Expression of human heat shock transcription factors 1 and 2 in HeLa cells and yeast.
We have examined reporter gene (beta-gal) expression directed by human heat shock transcription factors 1 and 2 (HSF1 and HSF2) in HeLa cells and in yeast (Saccharomyces cerevisiae). Transcriptional activation domains of both HSFs were mapped to the C-termini using chimeric proteins containing the GAL4 DNA binding domain (GAL4-DBD). Deletion analysis of HSF1 largely confirmed the mapping and expression pattern of activation domain 2 (AD2) previously reported by Green et al (1995) with the exception of the contribution of the oligomerization domain (hydrophobic region A) to basal repression in yeast, but not in HeLa cells. In addition, a C-terminal activation domain for HSF2 (amino acids 397 to 536) was identified by analysis in yeast. In contrast to HSF1, full length HSF2 and the isolated activation domain of HSF2 showed little activity in HeLa cells. Analysis of point mutations generated by low fidelity PCR within AD2 of HSF1 indicated that hydrophobic and charged amino acids in addition to proline, serine and threonine make critical contributions to transcriptional activity. Co-expression of GAL4-DBD fusions with AD2 of HSF1 and the C-terminal activation domain of HSF2 showed no evidence of synergism in the activation of transcription. Wild-type human HSF1 and HSF2 were both able to substitute for the endogenous yeast HSF under normal growth conditions.
Yuan
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CX
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611-0700, USA.
Czarnecka-Verner
E
E
Gurley
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WB
D002460:Cell Line; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D006367:HeLa Cells / Q000378:metabolism*; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D018869:Heat-Shock Response / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010449:Peptide Mapping; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016415:Sequence Alignment; D013696:Temperature; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics
D004268:DNA-Binding Proteins; C000619706:HSF1 protein, human; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C070389:HSF2 protein, human