Date of Award

12-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Ronald N. Laribee

Committee

Mondira Kundu Janet F. Partridge Lawrence M. Pfeffer Zhaohui Wu

Keywords

Chromatin, Epigenetics, Histone acetylation, Sirtuins, Sit4, Target of Rapamycin

Abstract

The gene expression profile of a eukaryotic cell is responsive to a variety of extracellular stimuli, including nutrient availability, which allows cells to toggle between anabolism and catabolism based on the favorability of their environment. Much of this information is relayed through signaling complexes, such as the target of rapamycin complex 1 (TORC1), to downstream chromatin modifying enzymes. These enzymes impact the gene regulatory process through altered histone post-translation modifications, changes in chromatin structure, and docking of chromatin regulatory complexes. Yet, despite preliminary studies suggesting that TORC1 affects epigenetic mechanisms, including histone H3 lysine 56 acetylation (H3K56ac), almost nothing is known about how the complex functions in this regard. In this report, we demonstrate that inhibition of TORC1 results in a site-specific reduction in acetylation on N-terminal residues of both histone H3 and H4. This effect is dependent on sirtuin histone deacetylases (HDACs), as inactivation of these enzymes, specifically Hst4, rescues the acetylation defect. We also find that this sirtuin-mediated deacetylation response requires a functional protein phosphatase 6 complex (PP6). PP6 is under direct negative regulation of TORC1, and relief of this inhibition initiates a rapid cytoplasmic to nuclear redistribution of Hst4 which correlates temporally with our observed loss of histone acetylation. The nuclear accumulation of Hst4 precedes an increase in Hst4 protein levels that occurs due to a reduction in Hst4 turnover. Notably, deletion of a subset of sirtuins (hst3Δ or hst4Δ) rescued the sensitivity of a non-essential TORC1 mutant (tco89Δ) to an array of TORC1 inhibitors. This result suggests the link between TORC1 and acetylation may play an essential role in cell cycle regulation and the DNA damage response. We further evaluated whether these TORC1-mediated acetylation marks contribute to the chromatin association of high mobility group proteins (HMGs). And while TORC1-dependent displacement of the HMGs coincides with vacuolar acidification, hyperactivation of TORC1, and significant cell death, it appears to occur independently of TORC1’s regulation of Hst4. We conclude by investigating mitochondrial function in a tco89Δ mutant and mapping the functional domains of Tco89 necessary to sustain TORC1 activity and respond to extracellular stress.

ORCID

http://orcid.org/0000-0002-9071-5207

DOI

10.21007/etd.cghs.2016.0577

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