From the UMR 144 CNRS, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Villin is an early marker of epithelial cells
from the digestive and urogenital tracts. Indeed villin is expressed in
the stem cells and the proliferative cells of the intestinal crypts. To
investigate the underlying molecular mechanisms and particularly those
responsible for the restricted tissue specificity, a large genomic
region of the mouse villin gene has been analyzed. A 9-kilobase (kb)
regulatory region of the mouse villin gene (harboring 3.5 kb upstream
the transcription start site and 5.5 kb of the first intron) was able
to promote transcription of the LacZ reporter gene in the small and
large intestines of transgenic mice, in a transmissible manner, and
thus efficiently directed subsequent Transgenic mice are routinely used to study the molecular and
cellular basis of normal and pathological states in intestinal mucosa
(1-5). The major limitation regarding the targeting of exogenous
transgenes in this tissue is that the epithelium of the mouse
intestinal mucosa is renewed every 2-5 days (6-8). The epithelial
cells arise from multipotent stem cells functionally anchored at the
base (more precisely in the lower third) of the proliferative
compartment of the epithelium, the crypts of Lieberkühn. These
crypts display a monoclonal organization because they are each derived
from a single progenitor cell (9). Descendants of stem cells multiply
in the middle portion of each crypt (10) and gradually differentiate
into four principal cell types. In the small intestine, absorptive
enterocytes (constituting >80% of the epithelial cells),
mucus-producing goblet cells, and enteroendocrine cells migrate upward
from the crypts to the apex of surrounding villi (whose colonic
counterparts are hexagonal-shaped cuffs) (11), where they become
apoptotic and are exfoliated into the gut lumen (12). In contrast,
antimicrobial peptides secreting Paneth cells migrate to the bottom of
the crypts, where they reside for about 20 days (13).
Given the remarkable protective effect of this epithelium, it is not
surprising that most previous studies aiming to induce neoplastic
transformation in intestinal mucosa of transgenic mice have failed (14,
15). In these reports, the use of promoter sequences that direct
oncogenes in nonproliferating enterocytes located in the upper third of
crypts produce only minor phenotypic abnormalities without tumorigenic
consequences in the gut epithelium, suggesting that the residence time
of these villus-associated cells may not be sufficient for the
oncogenes to exert their effects. Furthermore this suggests that
transgenic mouse models of neoplasia may require an efficient targeting
of oncogenes in crypt stem cells or their immediate descendants.
Villin is a cytoskeletal protein that is mainly produced in epithelial
cells that develop a brush border responsible for absorption as in the
digestive apparatus (epithelial cells of the large and small
intestines) and in the urogenital tract (epithelial cells of the kidney
proximal tubules). Because it is expressed in the proliferative stem
cells of the intestinal crypts (16, 17), it is believed to be an early
marker for committed intestinal cells. The multiple levels of
regulation control villin gene activity during mouse embryogenesis
(18-20) and account for the strict pattern of tissue-specific
expression observed in adults. Moreover, the expression of the villin
gene in intestinal epithelial cells is conspicuously maintained in
their corresponding carcinomas (21-24).
The specific expression pattern of villin suggests that it is an
appropriate candidate for the characterization of regulatory sequences
that could allow targeting of heterologous genes into a selected
population of cells in the mouse digestive tract. With this goal in
mind, the human villin gene has been isolated and characterized (25). A
2-kb1 5'-flanking region has
been found to contain sufficient regulatory elements to promote
tissue-specific expression of a reporter gene in intestinal and renal
cell lines (26). In transgenic mice, this regulatory region is able to
drive the expression of the human Ha-ras oncogene in the
tissues in which the endogenous gene is actively transcribed. However,
low levels of expression were observed that did not trigger malignant
tissue appearance into the gut of these
animals.2
These observations led us to further analyze an extended genomic region
of the mouse villin gene with the goal of mapping additional elements
localized 5' and/or 3' and involved in promoting high levels of
transgenic expression in the intestinal mucosa. Here we report the
analysis of tissue-specific expression of the mouse villin gene using:
(i) DNase I-hypersensitive sites assays, (ii) transient transfection
assays, and (iii) transgenic mice.
