Modulation of human heat shock factor trimerization by the linker domain.
Heat shock transcription factors (HSFs) are stress-responsive proteins that activate the expression of heat shock genes and are highly conserved from bakers' yeast to humans. Under basal conditions, the human HSF1 protein is maintained as an inactive monomer through intramolecular interactions between two coiled-coil domains and interactions with heat shock proteins; upon environmental, pharmacological, or physiological stress, HSF1 is converted to a homotrimer that binds to its cognate DNA binding site with high affinity. To dissect regions of HSF1 that make important contributions to the stability of the monomer under unstressed conditions, we have used functional complementation in bakers' yeast as a facile assay system. Whereas wild-type human HSF1 is restrained as an inactive monomer in yeast that is unable to substitute for the essential yeast HSF protein, mutations in the linker region between the DNA binding domain and the first coiled-coil allow HSF1 to homotrimerize and rescue the viability defect of a hsfDelta strain. Fine mapping by functional analysis of HSF1-HSF2 chimeras and point mutagenesis revealed that a small region in the amino-terminal portion of the HSF1 linker is required for maintenance of HSF1 in the monomeric state in both yeast and in transfected human 293 cells. Although linker regions in transcription factors are known to modulate DNA binding specificity, our studies suggest that the human HSF1 linker plays no role in determining HSF1 binding preferences in vivo but is a critical determinant in regulating the HSF1 monomer-trimer equilibrium.
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Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA. dthiele@umich.edu
Thiele
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D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018869:Heat-Shock Response; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020218:Response Elements; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; C000619706:HSF1 protein, human; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C070389:HSF2 protein, human