Identification of TNF-alpha-sensitive sites in HCMVie1 promoter.

TitleIdentification of TNF-alpha-sensitive sites in HCMVie1 promoter.
Publication TypeJournal Article
Year of Publication2001
AuthorsZhang H, Fu S, Busch A, Chen F, Qin L, Bromberg JS
JournalExp Mol Pathol
Volume71
Issue2
Pagination106-14
Date Published2001 Oct
ISSN0014-4800
KeywordsAntigens, 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.

DOI10.1006/exmp.2001.2391
Alternate JournalExp Mol Pathol
PubMed ID11599916
Grant ListAI 42842 / AI / NIAID NIH HHS / United States
Related Faculty: 
Lihui Qin, M.D., Ph.D.

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