Cell Culture and ex Vivo Transient Transfection--
Human colon
carcinoma enterocyte-like CaCo2 cells, pig kidney proximal
tubules-derived LLCPK1 cells, and distal tubules-derived Madin-Darby
canine kidney cells were cultured as described (26). Cells cultures
were cotransfected using 15 µl of Lipofectin reagent (Life
Technologies, Inc.) with 5 µg each of Primer Extension Analysis--
Total RNA was isolated from mouse
intestine with RNA NOW reagent (Biogentex). For primer extension assay,
2 ng of 32P-labeled oligonucleotide probe
(5'-GAGTGGTGATGTTGAGAGAGCCT-3') complementary to nucleotides +81 to
+103 of the murine villin cDNA (GenBankTM accession
number M98454) was hybridized with 30 µg of total RNA at 60 °C
(0.25 M KCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) for 90 min. Transcription with 5 units/µl of
Moloney murine leukemia virus reverse transcriptase (Life Technologies,
Inc.) was carried out at 37 °C for 90 min in 300 µl of a solution
containing 75 mM KCl, 3 mM MgCl2,
50 mM Tris-HCl (pH 8.3), 10 mM dithiothreitol, 0.75 mM deoxynucleoside triphosphates, 75 µg/ml
actinomycin D, and 0.3 units/µl RNasin. The primer extension products
were separated by electrophoresis in denaturing 8% polyacrylamide
gels. The full-length extension product (105 nucleotides) was obtained
by comparison with the length of the comigrating sequencing reaction
products. A primer extension control experiment was performed on the
same total RNA preparation using a 32P-labeled
oligonucleotide probe (5'-CATAGTTCTCGTTCCGGT-3') complementary to
nucleotides +63 to +80 of the mouse intestinal fatty acid-binding protein cDNA and generating an 81-nucleotide extension product (27).
DNase I-hypersensitive Site Analysis--
Tissues from 30 mice
were used per assay of intestine, kidney, liver, and spleen. Nuclei
preparation and DNase I digestion were performed as described (28) with
minor modifications. Nuclei were digested without or with 20 to 160 units of DNase I (DPRF Worthington) for 10 min at 0 °C. 10 µg of
purified DNA was digested overnight with restriction enzyme
(BamHI or BglII). The DNase I-hypersensitive
sites were analyzed by Southern blotting using the
BglII-PstI probe (0.5 kb) (as indicated in Fig.
2).
Plasmid Construction--
All constructs described were
subcloned into the pBluescript II KS vector (Stratagene) with fragments
isolated from a Transgenic Mice Generation--
The transgenes digested with
XhoI-NotI were injected into the pronuclei of the
fertilized eggs of the B6/D2 mice in collaboration with the Service
d'Experimentation Animale et de Transgénèse, CNRS
(Villejuif, France). Mice carrying transgenes were first identified by
PCR of genomic DNA to confirm the presence of the Reverse Transcription-PCR Analysis--
Total RNA was isolated
with SV Total RNA Isolation System (Promega). 20 ng of
pd(N)6 random primer (Pharmacia) were hybridized with 2 µg of total RNA at 70 °C for 10 min in distilled water. Reverse
transcription with 200 units of Moloney murine leukemia virus reverse
transcriptase (SuperScript II, Life Technologies, Inc.) was carried out
at 37 °C for 90 min in a 20-µl solution of 1× First Strand Buffer
(Life Technologies, Inc), 10 mM dithiothreitol, 0.5 mM deoxynucleoside triphosphate, and 0.4 units/µl RNasin. 2 µl of the resulting cDNAs were amplified by PCR reaction in 50 µl for 40 cycles. Each cycle consisted of 60 s at 94 °C,
60 s at 51 °C (for transgene and villin) and 57 °C (for
TFIID), and 30 s at 72 °C. For the transgene primers,
5'-CAACTTCCTAAGATCTCC-3' coding strand and 5'-ATTCAGGCTGCGCAACTGTT-3'
noncoding strand were used, generating a 250-bp product. For villin
amplification 5'-CAACTTCCTAAGATCTCC-3' coding strand primer and
5'-GCAACAGTCGCTGGACATCACAGG-3' noncoding strand primer were used,
generating a 473-bp product; for TFIID amplification
5'-CCACGGACAACTGCGTTGAT-3' coding strand primer and
5'-GGCTCATAGCTACTGAACTG-3' noncoding strand primer were used,
generating a 220-bp product. One-fifth of the PCR product was run on an
ethidium bromide-containing agarose gel.
Detection of Determination of the Transcription Start Site--
To determine
the transcriptional start site of the mouse villin gene, total RNA was
isolated from intestine and analyzed by primer extension assay using an
oligonucleotide complementary to the mouse villin cDNA downstream
of the ATG initiation codon. The efficiency of the reaction was
confirmed by primer extension of the mouse intestinal fatty
acid-binding protein gene (fabpi) from the same RNA
preparation (27). Analysis of the fabpi extension product on
a sequencing gel by comparison with a sequence ladder (Fig.
1A) revealed a strong signal
band of a size of 81 bp as expected. The extension product of villin
was 105 bp, indicating that the transcriptional start site (an
adenosine residue subsequently designed as nucleotide +1) was 57 nucleotides upstream of the translation initiation codon of the murine
villin cDNA (Fig. 1B). Comparison of the genomic
sequence encompassing 9 kb upstream from the ATG initiation codon with
the cDNA sequence, position of splice site consensus sequences in
the 9-kb genomic sequence (Fig. 1B), and determination of
the transcription start site reveal that the mouse villin gene has one
transcription start site that is separated from the ATG
initiation codon by a 5.5-kb intronic region (Fig. 1C).
