Date of Degree

2-2016

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Chang-Hui Shen

Advisor

Alejandra Alonso

Committee Members

Alejandra Alonso

Yu-Wen Hwang

Sebastien Poget

Jonathan Blaize

Subject Categories

Biology | Genetics | Molecular Genetics

Keywords

chromatin remodelers, histone acetyltransferases, INO1, SNF2, INO80, gene regulation

Abstract

It is known that histone acetylases (HATs) regulate gene expression, but only recently have new functional implications about remodelers’ acetylation emerged. For instance, the HAT, Gcn5p, is capable of acetylating the catalytic subunit of the nucleosome remodeling complex SWI/SNF, Snf2p, which results in the dissociation of the complex from chromatin. The implications of this acetylation and subsequent dissociation have yet to be explored with regard to transcriptional regulation and other possible mechanisms. To further understand the implications of remodeler acetylation, I used a yeast model system examining the expression of the inositol-3-phosphate synthase gene INO1. Through chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) assays, I demonstrated that remodeler Snf2p acetylation is required for dissociation of the subunit from the INO1 promoter. Furthermore, employing growth curve and mRNA analyses, along with ChIP targeting of nucleosomal components and polymerase, I showed this acetylation was not required for INO1 transcriptional activation. However, I observed that the chromatin remodeler, Ino80p, was unable to dissociate from the INO1 promoter in the absence of Snf2p acetylation. Even though HAT Gcn5p recruitment remained unaffected, acetylase Esa1p recruitment significantly decreased in the absence of Snf2p acetylation. Although a lack of Snf2p acetylation did not significantly impact the transcription of INO1, it did modify the occupancy/ recruitment of remodeler Ino80p and HAT Esa1p at the promoter region, suggesting Snf2p acetylation may promote the recycling of both chromatin remodelers.

In an attempt to understand the biological implications of Snf2p acetylation, I performed sensitivity assays. Our results showed that while DNA damage/replication, osmoregulation, and carbon source utilization were unaffected by acetylation, protection from copper toxicity was significantly impacted. In conditions where chromatin remodelers were highly concentrated at the INO1 promoter (ie unAcSnf2p cells in the absence or presence of inositol, as well as WT cells in the presence of inositol) copper toxicity defense was hindered, which suggests the possibility a novel link between INO1 and CUP1 expression, and remodeler recycling, previously unidentified. Since remodelers SWI/SNF, ISWI, and RSC are capable of being acetylated by HATs, I now demonstrated this acetylation could have significant distinct impact on pathways regulated by SWI/SNF.

I then expanded our study to INO80, which had yet to be shown as an acetylated remodeler. I demonstrated that Ino80p was acetylated, but lacked this acetylation when the DNA-binding HSA domain was deleted, which correlates to studies of other acetylated remodelers in that they tend to be acetylated in regions involved in DNA-binding. This may help explain why Ino80p accumulates at the INO1 promoter in either HAT mutant strain, gcn5∆ or esa1mt. To better characterize this acetylation, I then engineered a series of INO80-FLAG-tagged HAT deletion mutants and identified that HAT Esa1p was responsible for acetylating Ino80p. Furthermore, I found that the lack of Ino80p acetylation may cause a defect in DNA repair. As such, our findings for Snf2p and Ino80p have revealed insight into the mechanism of chromatin remodeler acetylation and its implications in gene expression regulation.

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