Characterization of the Schizosaccharomyces Pombe Hat1 Complex
Tong, Kevin. “Characterization of the Schizosaccharomyces Pombe Hat1 Complex”. PhD, Boston College, 2009. http://hdl.handle.net/2345/2222.
The Hat1 complex was characterized in S. pombe. Through tandem affinity purification and mass spectrometry, it was determined that Hat1 is associated with Mis16 (an orthologue of HAT2). Unlike HAT2 in S. cerevisiae, we confirm mis16 to be an essential gene in S. pombe. As expected, the S. pombe Hat1 complex was found to acetylate lysines 5 and 12 of histone H4. In contrast to budding yeast, deletion of hat1 alone resulted in the loss of telomeric silencing without concomitant mutations of the H3 N-terminal domain. Deletion of hat1 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 S. pombe. 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 in vivo. Our results support a model in which H2A/H2B exchange during transcription is mediated by the NAP1 chaperone.