The mating-type loci located at the ends of chromosome III in Saccharomyces cerevisiae are transcriptionally repressed by a region-specific but sequence-nonspecific silencing apparatus, mediated by cis-acting silencer sequences. Previous deletion analyses have defined the locations and organizations of the silencers in their normal context and have shown that they are composed of various combinations of replication origins and binding sites for specific DNA-binding proteins. We have evaluated what organization of silencer sequences is sufficient for establishing silencing at a novel location, by inserting individual silencers next to the MAT locus and then assessing expression of MAT. The results of this analysis indicate that efficient silencing can be achieved by inserting either a single copy of the E silencer from HMR or multiple, tandem copies of either the E or I silencer from HML. These results indicate that while all silencers are functionally equivalent, they have different efficiencies; HMR E is more active than HML E, which itself is more active than HML I. Both HMR E and HML E exhibit orientation-dependent silencing, and the particular organization of binding elements within the silencer domain is critical for function. In some situations, silencing of MAT is conditional: complete silencing is obtained when cells are grown on glucose, and complete derepression occurs when cells are shifted to a nonfermentable carbon source, a process mediated in part by the RAS/cyclic AMP signaling pathway. Finally, the E silencer from HMR is able to reestablish repression immediately upon a shift back to glucose, while the silencers from HML exhibit a lung lag in reestablishing repression, thus indicating distinctions between the two silencers in their reestablishment capacities. These results demonstrate that silencers can serve as nonspecific gene inactivation centers and that the attendant silencing can be rendered responsive to potential developmental cues.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology