The existence of multiple heat shock factor (HSF) genes in higher eukaryotes has prompted questions regarding the functions of these HSF family members, especially with respect to the stress response. To address these questions, we have used polyclonal antisera raised against mouse HSF1 and HSF2 to examine the biochemical, physical, and functional properties of these two factors in unstressed and heat-shocked mouse and human cells. We have identified HSF1 as the mediator of stress-induced heat shock gene transcription. HSF1 displays stress-induced DNA-binding activity, oligomerization, and nuclear localization, while HSF2 does not. Also, HSF1 undergoes phosphorylation in cells exposed to heat or cadmium sulfate but not in cells treated with the amino acid analog L-azetidine-2-carboxylic acid, indicating that phosphorylation of HSF1 is not essential for its activation. Interestingly, HSF1 and HSF2 overexpressed in transfected 3T3 cells both display constitutive DNA-binding activity, oligomerization, and transcriptional activity. These results demonstrate that HSF1 can be activated in the absence of physiological stress and also provide support for a model of regulation of HSF1 and HSF2 activity by a titratable negative regulatory factor.The induction of heat shock gene expression by heat and other forms of stress has been studied for several decades as a paradigm for inducible gene expression (25). The activation of heat shock gene transcription during the stress response is mediated by heat shock transcription factor (HSF), which binds to heat shock elements (HSE) in the promoters of heat shock genes (2,4,30,56,57). In the yeast Saccharomyces cerevisiae, HSF is bound to DNA in unstressed cells and undergoes heat shock-dependent phosphorylation concomitant with transcriptional competence (18,42,45). In unstressed Drosophila and vertebrate cells, however, HSF exists in a non-DNA-binding form which is rapidly converted to the DNA-binding form by elevated temperature and other stresses, such as treatment with cadmium sulfate or the amino acid analog L-azetidine-2-carboxylic acid (20,27,30,43,56,59). This conversion is accompanied by oligomerization (53). In addition, human HSF from heatshocked cells has been shown to be phosphorylated (22).Genes encoding HSF have been isolated from the yeasts S. cerevisiae and Kluyveromyces lactis and from fruit fly, tomato, human, mouse, and chicken cells (8, 19, 29, 33, 35-37, 45, 54 mains (33,(35)(36)(37). However, the two mouse HSFs, HSF1 and HSF2, have only 38% identity overall (35), and with the exception of DNA-binding activity, little is known about their functional properties. One functional difference that we have identified is that mouse HSF1 translated in vitro in a rabbit reticulocyte lysate displays heat-inducible DNA-binding activity, whereas mouse HSF2 binds DNA constitutively (35). These results are consistent with a potential role for HSF1 in the activation of heat shock gene transcription in response to stress.To determine the functional roles of HSF1 and HSF2 in...
We have cloned two distinct mouse heat shock transcription factor genes, mHSF1 and mHSF2. The mHSF1 and mHSF2 open reading frames are similar in size, containing 503 and 517 amino acids, respectively. Although mHSFI and mHSF2 are quite divergent overall (only 38% identity), they display extensive homology in the DNA-binding and oligomerization domains that are conserved in the heat shock factors of Saccharomyces cerevisiae, Kluyveromyces lactis, Drosophila, tomato, and human. The ability of these two mouse heat shock factors to bind to the heat shock element (HSE) is regulated by heat. mHSFI is expressed in an in vitro translation system in an inactive form that is activated to DNA binding by incubation at temperatures >41~ the same temperatures that activate heat shock factor DNA binding and the stress response in mouse cells in vivo. mHSF2, on the other hand, is expressed in a form that binds DNA constitutively but loses DNA binding by incubation at >41~ Both mHSFI and mHSF2 are encoded by single-copy genes, and neither is transcriptionally regulated by heat shock. However, there is a striking difference in the levels of mHSFI mRNA in different tissues of the mouse.
Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp7O, hsp9O, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp7O in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp7O gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shockinduced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp7O promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp7O gene transcription.Rapid transcriptional activation of heat shock genes and the consequent increase in the synthesis and accumulation of their encoded proteins are induced by environmental changes which damage cellular proteins, e.g., heat or incorporation of amino acid analogs (for reviews, see references 11, 15, and 18). Previous efforts to characterize the heat shock response have typically employed such inducers. These studies have revealed that transcriptional induction in mammalian cells is mediated by activation of a pre-existing heat shock transcription factor, HSF, which acquires the ability to bind to its recognition sequence in the promoter of heat shock genes (the heat shock element, HSE) and activate their transcription (4, 28, 48; for reviews, see references 22 and 40). By using a combination of gel mobility shift assays, transcription run-on analysis, and in vivo genomic footprinting, we have demonstrated that HeLa cells exposed to a 42°C heat shock exhibit a very close temporal correlation between levels of activated HSF, binding of HSF to the proximal HSE of the hsp7O promoter in vivo, and transcriptional induction of the hsp7O gene (1, 2) (39), is a result of HSFmediated transcriptional activation (43). Hemin, an ironcontaining protoporphyrin, is a normal cellular metabolite important for erythroid differentiation and has been shown to regulate translation initiation, to catalyze peroxidation reactions, and to exert a mitogenic effect on T cells (26,27,(31)(32)(33)41). In our previous study, we showed that treatment of K562 cells with hemin, which leads to erythroid differentiation of these cells (12), also results in elevated levels of activated HSF and hsp7O transcrip...
Two members of the heat shock transcription factor (HSF) family, HSF1 and HSF2, both function as transcriptional activators of heat shock gene expression. However, the inducible DNA-binding activities of these two factors are regulated by distinct pathways. HSF1 is activated by heat shock and other forms of stress, whereas HSF2 is activated during hemin-induced differentiation of human K562 erythroleukemia cells, suggesting a role for HSF2 in regulating heat shock gene expression under nonstress conditions such as differentiation and development. To understand the distinct regulatory pathways controlling HSF2 and HSF1 activities, we have examined the biochemical and physical properties of the control and activated states of HSF2 and compared these with the properties of HSF1. Our results reveal that the inactive, non-DNA-binding forms of HSF2 and HSF1 exist primarily in the cytoplasm of untreated K562 cells as a dimer and monomer, respectively. This difference in the control oligomeric states suggests that the mechanisms used to control the DNA-binding activities of HSF2 and HSF1 are distinct. Upon activation, both factors acquire DNA-binding activity, oligomerize to a trimeric state, and translocate into the nucleus. Interestingly, we find that simultaneous activation of both HSF2 and HSF1 in K562 cells subjected to hemin treatment followed by heat shock results in the synergistic induction of hsp7O gene transcription, suggesting a novel level of complex regulation of heat shock gene expression.The classical heat shock response occurs via transcriptional induction of heat shock genes mediated by activation of heat shock transcription factor (HSF) (for reviews, see references 21, 24, 26, and 49). HSF binds to a heat shock element (HSE), which consists of contiguous arrays of the alternately oriented pentanucleotide unit 5'-NGAAN-3' found in the promoter regions of heat shock genes (5,31,32,59). In addition to physiological stress, such as exposure to oxidants, heavy metals, amino acid analogs, and elevated temperatures, the hsp70 gene and other heat shock genes have long been known to be transcriptionally activated under a large number of circumstances, including early development and differentiation, bacterial and viral infections, and oncogenic activation (for reviews, see reference 25 and references therein). In part, some of this transcriptional regulation can be attributed to a complex array of basal promoter elements, which are responsible for growth-regulated and oncogene-activated transcription of the human hsp70 gene (4,23,51,56,58). Furthermore, the recent identification of a family of HSFs in larger eukaryotes, such as humans, mice, chickens, and tomato plants, suggests that the complexity of transcriptional regulation of heat shock genes may be attributable to the differential activation of distinct HSF family members (30,35,41,42,44).In mammalian cells, the transcription factors HSF1 and HSF2 have an overall 38% identity and contain highly conserved amino acid sequences corresponding to the DNA- Le...
In contrast to most genomic DNA in mitotic cells, the promoter regions of some genes, such as the stress-inducible hsp70i gene that codes for a heat shock protein, remain uncompacted, a phenomenon called bookmarking. Here we show that hsp70i bookmarking is mediated by a transcription factor called HSF2, which binds this promoter in mitotic cells, recruits protein phosphatase 2A, and interacts with the CAP-G subunit of the condensin enzyme to promote efficient dephosphorylation and inactivation of condensin complexes in the vicinity, thereby preventing compaction at this site. Blocking HSF2-mediated bookmarking by HSF2 RNA interference decreases hsp70i induction and survival of stressed cells in the G1 phase, which demonstrates the biological importance of gene bookmarking.
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