Genome-wide analysis of the biology of stress responses through heat shock transcription factor

Abstract

Heat shock transcription factor (HSF) and the promoter heat shock element (HSE) are among the most highly conserved transcriptional regulatory elements in nature. HSF mediates the transcriptional response of eukaryotic cells to heat, infection and inflammation, pharmacological agents, and other stresses. While HSF is essential for cell viability in Saccharomyces cerevisiae, oogenesis and early development in Drosophila melanogaster, extended life span in Caenorhabditis elegans, and extraembryonic development and stress resistance in mammals, little is known about its full range of biological target genes. We used whole-genome analyses to identify virtually all of the direct transcriptional targets of yeast HSF, representing nearly 3% of the genomic loci. The majority of the identified loci are heat-inducibly bound by yeast HSF, and the target genes encode proteins that have a broad range of biological functions including protein folding and degradation, energy generation, protein trafficking, maintenance of cell integrity, small molecule transport, cell signaling, and transcription. This genome-wide identification of HSF target genes provides novel insights into the role of HSF in growth, development, disease, and aging and in the complex metabolic reprogramming that occurs in all cells in response to stress.

FIG. 1.

Determination of threshold for HSF targets. Moving-window average analysis shows the relationship of HSF binding to downstream gene expression in heat shock experiments. Expression data are derived from previously published work (12). Genomic enrichment E values of loci indicating potential association with HSF based on 20 independent heat shock experiments are plotted on the x axis. The corresponding gene expression value of genes in a sliding window of size 100 is plotted on the y axis with the use of two different measures. (A) Average peak expression value (with log₂ scale) after heat shock relative to time zero in two independent data sets. (B) Fraction of genes within the window that are induced by at least threefold in at least two time points in the expression data sets. The threshold E value of 97.7 for designating a locus as an HSF target is indicated by the gray dashed line.

FIG. 2.

Genome-wide binding distribution of HSF. Cy5/Cy3 ratios of genomic fragments with enrichment values above the threshold are displayed using a red and green color scale. Gray cells indicate missing data that were filtered out. (A) Each column represents an IP performed after independent HSF cross-linking carried out under 30°C unstressed conditions or after a 20-min heat shock at the indicated temperature. Genes downstream of selected target loci are shown on the right. The complete list of target loci and their ratio values are included in supplemental data. (B) Binding of HSF to all targets during the heat shock time course. HSF ChIP samples prepared at the indicated times during the 39°C heat shock were analyzed by DNA microarrays.

FIG. 3.

Correlation between heat-inducible binding of HSF and mRNA expression. (A) Verification of HSF binding to selected targets by PCR. The same IP DNA used for microarrays as shown in Fig. 2B was used to verify binding of HSF to selected promoters by PCR. SSA1, HSP82, and SSA3 were previously known targets of HSF, and the remainder were targets newly identified in this study. The PHO5 promoter was used as a negative control. WCE, whole-cell extract. (B) Kinetics of HSF target gene expression by heat shock. Expression levels for the indicated genes were detected by RNA blot analysis. ENO1 was used as a negative control.

FIG. 4.

Expression profiles of HSF targets. Expression during multiple distinct stress conditions of all HSF targets identified here is represented using a red and green scale. Expression data are taken from the work of Gasch et al. (12). Targets that showed the smallest difference in HSF binding between 30°C and heat shock conditions are listed on top. The subset of target promoters used for motif discovery is indicated on the right.

FIG. 5.

Validation of microarray results. Promoter-specific PCR for the indicated genes was performed using ChIP samples that had earlier been analyzed on microarrays. (A) Genes predicted as targets by microarrays and containing a consensus HSE in their promoter. (B) Genes predicted as targets by microarrays and lacking a consensus HSE in their promoter. (C) Genes identified as nontargets by microarrays, containing a consensus HSE in their promoter. ILV2, ATC1, ICS2, and BSC5 were not appreciably induced by heat shock. WCE, whole-cell extract.

FIG. 6.

The constellation of HSF target genes in the yeast genome. Representative HSF targets are categorized according to their known or hypothetical functions based upon sequence homology. See supplemental data at http://www.iyerlab.org/hsf for the entire set of HSF targets identified in this work.