title: Identification and functional analysis of a second RBF-2 binding site within the HIV-1 promoter.
abstract: Transcription from the HIV-1 long terminal repeat (LTR) is mediated by numerous host transcription factors. In this study we characterized an E-box motif (RBE1) within the core promoter that was previously implicated in both transcriptional activation and repression. We show that RBE1 is a binding site for the RBF-2 transcription factor complex (USF1, USF2, and TFII-I), previously shown to bind an upstream viral element, RBE3. The RBE1 and RBE3 elements formed complexes of identical mobility and protein constituents in gel shift assays, both with Jurkat T-cell nuclear extracts and recombinant USF/TFII-I. Furthermore, both elements are regulators of HIV-1 expression; mutations in LTR-luciferase reporters and in HIV-1 molecular clones resulted in decreased transcription, virion production, and proviral expression in infected cells. Collectively, our data indicate that RBE1 is a bona fide RBF-2 binding site and that the RBE1 and RBE3 elements are necessary for mediating proper transcription from the HIV-1 LTR.
authors.affiliation: Department of Biochemistry and Molecular Biology, Molecular Epigenetics, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, B.C. Canada. dahabieh@interchange.ubc.ca
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Dahabieh
authors.forename: Marcel
authors.initials: M
authors.lastname: Ooms
authors.forename: Tom
authors.initials: T
authors.lastname: Malcolm
authors.forename: Viviana
authors.initials: V
authors.lastname: Simon
authors.forename: Ivan
authors.initials: I
authors.lastname: Sadowski
mesh_terms: D001665:Binding Sites; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D017930:Genes, Reporter; D015497:HIV-1 / Q000235:genetics*; D054884:Host-Pathogen Interactions; D006801:Humans; D019169:Jurkat Cells; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019473:Transcription Factors, TFII / Q000235:genetics / Q000378:metabolism*; D051720:Upstream Stimulatory Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: C104259:GTF2I protein, human; D011994:Recombinant Proteins; D019473:Transcription Factors, TFII; C497181:USF1 protein, human; C497184:USF2 protein, human; D051720:Upstream Stimulatory Factors; D008156:Luciferases