THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 277, No. 36, Issue of September 6, pp. 33275–33283, 2002
Printed in U.S.A.
cis Elements of the Villin Gene Control Expression in Restricted
Domains of the Vertical (Crypt) and Horizontal (Duodenum, Cecum)
Axes of the Intestine*
Received for publication, May 20, 2002, and in revised form, June 10, 2002
Published, JBC Papers in Press, June 13, 2002, DOI 10.1074/jbc.M204935200
Blair B. Madison‡, Laura Dunbar, Xiaotan T. Qiao, Katherine Braunstein, Evan Braunstein,
and Deborah L. Gumucio§
From the Department of Cell and Developmental Biology, The University of Michigan Medical School,
Ann Arbor, Michigan 48109-0616
Much has been learned about the process of organogenesis
through analysis of the regulation of structural genes that are
expressed tissue-specifically. For example, the study of globin
gene expression provided important insights into red cell development (1, 2), whereas the study of albumin expression led
to an improved understanding of the factors that regulate
development of the liver (3). Similarly, we propose that the
actin bundling protein, villin, is an excellent model gene for the
study of intestinal epithelial organogenesis.
The villin gene is first activated at 9.0 days post coitum
(dpc)1 in the presumptive intestinal hindgut endoderm during
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
‡ Supported by the Training Program in Organogenesis
(T32HL07505).
§ Supported by the Roger McDermitt Research Fund and by a Cancer
Innovation Grant, both from the University of Michigan Comprehensive Cancer Center. To whom correspondence should be addressed:
Dept. of Cell and Developmental Biology, University of Michigan Medical School, 5704 Medical Science II, Ann Arbor, MI 48109-0616. Tel.:
734-647-0172; Fax: 734-647-9559; E-mail: [email protected].
1
The abbreviations used are: dpc, days post coitum; LCR, locus
control region; PBS, phosphate-buffered saline; RT, reverse tranThis paper is available on line at http://www.jbc.org
gut tube closure (4 – 6). The early (10 dpc) expression domain of
villin rapidly extends throughout the small and large intestinal
endoderm and includes the distal stomach (4, 6). At 14 –15 dpc,
when the intestinal epithelium is dramatically remodeled and
villi are formed, villin gene expression is up-regulated (6, 7). At
16 dpc, a sharp, one-cell-thick border of villin expression becomes apparent in the pyloric epithelium; intestinal cells express villin at high levels whereas neighboring stomach cells
exhibit low or barely detectable expression of villin (4). Once
intestinal crypts are established, villin is expressed in all cells
of the crypt-villus axis, with an increasing gradient from crypt
to tip (6). Thus, the villin gene appears to respond to the
various morphogenetic cues that dictate key events of intestinal development at multiple time points both in utero and
postnatally. It is therefore likely that the molecular factors that
dictate this pattern of villin expression are the same factors
that coordinate these key events of intestinal development.
Villin regulatory sequences have been investigated in two
previous studies (8, 9). However, because the in vivo transcriptional start site of villin was initially erroneously mapped to
the same exon as the translational start codon (9), an early cell
culture study analyzed a villin “promoter” fragment that actually consisted entirely of intronic sequences (9). Later work
showed that transcription actually begins at a 5⬘ non-translated exon that is separated from exon 1 (the ATG-containing
exon) by 5.6 kb in the mouse (8, 10). A 9-kb fragment, containing this entire intron plus 3.4 kb of additional upstream 5⬘flanking sequence, was recently used to generate transgenic
mice (8). This 9-kb fragment drove reporter gene expression in
the intestine of adult mice, although four of five founders
showed “heterogeneous” expression. Because this analysis was
limited to the study of founders, embryonic expression of the
transgene was not investigated and the possible effects of
transgene mosaicism versus position effects were not assessed
(8).
Here, we utilized a larger transgene construct and analyzed
both fetal and adult transgene expression in multiple established lines. We report that a 12.4-kb region from the mouse
villin gene directs intestine-specific expression of the bacterial
␤-galactosidase gene in a manner that largely recapitulates the
normal expression pattern of villin in the intestine. In 75% of
the established lines, including two single copy lines, this
transgene directs continuous (not variegated) expression in the
entire intestinal epithelium. This suggests that this 12.4-kb
transgene contains a locus control region (LCR), a chromatin-
scriptase; UTR, untranslated repeat; p, postnatal day; PEV, position
effect variegation; E64, trans-epoxysuccinyl- L -leucylamido-(4guanidino)butane.
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Villin, an actin bundling protein found in the apical
brush border of absorptive tissues, is one of the first
structural genes to be transcriptionally activated in the
embryonic intestinal endoderm. In the adult, villin is
broadly expressed in every cell of the intestinal epithelium on both the vertical axis (crypt to villus tip) and the
horizontal axis (duodenum through colon) of the intestine. Here, we document that a 12.4-kilobase region of
the mouse villin gene drives high level expression of two
different reporter genes (LacZ and Cre recombinase)
within the entire intestinal epithelium of transgenic
mice. Deletion of a portion of this transgene results in
reduction of ␤-galactosidase activity in restricted domains of the small intestine (duodenum) and large intestine (cecum). In addition, expression is reduced in
the crypt compartment throughout the intestine. Thus,
the global expression pattern of villin in the intestine is
apparently the consequence of an amalgam of distinct
and individual domain-specific control processes. That
is, expression of villin in the duodenum and cecum requires different regulatory sequences than the rest of
the intestine, and the expression of villin in crypts is
regulated by different circuitry than expression of villin
on villus tips.
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Domain-specific Regulation of the Villin Gene
remodeling cis element that facilitates position-independent
expression (11–15). We also demonstrate that this 12.4-kb regulatory fragment can be used to direct expression of Cre recombinase in the large and small intestines of transgenic mice.
Analysis of a deletion derivative of this 12.4-kb regulatory
fragment revealed the presence of one or more multifunctional
enhancers within a 4.2-kb region of the first intron. This enhancer region appears to be at least partially responsible for
mediating LCR-like activity and is necessary for high level
expression of ␤-galactosidase in specific regions of the intestine, including the duodenum and cecum. In addition, the same
4.2-kb fragment is important for transgene expression in nascent and developed crypts along the entire cephalocaudal axis
of the intestine. Together, these observations indicate that the
continuous expression of villin throughout the small and large
intestinal epithelium is due to the sum of individual cis elements that confer transcriptional activity to discrete domains
along the cephalocaudal axis and crypt-villus axis of the
intestine.
