Regulation of the HSR by sirtuins. (A) Effect of the sirtuin inhibitor nicotinamide on chaperone gene expression. HeLa cells were treated with nicotinamide (NAM) before exposure to heat shock (HS), celastrol, CdCl₂, or MG132, and reverse transcription (RT)–PCR analysis was performed with the indicated primers. (B) SIRT1 siRNA inhibits transcription of hsp70. HeLa cells transfected with siRNA against SIRT1 or a control siRNA were treated with heat shock for the indicated times. RT-PCR analysis was performed and fold increase in hsp70 mRNA abundance was determined by densitometry and normalized to 18S ribosomal RNA. (C) SIRT1 siRNA inhibits HSF1 binding to the hsp70 promoter. HeLa cells were treated as described above (B). ChIP analysis was performed using an HSF1 antibody and qPCR, and the results were normalized to reactions performed with 1% of input. Experiments were performed in triplicate, and error bars indicate ±SD.

Regulation of stress-induced acetylation of HSF1 by SIRT1. (A) Acetylation of HSF1 in response to HSR inducers. 293T cells transfected with Flag-HSF1 and p300 were treated with heat shock (HS), celastrol, CdCl₂ or MG132. Cell lysates were analyzed by acetylation assay using immunoprecipitation and Western blotting (). Asterisk (*) indicates HSF1 that is slowly migrating because of increased phosphorylation (). (B) Effects of nicotinamide and trichostatin A on HSF1 acetylation. 293T cells transfected with Flag-HSF1 and p300 were treated with trichostatin A (TSA), nicotinamide (NAM), or both, and exposed to heat shock, then cell lysates were analyzed by acetylation assay. (C) Wild-type SIRT1, but not a catalytic mutant, inhibits HSF1 acetylation. 293T cells were transfected with Flag-HSF1, p300, and either SIRT1 WT or a SIRT1 H363Y mutant before treatment with heat shock and analysis by acetylation assay. (D) HSF1 acetylation in response to heat shock. Cos7 cells transfected with Myc-HSF1 were treated with heat shock for the indicated times and analyzed by acetylation assay. SIRT1 and HSF1 were detected with an antibody to SIRT1 or a Myc-specific antibody. Heat-shock cognate protein 70 (Hsc70) was a loading control. (E) Effects of resveratrol. HeLa cells were treated with ethanol solvent (EtOH) or resveratrol before heat-shock treatment for the indicated times. Cell extracts were analyzed by oligonucleotide pull-down assay and Western blotting. Increased phosphorylation of HSF1 is indicated by an asterisk. (F) SIRT1 overexpression effect on HSF1 DNA binding. 293T cells were transfected with SIRT1 WT and then subjected to heat shock for the indicated times. ChIP analysis was performed using an HSF1 antibody and qPCR. Experiments in (A) to (G) were performed in triplicate, and error bars indicate ±SD.

Mutation of HSF1 K80 inhibits the HSR. (A) Mutation of HSF1 at K80 disrupts DNA binding activity. EMSA reactions were performed with extracts from hsf1^−/− cells transfected with the indicated HSF1 constructs treated with or without heat shock (HS) (top). The EMSA probe contains the proximal HSE from the human hsp70 promoter. Western blot analysis was performed on the same samples to show HSF1 and Hsc70 levels. (B) Mutation of recombinant HSF1 at K80 disrupts DNA binding ability. EMSA reactions with increasing amounts (5, 20, 40, 80, or 120 ng) of recombinant WT HSF1 or HSF1 K80Q and a probe containing an HSE are shown (top). A sample without (−) HSF1 protein was a control. Western blot analysis was performed on the same samples to show HSF1 expression levels. (C) Failure of HSF1 mutated at K80 to rescue the HSR in hsf1^−/− cells. hsf1^−/− cells were transfected with the indicated versions of human HSF1 and treated with or without heat shock. RNA was quantified using qPCR with primers for the indicated genes. Data are normalized to values obtained for glyceraldehyde 3-phosphate dehydrogenase and are relative to the abundance of each mRNA in WT HSF1 cells treated without heat shock (value set as 1). Experiments in (A) to (C) were performed in triplicate, and error bars indicate ±SD.

Biological effects of SIRT1 on the HSR. (A) Protection of cells from severe stress by overexpressed SIRT1. 293T cells were transfected with empty vector (mock) or SIRT1 and treated with a 45°C heat shock for the indicated times, followed by recovery at 37°C. After 24 hours, cell death was determined by trypan blue uptake. The experiment was performed three times in triplicate, and error bars indicate ±SD. (B) Correlation of the age-dependent decline in the HSR with decreased abundance of SIRT1. Cell extracts from early passage (passage 21) and senescent (passage 44) WI-38 fibroblasts were treated with heat shock (HS) or heat shock followed by a 3-hour recovery at 37°C (R) and analyzed by EMSA with a probe containing an HSE (top). Western blot analysis was done on the same samples to show Hsp70, HSF1, and SIRT1 expression levels (bottom). (C) Model of the HSF1-SIRT1 regulatory network. The regulation of SIRT1 by aging and cellular metabolic state affects the activity of a network of transcription factors, including HSF1, to result in increased longevity and stress resistance.