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 w ith 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 reg ions, USF and NFIL-6, and their corresponding transcription factors in nucl ear extracts. By bandshift analysis, no GATA binding activity could be dete cted 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 re vealed 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 bo th huGATA-3 and USF factors. Finally, the presence of USF and huGATA-3 prot eins in the complexes formed with LTR NRE was ascertained using specific an ti-huGATA-3 and anti-USF2 polyclonal antibodies. (C) 1999 Academic Press.