Transcriptional regulation of pig lactase-phlorizin hydrolase: Involvement of HNF-1 and FREACs

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.

Section: Discussionmentioning

confidence: 99%

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

Isshiki

Kudo

Nishihara

et al. 2003

“…We therefore hypothesize that coexpression of GATA factors and HNF-1␣ is necessary, but not sufficient, for high levels of cellspecific LPH gene expression. Interaction with other tissuerestricted transcription factors and cofactors such as SP-1 (44), FOG-2 (62, 63), Cdx-2 (20), and the dimerization cofactor of HNF-1␣ (64) as well as other information farther upstream in the LPH promoter (18,65) must be considered when characterizing specific expression of the LPH gene in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…Identification of specific binding sites within the first 100 bp of 5Ј-flanking region in the LPH genes of several species has led to the demonstration that GATA, hepatocyte nuclear factor-1␣ (HNF-1␣), and Cdx-2 transcription factors are activators of the LPH promoter (15)(16)(17)(18)(19)(20)(21). Due to the close proximity of GATA and HNF-1 binding sites on the LPH promoter and the nearly exclusive coexpression of these two families of transcription factors in the intestinal epithelium (22)(23)(24)(25)(26)(27)(28)(29), we hypothesized that members of the GATA and HNF-1 families of transcription factors interact to modulate LPH gene expression.…”

mentioning

confidence: 99%

Physical Interaction between GATA-5 and Hepatocyte Nuclear Factor-1α Results in Synergistic Activation of the Human Lactase-Phlorizin Hydrolase Promoter

Wering

Huibregtse

Zwan

et al. 2002

GATA-4, -5, and -6 zinc finger and hepatocyte nuclear factor-1␣ (HNF-1␣) homeodomain transcription factors are expressed in the intestinal epithelium and synergistically activate the promoter of intestinal genes. Here, we demonstrate that GATA-5 and HNF-1␣ physically associate both in vivo and in vitro and that this interaction is necessary for cooperative activation of the lactasephlorizin hydrolase promoter. Furthermore, physical association is mediated by the C-terminal zinc finger of GATA factors and the homeodomain of HNF-1␣. Deletion of HNF-1␣ activation domains or interruption of HNF-1-binding sites in the lactase-phlorizin hydrolase promoter resulted in a complete loss of cooperativity, whereas deletion of GATA-5 activation domains or interruption of GATA-binding sites resulted in a reduction, but not an elimination, of cooperativity. We hypothesize that GATA/HNF-1␣ cooperativity is mediated by HNF-1␣ through its activation domains, which are oriented for high levels of activation through binding to DNA and physical association with GATA factors. These data suggest a paradigm whereby intestine-specific gene expression is regulated by unique interactions among tissuerestricted transcription factors coexpressed in the intestine. Parallel mechanisms in other tissues as well as in Drosophila suggest that zinc finger/homeodomain interactions are an efficient pathway of cooperative activation of gene transcription that has been conserved throughout evolution.The intestinal epithelium is a dynamic structure that undergoes a highly regulated process of cell division, migration, cell fate determination, and differentiation (1-3). During intestinal development, interactions between visceral endoderm and mesoderm at E8 1 in mice result in the formation of a primitive foregut that rapidly undergoes cytodifferentiation, so that by E19, an epithelial monolayer overlies nascent villi. During the first 2 weeks of postnatal life, a proliferating compartment develops into the crypts of Lieberkü hn. Stem cells located near the base of crypts rapidly divide and give rise to four terminally differentiated cell types, which migrate both basally and apically. Cells migrating to the base of crypts become Paneth's cells, whereas those migrating up the crypt toward villi become absorptive enterocytes, goblet cells, and enteroendocrine cells. At the crypt-villus junction, the proliferative phase ends, and cells acquire a differentiated phenotype characterized by the synthesis of functionally relevant proteins. The cells continue to migrate up the villi, enter an apoptotic cycle, and are shed into the intestinal lumen ϳ3 days after their initial appearance on villi. The molecular mechanisms underlying the dynamic processes of intestine-specific gene expression and cellular differentiation during development are poorly understood.Absorptive enterocytes compose ϳ95% of epithelial cells on villi and are the cells responsible for the terminal digestion and absorption of nutrients. Lactase-phlorizin hydrolase (LPH), the enzyme critical ...

“…Characterization of the lactase promoter in intestinal cell culture has led to the identification of several nuclear proteins involved in regulating intestine-specific transcription including Cdx2 (12, 13), GATA-4/5/6 (14, 15), and HNF-1 (36,37). The characterization of these regulatory elements and factors has been studied predominantly in cell culture with two previous exceptions.…”

Section: Discussionmentioning

confidence: 99%

Regulation of Intestine-specific Spatiotemporal Expression by the Rat Lactase Promoter

Lee

Wang

Lin

et al. 2002

Lactase gene transcription is spatially restricted to the proximal and middle small intestine of the developing mouse. To identify regions of the lactase gene involved in mediating the spatiotemporal expression pattern, transgenic mice harboring 0.8-, 1.3-, and 2.0-kb fragments of the 5-flanking region cloned upstream of a firefly-luciferase reporter were generated. Transgene expression was assessed noninvasively in living mice using a sensitive low light imaging system. Two independent, 1.3-and 2.0-kb, lactase promoter-reporter transgenic lines expressed appropriate high levels of luciferase activity in the small intestine (300 -3,000 relative light units/g) with maximal expression in the middle segments. Post-weaned 30-day transgenic offspring also demonstrated an appropriate 4-fold maturational decline in luciferase expression in the small intestine. The pattern of the 2.0-kb promoter transgene mRNA abundance most closely mimicked that of the endogenous lactase gene with respect to spatiotemporal restriction. In contrast, a 0.8-kb promoter-reporter construct expressed low level luciferase activity (<25 relative light units/g) in multiple organs and throughout the gastrointestinal tract in transgenic mice. Thus, a distinct 5-region of the lactase promoter directs intestine-specific expression in the small intestine of transgenic mice, and regulatory sequences have been localized to a 1.2-kb region upstream of the lactase transcription start site. In addition, we have demonstrated that in vivo bioluminescence imaging can be utilized for assessment of intestinal expression patterns of a luciferase reporter gene driven by lactase promoter regions in transgenic mice.Intestinal lactase-phlorizin hydrolase (LPH, 1 lactase) is the absorptive enterocyte membrane glycoprotein essential for digestive hydrolysis of lactose in milk. Lactase is present predominantly along the brush-border membrane of differentiated enterocytes lining the villi of the small intestine. Expression of the lactase gene is spatially restricted along the longitudinal axis of the gut (1). The lactase gene is expressed maximally in the proximal and middle small intestine and declines significantly in the distal segments of the intestine. Lactase gene expression is also temporally restricted in the gut during intestinal maturation. Enzyme activity is maximal in the small intestine of pre-weaned mammals and declines markedly during maturation. The maturational decline in lactase activity contrasts with a maturational increase in enzymatic activity of other intestinal hydrolases essential for digestion of solid foods (for comprehensive review see Ref. 2).Although the mechanisms regulating the spatial and temporal restriction of lactase gene expression have not been fully defined, lactase spatiotemporal restriction is largely regulated at the level of gene transcription. This is suggested by colocalization of lactase protein along the longitudinal axis of the gut and lactase mRNA transcripts detected by Northern blot and in situ hybridization (1). With...