Anemia is a common medical condition associated with significant morbidity and mortality, especially if present with other diseases such as heart failure. The development of recombinant Erythropoietin (Epo), a pro-erythrocyte hormone produced in adult kidney and liver during anemia, revolutionized anemia treatment. Unfortunately, non-physiological bolus Epo administration also promotes thrombosis, hypertension, and possibly cancer growth. This occurs in part because administration of exogenous Epo lacks the normal feedback regulatory features of endogenous Epo, which is rapidly down-regulated even before erythrocytosis is evident. Endogenous Epo production and iron uptake are tightly controlled by the stress-responsive, heterodimeric transcription factor Hypoxia Inducible Factor 2 (HIF-2), whose actions are regulated by oxygen-dependent and oxygen-independent post-translational modifications (PTM). The oxygen-dependent PTM include hydroxylation of prolyl residues in specific elements of the HIF alpha proteins mediated by oxygen-dependent prolyl hydroxylases (PHD), a family of three related proteins that exhibit differential recognition of HIF-1 and HIF-2 alpha subunits. Development of PHD inhibitors (PHDi) have recently come to market after a concerted development effort over nearly two decades, but their long-term safety remains unknown. The mechanism of action for PHDi involves stabilization and/or enhanced activity of HIF-2 signaling. However, HIF-1 signaling is likely activated as well given the lack of complete specificity for the PHDi as well as cross-talk that PHD proteins have for HIF themselves. Although PHDi may represent an improvement over exogenous bolus Epo administration, PHDi treatment must still be tightly controlled as they also lack feedback behaviors associated with control of endogenous Epo production. Furthermore, because these agents have only recently come to the market and are being used in select regions of the world, side effects associated with their use in real world settings are only now coming to light. There have been case reports of increased thrombosis with their use and there is at least a theoretical concern for development of iron overload, given that prolonged augmentation of HIF-2 signaling in the gut may also stimulate iron uptake and utilization. In order to leverage the normal feedback regulatory features of Epo expression, we must understand how HIF-2 signaling occurs in a dynamic and temporal sense during hypoxia. One mechanism for enhancing HIF-2 signaling involves cyclical acetylation and deacetylation post- translational modifications. A rate-limiting step in this process is provision of acetyl CoA for use in acetylation of HIF-2, which is generated by the acetate-dependent acetyl CoA generator, acyl CoA synthetase 2 (Acss2). Acss2 is active in the early phase of hypoxia when cells generate acetate in response to this environmental stress. In this proposal, we will define the interactome surrounding ...