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(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, ( 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-2
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). (
Volume 271,
Number 1,
Issue of January 5, 1996 pp. 595-602
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)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).
Plasmid Constructs
The E-cadherin promoter-CAT
constructs are derivatives of the deletion -178/+92 cloned
in the pCAT basic vector (Behrens et al., 1991). To generate
the -94 or -78 deletion constructs, a HindIII/PstI fragment (-178 to -79) was
replaced by a double-stranded oligonucleotide, or the blunt-ended
vector was directly religated, respectively. To obtain mutants of
E-Pal, the CCAAT-box, or the GC-rich region, restriction fragments of
the -178/+92 construct were replaced by corresponding mutant
oligonucleotides. The restriction sites PstI(-99), NarI(-54), EclXI(-22), or KspI
(+18), were used for the exchange. The CCAAT sequence was mutated
to TCCG, and the AP-2 binding sites in subregions GCI and GCII were
mutated to GCCGTTTTC and TTCTGCGGG, respectively.
TA (Williams and Tjian,
1991), nucleotides 153-413 were removed. All sequences were
confirmed by dideoxy sequencing.Cells, Transfections, and CAT Assays
Cells were
cultured in Dulbecco's modified Eagle's medium containing
10% fetal calf serum. Cell lines are described in Frixen et
al.(1991) and Behrens et al.(1991). For transfection
experiments, cells were seeded at a density of 10
per 10-cm
tissue culture dish and transfected with usually 5 µg of the
various promoter constructs using the calcium phosphate coprecipitation
method (Gorman et al., 1982). The amounts of cell extracts for
the CAT assays were adjusted according to 
-galactosidase activity
from cotransfected plasmids Rous sarcoma virus lacZ (Behrens et
al., 1991) or pCH110 (Pharmacia).In Vitro DNase I Footprint Assays
Nuclear extracts
were prepared as described (Behrens et al., 1991). For DNase I
footprint assays, an XbaI/KspI fragment of the
E-cadherin promoter (positions -178 to +17) or the intronic
enhancer (positions 672-311 from the BamHI site in
intron 1) were labeled with Klenow enzyme. For the AP-2 footprint
assays, 30 ng of purified recombinant transcription factor AP-2
(Promega) were employed. In the competition footprint assays of the
enhancer region, 50 ng of the double-stranded oligonucleotides were
added to the reaction: E II,
5`-GATCCCCCTGTGTGCCCTGAGGGGGGTCCTCCCCACTAG-3`; E III,
5`-GATCCACTAATCCCGGCGCCCGAGGCGAGGGAAGGGTTACCCTTG-3`; AP-2,
5`-GATCCAAAGTCCCCAGGCTCCCCAG-3`, SP-1,
5`-GATCCTGGGCGGAGTTAGGGGCGGGACTG-3`.
Characteristics of the E-cadherin Promoter
We
and others have previously analyzed the E-cadherin promoter and found
epithelial specificity in a -178 bp fragment upstream of the
transcription start site (Behrens et al., 1991, Ringwald et al., 1991, Bussemakers et al., 1994). This
fragment contains a GC-rich region with subregions GCI and GCII, a
CCAAT-box, and a 12-bp palindromic element that we named E-Pal (Fig. 1A). We demonstrate here that deletion or mutation of
E-Pal resulted in a 5-fold increase of promoter activity in mesenchymal
cells, e.g. fibroblasts or smooth muscle cells (Fig. 1B). Similarly, mutation of E-Pal leads to an
increase (2-4-fold) of promoter activity in E-cadherin-negative
carcinoma cells (Table 1). A slight decrease of promoter activity
was observed when E-Pal was mutated or deleted in E-cadherin-expressing
carcinoma cell lines. In the mouse mammary epithelial cell line
Ep-FosER, which looses E-cadherin expression following activation by
estrogen, the E-Pal mutation also leads to an increase of promoter
activity (Table 1). These data demonstrate that E-Pal is a
negative regulatory element in mesenchymal (E-cadherin-negative) cells
but has a weak positive activity in epithelial cells.
Characteristics of the Intronic Enhancer of the
E-cadherin Gene
In a recent in vivo analysis, we
identified DNase I-hypersensitive sites in the first and second introns
of the E-cadherin gene that are specific for E-cadherin-expressing
cells (Hennig et al., 1995). The respective genomic regions
were here examined for enhancer activity on the TK minimal promoter (Fig. 5). The used 1.9-kilobase BamHI fragment of the
first and part of the second intron enhanced promoter activity 4.5-fold
in epithelial cells; deletion of intron 2 sequences to position 672 led
to a further (12.5-fold) enhancement of activity, which may indicate
the presence of a negative regulatory region between positions 1100 and
672. Further deletion abolished activity, thus localizing the enhancer
to a region of 200 bp of intron 1 (compare the activity of constructs
672 and 475 in Fig. 5). Similar results were obtained when the
deletion fragments were examined with the SV40 minimal promoter.
