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

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.
Mesh Terms:
Aryl Hydrocarbon Receptor Nuclear Translocator, Basic Helix-Loop-Helix Transcription Factors, Cell Line, Tumor, Fluorescence Resonance Energy Transfer, Fluorescent Dyes, Gene Expression Regulation, Genetic Variation, Humans, Male, Prostatic Neoplasms, Protein Isoforms, Protein Multimerization, RNA Interference, RNA, Small Interfering
Exp. Cell Res.
Date: Aug. 15, 2015
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