DNase I-hypersensitive Sites in the Mouse Villin Gene--
To
characterize the key regulatory regions involved in the specific
control of villin expression, we have mapped the DNase I-hypersensitive
sites (31) in the mouse villin gene (along a region extending 9 kb
upstream and 4.4 kb downstream from the translation initiation codon,
as represented in Fig. 2A).
The chromatin form of the mouse villin gene in different tissues
(intestine, kidney, liver, and spleen) was submitted to limited DNase I
digestion and subsequently digested with the appropriate restriction
enzymes. Using BglII digestion and a 0.5-kb probe homologous
5' of the 7.5-kb BglII fragment (Fig. 2B), two
sets of DNase I incubation-related fragments were detected, migrating
at 5.5 and 2.7 kb, and corresponding to hypersensitive sites designated
as HS I (located at approximately +5.5 kb downstream from the
transcription start (+1) site, just upstream from the ATG initiation
codon) and HS II (located at approximately +3 kb downstream from the
(+1) site), respectively. HS I was observed in nuclei isolated from
intestine, kidney, and liver, whereas HS II was only present in
intestinal tissue. No specific hypersensitive sites were detected in
nuclei isolated from spleen. The presence and location of these
hypersensitive bands were confirmed by hybridization with the 0.8-kb
probe (Fig. 2A) homologous to the 3' end of the 7.5-kb
BglII fragment (data not shown). Using BamHI
digestion and the 0.5-kb probe (Fig. 2C), five sets of DNase
I-treated nuclei-related fragments were detected, migrating at 3.4, 4.3, and 4.7 kb and approximately 10 and 15 kb, corresponding to the
hypersensitive sites HS II, HS III (located at
In conclusion, four major distinct DNase I-hypersensitive sites (HS I
to HS IV) were shown to be present in the region extending from Analysis of Promoter Activity by Transient Expression--
To test
the effects of the segments containing the DNase I-hypersensitive sites
(Fig. 3A) on transcriptional activity and to define more
precisely the element(s) controlling villin gene expression in the
intestine, segments were subcloned upstream of a promoterless LacZ
plasmid (Fig. 3B). The construct pA1 contained all the
subcloned regions downstream from the ATG initiation codon, encompassing the four DNase I-hypersensitive sites (HS I to HS IV)
described above and the 5.5-kb intronic sequence, intron 1. Plasmids
pA2 and pA3 were identical to pA1 except for the presence of
intestine-specific hypersensitive site HS II and intron 1, respectively. Plasmid pB1 and plasmid pC1 were similar to plasmid pA1
but lacked the regions extending from
Transient transfections with these recombinant plasmids were performed
in CaCo2 and LLCPK1 cell lines, which express villin, and in
Madin-Darby canine kidney epithelial cells, in which no villin
expression is detected. Transcription from the villin promoter was
measured by assaying
To test specificity, the villin promoter-related constructs were
transfected in Madin-Darby canine kidney cells, which do not express
villin. After transfection, these cells showed only base-line levels of
Analysis of Transgene Expression in Mice--
Because the
To examine the precise cellular distribution of transgene expression
within the tissues, cryostat sections of small intestine, colon, and
kidney were prepared and subsequently stained for
These results demonstrate that (i) the 3.5-kb regulatory region
upstream from the transcription start site of the mouse villin gene is
necessary and sufficient to sustain expression strictly in small
intestine of transgenic mice, (ii) the first intron of the mouse villin
gene is required for colon and kidney expression in transgenic mice.
Concerning the pB3 and pC3 transgenic mice, no transgene expression was
observed in all tissues examined, including small intestine, colon, and
kidney. Thus, the key cis-acting elements of the villin gene required
for intestinal and/or kidney-related expression of transgene(s) in
transgenic mice are not located only within the region encompassing
In this report, we demonstrate that cis-acting sequences located
within a 9-kb region (-3.5 to +5.5 kb from the start site of
transcription) of the mouse villin gene are sufficient to direct both
correct tissue-specific and high expression level of the In the kidney, for only one animal of five analyzed, mouse reporter
gene expression was restricted to epithelial cells of the proximal
tubules recapitulating the villin expression pattern in this tissue.
