Biochemical and Biophysical Research Communications
Upregulation of MUC6 mucin gene expression by NFκB and Sp factors
Section snippets
Materials and methods
Oligo-capping method. To determine the transcriptional start point for the human MUC6 gene, we used the oligo-capping method [18]. To amplify the 5′-end of MUC6 cDNA, we used an oligo-capping cDNA, Cap Site cDNA dT, derived from a stomach tumor (Nippon Gene, Tokyo, Japan), and designed two MUC6-specific downstream primers: oligo-1D, 5′-TGTCCTGGAAGGTGCAGATCTCG-3′, and oligo-2D, 5′-TGACAAACTCGTTGGTCACCTTC-3′. In one set, the primers, 1RDT, for the sequence of the cap structure (Nippon Gene) and
Identification of the 5′-end of MUC6 gene transcripts
To isolate regulatory regions of the MUC6 gene, we determined the transcription start site by oligo-capping PCR using a Cap Site cDNA. We detected only one 5′-end of the transcripts in gastric carcinoma cDNA as a template (Fig. 1). An alternative variant was not found within the region among the primers. Then, we compared the cDNA sequence of the band with the human genomic sequence of chromosome 11 (AC139749), which indicated that the transcription start site was located at 57 bp upstream of
Discussion
The transcription start site of the MUC6 gene was determined by oligo-capping PCR and 5′-MUC6 was identified. The shorter the MUC6 reporter plasmids were, the lower their luciferase activities became. A TATA box was located at −35 to −29 bp of 5′-MUC6, indicating that the transcription start site determined in our experiment is critical. There are a putative NFκB binding site and two putative Sp family binding sites in the regulatory region for MUC6 expression. The recently submitted sequence of
Acknowledgments
We thank Drs. S. Smale and J.M. Horowitz for providing the Sp1 and Sp3 expression plasmids, respectively. We also thank Drs. T. Takada, R. Iwanaga, and T. Nakamura for the valuable technical advice and helpful discussions. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Present address: Drug Resistance Laboratory, Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast, Northern Ireland.