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J Biol Chem, Vol. 273, Issue 48, 31939-31946, November 27, 1998
From The calbindin-D9K gene encodes a
vitamin D-induced calcium-binding protein that is expressed as a marker
of small intestine differentiation. We have shown that 4580 base pairs
of its 5' DNA regulatory region can target reporter transgene
expression in the intestine and cause this transgene to respond like
the endogenous gene to vitamin D active metabolite and that the
homeoprotein Cdx2 is bound to the TATA box in the intestine. We now
show that the 4580 base pairs construct confers a differentiated
pattern of reporter transgene expression in the intestine and that
cooperation between the proximal promoter and a distal element located
in an opened chromatin structure is responsible for the intestinal expression and vitamin D responsiveness of the transgene. Gel shift and
footprinting assays using duodenal nuclear extracts indicate that this
distal element contains a Cdx2-binding site. Finally, a mutation in
this distal Cdx2-binding site dramatically decreases intestinal
expression in transgenic mice. This report, using an in
vivo approach, demonstrates the crucial role of Cdx2 for the
transcription of an intestinal gene.
The active cell proliferation and turnover of the intestinal
epithelium makes this tissue a good experimental model for
investigating the nature of regulatory networks that control events
such as cell lineage commitment and differentiation. All mature
epithelial cell types found in the adult intestine are derived from a
nonmigratory stem cell pool located at the base of the crypts of
Lieberkühn (see Ref. 1 for a review). The enterocytes are the
most abundant cells in the intestine, accounting for approximately 95%
of the mucosal cells of the small intestine. As the enterocyte
precursors migrate from the crypts, they stop proliferating and acquire
differentiated functions, governed by specific patterns of gene
expression, that depend on the cell lineage, state of proliferation or
differentiation, and spatial location along the crypt-villus and
cephalocaudal (duodenum/colon) axes. But the molecular basis of this
diversity is still poorly understood. Determining the sequences
involved in the regulation of genes expressed in a subset of intestinal epithelial cells is a first step toward defining these factors. Traber
and co-workers (2) used this approach to identify Cdx2, a homeoprotein
involved in the control of the sucrase-isomaltase gene. Cdx2 is one of
the very few transcription factors that is active only in the adult
intestinal epithelium (3), and it appears to be a key element
controlling the differentiation of the intestinal epithelium
(4-6).
The complexity of the architecture of the intestinal epithelium is such
that transgenic mouse technology is a powerful tool for characterizing
the complexity of the cis-acting elements involved in the
regulation of intestinal gene transcription (for reviews, see Refs.7
and 8).
We have used the calbindin-D9K
(CaBP9K) gene as a marker of intestinal differentiation in
studies on the regulatory elements involved in its intestine-specific
pattern of expression. The CaBP9K gene is expressed only in
the differentiated enterocytes of the proximal region of the small
intestine (mainly in the duodenum) and the large intestine (mainly in
the cecum) (9-11). We have shown that the CaBP9K gene
expression is under positive transcriptional control of the vitamin
D3 active metabolite, 1,25(OH)2D3,
in the intestine (10, 12). We used a DNase I hypersensitivity analysis to define two potential regulatory regions (13). The proximal region
contains a major intestine-specific DNase I
HS,1 HS4, close to the
promoter. In vitro footprinting and gel shift assays showed
that a combination of an ubiquitous factor (NF1), liver-enriched
factors (HNF1, C/EBP, and HNF4), and the Cdx2 homeoprotein bind to this
region and may be important for controlling CaBP9K gene
transcription in the intestine (14). The distal region, HS1, is a
duodenum-specific HS located 3.5 kbp upstream from the start site. We
have recently shown by transient transfection assays (14), as well as
by creating different transgenic mouse lines (15), that 9K/ The present study further examines the mechanisms that restrict
CaBP9K gene transcription to the epithelium of the villus and shows that the 9K/ Construction of Hybrid Genes and Transgenic Mice--
The
plasmid (9K/ Animal Treatments--
Mice were made vitamin
D3-deficient and treated with
1,25(OH)2D3 as in (15). The effect of the
vitamin D3-depleted diet was verified by Northern blotting
using duodenal mRNA hybridized with a CaBP9K probe (data
not shown).
CAT Assays--
Various tissues were dissected out from young
adult (5-7-week-old) F1 transgenic mice for CAT assays. Cell lysates
were obtained (15). CAT activity was measured according to TLC standard
protocols, with 5-300 µg of protein and reaction times of up to
4 h to keep the enzyme activity in a linear range.
