Arg82 kinase activity and IP₄ and/or IP₅ are important for PHO5 transcription and chromatin remodeling. (A) Pathway for the synthesis of soluble inositol polyphosphates (13, 14). (B) Northern analysis or (C) Cla I restriction enzyme accessibility assay of pho81δ pho80^tswild-type (WT) and arg82^D131A pho81δ pho80^ts strains grown under repressing (25°C) or shifted to inducing (37°C) conditions for 1 hour. PHO5 and PHO84 mRNA amounts in the arg82^D131A strain were reduced fivefold and 1.6-fold under inducing conditions. % cut indicates the amount of accessible fragment divided by the sum of accessible and protected fragments. A schematic model of the PHO5 promoter region is shown (bottom), indicating the nucleosomes that are perturbed during induction as open circles, the Pho4 and Pho2 binding sites as dark ovals, the TATA box as an open square, and the Cla I site. (D) Northern analysis of pho81δ pho80^ts strains with the relevant genotypes indicated. PHO5 and PHO84 mRNA amounts in the plc1, arg82, ipk1, and kcs1 mutants were 6% and 77%, 4% and 76%, 61% and 64%, and 45% and 50%, respectively, of the wild-type amount under inducing conditions. See fig. S1 for methods.

The functions of INO80 and SWI/SNF at the PHO5 and PHO84 promoters are regulated by Arg82. (A) Northern analysis of Pho85^F82G-expressing strains with the relevant genotypes indicated. Strains were grown under repressing conditions (-) or shifted to inducing conditions (+) by adding 10 μM 1-NaPP1 for one hour. (B) Cla I restriction enzyme accessibility assay for the PHO85^F82G, arp8δ PHO85^F82G, and snf6δ PHO85^F82G strains, grown under repressing conditions (-) or shifted to inducing conditions (+) by adding 10 m μM 1-NaPP1 for 30 min. % cut, as in Fig. 1C. ChIP using a monoclonal antibody to (C) Ino80-HA or (D) Snf2-Myc in Pho85^F82G-expressing strains with the relevant genotypes indicated. Immunoprecipitate efficiency for PHO5 or PHO84 DNA relative to ACT1 DNA is represented by (immunoprecipitated DNA/input DNA)/(ACT1 immunoprecipitated DNA/ACT1 input DNA). Values are the averages of three independent experiments; error bars represent standard error of the mean. DMSO, dimethyl sulfoxide. See fig. S3 for ChIP method.

Pho4 binds to UASp1 in mutants impaired for PHO5 chromatin remodeling. PHO5/ACT1, as in Fig. 2C. (A) ChIP using affinity-purified polyclonal antibodies to Pho4 in Pho85^F82G-expressing strains with the relevant genotypes indicated. DMSO, as in Fig. 2. (B) ChIP of Pho4 in PHO85^F82G, arg82δ PHO85^F82G, arp8δ PHO85^F82G, and snf6δ PHO85^F82G strains carrying substitutions in the Pho4-binding sites of UASp1 and UASp2 in the PHO5 promoter grown under inducing conditions.