(Received for publication, August 14, 1995; and in revised form, October 24, 1995)
From the
Epithelium-specific gene expression is fundamental in both embryogenesis and the maintenance of adult tissues, and impairment of epithelial characteristics contributes to diseases such as cancer. We have here analyzed the 5`-region of the epithelial (E-) cadherin gene in order to understand mechanisms of epithelium-specific transcription and loss of expression during epithelial-mesenchymal transitions. The regulatory region of the mouse epithelial cadherin gene is composed of a promoter (from position -94 to the transcription start site) and a 150-base pair enhancer located in the first intron. The 5`-promoter consists of positive regulatory elements (a CCAAT-box and two AP-2 binding sites in a GC-rich region) and the palindromic element E-Pal that activates and represses transcription in epithelial and mesenchymal cells, respectively. The enhancer of the first intron stimulates the activity of heterologous promoters exclusively in epithelial cells. This epithelium-specific enhancer consists of three elements (E I to E III; E II and E III bind AP-2) that are necessary and sufficient for activity. We thus propose two regulatory mechanisms by which epithelial specificity of epithelial cadherin expression is determined: suppression of promoter activity in mesenchymal cells by E-Pal and enhancement of activity in epithelial cells by both E-Pal and the epithelium-specific enhancer.
Epithelia are essential and abundant tissues in most eukaryotic organs. Epithelial cells are the first identifiable embryonic cell type, which appears during compaction of the morula early in development (Fleming et al., 1993). During gastrulation, epithelial-mesenchymal transitions take place, and in this process epithelium-specific genes are repressed, and genes of the mesenchymal (and neuronal) lineages are activated (Cunningham and Edelman, 1990; Jessell and Melton, 1992). New epithelia usually derive from existing ones, i.e. from the ectoderm or endoderm, but can also be formed from the mesoderm by mesenchymal-epithelial transitions (e.g. during development of the kidney) (Saxen, 1987). In development, epithelial-mesenchymal and mesenchymal-epithelial transitions take place in a temporally and spatially controlled manner (Valles et al., 1991; Boyer and Thiery, 1993; Birchmeier and Birchmeier, 1993), whereas in tumors these changes are highly uncontrolled; loss of epithelial character is typically observed late in progression of carcinomas and correlates there with the acquisition of invasive and metastatic potential (Birchmeier and Behrens, 1994; Reichmann, 1994).
Epithelial cells form continuous cell layers, and they are generally polar. In single-layered epithelia (e.g. the mature intestine), apical and basolateral cell surface are separated by tight junctions (Citi, 1993). An example of a multilayered epithelium is the skin, where basal cells (stem cells) are covered by layers of gradually differentiating cells (Fusenig et al., 1994). Thus, epithelia are extremely complex tissues, and they are highly variable in type and degree of differentiation. Typical structures in epithelia are adherens junctions and desmosomes (Buxton and Magee, 1992; Tsukita et al., 1993; Hülsken et al., 1994a), which are organelles responsible for strong intercellular adhesion; epithelial cells also form hemidesmosomes to the basement membranes at their basal side (Timpl, 1989; Sonnenberg et al., 1991). Polar epithelial cells developed special mechanisms that allow the transport of membrane proteins to either the apical or basolateral surface (Eaton and Simons, 1995). Epithelial cells express characteristic genes that are responsible for the maintenance of the epithelial phenotype; for example, components of junctions or keratins (Birchmeier and Behrens, 1994; Kouklis et al., 1994; Buxton et al., 1993), specific epithelial products (e.g. albumin in the liver) (Cereghini et al., 1987), and specific transcription factors (e.g. LFB-3 in the liver or kidney) (De Simone et al., 1991).