EXPERIMENTAL PROCEDURES
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Plasmid and Transgene Construction—The 12.4-kb promoter fragment from the villin gene was isolated from cosmid clones kindly
provided by Dr. Philippe Gros, McGill University, Montreal, Quebec,
Canada. After digestion with EcoRI (6.7 kb 5⬘ of the transcriptional
start site) and XmaI (in exon 1), a 12.4-kb promoter fragment was
isolated and ligated into EcoRI and XmaI sites of the pBluescript II
SK⫹ (pBSII SK⫹) plasmid (Stratagene) to create the plasmid pBSII12.4kbVill. A deletion in intron 1 of this fragment was generated by
digestion of the pBSII-12.4kbVill plasmid with NheI followed by religation of the plasmid to create the plasmid pBSII-⌬NheVill. The LacZ
coding region was removed from the pCH110 vector (CLONTECH) by
digestion with KpnI and EcoRI and ligated into a modified pGEM7
vector (Promega) in which the XbaI site was replaced with a XmaI site.
The LacZ insert was removed from this vector by digestion with XmaI
and ligated into XmaI sites in pBSII-12.4kbVill and pBSII-⌬NheVill
plasmids. This created the pBSII-12.4KbVilLacZ and pBSII-⌬NheVilLacZ plasmids. In both final plasmids, the ␤-galactosidase protein (with
the first 39 amino acids removed) is fused to the first 18 amino acids of
the villin protein.
To make the pBSII-12.4kbVill plasmid a more useful tool for expression of any suitable cDNA (including Cre recombinase), the villin ATG
and first 18 amino acids were removed. A 2.0-kb region of the villin
intron was amplified using a forward primer specific for sequences
flanking a unique BamHI site and a reverse primer specific for the first
11 bp of exon 1. The reverse primer in exon 1 excluded the villin ATG
and contained a linker with restriction sites for SmaI, EcoRI, EcoRV,
and NotI. The fragment was then cloned into the pBSII-12.4kbVill
plasmid at the BamHI and NotI sites, creating the pBSII-12.4kbVill/
⌬ATG plasmid. A Cre recombinase cDNA with a metallothionein
poly(A) signal was isolated as a XhoI/HindIII fragment from the
pBS185 plasmid (Invitrogen) and cloned into pBS (Invitrogen) to create
the pBS-Cre plasmid. A SalI/NotI fragment from the pBSII-12.4kbVill/
⌬ATG plasmid was cloned into the SalI/NotI sites of the pBS-Cre
plasmid to create the pBS-12.4KbVilCre plasmid. All engineered regions were sequenced to ensure fidelity.
Generation of Transgenic Mice—DNA fragments containing villin
regulatory sequences linked to LacZ were prepared by digestion of
pBSII-12.4KbVilLacZ and pBSII-⌬NheVilLacZ plasmids with EcoRI
and XhoI. The 12.4KbVilCre transgene construct was liberated from
the pBS-VillinCre vector sequences using KpnI. After purification,
transgene DNA was injected into the pronuclei of fertilized ova
(C57BL/6J x SJL/J) by the University of Michigan Transgenic Animal
Core. Founders were identified by PCR amplification of tail DNA with
specific primers for each transgene: 5⬘-TGCACTGGCCTAAAGCTCAC-3⬘ and 5⬘-CGACAGTATCGGCCTCAGG-3⬘ for the 12.4KbVilLacZ
transgene; 5⬘-TGGAACTAAAACCCACGG-3⬘ and 5⬘-CTGTTACACAGCCCAGCACT-3⬘ for the ⌬NheVilLacZ transgene; 5⬘-GTGTGGGACAGAGAACAAACCG-3⬘ and 5⬘-TGCGAACCTCATCACTCGTTGC-3⬘ for
the 12.4KbVilCre transgene. Founders were mated to C57BL/6 mice to
obtain F1 progeny for analysis of transgene expression. Embryos were
obtained by mating these F1 mice to C57BL/6 mice. Embryos were
staged by standard criteria (16, 17), and a small piece of tissue (tail,
yolk sac, or liver) was removed for genotyping by PCR using the primers
described above.
Tissue Fixation and Staining for ␤-Galactosidase—Dissected embryos or tissues were fixed on ice for 10 min in 4% paraformaldehyde
containing 1.25 mM EGTA, 2 mM MgCl2 in PBS), then washed twice in
PBS and placed in X-gal (5-bromo-4-chloro-3-indoyl-␤-D-galactoside)
staining solution (1 mg/ml X-gal in N,N-dimethylformamide, 5 mM
K3Fe(CN)6, 5 mM K4Fe(CN)6-3H2O, 2 mM MgCl2, 1.25 mM MgCl2 in
PBS) for 2–10 h at 37 °C, depending on tissue size and embryo stage.
Stained tissues were washed twice in PBS, post-fixed for 10 min at room
temperature with 0.2% glutaraldehyde (1.25 mM EGTA, 2 mM MgCl2 in
PBS), washed again in PBS, and further fixed in 4% paraformaldehyde
for 4 h at 4 °C. Tissues were stored in 70% ethanol after partial dehydration in a graded ethanol series (30%, 50%, and 70%, 5 min each).
Whole mount photographs were taken at this point on a Zeiss inverted
dissecting microscope.
Cryosectioning of Intestinal Tissue—Intestinal tissue was dissected
and washed with 0.02% Triton X-100 in 1⫻ PBS 10 min at 4 °C, fixed 10
min on ice in 4% paraformaldehyde containing 1.25 mM EGTA, 2 mM
MgCl2 in PBS, embedded in OCT (TissueTek), and snap frozen in liquid
nitrogen. Tissue blocks were warmed to ⫺20 °C, and 8-␮m sections
were generated on a Microm HM 500M cryostat, dried for 5 min at
25 °C, and placed in ice-cold 1⫻ PBS. As previously described (4),
sections were stained with X-gal for 2–14 h, dehydrated in a graded
ethanol series, and coverslipped. Slides were analyzed, and digital
images were captured using a Zeiss Axiophot microscope.
␤-Galactosidase Quantitative Solution Assay—For quantitative analysis of ␤-galactosidase activity, protein extracts were made from tissue
of F1 transgenic mice. Tissues were dissected, washed for 10 min with
0.01% Triton X-100 in PBS at 4 °C with agitation, washed with ice-cold
PBS, then homogenized on ice in 6 volumes of homogenization buffer
(PBS containing 40 ␮M phenylmethylsulfonyl fluoride, 3.13 mg/ml benzamidine, 20 ␮g/ml leupeptin, 20 ␮g/ml E64) per milligram of tissue.