This suggests that transcriptional mechanisms specifying gene
expression to intestine and kidney tissues are in the The construct that entirely lacks the first intron of 5.5 kb but that
harbors 3.5 kb 5' to the start site of transcription of the mouse
villin gene, placed in front of the Constructs harboring only the first 480 and 100 bp 5' to the start site
of transcription, in combination with the lack of the first intron,
placed in front of the The inability of shorter regulatory sequences of the mouse villin gene
to direct correct expression of the reporter gene in the whole
intestine of transgenic mice might also be explained by spatial
rearrangement of chromatine structure due to the lack of the entire
first intron. In fact, the results described here are reminiscent of
those of the adenosine deaminase gene (34) and the aldolase B gene
(35), in which elements located in the first intron are required for
transgene expression in vivo, because they may contain
cis-acting tissue-specific enhancer elements and/or elements involved
in promoting decondensation of the chromatin structure, allowing the
accessibility for transcription factors and RNA polymerase. Further
investigations will be required to elucidate the precise role of the
first intronic region of the mouse villin gene.
To explain the discrepancy seen in the ability of the mouse villin gene
regulatory elements to promote transcription of the reporter gene in
cell cultures versus transgenic animals, we may argue that
the regulation of gene expression in the intestinal epithelium occurs
as cells differentiate and migrate along the crypt-villus axis. This
process depends on the contacts that these cells maintain with other
neighboring cells on one hand and with the extracellular matrix on the
other hand (36). Thus, an ex vivo system as the
intestine-derived CaCo2 cell line used in our study, is limited by its
weak ability to recapitulate the temporal and spatial complexities of
this epithelium and emphasizes the importance of using in
vivo models to define a function for specific regulatory sequences
(37, 38).
Previous studies carried out in transgenic mice to map transcriptional
regulatory elements responsible for intestinal expression have been
performed using cis-acting sequences of genes expressed in villus
associated-enterocytes of small intestine (4, 5, 38-40). In some of
these cases, precocious activation in the crypts in combination with
extended expression in the colon occurs in an inappropriate manner.
Thus, to our knowledge, the 9-kb regulatory region of the mouse villin
gene represents the only characterized cis-acting sequences reported
today that allow the expression of a heterologous gene in small
intestine and colon epithelial cells of transgenic mice reproducing
with great fidelity the tissue-specific and cell-specific pattern of
expression when compared with that of the endogenous gene itself. In
addition, the mice lines that drive a transgenic expression exclusively
restricted to the intestinal mucosa could already be studied after
selection of those that will not display expression into the kidney
because of the positional effects.
The ability to target genes of interest in transgenic mice following
the villin-restricted pattern of expression and particularly in the
crypt stem cells should lead to the development of targeted genes in
animal models. Experimental mouse models reproducing several steps of
human colorectal carcinogenesis (a possible genetic pathway has been
proposed by Fearon and Vogelstein (41)) could for instance be obtained
by efficiently targeting the associated oncogenes or mutated tumor
suppressor genes to colonocytes using the villin regulatory region.
Another use could lie in the establishment of new cell lines derived
from the digestive tract by targeting a thermosensitive SV40 T antigen
to the crypt-resident progenitors of intestinal cells, as used in other
systems (42-44).
-galactosidase expression in
epithelial cells along the entire crypt-villus axis. In the kidney, the
transgene was also expressed in the epithelial cells of the proximal
tubules but is likely sensitive to the site of integration. A construct
lacking the first intron restricted
-galactosidase expression to the
small intestine. Thus, the 9-kb genomic region contains the necessary cis-acting elements to recapitulate the tissue-specific expression pattern of the endogenous villin gene. Hence, these regulatory sequences can be used to target heterologous genes in immature and
differentiated epithelial cells of the small and/or large intestinal mucosa.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-galactosidase reporter plasmid construct and the control plasmid pRSVLuc, which contains the
luciferase gene under the control of the Rous sarcoma virus promoter.
Cells were harvested 48 h later, and cell extracts were assayed by
chemiluminescent detection of both
-galactosidase (Galacto-Light,
Tropix, Inc.) and luciferase (Luciferase Assay Kit, Tropix, Inc.)
activities using a luminometer (Berthold).
-Galactosidase activity
(light units) was corrected for variations in transfection efficiencies
as determined by luciferase activity. All transfections were repeated
at least three times. Results are expressed as fold induction over that
of the vector without promoter, pBasic.
