Molecular and functional interactions of transcription factor USF with the long terminal repeat of human...

The human transcription factor USF, purified from HeLa cells, and its recombinant 43-kDa component bind to the long terminal repeat (LTR) of human immunodeficiency virus type 1. The proteins footprint over nucleotides from position -173 to -157 upstream of the transcription start site, generating strong DNAse I hypersensitivity sites at the 3' sides on both strands. As detected by methylation protection studies, the factor forms symmetric contacts with the guanines of the palindromic CACGTG core of the recognized sequence. Its binding ability is abolished by the mutation of this core sequence and is strongly reduced by the cytosine methylation of the central CpG dinucleotide. Upon binding, both recombinant and purified USFs bend the LTR DNA template. The role of USF in the control of transcription initiation from the LTR was tested by in vitro transcription assays. Upon addition of the protein, transcription from constructs containing an intact binding site is increased, while the responsiveness in constructs with a mutated sequence is abolished. Furthermore, the addition of a decoy plasmid which contains multiple repeats of the target sequence results in downregulation of transcription from the LTR. These results suggest that USF is a positive regulator of LTR-mediated transcriptional activation.