Figure 7: SSBP1 enhances the expression of mitochondrial and cytoplasmic/nuclear chaperones.

(a) Chaperone-related terms are over-represented in gene ontology enrichment analysis of heat-inducible genes. MEF cells, which were infected for 72 h with Ad-sh-mHSF1-KD2, Ad-sh-mSSBP1-KD1 or Ad-sh-SCR, were untreated (C) or treated with heat shock at 42 °C for 1 h (HS). Microarray analysis was performed using total RNA isolated from these cells. We identified 154 heat-inducible genes (fold change>+1.5, P<0.05; n=3) in scrambled RNA-treated cells, and this gene set was subjected to gene ontology enrichment (Partek Genomics Suite 6.5). The enriched biological process categories (P<0.0001, determined by Fisher’s exact test) are shown. (b) Summary of heat-inducible chaperone genes. All cytoplasmic/nuclear chaperones and mitochondrial chaperones among 154 heat-inducible genes from a are shown. Fold-changes (HS versus Cont.) were calculated by comparing mRNA levels in heat-shocked cells with those at control levels in HSF1 or SSBP1 knockdown cells (n=3). (c) Expression of mitochondrial chaperones during heat shock in SSBP1 or HSF1 knockdown cells. MEF cells were infected with Ad-sh-SCR, Ad-sh-mSSBP1-KD1 or Ad-sh-mHSF1-KD2, and heat shocked at 42 °C for the indicated periods. HSP60 and HSP10 mRNAs were quantified by RT-qPCR (n=3). Mean±s.d. is shown. Asterisks indicate P<0.05 by analysis of variance. (d) Protein levels of mitochondrial as well as cytoplasmic/nuclear chaperones in SSBP1 knockdown cells. Cells infected with Ad-sh-SCR, Ad-sh-mSSBP1-KD1 or Ad-sh-mHSF1-KD2, were heat shocked at 42 °C for 1 h and then allowed to recover for the indicated periods. Western blotting was performed. (e) HSF1–SSBP1 complex promotes the recruitment of BRG1 to the HSP60/HSP10 promoter. Cells, in which endogenous HSF1 was replaced with each interaction mutant, were untreated (Cont.) or treated with heat shock at 42 °C for 30 min (HS). ChIP-qPCR analyses in HSP60/HSP10 promoter were performed (n=3). Mean±s.d. is shown. Asterisks indicate P<0.05 by Student’s t-test. In schemes of promoter regions, each HSE consensus sequence, nGAAn, is underlined.