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• W1991456063 abstract "In response to elevated temperatures, cells from many organisms rapidly transcribe a number of mRNAs. In Saccharomyces cerevisiae, this protective response involves two regulatory systems: the heat shock transcription factor (Hsf1) and the Msn2 and Msn4 (Msn2/4) transcription factors. Both systems modulate the induction of specific heat shock genes. However, the contribution of Hsf1, independent of Msn2/4, is only beginning to emerge. To address this question, we constructed an msn2/4 double mutant and used microarrays to elucidate the genome-wide expression program of Hsf1. The data showed that 7.6% of the genome was heat-induced. The up-regulated genes belong to a wide range of functional categories, with a significant increase in the chaperone and metabolism genes. We then focused on the contribution of the activation domains of Hsf1 to the expression profile and extended our analysis to include msn2/4Δ strains deleted for the N-terminal or C-terminal activation domain of Hsf1. Cluster analysis of the heat-induced genes revealed activation domain-specific patterns of expression, with each cluster also showing distinct preferences for functional categories. Computational analysis of the promoters of the induced genes affected by the loss of an activation domain showed a distinct preference for positioning and topology of the Hsf1 binding site. This study provides insight into the important role that both activation domains play for the Hsf1 regulatory system to rapidly and effectively transcribe its regulon in response to heat shock. In response to elevated temperatures, cells from many organisms rapidly transcribe a number of mRNAs. In Saccharomyces cerevisiae, this protective response involves two regulatory systems: the heat shock transcription factor (Hsf1) and the Msn2 and Msn4 (Msn2/4) transcription factors. Both systems modulate the induction of specific heat shock genes. However, the contribution of Hsf1, independent of Msn2/4, is only beginning to emerge. To address this question, we constructed an msn2/4 double mutant and used microarrays to elucidate the genome-wide expression program of Hsf1. The data showed that 7.6% of the genome was heat-induced. The up-regulated genes belong to a wide range of functional categories, with a significant increase in the chaperone and metabolism genes. We then focused on the contribution of the activation domains of Hsf1 to the expression profile and extended our analysis to include msn2/4Δ strains deleted for the N-terminal or C-terminal activation domain of Hsf1. Cluster analysis of the heat-induced genes revealed activation domain-specific patterns of expression, with each cluster also showing distinct preferences for functional categories. Computational analysis of the promoters of the induced genes affected by the loss of an activation domain showed a distinct preference for positioning and topology of the Hsf1 binding site. This study provides insight into the important role that both activation domains play for the Hsf1 regulatory system to rapidly and effectively transcribe its regulon in response to heat shock. The ability to respond to a large number of environmental stresses is crucial for the survival of all organisms (1Morimoto R.I. Kline M.P. Bimston D.N. Cotto J.J. Essays Biochem. 1997; 32: 17-29PubMed Google Scholar). Heat shock is among a highly diverse group of environmental conditions that alter gene expression in prokaryotic and eukaryotic cells. The response to heat shock is characterized by a rapid induction of a conserved group of heat shock proteins (HSPs). 