Structural survey of HIF-2α reveals regulation of its subcellular localization and protein interactome

Hypoxia-inducible factor 2α (HIF-2α) is a central regulator of cellular homeostasis and a known oncogenic driver in multiple cancers. Although HIF-2α is canonically defined as a nuclear transcription factor, its cytoplasmic presence and non-canonical functions remain poorly understood. Here, we performed a structural survey of HIF-2α to determine the mechanisms underlying subcellular localization, protein abundance, and activity using a deletion-construct library, transcriptional assays, and in vivo xenograft models. We found that the oxygen-dependent degradation domain (ODD), the N-terminal intrinsically disordered region (n-IDR) and the N-terminal transactivation domain (NTAD) promote cytoplasmic localization, whereas the C-terminal IDR drives nuclear accumulation. Surprisingly, we found that HIF-2α nuclear localization occurs also in the absence of PAS A and B, the domains required for ARNT (HIF-1β) dimerization, resolving the long-standing question in the field. These data suggest a dominant role for non-canonical cytoplasmic mechanisms in HIF-2α-driven tumorigenesis. Strikingly, neither NTAD nor the C-terminal CTAD was required for tumor growth in vivo , in coherence with our transcriptional assays indicating that CTAD is dispensable for transactivation and NTAD functions as a suppressor rather than an activator of transcription. Proteomic analyses reveal HIF-2α interactions with regulators of mitochondrial function, translation initiation, RNA splicing, vesicular transport, and DNA replication. Together, these findings uncover previously unrecognized structural and functional complexity of HIF-2α compartmentalization and expand its role beyond canonical transcriptional regulation.