Identification and Characterization of a Tannic Acid-responsive Negative Regulatory Element in the Mouse Mammary Tumor Virus Promoter

Abstract

Tannic acid, which comprises polyphenolic compounds from tea leaves, suppresses the glucocorticoid-induced gene expression of mouse mammary tumor virus (MMTV) integrated into 34I cells. To investigate whether this suppression is due to promoter responsiveness to tannic acid, we performed chloramphenicol acetyltransferase analysis transfecting a MMTV promoter containing a chloramphenicol acetyltransferase expression vector into mouse fibroblast L929 cells. Deletion analysis of the promoter region revealed that a 50-base pair (bp) region located downstream of the TATA element is responsible for the suppressive effect of tannic acid. The tannic acid-sensitive suppressibility was introduced into a thymidine kinase promoter by inserting the 50-bp region into the region on the 5'-upstream side of the promoter. Detailed point mutation analyses revealed that two elements, a 13-bp element and an ACTG motif in the 50-bp region, contribute to tannic acid sensitivity and promoter repressibility, respectively. Interestingly, this repressive ACTG motif is found in the human immunodeficiency virus promoter, the activity of which is also suppressed by tannic acid (Uchiumi, F., Maruta, H., Inoue, J., Yamamoto, T., and Tanuma, S. (1996) Biochem. Biophys. Res. Commun. 220, 411-417). Furthermore, electrophoretic mobility shift analysis revealed that a protein factor(s) in nuclear extracts from L929 cells binds to the 50-bp region in a sequence-specific manner and that the amount of DNA-protein complex is increased by tannic acid treatment. Moreover, the negative regulatory sequence ACTG and the tannic acid-sensitive 13-bp element in this region were shown to be responsible for the formation of the DNA-protein complex by electrophoretic mobility shift analysis and footprint analyses. These findings suggest that the suppressive effect of tannic acid on MMTV gene expression is mediated by a protein factor(s) that binds to the negative regulatory element containing the common ACTG motif in a cooperative manner with the tannic acid-sensitive 13-bp element.