2The abbreviations used are: HSP, heat shock protein; Hsf1, heat shock transcription factor; Msn2/4, Msn2 and Msn4 transcription factor; HSE, heat shock element; NAD, N-terminal activation domain; CAD, C-terminal activation domain; STRE, stress response element; MOPS, 4-morpholinepropanesulfonic acid; ORF, open reading frame; MES, 4-morpholineethanesulfonic acid; ChIP, chromatin immunoprecipitation; SGD, Saccharomyces Genome Database; QT, quality threshold; TAF, TATA-binding protein-associated factor. In Saccharomyces cerevisiae, two regulatory systems are involved in this response: the heat shock transcription factor (Hsf1) (2Sorger P.K. Pelham H.R. Cell. 1988; 54: 855-864Abstract Full Text PDF PubMed Scopus (570) Google Scholar, 3Wu C. Annu. Rev. Cell Dev. Biol. 1995; 11: 441-469Crossref PubMed Scopus (982) Google Scholar) and the Msn2 and Msn4 (Msn2/4) transcription factors (4Estruch F. Carlson M. Mol. Cell. Biol. 1993; 13: 3872-3881Crossref PubMed Scopus (201) Google Scholar, 5Kobayashi N. McEntee K. Mol. Cell. Biol. 1993; 13: 248-256Crossref PubMed Scopus (169) Google Scholar). Yeast Hsf1 is an essential protein that binds to inverted repeats of nGAAn called heat shock elements (HSEs) within the promoters of many HSPs and activates their transcription. Hsf1 is composed of several well defined domains that are important for its function. They include the highly conserved central core, which is made up of the winged-helix-turn-helix DNA-binding domain (6Harrison C.J. Bohm A.A. Nelson H.C.M. Science. 1994; 263: 224-227Crossref PubMed Scopus (223) Google Scholar, 7Littlefield O. Nelson H.C.M. Nat. Struct. Biol. 1999; 6: 464-470Crossref PubMed Scopus (140) Google Scholar) and the hydrophobic coiled-coil region essential for the regulation of homotrimer formation (8Peteranderl R. Nelson H.C.M. Biochemistry. 1992; 31: 12272-12276Crossref PubMed Scopus (121) Google Scholar, 9Peteranderl R. Rabenstein M. Shin Y.K. Liu C.W. Wemmer D.E. King D.S. Nelson H.C.M. Biochemistry. 1999; 38: 3559-3569Crossref PubMed Scopus (80) Google Scholar, 10Sorger P.K. Nelson H.C.M. Cell. 1989; 59: 807-813Abstract Full Text PDF PubMed Scopus (289) Google Scholar). In addition, yeast Hsf1 has two trans-activation domains, one at the N terminus and the other at the C terminus (11Nieto-Sotelo J. Wiederrecht G. Okuda A. Parker C.S. Cell. 1990; 62: 807-817Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 12Sorger P.K. Cell. 1990; 62: 793-805Abstract Full Text PDF PubMed Scopus (189) Google Scholar). Although the structure and function of HSF is generally conserved from various organisms, there is variability in the number and importance of HSF genes in any particular organism. The yeasts, S. cerevisiae and Schizosaccharomyces pombe, have one HSF gene that is essential for cell survival (2Sorger P.K. Pelham H.R. Cell. 1988; 54: 855-864Abstract Full Text PDF PubMed Scopus (570) Google Scholar, 13Gallo G.J. Prentice H. Kingston R.E. Mol. Cell. Biol. 1993; 13: 749-761Crossref PubMed Scopus (72) Google Scholar, 14Wiederrecht G. Seto D. Parker C.S. Cell. 1988; 54: 841-853Abstract Full Text PDF PubMed Scopus (271) Google Scholar), whereas Drosophila melanogaster has a single HSF gene that is essential for oogenesis, early larval development, and survival in response to acute stress but is unnecessary for cell growth and viability (15Jedlicka P. Mortin M.A. Wu C. EMBO J. 1997; 16: 2452-2462Crossref PubMed Scopus (236) Google Scholar). In contrast, chickens and mammals have 3 HSF genes that vary in their cellular and physiological roles (16Pirkkala L. Nykanen P. Sistonen L. FASEB J. 2001; 15: 1118-1131Crossref PubMed Scopus (835) Google Scholar), whereas Arabidopsis thaliana has 21 HSF genes that are tightly regulated into a network of interacting proteins (17Baniwal S.K. Bharti K. Chan K.Y. Fauth M. Ganguli A. Kotak S. Mishra S.K. Nover L. Port M. Scharf K.D. Tripp J. Weber C. Zielinski D. von Koskull-Doring P. J. Biosci. 2004; 29: 471-487Crossref PubMed Scopus (418) Google Scholar). The existence of multiple HSF species in higher eukaryotes suggests that HSF isoforms may have specific functions that are triggered by distinct stimuli or activate specific target genes. So how does S. cerevisiae, with one Hsf1 isoform, regulate such a complex response to environmental stress? Yeast Hsf1 differs from metazoan HSFs in several important ways (16Pirkkala L. Nykanen P. Sistonen L. FASEB J. 2001; 15: 1118-1131Crossref PubMed Scopus (835) Google Scholar). First, S. cerevisiae utilizes the single essential Hsf1 to activate the expression of a wide variety of genes under normal physiological conditions and in response to environmental stress. Metazoan cells have three heat shock factors that only function under specific developmental or environmental conditions. Second, in