Fig. 1

Potential of vertebrate HSF1/3 to maximally induce HSP70 expression is closely related to homeothermic capacity. (A) The percent identity between cHSF1 or cHSF3 and its vertebrate orthologs was established. The number of amino acids of each HSF is shown. DBD DNA-binding domain, HR hydrophobic heptad repeat, DHR downstream of HR-C, Region X a region between the HR-A/B and HR-C domains, Region Y a C-terminal region downstream of the DHR domain⁴⁵. The red box in mHSF3 indicates an HR-C-like domain. The transcriptional activation domains (ADs) of human HSF1 (amino acids 372–529), mouse HSF1(amino acids 395–525), chicken HSF1 (amino acids 313–491), and chicken HSF3 (amino acids 261–313) are indicated as red bars. We herein refer to the C-terminal regions containing the Y region and DHR in HSF1 proteins as “core ADs”. The C-terminal region of platypus HSF3 has the potential to activate GAL4-driven luciferase reporter gene (see Supplementary Fig. S4). (B) Phylogenetic trees of HSF1 and HSF3 proteins in vertebrates. The phylogenetic tree was generated in CLUSTAL W⁷⁹. Gaps were excluded from all phylogenetic analyses. The unrooted tree was drawn using the program TREEVIEW⁸⁰. Bootstraps of 1000 replicates were performed. The bar represents 0.1 substitutions per site. The amino acid sequences used in tree construction are described in the “Methods” section. HSF proteins in monotremes and aves are indicated in red and blue, respectively. (C) Potential of monotreme HSF1/3 to induce HSP70 expression. HSF1-null MEF cells or wild-type cells (WT, lanes 1 and 2) were infected with adenovirus expressing each HSF protein or adenovirus expressing GFP as the control. These cells were untreated (C) or treated with heat shock at 42 °C for 1 h and then allowed to recover for 3 h (HS). Cell extracts were prepared, and aliquots were subjected to Western blotting using anti-HSP70 (W27), anti-HSF1 (anti-cHSF1x plus anti-mHSF1n), anti-HSF3 (anti-XtHSF3-2 or anti-mHSF3-1), anti-GFP, or anti-β-actin antibody. Human HSF1 (hHSF1), but not mouse HSF3 (mHSF3), showed the strong potential to induce HSP70 expression⁴¹. Arrows indicate major bands of HSF1/3. Asterisks indicate non-specific bands. Original blots are presented in Supplementary Fig. S5A. (D) Potential of avian HSF1/3 to induce HSP70 expression. HSF1-null cells or wild-type cells (WT) were infected with each adenovirus, and were treated as described in (B). Cell extracts were prepared, and aliquots were subjected to Western blotting using anti-HSP70 (W27), anti-HSF1 (anti-cHSF1x plus anti-mHSF1n), or anti-HSF3 (anti-XtHSF3-2) antibody. Arrows indicate major bands of HSF1 and HSF3 proteins. Asterisks indicate non-specific bands. Original blots are presented in Supplementary Fig. S5B.