Isolation and Functional Characterization of the Mouse p75 TNF Receptor Promoter
详细信息 查看全文
文摘
Tumor necrosis factor (TNF) is a pleiotropic cytokine that plays an important role in immunological and inflammatory responses. It exerts its biological effects via two distinct membrane receptors of apparent molecular weight of 55 (p55TNFR) and 75 kDa (p75TNFR), respectively. Most cell lines and primary tissues express both receptor types. While the p55TNFR gene is constitutively expressed at rather low levels, the transcription of p75TNFR is strongly modulated by a number of stimulatory agents. To characterize the mouse p75TNFR gene expression on a molecular level, we screened a mouse genomic library using the 5′ end of the p75TNFR cDNA as a probe. A 6.3-kb genomic clone containing about 6 kb of 5′ flanking region and 300 bp of 3′ sequence including the translational start site and the first exon was isolated and subcloned. Primer extension analysis revealed three transcriptional start sites located at −35, −39, and −564 bp upstream of the ATG-containing first exon. To determine whether the 5′ flanking region exerts functional promoter activity, we generated deletion mutants fused to the luciferase reporter gene. Transfection of mouse fibroblasts (NIH3T3) with these constructs showed functional promoter activity of the isolated 5′ region. By further sequence analysis of the 5′ flanking region a number of putative DNA-binding sites for transcription factors, e.g., Sp1, CREB, Yi, YY1, and IFNγ-responsive element, were identified.