Fig. 4.

HIF-1α is a transcriptional repressor of IRF5 and IRF3. (A and B) HIF-1α down-regulated IRF5 and IRF3 transcripts in human monocytes. HIF-1α encoding plasmid or HIF-1α siRNA was transfected into primary human monocytes and IRF5 or IRF3 mRNA was measured 24 h after transfection, mean ± SEM, n = 4. Unpaired t test. *P ≤ 0.05; **P ≤ 0.001. (C and D) HIF-1α binds the IRF5 promoter and IRF3 promoter. ChIP assay coupled to PCR was performed in HIF-1α–overexpressing monocytes. Anti-GFP, histone H3, and HIF-1α antibodies and primers covering the putative HIF-1α binding site on the IRF5 or IRF3 promoter regions were used. Data represent one of the three independent experiments. (E and F) IRF5 or IRF3-promoter constructs and HIF-1α–encoding plasmid or HIF1α siRNA were transfected into human monocytes. Gaussia luciferase (Gluc) and SEAP activities were measured from the cell supernatant (24 h). The ratio of luciferase to SEAP was calculated. Floating bars (minimum to maximum), each symbol represents an individual experiment. Unpaired t test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. n = 4. (G) Schematic of HIF1α binding site on IRF5 promoter and the constructed mutants. (H) The luciferase assay determined that HIF1α suppressed the IRF5 promoter through the HRE, GCGTG. The ratio of luciferase to SEAP was measured at 24 h. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. n = 4. (I) HIF-1α binds wild-type IRF5 promoter (IRF5p^wt) and IRF5 promoter mutant 1 (IRF5p^mut1) but not HRE mutants of the IRF5 promoter (IRF5p^mut2 and IRF5p^mut3). The transient ChIP assay from HEK293T cells was performed with the mutant constructs. Data represent one of the three independent experiments.