Investigating the mechanism of the Tup1-Cyc8 (Ssn6) Co-Repressor complex in the yeast Saccharomyces cerevisiae

The Tup1-Cyc8 (Ssn6) complex is a powerful epigenetic repressor of genes in the yeast Saccharomyces cerevisiae. The highly conserved complex brings about a repressive chromatin structure at regulatory regions of its target genes or prevents the recruitment of factors needed for activation of transcription. A gap in the current understanding is whether each of the subunits contribute differently to repression. The FLO family of genes are repressed by the Tup1-Cyc8 complex, these genes encode the proteins required for flocculation, a stress response in yeast where the cells aggregate, or form flocs, to protect cells within the floc. Interestingly, each mutant strain has a distinct flocculant phenotype. The tup1Δ strain displays large, dense flocs compared to smaller, more dispersed flocs associated with the cyc8Δ strain. RT-qPCR showed that FLO1 is highly de-repressed in the tup1Δ strain whereas it is de-repressed to a significantly lower level in the cyc8Δ strain. Using the Anchor Away (AA) technique, which allows for a nuclear protein to be conditionally sequestered to the cytoplasm, I am investigating differences in the sequence of events at the FLO1 promoter when Tup1p or when Cyc8p is removed from the nucleus. Six hours after Cyc8p is removed from the nucleus transcription of FLO1 almost reaches the maximum transcription seen in the cyc8Δstrain. However, six hours after removing Tup1p the level of transcription of FLO1 is still over ten times lower than the maximum transcription in tup1Δ. This difference indicates that each of the subunits have independent functions within the complex.