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Identification and characterization of a tannic acid-responsive negative regulatory element in the mouse mammary
1Department of Biochemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Shinjuku-ku, Tokyo 162, Japan.
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.
Insights
Tannic acid from tea leaves suppresses gene expression by affecting a specific DNA region. This region, containing an ACTG motif, binds proteins, with tannic acid enhancing this binding for gene repression.
Area of Science:
- Molecular Biology
- Gene Regulation
- Biochemistry
Background:
- Tannic acid, a polyphenol from tea, is known to suppress gene expression.
- The mechanism by which tannic acid affects gene expression, particularly glucocorticoid-induced genes like mouse mammary tumor virus (MMTV), requires detailed investigation.
Purpose of the Study:
- To elucidate the molecular mechanism underlying tannic acid's suppression of MMTV gene expression.
- To identify specific DNA sequences and protein interactions responsible for tannic acid's regulatory effects.
Main Methods:
- Chloramphenicol acetyltransferase (CAT) assays were used to analyze MMTV promoter activity.
- Deletion and point mutation analyses identified critical DNA elements within the MMTV promoter.
- Electrophoretic mobility shift assays (EMSA) and footprint analyses investigated protein-DNA interactions.
Main Results:
- A 50-base pair (bp) region downstream of the TATA element in the MMTV promoter mediates tannic acid's suppressive effect.
- This 50-bp region contains a 13-bp element crucial for tannic acid sensitivity and an ACTG motif involved in promoter repression.
- Tannic acid treatment increased the binding of nuclear protein factors to this 50-bp region, specifically to the ACTG motif and the 13-bp element.
Conclusions:
- The suppressive effect of tannic acid on MMTV gene expression is mediated by protein factors binding to a specific negative regulatory element.
- This binding is cooperative between a tannic acid-sensitive 13-bp element and an ACTG motif, which is also found in the human immunodeficiency virus promoter.