Real-time study of interactions between a composite DNA regulatory region (HIV-1 LTR NRE) and several transcription factors of nuclear extracts.
Here we describe the first real-time study of nuclear protein interaction with a composite DNA regulatory region. We studied the interplay between the three target sites of the negative regulatory element (NRE) of HIV-1 LTR, comprising a noncanonical GATA site overlapping two negative regulatory regions, USF and NFIL-6, and their corresponding transcription factors in nuclear extracts. By bandshift analysis, no GATA binding activity could be detected between LTR NRE and different nuclear extracts, although evidenced by in vitro footprinting. Additionally, the LTR NRE and a USF oligonucleotide showed identical retarded complexes. BIAcore study of these interactions revealed the binding of huGATA-3, as well as USF, to the immobilized LTR NRE oligonucleotide. Competition analyses, performed with GATA, USF, and NFIL-6 oligonucleotides, clearly showed that this regulatory region could bind both huGATA-3 and USF factors. Finally, the presence of USF and huGATA-3 proteins in the complexes formed with LTR NRE was ascertained using specific anti-huGATA-3 and anti-USF2 polyclonal antibodies.
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CNRS UPR 0415, ICGM, 22 rue Méchain, Paris, 75014, France.
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Vaquero
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D051743:CCAAT-Enhancer-Binding Protein-delta; D022762:CCAAT-Enhancer-Binding Proteins; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D050990:GATA3 Transcription Factor; D016325:HIV Long Terminal Repeat / Q000502:physiology*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D051720:Upstream Stimulatory Factors
D022762:CCAAT-Enhancer-Binding Proteins; C497252:CEBPD protein, human; D004268:DNA-Binding Proteins; D050990:GATA3 Transcription Factor; C494749:GATA3 protein, human; D009687:Nuclear Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C497181:USF1 protein, human; C497184:USF2 protein, human; D051720:Upstream Stimulatory Factors; D051743:CCAAT-Enhancer-Binding Protein-delta