A redox mechanism controls differential DNA binding activities of hypoxia-inducible factor (HIF) 1 alpha 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) hom ology transcription factors that undergo dramatically increased function at low oxygen levels. Despite strong similarities in their activation mechani sms (e.g, they both undergo rapid hypoxia-induced protein stabilization, bi nd identical target DNA sequences, and induce synthetic reporter genes to s imilar degrees), they are both essential for embryo survival via distinct f unctions during vascularization (HIF-1 alpha) or catecholamine production ( HLF). It is currently unknown how such specificity of action is achieved. W e 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-hyb rid 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 absenc e of Ref-1, we found that the C-terminal region containing the transactivat ion domain requires Ref-1 for fall activity. Our data reveal that the hypox ia-inducible factors are subject to complex redox control mechanisms that c an 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.