Immunocytochemical Studies--
CAT activity was revealed
immunocytochemically using an anti-rabbit polyclonal affinity-purified
antibody to CAT (5 Prime - 3 Prime, Inc., Paoli, PA) (18). The duodena
and colons from 9K/ Northern Blots--
Total RNA was prepared from several tissues
of young adult B6/CBA mice by the guanidium thiocyanate single-step
procedure (19) and analyzed by Northern blotting (15).
Gel Mobility Shift and DNase I Footprinting Assays--
Duodenal
nuclear extracts were prepared (14). Gel mobility shift assays and
DNase I footprinting assays were performed as before. The DNA fragments
used as probes for footprinting assays were prepared by subcloning
polymerase chain reaction products from the
The complementary oligonucleotides used for gel shift assays were
hybridized and end-labeled using [ Correct Crypt-villus Patterning Directed by the 9K/ Cooperation between the Proximal Promoter and a Distal Region (HS1)
Is Necessary for Directing Intestinal Expression and Responsiveness to
Vitamin D--
The mechanisms underlying the intestine-specific
expression of the CaBP9K gene along the cephalocaudal axis
were determined by identifying the minimal DNA regulatory regions
needed for intestinal specificity in transgenic mice. We created new
transgenes with various deletions. As there is a distal
intestine-restricted DNase I hypersensitive site (HS1) 3500 bp upstream
of the transcription start site (13), we explored its activating
potential by generating two types of transgenic mouse lines (Fig.
2A). The 9K/
In agreement with our previous data obtained with 9K/
Thus, the distal element HS1 contains sequences that cooperate with the
proximal CaBP9K promoter to stimulate specific transcription in the intestine; hence, the HS-1 box can be defined as an intestinal activator region.
We measured the activity of this distal activator element on a
heterologous promoter. For this, we cloned the distal HS1 element in
front of the ubiquitous minimal promoter of the herpes simplex virus
thymidine kinase gene, to obtain the HS1/thymidine kinase-CAT construct
(Fig. 2A). The four transgenic lines obtained showed no CAT
activity in the intestine or in the other tissues tested (Fig. 2, Table
I, and data not shown). Thus the distal activator HS1 did not confer
intestinal expression on a heterologous promoter in vivo and
acts only on its own promoter. This suggests that there must be
cooperation between the distal HS1 box and the minimal promoter to
activate the CaBP9K promoter in vivo in the intestine.
As the responsiveness of the CaBP9K gene to vitamin
D3 is an important feature of its intestinal expression,
and as the 9K/
The 9K/HS1-117 sequences thus contain all of the information needed to
direct the correct intestinal expression of the CaBP9K gene
along the horizontal axis of the small intestine and to mediate responsiveness to vitamin D3. However, a computer sequence
analysis of the entire 9K/HS1-117 region (Fig.
4) does not indicate the presence of a
consensus vitamin D-responsive element (VDRE), as previously
established by an in vitro approach (26), or a VDRE similar
to other previously identified VDREs (for a review, see Ref. 27).
A Binding Site for Cdx2 in the Distal HS1 Activator--
The HS1
cis-acting sequences that can cooperate with the minimal
promoter in stimulating the intestinal activity of the
CaBP9K gene promoter were identified by sequencing the
CaBP9K DNA regulatory sequences from
The caudal-related transcription factors appear to regulate the
transcription of several intestinal genes (2, 14, 28-31). We therefore
looked for a caudal motif in the distal activator HS1 and found a
sequence at
In vitro footprinting assays were performed to delineate the
Cdx2-related protection (Fig.
6A). We used two types of
polymerase chain reaction-derived probes: DNA fragments from Effect of the Distal Cdx2-binding Site on Intestinal Expression of
the 9K/CAT Transgene--
The functional role of the distal Cdx2 motif
in vivo was tested by introducing the mutated Cdx2 motif
(MUT HS1) into the 9K/
Last, we analyzed the profile of transgene expression along the length
of the intestine in the 9K/ We have further examined the mechanisms that restrict
CaBP9K gene transcription in the intestine. Previously, we
showed that nucleotides Cooperation between the HS1 Activator Region and the Inactive
Minimal Promoter of the CaBP9K Gene in the Intestine--
Transgenic
mice have been used to study other intestinal genes and help reveal the
complexity of the regulatory processes that govern intestinal gene
expression (see Refs. 7 and 8 for reviews). They indicate that multiple
functional cis-acting DNA elements are needed to mimic the
correct patterns of intestinal genes activities. However, studies on
these intestinal genes in transgenic mice (the genes for the liver and
intestinal fatty acid binding protein, the ileal binding protein, and
sucrase-isomaltase) have shown that the intestinal specificity is
conferred by the proximal promoter region of the gene, and that longer
fragments of 5'-flanking region of the gene modulate and confer the
correct gradient of reporter expression along the various axis of the intestine (33-40). In contrast, our data show that the minimal promoter of the rat CaBP9K gene cannot, alone, direct any
significant transgene expression in the intestine, although this
minimal promoter contains binding sites for Cdx2 and HNF-1 (14), which
are believed to be important transcription factors for many genes
expressed in the intestinal epithelium (31).
