Select early growth response (Egr) isoforms augment hypoxia inducible factor 2 (HIF-2) regulation of erythropoietin (Epo) gene expression in mammals.

Hypoxia inducible factors (HIFs) are heterodimeric, oxygen-sensitive, stress-responsive transcription factors in vertebrates composed of one of three alpha components conferring oxygen sensitivity and one of four beta components required for dimerization and DNA binding. The founding alpha member, HIF-1α, is ubiquitously expressed and regulates numerous target genes required for cellular function under physiological hypoxic states. In comparison, HIF-2α is more restricted in its expression, but nevertheless is also required for normal physiological function. The prototypical HIF-2 target gene is erythropoietin (Epo), one of the most highly hypoxia-inducible genes in mammals and in Hep3B cells, a model cell line used to study hypoxia-inducible gene regulation. However, despite cell culture and in vivo data supporting Epo as a preferential HIF-2 target, the molecular basis for selective activation of Epo by HIF-2 remains unknown. In this study, we report identification of novel evolutionary conserved cis-acting elements in the mammalian 3' Epo enhancer region that includes recognition sites for stress-responsive early growth response (Egr) transcription factors, demonstrate that select Egr2 and Egr3 isoforms augment HIF-2 activation of a reporter containing the extended 3' Epo enhancer with Egr binding sites, reveal stable Egr2/HIF-2 complex formation in hypoxic Hep3B cells, and provide conditional knockout data from mice supporting an in vivo role for Egr2 in Epo gene regulation. These results provide insights into HIF-2 selective signaling mechanisms with ramifications that extend well beyond Epo regulation.