Date of Degree

9-2016

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Chang-Hui Shen

Committee Members

Jimmie Fata

Eugenia Naro-Maciel

Gary Wen

Jonathan Blaize

Subject Categories

Biology | Molecular Genetics

Keywords

PAH1, gene regulation, chromatin remodelers, vacuole, V-ATPase

Abstract

The Saccharomyces cerevisiae gene, PAH1, encodes a phosphatidate (PA) phosphatase that plays a fundamental role in lipid metabolism. PA phosphatases are key enzymes that catalyze the PA dephosphorylation reaction to form diacylglycerides, the first step in the synthesis of triacylglycerols. Pah1p, one of the main PA phosphatases in yeast, has not only emerged as a key player in lipid biosynthetic pathways, but also acts as an important regulator of nuclear membrane biogenesis, the transcriptional regulation of many inositol-sensitive upstream activating sequence (UASINO)containing genes needed for phospholipid synthesis, vacuole homeostasis, and lipid droplet formation. Due to its crucial role in lipid and overall cell homeostasis, this thesis aimed to elucidate the role and regulation of the PAH1 gene. In the first part of the study, we tried to evaluate how PAH1 affects other genes in the lipid biosynthetic pathway as well as assess the impact that the crucial phospholipid precursors, inositol and choline, have on cellular growth. Results showed that while there was not a large difference between the growth rates of WT and pah1∆ strains when exposed to different concentrations of the precursors, the pah1∆ strain did tend to grow slightly better than WT in the presence of inositol. Furthermore, our RNA analysis showed that UASINO genes were upregulated in the pah1∆ strain when compared to WT cells, strongly suggesting the role of PAH1 as a negative regulator in the lipid biosynthesis pathway. Interestingly, the HXK2 gene was the only gene tested that was downregulated in pah1Δ cells. Since HXK2 is involved in preventing apoptosis, this implied that Pah1p might be involved in cellular apoptosis. Additional growth experiments on pah1∆ were conducted in the presence of acetic acid and hydrogen peroxide. Results showed that pah1Δ cells fared significantly better than WT cells after exposure to these apoptotic reagents. RNA analysis confirmed these results, showing a significant upregulation of anti-apoptotic genes in pah1∆. As such, this aim demonstrated that PAH1 plays an important regulatory role in phospholipid biosynthesis and suggested that it also plays a role in apoptosis by regulating anti-apoptotic genes as well.

For the second part of our study, we wanted to elucidate the conditions of PAH1 induction, as well as the ways in which it itself is regulated. Our experiments showed that PAH1 undergoes induction during the stationary phase of growth when inositol is present. These findings were subsequently used to help determine the chromatin remodelers involved in PAH1 gene expression. Chromatin remodelers play a significant role in modifying and restructuring the nucleosome and therefore play an essential role in the regulation of gene expression. Using growth curve analysis, qRT-PCR, and ChIP, we set out to determine which chromatin remodelers impact PAH1 gene expression. Our results showed that Snf2p plays a role in PAH1 gene induction and localizes at its promoter region. Interestingly, Snf2p is one of the chromatin remodelers important for INO1 regulation, one of the most crucial genes in inositol production, and a gene which is heavily regulated by PAH1. Taken together, these findings may indicate another mode by which PAH1 can affect INO1 expression; by possibly using the same remodeler to help keep gene expression in check. Overall, these finding give a better understanding of how PAH1 gene regulation is controlled at the chromatin level.

For the last part of our study, we decided to look into another organelle that PAH1 impacts, the vacuole. Since PAH1 has been previously documented to influence vacuole morphology by regulating the proteins involved in vacuole fusion, we wanted to determine if its presence also affects proper vacuole homeostasis, particularly the maintenance of acidification and the function of V-ATPase pumps. Using electron microscopy, we determined the vacuolar phenotype of pah1Δ cells, which consisted of fragmented vacuoles in both exponential and stationary phases of growth. This was followed by RNA analysis of V-ATPase genes. Our results demonstrated that all genes remained at similar or greater levels than WT in pah1Δ cells, suggesting that V-ATPase pump activity is not implicated despite the morphological defect. Growth experiments and vacuolar pH measurements confirmed this finding. In order to see if other important genes involved in vacuole fission and fusion were potential contributors to the mutant phenotype in pah1Δ cells, we performed qRT-PCR to measure gene expression. Results showed that the overexpression of genes like FAB1 and ATG18, which are crucial for normal fragmentation, in pah1Δ cells can be a contributing factor to the fragmented vacuole phenotype.

Overall, our study has looked into three important areas in the cell with respect to PAH1. We have elucidated the ways in which it impacts the lipid biosynthetic pathway, the modes in which it itself is regulated, and the influence it has on vacuolar morphology and function. These findings can be used to further understand the functional role of PAH1 and can provide insights into the ways in which PAH1 impacts cell homeostasis.

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