The homeodomain protein Pho2 and the basic-helix-loop-helix protein Pho4 bind DNA cooperatively at the yeast PHO5 promoter

Two transcription factors, the bHLH protein Pho4 and the homeodomain protein Pho2, are required for transcriptional activation of the PHO5 promoter in Saccharomyces cerevisiae, There are two essential Pho4 binding sites, corresponding to the regulatory elements UASp1 and UASp2 at the PHO5 promoter, but only a single, dispensable Pho2 binding site had previously been identified, We have reinvestigated binding of Pho2 to the PHO5 promoter using purified recombinant protein and have found multiple Pho2 binding sites of different affinities along the promoter. One of the high affinity Pho2 sites largely overlaps the Pho4 binding site at UASp1, Cooperative DNA binding of the two proteins to their overlapping sites, resulting in a high-affinity ternary complex, was demonstrated, Pho2 and Pho4 also bind DNA cooperatively at UASp2 where two Pho2 sites flank the Pho4 site, Finally, Pho2 facilitates binding of Pho4 to a third, cryptic Pho4 binding site which binds Pho4 with lower affinity than UASp1 or UASp2, These results suggest that cooperative DNA binding with Pho4 is integral to the mechanism by which Pho2 regulates transcription of the PHO5 gene.