A heat shock-responsive domain of human HSF1 that regulates transcription activation domain function.

Human heat shock factor 1 (HSF1) stimulates transcription from heat shock protein genes following stress. We have used chimeric proteins containing the GAL4 DNA binding domain to identify the transcriptional activation domains of HSF1 and a separate domain that is capable of regulating activation domain function. This regulatory domain conferred heat shock inducibility to chimeric proteins containing the activation domains. The regulatory domain is located between the transcriptional activation domains and the DNA binding domain of HSF1 and is conserved between mammalian and chicken HSF1 but is not found in HSF2 or HSF3. The regulatory domain was found to be functionally homologous between chicken and human HSF1. This domain does not affect DNA binding by the chimeric proteins and does not contain any of the sequences previously postulated to regulate DNA binding of HSF1. Thus, we suggest that activation of HSF1 by stress in humans is controlled by two regulatory mechanisms that separately confer heat shock-induced DNA binding and transcriptional stimulation.

In vitro activation of purified human heat shock factor by heat.

A major regulatory step in the heat-induced transcription of heat shock protein (hsp) genes in eukaryotes is the activation of heat shock factor (HSF). In metazoans and Schizosaccharomyces pombe, HSF is present in unstressed cells but is unable to bind to its target DNA sequence element, the heat shock element (HSE). Heat induction of the DNA binding activity of HSF is a critical component required for activation of heat shock genes. Inactive HSF in extracts of non-heat shocked human cells can be heated in vitro to activate HSF, suggesting the factors required to sense temperature and activate HSF are soluble factors [Larson, J. S., Schuetz, T. J., & Kingston, R. E. (1988) Nature 335, 372-375]. We utilized the ability to purify human HSF in the active form to characterize further the in vitro activation of HSF. Here we have developed a procedure to deactivate the DNA binding ability of HSF. When purified and deactivated HSF is heated, the DNA binding ability of HSF is activated. This activation occurs most efficiently at 43 degrees C (heat shock temperature), but, in contrast to activation in the crude system, some activation of HSF is observed at 37 degrees C (non-heat shock temperature). We show that purified and deactivated HSF is similar to natural inactive HSF in both size and shape. Thus, the monomer to trimer transition that activates HSF can occur in a temperature-dependent fashion in the absence of other proteins. It is possible that these biochemical properties of HSF contribute to the ability of HSF to respond to heat in vivo.

Isolation of a cDNA for HSF2: evidence for two heat shock factor genes in humans.

The heat shock response is transcriptionally regulated by an evolutionarily conserved protein termed heat shock factor (HSF). We report the purification to homogeneity and the partial peptide sequence of HSF from HeLa cells. The peptide sequence was used to isolate a human cDNA with a predicted open reading frame that has homology to the DNA binding domains of both Saccharomyces cerevisiae and Drosophila HSFs. The cDNA directs the synthesis of a protein that binds to the heat shock element with specificity identical to HeLa HSF and stimulates transcription from a heat shock promoter. The expressed protein cross-reacts with anti-HSF antibodies. Surprisingly, however, this cDNA does not encode all of the peptides obtained from purified HeLa HSF. These peptides are encoded by a distinct human cDNA, HSF1, described by Rabindran et al. [Rabindran, S. K., Giorgi, G., Clos, J. & Wu, C. (1991) Proc. Natl. Acad. Sci. USA 88, 6906-6910.] It therefore appears that there is a human heat shock factor gene family and that at least two separate but related HSF proteins regulate the stress response in humans.

Facilitated binding of GAL4 and heat shock factor to nucleosomal templates: differential function of DNA-binding domains.

Regulatory factors must contend with chromatin structure to function. Although nucleosome structure and position on promoters can be important in determining factor access, the intrinsic ability of factors to bind to nucleosomal DNA might also play an essential regulatory role. We have used templates where nucleosomes were either randomly positioned or rotationally phased to demonstrate that two transcription factors, heat shock factor (HSF) and GAL4, differ significantly in their ability to bind to nucleosomes. GAL4 was able to bind to nucleosomal templates. Surprisingly, in contrast to its behavior on naked DNA, GAL4 bound better to multiple GAL4 sites than to a single GAL4 site on these templates. HSF alone was not able to bind to nucleosomal templates. HSF was able to bind to nucleosomal templates, however, when the TATA-binding factor TFIID was present. Consequently, binding to nucleosomal templates could be facilitated by adjacent binding of the same protein in the case of GAL4 but required binding of a second protein in the case of HSF. Taken together, these data demonstrate that regulatory factors differ in their inherent ability to bind to nucleosomal templates. These differences are likely to be important to the function of these factors in vivo.

Regulation of heat shock factor in Schizosaccharomyces pombe more closely resembles regulation in mammals than in Saccharomyces cerevisiae.

The heat shock response appears to be universal. All eucaryotes studied encode a protein, heat shock factor (HSF), that is believed to regulate transcription of heat shock genes. This protein binds to a regulatory sequence, the heat shock element, that is absolutely conserved among eucaryotes. We report here the identification of HSF in the fission yeast Schizosaccharomyces pombe. HSF binding was not observed in extracts from normally growing S. pombe (28 degrees C) but was detected in increasing amounts as the temperature of heat shock increased between 39 and 45 degrees C. This regulation is in contrast to that observed in Saccharomyces cerevisiae, in which HSF binding is detectable at both normal and heat shock temperatures. The S. pombe factor bound specifically to the heat shock element, as judged by methylation interference and DNase I protection analysis. The induction of S. pombe HSF was not inhibited by cycloheximide, suggesting that induction occurs posttranslationally, and the induced factor was shown to be phosphorylated. S. pombe HSF was purified to near homogeneity and was shown to have an apparent mobility of approximately 108 kDa. Since heat-induced DNA binding by HSF had previously been demonstrated only in metazoans, the conservation of heat-induced DNA binding by HSF among S. pombe and metazoans suggests that this mode of regulation is evolutionarily ancient.

Activation in vitro of sequence-specific DNA binding by a human regulatory factor.

The human heat-shock factor (HSF) regulates heat-shock genes in response to elevated temperature. When human cells are heated to 43 degrees C, HSF is modified post-translationally from a form that does not bind DNA to a form that binds to a specific sequence (the heat-shock element, HSE) found upstream of heat-shock genes. To investigate the transduction of the heat signal to HSF, and more generally, how mammalian cells respond at the molecular level to environmental stimuli, we have developed a cell-free system that exhibits heat-induced activation of human HSF in vitro. Comparison of HSF activation in vitro and in intact cells suggests that the response of human cells to heat shock involves at least two steps. First, an ATP-independent, heat-induced alteration of HSF allows it to bind the HSE; the temperature at which activation occurs in vitro implies that a human factor directly senses temperature. Second, HSF is phosphorylated. It is possible that similar multi-step activation mechanisms play a role in the response of eukaryotic cells to a variety of environmental stimuli, and that these mechanisms evolved to increase the range and flexibility of the response.

Heat-inducible human factor that binds to a human hsp70 promoter.

A factor found in nuclear extracts of human cells bound to the heat shock element of a human heat shock protein 70 gene. The level of this factor was significantly increased after heat shock. This induction was rapid and was not blocked by cycloheximide, suggesting that an initial event in the response of a human cell to heat is the activation of a preexisting regulatory factor.