The action of HIF-3α variants on HIF-2α–HIF-1β heterodimer formation is directly probed in live cells

• We present a live-cell assay to probe direct interaction between HIF-2α and HIF-1β.
• Depletion of HIF-1α enhances the heterodimer formation of HIF-2α and HIF-1β.
• HIF-3α variants do not affect the localization of HIF-2α and HIF-1β.
• HIF-3α variants inhibit the heterodimer formation of HIF-2α and HIF-1β.

Hypoxia-inducible factors (HIFs), consisting of α and β subunits, activate various genes to adapt to low oxygen environments through their heterodimeric complex formation in the nucleus. While most of the studies have been extensively focused on the HIF-1α isoform, the effect of HIF-α isoforms on the complex formation between HIF-2α and HIF-1β in live cells has not been reported in detail. To probe these interactions in a physiological condition, we established a fluorescence resonance energy transfer (FRET) assay by introducing fluorescent reporter proteins onto the N-termini of HIF-2α and HIF-1β in live PC3 cells. After thorough validations of our FRET assay system, we showed that both HIF-1α and HIF-3α variants likely function as negative regulators on the heterodimer formation of HIF-2α with HIF-1β in cells. We also characterized the localization and stabilization of HIF-3α variants and measured the interaction between HIF-3α variants and other HIF isoforms in live cells. In contrast to the previous results showing HIF-3α-mediated blockage of HIF-1α translocation, the presence of HIF-3α did not affect the localization of HIF-2α, suggesting distinct roles of HIF-3α in regulation of two HIF-α isoforms.