Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter - Basal transcription is mediated by Sp1 and Sp3

Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is known to be associated with anti-tumorigenic activity and belongs to the transforming growth factor-beta superfamily. In the present study, we cloned the promoter region (-3500 to +41) and investigated the transcriptional regulatory mechanisms of the basal expression of the human NAG-1 gene. Several potential transcription factor-binding sites in this region were identified. Based on the results from clones of nested deletions, the construct between -133 and +41 base pairs contains three Sp1-binding sites (Sp1-A, Sp1-B, and Sp1-C, which confer basal transcription specific activity of NAG-1 expression. When the Spi-C site was mutated (GG to TT), a 60-80% decrease in promoter activity was observed in HCT-116 cells. Gel shift, cotransfection, and chromatin immunoprecipitation assays showed that the Sp transcription factors bind to the Spl-binding sites and transactivate NAG-1 expression. In addition, chicken ovalbumin upstream promoter transcription factor I can interact with the C-terminal region of Sp1 and Sp3 proteins and induce NAG-1 promoter activity through SpI and Sp3 transcription factors. These results identify the critical regulatory regions for the human NAG-1 basal promoter. Furthermore, the results suggest that the level of expression of the NAG-1 gene will depend on the availability of Sp proteins and on co-factors such as chicken ovalbumin upstream promoter-transcription factor 1.