Upregulation of MUC6 mucin gene expression by NFκB and Sp factors

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Abstract

To clarify the mechanism underlying regulation of MUC6 expression, we isolated the 5′ flanking region of the MUC6 gene (5′-MUC6). We determined the transcription start site of the MUC6 gene, and found a TATA box at −35 to −29 bp, a putative NFκB consensus sequence at −173 to −164 bp, and putative Sp family binding sites at −530 to −521 and −847 to −838 bp. The luciferase activity of 5′-MUC6 gradually decreased with deletion of these sites. NFκB inhibitory factor IκB decreased the luciferase activity, and forced expression of NFκB induced MUC6 transcription. An inhibitor of Sp family binding, mithramycin A, suppressed MUC6 transcripts, and Sp1 and Sp3 overexpression up-regulated them. Binding of Sp family members to their putative sites was confirmed by electrophoretic mobility shift assays. Our results suggest that MUC6 transcription is regulated by NFκB and Sp family members.

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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