Identification of TNF-alpha-sensitive sites in HCMVie1 promoter.
Viral vectors using the human cytomegalovirus immediate-early promoter (HCMVie1 promoter) are potentially efficient tools for gene delivery in vivo to diverse cell types. We previously demonstrated that two cytokines, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (INF-gamma), inhibited transgene expression from this promoter in skeletal and cardiac myocytes. In this study, electrophoretic mobility shift assays (EMSAs) were performed to identify the TNF-alpha response elements from the HCMVie1 promoter. The results show that TNF-alpha enhances the interaction of nuclear proteins from the C2C12 myocyte line with a single restricted segment of the HCMVie1 promoter. In vitro DNase I footprinting defined precisely the sites of interaction to two elements: nucleotides -1 to 0 and +24 to +36 relative to a transcription initiation cap homologous in the HCMVie1 promoter. These sites contain homologous sequences for cap initiation site (82%) and NFkappaB (62%) sites, respectively. Specificity was further ascertained by competitive EMSAs with wild-type and mutant oligonucleotide probes. Southwestern blotting showed that three proteins (45, 30, and 20 kDa) bound to this TNF-alpha-sensitive element, separately. However, EMSAs failed to prove a role for Yin Yang-1 (YY-1), NFkappaB (p65), or NFkappaB (p50) in binding to these sites. Our results provide evidence for two novel sites in the HCMVie1 promoter that are targets for TNF-alpha enhanced binding of transcription factors.
Zhang
H
H
The Institute for Gene Therapy and Molecular Medicine and the Recanati/Miller Transplant Institute, New York, New York 10029, USA.
Fu
S
S
Busch
A
A
Chen
F
F
Qin
L
L
Bromberg
J S
JS
D000956:Antigens, Viral / Q000378:metabolism*; D026501:Blotting, Southwestern; D002999:Clone Cells; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011485:Protein Binding; D023302:Protein Footprinting; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000956:Antigens, Viral; D017931:DNA Primers; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; C044582:immediate-early proteins, cytomegalovirus; D003850:Deoxyribonuclease I