Recently, much progress has been made in the
elucidation of the molecular basis of epithelial junction formation
(Tsukita et al., 1993; Garrod, 1993; Citi, 1993;
Hülsken et al., 1994b). Adherens junctions
are specialized structures containing the transmembrane cell adhesion
molecule epithelial cadherin E-cadherin, ()that recognizes
and binds E-cadherin present on the neighboring cells in a
Ca
-dependent manner. The cDNA of E-cadherin codes for
a signal peptide and a presequence at the amino terminus, a large
extracellular domain with four repeated domains important in
Ca
-binding, a single transmembrane sequence, and a
short cytoplasmic domain (Takeichi, 1991; Kemler, 1993). E-cadherin is
the prototype of a family of Ca
-dependent cell
adhesion molecules and is expressed in all embryonal and adult
epithelial tissues. In development, E-cadherin expression is
down-regulated during epithelial-mesenchymal transitions and reappears
during reversion to the epithelial phenotype. For example, E-cadherin
disappears during differentiation of the dorsal ectoderm into the
neural tube (Thiery et al., 1982; Nose and Takeichi, 1986),
and it is induced in epithelial cells that develop from mesenchyme
during morphogenesis of kidney tubules (Vestweber et al.,
1985). Overall, E-cadherin is thus a faithful component in all
epithelia and plays a functional role that is essential for the
maintenance of the epithelial phenotype (Imhof et al., 1983;
Behrens et al., 1989). Accordingly, homozygous mutations of
E-cadherin introduced into mice by homologous recombination disturbed
early embryogenesis: The individual cells of the morulae lose their
morphologic polarization and do not form a blastocoel. The mutant
embryos cannot leave the zona pellucida and, therefore, do not implant
into the uterus (Larue et al., 1994; Riethmacher et
al., 1995).
Since down-regulation of E-cadherin expression is a frequent event late in progression of human carcinomas and since modulation of E-cadherin expression plays a major role during development, we and others have begun to analyze the E-cadherin promoter and have found epithelial specificity in a fragment 178 bp upstream of the transcription start site (Behrens et al., 1991; Ringwald et al., 1991; Bussemakers et al., 1994). This promoter fragment contains a GC-rich region, a CCAAT-box, and a 12-bp palindromic element, which we named E-Pal. We have furthermore found that epithelium-specific transcription correlates with factor binding to these elements in vivo and to a loosening of chromatin structure in the promoter region (Hennig et al., 1995). Other epithelium-specific promoters have recently also been examined: The upstream regulatory regions of the epithelium-specific human papilloma viruses (HPV) 16 and 18 contain several binding sequences for ubiquitous cellular transcription factors (cf. Cripe et al., 1990; Hoppe-Seyler and Butz, 1994; Bernard and Apt, 1994). Epithelial specificity thus appears to be achieved by different combinations of these cellular factors. Specific factors have also been characterized that contribute to epithelial specificity; for instance, a mesenchyme-specific member of the NF-1 family represses transcription in fibroblasts but not in epithelial cells (Apt et al., 1993). Other epithelium-specific activators and repressors such as KRF-1, a coactivator of transcriptional enhancer factor-1 and YY1 have recently been described (Mack and Laimins, 1991; Ishiji et al., 1992, Bauknecht et al., 1992).
Here we report that epithelium-specific expression of the E-cadherin gene is achieved by two different mechanisms; E-Pal in the upstream promoter activates or suppresses transcription in epithelial or mesenchymal cells, respectively. A new epithelium-specific enhancer (ESE) was discovered in the first intron of the gene, that enhances transcription in a tissue-specific manner and binds nuclear factors specifically in epithelial cells.
For analysis of
the intronic enhancer, a 1.9-kilobase genomic BamHI fragment
containing intron 1 and parts of intron 2 was ligated into the TK-CAT
construct (pBLCAT2) (Luckow and Schütz, 1987).
Deletions of the enhancer were made with Exonuclease III (Pharmacia
Biotech Inc.), and PCR fragments were ligated into TK-CAT. The AP-2
expression construct SPRSV AP-2 was kindly provided by Dr. T. Williams
(New Haven, CT). In the construct AP-2TA (Williams and Tjian,
1991), nucleotides 153-413 were removed. All sequences were
confirmed by dideoxy sequencing.
Figure 1: Deletion and mutation of the E-Pal element increases E-cadherin promoter activity in mesenchymal cells. A, schematic representation of the E-cadherin promoter indicating the elements E-Pal, the CCAAT-box, the GC-rich region (with subregions GCI and GCII), and the transcription start site (arrow). The sequences of E-Pal and a mutation involving the two central nucleotides (mut) are indicated. B, activities of promoter fragments (as indicated in panel A) in ras3T3 fibroblasts. Shown are results from duplicate experiments.
