Expanding the HIF-2α proteomic interactome with TurboID proximity labeling

The transcription factor HIF-2α is a central regulator of cellular adaptation to hypoxia, orchestrating gene expression programs that control angiogenesis, erythropoiesis, and metabolic reprogramming, and is famously overexpressed in cancers such as clear cell Renal Cell Carcinoma (ccRCC). While the gene targets of HIF-2α have been extensively characterized, the composition and dynamics of its protein interaction network remain less well defined. In this study, we sought to systematically characterize proteins associated with HIF-2α using two distinct proteomic approaches, enabling the identification of both stable binding partners and transient or indirect interactors. We integrated affinity purification–mass spectrometry with proximity labeling strategies, thus expanding the existing interactome of HIF-2α to include uniquely identified proteins and complexes such as members of the nuclear pore complex. Across both immunoprecipitation mass spectrometry (IP-MS) and TurboID-MS, we identified 888 and 1,048 proteins, respectively. We applied stringent filtering criteria to refine both datatsets, yielding a dataset of 329 HIF-2α interacting proteins, approximately 75% of which have yet to be documented as HIF-2α interactors. We queried this set across publicly available cancer genomics and proteomics datasets and detected expression of our interactors in breast cancer and ccRCC tumor proteomes. These findings led us to identify GRHL2 and KIF4A as proteomic interactors that contribute to poor patient survival in breast cancer. Taken together, we herein provide a comprehensive resource of HIF-2α interacting proteins which provide insights into the mechanisms by which HIF-2α drives disease progression.