Protein concentrations were determined in triplicate by the Lowry
method using the Bio-Rad DC Protein Assay kit. ␤-Galactosidase assays were performed in triplicate with 10 –75 ␮g of total protein tissue
extracts by diluting with reaction buffer (0.1 M sodium phosphate
buffer, pH 7.4, 1 mM MgCl2) containing 0.88 mg/ml O-nitrophenyl-␤-Dgalactopyranoside) to a total volume of 300 ␮l. Samples were incubated
at 37 °C for 30 or 50 min (for kidney and yolk sac extracts), and
reactions were stopped by addition of 0.5 ml of 1 M Na2CO3. Samples
were then read on a spectrophotometer for absorbance at 420 nm.
Southern Blot Analysis of Transgene Copy Number—Genomic DNA
(6 – 8 ␮g) isolated from tails or liver was digested with KpnI and NheI
restriction enzymes and subjected to electrophoresis on a 1.25% agarose
gel. Gels were blotted overnight to Hybond N⫹ membranes (Amersham
Biosciences). Membranes were cross-linked by exposing to 1200 ␮J of
UV radiation in a UV Stratalinker (Stratagene) and prehybridized for
at least 1 h at 60 °C with 10 ml (per 200 cm2 of membrane) of ExpressHyb solution (CLONTECH) supplemented with 150 ␮g/ml singlestranded sheared salmon sperm DNA. The Southern blot probe was
prepared by PCR amplification of a region in intron 1 (between ⫹5258
and ⫹5614, in reference to start site of ⫹1) using specific primers:
5⬘-CCAATGGAGGGTTCTTTTTGTG-3⬘ and 5⬘-AAACTGGCTTCCTATGGGGGTC-3⬘. Labeled probe was denatured at 95 °C for 5 min, quickcooled on ice, diluted to 2 ⫻ 106 cpm/ml with ExpressHyb solution
(CLONTECH) supplemented with 150 ␮g/ml single-stranded sheared
salmon sperm, and added to membranes (10 ml per 200 cm2 of membrane). Hybridization was carried out for 14 –16 h at 60 °C. Band
intensity was assessed on a PhosphorImager (Amersham Biosciences).
RT-PCR Analysis of Transgene mRNA Expression—Total RNA was
isolated from tissues using the TRIzol reagent (Invitrogen), and concentrations were determined using a spectrophotometer. One microgram of total RNA was used for reverse transcriptase (RT)-mediated
synthesis of cDNA using Superscript II RT (Invitrogen) and 1 ng of
random hexamer primers. To assess the RT reaction and quality of
synthesized cDNA, PCR amplification of the hypoxanthine phosphoribosyl transferase cDNA was performed using the specific primers: forward 5⬘-CACAGGACTAGAACACCTGC-3⬘ and reverse 5⬘-GCTGGTGAAAAGGACCTCT-3⬘. Primers for the transgene mRNA were
designed to flank the villin transgenic intron 1 with the forward primer
located in exon 1 (5⬘-GATCTCCCAGGTGGTGGCTGCCTCTTCC-3⬘)
and the backward primer located in the coding region of LacZ (5⬘TCACTCCAACGCAGCACCATCACCG-3⬘). These primers amplify the
same 744-bp fragment from both transgenes. PCR amplification was
performed in a thermocycler for 35 cycles with 30-s cycles of denaturation at 94 °C and 45 s of polymerase extension at 72 °C. Annealing
temperatures were performed as follows: for the first five cycles the
annealing step was programmed at 68 °C for 30 s followed by 10 cycles
Domain-specific Regulation of the Villin Gene
in which the temperature was lowered 1 °C each cycle. The final 20
cycles were performed at an annealing temperature of 58 °C.
Luciferase Assays—Genomic fragments from the promoter region of
the mouse villin gene were cloned into the luciferase reporter vector
pGL2-Basic (Promega) and transfected by calcium phosphate precipitation into the human intestine-like cell line CaCo2 or Rat2 fibroblasts.
Luciferase vectors (5 ␮g) were co-transfected with 1 ␮g of a pSV-␤galactosidase control vector (Promega). Twenty-four hours after transfection, cells were harvested for the luciferase assay (Promega) and
relative activities were normalized to ␤-galactosidase activity.
RESULTS
Transient Transfection of Mouse Villin Gene Sequences—As
shown in Fig. 1A, the villin gene locus contains a 5⬘-untranslated region (UTR) that is separated from the first coding exon
by a 5.6-kb intron. The constructs used in these transfection
experiments are shown in Fig. 1B, whereas luciferase assay
results (normalized by co-transfected ␤-galactosidase activity)
are shown in Fig. 1C.
A construct containing 1236 bp of 5⬘-flanking sequence (⫺1
to ⫺1236 from the transcriptional start site) reliably directs
luciferase expression at high levels in CaCo2 cells, and at 5- to
6-fold lower levels in Rat2 fibroblasts (⫺1236, Fig. 1C). Removal of the 5⬘-most 682 bp significantly reduces transcriptional activity in both cell types (⫺554, Fig. 1, B and C).
Removal of an additional 88 bp (⫺466) results in a further
2-fold reduction of reporter activity in CaCo2 cells. Thus, critical activating sequences appear to lie between ⫺1236 and
⫺554 bp, as well as between ⫺554 and ⫺466 bp. This confirms
and refines earlier studies indicating the presence of activators
between ⫺3.5 kb and ⫺480 bp and between ⫺480 and ⫺100bp
of the mouse villin gene (8).
Addition of the 5.6-kb first intron to the ⫺554 construct
enhances promoter activity nearly 4-fold in CaCo2 cells,
whereas promoter activity is unchanged in Rat2 fibroblasts
(compare ⫺544 to ⫺554/intron, Fig. 1, B and C). Interestingly,
Pinto et al. (8) also tested the effect of adding this intron and
noted a 2-fold enhancement when the intron was linked to 3.5
kb of upstream sequence but no effect (or slight diminution)
when the intron was linked to ⫺480 bp of upstream sequence.
Together, the data from the two studies indicate that elements
within the first intron may interact with or synergize with
5⬘-flanking sequences between ⫺480 and ⫺544 bp.
Deletion of a 4.2-kb region between two NheI sites in intron
1 results in a 2-fold reduction in reporter activity in CaCo2
cells, compared with the construct containing the entire intron
(⫺554/⌬Nhe, Fig. 1, B and C). In contrast, this deletion increases expression in Rat2 fibroblasts by more than 4-fold.
Thus, enhancer sequences within this 4.2-kb region appear to
facilitate expression in intestinal-like cells while repressing
expression in non-intestinal cells.
To further explore the in vivo function of the putative intestinal cell-type-specific enhancer in intron 1 of the villin gene,
two transgene constructs were prepared in which villin regulatory sequences were linked to a ␤-galactosidase reporter.