The regulation for the CaBP9K gene in the intestine may be
special because the intestine-specific transcriptional factor Cdx2 is
bound to the TATA box (14). This situation is reminiscent of the
chicken
Our data show that deleting DNA bases An Unusual Vitamin D3 Activation Pathway in the
Intestine--
The rat CaBP9K gene is under the positive
control of the active metabolite of vitamin D in the intestine (10).
This control involves the vitamin D receptor, as mice lacking the
vitamin D receptor have much less CaBP9K mRNA than normal and the
CaBP9K gene no longer responds to vitamin D (43-45).
However, we have found no VDRE implicated in this hormonal control by
transfection assays (46). The responsiveness to vitamin D only occurs
in transgenic mice (15), (Fig. 3). This result suggests that regulation of CaBP9K gene expression by
1,25(OH)2D3 involves tissue-specific factors.
Several exemples of tissue-restricted hormonal control have been
described (25, 47-49). We find that the 9K/HS1-117 construct, which
contains information needed to direct expression in the duodenum, also
contains the information needed to confer vitamin D3
responsiveness on the CaBP9K promoter. It will be of interest to see whether the tissue-specific element(s) and the element(s) implicated in the vitamin D regulation, within the 9K/HS1-117-CAT construct, can be separated or whether they act in
concert to impart the specific expression of the CaBP9K gene seen in the duodenum.
Careful analysis of the sequence of the regulatory element in the
9K/HS1-117-CAT construct has not revealed any consensual VDRE. This
suggests that the regulation of the rat CaBP9K gene by
1,25(OH)2D3 is complex and requires a
nonconsensual vitamin D-responsive element, as does the
c-fos gene. In this gene, a 34-bp DNA element binds vitamin
D receptor, 9-cis-retinoic acid receptor, and a member of
the NF1 family, which cooperate to produce a full response to vitamin D
(50). Nevertheless, a weak VDRE for the rat CaBP9K gene has
been found by transfection experiments (51), but its in vivo
relevance remains to be determined, because it is not included in our
smallest transgene construct that can confer vitamin D3 responsiveness.
Cdx2 and Transcription of the CaBP9K Gene--
Our examination of
the cis-acting DNA element in the distal activator region,
HS1, that confers intestinal specificity on the 9K/CAT constructs, has
focused on a Cdx2 binding element in this activator. Several lines of
evidence suggest that Cdx2 is crucial for regulating intestinal gene
transcription (2, 28-31, 52, 53) and intestinal differentiation: Cdx2
causes the morphological and molecular differentiation of
undifferentiated intestinal cell lines (5, 6), and Cdx2-null
heterozygote mice develop multiple tumors of the colon (4). This
suggests that Cdx2 must be continuously active to keep intestinal cells
differentiated, because the intestinal epithelial cells are constantly
being renewed from the stem cell pool in the crypts.
We have shown that Cdx2 binds to a caudal motif present in the
activator HS1. A mutation in this HS1 distal Cdx2-binding site greatly
decreases the activating function of HS1 in transgenic mice. However,
Cdx2 alone cannot explain the great expression of the
CaBPD9K gene in the duodenum. The restricted expression of
the transgene of the 9K/CAT constructs in the duodenum in the small
intestine could be due to a duodenal transcription factor(s) binding to
a regulatory site(s) in the distal activator HS1 or in the proximal
promoter that cooperates with Cdx2. This idea is supported by our
finding of a duodenum-specific footprint in the promoter region of the
CaBP9K (footprint B, nucleotides
In summary, we have obtained direct evidence that Cdx2 is required for
the intestinal expression of the CaBP9K gene by terminally differentiated enterocytes. This is yet another indication that this
homeoprotein plays a key role in the maintenance of the differentiated phenotype of the intestinal epithelium.
We are grateful to Prof. P. G. Traber
(University of Pennsylvania, Philadelphia, PA) for vector and antibody.
