Lewis Type 1 Antigen Synthase (β3Gal-T5) Is Transcriptionally Regulated by Homeoproteins

Abstract

The type 1 carbohydrate chain, Gal␤1-3GlcNAc, is synthesized by UDP-galactose:␤-N-acetylglucosamine ␤1,3-galactosyltransferase (␤3Gal-T). Among six ␤3Gal-Ts cloned to date, ␤3Gal-T5 is an essential enzyme for the synthesis of type 1 chain in epithelium of digestive tracts or pancreatic tissue. It forms the type 1 structure on glycoproteins produced from such tissues. In the present study, we found that the transcriptional regulation of the ␤3Gal-T5 gene is controlled by homeoproteins, i.e. members of caudal-related homeobox protein (Cdx) and hepatocyte nuclear factor (HNF) families. We found an important region (؊151 to ؊121 from the transcription initiation site), named the ␤3Gal-T5 control element (GCE), for the promoter activity. GCE contained the consensus sequences for members of the Cdx and HNF families. Mutations introduced into this sequence abolished the transcriptional activity. Four factors, Cdx1, Cdx2, HNF1␣, and HNF1␤, could bind to GCE and transcriptionally activate the ␤3Gal-T5 gene. Transcriptional regulation of the ␤3Gal-T5 gene was consistent with that of members of the Cdx and HNF1 families in two in vivo systems. 1) During in vitro differentiation of Caco-2 cells, transcriptional up-regulation of ␤3Gal-T5 was observed in correlation with the increase in transcripts for Cdx2 and HNF1␣. 2) Both transcript and protein levels of ␤3Gal-T5 were determined to be significantly reduced in colon cancer. This down-regulation was correlated with the decrease of Cdx1 and HNF1␤ expression in cancer tissue. This is the first finding that a glycosyltransferase gene is transcriptionally regulated under the control of homeoproteins in a tissue-specific manner. ␤3Gal-T5, controlled by the intestinal homeoproteins, may play an important role in the specific function of intestinal cells by modifying the carbohydrate structure of glycoproteins.

Comparative Analysis of Retroviral and Native Promoters Driving Expression of β1,3-Galactosyltransferase β3Gal-T5 in Human and Mouse Tissues

“…Moreover, the total amount of ␤3Gal-T5 transcript measured in the sample pairs varied from 20 to 150% (Fig. 2), as previously reported in human colon (3,6).…”

“…6D). The LTR sequence completely abolished both the HNF-1 complex and the minor complex, suggesting that the binding is due to a Cdx binding site (6). Identical complexes were found with the LTR probe, but only the major complex was inhibited by a 200-fold molar excess of the Ϫ614 to Ϫ574 sequence, suggesting a lower affinity of the Ϫ614 to Ϫ574 sequence for the minor complex.…”

“…A comparison of these two sequences (Fig. 6F) further revealed a string of nine identical nucleotides that includes the HNF-1 binding site and the overlapping sequence suggested to be a Cdx binding site (6). This same sequence is also present in the chimpanzee ortholog located in chromosome 22 (19).…”

“…In competitive EMSAs (Fig. 6C), formation of the major complex was prevented by the addition of the HNF-1 binding sequence of the SIF3 oligonucleotide as well as by an antibody specific to HNF-1, indicating the presence of an HNF-1 binding site, as reported for the LTR promoter (6,7). To directly compare these two promoters, competitive EMSAs were performed using both sequences (Fig.…”

“…␤3Gal-T5 is highly expressed in human colon mucosa, down-regulated in colon cancers (3), and large amounts of its reaction products are found in the bile and small intestine (4,5). In some colon cancer cells, the first exon (exon 1) in the 5Ј-UTR of the ␤3Gal-T5 transcript is under the control of a promoter located ϳ150 bp upstream and regulated primarily by the transcription factors HNF-1 and Cdx (6). Interestingly, the entire exon 1, as well as the HNF-1/Cdx binding motif, belongs to a retroviral long terminal repeat (LTR) sequence that is the dominant promoter for the ␤3Gal-T5 gene in the colon but is inactive in other tissues where ␤3Gal-T5 is expressed (7).…”