Because earlier work (8) had shown that a transgene construct
encompassing 9 kb of the mouse villin gene, including the first
intron, was not sufficient to impart position-independent expression, we began with a larger segment of 5⬘ sequence. A
transgene construct, 12.4KbVilLacZ, was prepared containing
6.7 kb of sequence upstream of the transcriptional start site,
the untranslated exon (exon ⫺1), the entire first intron, and the
first 65 base pairs of exon 1, fused in-frame to the bacterial
␤-galactosidase gene (Fig. 2A). A second transgene construct,
⌬NheVilLacZ, was also generated, in which the same 5⬘-flanking sequences were included, but the 4.2-kb region of intron 1
containing the putative intronic enhancer identified in cell
culture studies was deleted (Fig. 2A).
␤-Galactosidase Expression in Transgenic Mice—Eight
founders carrying the 12.4KbVilLacZ transgene and five
founders carrying the ⌬NheVilLacZ transgene were bred to
obtain transgenic lines for analysis. At 4 to 8 weeks of age,
tissues from F1 mice obtained from founders crossed to
C57BL/6 mice were analyzed for ␤-galactosidase expression by
staining with X-gal. Tissues analyzed included small intestine,
colon, kidney, heart, lung, liver, spleen, skeletal muscle, stomach, thymus, thyroid, brain, uterus, ovaries, and testes.
In all eight lines of mice carrying the 12.4KbVilLacZ construct, ␤-galactosidase activity was detectable exclusively in
the small intestine and colon. In most lines, the highest intensity of ␤-galactosidase staining was seen in the duodenum with
progressively diminished staining in the ileum and colon (Fig.
2B), a gradient pattern that was confirmed by a quantitative
␤-galactosidase enzymatic solution assay (data not shown). In
one very highly expressing line (Fig. 2C), ␤-galactosidase activity was similar along the entire horizontal axis of the intestine. The decreasing gradient of villin expression seen in most
lines mimics the expression pattern of villin in the small intestine and colon seen in genetically modified mice in which a
␤-galactosidase cDNA was inserted into the endogenous villin
locus (10). Thus, the 12.4-kb regulatory fragment contains the
necessary cis sequences for the cephalocaudal expression pattern of villin in the intestine. The pyloric border of ␤-galactosidase expression was also appropriately abrupt. No expression
of the reporter was observed in stomach, but adjacent intestinal cells were intensely stained (Fig. 2, G and H).
In six of eight lines, including two lines that contained a
single copy of the transgene (see below), ␤-galactosidase ex-
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FIG. 1. Luciferase assays identify key cis regulatory elements
in the villin gene. A, schematic of the endogenous villin locus. The
locus contains an upstream untranslated exon (UTR) and a 5.6-kb
region corresponding to intron 1. The start codon for protein translation
is located in exon 1. B, constructs used in transient transfection assays.
C, relative luciferase activity for each construct in CaCo2 intestinal
cells and Rat2 fibroblasts. Luciferase activity was assayed for each
construct, and the results were normalized to ␤-galactosidase activity.
Transfections were repeated four to six times for each construct. The
error bars represent standard error of the mean.
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Domain-specific Regulation of the Villin Gene
pression was continuous throughout the small and large intestine, showing no evidence of mottled or variegated expression
(Fig. 2, B and C). In the intestine, position effect variegation is
expected to cause mottled expression in the adult, because
crypts are monoclonal (18). Stochastic silencing of the transgene in some crypts and activation in others would result in an
all or none expression pattern with some crypts being totally
␤-galactosidase-positive and others ␤-galactosidase-negative.
This type of patchy expression pattern was observed in two of
the eight lines studied; both were high copy number lines (see
below). The fact that only 25% of 12.4KbVilLacZ lines showed
evidence of patchy expression while 80% of founders carrying a
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FIG. 2. ␤-Galactosidase activity in the intestines of transgenic
mice carrying 12.4KbVilLacZ or ⌬NheVilLacZ constructs. A,
schematic representation of the two constructs used in these studies.
Both constructs contain 6.7 kb of 5⬘ sequence upstream of the start of
transcription. The 12.4KbVilLacZ construct contains all of intron 1,
whereas the ⌬NheVilLacZ construct contains a 4.2-kb deletion demarcated by two NheI sites. B, 12.4KbVilLacZ transgenic line 12.4-8 expresses ␤-galactosidase in duodenum (d), ileum (i), and colon (c). A
decreasing cephalocaudal gradient of ␤-galactosidase is apparent. C,
12.4KbVilLacZ transgenic line 12.4-1 also expresses LacZ in duodenum
(d), ileum (i), and colon (c), but the higher expression in this line seems
to abolish evidence of the cephalocaudal gradient. D, the ⌬NheVilLacZ
line N2 expresses ␤-galactosidase in small intestine and large intestine
but exhibits greatly diminished expression in proximal duodenum (asterisk). E and F, reduced and variegated expression is observed in
duodenum of ⌬NheVilLacZ-expressing lines N5 and N3, respectively.
Reduced expression is most predominant in the most proximal duodenal region (asterisks). G, a sharp boundary of expression is seen in
12.4KbVilLacZ lines at the pyloric border; intestinal epithelium expresses ␤-galactosidase, whereas no expression is seen in stomach. The
photograph shows the mucosal surface of whole mount tissue at the
stomach/duodenal boundary, stained for ␤-galactosidase. H, a discrete
border of ␤-galactosidase expression is evident in frozen-sectioned pyloric tissue from 12.4KbVilLacZ lines, where ␤-galactosidase-positive
intestinal epithelial cells abut ␤-galactosidase-negative stomach epithelium. I, expression of LacZ is variegated at the pylorus of ⌬NheVilLacZ
line N2, but no expression is observed in stomach. Similar results were
obtained for all three expressing ⌬NheVilLacZ lines.
transgene driven by a 9-kb villin fragment (8) exhibited this
pattern suggests that the extra 3.4 kb of 5⬘-flanking sequence
used here are important for suppression of chromosomal position effects.
The expression of ␤-galactosidase was also analyzed in 4- to
8-week-old ⌬NheVilLacZ transgenic mice. Highly variegated
expression was seen in the intestine in two of five lines
(N3, N5), whereas two lines (N1, N4) showed no expression at
all in any tissue, including the intestine. One line (N2), exhibited non-variegated expression, except in the duodenum just
distal to the stomach (Fig. 2D, asterisk), where expression was
both reduced and variegated. The lack of expression in some
⌬NheVilLacZ lines and variegated expression in others suggests that this transgene is susceptible to position effect silencing. This is in dramatic contrast to the 12.4KbVilLacZ transgenic mice in which variegated expression was only observed in
two out of eight lines. Thus, the deleted 4.2-kb region of intron
1 may contain sequences important for LCR-like activity.