DASHII phage containing a 16.3-kb region (9 kb
upstream and 7.3 kb downstream from the translation initiation codon)
of the mouse villin gene (kindly furnished by G. Tremp,
Rhône-Poulenc Rorer) (29). The pD1 construct (as described in the
legend to Fig. 3B) was prepared by ligating a
BamHI fragment of 5.1 kb (1.8 kb upstream from the ATG
translation initiation codon of the mouse villin gene, subcloned 5' to
the nuclear localization signal-
-galactosidase gene-SV40 polyadenylation site, using a polymerase chain reaction (PCR) strategy)
at the BamHI site in a plasmid containing the 3.7-kb region
of the mouse villin gene (immediately 5' to the 1.8-kb region described
above). The pA1 and pA2 (containing an internal 1-kb deletion)
constructs have resulted from several steps based on the
BstEII sites present in the 3.7-kb region described above and in a plasmid containing the 3.5-kb region of the mouse villin gene
(immediately 5' to the 3.7-kb region). The pC1 and pC2 constructs were
derived from the pA1 and pA2 plasmids cut with ApaI and
re-ligated, respectively. To generate the pB1 construct, a
BglII fragment (480 bp) from the 3.5-kb region described
above was excised and cloned into the KpnI site of the pC1
plasmid. The pA3, pB3, and pC3 constructs correspond to the pA1, pB1,
and pC1 deleted from the intron 1 (see Fig. 3B). The
sequence between the transcription initiation start site and the
translation initiation codon, excluding the intron 1, was deduced from
that of the murine villin cDNA and was introduced into the
BglII-NcoI sites of the pC1 construct by using a
dimerized oligodimer made of a coding-strand oligonucleotide (5'-GATCTCCCAGGTGGTGGCTGCCTCTTCCAGACAGGCTCGTCCAC-3') and a
noncoding-strand oligonucleotide
(5'-CATGGTGGACGAGCCTGTCTGGAAGAGGCAGCCACCACCTGGGA-3'), resulting
in the pB3 construct. The pA3 and the pC3 constructs were derived from
the pB3 plasmid by ligating an ApaI fragment (3.1 kb) and a
BglII fragment (480 bp), both from the 3.5-kb region described above, at the ApaI site in the pB3 plasmid,
respectively. Subcloning steps were confirmed by DNA sequencing.
-galactosidase
gene and then analyzed by Southern blotting to determine the copy
number of the integrated transgene. Each founder animal harbored one
copy of the transgene per genome. Small intestine, colon, kidney,
stomach, liver, heart, lung, thymus, brain, spleen, and muscle were
dissected from transgenic mice and either prepared for total RNA
extraction or embedded to perform cryosections.
-Galactosidase Activity--
Cryosections (5 µm) from the tissues were fixed with 3% paraformaldehyde for 5 min
and incubated in a staining solution that contained 0.4 mg of
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside/ml, 4 mM potassium ferricyanide, 4 mM potassium
ferrocyanide, 2 mM MgCl2 at 37 °C for 8 h.
RESULTS
View larger version (33K):
[in a new window]
Fig. 1.
Determination of the transcription start site
of the mouse villin gene by primer extension. A, primer
extension analysis was performed with mouse intestinal total RNA (30 µg) and with either the end-labeled villin oligonucleotide
(generating a 105-nuleotide extension product) or the end-labeled mouse
intestinal fatty acid-binding protein gene (fabpi)
oligonucleotide used as a positive control (generating an 81-nucleotide
extension product). The sizes of the fragments obtained by primer
extension are shown at the right. The unrelated sequence
ladder that was run in the same gel is used as a size marker.
B, nucleotide sequence between the transcription start site
(the bold adenosine designated as +1) and the initiation
codon (the bold underlined ATG codon) of the
mouse villin cDNA. Each of the splice junctions present in the
intron 1 (indicated at the bottom of the panel)
conforms to the consensus splice donor (the italic GT
nucleotides) and acceptor (the italic AG nucleotides)
patterns, described by Breathnach and Chambon (30). C,
schematic representation of the organization of the 5'-flanking region
of the murine villin gene. The open box represents the
untranslated exon, and the shaded box represents the first
coding exon. The sizes of the exon and the intron are indicated.
0.5 kb upstream from
the (+1) site), HS IV (located at
1 kb upstream from the (+1) site),
HS V (located at
10 kb upstream from the (+1) site), and HS VI
(located at
15 kb upstream from the (+1) site), respectively. HS III
was observed in nuclei isolated from both intestine and kidney, whereas
HS IV was only present in intestinal tissue as HS II. The
hypersensitive sites HS V and HS VI were only present in liver tissue
(in which villin is weakly expressed) and were located far upstream
from the transcription start site in regions (i) that have not been
subcloned and (ii) that could belong to an adjacent gene; for these
reasons, these hepatic-specific hypersensitive sites were not analyzed
further. As for BglII digestion, no specific hypersensitive
sites were detected in nuclei isolated from spleen. Using other
independant restriction digestions (EcoRI and
HindIII) and the 0.5, 0.8, and 1.25 kb probes (as
represented in Fig. 2A), similar results were obtained (data
not shown).
View larger version (38K):
[in a new window]
Fig. 2.
DNase I hypersensitivity in the mouse villin
gene. A, a partial restriction map diagram of the mouse
villin gene regions subcloned ( 3.5 to +9.9 kb in respect to the
transcription start site, indicated by an arrowhead).