We thank Isabelle Lagouthe, Arlette Dell'Amico, Hervé Gendrot,
and Pascal Lacourte for skillful care of the numerous mice used in this
study. We are indebted to Franck Letourneur and Emmanuel Gomas for
sequence analysis.
*
This work was supported by the Association de Recherche
contre le Cancer and the Ligue Nationale contre le Cancer.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X16635.
§
These authors contributed equally to this work.
The abbreviations used are:
HS, hypersensitive
site; CAT, chloramphenicol acetyltransferase; bp, base pair(s); VDRE, vitamin D-responsive element; SIF1, sucrase isomaltase factor 1; WT, wild type; MUT, mutant.
Intestinal Expression of the Calbindin-D9K Gene
in Transgenic Mice
REQUIREMENT FOR A Cdx2-BINDING SITE IN A DISTAL ACTIVATOR
REGION*
§,
,,
INSERM U458, Hôpital Robert Debré,
48 boulevard Sérurier, 75019 Paris, France, ¶ INSERM
U129, Institut Cochin de Génétique Moléculaire,
24 rue du faubourg St Jacques, 75014 Paris, France, and ** INSERM U246,
Faculté de Médecine Xavier Bichat, Institut
Fédératif de Recherche 02, B.P. 416, 75810 Paris Cedex
18, France
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
4580
sequences of the CaBP9K gene (from 4580 to +365)
containing the two potential HS are necessary to direct active
transgene expression in the duodenum in a vitamin D3
dependent manner. In addition, a short construct, 9K/117 (from 117
to +365), containing only HS4, is not sufficient to direct expression of the transgene in the intestine (15).
4580-chloramphenicol acetyltransferase (CAT)
construct confers a correct crypt-villus pattern of transgene expression. We have identified the minimal regulatory elements needed
to confer this intestinal expression on the CaBP9K gene and
demonstrated that there must be cooperation between the HS1 distal
activator and the minimal promoter containing a Cdx2-binding site for
the correct targeting of cephalocaudal transgene expression and vitamin
D responsiveness in the small intestine. Last, we have described a
potential second Cdx2-binding site in the distal activator HS1 and
tested in vivo the functional relevance of this site by
mutagenesis in transgenic mice.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
4400-CAT) has been previously described (15). Sequence
analysis has revealed that it contained 4580 base pairs of the 5'
regulatory sequences of the rat CaBP9K gene (4580 to +365)
placed in front of the coding sequences of the CAT gene. Therefore,
this plasmid has been termed 9K/4580-CAT in this report. The
transgene 9K/117-CAT was isolated by HindIII digestion of the plasmid 9K/4580-CAT. This fragment contains the promoter region
(beginning at 117), the first exon, the first intron, and the
beginning of the second exon in front of the CAT gene. A
SmaI-PvuII CaBP9K fragment (3735 to 2891) was
inserted upstream of a blunted BamHI site of pBLCAT2 to
generate 9K/HS1-thymidine kinase-CAT, and insertion upstream of a
blunted HindIII site of 9K/117-CAT generated
9K/HS1-117-CAT. The Kunkel method (16) was used for
oligonucleotide-directed in vitro mutagenesis after subcloning the 4580 to 2200 CaBP9K fragment in the KS
Bluescript phagemid and transformation in RZ1032 host cells (Promega).
The oligonucleotide primer used was
5'-AAACATGCCCATAGGTGCAGGGTGTGT-3'. The entire wild type and mutant
regions were sequenced on both strands (17) to verify that only the
desired mutation was present. The mutant fragment was then subcloned in
9K/4580-CAT-KS instead of the wild type fragment to obtain the
4580Cdxmut-CAT plasmid. Microinjection fragments were linearized and
purified. Transgenic mice were generated, identified, and propagated
(15).
4580-CAT transgenic mice were removed, cut into
small pieces and rapidly frozen. Frozen sections (7 µm) were cut with
a cryostat and incubated with the anti-CAT antibody (dilution, 1:1000)
at room temperature for 1 h. The sections were rinsed in
phosphate-buffered saline, incubated with anti-rabbit IgG-digoxigenin
plus F(ab')2 fragment (dilution, 1:400; Boehringer
Mannheim) for 30 min at room temperature, and rinsed three times with
phosphate-buffered saline. The sections were then incubated with
anti-digoxigenin-fluorescein isothiocyanate (dilution, 1:400;
Boehringer Mannheim) for 30 min at room temperature. Sections were
rinsed in phosphate-buffered saline, mounted, and examined under a
Zeiss microscope equipped with epifluorescence optics. Control
sections were processed as above but without the anti-CAT antibody.