We attempted to identify regulatory factors that control the E-cadherin promoter through the E-Pal element, by comparing the functional effects of specific mutations of E-Pal with the capacity of nuclear factor binding in gel retardation assays. Mutations in the center or in the 3`-half of E-Pal (mut 1 to mut 4) increased promoter activity in fibroblasts (Fig. 2A), in contrast to a mutation of the 5`-side (mut 5). A specific nuclear factor from fibroblasts was found to bind to the E-Pal element in gel retardation assays (Fig. 2B), which could be competed only by the wild-type oligonucleotide and the one mutated in the 5`-half of E-Pal. This suggests that binding of a specific factor (repressor) of fibroblasts to E-Pal correlates with suppression of promoter activity. However, we are aware of the fact that a similar band shift is seen when nuclear extracts from epithelial cells are examined (not shown, but see Behrens et al.(1991)). We next examined the contribution of the CCAAT-box and the GC-rich region to transcriptional activity of the E-cadherin promoter. Mutation of either the core sequence of the CCAAT-box or one of the two consensus binding sites for the transcription factor AP-2 (cf. Williams and Tjian, 1991) strongly reduced promoter activity in epithelial cells (Fig. 3A). These mutations also reduced the activity of the promoter with a mutated E-Pal in fibroblasts (Fig. 3B). Mutation of all three elements completely abolished promoter activity. These data show that both the CCAAT-box and the GC-rich region represent positive regulatory elements in both epithelial and mesenchymal cells.
Figure 2: Activity and binding specificity of the E-Pal element. A, effect of various mutations of E-Pal on the activity of E-cadherin promoter-CAT constructs in ras3T3 fibroblasts. Results are expressed relative to the CAT activity of the wild-type -178 bp promoter. B, binding of a specific nuclear factor from ras3T3 fibroblasts to the E-Pal oligonucleotide (arrowhead) in a gel retardation assay and competition with an excess of unlabeled oligonucleotides (cf. panel A). -, no competitor. Gel retardation assays were performed as described in Behrens et al.(1991).
Figure 3: CCAAT-box and GC-rich region are positively acting elements of the E-cadherin promoter. Left, schemes of the wild type and mutant constructs of the -178 bp E-cadherin promoter. Point mutations of the elements are marked by crosses (for sequences see ``Materials and Methods''). Right, CAT activities of the various mutant constructs in MCF-7 epithelial cells (A) and ras3T3 fibroblasts (B). Activities are expressed relative to the wild type promoter.
The GC-rich region of the E-cadherin promoter binds the transcription factor AP-2, as revealed by footprint analysis (Fig. 4). Mutation of each of the two AP-2 binding sites in the subregion GCI or GCII narrowed the footprint with both recombinant AP-2 and nuclear extracts on the respective sides. Footprint formation at both sites was inhibited by an oligonucleotide containing the AP-2 binding site of the SV40 enhancer (data not shown; cf. Imagawa et al. (1987)). Furthermore, a cotransfected dominant-negative mutant of AP-2 that lacks the transactivation domain inhibited activity of both the -178 and -58 bp promoters in a concentration-dependent fashion (Table 2). These data indicate that AP-2 or a closely related factor regulate the E-cadherin promoter by binding in a tandem arrangement to the GC-rich region.
Figure 4: Binding of recombinant transcription factor AP-2 and nuclear factors to the GC-rich region. DNase I footprint analysis of the -178 E-cadherin promoter fragment containing the wild type GC-rich region or the mutants of the AP-2 binding sites in GCI or GCII (cf. ``Materials and Methods'') in the presence of recombinant AP-2 (A) or nuclear extracts from MCF-7 epithelial cells (B). G + A, Maxam-Gilbert sequencing reaction of the wild type -178/+17 fragment; - or +, DNase I digestion in the absence or presence of factors. Footprint boundaries observed with the various mutations are indicated on the right; subregions GCI and GCII are marked on the left.
