Visualization of Hypoxia-Inducible Factor 1 alpha-p300 Interactions in Live Cells by Fluorescence Resonance Energy Transfer

Abstract

Hypoxia-inducible factor (HIF)-1 mediates the hypoxia response signaling pathway essential for maintaining cellular homeostasis in low oxygen environments through its complex formation with CBP/p300 in the nucleus. Employing fluorescence resonance energy transfer (FRET), we devised a live-cell interaction assay based on reporter proteins by tagging fluorescent proteins onto the carboxy termini of HIF-1 and p300. The nature of the constructed reporter protein was verified by observing localized distribution, degradation, and stabilization kinetics in cells transfected with the HIF-1 containing plasmid. A mutant HIF-1 incapable of binding to p300 was then utilized to demonstrate insignificant FRET efficiency, thereby confirming that our constructs could effectively probe the direct interaction between HIF-1 and p300. We further examined the effects of small molecules known to modulate the HIF-1-p300 interaction and transcriptional activity on FRET. Finally, by inhibiting activities of two HIF-specific hydroxylases, HIF-specific prolyl hydroxylase (PHD) 2 and factor inhibiting HIF-1 (FIH-1) with their specific siRNAs, we explored how these HIF-specific hydroxylases contribute to the HIF-1-p300 interaction by FRET measurements along with HIF-1 mediated transcriptional activation. Therefore, this technique would provide a way to study selective inhibition of either PHD2 or FIH-1 within living cells, and to screen specific inhibitors of HIF-mediated transcription activity for therapeutic applications. J. Cell. Biochem. 115: 271-280, 2014. (c) 2013 Wiley Periodicals, Inc.