Title | Identification of TNF-alpha-sensitive sites in HCMVie1 promoter. |
Publication Type | Journal Article |
Year of Publication | 2001 |
Authors | Zhang H, Fu S, Busch A, Chen F, Qin L, Bromberg JS |
Journal | Exp Mol Pathol |
Volume | 71 |
Issue | 2 |
Pagination | 106-14 |
Date Published | 2001 Oct |
ISSN | 0014-4800 |
Keywords | Antigens, Viral, Blotting, Southwestern, Clone Cells, Deoxyribonuclease I, DNA Primers, DNA-Binding Proteins, Electrophoretic Mobility Shift Assay, Gene Expression Regulation, Humans, Immediate-Early Proteins, NF-kappa B, Plasmids, Promoter Regions, Genetic, Protein Binding, Protein Footprinting, Transcription, Genetic, Tumor Necrosis Factor-alpha |
Abstract | 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. |
DOI | 10.1006/exmp.2001.2391 |
Alternate Journal | Exp Mol Pathol |
PubMed ID | 11599916 |
Grant List | AI 42842 / AI / NIAID NIH HHS / United States |
Related Faculty:
Lihui Qin, M.D., Ph.D.