Figure 5: Localization of the intronic E-cadherin enhancer by deletion analysis. Left, deletion fragments in front of the TK promoter. Right, promoter activity of the deletions in MCF-7 epithelial cells; basal activity of the TK promoter is set to 1.0. Numbers in brackets indicate distances in bp from the BamHI site of intron 1. The epithelium-specific enhancer is marked by a shaded ellipse.
Footprint analysis of the enhancer in intron 1 revealed binding of nuclear factors to three subregions, E I to E III (Fig. 6). The sequences E II and E III are specifically protected by nuclear extracts from E-cadherin-expressing carcinoma cell lines (MCF-7 and RT 112) but not from E-cadherin-negative carcinoma cells (MDA-MB-231 and T 24); region E I was protected by extracts from both cell types. DNA sequencing of the protected areas revealed that E I to E III are GC-rich; E II and E III contain sequences that match the AP-2 consensus site (Fig. 7; cf. also Williams and Tjian(1991)).
Figure 6: DNase I footprinting analysis of the E-cadherin enhancer. A fragment of intron 1 from position 672 to the KpnI site of intron 1 (cf. Fig. 5) was used for footprinting analysis using nuclear extracts from E-cadherin expressing (MCF-7 and RT 112) and nonexpressing cells (MDA-MB-231 and T 24) as indicated. G and G + A, Maxam-Gilbert sequence reactions; -, absence of extract. The protected regions E I to E III are marked on the right.
Figure 7: Genomic sequence of the E-cadherin enhancer region. The sequence encompasses the KpnI site of intron 1 (position 311, cf. Fig. 5), the regions E I to E III (boxes), and part of exon 2 (boldface letters). Numbering is from the BamHI site of intron 1. Arrows indicate primers used for PCR cloning of enhancer fragments. The putative AP-2 and H4TF-1 binding sites are marked by dots and a dashed line, respectively. The sequence of the ESE has been submitted to the EMBL data bank, accession number X90561.
The contribution of the individual elements E I to E III to the function of the intronic enhancer was examined by using various PCR-generated subfragments (Fig. 8): a 149-bp fragment comprising all three elements exhibited strong enhancer activity in both orientations on the TK promoter in MCF-7 epithelial cells. Two copies of the enhancer showed 40-fold enhancement of activity (not shown). Interestingly, removal of any of the individual elements abolished enhancer activity, indicating that the integrity of the whole E I to E III cluster is sufficient and necessary to confer enhancer activity. The fragment of 149 bp also confers enhancer activity to the TK promoter in several other E-cadherin-expressing cell lines (Table 3). No enhancer activity was found in fibroblasts and dedifferentiated carcinoma cells. We therefore named the 149-bp region ESE, i.e. epithelium-specific enhancer. Detailed footprint analysis of the ESE element with various nuclear extracts revealed general protection of the elements E I to E III in E-cadherin-expressing carcinoma cells; the element E I is protected in E-cadherin-negative carcinoma cells, and the elements E II and E III are protected in fibroblasts (Table 3). Region E I contains a consensus binding sequence for the transcription factor H4TF-1 (cf. Dailey et al.(1988); we have not examined this factor any further). Binding of nuclear factors of MCF-7 cells to both regions E II and E III was competed by an AP-2 but not by an SP-1 binding site oligonucleotide (Fig. 9; Kadonaga et al.(1987)). Oligonucleotides containing the sequences of either E II or E III interfered with factor binding to both regions. Moreover, regions E II and E III were also protected by recombinant AP-2 (Fig. 9).
Figure 8: Fine analysis of the E-cadherin enhancer. PCR-generated fragments containing the elements E I to E III (left) were tested for enhancer activity in combination with the TK promoter in epithelial MCF-7 cells. Positions are from the BamHI site of the first intron. Activities are given as -fold induction with respect to the minimal TK promoter (right).
Figure 9: Analysis of AP-2 binding to the E-cadherin enhancer by in vitro footprinting. Footprinting analysis was performed using nuclear extracts from epithelial MCF-7 cells or recombinant AP-2 protein. Competitor oligonucleotides with binding sites for AP-2 and SP-1 as well as E II and E III are indicated. -, no oligonucleotide or extract was added. Protected regions E I to E III are marked on the left.
