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Originally published In Press as doi:10.1074/jbc.M101814200 on July 9, 2001

J. Biol. Chem., Vol. 276, Issue 36, 33384-33392, September 7, 2001
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Molecular Cloning and Characterization of Human Nonsteroidal Anti-inflammatory Drug-activated Gene Promoter
BASAL TRANSCRIPTION IS MEDIATED BY Sp1 AND Sp3*

Seung Joon BaekDagger , Jonathan M. Horowitz§, and Thomas E. ElingDagger

From the Dagger  Laboratory of Molecular Carcinogenesis, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and the § Department of Anatomy, Physical Science, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606

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.

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF305420.

To whom correspondence should be addressed: Lab. of Molecular Carcinogenesis, 111 TW Alexander Dr., Research Triangle Park, NC 27709. Tel.: 919-541-3911; Fax: 919-541-0146; E-mail: Eling@niehs.nih.gov.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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