3537 to 3281 region of
9K/4580-CAT and 4580Cdxmut-CAT plasmids to generate wild type (WT
HS1) and mutated probe (MUT HS1). The annealed oligonucleotides used as
probes or competitors were as follows: SIF1 (59 to 31),
5'-GAGGGTGCAATAAAACTTTATGAGTAGGT-3' from human sucrase-isomaltase
promoter (20); 9KCdxd (3441 to 3422), 5'-TGCATTTATGGGCATGTTTCC-3';
and MUT9KCdxd 5'-TGCACCTATGGGCATGTTTCC-3'.
-32P]T4
polynucleotide kinase. For supershift assays, binding reactions were
incubated for 15 min at room temperature with anti-Cdx2 antibody (a
gift from P. G. Traber) with gentle agitation. The radiolabeled DNA
was then added (14).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
4580-CAT
Fragment--
The mouse and rat CaBP9K genes are mainly
expressed in the proximal region of the small intestinal epithelium
(duodenum) and only in the enterocytes (11, 21, 22) (see Fig.
2E). The CaBP9K gene is expressed in the cecum
and weakly in the proximal colon of the large intestine (10) (see Fig.
2E). The 9K/4580 sequences of the rat CaBP9K
gene contain the information necessary for correct transgene expression
along the cephalocaudal axis in the small intestine, but this gene
causes also ectopic expression of the transgene in the distal colon
(15). Immunocytochemical studies on 9K/4580-CAT mice showed that the
transgene was expressed along the crypt-villus axis within the duodenum
and the colon (Fig. 1). Analysis of
several sections of duodena from transgenic mice revealed that CAT
transgene is expressed along the length of the villus, in the
epithelial layer but not in the crypt (Fig. 1A). The
staining intensity is consistent with the amount of the CaBP9K in the
enterocytes, which constitute 95% of the cellular population in the
villus epithelium. But the difficulty of immunolocalizing the CAT
transgene made it difficult to be sure that transgene expression was
restricted to the enterocytes. The same gradient of CAT expression
occurred in the distal colon (Fig. 1C). Thus, the 9K/4580
regulatory sequences of the CaBP9K gene contain the information needed for expression of the transgene in the
differentiated epithelial cells in the small and large intestine.
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Fig. 1.
Immunohistochemical detection of the CAT
protein in the duodenum and colon of 9K/ 4580-CAT transgenic
mice. The distribution in the duodenum (A and
B) and colon (C and D) of the CAT gene
product detected using an anti-CAT antibody. Gene products revealed by
positive immunostaining (A) were detected in the duodenal
epithelial cells in the upper part of the villi; cells in the crypt
were not stained. The colon contains positive immunostaining in cells
bordering the lumen (C). Controls show no positive staining
(B and D) when tissue sections were processed
omitting the anti-CAT primary antibody. Bars, 1 µm.
117-CAT construct was prepared to confirm results previously obtained using the
9K/117-AgT construct, in which 9K/117 sequences of the
CaBP9K promoter (the minimal promoter) were unable to target expression of the SV40 large T antigen in the intestine (15). We
prepared the 9K/HS1-117-CAT construct containing an internal deletion
that placed the distal HS1 box just in front of the minimal promoter.
We generated seven lines of mice with the 9K/117-CAT transgene and
six lines with the 9K/HS1-117-CAT transgene (Table I and Fig. 2, B and
C). The CAT activities in the duodenum and other parts of
the intestine were compared with those obtained with mouse transgenic
line 94 harboring the 9K/4580-CAT construct (15). Differences between
individuals in each transgenic line were relatively large, so that we
were obliged to examine many mice from each transgenic line (Table
I).
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Fig. 2.
CAT activities of the distal and proximal
CaBP9K promoter in the intestine of transgenic mice.
A, diagram showing the transgenes used. The nucleotide
positions are numbered from the CaBP9K transcription start
site (+1). Boldface arrows denote the major DNase I
hypersensitive sites (HS1 and HS4); lightface arrows
indicate minor HSs (HS2, 3, 5). The broken line indicates a
DNA deletion. B, CAT expression of transgenes in the duodena
of transgenic mice. Bars represent the mean activities of
each line, and each black diamond indicates the value of a
single expressing mouse line (values are given in Table I and are the
means of at least three individuals). C and D,
CAT expression pattern along the length of the intestine (duodenum to
colon). D, duodenum; J, jejunum; I,
ileum; C, cecum; PC, proximal colon;
DC, distal colon. Bars and diamonds
are as described for B. E, Northern blot analysis
of CaBP9K mRNA along the intestine.
