Date of Award
Doctor of Philosophy (PhD)
Cell & Molecular Biology
Shelley L. Berger
DNA exists within the cell as part of a complex structure called chromatin which is comprised of many proteins, including histones, and participates in and influences every DNA-related process. Chromatin's proteins are modified post-translationally and this impacts their functions and in turn, the DNA processes in which they participate. However, the repertoire of post-translational modifications (PTMs), the enzymes that create and remove them, and their roles in chromosome biology are not fully understood. We have used the budding yeast Saccharomyces cerevisiae to investigate chromatin PTMs, specifically lysine modifications, through three avenues: the enzymes that regulate lysine PTMs, the histone chromatin lysine PTMs, and the non-histone chromatin lysine PTMs. First, we demonstrated that the JmjC domain-containing protein Kdm5 demethylates methylated lysine 4 of histone H3 in vitro and in vivo, refuting the longstanding hypothesis that lysine methylation is irreversible. Second, we confirmed and characterized monomethylation of lysine 20 of histone H4. Its abundance is highest at heterochromatic locations including rDNA, the silent mating type loci, and subtelomeres, lowest at euchromatic locations including centromeres and promoter/5' regions of genes, and intermediate inside genes. We observed a correlation between the locations of H4 K20A-mediated derepression and H4 K20me1 enrichment. Additionally, H4 K20me1 decreases globally during replicative ageing and may participate in survival during DNA damage. Our results refute the longstanding hypothesis that this PTM is not conserved in S. cerevisiae and potentially identify the first repressive budding yeast histone lysine methylation. Third, we investigated NuA4's lysine acetylation of the chromatin protein Spt16, identified by a previously published in vitro acetyltransferase screen. Mass spectrometry identified three acetylation sites in vivo and their substitution with unacetylatable arginines, but not the acetyllysine mimics glutamine, causes moderate heat-sensitivity. Our results suggest that Spt16 acetylation occurs in vivo and is necessary for heat-tolerance.
Edwards, Christopher Richard, "Lysine Post-Translational Modifications of Saccharomyces Cerevisiae Chromatin Proteins" (2012). Publicly Accessible Penn Dissertations. 504.