NF-kappa B site-mediated negative regulation of the HIV-1 promoter by CCAAT/enhancer binding proteins in brain-derived cells.
Several transcription regulatory elements that interact with cellular DNA-binding proteins have been identified in the HIV-1 long terminal repeat (LTR). We have identified two sequence motifs in the U3 region of the LTR that are similar to the consensus 9-bp DNA-binding element of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. One of the sequences (promoter-proximal) mapped immediately upstream of the NF-kappa B element, whereas the other (promoter-distal) completely overlapped the upstream stimulatory factor (USF) binding site. In this study, we investigated the role of the enhancer-proximal consensus C/EBP binding sequence in the expression of the HIV-1 LTR. In cotransfection assays we found that although this sequence is a functional C/EBP-responsive element, the regulation of the HIV promoter by C/EBP is very complex. C/EBP isoforms inhibited the phorbol 12-myristate 13-acetate (PMA)-stimulated HIV-1 promoter activity in human glioblastoma U138MG and neuroblastoma SHSY5Y cells, but not in HeLa epithelial cells, and this inhibition required the NF-kappa B element. C/EBP also downregulated the HIV NF-kappa B element-containing SV40 early promoter activity, regardless of the presence of the flanking C/EBP-binding sequences, in the two brain-derived cells. In electrophoretic mobility shift assays with nuclear extracts from HeLa and U138MG cells, purified C/EBP markedly increased the complex formation between endogenous proteins and the NF-kappa B DNA probe without detectable association with the complex. However, with extracts from U138MG cells but not from HeLa cells, a slow migrating complex was observed. Our data suggest that the C/EBP family of transcription factors can downregulate the HIV-1 promoter activity in CNS-derived cells through the NF-kappa B binding elements.
Mondal
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D
Department of Molecular Oncology, Alton Ochsner Medical Foundation, New Orleans, LA 70121, USA.
Alam
J
J
Prakash
O
O
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000166:cytology*; D022762:CCAAT-Enhancer-Binding Proteins; D016384:Consensus Sequence; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D005909:Glioblastoma / Q000473:pathology; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000502:physiology*; D009447:Neuroblastoma / Q000473:pathology; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D022762:CCAAT-Enhancer-Binding Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D016328:NF-kappa B; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D013755:Tetradecanoylphorbol Acetate