the absence of heat shock, S. cerevisiae Hsf1 exists in the nucleus as a trimer bound to HSEs. In contrast, mammalian HSF1 exists in the cytoplasm as an inactive monomer. In response to heat shock, mammalian HSF undergoes nuclear localization, trimerizes, and binds to HSEs. Third, S. cerevisiae Hsf1 has two trans-activation domains, whereas the metazoan HSF has only one located at the C terminus. The activation domains of the S. cerevisiae Hsf1 appear to mediate different temporal aspects of the heat shock response, which can be divided into two phases: the transient response and the sustained response (11Nieto-Sotelo J. Wiederrecht G. Okuda A. Parker C.S. Cell. 1990; 62: 807-817Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 12Sorger P.K. Cell. 1990; 62: 793-805Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 18Young M.R. Craig E.A. Mol. Cell. Biol. 1993; 13: 5637-5646Crossref PubMed Scopus (34) Google Scholar). The transient response is characterized as how cells tolerate an increase in temperature over 35 °C for <1 h, whereas the sustained response is characterized as how cells acclimate to the higher temperatures for long periods of time. The N-terminal activation domain (NAD), found in the first 65 amino acids, is thought to mediate the transient response, whereas the C-terminal activation domain (CAD), found between residues 595 and 783, is thought to be responsible for the sustained response to stress. Although there have been no detailed studies of the genes effected by the NAD, the effect of the CAD has been studied for specific genes. The CAD is critical for the heat-induced expression of CUP1, HSP82, and HSP26, whereas the loss of the CAD has no effect on the heat-induced expression of SSA1, SSA3, and HSP104 (19Amoros M. Estruch F. Mol. Microbiol. 2001; 39: 1523-1532Crossref PubMed Scopus (109) Google Scholar, 20Santoro N. Johansson N. Thiele D.J. Mol. Cell. Biol. 1998; 18: 6340-6352Crossref PubMed Scopus (90) Google Scholar, 21Tamai K.T. Liu X. Silar P. Sosinowski T. Thiele D.J. Mol. Cell. Biol. 1994; 14: 8155-8165Crossref PubMed Scopus (123) Google Scholar). The second regulatory system of the heat shock response, Msn2 and Msn4, has not been conserved between yeasts and humans. These proteins are non-essential transcription factors that bind to stress response elements (STREs) found in the promoters of most HSPs (22Estruch F. FEMS Microbiol. Rev. 2000; 24: 469-486Crossref PubMed Google Scholar). Msn2 and Msn4 are 41% identical in amino acid sequence and 100% conserved at three residues that are pertinent for DNA sequence recognition (23Martinez-Pastor M.T. Marchler G. Schuller C. Marchler-Bauer A. Ruis H. Estruch F. EMBO J. 1996; 15: 2227-2235Crossref PubMed Scopus (861) Google Scholar). Under constitutive conditions, Msn2/4 proteins are localized to the cytoplasm; however, during heat shock, they are transported to the nucleus where they activate transcription of HSPs (24Gorner W. Durchschlag E. Martinez-Pastor M.T. Estruch F. Ammerer G. Hamilton B. Ruis H. Schuller C. Genes Dev. 1998; 12: 586-597Crossref PubMed Scopus (604) Google Scholar). The contributions of Msn2/4 and Hsf1 proteins to the yeast heat shock response have been addressed previously (25Boy-Marcotte E. Lagniel G. Perrot M. Bussereau F. Boudsocq A. Jacquet M. Labarre J. Mol. Microbiol. 1999; 33: 274-283Crossref PubMed Scopus (140) Google Scholar, 26Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3763) Google Scholar, 27Hahn J.S. Hu Z. Thiele D.J. Iyer V.R. Mol. Cell. Biol. 2004; 24: 5249-5256Crossref PubMed Scopus (326) Google Scholar, 28Treger J.M. Schmitt A.P. Simon J.R. McEntee K. J. Biol. Chem. 1998; 273: 26875-26879Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). These studies, although not exhaustive, establish that each regulatory system contributes differently to the expression of genes. For some genes, such as HSP26 and HSP104, their expression is regulated by cooperation between both Msn2/4 and Hsf1 (19Amoros M. Estruch F. Mol. Microbiol. 2001; 39: 1523-1532Crossref PubMed Scopus (109) Google Scholar, 29Grably M.R. Stanhill A. Tell O. Engelberg D. Mol. Microbiol. 