cis‐Acting Elements and Transcription Factors Involved in the Intestinal Specific Expression of the Rat Calbindin‐D9k Gene

Abstract

The calbindin-D9k (CaBP9k) gene is mainly expressed in differentiated duodenal epithelial cells and is used as a model for studying the molecular mechanisms of intestine-specific transcription. The gene has been cloned, two major DNase-I-hypersensitive sites in the duodenum have been described, and a vitamin-D-response element has been identified. We have now analysed the transcription factors and regulatory sequences involved in the transcription of the CaBP9k gene in the intestine in ex vivo and in vitro experiments. Transfection experiments in intestinal (CaCo-2) and non-intestinal (HeLa) cell lines defined two regions in the 5'-flanking sequences of the rat CaBP9k gene. A minimal proximal region (-117 to +20) promoted transcription in both intestinal expressing and non-expressing cell lines. Tissue specificity was conferred by the sequences situated further upstream, which are responsible for complete repression in the non-intestinal cells. Intestinal transcription was specified by the proximal region, containing a specialized TATA box, and a distal region, which contains a previously described intestinal DNase-I-hypersensitive site. In vitro DNase I footprinting, electrophoretic mobility shift assays and antibody supershift assays were used to examine the factors bound to the proximal promoter region (-800 to +80 bp). Rat duodenal nuclear extracts protected 12 sites. Some of them appear to be binding sites for ubiquitous (nuclear factor 1) or hepatic-enriched sites (hepatocyte nuclear factors 1 and 4, enhancer binding protein a and p) factors. DNA binding studies and transfection experiments indicated that an intestine-specific transcription factor, caudal homeobox-2, binds to the TATA box of the rat CaBP9k gene. These data contribute to our understanding of the control of the intestinal transcription of the CaBP9k gene and demonstrate that several trans-acting factors, other than the vitamin D receptor, may be factors for intestine-specific CaBP9k gene expression.Keywords: intestine ; homeodomain protein ; transcription ; calbindin-D9k gene promoter; TATA box, Relatively few of the genes expressed in the intestine have been analysed and the way in which gene transcription is regulated in the intestine is not clearly understood. Hence, the characterization of the elements required for the expression of a gene in the intestine, particularly in enterocytes, is a necessary first step in the development of molecular tools for use in the intestine. The transcription factors implicated in the expression of few intestinal genes have been identified, including those for Correspondence to M. Thomasset, lNSERM U120, HBpital Robert

Control of differentiation-induced calbindin-D9kgene expression in Caco-2 cells by cdx-2 and HNF-1α

“…This is consistent with earlier in vivo data (24,38,49,53). Previous research implicated two cdx-2 response elements as the important determinants for intestinal expression of the calbindin D 9k gene (9,34). Both elements bind to cdx-2 by ESMA (1, 9, 34), and mutation of the distal element at Ϫ3500 bp almost completely eliminated expression of a 4.6-kb rat calbindin D 9k promoter-CAT reporter gene construct in transgenic mice (9).…”

“…In addition to vitamin D regulation, calbindin D 9k expression is restricted to the differentiated villus compartment (24,38,49,53), indicating that it is under the control of factors programming intestinal cell differentiation; mutation of a cdx-2 response element at Ϫ3.5 kb decreased the intestinal expression of a calbindin D 9k reporter gene construct by 99% in transgenic mice (9). Several other potential regulatory sites in the proximal rat calbindin D 9k promoter have been reported on the basis of footprinting and gel-shift assays (1,34), i.e., a proximal cdx-2 element and sites for HNF-1, HNF-4, and CCAAT enhancer binding protein (C/EBP). None of those sites has been assigned a functional role in the regulation of calbindin D 9k promoter activity.…”

Gastrointestinal differentiation marker Cytokeratin 20 is regulated by homeobox gene CDX1

“…A number of intestine-specific genes, including lactasephlorizin hydrolase, calbindin-D9K, carbonic anhydrase, gutenriched Kruppel-like factor, MUC2, sucrase isomaltase, and liver intestine (L1)-cadherin have been characterized as being regulated by CDX2 (32)(33)(34)(35)(36)(37)(38)(39)(40)(41). The relationship of these downstream targets regulated by CDX1 and CDX2 is not clear.…”

Functional Characterization of the CCL25 Promoter in Small Intestinal Epithelial Cells Suggests a Regulatory Role for Caudal-Related Homeobox (Cdx) Transcription Factors

“…The presence of putative Cdx-1 and Cdx-2 binding sites within the murine CCL25 promoter was of interest because Cdx-1 and Cdx-2 expression is restricted to the gut epithelium in adult mice (46), and these transcription factors have been implicated in regulating expression of intestinal specific genes (47)(48)(49)(50)(51)(52). Importantly, although freshly isolated small intestinal epithelial cells expressed Cdx-1 and Cdx-2 mRNA (Fig.…”