The roles of histone H3 methylation in gene regulation, heterochromatin assembly, and DNA elimination in Tetrahymena thermophila

Eukaryotic chromatin is organized into regions of transcriptionally-active euchromatin and transcriptionally-silent heterochromatin, states that can be propagated in an epigenetic fashion, independent of their DNA sequence. Recent studies suggest that cells can use post-translational histone modifications as a mechanism to transmit this information to the chromatin of subsequent generations. In this dissertation, I present evidence that H3 histone methylation on lysine 9 [methyl(Lys9)H3] serves as such a mark for heterochromatin-dependant processes. I also discuss a different H3 methylation mark lysine 4 [methyl(Lys4)H3], and its role in transcription.; In chapter 1, I determine that methyl(Lys9)H3 acts as an mark for heterochromatin-dependent gene silencing. Through a variety of biophysical studies, I demonstrate that this effect is mediated by the specific binding of the modification to the chromodomain of the heterochromatin-associated protein HP1. In chapter 2, I show that Tetrahymena has adapted this interaction for use in specialized heterochromatic environments wherein chromodomain-containing machinery, Pddps (programmed DNA degradation), eliminate specific regions of DNA in a developmentally regulated fashion. Through this research, heterochromatin formation and Lys9 methylation were also linked to the RNA interference pathway, an evolutionarily conserved dsRNA dependent mechanism of posttranscriptional gene silencing thought to protect against viral invasion. In chapter 3, I examine the correlation of methyl(Lys4) with transcriptional activation. Based on studies in Tetrahymena I suggest distinct roles for the mono-, di-, and tri-methylation that can occur on this one lysine. Furthermore, I suggest a mechanistic explanation for why some assays for methyl(Lys4) function seem to suggest a link to gene silencing, based on drug sensitivity of yeast strains mutated in pathways linked to these methylation events. Future biochemical experiments involving Tetrahymena and yeast should further address the biological role of methyl(Lys4).