2002; 44: 21-35Crossref PubMed Scopus (48) Google Scholar), whereas other genes appear to be regulated by only one system. The Msn2/4 regulatory system controls the expression of genes classified as chaperones, carbon metabolism, and oxidative stress (25Boy-Marcotte E. Lagniel G. Perrot M. Bussereau F. Boudsocq A. Jacquet M. Labarre J. Mol. Microbiol. 1999; 33: 274-283Crossref PubMed Scopus (140) Google Scholar, 28Treger J.M. Schmitt A.P. Simon J.R. McEntee K. J. Biol. Chem. 1998; 273: 26875-26879Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), whereas the Hsf1 regulatory system controls the expression of genes classified as chaperones, cell wall maintenance, and energy regeneration (26Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3763) Google Scholar, 27Hahn J.S. Hu Z. Thiele D.J. Iyer V.R. Mol. Cell. Biol. 2004; 24: 5249-5256Crossref PubMed Scopus (326) Google Scholar, 30Yamamoto A. Mizukami Y. Sakurai H. J. Biol. Chem. 2005; 280: 11911-11919Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In this study, we used DNA microarrays to quantify the contribution that the Hsf1 regulatory system makes to the transient heat shock response in the absence of the Msn2/4 transcription factors. In particular, we focused on the roles of the activation domains of Hsf1 by analyzing the expression patterns of strains with NAD or CAD deletions. We applied clustering algorithms to our data to illuminate physiological and mechanical relationships among genes. Promoter analysis showed architectural distinctions between the different clustered genes. These data show that Hsf1, its activation domains, and HSEs all play distinctive roles in the reorganizing and orchestrating of cellular events during the heat shock response. Yeast Strains and Growth Conditions—All strains were constructed in our laboratory and derived from the S. cerevisiae strain W303-1A (MATa ade2-1 trp-1 can-1 leu2, 3-112 his-11, 15 ura3-1). We used the KanMX cassette in combination with cre-loxP recombination technology to create the msn2::loxP msn4::loxP null alleles (31Ferguson S.B. Anderson E.S. Harshaw R.B. Thate T. Craig N.L. Nelson H.C. Genetics. 2005; 169: 1203-1214Crossref PubMed Scopus (50) Google Scholar). Creation of the activation domain deletion strains has been previously described (32Ferguson S.B. Negative Regulation of the Heat Shock Transcription Factor by Protein Kinase A in Saccharomyces cerevisiae, Ph.D. dissertation, Cell and Molecular Biology. University of Pennsylvania, Philadelphia, PA2005Google Scholar). Briefly, URA3-marked integration plasmids that contained Hsf1 genes deleted for either the N- or C-terminal activation domains were constructed. These plasmids were used to replace the wild-type HSF1 allele with either the hsf1(66-833) or hsf1(1-583) alleles in both MSN2/4 and msn2/4Δ strains. Strains with the wild-type HSF1 were designated HSF, strains with hsf1(66-833) allele were designated HSFΔNAD, and strains with the hsf1(1-583) allele were designated HSFΔCAD. Cells were grown in yeast extract-peptone-dextrose medium supplemented with adenine (YPAD) at 30 °C to mid-log phase (A600 of 0.5). RNA Preparation and Northern Blot Analysis—The culture was split into two, with one half remaining at standard growing conditions, while the other half was heat shocked at 37 °C for 15 min. Cells were harvested by centrifugation at 258 × g at 4 °C for 5 min, flash frozen in liquid nitrogen, and stored at -80 °C. Total RNA was isolated using the hot acidic phenol method (33Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates, Inc., and John Wiley & Sons, Inc., 2004: 13-12.1-13-12.5Google Scholar). Pellets were resuspended in RNase-free dH2O, quantified, and store at -80 °C until needed. RNA samples (7 μg/lane) were incubated in a 1:1 ratio of loading buffer (54% formamide, 19% formaldehyde, 40 mm MOPS, pH 7, 10 mm sodium acetate, 4.2% glycerol, 0.03% xylene cyanol, 0.03% bromphenol blue, 1 mm EDTA, pH 8) at 65 °C for 10 min. The RNA samples were resolved on formaldehyde-agarose gels and then transferred onto ZetaProbe Nylon membrane (Bio-Rad). The membrane was cross-linked in the UV Stratalinker 2400 (Stratagene) and washed in 0.1× SSC at room temperature. The membrane was pre-hybridized in RapidHyb buffer (Amersham Biosciences) at 55 °C for a minimum of 2 h. Probes were