Partially phosphorylated Pho4 activates transcription of a subset of phosphate-responsive genes

Abstract

A cell's ability to generate different responses to different levels of stimulus is an important component of an adaptive environmental response. Transcriptional responses are frequently controlled by transcription factors regulated by phosphorylation. We demonstrate that differential phosphorylation of the budding yeast transcription factor Pho4 contributes to differential gene expression. When yeast cells are grown in high-phosphate growth medium, Pho4 is phosphorylated on four critical residues by the cyclin-CDK complex Pho80-Pho85 and is inactivated. When yeast cells are starved for phosphate, Pho4 is dephosphorylated and fully active. In intermediate-phosphate conditions, a form of Pho4 preferentially phosphorylated on one of the four sites accumulates and activates transcription of a subset of phosphate-responsive genes. This Pho4 phosphoform binds differentially to phosphate-responsive promoters and helps to trigger differential gene expression. Our results demonstrate that three transcriptional outputs can be generated by a pathway whose regulation is controlled by one kinase, Pho80-Pho85, and one transcription factor, Pho4. Differential phosphorylation of Pho4 by Pho80-Pho85 produces phosphorylated forms of Pho4 that differ in their ability to activate transcription, contributing to multiple outputs.

Figure 1. Whole-Genome Expression DNA Microarrays of PHO4^SA1234WT6 and PHO4^SA1234PA6 Strains

(A) Schematic showing phosphorylation sites (O'Neill et al. 1996), the transcriptional activation domain (McAndrew et al. 1998), the nuclear localization sequence (Kaffman et al. 1998b), the Pho2-binding domain (Hirst et al. 1994), and the DNA-binding domain of Pho4 (Ogawa and Oshima 1990). Pho4 is phosphorylated on five sites (referred to as sites 1, 2, 3, 4, and 6) by Pho80–Pho85 (O'Neill et al. 1996). Site 1 is phosphorylated inefficiently in vivo and in vitro, and no functional consequence has been attributed to its phosphorylation (O'Neill et al. 1996; Komeili and O'Shea 1999). (B) Color scheme denoting the fold induction/repression for all of the microarray experiments. (C) Expression of phosphate-responsive genes in strains containing Pho4^SA1234WT6 and Pho4^SA1234PA6 grown in high-phosphate medium. Cy5-labeled samples are colored red, and Cy3-labeled samples are colored green. The percent of induction of each gene in the Pho4^SA1234WT6 strain compared to its maximal induction in the Pho4^SA1234PA6 strain is presented on the right. Several phosphate-responsive genes (PHO5, PHO8, PHO11, and PHO12) share large regions of homology. Cross-hybridization leads to similar response profiles even though the genes probably respond differently.

Figure 2. Growth of Yeast Cells in Intermediate-Phosphate Medium Leads to Differential Phosphorylation of Pho4 and Differential Expression of PHO5 and PHO84

(A) Fluorescence microscopy of yeast cells containing Pho4–GFP grown in no, 50 μM, 100 μM (intermediate [int]), 300 μM, or 10,000 μM (high) phosphate medium. (B) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1 in wild-type cells grown in medium containing different concentrations of phosphate. (C) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1, in wild-type cells grown for 1, 2, or 5 h in intermediate-phosphate medium. (D) Expression of genes in the phosphate-responsive cluster (Carroll et al. 2001) for a wild-type strain grown in intermediate- or no-phosphate medium compared to wild-type cells grown in high-phosphate medium. Cy5 and Cy3 samples are colored red and green, respectively. The percent of induction of each gene in intermediate-phosphate medium compared to its maximal induction in no-phosphate medium is presented on the right. (E) Analysis of Pho4 protein and phosphorylation by Western blotting for wild-type cells grown in no-, intermediate-, and high-phosphate medium. Samples were probed with phosphopeptide antibodies specific to sites 2, 3, and 6 of Pho4 and by a polyclonal antibody that recognizes Pho4. (F) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1, in a PHO4^WT1234SD6 strain.