Strikingly, in all three ⌬NheVilLacZ lines that expressed
␤-galactosidase, expression was most severely reduced in the
proximal duodenum (Fig. 2, D–F, asterisks). This indicates
that, in addition to possible LCR-like function, the 4.2-kb region of intron 1 is also required for region-specific expression in
the duodenum. Close examination of the pyloric region in
12.4KbVilLacZ (Fig. 2, G and H) and in ⌬NheVilLacZ mice (Fig.
2I) shows that sequences important for the establishment of
the discrete epithelial border between stomach and intestine
are present in both constructs. Expression was not detected in
the stomach in any of the ⌬NheVilLacZ lines or the 12.4KbVilLacZ lines.
␤-Galactosidase Expression during Development of Transgenic Embryos—␤-Galactosidase activity was evaluated at
10.5, 12.5, and 14.5 dpc in multiple F2 embryos representing
three separate lines for each transgene. In both groups of
transgenic mice, expression was first detectable in the embryonic midgut and hindgut at 12.5 dpc (data not shown). This is
3 days later than that observed in the LacZ knock-in model (4)
and indicates that some critical sequences necessary for transgene activation at the proper time are missing from both constructs. For 12.4KbVilLacZ transgenic mice, embryonic expression was restricted to the midgut and hindgut with expression
decreasing distally along the axis of the hindgut (Fig. 3A),
identical to the embryonic expression pattern seen in the Villin
LacZ knock-in model (4). Interestingly, the further developmentally programmed postnatal up-regulation of ␤-galactosidase (concomitant with endogenous villin up-regulation) apparently causes this gradient to disappear in adults of this
particular line; in other transgenic lines the gradient is still
visible in the adult (Fig. 2, B versus C).
In ⌬Nhe embryos, however, expression was consistently diminished in the presumptive proximal duodenal area just distal to the stomach (Fig. 3B, asterisk). Thus, in both embryos
and adults carrying the ⌬Nhe transgene, ␤-galactosidase expression is reduced in the duodenum.
Also apparent in Fig. 3B is the loss of ␤-galactosidase staining in the presumptive cecum of ⌬Nhe mice. Like the reduction
in duodenal staining, this is a reproducible finding, seen in
three independent ⌬Nhe lines. However, unlike the duodenal
pattern, where reduced staining persisted into adulthood, cecal
expression was partially regained in adults (data not shown).
Although the endogenous villin gene is also expressed at
high levels in the yolk sac and in the proximal tubules of the
kidney (4, 6, 7), neither the 12.4KbVilLacZ nor the ⌬NheVilLacZ transgene was expressed in these tissues. This was determined by ␤-galactosidase solution assay and by RT-PCR
analysis (data not shown). Thus, the 12.4-kb regulatory frag-
Domain-specific Regulation of the Villin Gene
ment appears to lack cis elements necessary for both kidney
and yolk sac expression. Furthermore, no ␤-galactosidase activity was detected (above background staining) in any other
developing tissue of 12.4KbVilLacZ embryos at any of these
stages. F2 embryos from ⌬NheVilLacZ transgenic mice, however, demonstrated ectopic reporter expression in tissues such
as the developing brain and limbs (data not shown). This is in
accord with the transient transfection data indicating that
sequences within the 4.2-kb NheI fragment are required for
suppression of expression in Rat2 fibroblasts.
Copy Number-dependent Expression of the 12.4KbVilLacZ
Transgene—Judging from the continuous expression pattern
and lack of variegation in the majority (75%) of the 12.4KbVilLacZ founder lines and the contrasting highly variegated expression seen in most ⌬Nhe lines, we predicted that the mouse
villin gene contains an LCR-like activity located in intron 1. To
test this prediction, we examined copy number-dependent expression, a hallmark of LCR activity. ␤-Galactosidase expression levels were determined by solution assay in extracts of
whole intestine from each line. Genomic DNA from the six
non-variegated 12.4KbVilLacZ lines was loaded onto an agarose gel, left to right, in the rank order of ␤-galactosidase
activity (Fig. 4B). DNA from the two variegated lines (12.4-6
and 12.4-7), both of which expressed ␤-galactosidase at lower
levels than the continuous lines, was loaded in the last two
FIG. 4. Copy number determination for transgenic lines. Transgene copy number was determined for each transgenic founder line by
Southern blotting. A, a schematic of a portion of the endogenous villin
locus (top line), the 12.4KbVilLacZ transgene (middle line), and the
⌬NheVilLacZ transgene (lower line). The transgene copy number for
each transgenic line was determined by digestion of F1 genomic DNA
with NheI and KpnI. Blots were hybridized with a probe (shown beneath
the ⌬NheVilLacZ construct) that recognizes a 1.0-kb NheI/KpnI fragment from the endogenous villin locus and a 0.5-kb NheI/KpnI fragment
from both the 12.4KbVilLacZ and ⌬NheVilLacZ transgenes. B, Southern blots. ␤-Galactosidase activity was determined by solution assay.
DNA from continuously expressing lines was loaded in the order of their
␤-galactosidase activity (left to right, lowest to highest). DNA from the
two variegated 12.4KbVilLacZ lines, which express ␤-galactosidase at
lower levels than the continuously staining lines, was loaded in the last
two lanes (marked PEV). Copy number (reported below each lane) was
determined by densitometric analysis of the blots after normalizing the
band from the endogenous villin locus to two copies for each lane. Lines
12.4-5 and 12.4-3 were single copy lines. Two of five ⌬NheVilLacZ lines
(N1 and N4) had no detectable ␤-galactosidase expression (NE). Two
lines (N3 and N5) exhibited mottled or patchy expression throughout
the intestine, indicative of position effect variegation (PEV); these two
lines had similar ␤-galactosidase activity. Finally, one line, N2, exhibited continuous expression at a high level, although expression was
variegated in the duodenum. C, correlation between copy number and
␤-galactosidase activity in 12.4KbVilLacZ transgenic lines, as measured in tissue extracts in duodenum (duod.), ileum (il.), and colon (col.)
(average r2 ⫽ 0.723).
lanes (marked PEV). Copy numbers were determined by
scanning the Southern blot of this gel (Fig. 4B, left) on a
PhosphorImager, and results were plotted against the level of
␤-galactosidase expression for all six continuously expressing
12.4KbVilLacZ lines (Fig. 4C). A clear correlation between copy
number and expression was observed (r2 ⫽ 0.723).
Copy number dependence was similarly assessed in
⌬NheVilLacZ lines. As shown in the right panel of Fig. 4B, one
of the two non-expressing lines (NE) contained a high transgene copy number of 32, whereas the other contained a relatively low copy number of 6. The two variegated lines (PEV)
expressed ␤-galactosidase at similar levels, but contained different copy numbers (5 and 10 copies). Furthermore, two of five
⌬NheVilLacZ lines failed to express at all, indicative of 100%
silencing in these chromatin environments. Thus, variegated
expression or failure to express was observed in the majority of
⌬NheVilLacZ lines (4 of 5). These data suggest that the ⌬Nhe
deletion in intron 1 removes sequences that suppress position
effect silencing.
