GSBS Dissertations and Theses

Approval Date

June 1994

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Molecular Genetics and Microbiology

Subjects

Centromere; DNA-Binding Proteins; Saccharomyces cerevisiae; Academic Dissertations; Dissertations, UMMS

Abstract

CP1 (encoded by the gene CEP1) is a sequence-specific DNA-binding protein of Saccharomyces cerevisiae that recognizes a sequence element (CDEI) found in both yeast centromeres and gene promoters. Strains lacking CP1 are viable but exhibit defects in growth, chromosome segregation, and methionine biosynthesis. To investigate the basis of the methionine requirement, a YEp24-based yeast genomic DNA library was screened for plasmids which suppressed the methionine auxotrophy of a cep1 null mutant. The suppressing plasmids contained either CEP1 or DNA derived from the PHO4 locus. PHO4 encodes a factor which positively regulates transcription of genes involved in phosphate metabolism via an interaction with CDEI-like elements within the promoters of these genes. Subcloning experiments confirmed that suppression correlated with increased dosage of PHO4. PHO4c, pho80, and pho84 mutations, all of which lead to constitutive activation of the PHO4 transcription factor, also suppressed cep1 methionine auxotrophy. The suppression appeared to be a direct effect of PHO4, not a secondary effect of PHO regulon derepression, and was dependent on a second transcriptional regulatory protein encoded by PHO2. Spontaneously arising extragenic suppressors of the cep1 methionine auxotrophy were also isolated; approximately one-third of the them were alleles of pho80. While PHO4 overexpression suppressed the methionine auxotrophy of a cep1 mutant, CEP1 overexpression failed to suppress the phenotype of a pho4 mutant; however, a cep1 null mutation suppressed the low-Pi growth deficiency of a pho84 mutant. The results suggest that CP1 functions as a transcriptional regulator of MET genes, and that activation of PHO4 restores expression to those genes transcriptionally-disabled by the cep1 mutation. The results also suggest the existence of a network that cross-regulates transcription of genes involved in methionine biosynthesis and phosphate metabolism.

A direct molecular approach to investigate CP1's role in MET gene expression was also taken. CDEI sites are associated with the promoter regions of most MET genes, but only MET16, the gene encoding PAPS reductase, has been shown to require CP1 for expression; both PAPS reductase activity, and MET16 mRNA are absent in cep1 mutants. Results of the present study demonstrate that CP1 participates in two systems which regulate expression of MET16, one triggered by methionine starvation and requiring the transactivator MET4 (pathway-specific control), and the other triggered by starvation for many different amino acids and requiring GCN4 (general control). CP1 was shown to mediate its regulatory function through the upstream CDEI site, and to act directly or indirectly to modulate the chromatin structure of the MET16 promoter. In addition, the pho80 mutation was found to partially restore MET16 expression to the cep1 strain, confirming the proposed nature of PHO4 suppression. A second methionine biosynthetic gene MET25, was also analyzed. Like MET16, MET25 was found to be regulated by both pathway-specific and general control mechanisms, but in contrast to MET16, CP1 only participated in the pathway-specific response of this gene. The results demonstrate that CP1, possibly by modulating changes in chromatin structure, assists the regulatory proteins MET4 and GCN4 in activating transcription of MET genes.

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