Enhancer activity was also observed when the 1.9-kilobase fragment was
tested in the reverse orientation (in the TK and SV40 promoter) or at a
distance of 2.7 kilobases from the transcription start site of the SV40
promoter (data not shown).
Restriction of E-cadherin Promoter Activity to Epithelial Cells
Is in Part Due to Cell Type-specific Repression
We describe here
three cis-acting elements in the E-cadherin promoter that exhibit
positive regulatory activity in epithelial cells: E-Pal, the CCAAT-box,
and a GC-rich region. We have previously shown that the E-Pal element
also confers positive regulatory activity to a SV40 promoter in
epithelial cells (Behrens et al., 1991). In nonepithelial
cells, CCAAT-box and GC-rich region exhibit positive transcriptional
potential, but this activity is masked by E-Pal, i.e. becomes
apparent only when E-Pal is mutated or deleted. We also provide here
evidence that a specific factor binds to E-Pal in fibroblasts and that
binding specificity of this factor correlates with biological activity.
Transcriptional repressors that act via binding to a specific promoter
element have been described by others (see Renkawitz(1990) and
Johnson(1995) for reviews). For instance, the Wilms' tumor
suppressor gene wt1 suppresses transcription by binding to a
GC-rich promoter sequence, and a protein domain of WT1 could be
identified that retained inhibitory activity when combined with a
heterologous DNA-binding domain (Madden et al., 1991; Drummond et al., 1992). Furthermore, the inhibitory activity of WT1
depends on p53; in its absence, WT1 acts as a transcriptional activator
(Maheswaran et al., 1993). Thus, the dual function of WT1 is
reminiscent of the opposite activities of E-Pal and its putative
binding factor in epithelial and mesenchymal cells.Properties of the Novel ESE in the E-cadherin
Gene
The ESE of the E-cadherin gene was discovered by the
analysis of DNase I-hypersensitive sites followed by analysis of
deletion constructs and PCR fragments for enhancer activity. The
enhancer region corresponds to hypersensitive site 4 in the first
intron of the E-cadherin gene, as described previously (Hennig et
al., 1995). Transcriptional enhancers are generally associated
with loosened chromatin structure, as in the case of the SV40 and
polyoma virus enhancer; these enhancers are characterized as
nucleosome-free regions and are hypersensitive toward DNase I (Serfling et al., 1985). The minimal region of the ESE of the E-cadherin
gene (150 bp) activates transcription from heterologous promoters by a
factor of 15 in MCF-7 cells (40-fold when used in a tandem arrangement)
that is independent of orientation and distance. Detailed analysis of
the ESE revealed three elements, E I to E III, which are all GC-rich.
These elements are necessary and sufficient for enhancer activity in
several E-cadherin-expressing epithelial cell lines. In contrast, the
ESE is inactive in all E-cadherin-negative cell lines such as
fibroblasts and dedifferentiated carcinoma cells. The ESE therefore has
a modular structure, which is characteristic for several other
enhancers, e.g. the CD2 or SV40 enhancers (Serfling et
al., 1985; Lake et al., 1990). In the epithelium-specific
enhancer of papilloma viruses, numerous elements contribute to enhancer
activity, but the involved sequences are generally bound by ubiquitous
transcription factors (see Hoppe-Seyler and Butz(1994) for a review).
We found that the ESE of the E-cadherin gene is active in cells that
express all three factors that bind to E I to E III, but is inactive
when one factor is lacking. We also demonstrated that AP-2 or related
factors bind to E II and E III; again epithelium-specificity cannot be
explained by the action of AP-2 only. It is possible that recently
identified splice variants of AP-2 (Meier et al., 1995) play a
role in the specific regulation of ESE.
)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.
