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

Springer M, Wykoff DD, Miller N, O'Shea EK - PLoS Biol. (2003)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of California, San Francisco, USA.

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.

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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 Pho4SA1234WT6- and Pho4SA1234PA6-expressing strains are normalized to the maximal amount of enrichment in a strain expressing Pho4SA1234PA6 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, PHO4SA1234WT6, and PHO4SA1234PA6). 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, Pho4SA1234WT6 and Pho4SA1234PA6 strains grown in high-phosphate medium, and pho2Δ Pho4SA1234WT6 and pho2Δ Pho4SA1234PA6 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, PHO4SA1234WT6, pho2Δ PHO4SA1234WT6, PHO4SA1234PA6, and pho2ΔPHO4SA1234PA6). 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.
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pbio.0000028-g003: 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 Pho4SA1234WT6- and Pho4SA1234PA6-expressing strains are normalized to the maximal amount of enrichment in a strain expressing Pho4SA1234PA6 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, PHO4SA1234WT6, and PHO4SA1234PA6). 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, Pho4SA1234WT6 and Pho4SA1234PA6 strains grown in high-phosphate medium, and pho2Δ Pho4SA1234WT6 and pho2Δ Pho4SA1234PA6 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, PHO4SA1234WT6, pho2Δ PHO4SA1234WT6, PHO4SA1234PA6, and pho2ΔPHO4SA1234PA6). 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.

Mentions: To determine whether differential gene expression results from differential occupancy of Pho4 at PHO5 and PHO84, we examined the binding of Pho4 to these promoters using chromatin immunoprecipitation. There was little enrichment of Pho4 at the PHO5 or PHO84 promoters when cells were grown in high-phosphate medium, whereas in no-phosphate medium, Pho4 was significantly enriched at both the PHO5 and PHO84 promoters (Figure 3B, left). When cells were grown in intermediate-phosphate medium, Pho4 was enriched at the PHO84 promoter, but was not significantly enriched at the PHO5 promoter. Furthermore, chromatin immunoprecipitation experiments carried out with PHO4SA1234WT6 and PHO4SA1234PA6 strains grown in high-phosphate medium resembled immunoprecipitations carried out with wild-type cells grown in intermediate and no-phosphate medium, respectively (Figure 3B, right). Thus, differential expression of PHO5 and PHO84 correlates with differential Pho4 binding to these promoters.


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

Springer M, Wykoff DD, Miller N, O'Shea EK - PLoS Biol. (2003)

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 Pho4SA1234WT6- and Pho4SA1234PA6-expressing strains are normalized to the maximal amount of enrichment in a strain expressing Pho4SA1234PA6 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, PHO4SA1234WT6, and PHO4SA1234PA6). 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, Pho4SA1234WT6 and Pho4SA1234PA6 strains grown in high-phosphate medium, and pho2Δ Pho4SA1234WT6 and pho2Δ Pho4SA1234PA6 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, PHO4SA1234WT6, pho2Δ PHO4SA1234WT6, PHO4SA1234PA6, and pho2ΔPHO4SA1234PA6). 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.
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pbio.0000028-g003: 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 Pho4SA1234WT6- and Pho4SA1234PA6-expressing strains are normalized to the maximal amount of enrichment in a strain expressing Pho4SA1234PA6 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, PHO4SA1234WT6, and PHO4SA1234PA6). 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, Pho4SA1234WT6 and Pho4SA1234PA6 strains grown in high-phosphate medium, and pho2Δ Pho4SA1234WT6 and pho2Δ Pho4SA1234PA6 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, PHO4SA1234WT6, pho2Δ PHO4SA1234WT6, PHO4SA1234PA6, and pho2ΔPHO4SA1234PA6). 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.
Mentions: To determine whether differential gene expression results from differential occupancy of Pho4 at PHO5 and PHO84, we examined the binding of Pho4 to these promoters using chromatin immunoprecipitation. There was little enrichment of Pho4 at the PHO5 or PHO84 promoters when cells were grown in high-phosphate medium, whereas in no-phosphate medium, Pho4 was significantly enriched at both the PHO5 and PHO84 promoters (Figure 3B, left). When cells were grown in intermediate-phosphate medium, Pho4 was enriched at the PHO84 promoter, but was not significantly enriched at the PHO5 promoter. Furthermore, chromatin immunoprecipitation experiments carried out with PHO4SA1234WT6 and PHO4SA1234PA6 strains grown in high-phosphate medium resembled immunoprecipitations carried out with wild-type cells grown in intermediate and no-phosphate medium, respectively (Figure 3B, right). Thus, differential expression of PHO5 and PHO84 correlates with differential Pho4 binding to these promoters.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of California, San Francisco, USA.

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.

Show MeSH
Related in: MedlinePlus