1 Department of Molecular Embryology, Max-Planck Institute of Immunobiology,
Stuebeweg 51, D-79108 Freiburg, Germany
2 Andreas Hecht, Institute of Molecular Medicine and Cell Science, University of
Freiburg, Stefan-Meier-Strasse 17, D-79104 Freiburg, Germany
* Author for correspondence (e-mail: kemler{at}immunbio.mpg.de)
Accepted 22 December 2004
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SUMMARY |
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Key words: Cell adhesion, Knock-in, Transcription, Gene regulation, Gene expression, Mouse embryo, LCR, Comparative genomics
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Introduction |
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Downregulation of E-cadherin is also a frequent event in tumorigenesis
(Berx et al., 1998;
Thiery, 2002
), when the
epithelial cell phenotype is lost during tumor progression. In many cases, the
loss of E-cadherin, either by mutation within the coding sequence or by
transcriptional downregulation, is a necessary step that promotes invasiveness
(Berx et al., 1998
;
Perl et al., 1998
;
Thiery, 2002
).
Although much information has been gathered about E-cadherin protein during
development, organogenesis and tumor formation, little is known about the
trancriptional regulation of E-cadherin, particularly how expression is
activated and maintained in a developmentally and cell-type-specific manner.
Several transcriptional repressors, all binding to the E-cadherin promoter
region, have been identified that are able to downregulate the E-cadherin gene
in specific contexts. The zinc-finger proteins Snail, Slug, EF1/ZEB-1
and Sip-1/ZEB-2, and the basic helix-loop-helix transcription factors Twist
and E12/E47 inhibit E-cadherin expression
(Batlle et al., 2000
;
Cano et al., 2000
;
Carver et al., 2001
;
Comijn et al., 2001
;
Conacci-Sorrell et al., 2003
;
Grooteclaes and Frisch, 2000
;
Peinado et al., 2004
;
Perez-Moreno et al., 2001
;
Yang et al., 2004
). These
regulatory factors bind to a common DNA sequence known as the E-box motif,
present three times in the E-cadherin promoter. In addition, mediators of Wnt
signaling, namely ß-catenin and Lef-1, downregulate E-cadherin in hair
follicle bud formation (Jamora et al.,
2003
). Lef-1 binds to a single Lef/Tcf motif upstream of the
E-boxes (Huber et al., 1996b
).
Besides these precisely defined cis-regulatory elements at the promoter, an
enhancer element in intron 1 has been identified
(Behrens et al., 1991
;
Bussemakers et al., 1994
;
Hennig et al., 1995
;
Hennig et al., 1996
;
Ringwald et al., 1991
;
Sorkin et al., 1993
).
Recently, we provided evidence that the above mentioned elements are
insufficient to give E-cadherin-specific expression in transgenic mice
(Stemmler et al., 2003
). In
addition, we identified sequences in the first third of intron 2 (15 kb), that
conferred some cell-type-specific gene activation
(Stemmler et al., 2003
).
Although promising, the use of large fragments of the E-cadherin gene (between
-6 and +16 kb from the transcription start) still did not recapitulate the
complete endogenous expression pattern, indicating that important regulatory
elements were missing in this analysis. However, this work pointed to the
possibility that important regulatory sequences may be located in intron 2 of
the E-cadherin gene.
Here, we have investigated the function of intron 2 sequences in proper E-cadherin gene regulation by deleting the entire intron 2 of E-cadherin by gene targeting in ES cells. We show that these sequences are essential for gene activation in early embryonic development. During late embryogenesis, intron 2 strongly enhances transcription. Additionally, we show that intron 2 is required for maintenance of E-cadherin expression after initial transcriptional activation.
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Materials and methods |
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Generation of teratomas
ES cells grown on embryonic fibroblasts were trypsinized and resuspended in
PBS. Of these, 107 cells in a volume of 100 µl were injected
peritoneally into 129/Sv mice. After 3 weeks, teratomas were isolated and
stained with X-gal for ß-galactosidase activity.
