The Regulation of the Phosphatidate Phosphatase Gene <em>PAH1</em> and Its Regulatory Role on Cell Homeostasis

<p>The <em>Saccharomyces cerevisiae</em> gene, <em>PAH1</em>, 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 (UAS<em>_INO</em>)<em></em>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 <em>PAH1 </em>gene. In the first part of the study, we tried to evaluate how <em>PAH1</em> 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 <em>pah1∆</em> strains when exposed to different concentrations of the precursors, the <em>pah1∆</em> strain did tend to grow slightly better than WT in the presence of inositol. Furthermore, our RNA analysis showed that UAS<em>_INO</em> genes were upregulated in the <em>pah1∆</em> strain when compared to WT cells, strongly suggesting the role of <em>PAH1</em> as a negative regulator in the lipid biosynthesis pathway. Interestingly, the <em>HXK2</em> gene was the only gene tested that was downregulated in <em>pah1Δ </em>cells. Since <em>HXK2 </em>is involved in preventing apoptosis, this implied that Pah1p might be involved in cellular apoptosis. Additional growth experiments on <em>pah1∆</em> were conducted in the presence of acetic acid and hydrogen peroxide. Results showed that <em>pah1Δ </em>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 <em>pah1∆</em>. As such, this aim demonstrated that <em>PAH1 </em>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.</p> <p>For the second part of our study, we wanted to elucidate the conditions of <em>PAH1</em> induction, as well as the ways in which it itself is regulated. Our experiments showed that <em>PAH1 </em>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 <em>PAH1</em> 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 <em>PAH1</em> gene expression. Our results showed that Snf2p plays a role in <em>PAH1</em> gene induction and localizes at its promoter region. Interestingly, Snf2p is one of the chromatin remodelers important for <em>INO1 </em>regulation, one of the most crucial genes in inositol production, and a gene which is heavily regulated by <em>PAH1</em>. Taken together, these findings may indicate another mode by which <em>PAH1</em> can affect <em>INO1</em> expression; by possibly using the same remodeler to help keep gene expression in check. Overall, these finding give a better understanding of how <em>PAH1</em> gene regulation is controlled at the chromatin level.</p> <p>For the last part of our study, we decided to look into another organelle that <em>PAH1 </em>impacts, the vacuole. Since <em>PAH1</em> 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 <em>pah1Δ </em>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<em> </em>in <em>pah1Δ </em>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 <em>pah1Δ </em>cells, we performed qRT-PCR to measure gene expression. Results showed that the overexpression of genes like <em>FAB1 </em>and <em>ATG18</em>, which are crucial for normal fragmentation, in <em>pah1Δ </em>cells can be a contributing factor to the fragmented vacuole phenotype.</p> <p>Overall, our study has looked into three important areas in the cell with respect to <em>PAH1</em>. 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 <em>PAH1 </em>and can provide insights into the ways in which <em>PAH1</em> impacts cell homeostasis.</p>

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biology

Grantor

The Graduate School and University Center of The City University of New York

Year dc:date.available

2016

Author dc:creator

• Sherr, Goldie Libby

Advisor dc:contributor.advisor

• Chang-Hui Shen

Committee members dc:contributor.committeemember

• Jimmie Fata
• Eugenia Naro-Maciel
• Gary Wen
• Jonathan Blaize

• Biology
• Molecular Genetics
• PAH1
• gene regulation
• chromatin remodelers
• vacuole
• V-ATPase

Repository record dc:identifier

https://academicworks.cuny.edu/gc_etds/1599

OAI identifier oai:identifier

oai:academicworks.cuny.edu:gc_etds-2607