Transcriptional repression by the Pho4 transcription factor controls the timing of SNZ1 expression

Abstract

Nutrient-sensing kinases play important roles for the yeast Saccharomyces cerevisiae to adapt to new nutrient conditions when the nutrient status changes. Our previous global gene expression analysis revealed that the Pho85 kinase, one of the yeast nutrient-sensing kinases, is involved in the changes in gene expression profiles when yeast cells undergo a diauxic shift. We also found that the stationary phase-specific genes SNZ1 and SNO1, which share a common promoter, are not properly induced when Pho85 is absent. To examine the role of the kinase in SNZ1/SNO1 regulation, we analyzed their expression during the growth of various yeast mutants, including those affecting Pho85 function or lacking the Pho4 transcription factor, an in vivo substrate of Pho85, and tested Pho4 binding by chromatin immunoprecipitation. Pho4 exhibits temporal binding to the SNZ1/SNO1 promoter to down-regulate the promoter activity, and a Deltapho4 mutation advances the timing of SNZ1/SNO1 expression. SNZ2, another member of the SNZ/SNO family, is expressed at an earlier growth stage than SNZ1, and Pho4 does not affect this timing, although Pho85 is required for SNZ2 expression. Thus, Pho4 appears to regulate the different timing of the expression of the SNZ/SNO family members. Pho4 binding to the SNZ1/SNO1 promoter is accompanied by alterations in chromatin structure, and Rpd3 histone deacetylase is required for the proper timing of SNZ1/SNO1 expression, while Asf1 histone chaperone is indispensable for their expression. These results imply that Pho4 plays positive and negative roles in transcriptional regulation, with both cases involving structural changes in its target chromatin.

FIG. 1.

Requirement of Pho85 for SNZ/SNO gene expression. (A) Time course of SNZ1/SNO1 expression as determined by GeneChip analysis (based on data from reference 18). The extents of SNZ1 (squares) and SNO1 (circles) expression are shown as log₂(signal ratio) values. Solid and broken lines designate expression in the wt and a Δpho85 mutant, respectively. (B) Time course of SNZ2/SNO2 expression by GeneChip analysis (18). SNZ2 (triangles) and SNO2 (diamonds) expression levels in the wt and a Δpho85 mutant are shown as described for panel A. (C) Northern analysis of SNZ1, SNO1, SNZ2, and SNO2 expression in the wt and various pho mutants and under high (H)- or low (L)-P_i conditions. Total RNA was extracted from the yeast cells (designated at the top of the panel) grown for 12 (SNZ1 only) or 30 h and was subjected to Northern analysis as described in Materials and Methods, and then the blot was hybridized with the respective digoxigenin-labeled probe. To analyze PHO5 expression, yeast cells were incubated in high- or low-P_i medium for 5 h before RNA isolation. ACT1 was detected with RNA prepared from a 12-h culture. (D) Effect of 3-AT on SNZ1 expression. Yeast strains, as designated at the top of the panel, were grown to mid-log phase, resuspended in SD medium lacking histidine and with (+) or without (−) 100 mM 3-AT, and then incubated for 1 h before RNA isolation.

FIG. 2.

Pho4 binding to the SNZ1 promoter and involvement of the Pho4-binding site in transcriptional regulation of SNZ1. (A) In vivo Pho4 binding to the SNZ1 promoter and the SNO2 ORF demonstrated by ChIP followed by gene-specific PCR. Total DNA in the extract (Input) and the immunoprecipitated chromatin fragment prepared from the wt (MFY376) cells grown under low- or high-P_i conditions were subjected to PCR with either the SNZ1 or PHO5 promoter-specific or SNO2 ORF-specific primers, and the DNA was amplified by 25 or 30 cycles of the reaction. (B) A schematic representation of SNZ1 and its mutant (SNZ1mut) promoters, showing the Pho4-binding site (black box) with the wt (AACGTG) or mutant (AAGCTT) sequences and the three Gcn4-binding sites (shaded boxes). The activities of the wt and SNZ1mut promoters, represented by β-galactosidase activity data, are shown on the right side. The wt and various mutant strains harboring the reporter plasmid were grown in high-P_i medium for 30 h prior to the assay of the reporter activity. The values represent averages and standard errors of the results of three independent assays. (C) Northern analysis of chromosomal SNZ1 expression in the SNZ1mut and SNZ1mut Δpho85 double mutant. Total RNA was isolated from cells grown for 30 h in YPAD medium and was subjected to Northern analysis as described in Materials and Methods. ACT1 is a loading control.

FIG. 3.

(A) Northern analysis of the time course of SNZ1, SNO1, SNZ2, and SNO2 gene expression in the wt and in various mutants affecting the PHO system or chromatin structure. Total RNA was isolated from cells grown in YPAD medium at 12, 18, 24, and 36 h and was subjected to Northern analysis as described in Materials and Methods. ACT1 is shown as a loading control. (B) Temporal Pho4 binding to the SNZ1 promoter in the wt (MFY376) and Δpho85 (MFY377) strains as demonstrated by ChIP and PCR. Total DNA in the extract (Input) and the immunoprecipitated chromatin fragments prepared from the wt and mutant cells at the designated times were subjected to PCR with the SNZ1 promoter-specific primer and were amplified by 30 cycles of the reaction.

FIG. 4.

A high-resolution analysis of chromatin structure in the SNZ1 promoter region. (A) Chromatin DNA was isolated from the strains designated at the top of the panel, digested with MNase (0.1 or 0.2 units/μl) as described in Materials and Methods, and subjected to a primer extension reaction using a ³²P-labeled primer (−495 to −461). A schematic representation of the SNZ1 promoter region, showing the TATA box and the Pho4- and Gcn4-binding sites, is on the left side of the panel. Numerals designate nucleotide distance, taking the A of ATG of the SNZ1 ORF as +1. D, purified DNA digested with MNase. The arrows indicate enhanced MNase cleavage signals near the Pho4-binding site observed in the chromatin samples from Δpho4 and SNZ1mut strains. Asterisks are for those observed between the two downstream Gcn4-binding sites (at −255 and −180) and near the upstream Gcn4 site at −305 compared to those in the wt and Δpho85 strains. Possible nucleosome positions are designated by gray ellipses on the right-hand side of the panel. (B) A densitometric analysis of the gel images. The lanes containing the chromatin samples digested with 0.1 U/μl MNase were analyzed (lanes 2, 5, 8, 11, and 14 in Fig. 4A). The intensity of the bands was normalized with reference to the strongly cleaved band at −255 (bold arrow). The asterisks and arrows designate the peaks of the corresponding bands in panel A. A schematic representation of the SNZ1 promoter is shown at the bottom.