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Date of Award
The basic helix-loop-helix (bHLH) family of proteins is among the most thoroughly studied and functionally important protein classes in eukaryotes. Members of this protein family are known to regulate gene expression at key steps in a variety of developmental and metabolic processes in a wide array of organisms. The INO2 gene of the yeast Saccharomyces cerevisiae encodes a member of the bHLH family. The INO2 gene product, Ino2p, heterodimerizes with another bHLH family member (Ino4p) and activates transcription of several genes in the phospholipid biosynthetic pathway in response to inositol deprivation by binding the UASINO promoter element (5'-CATGTGAAAT-3') found in their promoters. Expression of INO2 is itself auto-regulated and induced in response to inositol deprivation. Induction of INO2 is required for transcription of target genes such as INO1, CHO1 and CHO2. However, transcription of target genes is still repressed by the presence of inositol when cells express INO2 constitutively. This suggests that repression of transcription in response to inositol is mediated by a post-transcriptional mechanism. Ino2p is a likely candidate for this regulation because its expression is highly regulated and it is multiply phosphorylated. Therefore, the goal of this dissertation is to investigate the mechanisms by which Ino2p exerts its control on the phospholipid biosynthetic genes in response to inositol. Using ChIP (Chromatin I&barbelow;mmunop&barbelow;recipitation) analysis, we have identified that the presence of inositol does not inhibit the ability of the Ino2p:Ino4p heterodimer to bind the UASINO promoter element. Additionally, we have determined that significant levels of Ino2p are present in the cell during the time-period that INO1 transcription is repressed in response to the addition of inositol to the growth medium. Together, these results suggest that Ino2p is involved in the inositol-mediated repression mechanism. Further support for this hypothesis lies in the fact that we were able to isolate five mutations in Ino2p which cause deficiencies in inositol-mediated repression. Lastly, a study was conducted to identify whether the Ino2p:Ino4p heterodimer has an orientation binding preference for its interaction with the non-palindromic UASINO promoter element. The results suggest that the Ino2p:Ino4p heterodimer binds the UASINO promoter element in either of the two possible orientations. Therefore, the work presented in this dissertation furthers our understanding of the role of both Ino2p and the Ino2p:Ino4p heterodimer in inositol-mediated regulation of the phospholipid biosynthetic genes.
Gardenour, Kyle R., "Analysis of the function of INO2p, a basic helix-loop-helix protein in Saccharomyces cerevisiae" (2003). Wayne State University Dissertations. 3319.