We report here that epithelium-specific regulation of the E-cadherin gene is controlled by two different mechanisms. First, the E-Pal element in the promoter 5` of the transcription start site acts as a positive or negative element in epithelial or mesenchymal cells, respectively. Second, a tissue-specific enhancer (ESE) in the first intron promotes transcription exclusively in epithelial cells. We also demonstrate that both these mechanisms are disturbed in carcinoma cells that have progressed to a less differentiated state and are E-cadherin-negative. We suspect that the E-cadherin gene is activated and repressed by similar mechanisms during mesenchymal-epithelial transitions in development.
Both half-sites of E-Pal are similar to the consensus sequence for binding of helix-loop-helix (HLH) transcription factors; it is therefore suggested that HLH factors play a role in the regulation of the E-cadherin promoter by the E-Pal element. Mutation analysis showed that the right HLH binding motif in E-Pal is necessary for function, whereas integrity of the left HLH site is less essential. Interestingly, only the right half of E-Pal is conserved in the human E-cadherin promoter (Bussemakers et al., 1994). We have also found that the HLH proteins Myc and Max bind to E-Pal in vitro; however, no modulation of the activity of the E-cadherin promoter could be demonstrated (data not shown). HLH proteins have frequently been implicated in tissue-specific gene expression (Weintraub et al., 1991; Lee et al., 1995). It is therefore conceivable that yet unknown HLH transcription factors play a role in epithelium-specific regulation of the E-cadherin promoter. Interestingly, expression of the muscle-specific HLH transcription factor myoD in keratinocytes induces morphological dedifferentiation of the cells and loss of epithelial markers (Boukamp et al., 1992).
We also demonstrate here that the CCAAT-box and the GC-rich region are positively acting elements in the E-cadherin promoter and have little cell type specificity of their own. CCAAT-box binding proteins that serve tissue-specific functions have been described previously (Umek et al., 1991; Katz et al., 1993). We found three complexes of factors that bind to the CCAAT-box in gel retardation assays, and these did not appear when the CCAAT sequence was mutated. Competition by specific oligonucleotides revealed that various members of the CCAAT-box family of proteins such as CP-1 and C/EBP (Chodosh et al., 1988; Landschulz et al., 1988) are candidates for activation of the E-cadherin promoter (data not shown). We have not analyzed the CCAAT-box of the E-cadherin promoter any further. Binding and functional data suggest that the transcription factor AP-2 or closely related factors regulate the activity of the GC-rich region of the E-cadherin promoter. (i) In DNase I footprint assays, two subregions GCI and GCII were identified that bound purified AP-2 with similar characteristics as factors present in nuclear extracts. Binding to both sites GCI and GCII could be competed with a single AP-2 specific oligonucleotide. (ii) In transient transfection experiments, AP-2 that lacks the transactivation domain suppressed the activity of the E-cadherin promoter in a concentration-dependent manner. Cis-elements representing AP-2 binding sites have recently been implicated in the regulation of promoters of various epidermal cytokeratins (Leask et al., 1991, Snape et al., 1991, Byrne and Fuchs, 1993). However, it is unlikely that AP-2 has an exclusive role in epithelium-specific gene expression since it is found in ectodermal derivatives but not in several other epithelial tissues that express E-cadherin (Mitchell et al., 1991).
The expression of the
E-cadherin gene is thus under the control of an epithelium-specific
promoter plus an epithelium-specific enhancer. We suggest that the
combination of these two regulatory mechanisms provides additional
specificity and strength of expression of the E-cadherin gene. This is
thus different to the regulation of the related L-CAM gene (a chicken
homologue of E-cadherin). There, a low activity and nonspecific
promoter gains tissue specificity when combined with an enhancer in the
second intron (Sorkin et al., 1993). In the P-cadherin gene,
an enhancer is also located in the second intron (Hatta et
al., 1994). ()In addition promoter and enhancer of the
E-cadherin gene act differently in mesenchymal cells. Here the enhancer
is inactive, but the promoter contributes to repression through the
E-Pal element. To our knowledge, a combination of an
epithelium-specific enhancer with an epithelium-specific promoter that
acts as a repressor in mesenchymal cells has not previously been found.