Genetic Selection for Constitutively Trimerized Human HSF1 Mutants Identifies a Role for Coiled-Coil Motifs in DNA Binding

Abstract

Human heat shock transcription factor 1 (HSF1) promotes the expression of stress-responsive genes and is a critical factor for the cellular protective response to proteotoxic and other stresses. In response to stress, HSF1 undergoes a transition from a repressed cytoplasmic monomer to a homotrimer, accumulates in the nucleus, binds DNA, and activates target gene transcription. Although these steps occur as sequential and highly regulated events, our understanding of the full details of the HSF1 activation pathway remains incomplete. Here we describe a genetic screen in humanized yeast that identifies constitutively trimerized HSF1 mutants. Surprisingly, constitutively trimerized HSF1 mutants do not bind to DNA in vivo in the absence of stress and only become DNA binding competent upon stress exposure, suggesting that an additional level of regulation beyond trimerization and nuclear localization may be required for HSF1 DNA binding. Furthermore, we identified a constitutively trimerized and nuclear-localized HSF1 mutant, HSF1 L189P, located in LZ3 of the HSF1 trimerization domain, which in response to proteotoxic stress is strongly compromised for DNA binding at the Hsp70 and Hsp25 promoters but readily binds to the interleukin-6 promoter, suggesting that HSF1 DNA binding is in part regulated in a locus-dependent manner, perhaps via promoter-specific differences in chromatin architecture. Furthermore, these results implicate the LZ3 region of the HSF1 trimerization domain in a function beyond its canonical role in HSF1 trimerization.

Regulation of heat shock transcription factors and their roles in physiology and disease

“…Evidence has demonstrated that increased temperature causes intrinsic structural changes in HSF1 that may facilitate oligomerization and activation 9 . This study closely examined the structural changes in heptad repeat A (HR-A) and HR-B and the repressive properties of HR-C, which is proposed to intra-molecularly repress HSF1 oligomerization via hydrophobic and ionic interactions with HR-A and HR-B (BOX 1) 10 . The mutagenesis of key residues predicted to be required for this interaction results in constitutively oligomeric species, both in vivo and in vitro 10–12 .…”

Remote Control of Mammalian Cells with Heat-Triggered Gene Switches and Photothermal Pulse Trains

“…39,40 The exact mechanism may be elucidated by examining the heat-response of substitution or deletion mutations within the hydrophobic domains that govern and regulate HSF1 trimerization. 39,41–44 …”

High-throughput screening system for inhibitors of human Heat Shock Factor 2

“…This defect has previously been attributed to the inability of hHSF1 to trimerize in yeast cells (Liu et al 1997). Recently, a number of mutant forms of human HSF1 capable of supporting yeast cell growth have been reported (Neef et al 2013). These mutant forms may be utilized in secondary discriminating assays, provided they manifest a slow-growth phenotype when overexpressed.…”

“…4) further supports this concept. The existence of three specialized isoforms of human HSFs and their abilities to functionally substitute for the single essential yeast HSF, with the caveat that hHSF1 requires modest mutation (Liu et al 1997;Neef et al 2013), provide flexibility for further development of our high-throughput screening assay.…”

“…Surprisingly, substitution with human HSF1 (hHSF1) is impossible, presumably due to a defect in trimerization of hHSF1 in yeast. This deficit is ameliorated by three point mutations in hHSF1 that give rise to a constitutively trimerized hHSF1 (Liu et al 1997;Neef et al 2013). The high conservation of HSF function and the interchangeability of human and yeast HSFs open the possibility of creating a screening system for HSF inhibitors using humanized yeast strains.…”