Fig 3. Hypoxia-inducible factor (HIF) binding to the hypoxia response element (HRE) induced activation of the macrophage migration inhibitory factor (MIF) gene promoter under hypoxia, that was abrogated by the allelic variant of the HRE.

A. The effects of hypoxia on HIF binding at the MIF gene promoter in primary cultured astrocytes (PCAs). PCAs were incubated for 6, 24, or 48 h in 21.0% or 0.1% O₂. HIF-1α binding to the MIF gene promoter was analyzed by chromatin immunoprecipitation (ChIP) assay. Real-time PCR was performed on DNA purified from each of the ChIP reactions by using a primer set specific for the MIF gene promoter. The % input values indicate the ratio of immunoprecipitated DNA fragments to the input DNA fragments (Student’s t-test after logarithmic transformation; n = 6). B. The allelic variant of the HRE abrogates MIF induction by hypoxia in PCAs. PCAs were transfected with a reporter plasmid containing the MIF genomic region (−335 to −1) upstream of the Nanoluc luciferase reporter gene and a control plasmid containing the firefly luciferase reporter gene. Where indicated, the wild type (WT) HRE sequence (CACGT) was mutated to AACGT (SNP), CTAGC (mutHRE), or deletion (delHRE). At 24 h after transfection, PCAs were incubated for 24 h in 21.0% or 0.1% O₂. The graph represents the corrected luciferase activity values of each construct in cells exposed to hypoxia over the luciferase activity obtained in normoxic cells (Dunnett’s test; n = 9–14). The data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, or ***p < 0.001.