BamHI (B), BglII (Bg),
EcoRI (E), and HindIII (H)
restriction sites, ATG initiation codon, and the probes used to map the
hypersensitive sites (0.5, 0.8, and 1.25 kb) are shown. B
and C, intestine, kidney, liver, and spleen nuclei were
digested with increasing amounts of DNase I at 0 °C for 10 min (0, 20, 40, 80, and 160 units). 10 µg of purified genomic DNA was
digested with BglII (panel B) and
BamHI (panel C), electrophoresed, and transferred
to a nylon membrane. Hypersensitive sites were revealed by probing with
a 32P-labeled fragment of 0.5 kb. Positions of
coelectrophoresed molecular mass markers are indicated at the
left, and the hypersensitive bands are marked by
arrows at the right. The maps represented at the
bottom of panels B and C show the
position of restriction sites, the deduced DNase I-hypersensitive sites
(indicated by arrows), and the 0.5-kb probe used.
1 kb
to +5.5 kb in respect to the transcription start site (Fig.
3A) of the mouse villin gene.
These sites were detected in intestine (HS I to HS IV), kidney (HS I
and HS III), and liver (HS I), tissues in which villin is expressed,
but they were not found in spleen, a tissue that does not produce
villin. These findings correlate with the tissue-specific control of
villin gene expression and suggest that the putative critical
regulatory elements lie within these regions. HS II and HS IV were only
detected in intestine and are probably associated with tissue-specific transcription factor-binding sites involved in the positive control of
villin gene intestinal expression.
View larger version (26K):
[in a new window]
Fig. 3.
Transient transfection analysis of the mouse
villin promoter. A, above a partial restriction map
diagram of the mouse villin gene from 9 kb with respect to the
translation initiation codon. ApaI (A),
BamHI (B), BglII (Bg),
BstEII (Bs), DrdI (D),
NcoI (N), and XbaI (X)
restriction sites are shown. The schematic representation at the
bottom of panel A shows the location of the four
hypersensitive sites (I-IV) as well as the 5.5-kb intron (represented
by a hatched rectangle) separating the transcription start
site (indicated by an arrowhead) and the translation
initiation codon. B, diagrams of the various constructs
generated by deletion. Different portions of the 5'-flanking region of
the mouse villin gene were fused with the Escherichia coli
-galactosidase gene containing the nuclear localization signal
(nls). C,
-galactosidase activities resulting
from transient transfections into CaCo2 colon cells (shaded
bars) or LLCPK1 kidney cells (open bars) with the
reporter constructs generated (represented in panel B).
Basal activity resulting from the promoterless pBasic plasmid was set
arbitrarily at 1. Values indicate the averages of at least three
independent transfections.
480 bp to
3.5 kb and
100 bp
to
3.5 kb according to the transcription start site, respectively.
Plasmid pC2 was identical to pA2 but lacked the region extending from
100 bp to
3.5 kb. Plasmids pB3 and pC3 were identical to pB1 and
pC1 except for the presence of intron 1, respectively. The plasmid pD1
was identical to pA1 except for the presence of the transcription start
site and the region extending upstream from this site. The plasmid
pBasic, which does not contain a promoter or enhancer, and a pControl
plasmid, which possesses the SV40 promoter, were also tested in each experiment.
-galactosidase activity in extracts made from
the transfected cells, and the results were expressed as fold induction
over that of the promoterless vector, pBasic (Fig. 3C). High
levels of
-galactosidase activity in the pControl transfected cell
lines (CaCo2 cells, 50-fold over that of pBasic; LLCPK1 cells, 98-fold)
demonstrated the presence of efficient general
transcription/translation mechanisms in these cells (data not shown).
Very low levels of
-galactosidase activity in pD1 both transfected
cells compared with pBasic transfected cells showed that the
transcription start site was necessary for an efficient specific
transcription of the reporter gene and that nonspecific transcription
was not initiated elsewhere in the villin regulatory sequences. The
construct pA1 expressed the
-galactosidase gene at the highest level
in CaCo2 cells (8-fold over pBasic) as compared with LLCPK1 cells
(1.5-fold over pBasic), suggesting that the four DNase I-hypersensitive
sites together with the first intron are necessary to efficiently
promote transcription in cells of intestinal origin. Deletion of the
fragment containing the intestinal-specific hypersensitive site HS II
(pA2) dramatically decreased
-galactosidase expression in CaCo2
cells (2-fold over pBasic) to about 25% of that of pA1, demonstrating
that a major element that confers intestinal activity was confined
within this fragment. Similar results were obtained when the region
upstream from the transcription start site (encompassing HS III and HS IV) was almost wholly deleted with or without HS II (pC1 and pC2, respectively). The deletion of the intronic region alone (pA3) or in
combination with deleted sequences upstream from the transcription start site (pB3 and pC3 extend only from
480 and
100 bp,
respectively) affected to a lesser extent
-galactosidase expression
in the same intestinal cells (5.5-fold over pBasic), with a decrease to
only about 65% of that of pA1, demonstrating that the regulatory elements that lay within 100 bp were sufficient to promote
transcription in cultured cells. However, the level of
-galactosidase activity increased strongly when the plasmids pA3,
pB3, and pC3 were transfected in LLCPK1 cells (10-, 44-, and 45-fold
over pBasic, respectively), showing that the absence of the first
intron, in combination with the lack of intestine-specific HS IV, was
able to promote transcription in a kidney cell line. This would suggest
that negative elements that confer repression in kidney transcription
are confined in these elements.
