A redox mechanism controls differential DNA binding activities of hypoxia-inducible factor (HIF) 1α and the HIF-like factor

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the HIF-like factor (HLF) are two highly related basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) homology transcription factors that undergo dramatically increased function at low oxygen levels. Despite strong similarities in their activation mechanisms (e.g, they both undergo rapid hypoxia-induced protein stabilization, bind identical target DNA sequences, and induce synthetic reporter genes to similar degrees), they are both essential for embryo survival via distinct functions during vascularization (HIF-1 alpha) or catecholamine production (HLF). It is currently unknown how such specificity of action is achieved. We report here that DNA binding by HLF, but not by HIF-1 alpha, is dependent upon reducing redox conditions. In vitro DNA binding and mammalian two-hybrid assays showed that a unique cysteine in the DNA-binding basic region of HLF is a target for the reducing activity of redox factor Ref-1. Although the N-terminal DNA-binding domain of HIF-1 alpha can function in the absence of Ref-1, we found that the C-terminal region containing the transactivation domain requires Ref-1 for fall activity. Our data reveal that the hypoxia-inducible factors are subject to complex redox control mechanisms that can target discrete regions of the proteins and are the first to establish a discriminating control mechanism for differential regulation of RIF-la and HLF activity.