and less stable than H2A.Z-containing NCPs reconstituted with non-acetylated histones. Acetylated H2A.Z NCPs are nevertheless more compact and stable than acetylated H2A-containing NCPs. We have also identified the presence of two H2A.Z protein isoforms in vertebrates, H2A.Z-1 and H2A.Z-2, and characterized the sites and abundances of their N-terminal peptide acetylation. Further characterization of the human H2A.Z isoforms is presented in Chapter 3 and indicates that they are expressed across a broad range of human tissues, and that they exhibit a similar but non-identical distribution within chromatin. Our results suggest that H2A.Z-2 preferentially associates with H3 trimethylated at lysine 4 compared to H2A.Z-1, and the phylogenetic analysis of the promoter regions of H2A.Z-1 and H2A.Z-2 indicate that they have evolved separately during vertebrate evolution. Overall, these data suggest that the two isoforms of H2A.Z present in vertebrates may have acquired a degree of functional independence. In Chapter 4, we show that H2A.Z and an N-terminally acetylated form of H2A.Z associate with the prostate specific antigen (PSA) gene promoter and the levels of these proteins are reduced upon induction of the gene with androgen. Furthermore, H2A.Z protein levels increase in response to treatment with androgen which correlates with an increase in the mRNA expression levels of the H2A.Z-1 gene. Preliminary Western Blot and quantitative PCR analysis of H2A.Z (-1 and -2) levels in a tumor progression model of prostate cancer indicate that increased H2A.Z expression may be involved in the development of androgen independent prostate cancer. Collectively, our results contribute to our understanding of H2A.Z biology in vertebrates and support a role for this protein and its acetylated forms in poising promoter chromatin for subsequent gene transcription.



chromatin, histone variants