-galactosidase activity when compared with pBasic-related activity
(data not shown), demonstrating that the villin regulatory sequences
were unable to promote efficient transcription in nonexpressing villin
cells and that consequently the expression of the reporter gene in
CaCo2 and LLCPK1 cells is specifically dependent upon these regulatory
sequences. Taken together, these results from transient transfection of
cultured cells demonstrate that (i) the mouse villin genomic sequence,
extending from
3.5 to +5.5 kb, specifically directs an efficient
level expression of the
-galactosidase reporter gene in
intestine-derived cells, (ii) this level is dramatically reduced when
the intronic intestine-specific hypersensitive site HS II or the region
upstream from the (+1) site is deleted, (iii) lack of the entire first
intron seems to partially restore the intestine-related ability to
promote transcription, and (iv) lack of the entire first intron in
combination with intestine-specific hypersensitive site HS IV is
correlated with a strong increase of ability in promoting transcription
in kidney-derived cells.
3.5 to
+5.5-kb region of the mouse villin contained the
enterocyte-like-specific promoter/enhancer activity in transient
transfection assays, we examined the ability of this region to drive
intestine-specific expression of the
-galactosidase reporter gene in
transgenic mice. Five founder animals that contained the pA1 construct
as a transgene were obtained. The founder mice were analyzed for
mRNA reporter gene expression in several adult tissues by reverse
transcription-PCR analysis. From the same cDNA samples, products
encoding
-galactosidase, villin, and TFIID were analyzed. The PCR
assays enabled only the detection of spliced transcribed mRNA,
excluding that from genomic DNA itself, by means of an exon connection
strategy by combination of a 5' PCR primer from within the mouse villin
promoter sequence just upstream of the splice donor site and the 3'
primers from within the
-galactosidase gene or the villin gene. For
each founder, no reporter gene expression was detected in the tissues
in which villin mRNAs were not detected using the PCR assay (Fig.
4). For all founder mice, the reporter gene transcription was detected along the cephalocaudal axis of the gut
(duodenum, jejunum, ileum, proximal, and distal colon) following the
intestine-specific expression of the villin gene (Fig. 4). In the
kidney, the transgene was only transcribed in one founder of five
animals obtained (Fig. 4). TFIID mRNA was present in all samples
from tissues in which the reporter gene expression could not be
detected (Fig. 4), confirming the quality of RNA from these
tissues.
View larger version (30K):
[in a new window]
Fig. 4.
Expression pattern of the transgene.
Transgene-specific ( -Gal) transcripts were detected by
reverse transcription-PCR in a ethidium bromide-containing agarose gel.
Above each lane are the different tissues tested and the controls. +,
kidney mRNA from a mouse in which the
-galactosidase was
inserted at the villin locus (32);
, distilled H2O as a
template. Reverse transcription-PCR were also performed on mRNAs of
the endogenous villin gene and the ubiquitous TFIID gene.
-galactosidase enzyme activity. Similar results were obtained by immunofluorescence analysis of
-galactosidase expression (data not shown). For four of
five transgenic mice, a heterogeneous pattern of expression in small
intestine and colon was observed. This heterogeneity might be due to
mosaicism because we examined founder animals. The expression was
confined to the nucleus of the epithelial cells, as expected because
the
-galactosidase gene contains a nuclear localization sequence
signal (Fig. 5). The staining was
detected by a stronger signal in the villi migrating cells when
compared with the crypt cells, of both small intestine (Fig.
