EPIGENETIC SWITCHING OF TRANSCRIPTIONAL STATES - CIS-ACTING AND TRANS-ACTING FACTORS AFFECTING ESTABLISHMENT OF SILENCING AT THE HMR LOCUS IN SACCHAROMYCES-CEREVISIAE

In this study, we used the ADE2 gene in a colony color assay to monito r transcription from the normally silent HMR mating-type locus in Sacc haromyces cerevisiae. This sensitive assay reveals that some previousl y identified cis- and trans-acting mutations destabilize silencing, ca using genetically identical cells to switch between repressed and dere pressed transcriptional states. Deletion of the autonomously replicati ng sequence (ARS) consensus element at the HMR-E silencer or mutation of the silencer binding protein RAP1 (rap1s) results in the presence o f large sectors within individual colonies of both repressed (Ade-, pi nk) and derepressed (Ade+, white) cells. These results suggest that bo th the ARS consensus element and the RA-P1 protein play a role in the establishment of repression at HMR. In diploid cells, the two copies o f HMR appear to behave identically, suggesting that the switching even t, though apparently stochastic, reflects some property of the cell ra ther than a specific event at each HMR locus. In the ADE2 assay system , silencing depends completely upon the function of the SIR genes, kno wn trans-acting regulators of the silent loci, and is sensitive to the gene dosage of two SIR genes, SIR1 and SIR4. Using the ADE2 colony co lor assay in a genetic screen for suppressors of rap1s, silencer ARS e lement deletion double mutants, we have identified a large number of g enes that may affect the establishment of repression at the HMR silent mating-type locus.