Organization and Regulatory Aspects of the Human Intestinal Mucin Gene (MUC2) Locus

Abstract

The human MUC2 gene maps to chromosome 11p15, where three additional mucin genes have been located, and encodes the most abundant gastrointestinal mucin normally expressed in the intestinal goblet cell lineage. However, in pathological conditions, including colorectal cancer, MUC2 can be abnormally expressed. Therefore, it is of considerable interest to understand the regulation of the MUC2 gene and how the mechanism is altered in colon cancer. Toward this goal, we have isolated a group of overlapping clones (contig) spanning 85 kilobases harboring the entire MUC2 locus, including sequences located upstream of the gene. Detection of two DNase I-hypersensitive sites in the 5 region of the MUC2 gene suggests the presence of DNA regulatory elements. To better characterize this region, we have sequenced 12 kilobases of the upstream region and analyzed it for functional activity by cloning portions of it into a luciferase reporter vector and assaying for promoter/enhancer activity using a transient transfection assay. A fragment from the AUG translational initiation codon ؉1 to ؊848 confers maximal transcriptional activity in several intestinal cell lines. Elements located further upstream exert a negative effect on the expression of the reporter gene when tested in conjunction with homologous or heterologous promoters. The same pattern of expression is observed when the MUC2/luciferase constructs are transfected into HeLa cells, which do not express the endogenous MUC2 gene. However, the level of activity in HeLa cells is at least an order of magnitude higher, suggesting that additional sequences singularly or in combination are responsible for the tissue-and cell lineage-specific expression of MUC2. Finally, we have identified an additional mucin-like gene (MUCX), located upstream of MUC2. We show that this MUCX gene, that is transcribed in opposite orientation to that of MUC2, is expressed with a pattern distinct from that of MUC2, yet similar to that of MUC5B and MUC6, two additional mucin genes located at chromosome 11p15. Recent information on the order of the mucin genes at chromosome 11p15 suggests that MUCX may be MUC6, one of the already identified mucin genes, or a novel one, yet to be fully characterized.Mucins are the major components of mucus, the visco-elastic substance that protects and lubricates epithelial mucosa, including that of the gastrointestinal tract. They are highly glycosylated molecules, and up to 80% of their mass consists of O-linked glycosyl residues. Recently, the cloning of full-length or partial cDNA sequences of mucins expressed in different tissues has greatly facilitated investigations of the polypeptide moieties (reviewed in Refs.

Altered phenotype of HT29 colonic adenocarcinoma cells following expression of the DCC gene

“…Individual colonies were isolated, expanded and further characterized. Induction of cells with TPA and forskolin was for 8 h as previously described (Velcich and Augenlicht, 1993).…”

“…Therefore, we investigated whether expression of DCC was mechanistically linked to the elaboration of the goblet cell phenotype. We have previously shown that HT29 cells can be induced to express a high level of MUC2 mRNA and protein, which represents the major form of colonic mucin, in response to forskolin and TPA (Velcich and Augenlicht, 1993). Thus, we determined whether the DCC + clones expressed MUC2 mRNA constitutively, or whether DCC could potentiate the responses to treatment with forskolin and TPA.…”

“…Thus, we investigated whether the expression of DCC caused an increase in the rate of apoptosis in the monolayer of Figure 3 MUC2 expression is not a ected by DCC levels. HT29, Neo + , DCC3 and DCC11 cells were not treated or treated with forskolin (F) or TPA for 8 h, as previously reported (Velcich and Augenlicht, 1993). Total RNA was isolated and levels of MUC2 mRNA were determined by Northern blot analysis.…”

“…Our previous work has utilized HT29 cells to study the regulation and structure of a human gastrointestinal mucin gene, MUC2 (Velcich and Augenlicht, 1993;Velcich et al, 1997), which encodes the major form of intestinal mucins and is expressed predominantly in goblet cells (Chang et al, 1994;Gum et al, 1994;Gendler and Spicer, 1995). We have shown that in HT29 cells MUC2 gene expression can be induced (Velcich and Augenlicht, 1993), however, the cells do not acquire a fully di erentiated goblet phenotype, as they fail to polarize and secrete a mucus gel (Velcich et al, 1995).…”

“…We have shown that in HT29 cells MUC2 gene expression can be induced (Velcich and Augenlicht, 1993), however, the cells do not acquire a fully di erentiated goblet phenotype, as they fail to polarize and secrete a mucus gel (Velcich et al, 1995). In addition, we have shown that the MUC2 gene is deregulated in mucinous tumor cell lines (Velcich and Augenlicht, 1993).…”