Real-time quantitative RT-PCR
RNA was isolated from embryonic halves of E7.5 embryos with an RNeasy Kit
(Qiagen) and from yolk sacs with RNA-Bee reagent (ams biotechnology). RNA of
one or two embryos or 2 µg total RNA was used to synthesize cDNA with
oligo(dT)-primer and a Superscript II Kit (Invitrogen). Amplification of
betageo RNA was carried out with the primer pair
5'-TTACTGCCGCCTGTTTTGAC-3' and
5'-TAGCCGAATAGCCTCTCCAC-3', and that of Gapd with the
primer pair 5'-ACCACAGTCCATGCCATCACT-3' and
5'-GTCCACCACCCTGTTGCTGTA-3' [in both cases using FastStart DNA
MasterPLUS (Roche) in the LightCycler Instrument (Roche) according
to the manufacturer's instructions]. Transcripts were normalized to
Gapd expression. Values in arbitrary units are the mean of three
separate experiments comparing Ecad-In2flox and Ecad-In2floxdel samples.
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Results |
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A scheme for the deletion of the entire intron 2 of the E-cadherin gene (45
kb genomic sequence) is depicted in Fig.
1A. Two independent homologous recombination events were used to
insert loxP sites 5' and 3' of intron 2. Additionally, we
inserted a betageo reporter gene at the start codon of E-cadherin to
monitor the transcriptional activity of the targeted locus (TV1,
Fig. 1A). More than 80% of
ES-cell clones were homologously recombined
(Fig. 1B) after electroporation
of TV1. A 6.2 kb wild-type fragment and a 9 kb fragment of the mutated allele
were detected with probe a in Southern blot analysis after
BamHI digestion (Fig.
1B). One recombined ES-cell clone was taken for the second gene
targeting. The 3' loxP site was inserted by homologous
recombination at exon 3 with targeting vector 2 (TV2,
Fig. 1A, right side). Southern
blot analysis showed homologous recombination at the 3' end of the locus
with a frequency of 10% (Fig.
1C). A BamHI digest probed with probe f revealed
a 12 kb wild-type fragment and a 7 kb fragment of the mutated allele due to
the insertion of a BamHI site at the loxP site. To identify
recombination events which had occurred on the same allele, pulse-field gel
electrophoresis separation and Southern blot analysis were performed.
Hybridization with probes e and c
(Fig. 1A,D) revealed a fragment
that migrates at the predicted size corresponding to recombination in cis
(clones 2, 6, 8-11, arrowhead, Fig.
1D). By contrast, in addition to the wild-type fragment of
400 kb (arrow in Fig. 1D),
a fragment of
300 kb with probe e
(Fig. 1D, left) and of 100 kb
with probe c (Fig. 1D,
right) appeared in cases where the homologous recombination event occurred in
trans (clones 3-5, 7, open arrow, Fig.
1D). Three ES-cell clones with both homologous recombination
events in cis were used to generate transgenic mice. Neither a potential fused
mRNA between betageo and E-cadherin sequences as a result of the
knock-in nor a hypomorphic fusion protein was detected in heterozygous mice
(data not shown). Because of the betageo insertion at the ATG codon
of E-cadherin, the targeted allele should result in a null phenotype.
Consistent with the null having an early lethal phenotype
(Larue et al., 1994
;
Riethmacher et al., 1995
),
interbreeding of mice heterozygous for the targeted allele failed to generate
any viable homozygous knock-in offspring (data not shown).
Deletion of intron 2 leads to loss of reporter gene expression in ES cells
First insights into the regulatory function of sequences in intron 2 were
obtained with the targeted ES cells (Ecad-In2flox), which, after transient
transfection with a Cre expression vector
(Gu et al., 1993), removed
intron 2 (Ecad-In2floxdel), as demonstrated by PCR and Southern blot
(Fig. 2A,B). X-Gal staining of
Ecad-In2flox ES cells revealed ß-galactosidase (ß-gal) activity,
albeit in a heterogeneous pattern (Fig.