generated by incorporating Biotin-16-dUP nucleotide (Roche Applied Science) into PCR products of the HSP12, HSP26, HSP82, HSP104, SSA3, SSA4, and SSE2 open reading frames (ORFs). Hybridization was carried out with denatured probes in the same buffer at 55 °C overnight. The following day, the filters were washed three times in 300 mm NaCl; 30 mm sodium citrate, 0.1% SDS at 55 °C for 20 min. The protocol from the North2South Chemiluminescent Detection Kit (Pierce) was used for probe detection and substrate development. Signals were then quantified on a Fluorchem 8800 cooled charge-coupled device detection system (Alpha Innotech Corp.). Microarray Hybridization—The Affymetrix yeast genome YG_S98 microarrays used in this study contain ∼6400 known or predicted open reading frames as described in the Saccharomyces Genome Database (SGD) and additional probe sets representing putative ORFs identified by SAGE analysis, mitochondrial proteins, and TY proteins (Affymetrix). All protocols were conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly, 10 μg of total RNA was incubated with Superscript II reverse transcriptase primed by a poly(T) oligomer that incorporated the T7 promoter to convert the RNA into first-strand cDNA. Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP. The cRNA products were fragmented to an average of 200 nucleotides, heated at 99 °C for 5 min, and hybridized for 16 h at 45 °C to the Affymetrix microarrays at the University of Pennsylvania Microarray Core Facility. The microarrays were then washed at low (0.9 m NaCl, 60 mm Na H2PO4, pH7.5, 6 mm EDTA, 0.005% Triton X-100) and high (100 mm MES, 0.1 m NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain. A confocal scanner was used to collect fluorescence signal at 3-μm resolution after excitation at 570 nm. The average signal from two sequential scans was calculated for each microarray feature. Gene expression values were calculated using Affymetrix Microarray Suite, version 5.0, and exported as “chp” files. The data were imported and analyzed using GeneSpring GX version 7.2 (Agilent Technologies). The chp files were pre-normalized (centered on a trimmed mean of 150). In GeneSpring, the values were further normalized “per chip” by dividing each intensity value by the median intensity of the entire chip, and then “per gene” by dividing the signal levels of a given gene across all 18 samples by the median signal across the same samples for that gene. Data Acquisition and Analysis—First, an experiment was set up to group the three independent replicates of each condition and strain together. This generated an average signal log ratio value, which reflects the abundance of each Affymetrix probe set. At the end of this summarization process, there were six normalized values for each replicate group, one for each strain and condition tested. Then, a pair-wise comparative analysis was performed between the experimental heat shock and the baseline constitutive microarrays for each of the three strains. The probe sets were designated present or induced if they were enriched by a minimum of 2-fold (normalized fold change (NFC) = 2.0). The loci that pass the filter were saved as a gene list. In parallel with the above analysis, normalized signal values for all probe sets were statistically analyzed to identify probe sets that changed in each of the strains in response to heat shock. We applied Significance Analysis of Microarrays, version 2.2 (SAM) (34Tusher V.G. Tibshirani R. Chu G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5116-5121Crossref PubMed Scopus (9801) Google Scholar) to the experimental heat shock and the baseline constitutive microarrays for each of three strains in two-sample unpaired mode, with no fold-change cutoff. The stringency for calling loci significant was set at a moderate threshold (Δ) of 0.6. This analysis generated a differentially expressed probe set list for each of the three comparisons. The false discovery rates for the lists were 3.6% for strain HSF, 4.2% for strain HSFΔNAD, and 3.9% for strain HSFΔCAD. We then imported the gene lists of statistically significant probe sets identified by SAM into GeneSpring and compared it to the gene list with >2-fold change