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Identification of the Promoter and a Transcriptional Enhancer of The

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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11356-11360, December 1993 Developmental Biology Identification of the promoter and a transcriptional enhancer of the gene encoding L-CAM, a calcium-dependent cell adhesion molecule (gene expression/regulatory sequences/morphogenesis/cadherins) BARBARA C. SORKIN, FREDERICK S. JONES, BRUCE A. CUNNINGHAM, AND GERALD M. EDELMAN The Department of Neurobiology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037 Contributed by Gerald M. Edelman, August 20, 1993 ABSTRACT L-CAM is a calcium-dependent cell adhesion expressed in the dermis, but rather in the adjacent epidermis molecule that is expressed in a characteristic place-dependent (6). pattern during development. Previous studies of ectopic ex- L-CAM mediates calcium-dependent cell-cell adhesion (7) pression of the chicken L-CAM gene under the control of via a homophilic mechanism; i.e., L-CAM on one cell binds heterologous promoters in transgenic mice suggested that directly to L-CAM on apposing cells (8). In all tissues where cis-acting sequences controlling the spatiotemporal expression the molecule is expressed, L-CAM is detected as a trans- patterns ofL-CAM were present within the gene itself. We have membrane protein of 124 kDa (7). Mammalian proteins now examined the L-CAM gene for sequences that control its uvomorulin (9), E-cadherin (10), cell-CAM 120/80 (11), and expression and have found an enhancer within the second Arcl (12) are similar to L-CAM in their biochemical and intron of the gene. A 2.5-kb Kpn I-EcoRI fragment from the functional properties and tissue distributions. The calcium- intron acted as an enhancer of a simian virus 40 minimal dependent CAMs also include B-cadherin (13), M-cadherin promoter driving a chloramphenicol acetyltransferase (CAT) (14), N-cadherin (15), P-cadherin (16), R-cadherin (17), and reporter gene and produced 14.0-fold induction of CAT activ- T-cadherin (18). ity in MDCK cells. To narrow down the region responsible for The chicken L-CAM gene is "10 kb in length and contains enhancer activity and to determine whether the enhancer could 16 exons (19). A search for the upstream sequences that function in a cell type-specific manner, a number of smaller regulate expression of the L-CAM gene revealed another restriction fragments from the intron were tested for activity in gene, designated K-CAM, encoding a calcium-dependent two chicken cell lines, the LMH hepatoma line, which produces CAM very similar to L-CAM only 700 bp upstream of the high levels of L-CAM, and the SL-29 fibroblast line, which L-CAM translation initiation codon (20). Sequence analyses produces little, if any, L-CAM. Four L-CAM enhancer plas- and comparisons have indicated that K-CAM and B-cadherin mids containing shorter segments derived from the intron (13) are the same molecule. showed enhanced CAT activity levels (between 9.4- and 16.5- Studies of the ectopic expression of the chicken L-CAM fold) in extracts from transfected LMH cells but not from SL-29 gene in transgenic mice directed by either the rat insulin cells. DNA sequence analysis of the L-CAM enhancer region promoter or the mouse neurofilament promoter suggested revealed putative binding sites for the transcription factors that the L-CAM gene contained cis-regulatory sequences SP1, E2A, and AP-2. In addition, LE-9, the smallest L-CAM within its introns (21). This conclusion was prompted by the enhancer segment (310 bp), contained a consensus binding site observation that, although its expression was directed to for the liver-enriched POU-homeodomain transcription factor, tissue sites normally expected for the heterologous promot- HNF-1. Tests of upstream sequences showed that a 630-bp ers, the chicken L-CAM gene was also expressed in some fragment, corresponding to nearly the entire intergenic region tissues where the heterologous promoter was not expected to between L-CAM and its neighboring CAM gene, K-CAM, be active, but where L-CAM is normally expressed (kidney, could function as a promoter. In combination with the L-CAM liver, intestine, and lung). These results were reproduced in enhancer, this fragment directed cell type-specific expression of at least two independently derived pedigrees for each L-CAM the CAT reporter gene in LMH cells at a level comparable to construct. This suggested that regulatory elements within the that observed with enhancer constructs using the simian virus introns of the L-CAM gene, acting in conjunction with the 40 minimal promoter. These combined observations derme a ectopic promoter, were creating combinatorial patterns of promoter and an enhancer for the chicken L-CAM gene. They expression independent of the chromosomal integration site raise the possibility that these cis-acting regulatory sequences of the transgene. may be instrumental in directing specific place-dependent In the present study, we describe the identification and expression of the L-CAM gene in the chicken. characterization ofan enhancer within the second and largest intron of the L-CAM gene.