Genomewide identification of Sko1 target promoters reveals a regulatory network that operates in response to osmotic stress in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, the ATF/CREB transcription factor Sko1 (Acr1) regulates the expression of genes induced by osmotic stress under the control of the high osmolarity glycerol (HOG) mitogen-activated protein kinase pathway. By combining chromatin immunoprecipitation and microarrays containing essentially all intergenic regions, we estimate that yeast cells contain approximately 40 Sko1 target promoters in vivo; 20 Sko1 target promoters were validated by direct analysis of individual loci. The ATF/CREB consensus sequence is not statistically overrepresented in confirmed Sko1 target promoters, although some sites are evolutionarily conserved among related yeast species, suggesting that they are functionally important in vivo. These observations suggest that Sko1 association in vivo is affected by factors beyond the protein-DNA interaction defined in vitro. Sko1 binds a number of promoters for genes directly involved in defense functions that relieve osmotic stress. In addition, Sko1 binds to the promoters of genes encoding transcription factors, including Msn2, Mot3, Rox1, Mga1, and Gat2. Stress-induced expression of MSN2, MOT3, and MGA1 is diminished in sko1 mutant cells, while transcriptional regulation of ROX1 seems to be unaffected. Lastly, Sko1 targets PTP3, which encodes a phosphatase that negatively regulates Hog1 kinase activity, and Sko1 is required for osmotic induction of PTP3 expression. Taken together our results suggest that Sko1 operates a transcriptional network upon osmotic stress, which involves other specific transcription factors and a phosphatase that regulates the key component of the signal transduction pathway.

FIG. 1.

In vivo occupancy of Sko1 at various intergenic regions. From (HA)₃-Sko1-expressing strain MAP37 (HA-SKO1) and the untagged parental strain (control), cross-linked chromatin was immunoprecipitated with anti-HA antibodies followed by quantitative PCR analysis in real time of the indicated regions. Occupancy levels are measured compared to an internal region of the POL1 gene, which is not bound by Sko1. The intergenic regions are ordered by their Sko1 occupancy levels determined by the targeted chromatin immunoprecipitation assays. In the cases where the intergenic region corresponds to the upstream regulatory sequence of more than one gene, all gene names are listed. The P values are derived from the genomewide location analysis. In general, these P values are based on data from at least two or three independent experiments; P values based on one experiment are indicated by asterisks.

FIG. 2.

Sko1 regulates the osmoinducible expression of MSN2, MOT3, MGA1, and PTP3. Reverse transcriptaion analysis of the indicated mRNA levels under nonstress (−NaCl) and osmotic stress (+NaCl; 0.4 M NaCl for 10 min) conditions in wild-type (W303-1A) and sko1 (MAP19) and hog1 (MAP32) deletion strains. DNA regions were quantified by quantitative PCR in real time. Transcript levels are given relative to the TBP1 control.

FIG. 3.

Schematic overview of Sko1-bound genes and their biological functions. Only experimentally confirmed Sko1 targets are included with the exception of ZRC1, PRR1, and DDR48, which are bound with a probability P of <10⁻², but are not confirmed by standard chromatin immunoprecipitation.