Heterogeneous Staining on Villi and Reduced Expression of
␤-Galactosidase in the Crypt Compartment of ⌬NheVilLacZ
Mice—To assess whether these transgenes drive expression
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FIG. 3. Embryonic expression of ␤-galactosidase in transgenic
mice at 14.5 dpc. A, expression in 12.4KbVilLacZ lines is observed
throughout the length of the presumptive intestine, beginning at the
pylorus, and continuing throughout the midgut, cecum (arrow), and
hindgut where expression decreases caudally. B, expression of ␤-galactosidase at 14.5 dpc in ⌬NheVilLacZ lines (line N2 is shown) reveals
reduced expression in presumptive proximal duodenum (double-headed
arrow with asterisk) and no expression in the developing cecum (arrow).
33279
33280
Domain-specific Regulation of the Villin Gene
throughout the vertical axis of the intestine, we examined
␤-galactosidase staining in cryosections of intestine from transgenic mice. In mice carrying the 12.4KbVilLacZ construct, continuous expression of LacZ was observed in both crypt and tip
compartments (Fig. 5, A–C) of the small and large intestines,
with slightly lower expression in crypts, in concordance with
the reported expression gradient of villin along the crypt-villus
tip axis (6).
In lines carrying the ⌬NheVilLacZ transgene two differences
in epithelial staining were observed. First, the pattern of staining on the villi was heterogeneous, with neighboring cells exhibiting quite different levels of ␤-galactosidase staining (Fig.
5, D–F). This type of expression pattern has been seen earlier
for the endogenous L-FABP gene (Fabpl) (19) and for ILBP
transgenes (20). A second interesting difference seen only in
⌬Nhe mice was that ␤-galactosidase staining was severely reduced in crypts along the entire cephalocaudal axis of the
intestine (Fig. 5, D–F). To confirm that reduced crypt staining
was not simply due to an overall reduction in staining in the
epithelium to levels that leave crypt cells below the limits of
detection, we compared staining in N2 (Fig. 5E) and N5 mice
(Fig. 5F) with that seen in 12.4KbVilLacZ line 12.4-3, a single
copy line. Although the 12.4-3 line exhibits equal or lower
intensity of ␤-galactosidase staining on villi than either N2 or
N5, its crypts are still clearly ␤-galactosidase-positive (Fig. 5C),
whereas those of both ⌬Nhe lines are strikingly negative. Thus,
the data indicate that the 4.2-kb NheI fragment contains a
crypt-specific enhancer.
To determine when, during crypt development, cells with
reduced ␤-galactosidase are first observed, we examined the
expression of ␤-galactosidase in postnatal day 0 (p0), postnatal
day 7 (p7), and adult (⬎4-week-old) intestines. In newborn
mice, before the emergence of crypts, all villus and intervillus
cells of the small intestine are strongly ␤-galactosidase-positive
in both ⌬NheVilLacZ and 12.4KbVilLacZ newborn mice (Fig. 6,
A and B). However, in p7 ⌬Nhe mice, the first few invaginating
crypt cells of the small intestine show reduced or absent ␤-galactosidase staining (Fig. 6C), whereas invaginating crypts of
12.4KbVilLacZ mice are strongly ␤-galactosidase-positive (Fig.
6D). In adult ⌬Nhe mice, a clear boundary is visible between
the ␤-galactosidase-positive villus tip cells and the adjacent
crypt compartment where ␤-galactosidase staining is reduced
or absent (Fig. 6E, arrow). In contrast, all crypt cells of the
12.4KbVilLacZ lines are ␤-galactosidase-positive (Fig. 6F). The
same pattern is also seen in the large intestine: ␤-galactosidase
expression in all cells at all stages in 12.4KbVilLacZ mice (Fig.
6, G and H); reduction in expression specifically in the crypt
compartment of adult, but not newborn ⌬NheVilLacZ mice
(Fig. 6, I and J). These data indicate that a different genetic
program for the regulation of villin expression is present in
cells of the crypts and cells of the villi. The crypt program, but
not the villus program, requires cis elements within the 4.2-kb
deleted region.
Spatial Conservation of cis Elements in the Proximal Promoter and Intron 1 Region among Mice and Humans—The
nucleotide sequence of the human villin gene was obtained
from the NCBI data base of high throughput human genomic
sequence. Intron 1 of the human villin gene was cloned from
total genomic DNA, sequenced, and found to be identical with
sequence from the NCBI data base. The proximal promoter
region (obtained by PCR of BAC clone RPCI-11-378A13, GenBankTM accession number AC021016, kindly provided by the
BACPAC Resource Center at the Children’s Hospital Oakland
Research Institute, Oakland, CA) of the human villin gene was
re-sequenced for confirmation. The 12.4-kb regulatory fragment from the mouse villin gene was sequenced in its entirety.
An alignment was performed between human (⫺8.6 kb through
intron 1) and mouse (⫺6.7 kb through intron 1) sequence using
the Pustell DNA matrix algorithm on MacVector software. To
facilitate the alignment, Alu sequences were identified and
removed from the human sequence. Fig. 7A illustrates the
considerable sequence conservation in multiple short (20 – 400
bp) elements of the promoter and first intron. The straight diagonal line indicates that the spacing of conserved elements has
also been maintained. Conserved elements in intron 1 lie largely
within the region deleted in ⌬NheVilLacZ transgenic mice.
Use of the Villin 12.4-kb Regulatory Fragment to Drive Cre
Expression in the Intestine of Transgenic Mice—The reproduc-
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FIG. 5. ⌬NheVilLacZ transgenic lines lack cis elements necessary for expression of ␤-galactosidase in intestinal crypts. A,
duodenum of 4-week-old 12.4KbVilLacZ line 12.4-1 (and all other 12.4KbVilLacZ lines analyzed) shows expression in cells of villus tips and crypts,
with slightly lower levels of expression seen in crypts, in concordance with previous reports of a villin expression gradient along the crypt-villus
tip axis (6). B, jejunum of 12.4KbVilLacZ line 12.4-1, showing crypt staining. C, crypts are also stained in line 12.4-3, a single copy line that shows
weaker ␤-galactosidase staining on villus tips, but still retains staining in crypt cells. D, the duodenum of a 4-week-old ⌬NheVilLacZ pup from line
N2 exhibits heterogeneous ␤-galactosidase expression in cells of the villus tips, and severely reduced expression in crypts of distal duodenal
epithelium. The inset shows more proximal duodenum, where villus tip staining is further reduced on tips and absent in crypts. E, expression of
␤-galactosidase in the jejunum of ⌬NheVilLacZ (line N2) pups. Note reduced crypt staining (arrow points to base of crypt). F, crypt staining is also
reduced in the small intestine of ⌬NheVilLacZ line N5 (arrow points to base of crypt).
