Identification and functional validation of HSF1-binding site in the promoter of human <i>HYPK</i> gene.

<p>A. Different parts of human <i>HYPK</i> gene promoter cloned in pGL3 vector. HYPK_ups construct (−116 to +185) is the shortest construct that encompassed putative HSE (+69 to +83) flanked by some additional nucleotides on both sides. HYPK_ups_long_construct spanned the region −450 to +185. Compared to HYPK_ups construct, HYPK_ups_long contained additional nucleotides in the upstream region. HYPK_ups_short construct (−450 to +50) contained the upstream sequence same as HYPK_ups_long but lacked the HSE (+69 to +83). HYPK_ups_ΔHSE is a mutant HYPK promoter where two bases of HSE (G at position +70 and T at position +75) shown in shadow in the figure were deleted by mutagenesis. B. Luciferase reporter assay (n = 3) of the reporter constructs earlier described in HeLa cells undergoing no HS treatment (control) and HeLa cells subjected to HS at 42°C for 60 min followed by recovery at 37°C for 4 h. Luciferase activity of the above cells was normalized by the luciferase activity of the corresponding empty pGL3 vector transfected cells. C. Luciferase reporter assay (n = 3) of the reporter constructs in HeLa cells transiently transfected with empty pcDNA vector (control) and HSF1-pcDNA, exposed to HS at 42°C for 60 min followed by recovery at 37°C for 4 h. Luciferase activity of the above cells was normalized by the luciferase activity of the corresponding empty pGL3 vector transfected cells. D. Chromatin immunoprecipitation showing <i>in vivo</i> interaction of HSF1 and RNA polymerase II with the human <i>HYPK</i> and <i>hsp70</i> promoter and acetylation of histone H4 (AcH4) at the same site in HeLa cells subjected to no HS treatment and HeLa cells exposed to HS at 42°C for 60 min followed by recovery at 37°C for 4 h. Immunoprecipitation was carried out using anti-HSF1, anti-RNA polymerase II and anti-AcH4 antibody respectively and precipitated DNA was PCR-amplified using primers flanking the HSF1-binding sites present in human <i>HYPK</i> and <i>hsp70</i> promoter. Lane IN: PCR amplification was carried out using DNA isolated from HeLa cells subjected to no HS or HS treatment. Lane +Ab: PCR amplification was carried out using chromatin immunoprecipitated by anti-HSF1, anti-RNA polymerase II and anti-AcH4 (acetylated histone H4) antibodies. Lane IgG: PCR amplification was carried out using chromatin immunoprecipitated by IgG alone. Lane −Ve: PCR amplification was carried out without adding any template DNA. E. Quantitative analysis of ChIP assay. DNA from different samples described in D were amplified using primers specific for human <i>HYPK</i> and <i>hsp70</i> gene promoter on quantitative Real Time PCR machine. Amplification of a region of DNA bearing no putative HSF1-binding site (NS seq) was done in each experiment as a negative control. Quantification was done by normalizing the amount of immunoprecipitated DNA to the input DNA in each sample and fold enrichment was calculated by considering the normalized immunoprecipitated DNA in unstressed cells (control) as 1. <i>Error bars</i> indicate ± SD. The statistical significance level between various experimental pairs is indicated (*,<i>p</i><0.05; **,<i>p</i><0.01; ***,<i>p</i><0.001).