The E-cadherin promoter : Functional analysis of a GC-rich region and an epithelial cell-specific palindromic regulatory element ( epithelium-specific gene expression / initiator element / keratin promoter regulatory element / tumor differentiation / tutmor invasion )

The cell-cell adhesion molecule E-cadherin is specifically expressed in epithelia and is involved in the maintenance of the epithelial phenotype. Expression of E-cadherin is downregulated in many poorly differentiated carcinomas, which leads to higher motility and invasiveness of the cells. To examiine the mechanisms that regulate tissue-specific expression, we have characterized the promoter of the E-cadherin gene. We found that an upstream fragment (positions -178 to +92) mediates strong expression of a chloramphenicol acetyltransferase reporter gene in epithelial cells (i.e., 60% of the level obtained with simian virus 40 promoter/enhancer constructs), whereas in nonepithelial cells this promoter was either inactive or much less active. By DNase I footprinting and gel retardation analysis as well as through functional dissection of the regulatory sequences, we identified two regions that contribute to tissue-speciflic activity of the promoter: (i) a G-C-rich region between -25 and -58 that generates basic epithelial promoter activity, most likely in combination with an "initiator" element present at the single transcription start site ofthe gene, and (ii) a palindromic sequence between -75 and -86 (named E-pal) that potentiates the activity of the proximal E-cadherin promoter and confers epithelial cell-specific activity on a simian virus 40 promoter. The E-pal sequence is homologous to cis regulatory elements active in keratin gene promoters and competes with these elements for nuclear factor binding. Interestingly, the activity of the E-cadherin promoter was reduced in dedifferentiated breast carcinoma cells, indicating that the identified elements are subject to negative regulation during tumor progression. Epithelial tissues are characterized by unique structural properties, the most prominent being the tight association of individual cells through various junctional organelles and the polarized distribution of cytoplasmic and cell surface components. As a functional consequence, epithelial cells are often less mobile than cells of mesenchymal origin, they form selective permeability barriers, and they carry out vectorial transport processes in tissues (1). Several of these epithelial characteristics are controlled by the function of the cell-cell adhesion molecule E-cadherin, which is a 120-kDa transmembrane glycoprotein specifically expressed in epithelial tissues (see ref. 2 for a review). Antibodies against the extracellular domain of E-cadherin disturb its function and thereby lead to the loss of the epitheloid morphology of the cells. This treatment also enhances the motility and reduces the tight junction-mediated resistance of the cells (3-5). Furthermore, forced expression of E-cadherin cDNA in nonepithelial cells generates epithelium-like monolayers that exhibit polarized distribution of certain marker molecules (6-8). During tumor progression, a loss of E-cadherin expression can parallel dedifferentiation ofhuman carcinomas in vitro and in vivo; i.e., E-cadherin is present in well-differentiated noninvasive carcinomas, but its expression is often downregulated in poorly differentiated invasive tumors (4, 9, 10). Furthermore, invasiveness of dedifferentiated carcinoma cells can be prevented by transfection with E-cadherincDNA and restored by treatment of the transfected cells with dissociating anti-Ecadherin antibodies (9). From these studies it is evident that E-cadherin is a major determinant for the establishment and the maintenance ofthe normal epithelial phenotype and might act as an invasion suppressor in carcinomas.* MATERIALS AND METHODS A mouse genomic library in cosmid pWE15 (Stratagene) was screened for the putative E-cadherin promoter region with the 32P-labeled 145-base-pair (bp) Sac I-Acc I fragment of the 5' end of the mouse E-cadherin cDNA (6, 11). Primer extension analysis was performed with 2 gg ofpoly(A)+ RNA hybridized to 105 cpm of two 5' 32P-labeled oligonucleotide primers (a, complementary to positions +88 to +107; b, complementary to positions +133 to +157). S1 nuclease mapping was performed according to Sambrook et al. (12). The -3000 and -1400 E-cadherin promoter/chloramphenicol acetyltransferase (CAT) gene constructs were prepared by cloning appropriate restriction fragments into the polylinker of the promoterless plasmid pCAT-Basic (Promega). The -800, -178, -58, and -21 promoter/CAT constructs were prepared by BAL-31 nuclease (Boehringer Mannheim) digestion of the -3000 deletion fragment. All deletion fragments had a common 3' end at position +92. CAT activities ofthe various constructs were compared with those produced by CAT plasmids containing either the simian virus 40 (SV40) promoter/enhancer (pCAT-Control, Promega) or the Rous sarcoma virus promoter. Furthermore, two complementary oligonucleotides spanning the sequence from -92 to -69 (E-pal) were cloned via BamHI linkers into the Bgl II site of the CAT plasmid containing the SV40 promoter (pCATPromoter, Promega) or via blunt-end ligation into the Acc I site upstream of the -58 promoter/CAT construct. CAT assays were performed and activities were quantified as described (13) except that cells were transfected in suspension. To control for variations in transfection efficiency, plasmid pCH110 (a gift of G. Ryffel, University of Essen), containing the SV40 promoter and the Escherichia coli lacZ gene, was cotransfected and the amounts of cell extracts were adjusted according to the ,B-galactosidase activity. Nuclear extracts were prepared and gel retardation assays were performed as described (14, 15), except that 1 ug of poly(dI-dC) (Boehringer Mannheim) was used as nonspecific competitor. The following oligonucleotides and their complements were used: E-pal, 5'-GATCCGGCTGCCACCTGCAGGTGCGTCCCG-3'; KER-1, 5'-GATCCAAGTGAbbreviations: CAT, chloramphenicol acetyltransferase; SV40, simian virus 40; nt, nucleotide(s). *The sequence reported in this paper has been deposited in the GenBank data base (accession no. M81449). 11495 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 114% Biochemistry: Behrens et al. TAGCCTGCAGGCCCACACCG-3' (16); AP-2, 5'-GATCCAAAGTCCCCAGGCTCCCCAG-3' (17); KTF-1, 5'TCGACAACAAACACCCTGAGGCTACGTAG-3' (18). For DNase I footprinting, the -178/+92 bp promoter fragment was radioactively labeled at either the 5' end of the coding strand (with phage T4 polynucleotide kinase) or at the 3' end of the noncoding strand (with Klenow fragment of DNA polymerase I) and cleaved at position + 17 with Ksp I. Footprinting assays were performed as described (19) with 100-150 ttg of protein from crude nuclear extracts, 1 ptg of poly(dI-dC), and 1-2 ng of labeled probe (specific activity, 2 x 107 cpm/gg). For the Spl footprinting assay, 15 ng of purified recombinant transcription factor Spl (Promega) was employed.