Identification of Sp1 as insulin-stimulated transcription factor to bind HIF-1α promoter region.

<p>(A) EMSA was performed to determine HIF-1α promoter binding complexes. 3T3-L1 cells were treated for 4 h with insulin (30 nM). Nuclear extracts were mixed with a ³²P-labeled, double-stranded 24-mer probe containing putative Sp1 binding GC-rich region of HIF-1α promoter. Similarly, nuclear extracts were incubated with ³²P-labeled, 24-mer probe in the presence of 2 µl of anti-Sp1, anti-Sp3, or both for 30 min. Probe-bound complexes were resolved by 5% nondenaturing PAGE and visualized by autoradiography. The position of the DNA-protein complex is indicated by arrow. (B) To determine specificity of the binding radiolabeled probe was pre-mixed with unlabeled, annealed, 24-mer oligonucleotide competitor at 3- or 10-fold molar excess before addition to the nuclear extracts for wild-type (W). For mutant (M) 10- or 30-fold molar excess probes were used. After the incubation, mixtures were subjected to 5% nondenaturing PAGE and autoradiography. The position of the Sp1/Sp3 -DNA complex is indicated by arrow. (C) Nuclear extracts prepared from insulin (30 nM) treated and untreated cells were subjected to chromatin immunoprecipitation assays in which anti- p65 and anti-Sp1 antibodies were used. PCR products were analyzed on 1.5% agarose gel with ethidium bromide staining. (D) Cells were treated with mithramycin A (Mith A, 0.5 µM) for 30 min before insulin (30 nM) treatment for 4 h. Nuclear extracts were isolated and EMSA was performed using ³²P-labeled, double-stranded 24-mer probe containing Sp1 binding GC-rich region of HIF-1α promoter. All these results (A–D) are representative of one of three independent experiments with similar result. (E) Immunoblot analyses were performed in nuclear extracts as described in previous experiment (D) using HIF-1α (left upper panel) and actin (left lower panel) antibodies. Right panel shows SD of densitometric analysis of three independent experiments.