generated by the GeneSpring software. The overlapping probe sets that met both qualifications, Δ= 0.6 and normalized fold change (NFC) = 2.0, were used for further analysis. Quality threshold (QT) clustering was performed to uncover patterns of gene expression and the relationships between these patterns (35Heyer L.J. Kruglyak S. Yooseph S. Genome Res. 1999; 9: 1106-1115Crossref PubMed Scopus (830) Google Scholar). The algorithm builds a cluster by starting with a single locus and a cluster diameter of zero. One by one, other loci are added to the cluster and a new cluster diameter is calculated. If a locus cannot be placed into an existing cluster without sacrificing the quality or the diameter threshold, it is placed into a new cluster. As a starting point, we analyzed the loci induced in the HSF strain with the QT clustering algorithm of GeneSpring. When all of the loci had been placed, 18 clusters had been generated. We then manually varied the diameter from 0.3 to 0.7 to reduce the number of clusters and to be able to add more loci to each cluster while maintaining a well matched pattern within the cluster. The end result was a five-cluster pattern. To identify Hsf1 binding sites in the promoter sequences, we analyzed 1000 bp upstream of the start codon of the loci that represented verifiable ORFs. Sequences were retrieved from the SGD (36Ball C.A. Dolinski K. Dwight S.S. Harris M.A. Issel-Tarver L. Kasarskis A. Scafe C.R. Sherlock G. Binkley G. Jin H. Kaloper M. Orr S.D. Schroeder M. Weng S. Zhu Y. Botstein D. Cherry J.M. Nucleic Acids Res. 2000; 28: 77-80Crossref PubMed Scopus (80) Google Scholar, 37Balakrishnan R. Christie K.R. Costanzo M.C. Dolinski K. Dwight S.S. Engel S.R. Fisk D.G. Hirschman J.E. Hong E.L. Nash R. Oughtred R. Skrzypek M. Theesfeld C.L. Binkley G. Lane C. Schroeder M. Sethuraman A. Dong S. Weng S. Miyasato S. Andrada R. Botstein D. Cherry J.M. Saccharomyces Genome Database. 2005www.yeastgenome.orgGoogle Scholar) and analyzed using a simple pattern-identification program. We defined three types of HSEs, each having three nGAAn repeats in Perfect (PFT), GAP (GAP), and STEP (STP) arrangements. The perfect HSE (PFT) consists of three contiguous, inverted repeats of the nGAAn sequence, either nGAAnnTTCnnGAAn or nTTCnnGAAnnTTCn. The GAP HSE consists of an nGAAn repeat, followed by any 5 bp and 2 inverted nGAAn repeats (nGAAn-(5-bp)-nGAAnnTTCn) and its complement (nGAAnnTTCn-(5-bp)-nTTCn), as well as the related sequences nTTCn-(5-bp)-nTTCnnGAAn and nTTC-nnGAAn-(5-bp)-nGAAn. The STP HSE has a 5-bp insert between each of the 3 nGAAn repeats, yielding the sequences nGAAn-(5-bp)-nGAAn-(5-bp)-nGAAn and nTTCn-(5-bp)-nTTCn-(5-bp)-nTTCn (30Yamamoto A. Mizukami Y. Sakurai H. J. Biol. Chem. 2005; 280: 11911-11919Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). As per previous studies (27Hahn J.S. Hu Z. Thiele D.J. Iyer V.R. Mol. Cell. Biol. 2004; 24: 5249-5256Crossref PubMed Scopus (326) Google Scholar, 30Yamamoto A. Mizukami Y. Sakurai H. J. Biol. Chem. 2005; 280: 11911-11919Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), we also allowed a single mismatch (nGAR) in one of the three nGAAn repeats for PFT or GAP. Both Activation Domains of Hsf1 Contribute to the Transient Heat Shock Response—Yeast Hsf1 has two activation domains that play distinct temporal roles during the heat shock response (12Sorger P.K. Cell. 1990; 62: 793-805Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 18Young M.R. Craig E.A. Mol. Cell. Biol. 1993; 13: 5637-5646Crossref PubMed Scopus (34) Google Scholar). To compare their relative contributions in the initial response to heat stress, we constructed six isogenic strains, three in an MSN2/4 background and three in an msn2/4Δ background. In both backgrounds, we tested various truncations of Hsf1: HSF, wild-type Hsf1; HSFΔNAD, which eliminated the first 65 residues of Hsf1; and HSFΔCAD, which eliminated the last 250 residues of Hsf1 (Fig. 1). The six strains were subjected to a 15-min heat shock at a moderate (37 °C) temperature. Northern blot analysis was used to measure the constitutive and heat-induced levels of several HSF-controlled mRNAs. In all six strains, the constitutive levels of HSP104, HSP82, and HSP26 