Figure 6

HIF-1A and C/EBPs transcriptionally regulate the expression of adipocyte-specific genes. (A) Predicted binding sites of hypoxia-inducible factor 1 alpha (HIF-1A; blue boxes) and CCAAT enhancer-binding proteins (C/EBPs; red boxes) in the sequence approximately 1 kb upstream of the transcription start sites (TSSs). Luciferase activity was measured 48 hours after transfection. Co-transfection with a HIF-1A expression vector markedly increased the reporter expression rate of leptin, HIG2 and PGAR. Co-transfection with a C/EBPδ expression vector markedly increased the reporter expression rate of leptin, PGAR, CFD and LPL. HIF-1A and C/EBPβ coordinately upregulated the promoter activities of leptin and HIG2. The expression of leptin was also coordinately upregulated by HIF-1A and C/EBPδ. (B) Chromatin immunoprecipitation analysis of HIF-1A or C/EBPδ binding to the promoters was conducted. DNA released from the precipitated complexes was amplified by PCR and the PCR products were separated by agarose gel electrophoresis. Lane 1, DNA ladder; lanes 2 to 6, HIF-1A antibody or C/EBPδ antibody pull-down; lane 7, anti-RNA polymerase II; lane 8, immunoglobulin G control; lanes 9 to 13, input.