Abstract
Heat stress regulation of human heat shock genes is mediated by human heat shock transcription factor hHSF1, which contains three 4-3 hydrophobic repeats (LZ1 to LZ3). In unstressed human cells (37 degrees C), hHSF1 appears to be in an inactive, monomeric state that may be maintained through intramolecular interactions stabilized by transient interaction with hsp70. Heat stress (39 to 42 degrees C) disrupts these interactions, and hHSF1 homotrimerizes and acquires heat shock element DNA-binding ability. hHSF1 expressed in Xenopus oocytes also assumes a monomeric, non-DNA-binding state and is converted to a trimeric, DNA-binding form upon exposure of the oocytes to heat shock (35 to 37 degrees C in this organism). Because endogenous HSF DNA-binding activity is low and anti-hHSF1 antibody does not recognize Xenopus HSF, we employed this system for mapping regions in hHSF1 that are required for the maintenance of the monomeric state. The results of mutagenesis analyses strongly suggest that the inactive hHSF1 monomer is stabilized by hydrophobic interactions involving all three leucine zippers which may form a triple-stranded coiled coil. Trimerization may enable the DNA-binding function of hHSF1 by facilitating cooperative binding of monomeric DNA-binding domains to the heat shock element motif. This view is supported by observations that several different LexA DNA-binding domain-hHSF1 chimeras bind to a LexA-binding site in a heat-regulated fashion, that single amino acid replacements disrupting the integrity of hydrophobic repeats render these chimeras constitutively trimeric and DNA binding, and that LexA itself binds stably to DNA only as a dimer but not as a monomer in our assays.