The N-terminal Transactivation domain confers target gene specificity of hypoxia-inducible factors HIF-1α and HIF-2α

The basic helix-loop-helix-Per-ARNT-Sim proteins hypoxia-inducible factor (HIF)-1 alpha and HIF-2 alpha are the principal regulators of the hypoxic transcriptional response. Although highly related, they can activate distinct target genes. In this study, the protein domain and molecular mechanism important for HIF target gene specificity are determined. We demonstrate that although HIF-2 alpha is unable to activate multiple endogenous HIF-1 alpha-specific target genes (e.g., glycolytic enzymes), HIF-2 alpha still binds to their promoters in vivo and activates reporter genes derived from such targets. In addition, comparative analysis of the N-terminal DNA binding and dimerization domains of HIF-1 alpha and HIF-2 alpha does not reveal any significant differences between the two proteins. Importantly, replacement of the N-terminal transactivation domain (N-TAD) (but not the DNA binding domain, dimerization domain, or C-terminal transactivation domain [C-TAD]) of HIF-2 alpha with the analogous region of HIF-1 alpha is sufficient to convert HIF-2 alpha into a protein with HIF-1a functional specificity. Nevertheless, both the N-TAD and C-TAD are important for optimal HIF transcriptional activity. Additional experiments indicate that the ETS transcription factor ELK is required for HIF-2 alpha to activate specific target genes such as Cited-2, EPO, and PAI-1. These results demonstrate that the HIF-alpha TADS, particularly the N-TADS, confer HIF target gene specificity, by interacting with additional transcriptional cofactors.