Dual Mechanisms Regulating Alpha Subunit-Specific Activity in Hypoxia-Inducible Factor Signaling

The ability to adapt to and protect from environmental stresses is essential to survival and has played a major role in fitness selection during evolution. As oxygen is essential to most life, many organisms have developed a response to conditions of low oxygen availability. Throughout the animal kingdom, hypoxia-inducible factor has emerged as a master regulator of this response. These bHLH transcription factors enhance transcription of a variety of genes that work to maintain oxygen homeostasis and allow adaptation to decreased oxygen availability. Two homologues, HIF-1α and HIF-2α, have been extensively studied in this field. Though they have similar domain structures and amino acid sequences, display overlap in some gene targets, and share regulatory mechanisms, they also perform distinct roles. They differ in tissue expression patterns, both temporally during development and spatially, hypoxia-driven expression kinetics, target genes, and fold induction. To elucidate mechanisms of this differential behavior, I investigated two aspects of HIF-2α-specific regulation. Firstly, I explored the contribution of early growth response transcription factors, EGRs, to HIF-2α-directed erythropoietin expression. Through reporter assays and chromatin immunoprecipitation, these factors were determined to occupy the erythropoietin enhancer adjacent to the HIF-2α binding site. Overexpression analysis showed they could amplify HIF-2α transactivation of erythropoietin, while knockdown experiments showed they were necessary for full, endogenous expression. And co-immunoprecipitation studies revealed a physical interaction between EGRs and HIF-2α that was necessary for cooperative activity. Secondly, I investigated the mechanism by which modulation of HIF-2α activity by CBP/SIRT1-dependent acetylation was signaled. Our studies revealed ACSS2, an acetyl CoA synthetase, as the source of acetyl CoA required for HIF-2α complex formation with the acetyltransferase CBP, subsequent HIF-2α acetylation, and target gene activation. The ACSS2 substrate acetate is produced during hypoxia, and exogenous acetate supplementation to cell culture media induced this pathway independent of hypoxia. Acetate administration in mice also augmented the HIF-2α-influenced pathways of red blood cell production and tumor growth in an ACSS2-dependent manner. Thus, EGRs represent novel HIF-2α cofactors in erythropoietin induction, while acetate, through ACSS2, regulates HIF-2α acetylation-dependent activity.