Figure 6: HSF1–SSBP1 complex promotes recruitment of BRG1.

(a) SSBP1 does not affect DNA-binding activity of HSF1. MEF cells were infected for 72 h with Ad-sh-mSSBP1-KD1 or Ad-sh-SCR, and heat-shocked at 42 °C until 40 min (HS). Whole-cell extracts were prepared and subjected to EMSA using a ³²P-labelled ideal HSE-oligonucleotide in the presence of preimmune serum (PI) or HSF1 antibody (Anti-HSF1; upper). Western blotting was performed (lower). (b) Schematic representation of mouse HSP70.3 (HSPA1A) locus. Regions a–e in the HSP70.3 promoter were amplified by PCR using primer sets. Amplified proximal and distal HSE regions (pHSE and dHSE), and coding (coding) and intergenic regions (inter.) by real-time PCR are shown as grey boxes. (c) SSBP1 is recruited to dHSE containing region. ChIP-enriched DNA was prepared, using preimmune serum (PI) or antibody for HSF1 or SSBP1, from MEF cells untreated (C) or treated with heat shock at 42 °C for 30 min (HS). DNA fragments of the regions were amplified by PCR. (d) HSF1-dependent recruitment of SSBP1. MEF cells, in which HSF1 or SSBP1 was knocked down, were treated without (Cont.) or with heat shock at 42 °C for 30 min (HS). ChIP-qPCR analyses were performed in pHSE, dHSE and intergenic (Inter.) regions using HSF1 or SSBP1 antibody (n=3). Mean±s.d. is shown. Asterisks indicate P<0.05 by Student’s t-test. HSF1 and SSBP1 protein levels were examined by western blotting. (e) SSBP1 occupancy in the presence of HSF1 mutants. Cells, in which endogenous HSF1 was substituted with each interaction mutant, were untreated (Cont.) or treated with heat shock at 42 °C for 30 min (HS). ChIP-qPCR analyses were performed (n=3). Protein levels of HSF1 and its mutants were examined by western blotting. (f) SSBP1 promotes the recruitment of BRG1 and Pol II. SSBP1 knockdown cells were heat-shocked as described in d, and ChIP-qPCR analyses were performed using BRG1 antibody (n=3). Endogenous HSF1 was also substituted with each interaction mutant as described in e. ChIP-qPCR analyses in the dHSE and pausing, coding and intergenic regions were performed using BRG1 or Pol II antibody (n=3).