| Post-translational modification of histones and histone variants are among the conserved mechanisms for regulating chromatin dynamics and chromatin based processes. This thesis centered on the role of histone phosphorylation and histone variants in chromatin functions in Tetrahymena thermophila .;With detailed chromatin immunoprecipitation analyses, promoter mapping and functional studies, I demonstrated that unphosphorylated H1 was specifically localized to a restricted region of the CDC2 promoter that is essential for CDC2 expression only during starvation when CDC2 is down-regulated, but not in log phase CDC2 is highly expressed. These studies are the first demonstration that the phosphorylation state of H1 can be targeted to a specific promoter where it likely regulates transcription in a gene-specific manner.;I identified an essential, divergent H2A gene, HTAY, in the newly sequenced Tetrahymena genome and characterized its in viva functions. I demonstrated that this H2A gene encodes a chimeric histone H2A.Y consisting of a leucine-rich repeat (LRR) domain that has homology to a protein phosphatase 1 regulator, and a histone fold domain. I showed that H2A.Y has multiple functions, including regulation of transcription of a subset of genes, maintenance of nuclear integrity and dephosphorylation of mitosis-associated histone H3 S10 after mitosis, providing a striking case of "cross-talk" between a H2A variant and a specific post-translational modification of another histone, as well as the first example of a species-specific H2A variant which functions in gene regulation in vivo.;Collaborated with Qinghu Ren and Elizabeth Goneska (Dr. David Allis's lab), we found that one of the major histone H2As in Tetrahymena, H2A.X, is phosphorylated at serine residue 134 in the highly conserved SQ motif (to produce an isotope known as gamma-H2A.X) in response to DSBs induced by chemical agents and during meiosis. Using an antibody specific to gamma-H2A.X, together with a mutant strain (S134A) that abolished this phosphorylation site, we demonstrated that absence of phosphorylation of the SQ motif leads to accumulation of DSBs in macro- and micronuclei during gamma-H2A.X is important for repairing exogenous DNA damage, for normal micronuclear meiosis and mitosis, and to a lesser extent, for normal macronuclear division of Tetrahymena cells. |
