title: The action of HIF-3α variants on HIF-2α-HIF-1β heterodimer formation is directly probed in live cells.
abstract: 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.
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, South Korea; Department of Biomedical Engineering, Korea University of Science and Technology (UST), KIST Campus, Seoul 136-791, South Korea.
authors.forename: Seong Ho
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, South Korea; Department of Biological Chemistry, Korea University of Science and Technology (UST), KIST Campus, Seoul 136-791, South Korea.
authors.forename: Dohyeon
authors.initials: D
authors.lastname: Hwang
authors.affiliation: Department of Life Science, University of Seoul, Siripdae-gil 13, Dongdaemun-gu, Seoul 130-743, South Korea.
authors.forename: Hyunsung
authors.initials: H
authors.lastname: Park
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, South Korea; Department of Biological Chemistry, Korea University of Science and Technology (UST), KIST Campus, Seoul 136-791, South Korea.
authors.forename: Eun Gyeong
authors.initials: EG
authors.lastname: Yang
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, South Korea; Department of Biological Chemistry, Korea University of Science and Technology (UST), KIST Campus, Seoul 136-791, South Korea.
authors.forename: Hak Suk
authors.initials: HS
authors.lastname: Chung
authors.affiliation: Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, South Korea; Department of Biomedical Engineering, Korea University of Science and Technology (UST), KIST Campus, Seoul 136-791, South Korea. Electronic address: soyeonkim@kist.re.kr.
authors.forename: So Yeon
authors.initials: SY
authors.lastname: Kim
mesh_terms: D051017:Apoptosis Regulatory Proteins; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000378:metabolism*; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics; D055503:Protein Multimerization / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051017:Apoptosis Regulatory Proteins; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D005456:Fluorescent Dyes; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D000099162:Endothelial PAS Domain-Containing Protein 1; C497396:ARNT protein, human; C116973:HIF3A protein, human
keywords: FRET; HIF-α; HIF-β; Heterodimer formation