The action of HIF-3 alpha variants on HIF-2 alpha-HIF-1 beta heterodimer formation is directly probed in live cells

Abstract

Hypoxia-inducible factors (HIFs), consisting of alpha and beta 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-1 alpha isoform, the effect of HIF-alpha isoforms on the complex formation between HIF-2 alpha and HIF-1 beta 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-2 alpha and HIF-1 beta in live PD cells. After thorough validations of our FRET assay system, we showed that both HIF-1 alpha and HIF-3 alpha variants likely function as negative regulators on the heterodimer formation of HIF-2 alpha with HIF-1 beta in cells. We also characterized the localization and stabilization of HIF-3 alpha variants and measured the interaction between HIF-3 alpha variants and other HIF isoforms in live cells. In contrast to the previous results showing HIF-3 alpha-mediated blockage of HIF-1 alpha translocation, the presence of HIF-3 alpha did not affect the localization of HIF-2 alpha, suggesting distinct roles of HIF-3 alpha in regulation of two HIF-alpha isoforms. (C) 2015 Elsevier Inc. All rights reserved.