Genetic and epigenetic regulation of the cell wall glycoproteins, Flo10p and Flo11p, generate phenotypic variation in S. cerevisiae

Abstract

Many organisms, in response to selective pressures imposed by their environment, have evolved mechanisms that allow them to generate phenotypic variation. Such phenotypic variation can result from genetic regulation, in which changes in DNA sequence produce the variant phenotype, or epigenetic regulation, in which there are no changes in DNA sequence associated with the variant phenotype. This doctoral thesis describes the identification and analysis of novel phenotypic variation among populations of the bakers' yeast, Saccharomyces cerevisiae. This phenotypic variation, most easily identified as a switching between smooth and wrinkled colony morphologies, involves changes in several adhesive and morphological phenotypes. Experiments reveal that this phenotypic switch is the result of both genetic and epigenetic regulation. The genetic component of this phenotypic variation involves mutation at either of the two yeast Ras-GAP encoding genes, IRA] and IRA2. The IRA genes are hot spots for mutation, as loss-of-function mutations at these genes are much more frequent than mutations at other loci (- 10-6). Several factors regulate the genetic stability of these genes, including DNA double-strand break (DSB) repair pathways. Genetic analysis demonstrates that both homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways of DSB repair maintain genetic stability at the IRA loci. Since these pathways specifically process a DSB substrate, this suggests that directed DSB formation may be the initiating event for IRA+ to ira- switching. ira- mutations activate transcription of the yeast cell wall glycoprotein genes, FLOO0 and FLO11, producing the variant adhesive and morphological phenotypes described above.

(cont.) The epigenetic regulation of this phenotypic variation acts via the expression of FLOO and FLOl]. In addition to activation by ira' mutations, these FLO genes are regulated by epigenetic silencing, resulting in the variegated expression of both genes in a clonal population of cells. Silencing at FLOlO is regulated by the histone deacetylase (HDAC) proteins Hstlp and Hst2p, whereas silencing at the FLO]l gene requires the HDAC Hdalp. In addition, silencing of FLOO1 and FLOll is dependent on their positions in the genome, suggesting that their locations are relevant to their regulation and function. Epigenetic silencing also regulates differentiation. Upon nitrogen starvation, diploid S. cerevisiae strains will undergo a developmental transition from yeast to pseudohyphal forms, which is regulated by silencing at the FLO1] locus. In summary, our analysis of phenotypic variation in S. cerevisiae provides a new perspective into both the genetic and epigenetic mechanisms for generating diversity in eukaryotic organisms.