Investigating the role of Histone Post Translational Modifications in yeast Stationary Phase

Abstract

The aim of this thesis was to determine how the chromatin modifications, histone H2B ubiquitination (H2Bub) and H3 K36 methylation (H3K36me), influence the development and maintenance of cellular quiescence in the yeast, Saccharomyces cerevisiae. Single and double mutants deficient for H2Bub (htb-K123R�) and H3K36me (hht-K36A�) were therefore characterised during growth into stationary phase, during which time cellular quiescence normally develops. The aim was to test the hypothesis that H2Bub and H3K36me are required to ensure the proper transcriptional pathways occur to establish quiescent cells and maintain their transcriptional silencing and survival in the absence of glucose. A clear growth delay was seen due to loss of H3K36me, whereas even slower proliferation and lower final cell densities in rich medium were seen in the double mutant. However, glucose uptake was unaffected in any of the mutants. Hypersensitivity to DNA damage, cell wall stress, and osmotic shock was shown by the double mutant in stationary phase. All core histone proteins levels were similar to wild-type levels in each mutant during exponential growth and at the diauxic shift. However, H3 was abnormally elevated in stationary phase double mutant cells suggesting H2Bub and H3K36me contribute to correct histone stoichiometry after prolonged glucose depletion. Increased linker histone H1 (Hho1p) levels were seen during exponential growth in all mutants, suggesting that in transcriptionally active conditions these marks normally restrain H1 abundance. Together, these data suggest a role for H2Bub and H3K36me in maintaining correct chromatin structure during growth into stationary phase. High H3K14ac, H4ac4, and H3K9ac during exponential growth and repression of these marks in stationary phase was seen in wild-type cells. However, elevated H3K14ac and H4Ac4 levels were seen in all mutants during exponential growth, indicating hyperacetylation when H2Bub and/or H3K36me are lost. Furthermore, in stationary phase, the double mutant fails to repress these marks, indicating defective establishment of a repressed chromatin state occurs in the absence of both H2Bub and H3K36me. Stationary-phase development was perturbed in all mutants. However, a bias toward increased quiescent (Q) cell formation was seen in the H2Bub-deficient mutant and the population of quiescent cells that were formed showed progressive viability loss over time. Conversely, the non-quiescent (NQ) cells formed in the H2Bub-deficent mutant showed increased survival. This suggests a role for H2Bub in potentially opposing apoptosis in quiescent cells and promoting apoptosis in the non-quiescent cell population. Conversely, H3K36me loss resulted in a bias toward elevated NQ cell formation and reduced viability in both Q and NQ pools. A blurred Q/NQ identity was seen in the double mutant highlighting synergistic disruption of the chromatin-governed quiescence program. RNA-Seq analysis of day 7 purified quiescent cells revealed that the H3K36me-deficient mutant showed the greatest numbers of up and downregulated genes compared to the H2Bub-deficient mutant. However, the impact upon gene activation and repression in the H3K36me-deficient mutant was reduced when the site for H2Bub was additionally mutated in the double mutant. This suggests a dominant role for H3K36me in governing activation and repression of gene transcription in quiescent cells and that H2Bub contributes to the de-repression and repression of transcription evident in the absence of H3K36me. Overall, the data suggest that there is a complex non-additive epistasis occurring between H2Bub and H3K36me which is required to properly remodel chromatin in quiescence and correctly regulate transcription.