mRNAs were low, whereas the heat-induced levels of the same mRNAs increased to varying degrees (Fig. 2). When we compared the heat induction of HSP104 and HSP26 in the MSN2/4 strain versus the msn2/4Δ strain, there was only a slight increase, whereas HSP82 was significantly reduced in the same background comparison. When we analyzed the contribution of the NAD or the CAD in MSN2/4 strains, the results were similar to those obtained in the HSF strain. The effects of the deleted activation domains were masked and/or compensated for by the Msn2/4 regulatory system.FIGURE 2Removal of a single activation domain of Hsf1 reveals promoter-specific defects in heat shock induction of HSPs in the absence of msn2/4. A and B, total RNA was isolated from six different strains that were either maintained at constitutive conditions (30 °C) or heat shocked at 37 °C for 15 min. A representative Northern blot is shown for the transcripts HSP104, HSP12, HSP26, and ACT1 (loading control). The bar graph below illustrates heat induction, which was calculated as the -fold change of the normalized transcript between heat shock conditions versus constitutive conditions. The asterisks indicate significant differences between wild-type and the condition tested using Student's t test: **, p < 0.01; ***, p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) On the other hand, in the msn2/4Δ strains, each of the HSPs tested was affected differently by the loss of one of the Hsf1 activation domains (Fig. 2B). In the HSF strain, the heat-induced mRNA levels increased moderately for HSP104 (12-fold) and HSP82 (4-fold) and dramatically for HSP26 (37-fold). In the HSFΔNAD strain, the heat-induced mRNA levels increased moderately for HSP104 (7-fold) and HSP26 (7-fold) and dramatically for HSP82 (34-fold). In the HSFΔCAD strain, the heat-induced mRNA levels increased moderately for HSP104 (5-fold) and HSP82 (6-fold) but had no effect on the levels of HSP26 (0.9-fold). From these experiments we were able to confirm that both regulatory systems cooperate in the expression of many genes. More importantly, we could use the msn2/4 null strains to look specifically at the contribution of the Hsf1 regulatory system. Global Transcript Profiling of mRNAs Shows a Unique Response to Mutations in Hsf1 during Heat Shock—To understand the broader utilization of Hsf1 and its activation domains during the transient heat shock response, we used microarrays to examine genome-wide changes in transcription levels. For each msn2/4Δ strain, we isolated RNA from constitutive and heat-induced conditions in triplicate, resulting in 18 RNA samples. These samples served as templates to generate biotinylated antisense probes for hybridization to Affymetrix yeast genome YG_S98 microarrays. After spot quantitation and normalization, the signal log ratio for each locus on the microarray was determined. We compared the constitutive and heat shock conditions in each strain and identified loci that responded with significant changes in their transcript level. We then imported the list into the SGD to identify the loci that represented known or putative genes. In the HSF strain, 442 genes were heat-induced. The induction of these genes is significantly higher than would be expected if induction was not correlated with heat shock expression (p = 2.1 × 10-21). Of the 442 genes, 63% were increased by 2- to 3-fold, 28% increased by 3- to 6-fold, and 9% increased by more than 6-fold. The highest expression profile was for the HSP70 homolog SSA4, which increased its levels by 137-fold. In the HSFΔNAD strain, 329 genes were heat-induced (p = 4.3 × 10-16). Of the 329 genes, 63% were increased by 2- to 3-fold, 31% increased by 3- to 6-fold, and 6% increased by" @default.
• W1991456063 created "2016-06-24" @default.
• W1991456063 creator A5006676094 @default.
• W1991456063 creator A5048492570 @default.
• W1991456063 date "2006-10-01" @default.
• W1991456063 modified "2023-10-06" @default.
• W1991456063 title "Genome-wide Analysis Reveals New Roles for the Activation Domains of the Saccharomyces cerevisiae Heat Shock Transcription Factor (Hsf1) during the Transient Heat Shock Response" @default.
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