Identification of TNF-alpha-sensitive sites in HCMVie1 promoter
| Autor: | Hui Zhang, Shuang Fu, Annette Busch, Fanqing Chen, Lihui Qin, Jonathan S. Bromberg |
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| Rok vydání: | 2001 |
| Předmět: |
Transcription
Genetic Clinical Biochemistry Electrophoretic Mobility Shift Assay Biology Pathology and Forensic Medicine Immediate-Early Proteins Blotting Southwestern Transcription (biology) Deoxyribonuclease I Humans Electrophoretic mobility shift assay Protein Footprinting Binding site Southwestern blot Promoter Regions Genetic Molecular Biology Transcription factor Antigens Viral DNA Primers Regulation of gene expression Protein footprinting Tumor Necrosis Factor-alpha NF-kappa B Promoter Molecular biology Clone Cells DNA-Binding Proteins Gene Expression Regulation Plasmids Protein Binding |
| Zdroj: | Experimental and molecular pathology. 71(2) |
| ISSN: | 0014-4800 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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