2C). By contrast, no ß-gal staining was detectable in
Ecad-In2floxdel ES cells (Fig.
2D). Teratomas were produced in isogenic mice from Ecad-In2flox
and Ecad-In2floxdel ES cells and in both cases these tumors contained the
well-known typical variety of different tissues and cell types. Reporter gene
activity was observed throughout teratomas derived from Ecad-In2flox cells
(Fig. 2E) and was particularly
strong in cysts and polarized epithelia
(Fig. 2G). However, in
teratomas derived from Ecad-In2floxdel cells, only partial and weaker
ß-gal expression was observed (Fig.
2F), and this did not coincide with the locations of cysts
(Fig. 2H). Importantly,
epithelia of Ecad-In2floxdel teratomas did not stain for ß-gal
(Fig. 2H). These results
provide strong evidence that intron 2 is necessary for the expression of
E-cadherin in ES cells and in teratoma-derived differentiated epithelia. To
study the differences in gene activity that are due to the function of intron
2, we compared the abundance of betageo transcripts in Ecad-In2flox
versus Ecad-In2floxdel ES cells using a semi-quantitative PCR approach.
Transcripts for betageo were detected in Ecad-In2flox samples, and
these were much less abundant in Ecad-In2floxdel samples
(Fig. 2I, upper panel). This
result was verified by quantitative PCR, which showed a 95% reduction in gene
activity after deletion of intron 2 (Fig.
2I, lower panel), thus confirming the pivotal role for intron 2 in
activating E-cadherin gene expression.
|
|
|
Intron 2 sequences are not required for the E-cadherin reporter gene expression in the yolk sac
The results described above revealed that the presence of intron 2 had a
more global enhancing effect on activation of E-cadherin transcription,
particularly in later stages of development. During this analysis it became
apparent that the ß-gal expression in the yolk sac was independent of
intron 2 sequences. Whereas yolk sacs of wild-type embryos do not show
endogenous ß-galactosidase expression at E10.5
(Fig. 5A) and only faint
staining was observed at E12.5 (Fig.
5C), the yolk sacs of Ecad-In2flox and Ecad-In2floxdel embryos
showed high-level reporter gene-derived ß-gal expression
(Fig. 5B,D). Remarkably,
ß-gal expression was equally high in the yolk sacs of both genotypes,
although a clear difference was observed between the respective embryos
(Fig. 5B,D). Semi-quantitative
and real-time PCR corroborated the X-gal staining data showing intron
2-independent expression of ß-gal in yolk sacs at E10.5 and E16.5
(Fig. 5E).
|
|
Using CK14-Cre to recombine the Ecad-In2flox locus, no difference
in ß-gal expression between the Ecad-In2flox and
Ecad-In2flox/CK14-Cre was detected before E12.5 (data not shown). At
E12.5, a slight reduction in ß-gal expression was observed in the surface
ectoderm of Ecad-In2flox embryos carrying the CK14-Cre allele
(Fig. 7A, right, +/)
when compared with CK14-Cre negative embryos
(Fig. 7A, left, +/flox). This
difference became more evident at E13.5 and E14.5
(Fig. 7B,C). Interestingly,
ß-gal expression persisted in the lens and the gut loops of
Ecad-In2flox/CK14-Cre embryos (compare left/right
Fig. 7B), because the
CK14-Cre is not expressed in these tissues
(Hafner et al., 2004
;
Wang et al., 1997
). At E16.5,
intense ß-gal expression was visible in the skin of control embryos
(Fig. 7D, left), but only faint
staining was observed in the skin of Ecad-In2flox/CK14-Cre embryos
(Fig. 7D, right; compare with
Fig. 4D,I).