Domain-specific Regulation of the Villin Gene
33281
ible and high level expression of ␤-galactosidase in 12.4KbVilLacZ mice indicated that this regulatory fragment could be a
valuable tool for the generation of conditional genetic mutations. Therefore, we linked the 12.4-kb region to Cre recombinase, generated transgenic founders, and mated these mice
(12.4KbVilCre) to the Rosa26 conditional reporter strain, R26R
(21). Cre-mediated recombination at loxP sites surrounding a
neomycin gene at the Rosa26 locus results in excision of this
gene (and its linked polyadenylation sites) and subsequent
expression of ␤-galactosidase.
Intestines from F1 transgenic pups obtained from matings to
R26R mice were stained with ␤-galactosidase to assess the
efficacy and specificity of Cre expression. Continuous and high
level ␤-galactosidase expression was seen in the small and
large intestines of these pups in five of the seven lines already
examined (Fig. 8, A and B). Two other lines show patchy or
mottled expression indicative of Cre expression in a subset of
crypts (Fig. 8C). Although the continuous expression of Cre in
all crypts is required to generate a complete conditional gene
modification in the intestine, patchy expression of Cre recombinase will also be of value. Such a line will allow examination
of modified and unmodified crypts and villi in the same
intestine.
Interestingly, one of the seven Cre lines also exhibits Cre
expression in the kidney (data not shown). In an earlier study,
one of four founders carrying a 9-kb villin transgene exhibited
reporter gene activity in the kidney (8). It is likely, therefore,
that these villin regulatory sequences are able to drive expression in the kidney in the context of certain chromosomal
environments.
DISCUSSION
In this study, we describe a 12.4-kb fragment from the mouse
villin gene that reliably and reproducibly drives reporter gene
expression in the small and large intestines in a pattern that
closely resembles that of the endogenous mouse gene. Further-
FIG. 7. Identification of phylogenetically conserved elements
within intron 1 and the proximal promoter region of the human
and mouse villin genes. The entire 12.4-kb murine villin fragment
was sequenced, and intron and upstream sequences were aligned with
human intron sequences (after removal of Alu repeats). A, the diagonal
line generated by this comparison indicates the presence of scattered
short homologous elements located in a similar spatial arrangement
from ⫺200 bp through the first 3.3 kb of intron 1. B, a schematic
diagram of the mouse and human villin genomic loci is shown beneath
the homology matrix and aligned vertically with the matrix. The triangles on the human locus demarcate the positions of removed Alu sequences. The gray-shaded box indicates the region containing the majority of homology in the matrix. The 3⬘ regions of both genomic loci
indicated here, including the 3⬘-most two kilobases of mouse intron 1,
and the 3⬘-most 500 bp of human intron 1, show little or no homology.
Likewise, homology is reduced upstream of position ⫺206 (the upstream non-translated exon, UTR, begins at position ⫹1 for both sequences), although several short homologous elements have been found
between ⫺206 and ⫺800 (data not shown). It is clear that the NheI
deletion (shown as a bold line labeled ⌬Nhe) removes several homologous elements.
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FIG. 6. ␤-Galactosidase activity is reduced in emerging crypt cells during the formation of intestinal crypts 1 week after birth. A,
C, E, I, and J: ⌬NheVilLacZ lines. B, D, F, G, and H: 12.4KbVilLacZ lines. A and B, at birth (p0), the intervillus region is continuously stained in
small intestine of both ⌬Nhe line N2 (A) and in line 12.4-1 (B). Seven days later (p7), as initial crypt cells emerge, the nascent cells are
␤-galactosidase-negative in ⌬Nhe line N2 (C) and positive in line 12.4-1 (D). Arrows in C point to the boundary between ␤-galactosidase expression
and non-expressing cell populations. In adults, crypts of ⌬Nhe line N5 show severely reduced ␤-galactosidase expression (E), whereas those of line
12.4-1 exhibit robust staining in crypts (F). A similar pattern is noted in the colon (G–J). Pre-crypt cells of the colon of newborn (G) and adult (H)
12.4KbVilLacZ mice (line 12.4-1) are positive for ␤-galactosidase. Pre-crypt cells of the colon of newborn ⌬NheVilLacZ mice are also positive for
␤-galactosidase (I), but adults show reduced staining in crypts (J) with a distinct boundary between expressing and non-expressing cells (arrow).
33282
Domain-specific Regulation of the Villin Gene
more, we have used this regulatory fragment to generate a line
of mice in which Cre recombinase is expressed efficiently in all
cells of the intestine. We expect that this 12.4-kb regulatory
fragment will provide an important tool for a variety of future
studies on gut development and function.
The early (9.0 dpc) activation of reporter gene transcription
is one feature of endogenous villin gene expression that was not
recapitulated by the 12.4-kb villin regulatory fragment. Despite the moderately large size of this genomic fragment, it was
unable to direct expression earlier than 12.5 dpc, indicating
that villin cis-regulatory sequences necessary for early embryonic expression are missing. However, we note that the transgene is nevertheless activated relatively early in gut development; activation precedes (by 2 days) the major morphological
remodeling of the intestinal epithelium that occurs at 14 –15
dpc. Thus, this regulatory fragment can drive transgene expression during early stages of intestinal organogenesis.
The 12.4-kb regulatory fragment is largely specific for the
intestinal epithelium. One line of 12.4KbVilCre mice (but none
of the LacZ lines) exhibited Cre expression in the kidney. Moreover, none of the LacZ lines express the transgene in the yolk
sac (Cre lines remain to be tested for yolk sac expression). The
embryonic distal stomach (prior to 16.5 dpc) is another tissue
that normally expresses villin, and this region is clearly stained
in embryos in which the ␤-galactosidase cDNA is inserted into
the endogenous villin gene locus (4). However, distal stomach
did not exhibit detectable expression of the ␤-galactosidase
reporter in any of the eight 12.4KbVilLacZ lines studied and
did not exhibit Cre expression in any of seven 12.4KbVilCre
lines thus far analyzed. Thus, we conclude that the cis sequences responsible for expression of villin in the stomach are
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FIG. 8. A, ␤-galactosidase expression due to Cre recombinase activity
in 4-week-old mice produced by mating homozygous Rosa26 conditional
reporter mice to hemizygous transgenic founders carrying the
12.4KbVilCre construct. 12.4KbVilCre transgenic intestines exhibit
continuous expression of ␤-galactosidase: d, duodenum in the pyloric
region, showing the stomach/duodenum border, where a sharp boundary of expression is seen, with no expression in the stomach (arrow);
j, jejunum; i, ileum; c, colon. B, in all regions of the intestine, Cre was
expressed continuously as evidenced by non-variegated ␤-galactosidase staining; levels of Cre were sufficient to allow recombination in
every cell. This has been the case for five of seven lines thus far
examined; two additional lines exhibit mosaic expression (C), as
indicated by wholly positive crypts surrounded by wholly negative
ones. Villus architecture is also normal, indicating no apparent deleterious effects due to the expression of Cre recombinase in the
epithelium to this point in development. No ␤-galactosidase staining
was observed in non-transgenic littermates (not shown). One of the
seven lines showed staining in the kidney (data not shown).
different from those needed for intestinal expression and are
not contained within the 12.4-kb regulatory fragment tested.
