title: Structural and functional analysis of the related transcriptional enhancer factor-1 and NF-κB interaction.
abstract: The related transcriptional enhancer factor-1 (RTEF-1) increases gene transcription of hypoxia-inducible factor 1α (HIF-1α) and enhances angiogenesis in endothelium. Both hypoxia and inflammatory factor TNF-α regulate gene expression of HIF-1α, but how RTEF-1 and TNF-α coordinately regulate HIF-1α gene transcription is unclear. Here, we found that RTEF-1 interacts with p65 subunit of NF-κB, a primary mediator of TNF-α. RTEF-1 increased HIF-1α promoter activity, whereas expression of p65 subunit inhibited the stimulatory effect. By contrast, knockdown of p65 markedly enhanced RTEF-1 stimulation on the HIF-1α promoter activity (7-fold). A physical interaction between RTEF-1 and p65 was confirmed by coimmunoprecipitation experiments in cells and glutathione S-transferase (GST)-pull-down assays. A computational analysis of RTEF-1 crystal structures revealed that a conserved surface of RTEF-1 potentially interacts with p65 via four amino acid residues located at T347, Y349, R351, and Y352. We performed site-directed mutagenesis and GST-pull-down assays and demonstrated that Tyr352 (Y352) in RTEF-1 is a key site for the formation of RTEF-1 and p65-NF-κB complex. An alanine mutation at Y352 of RTEF-1 disrupted the interaction of RTEF-1 with p65. Moreover, expression of RTEF-1 decreased TNF-α-induced HIF-1α promoter activity, IL-1β, and IL-6 mRNA levels in cells; however, the effect of RTEF-1 was largely lost when Y352 was mutated to alanine. These results indicate that RTEF-1 interacts with p65-NF-κB through Y352 and that they antagonize each other for HIF-1α transcriptional activation, suggesting a novel mechanism by which RTEF-1 regulates gene expression, linking hypoxia to inflammation.
authors.affiliation: College of Life Science, Liaoning University, Shenyang, China;
authors.forename: Jieliang
authors.initials: J
authors.lastname: Ma
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Aaron R
authors.initials: AR
authors.lastname: Tipton
authors.forename: Jiaping
authors.initials: J
authors.lastname: Wu
authors.forename: Angela F
authors.initials: AF
authors.lastname: Messmer-Blust
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Philbrick
authors.forename: Yajuan
authors.initials: Y
authors.lastname: Qi
authors.forename: Song-Tao
authors.initials: ST
authors.lastname: Liu
authors.forename: Hongsheng
authors.initials: H
authors.lastname: Liu
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.forename: Shaodong
authors.initials: S
authors.lastname: Guo
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D000091262:TEA Domain Transcription Factors; D051996:Transcription Factor RelA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009124:Muscle Proteins; D000091262:TEA Domain Transcription Factors; C486309:TEAD4 protein, human; D051996:Transcription Factor RelA; D014157:Transcription Factors
keywords: hypoxia-inducible factor-1α; nuclear factor κB; related transcriptional enhancer factor-1; tumor necrosis factor-α