Figure 3. Deletion of PHO2 Abrogates Expression of PHO5 and PHO84 and Binding of Pho4 to These Promoters

(A) Quantitation of RNA levels by Northern blot analysis in pho2Δ strains grown in no-, intermediate-, or high-phosphate medium. (B) Chromatin immunoprecipitation analysis of Pho4. Pho4 was immunoprecipitated from extracts of wild-type cells grown in high-, intermediate-, or no-phosphate medium, from a strain lacking Pho4 and from the two mutant Pho4 strains grown in high-phosphate medium. Experiments using the Pho4^SA1234WT6- and Pho4^SA1234PA6-expressing strains are normalized to the maximal amount of enrichment in a strain expressing Pho4^SA1234PA6 in high-phosphate medium. The fold enrichment of PHO5 over ACT1 was 1.03, 0.99, 1.26, 2.84, 2.41, and 5.04 in lanes 1–6, respectively (pho4Δ, wt high, wt int, wt no, PHO4^SA1234WT6, and PHO4^SA1234PA6). The fold enrichment of PHO84 over ACT1 was 0.99, 1.75, 4.27, 6.28, 10.8, and 11.6 in lanes 1–6, respectively. (C) Chromatin immunoprecipitation analysis of Pho4. Pho4 was immunoprecipitated from extracts of wild-type cells grown in no-phosphate medium, a mutant lacking Pho2 in no-phosphate medium, Pho4^SA1234WT6 and Pho4^SA1234PA6 strains grown in high-phosphate medium, and pho2Δ Pho4^SA1234WT6 and pho2Δ Pho4^SA1234PA6 strains grown in high-phosphate medium. The fold enrichment of PHO5 over ACT1 was 2.84, 1.19, 2.41, 1.49, 5.04, and 2.06 in lanes 1–6, respectively (wt no, pho2Δ no, PHO4^SA1234WT6, pho2Δ PHO4^SA1234WT6, PHO4^SA1234PA6, and pho2ΔPHO4^SA1234PA6). The fold enrichment of PHO84 over ACT1 was 6.28, 2.0, 10.8, 2.4, 11.6, and 2.63 in lanes 1–6, respectively.

Figure 5. A Strain Expressing Pho4^SA14PA6 from a Low Copy Plasmid Differentially Expresses PHO5 and PHO84 in Intermediate-Phosphate Medium

(A) Fluorescence microscopy of yeast cells containing Pho4^SA14PA6–GFP grown in no, 100 μM, or 10 mM phosphate medium. (B) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1 from strains expressing Pho4^SA14PA6 or Pho4^WT1234PA6 from a low copy plasmid grown in no, 100 μM, or 10,000 μM phosphate medium.

Figure 4. A Strain Expressing Pho4 That Cannot Be Phosphorylated on Site 6 Does Not Induce PHO5 in Intermediate-Phosphate Medium

A strain expressing Pho4^WT1234PA6 grown for 2 h in no-, intermediate-, and high-phosphate medium was analyzed by Northern blot analysis (A) and chromatin immunoprecipitation (B). The fold enrichment of PHO5 over ACT1 was 1.71, 2.42, 10.49, 0.99, 1.26, and 2.84 in lanes 1–6, respectively (PHO4^WT1234WT6 high, PHO4^WT1234WT6 int, PHO4 ^WT1234WT6 no, wt high, wt int, wt no). The fold enrichment of PHO84 over ACT1 was 24.64, 27.44, 43.74, 1.75, 4.27, and 6.28 in lanes 1–6, respectively.

Figure 6. Models for Differential Gene Regulation

(A) Differential affinity of Pho4 for the PHO84 and PHO5 promoters can cause differential gene expression. Simulated curves of the percent occupancy of Pho4 at the PHO84 and PHO5 promoters, assuming Michaelian binding. Pho4 was modeled as having a K_d of 10 at the PHO84 promoter and of 100 at the PHO5 promoter. Phosphorylation of Pho4 was simulated as raising the K_d of Pho4 to 40 at the PHO84 promoter and to 400 at the PHO5 promoter. The nuclear concentration of Pho4 was assumed to be 7.5-fold higher in intermediate-phosphate medium than in high-phosphate medium and 10-fold higher in no-phosphate medium than in high-phosphate medium. (B) Kinetic diagram of the steps leading to active transcription at the PHO84 and PHO5 promoters. Differences in the kinetic mechanisms of activation of PHO84 and PHO5 can lead to differential gene expression. Even if promoter occupancy is high at the PHO5 promoter, if the transcriptional activation step is slow compared to the rate of Pho4 phosphorylation and inactivation, PHO5 will not be induced.