5A) and colon (Fig. 5C) epithelium, thus
confirming that the
3.5 to +5.5-kb region of the mouse villin gene is
able to recapitulate precisely the cellular pattern of expression,
along the crypt-villus differentiation axis, of the endogeneous villin
gene (17). A continuous labeling of all cells of the crypt (Fig. 5,
B and D) was observed, suggesting the expression
of the transgene in the stem cells (10). It is noteworthy that the
intensity of the
-galactosidase staining was similar to that of
intestinal sections from chimeric animals which possess a
-galactosidase gene integrated at the villin locus by homologous
recombination procedure (32), indicating that the
3.5 to +5.5-kb
region of the mouse villin gene was able to promote intestinal
transcription as efficiently as the mouse villin gene itself. In the
kidney of the founder mouse in which the transgene was detected by
reverse transcription-PCR, the staining was only observed in the
epithelial cells of the proximal tubules where the villin gene is
expressed (data not shown). The founder animals were able to transmit
the transgene to their offspring with a similar pattern of
-galactosidase expression (data not shown). In our attempt to direct
an efficient expression of the reporter gene in the intestinal
epithelium with shorter regulatory sequences, plasmids pA3, pB3, and
pC3 were used to generate transgenic mice, because these constructs
display efficient levels of
-galactosidase activity in
intestine-derived CaCo2 cells. The presence of the transgene assessed
by
-galactosidase staining procedure was observed in three of the
four independent lines of pA3 transgenic mice generated. These three
lines expressed the reporter gene only in the small intestine (in both
the immature and differentiated epithelial cells along the crypt-villus
axis), and all three lines failed to express the transgene in the other
tissues tested, particularly noteworthy is the lack of expression in
the colon and the kidney (data not shown).
View larger version (170K):
[in a new window]
Fig. 5.
-Galactosidase activity in
sections of small intestine and colon from transgenic mice.
Tissues were removed from transgenic mice, fixed, and stained for
-galactosidase activity with
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside as
described.
-Galactosidase activity was observed in the epithelial
cells, both immature and differentiated, along the crypt-villus axis in
the small intestine (A). Note that the differentiated cells
exhibited a strong signal as did the villus-associated cells and the
Paneth cells (arrows) localized to the bottom of the crypt
(B). The epithelial cells of the colon were also stained
(C), particularly all the cells in the crypt (D).
Bars, 100 µm (A and C) and 40 µm
(B and D).
480 bp upstream from the transcription start site, as observed in the
cultured epithelial cells.
DISCUSSION
-galactosidase reporter gene in transgenic mice, when compared with
the endogenous gene (19). Reporter gene expression is detected in the
whole intestinal tube and appropriately restricted to epithelial cells
along the crypt-villus axis of both small intestine and colon. In
addition, these regulatory elements can maintain a gradient of
-galactosidase gene expression from the crypts of Lieberkühn to the tips of villi that precisely reproduce the gradient exhibited by
the murine villin gene (17). Similarities between transgene and
endogenous gene expression were also noticed as judged by a comparison
with the staining intensity of
-galactosidase activity in intestinal
sections from our transgenic mice and mice in which the reporter gene
has been inserted at the natural villin locus by homologous
recombination (32).
3.5 to +5.5-kb
region of the mouse villin gene and that those related to kidney may be
sensitive to positional effects. Indeed it is known that the transgene
expression is dependent on the site of chromosomal integration and can
be influenced by regulatory regions in the vicinity, presumably acting
on chromatin conformation (33).
-galactosidase gene, restricts
the in vivo expression of the reporter gene only into the
epithelial cells along the crypt-villus axis of the small intestine.
The extinction of the reporter gene expression in the kidney might be
due to strong positional effects, as reported above, whereas the
extinction related to the colon might be due to the absence of
regulatory elements of the intron 1, such as the intestine-specific
DNase I-hypersensitive site HS II.
-galactosidase gene, both failed to drive
intestine-specific and kidney-specific expression of
-galactosidase,
suggesting that the intestine-specific DNase I-hypersensitive site HS
IV localized just upstream from the 480 bp might play an important role
in promoting reporter gene expression into the epithelial cells of the
small intestine. Thus, distinct and separable regulatory elements in
the mouse villin gene may direct transgene expression along the
cephalocaudal axis of the gut: the regulatory elements required for
transgene expression in the small intestine might be localized in the
3.5-kb region (i.e. the HS IV site) upstream from the
transcription start site, whereas those necessary for the colonic
expression might be localized in the first intron (i.e. the
HS II site).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Mireille Lambert and Christine Perret for contributions and helpful advice concerning the DNase I-hypersensitive sites assays and members of our laboratory for useful discussions. Thanks are also due to Dr. Italina Cerutti and members of the Service d'Experimentation Animale et de Transgénèse (Villejuif, France) for the collaboration in the generation of transgenic mice. We are grateful to Dr. Roy Golsteyn for critically reading the manuscript and for his constructive comments.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Centre National de la Recherche Scientifique, l'Institut Curie, l'Association pour la Recherche sur le Cancer, la Ligue Nationale contre le Cancer, and Rhône-Poulenc Rorer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a fellowship from the Association pour la Recherche
sur le Cancer.
§ Present address: INSERM U246, Faculté de Médecine X. Bichat, 16 rue H. Huchard, 75018 Paris, France.
¶ To whom correspondence should be addressed. Tel.: 33-142346378; Fax: 33-142346377; E-mail: dlouvard{at}curie.fr.
2 G. Tremp, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: kb, kilobase(s); PCR, polymerase chain reaction; bp, base pair(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|