Combinatorial repression of the hypoxic genes of Saccharomyces cerevisiae by DNA binding proteins Rox1 and Mot3

The hypoxic genes of Saccharomyces cerevisiae are transcriptionally repressed during aerobic growth through recruitment of the Ssn6/Tup1 general repression complex by the DNA binding protein Rox1. A second DNA binding protein Mot3 enhances repression of some hypoxic genes. Previous studies characterized the role of Mot3 at the hypoxic ANB1 gene as promoting synergy among one Mot3 site and two Rox1 sites comprising operator A of that gene. Here we studied the role of Mot3 in enhancing repression by Rox1 at another hypoxic gene, HEM13, which is less strongly regulated than ANB1 and has a very different arrangement of Rox1 and Mot3 binding sites. By assessing the effects of deleting Rox1 and Mot3 sites individually and in combination, we found that the major repression of HEM13 occurred through three Mot3 sites closely spaced with a single Rox1 site. While the Mot3 sites functioned additively, they enhanced repression by the single Rox1 site, and the presence of Rox1 enhanced the additive effects of the Mot3 sites. In addition, using a Rox1-Ssn6 fusion protein, we demonstrated that Mot3 enhances Rox1 repression through helping recruit the Ssn6/Tup1 complex. Chromatin immunoprecipitation assays indicated that Rox1 stabilized Mot3 binding to DNA. Integrating these results, we were able to devise a set of rules that govern the combinatorial interactions between Rox1 and Mot3 to achieve differential repression.