* In addition, we show that the intergenic region between the K-CAM and L-CAM genes The liver cell adhesion molecule (L-CAM) is expressed early functions as a promoter. When combined with the enhancer, in chicken embryonic development and persists in nonneural this region was found to regulate expression of a reporter epithelia in the adult (1). During development, changes in gene in a cell type-specific manner. L-CAM expression are spatiotemporally correlated with in- ductive events (2). Consistent with this observation, trans- fection and antibody perturbation studies (3-5) indicate that MATERIALS AND METHODS L-CAM plays a critical role in morphogenesis, particularly in The chicken fibroblast cell line SL-29 and the Madin-Darby the formation of epithelia. For example, antibodies to canine cell lines were obtained from the L-CAM disrupt feather development by altering pattern kidney (MDCK) formation in the dermis, despite the fact that L-CAM is not Abbreviations: SV40, simian virus 40; CAT, chloramphenicol ace- tyltransferase; CAM, cell adhesion molecule; L-CAM, liver CAM; The publication costs of this article were defrayed in part by page charge N-CAM, neural CAM. payment. This article must therefore be hereby marked "advertisement" *The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U02633). 11356 Downloaded by guest on September 30, 2021 Developmental Biology: Sorkin et aL Proc. Natl. Acad. Sci. USA 90 (1993) 11357 American Type Culture Collection and were cultured in K-CAM L-CAM Dulbecco's modified Eagle's medium containing 10%o (vol/ 16 2 vol) fetal bovine serum. The chicken hepatocellular carcino- ma-derived cell line LMH (22) was grown in dishes coated with a gelatin substrate in Waymouth's MB752/1 medium containing 10%o fetal bovine serum. 1 - 3 Western blots were performed as described (23). Confluent 60-mm dishes of cells were washed twice with phosphate- buffered saline containing 0.5 mM CaCl2 and 0.5 mM MgC92, and each dish was extracted with 0.5 ml of 2x Laemmli sample buffer (24). Samples were resolved by electrophoresis on a 7.5% polyacrylamide gel and transferred to nitrocellu- lose (23). L-CAM was detected on blot transfers using rabbit anti-L-CAM IgG followed by incubation with 1251-labeled protein A. 12 3 4 Enhancer constructs were prepared (25) from segments of the second intron of the L-CAM gene. Ten different restric- tion fragments (LE-1-LE-10) were inserted into theXba I site downstream ofthe chloramphenicol acetyltransferase (CAT) gene in the pCAT-Promoter vector (Promega). For promoter constructs, a 630-bp Mbo I-Kpn I fragment, corresponding to almost the entire intergenic region between the K-CAM and L-CAM genes, was cloned into the Xba I site upstream ofthe CAT gene in the promoterless CAT vector, pCAT-Basic (Promega). To assay the enhancer in the presence of the L-CAM promoter, the Sst I fragment from the L-CAM FIG. 1. A DNA fragmnent from the second intron of the L-CAM second intron (LE-6) was cloned into the BamHI site down- gene confers activation of SV40 promoter-mediated CAT gene stream of the CAT gene. expression in MDCK cells. (Upper) Diagram of the 16th exon (filled Cells cultured in 60-mm dishes were transfected in dupli- box) ofthe K-CAM gene and the first three exons (open boxes) ofthe cate with 25 .g ofCAT construct, 30 pl ofLipofectin reagent L-CAM gene. The 2.5-kb Kpn I-EcoRI fragment of the L-CAM (BRL) in serum-free medium (Optimem; GIBCO/BRL), and second intron was inserted into the Xba I site in the pCAT-Promoter 1 ug of the ,-galactosidase expression vector pCMVb vector. The vector contains an SV40) promoter (hatched) followed by (Promega). After a minimum of5 h, the medium was replaced the CAT gene. (Lower) One hundred-millimeter dis'hes of MDCK cells were transfected with pCAT-Promoter (lane 1), pCAT- with that containing 10%o serum, and cells were allowed to Promoter containing the 2.5-kb intron fragment in the foreword grow an additional 18-60 h. ,B-Galactosidase and CAT assays orientation (lane 2), pCAT-Promoter containing the 2.5-kb intron were performed as described (25). To normalize for differ- fragment in the reverse orientation (lane 3), and pSV2CAT, a ences in transfection efficiency, equal amounts of 3-galacto- construct in which the CAT gene is driven by both the SV40promoter sidase activity were used for each CAT assay. The 14C- and SV40 enhancer (lane 4). Cells were harvested after 60 h and labeled chloramphenicol and acetylated products were re- assayed for CAT activity.
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