engleski

Cooperative DNA binding of the homeodomain protein Pho2 and the basic-helix-loop-helix protein Pho4 is necessary to induce transcription at the yeast PHO5

Two transcription factors, the bHLH protein Pho4 and the homeodomain protein Pho2, are required for transcriptional activation of the PHO5 promoter in S. cerevisiae. There are two essential Pho4 binding sites, corresponding to regulatory elements UASp1 and UASp2 at the PHO5 promoter, but only a single, dispensable Pho2 binding site had previously been identified (Vogel et al., MCB 9, 2050 - 2057). 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 ternary complex, was demonstrated. Pho2 and Pho4 also bind DNA cooperatively at UASp2 where two Pho2 sites flank the Pho4 site (Barbarić et al., NAR 24, 4479 - 4486). These results suggest that cooperative DNA binding with Pho4 is integral to the mechanism by which Pho2 regulates transcription of the PHO5 gene. We have started to mutate single and multiple Pho2 binding sites around the UAS-elements and found correlations to the level of transcription in vivo. A mutation of the high affinity Pho2 site which largely overlaps the Pho4 binding site at UASp1, leads to a loss of cooperative DNA binding which in vivo correlates with a 2-3 fold decrease of transcriptional activity. Other mutations of moderate and weak Pho2 binding sites around UASp2 lead to a smaller decrease of transcriptional activity, but combining these mutations completely inactivates the PHO5 promoter. This is the first PHO5 promoter derivative inactivated by upstream cis mutations, which has complete intact Pho4 binding sites. Use of Pho4D200int, a Pho4 protein with an internal deletion (AA 201 - 246) of a domain which interacts in the two-hybrid system with Pho2 ( Hirst et al., EMBO J., 13, 5410 - 5420) leads to a dramatic loss of cooperative DNA binding at UASp1, but only a partial loss at UASp2, suggesting that protein binding and cooperativity are strongly dependent on the sequence of the DNA binding element and its microenvironment. These results demonstrate the in vivo importance of the intricate network of protein/protein and protein/DNA interactions that lead to the full transcriptional activation and chromatin remodeling of the PHO5 gene.

protein-DNA interactions; regulation of gene expression

nije evidentirano