))
We thank Beate Voss for technical assistance. We also
thank I. Wiznerowicz for excellent secretarial help.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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  S.-H. Park, L. W. T. Cheung, A. S. T. Wong, and P. C. K. Leung Estrogen Regulates Snail and Slug in the Down-Regulation of E-Cadherin and Induces Metastatic Potential of Ovarian Cancer Cells through Estrogen Receptor {alpha} Mol. Endocrinol., September 1, 2008; 22(9): 2085 - 2098. [Abstract] [Full Text] [PDF]
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 H.-J. Choi, T.-W. Chung, S.-J. Kim, S.-Y. Cho, Y.-S. Lee, Y.-C. Lee, J.-H. Ko, and C.-H. Kim The AP-2{alpha} transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of a PTEN gene Glycobiology, May 1, 2008; 18(5): 395 - 407. [Abstract] [Full Text] [PDF]
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 M. P. Stemmler, A. Hecht, and R. Kemler E-cadherin intron 2 contains cis-regulatory elements essential for gene expression Development, March 1, 2005; 132(5): 965 - 976. [Abstract] [Full Text] [PDF]
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 R. Alpatov, G. C. Munguba, P. Caton, J. H. Joo, Y. Shi, Y. Shi, M. E. Hunt, and S. P. Sugrue Nuclear Speckle-Associated Protein Pnn/DRS Binds to the Transcriptional Corepressor CtBP and Relieves CtBP- Mediated Repression of the E-Cadherin Gene Mol. Cell. Biol., December 1, 2004; 24(23): 10223 - 10235. [Abstract] [Full Text] [PDF]
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 C. Faleiro-Rodrigues, I. Macedo-Pinto, D. Pereira, and C. S. Lopes Prognostic value of E-cadherin immunoexpression in patients with primary ovarian carcinomas Ann. Onc., October 1, 2004; 15(10): 1535 - 1542. [Abstract] [Full Text] [PDF]
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 N. Wajapeyee and K. Somasundaram Cell Cycle Arrest and Apoptosis Induction by Activator Protein 2{alpha} (AP-2{alpha}) and the Role of p53 and p21WAF1/CIP1 in AP-2{alpha}-mediated Growth Inhibition J. Biol. Chem., December 26, 2003; 278(52): 52093 - 52101. [Abstract] [Full Text] [PDF]
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 J. E. Korkola, S. DeVries, J. Fridlyand, E. S. Hwang, A. L. H. Estep, Y.-Y. Chen, K. L. Chew, S. H. Dairkee, R. M. Jensen, and F. M. Waldman Differentiation of Lobular versus Ductal Breast Carcinomas by Expression Microarray Analysis Cancer Res., November 1, 2003; 63(21): 7167 - 7175. [Abstract] [Full Text] [PDF]
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S. Oesterreich, W. Deng, S. Jiang, X. Cui, M. Ivanova, R. Schiff, K. Kang, D. L. Hadsell, J. Behrens, and A. V. Lee Estrogen-mediated Down-Regulation of E-cadherin in Breast Cancer Cells Cancer Res., September 1, 2003; 63(17): 5203 - 5208. [Abstract] [Full Text] [PDF]
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 D. A. Tyas, H. Pearson, P. Rashbass, and D. J. Price Pax6 Regulates Cell Adhesion during Cortical Development Cereb Cortex, June 1, 2003; 13(6): 612 - 619. [Abstract] [Full Text] [PDF]
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 J. P. Bilello, E. E. Cable, and H. C. Isom Expression of E-Cadherin and Other Paracellular Junction Genes Is Decreased in Iron-Loaded Hepatocytes Am. J. Pathol., April 1, 2003; 162(4): 1323 - 1338. [Abstract] [Full Text] [PDF]
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 V. Bolos, H. Peinado, M. A. Perez-Moreno, M. F. Fraga, M. Esteller, and A. Cano The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors J. Cell Sci., February 1, 2003; 116(3): 499 - 511. [Abstract] [Full Text] [PDF]
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S. Decary, J. T. Decesse, V. Ogryzko, J. C. Reed, I. Naguibneva, A. Harel-Bellan, and C. E. Cremisi The Retinoblastoma Protein Binds the Promoter of the Survival Gene bcl-2 and Regulates Its Transcription in Epithelial Cells through Transcription Factor AP-2 Mol. Cell. Biol., November 15, 2002; 22(22): 7877 - 7888. [Abstract] [Full Text] [PDF]
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 J. Pellikainen, V. Kataja, K. Ropponen, J. Kellokoski, T. Pietilainen, J. Bohm, M. Eskelinen, and V.-M. Kosma Reduced Nuclear Expression of Transcription Factor AP-2 Associates with Aggressive Breast Cancer Clin. Cancer Res., November 1, 2002; 8(11): 3487 - 3495. [Abstract] [Full Text] [PDF]
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S. Guaita, I. Puig, C. Franci, M. Garrido, D. Dominguez, E. Batlle, E. Sancho, S. Dedhar, A. G. de Herreros, and J. Baulida Snail Induction of Epithelial to Mesenchymal Transition in Tumor Cells Is Accompanied by MUC1 Repression and ZEB1 Expression J. Biol. Chem., October 11, 2002; 277(42): 39209 - 39216. [Abstract] [Full Text] [PDF]
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J. J. Eloranta and H. C. Hurst Transcription Factor AP-2 Interacts with the SUMO-conjugating Enzyme UBC9 and Is Sumolated in Vivo J. Biol. Chem., August 16, 2002; 277(34): 30798 - 30804. [Abstract] [Full Text] [PDF]
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U. Werling and H. Schorle Transcription Factor Gene AP-2{gamma} Essential for Early Murine Development Mol. Cell. Biol., May 1, 2002; 22(9): 3149 - 3156. [Abstract] [Full Text] [PDF]
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 K. C. Mansky, K. Marfatia, G. H. Purdom, A. Luchin, D. A. Hume, and M. C. Ostrowski The microphthalmia transcription factor (MITF) contains two N-terminal domains required for transactivation of osteoclast target promoters and rescue of mi mutant osteoclasts J. Leukoc. Biol., February 1, 2002; 71(2): 295 - 303. [Abstract] [Full Text] [PDF]
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