CAT activity in intestinal tissues from 9K/CAT transgenic mice
117-AgT, the
9K/117-CAT construct did not target any detectable CAT expression in
any part of the intestine studied, whatever the transgene copy number
(Fig. 2, B and C, and Table I). By contrast, juxtaposition of HS1 box and the minimal promoter restored CAT activity
in the duodenum, but the activity was weaker than that obtained with
the 9K/4580-CAT construct (Fig. 2B). A compilation of our
previous (15) and present data clearly shows that except for one low
transgene copy number mouse line (line 84, two copies) that had little
CAT activity in the duodenum, the mice of all the transgenic lines
harboring the 9K/4580-CAT construct had high CAT activity in the
duodenum, the distribution of which was relatively uniform (Fig.
2B and Table I). By contrast, the CAT activity in the
duodena of the mice of the HS1-117-CAT transgenic lines was more
dispersed and independent of the transgene copy number, which is
consistent with an integration site effect on transgene expression
(Table I and Fig. 2B). This result suggests that the region
between 2894 and 117 is required to establish an open chromatin
structure for high activity, but not for the duodenal specificity. The
anterior-posterior distribution of transgene activity in 9K/4580-CAT
mice (line 94 was representative of the other lines tested (Ref. 15 and
data not shown) was maximal in the duodenum and distal colon (Fig.
2C). In contrast, transgene expression in the
9K/HS1-117-CAT mice was maximal in the duodenum and the cecum (Fig.
2D), which correlated more closely with the expression of
the endogenous CaBP9K gene (Fig. 2E). We obtained no data on the expression of the transgene along the crypt-villus axis
with the 9K/HS1-117-CAT construct; the duodenal CAT activity was below
the threshold of the immunocytochemical assay used.
4580 sequences confer vitamin D3-inducible
intestinal expression (15), we have looked for a response to this
hormone in four transgenic mouse lines harboring the 9K/HS1-117-CAT
construct. The 9K/HS1-117 sequences conferred responsiveness to
vitamin D3 on transgenic mice (Table I and Fig.
3). A single injection of 25 ng of
1,25(OH)2D3 caused vitamin
D3-deficient transgenic mice to increase their duodenal
expression of CAT 1.5-26-fold (Fig. 3). The CAT activities obtained
with the "norm D" mice fed a standard diet were in the same range
as those for the "D" mice fed a vitamin D3-deficient diet (Table I). This is probably because the standard chow diet (norm
D) is naturally deficient in vitamin D3, because the amount of CaBP9K mRNA in the duodena of mice fed a standard chow diet was
similar to that of the duodena of mice fed a vitamin
D3-deficient diet (data not shown). The degree of vitamin
D3 inducibility in each transgenic line was not correlated
with the level of transgene expression (Table I). A mouse line with a
high intestinal transgene activity (line 24) was poorly vitamin
D3-inducible (1.5-fold), whereas a line with less
intestinal transgene activity (line 43) was very vitamin
D3-inducible (26-fold). This probably reflects an
integration position effect. The position effects on both the basal
expression and hormonal inducibility of transgenes are well documented and probably reflect variegation-type phenomena
(i.e. the influence of the chromatin structure around the
integration site) and the presence of regulatory elements near the
integrated transgene (23-25).
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Fig. 3.
Response of the 9K/HS1-117-CAT
transgene to vitamin D3 in vivo. Vitamin
D3-deficient mice were given
1,25(OH)2D3 (+D) or vehicle ( D). This
histogram of +D/D ratios indicates stimulation of transgene activity
by 1,25(OH)2D3. The bars indicate
the mean stimulation by 1,25(OH)2D3 of four
mouse lines bearing 9K/HS1-117-CAT, and of line 94 for 9K/4580-CAT.
The values of single expressing lines are denoted by
diamonds (for details, see Table I).
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Fig. 4.
Sequence of the 2.3-kbp DNA fragment upstream
from the 2227 EcoRI site of the CaBP9K promoter
region. Nucleotides are numbered in the right and
left margins. The probable extent of HS1 is denoted by the
boxed sequence. The caudal motif is in boldface.
HindIII (4581) is the 5'-end of 9K/4580-CAT fragment.
SmaI (3735) and PvuII (2891) are the 5'- and
3'-ends of the HS1 fragment subcloned in the 9K/HS1-117-CAT construct.
The nucleotide sequence has been updated in the EMBL data library under
accession number X16635.
