Thesis advisor: Anthony T. Annunziato / The histone acetyltransferase Hat1 is an enzyme that specifically acetylates newly synthesized histone H4 at positions K5 and K12 (or their homologous positions) in all eukaryotes. In Schizosaccharomyces pombe, the deletion of hat1 presents a mutant phenotype. The telomeres in a hat1-del strain become permissive for transcription, as analyzed by a telomeric ura4 marker gene. In this study, we evaluate the efficacy of high copy suppression of this hat1 deletion. Due to high-frequency recombination events in the telomere, it became necessary to create a hat1-rad52 double deletion strain that also contains a telomeric ura4 reporter. High copy suppressor screens for recovery of telomeric silencing yielded several promising transformants. Multiple rounds of testing were performed to assess the recovery of transcriptional repression at the telomere. It was found that despite the anti-recombination effect of deleting rad52, the ura4 reporter was still lost from the telomere through recombination. Additional observation of the hat1-del rad52-del ura4-tel strain revealed a significant synthetic slow-growth phenotype. The double mutant displays a greatly decreased growth rate compared to hat1-del, as well as increased cellular length. Further study showed unique phenotypes on various media, and gene expression studies showed unique patterns of regulation in this double mutant when compared to both a wild- type and its single mutant counterparts (hat1-del, rad52-del). In summary, the telomeric ura4 marker in a hat1-del strain of S. pombe is not stable and is lost by recombination at a high frequency. This has led to the discovery of a double mutant (hat1-del rad52-del) that displays a severe synthetically sick phenotype. / Thesis (MS) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Anthony T. Annunziato / Thesis advisor: Charles Hoffman / The Hat1 complex was characterized in <italic>S. pombe</italic>. Through tandem affinity purification and mass spectrometry, it was determined that Hat1 is associated with Mis16 (an orthologue of HAT2). Unlike HAT2 in <italic>S. cerevisiae</italic>, we confirm <italic>mis16</italic> to be an essential gene in <italic>S. pombe</italic>. As expected, the <italic>S. pombe</italic> Hat1 complex was found to acetylate lysines 5 and 12 of histone H4. In contrast to budding yeast, deletion of <italic>hat1</italic> alone resulted in the loss of telomeric silencing without concomitant mutations of the H3 N-terminal domain. Deletion of <italic>hat1</italic> caused an increase of H4 acetylation at telomeres. Additionally, the hyperacetylation of histones also results in the loss of telomeric silencing. Loss of Hat1 did not affect silencing at the inner most repeat (imr) or outer repeat (otr) regions of the centromere, but did appear to increase silencing at the central core region (cnt) of the centromere. The experiments described herein demonstrate Hat1 to be essential for the establishment of proper telomeric silencing in fission yeast, and suggest that the timely acetylation of H4 during chromatin assembly is a unique factor in generating the correct epigenetic state at telomeres in <italic>S. pombe</italic>. Additionally, Hat1 and its acetylation of new H4 may have entirely different roles during telomeric silencing than during silencing at the centromeric central core. Our studies in HeLa cells demonstrated that transcription is involved in the exchange of H2A/H2B in acetylated chromatin regions. The finding that cytosolic H2A can be acetylated at lysine 5 is the first demonstration that cytosolic H2A can be specifically modified <italic>in vivo</italic>. Our results support a model in which H2A/H2B exchange during transcription is mediated by the NAP1 chaperone. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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