Visualization of hypoxia-inducible factor 1α-p300 interactions in live cells by fluorescence resonance energy transfer
- Visualization of hypoxia-inducible factor 1α-p300 interactions in live cells by fluorescence resonance energy transfer
- 김소연; 이명진; 나유란; 김상윤; 양은경
- HIF-1α; p300; FRET; HIF-1α-p300 interaction; TAD; PHD2; FIH-1
- Issue Date
- Journal of cellular biochemistry
- VOL 115, NO 2, 271-280
- Hypoxia-inducible factor (HIF)-1α 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-1α 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-1α containing plasmid. A mutant HIF-1α 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-1α and p300. We further examined the effects of small molecules known to modulate the HIF-1α-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-1α-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.
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