4580 to 2232.
Published data (13) indicate that HS1 is centered around position
3500 bp and extending 100 bp upstream and downstream this site (Fig.
4).
3437 (ATTTATG) that is very similar to known canonical
caudal motif (32). We characterized this element by electrophoretic
mobility shift assay using the probe 9KCdxd
5'-TGCATTTATGGGCATGTTTCC-3' (caudal motif is bold). A
specific DNA protein complex was revealed with duodenal nuclear extract
(Fig. 5A). A 10-fold excess of
unlabeled SIF1 element defined as the binding site for Cdx2 in the
sucrase-isomaltase gene (2) was sufficient to displace 9KCdxd (Fig.
5A), whereas a mutated 9KCdxd element (Mut,
5'-TGCACCTATGGGCATGTTTCC-3') competed poorly. In agreement with the competition experiment, the
mutated element (Mut9KCdxd) retained only very faint binding with
duodenal nuclear extract (Fig. 5A). The specific 9KCdxd
complex was shown to contain Cdx2 using an antibody against Cdx2 (a
gift from P. G. Traber), which supershifted the entire complex formed. As controls, the SIF1 monomeric and dimeric complexes previously described (2) were also supershifted (Fig. 5B), and an
unrelated antibody (anti-IPF1, a gift from Dr. M. Montminy) did not
supershift the retarded complex (data not shown). Therefore, the 9KCdxd
caudal motif is able to bind to the duodenal Cdx2 homeoprotein in
vitro.
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Fig. 5.
Binding activity of 9KCdxd caudal motif to
duodenal nuclear extracts. A, a double-stranded
oligonucleotide containing the distal Cdx2 motif (9KCdxd) was
radiolabeled and incubated with nuclear extracts. The complex is shown
by an arrow. The competitors of the 9KCdxd complexes, used
at 10- and 100-fold excess, were 9KCdxd itself (Cdxd), SIF1
(SIF), and MUT9KCdxd (MUT). The right
lane shows binding of the labeled MUT9KCdxd probe to duodenal
nuclear extract. B, supershift experiments with an anti-Cdx2
antibody. Black arrows indicate monomeric and dimeric
complexes obtained with the SIF1 probe (SIF) and duodenal
nuclear extract (Duo), and the specific complex obtained
with 9KCdxd probe and duodenal nuclear extract. Open arrows
show the supershifted complexes obtained with an anti-Cdx2 antibody
(Ab).
3537 to
3281 that contained the distal wild type Cdx2 motif (WT HS1) or the mutated distal Cdx2 motif (MUT HS1). Several protections were observed
with duodenal nuclear extracts, but only one was entirely affected by a
mutation in the caudal motif (Fig. 6, A and B). This protection is bordered by the caudal motif at the 5'-end of the
sense strand and extends over 21-24 bp, suggesting that other
transcription factors also bind to this sequence.
View larger version (69K):
[in a new window]
Fig. 6.
DNase I footprinting of the HS1 region of the
rat CaBP9K gene. A, Labeled wild type (WT HS1)
and Cdx2 mutated (MUT HS1) CaBP9K fragments on the coding
and noncoding strands were incubated without protein (DNA)
or with 35 µg of rat duodenal nuclear extracts (DUO).
G+A indicates Maxam/Gilbert sequences. The WT HS1-specific
protected area is denoted by a box. The two
asterisks represent the two mutated thymines (in cytosine)
in MUT HS1. The boundaries of the regions are numbered from the
transcriptional start site. B, the extent of the DNase I
footprint is indicated on the HS1 sequence by a box. The
beginning of the protection was difficult to locate, as indicated by
the dashed line, because DNase I did not attack these
sequences on naked DNA. The caudal motif is in boldface, and
the mutated nucleotides are shown.
4580-CAT construct and by studying the effect
of this mutation on the pattern of transgene expression along the
intestine (Fig. 7A). Six mouse lines carrying the 9K/4580Cdxmut-CAT fragment were obtained (Table I). Mutation of the distal Cdx2-binding site resulted in a dramatic drop of CAT activity in the duodenum to become about 100-fold decrease
than in 9K/4580-CAT mice (Fig. 7B). Only one mouse line harboring the 9K/4580Cdxmut-CAT construct (line 94) with a very high
copy number (about 150 copies) had greater transgene expression than
the other mouse lines. This CAT activity was only 4-fold that of the
transgenic mice (line 84) harboring only two copies of the
9K/4580-CAT construct (15) (Fig. 7B). The very weak expression of the transgene with the 4580Cdxmut-CAT construct probably
reflects the residual binding activity of Cdx2 to the 9KCdxdmut site.