|
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Discussion |
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Complexity of E-cadherin transcriptional regulation
The position of cis-regulatory elements on genomic DNA sequences can be
indicated by the presence of DNase-I-hypersensitive sites (DHSs). DHSs arise
from nucleosome-free chromatin that is highly accessible to DNaseI and result
from bound transcription factors. The occurrence of DHSs and the presence of
cis-regulatory elements correlate in other genes
(Harju et al., 2002;
Kintscher et al., 2004
;
Lefevre et al., 2001
;
Murakami et al., 2004
). At the
E-cadherin locus, only one DHS is found upstream of the transcription start
site at position -0.1 kb. The absence of additional DHSs further upstream of
the E-cadherin promoter region and the accumulated occurrence of DHSs in the
5' part of intron 2 in E-cadherin-expressing cells hint at
cis-regulatory elements in intron 2
(Stemmler et al., 2003
). A
high degree of sequence conservation in mouse, rat, human, chimp and dog
around these DNA elements in the part of intron 2 that has been analyzed for
DHSs (-15 to +18 kb) further supports this notion (see Fig. S1 in the
supplementary material). Because additional areas of significant sequence
conservation in intron 2 outside of the DHS-mapped region were found, this
suggested the existence of other regulatory elements spread over the entire
intron 2.
In our previous transgenic reporter gene approach, we had demonstrated that
a -6 to +0.1 kb promoter fragment is insufficient to drive E-cadherin
expression. The first 15 kb of intron 2 sequences were beneficial towards
properly regulating an E-cadherin transgene
(Stemmler et al., 2003). This
work also demonstrated that sequences required for E-cadherin-specific
expression in the endoderm are found between +0.1 and +11 kb of the E-cadherin
gene, a general enhancer between +11 and +16 kb, and a brain-specific enhancer
between -6 and -1.5 kb. Nevertheless, it became evident that not all
regulatory sequences have been covered by this analysis.
Nonetheless, encouraged by these findings and the fact that the entire intron 2 contained conserved sequences (see Fig. S1 in the supplementary material), we analyzed the function of these sequences in vivo by ablating the entire intron 2 using gene targeting. We were able to show that, if these sequences are deleted, E-cadherin expression is completely lost during early embryogenesis. Only during later embryonic development can the locus be activated without intron 2, but with significantly reduced expression levels. In addition, our analyses revealed even more complex regulatory functions of intron 2. In general, in expression domains that are affected by the absence of intron 2, these sequences are required for both activation of the locus and maintenance of expression. We found that, in the lens and the salivary glands, expression is absolutely controlled by cis-regulatory elements of intron 2, whereas, in the yolk sac, expression can be activated regardless of the presence of these sequences.
Based on our previous findings in transgenic mice
(Stemmler et al., 2003) and
the data presented here, we suggest the following model of regulating
E-cadherin gene activity (Fig.
8). Whereas E-boxes at the promoter contribute to downregulating
the locus (small red boxes, Fig.
8), E-cadherin gene activation is initiated and maintained due to
intron 2 sequences. Importantly, in Ecad-In2floxdel embryos the
endoderm-specific expression of the E-cadherin reporter gene was lost,
probably owing to the lack of sequences between +1.2 and +11 kb (endoderm,
Fig. 8). Entire loss of
ß-gal expression in Ecad-In2floxdel embryos can be partially ascribed to
the general enhancer between +11 and +16 kb (enh.,
Fig. 8). However,
ectoderm-specific expression is not at all detectable until E11.5 in
Ecad-In2floxdel embryos nor was it consistently observed in the transgenic
analysis (Stemmler et al.,
2003
). This indicates that additional, so far undescribed
cis-regulatory elements in intron 2 are present between +18 and +47 kb to
drive expression in the ectoderm (indicated by `tse' in
Fig. 8). E-cadherin-specific
reporter gene expression in the brain due to the function of cis-regulatory
elements between -6 and -1.5 kb (brain,
Fig. 8) needs to be restricted
to the E-cadherin expression domain by an as yet unknown brain-specific
silencer (sil., Fig. 8),
because we observed additional ectopic ß-gal activity in the brain of
transgenic embryos (Stemmler et al.,
2003
). Because this was not the case in Ecad-In2floxdel embryos,
we conclude that the postulated brain-specific silencer must be located
outside of intron 2.
