The transcriptional activators of the PHO regulon, Pho4p and Pho2p, interact directly with each other and with components of the basal transcription machinery in Saccharomyces cerevisiae.

The transcriptional regulators Pho4p and Pho2p are involved in transcription of several genes in the PHO regulon of Saccharomyces cerevisiae. Genetic evidence with temperature-sensitive pho4 and pho2 mutants suggested that Pho4p and Pho2p interact with each other. Immunoprecipitation experiments showed that Pho4p and Pho2p form a complex on a 36-bp sequence bearing an upstream activation site (UAS) and protein binding assays indicated that these proteins interact directly. DNA-binding experiments with crude extracts prepared from yeast strains expressing T7-PHO4, encoding Pho4p tagged with the T7 epitope, indicated that Pho2p interacts with T7-Pho4p and enhances the binding affinity of T7-Pho4p to the UAS. Protein binding experiments also showed that both Pho4p and Pho2p could bind with the general transcription factors, TBP, TFIIB, and TFIIEbeta, suggesting that the Pho4p-Pho2p complex bound to the UAS activates transcription of the PHO genes by direct interaction with the general transcription factors.

The homeodomain protein Pho2p binds at an A/T-rich segment flanking the binding site of the basic-helix-loop-helix protein Pho4p in the yeast PHO promoters.

Transcription of the genomic PHO5, PHO81 and PHO84 genes of the PHO regulon requires Pho4p and Pho2p activity, whereas transcription of PHO8 is directed by Pho4p alone. Pho4p binds to two 9-bp motifs, 5'-GCACGTGGG-3' (type 1. e.g. UASp2 of PHO5 and site D of PHO84) and 5'-GCACGTTTT-3' (type 2, e.g. UASp1 of PHO5 and site E of PHO84) in the PHO promoter. Experiments were performed to evaluate the ability of these 9-bp motifs to function as upstream activation sites (UASs) by insertion of various 36-bp fragments bearing the 9-bp motif in a CYC1-lacZ fusion gene. No expression of the lacZ gene was detected with the 36-bp fragment bearing UASp2 of PHO5, whereas similar 36-bp fragments bearing UASp1 of PHO5 and sites D and E of PHO84 showed UAS activity in response to Pi concentration in the medium and to the pho2 mutation. The Pho2p-responsive UASs are flanked by one or two copies of an A/T-rich segment, whereas UASp2 is not. Gel retardation and competition experiments performed using a T7-Pho2p-His chimeric protein showed that Pho2p binds to the 36-bp fragments bearing A/T-rich segment(s) but not appreciably to the 36-bp fragments not bearing such segment(s). Thus, the A/T segments flanking the PHO UASs are Pho2p binding sites and play an important role in PHO regulation.

Structure and distribution of specific cis-elements for transcriptional regulation of PHO84 in Saccharomyces cerevisiae.

Transcription of the PHO84 gene encoding a Pi transporter in Saccharomyces cerevisiae is regulated by the Pi concentration in the medium. The promoter region of PHO84 bears five copies of the motif 5'-CACGT(G/T)-3', a candidate for the upstream activation site (UAS) that binds the transcriptional activator protein of the phosphatase regulon, Pho4p. These motifs are found at nucleotides -880 (site A), -587 (B), -436 (C), -414 (D), and -262 (E) relative to the putative ATG codon of PHO84. The Pho4p binds to all five 6-bp motifs with various affinities. Deletion analysis of the PHO84 promoter using a PHO84-lacZ fusion gene and base substitutions in the 6-bp motif revealed that two copies of the 6-bp motif, either C or D, and E, are necessary and sufficient for full regulation of the PHO84 gene. Results of expression studies with a CYC1-lacZ fusion gene with various 36-bp oligonucleotides including the 30-bp sequences around site D or E, or with modified sequences, inserted in the CYC1 promoter region indicated that the 6-bps motif flanked by a thymine nucleotide at its 5' end is much less effective as a UAS site for Pho4p in vivo than other versions. Thus, the consensus sequences for phosphatase regulation are 5'-GCACGTGGG-3' and 5'-GCACGTTTT-3' which differ from the binding sequences for the Cpflp protein required for transcription of the genes in methionine biosynthesis and for centromere function. However, Pho4p binding in vitro was unaffected by modification of the 5' or 3' flanking sites of the 6-bp motif, while modification inside the 6-bp motif affected it severely. The UAS function of the GCACGTTTT motif with respect to the Pi signal depends on its orientation in the promoter sequence.