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 betwe en 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 affini ty. To dissect regions of HSF1 that make important contributions to the sta bility of the monomer under unstressed conditions, we have used functional complementation in bakers' yeast as a facile assay system. Whereas wild-typ e human HSF1 is restrained as an inactive monomer in yeast that is unable t o substitute for the essential yeast HSF protein, mutations in the linker r egion between the DNA binding domain and the first coiled-coil allow HSF1 t o homotrimerize and rescue the viability defect of a hsf Delta strain. Fine mapping by functional analysis of HSF1-HSF2 chimeras and point mutagenesis revealed that a small region in the aminoterminal portion of the HSF1 link er is required for maintenance of HSF1 in the monomeric state in both yeast and in transfected human 293 cells. Although linker regions in transcripti on factors are known to modulate DNA binding specificity, our studies sugge st that the human HSF1 linker plays no role in determining HSF1 binding pre ferences in vivo but is a critical determinant in regulating the HSF1 monom er-trimer equilibrium.