|
Two mechanisms to initiate and maintain E-cadherin expression
We observed that, despite the lack of intron 2, the E-cadherin locus was
activated in many cell types of epithelial origin during late embryogenesis
after E10.5. This suggests that the E-cadherin locus can be activated by two
independent mechanisms. One mechanism acts during early embryogenesis and
requires intron 2 for the onset of expression, and the second one functions at
later stages. This second mechanism initiates E-cadherin expression
independently of intron 2, although for high-level expression the support of
the intron 2 enhancer elements is still required. The onset of the second wave
of expression becomes apparent around E12.5 in the surface ectoderm
(coinciding with the differentiation of the surface ectoderm and ongoing skin
development) and in the gut endoderm. Presumably, this second, alternative
activation mechanism is regulated by a common subset of transcription factors
active in the specialized epithelia and might be achieved at the promoter or
the intron 1 enhancer (Fig.
8).
Different requirements of intron 2 sequences in certain specialized epithelia
Even more complexity of E-cadherin gene regulation emerged from the
analysis of expression in the yolk sac, lens and salivary glands. The
initiation of high-level reporter gene expression in the yolk sac is achieved
independently of cis-regulatory elements of intron 2 and could reflect a
gene-regulation mechanism specific to extra-embryonic tissues. By contrast,
E-cadherin expression in the lens and the salivary glands is absolutely
dependent on intron 2. Surprisingly, E-cadherin expression differs in tissues
that originate from similar germ-layers. The lens develops from the lens
placode, which is derived from surface ectoderm from E9.5 onwards. Whereas
E-cadherin reporter gene expression is initiated by the second wave of
expression in Ecad-In2floxdel embryos in the surface ectoderm of later stage
embryos, no gene activation was found in the lens. Similarly, in epithelia of
salivary glands of Ecad-In2floxdel embryos ß-gal was never expressed,
although they share the same germ-layer origin with epithelia of other inner
organs. The postulated factors that are able to initiate E-cadherin
transcription in later embryogenesis without intron 2 do not seem to be
present in epithelia of salivary glands or in the lens. To explain the intron
2-dependent and independent E-cadherin expression, we propose that different
tissue-specific enhancers probably exist that mediate E-cadherin expression in
the yolk sac or in the lens and the salivary glands. This difference probably
coincides with the different functions of specialized epithelia.
The role of intron 2 in tumor progression
The data presented here reveal and emphasize the pivotal role of intron 2
in E-cadherin gene regulation during embryonic development. The importance of
intron 2 sequences in gene regulation may also have an impact on
tumorigenesis. The invasive property of cancer cells is often linked to loss
of E-cadherin expression, in several cases owing to transcriptional
downregulation (Berx et al.,
1998). Accordingly, dysregulated expression of E-cadherin may be
linked to mutations in intron 2 in cancer cells in which no mutation in the
promoter or the coding sequence and no activation of a transcriptional
repressor could be described. In some tumor cell lines, CpG-hypermethylation
of the E-cadherin gene was discovered, but no mutation was found that might be
responsible for this epigenic inactivation of the locus
(Berx et al., 1998
;
Yoshiura et al., 1995
). The
mutations that are responsible for E-cadherin downregulation and subsequent
CpG-hypermethylation may be located in intron 2. The identification of intron
2 mutations would underline the role of intron 2 in gene regulation in
tumorigenesis. To be able to assess the impact of such mutations, a more
precise description of the location and architecture of regulatory elements in
intron 2 is required. Further gene targeting or transgenic mouse studies will
concentrate on locating single tissue-specific cis-regulatory elements. An
integrated in silico search for transcription factor binding sites can be used
to determine which transcription factors bind to the putative regulatory
sequences of intron 2. Together, these approaches will lead to better
understanding of the complex interplay of multiple regulatory regions
dispersed throughout large parts of the E-cadherin locus.
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Supplementary material |
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ACKNOWLEDGMENTS |
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