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 Sp1-C site was mutated (GG to TT), a 60-80% decrease in promoter activity was observed in HCT-116 cells. Gel shift, co-transfection, and chromatin immunoprecipitation assays showed that the Sp transcription factors bind to the Sp1-binding sites and transactivate NAG-1 expression. In addition, chicken ovalbumin upstream promoter-transcription factor 1 can interact with the C-terminal region of Sp1 and Sp3 proteins and induce NAG-1 promoter activity through Sp1 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.
Baek
S J
SJ
Laboratory of Molecular Carcinogenesis, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Horowitz
J M
JM
Eling
T E
TE
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D051838:COUP Transcription Factor I; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003001:Cloning, Molecular; D016207:Cytokines / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D017353:Gene Deletion; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D055436:Growth Differentiation Factor 15; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D000894:Anti-Inflammatory Agents, Non-Steroidal; D051838:COUP Transcription Factor I; D002843:Chromatin; D016207:Cytokines; D004268:DNA-Binding Proteins; C526140:GDF15 protein, human; D055436:Growth Differentiation Factor 15; C497589:NR2F1 protein, human; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D005982:Glutathione Transferase