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Mucin Biosynthesis

Identification of the cis-Regulatory Elements of Human C2GnT-M Gene

Department of Biochemistry and Molecular Biology, College of Medicine, and Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska

Correspondence and requests for reprints should be addressed to Pi-Wan Cheng, Ph.D., Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, NE 68198–5870. E-mail: pcheng{at}unmc.edu

Mucin glycan is the primary determinant of mucin functions. These functions are expanded by three branch structures, including core 2, core 4, and blood group I, which are synthesized by core 2 1,6 N-acetylglucosaminyltransferase-M (C2GnT-M). Alteration of C2GnT-M gene expression is expected to have a profound effect on mucin functions, which prompted us to study the regulation of this gene. Quantitative real-time PCR analysis of the expression of this gene in 24 human tissues and airway epithelial cells showed that this gene was expressed primarily in mucus-secretory tissues. 5' Rapid amplification of cDNA ends analysis, coupled with sequence alignment with human genome database, revealed that this gene was comprised of three exons and two introns. Northern blotting using exon 1 probe showed the presence of this exon in all transcripts, suggesting the presence of cis-regulatory elements in the proximal region upstream of and/or near the transcription initiation site (+1). Analysis of this DNA region (–417/+187) by a promoter-reporter transient transfection assay, coupled with serial deletion and linker scanning mutagenesis, revealed two positive regulatory regions, including –291/–282, and –62/–43. Further, the promoter activity was enhanced by all-trans retinoic acid (ATRA) and IL-13. Thus, the promoter region is specific to hC2GnT-M gene and subject to regulation by ATRA and IL-13. These cis-regulatory elements may be useful for construction of a mucus cell–specific vector for therapy of mucus hypersecretory diseases.

Key Words: mucin • C2GnT-M • quantitative real-time PCR • cis-regulatory elements • promoter

CLINICAL RELEVANCE Identification of the cis-regulatory elements of the hC2GnT-M gene would afford an opportunity for constructing a mucus cell-specific vector for gene therapy of mucus hypersecretory diseases.