Thus mutation of the distal Cdx2-binding site leads to a strong
reduction of transgene expression in the duodenum, suggesting that Cdx2
is critical for the CaBP9K promoter activity in the
intestine.
View larger version (31K):
[in a new window]
Fig. 7.
CAT activity in the intestine of transgenes
containing a distal mutated Cdx2 site. A, diagram of
the transgenes used. The mutation in the Cdx2 site shown by a
black X on this site. B, CAT activity of the
transgenes in the duodenum. Bars represent the mean activity
of the different mouse lines, and each black diamond
indicates the value of a single expressing line (detailed in Table I).
C and D, CAT activity along the length of the
intestine. D, duodenum; J, jejunum; I,
ileum; C, cecum; PC, proximal colon;
DC, distal colon. Bars and diamonds
are as in B. E, Northern blot analysis of Cdx2
mRNA along the intestine. The same blot as Fig. 2E was
hybridized with a Cdx2 cDNA fragment probe from P. G.
Traber.
4580Cdxmut-CAT mice. The CAT activity in
these mice was somewhat similar to that of 9K/4580-CAT, with great
activity in the duodenum and distal colon (Fig. 7, C and
D). These results and the abundance of the endogenous Cdx2 mRNA that did not parallel the pattern of 9K/4580-CAT transgene expression (the maximal level of Cdx mRNA is in the cecum and proximal colon (Fig. 7E)) strongly suggest that factors
other than Cdx2 are involved in determining the gradient of expression of the transgene in the intestine.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
4580 to +365 of the rat CaBP9K
gene recapitulate most of the intestine-specific CaBP9K profile (15).
This study shows that this 5' flanking region also maintains the
crypt-villus expression pattern, limiting transgene expression to the
epithelium of the villus. The intestinal specificity and vitamin
D3 responsiveness of the rat CaBP9K gene is
conferred by cooperation between the minimal promoter and the distal
activator region (HS1). This distal element contains a binding site for
Cdx2, mutation of which causes a great reduction in the activating
function of HS1 in transgenic mice. Thus, Cdx2 plays a key role in the
intestine-specific transcription of the CaBP9K gene.
A globin gene. The promoter of this gene binds
several transcription factors and the erythroid-specific factor GATA-1
is bound to the TATA box. Thus, as for the rat CaBP9K gene,
transcription of the A globin gene in a
erythroid-specific fashion depends on the presence of a distal
activator containing a GATA-1 site (41). An in vitro transcription assay was recently used to show that the distal activator
acts via derepression of the inactive A
globin promoter (42). This derepression depends on the specific promoter of the A globin gene and does not occur on a
consensus TATA box. Thus, the transcription of the CaBP9K
gene in the intestine may require the presence of the distal activator
HS1 to derepress the inactive CaBP9K promoter produced by
binding a tissue-specific factor to the TATA box. Alternatively, the
proximal Cdx2-containing transcription complex alone could be by itself
insufficient for chromatin opening and significant transcription in the
absence of a loop structure, allowing specific cooperation between HS1
and HS4 through elective cooperation between distal and proximal Cdx2.
This would explain why the distal activator HS1 cannot be considered to
be classical enhancer, because it is unable to enhance the promoter
activity of a heterologous promoter or give it intestinal specificity.
2891 to 117 does not affect
the profile of expression of the transgene in the small intestine and
thus imply that the information needed to direct expression of the
CaBP9K gene in the duodenum is contained in the
9K/HS1-117-CAT construct. This construct can trigger expression of the
transgene in the cecum and decrease transgene activity in the distal
colon, which more closely reproduces the pattern of expression of the
endogenous gene. This result indicates that the region between 2891
and 117 contains DNA regulatory elements implicated in the spatial
control of the CaBP9K gene in the large intestine. Others
have suggested such a combination of the DNA regulatory elements to
explain the appropriate pattern of expression of intestinal genes
(33-40).
76 to 46) (14). Cdx2
may require specific coactivators or posttranslational modification
before it can activate the CaBP9K gene in the duodenum. Taylor et al. (53) have proposed that cell specific
mechanisms determine the ability of Cdx2 to activate transcription when
it is present in an enhancer context.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: INSERM U440, 17 rue du fer à Moulin,
75005 Paris, France.
To whom correspondence should be addressed. Tel.:
33-1-44-41-24-12; Fax: 33-1-44-41-24-21; E-mail:
perret{at}icgm.cochin.inserm.fr.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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