Fig. 2: Phosphorylated HSF1 at S419 recruits TRRAP to HSP72 promoter.

a, b Schematic representation of phosphorylation sites in hHSF1 and its mutants. DBD (red box), DNA-binding domain; HR (orange box), hydrophobic heptad repeat; DHR (light green box), downstream of HR-C (yellow box). Extracts of heat-shocked HSF1-null MEF cells expressing these mutants or GFP were co-immunoprecipitated using anti-TRRAP and subjected to immunoblotting. HeLa cells were treated with heat shock, and cell extracts were subjected to HSF1 immunoprecipitation and immunoblotting using antibody for HSF1 phospho-S419 or HSF1 (c). Some extracts were incubated without (−) or with lambda protein phosphatase (λPPase) (d). Blue arrows indicate the positions of HSF1-S419 phosphorylated bands. The intensity of the upper band was markedly enhanced during heat shock. e Alignment of amino acid sequences for the DHR domain containing S419 in human, mouse, chicken, lizard (Anolis sagrei, As), and frog (Xenopus tropicalis, Xt) HSF1. This domain is characterized by the heptad repeats of hydrophobic amino acids (black circles and triangles), which are conserved in vertebrate HSF family members (HSF1 to HSF4). A red circle indicates the position of HSF1-S419. f Cells, in which endogenous HSF1 was replaced with hHSF1-HA, S419A-HA or S419G-HA, were heat-shocked, and HSP72 mRNA levels were quantified. Extracts from these cells were subjected to immunoblotting. Occupancy of TRRAP, HSF1 (g), or Pol II (h) in cells expressing HSF1-S419 mutants. Cells were treated as described in f, and ChIP assay was performed. d, distal; p, proximal; and inter, intergenic. Norminal p-values were determined by one-way ANOVA, followed by Tukey-Kramer test in f–h. Error bars indicate SEM (n = 3) in f–h. Experiments were repeated two times for a–d.