title: Visualization of hypoxia-inducible factor 1α-p300 interactions in live cells by fluorescence resonance energy transfer.
abstract: 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.
authors: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, 136-791, Republic of Korea.
authors: So Yeon
authors: SY
authors: Kim
authors: Myong Jin
authors: MJ
authors: Lee
authors: Yu-Ran
authors: YR
authors: Na
authors: Sang Yoon
authors: SY
authors: Kim
authors: Eun Gyeong
authors: EG
authors: Yang
mesh_terms: D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D050881:E1A-Associated p300 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D060066:Protein Interaction Maps / Q000235:genetics; D015398:Signal Transduction; D015533:Transcriptional Activation / Q000235:genetics*
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D050881:E1A-Associated p300 Protein; C494360:EP300 protein, human
keywords: FIH-1; FRET; HIF-1α; HIF-1α-p300 INTERACTION; PHD2; TAD; p300