The data presented here indicate that a multifunctional enhancer (or multiple enhancers) within intron 1 of villin directs
expression in discrete areas of the horizontal (duodenum and
cecum) and vertical (crypt) axis of the intestine. Thus, different
regulatory circuits control villin expression in the duodenum
versus the rest of the small intestine, in the cecum versus the
rest of the large intestine, and in crypts versus villus tips. This
type of region-specific control is reminiscent of the regulation of
the Drosophila even-skipped gene, in which several small discrete enhancers control expression of eve in each of the seven
different parasegment stripes that comprise its expression domain (22). Region-specific control directed by discrete regulatory elements is also seen in the adenosine deaminase gene
(23–24). This ubiquitously expressed gene is controlled in specific tissues by distinct enhancers; T cell-, placenta-, and duodenum-specific enhancers have been described.
The results presented here differ in two aspects from the
results of a previous transgenic study in which villin regulatory
sequences were used to drive ␤-galactosidase (8). First, Pinto et
al. reported that deletion of the entire first intron from the 9-kb
genomic fragment (their pA3 transgene) resulted in loss of
colon expression in three expressing founders (one founder
showed no expression at all). In our study, deletion of the 4.2-kb
fragment from intron 1, which removes most, but not all of this
intron, did not compromise colon expression, although it did
increase position effects in accord with the Pinto et al. results
(8). Together, these data suggest that elements important for
colon-specific expression of villin may lie within intron 1 but
outside of the NheI deletion region. Second, the pA3 transgenic
founders, which lack intron 1, were reported to express ␤-galactosidase in the entire intestinal epithelium, including the
crypt compartment, although these data were not shown. This
result is hard to reconcile with our findings that deletion of a
portion of this intron leads to reduced expression in the crypts.
This result is not secondary to position effects in our mice,
because we observed the same phenotype in multiple offspring
of two independently bred lines. Although the different genetic
backgrounds of the transgenic lines produced in the two studies
could play some modulatory role, the reason for this disparity
in results is presently not clear.
Several attributes of the 12.4-kb regulatory fragment suggest that it contains LCR-like activity. First, expression of
␤-galactosidase was observed in all founders isolated, including
two single-copy number lines. Second, expression was continuous in all cells of six out of eight founders, with no evidence of
mottled or patchy expression. In the adult intestine, position
effect variegation normally presents itself in the form of a
mottled expression pattern. This is a direct reflection of the fact
that crypts are monoclonal and that stochastic silencing of
transgene expression occurs in some proportion of crypts (18).
Third, expression was copy number-dependent for all six low
copy number lines carrying the 12.4KbVilLacZ construct. Finally, specific regions of the villin 12.4-kb genomic fragment
can be implicated in LCR-like function. The region between
⫺6.7 and ⫺3.5 kb appears to play a role, because a 9-kb region
of the villin gene (from ⫺3.5 kb through the first intron)
showed patchy or heterogeneous expression in 80% of founders
studied (8), whereas addition of the three extra kilobases on our
construct reduced the proportion of heterogeneously expressing
lines to 25%. In addition, deletion of the 4.2-kb region of intron
1 resulted in the apparent loss of copy number dependence and
greater variability in transgene expression, indicating that this
region also harbors sequences with LCR-like activity. It is
possible that the upstream and intronic regions act together to
Domain-specific Regulation of the Villin Gene
regulatory activities within the first intron of the villin gene,
and the recognition of significant evolutionary conservation
within this region, will aid in the future investigation of the cis
and trans factors involved in a number of spatial patterning
pathways important in intestinal organogenesis.
Acknowledgments—B. B. M. is grateful for support from the Training
Program in Organogenesis. D. L. G. acknowledges support from the
Roger McDermitt Research Fund, and from a Cancer Innovation Grant,
both from the University of Michigan Comprehensive Cancer Center.
We are grateful to Christine Babcock for help with animal breeding and
genotyping. We also thank the University of Michigan Transgenic Animal Core for transgenic mouse production and Dr. Stuart Orkin and
members of his laboratory for assistance with 12.4KbVilCre construct
assembly.
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ensure position independence, similar to what has been observed in the ␤ globin locus (26).
There is one other description of an intestinal LCR. A duodenum-specific enhancer was recently described in the second
intron of the human adenosine deaminase gene, but a promoter
fragment, including this intron, was unable to drive copy numberdependent expression of a chloramphenicol acetyltransferase
reporter in transgenic mice (24). Interestingly, a T-cell-specific
enhancer of the human adenosine deaminase gene, located in
the first intron, was able to direct copy number-dependent
expression of chloramphenicol acetyltransferase in the thymus
and in the duodenum when in the presence of a genomic fragment containing the duodenum-specific enhancer (24).
The villin crypt element identified here appears to be necessary for efficient activation of reporter gene expression in the
crypt compartment of the entire intestine. Indeed, as this compartment emerges from the intervillus epithelium in the week
after birth, an immediate and discrete compartment boundary,
as measured by a change in ␤-galactosidase staining in the
epithelium of ⌬NheVilLacZ mice, is visible. It is also of interest
that, in differentiated villus tip cells, the 4.2-kb fragment is
required for region-specific expression (duodenum and cecum),
whereas its activity in crypt cells is not region-specific but
similar throughout the large and small intestines. Thus, a
regulatory circuitry, to which the villin gene responds, may be
held in common by crypts along the entire intestinal tract.
Alternatively, there may be more than one genetic program for
establishment of villin expression in the crypt compartment
across the length of the intestine, and multiple cis elements
within the 4.2-kb NheI fragment may exist that are responsive
to each of these programs. A more precise identification of one
or more specific cis response elements for such regulatory networks will facilitate the elucidation of the trans factors involved in this pathway. For future studies, it will be important
to determine whether this 4.2-kb fragment (or subfragments
thereof) is both necessary and sufficient to confer crypt-specific
expression on a heterologous transgene.
In summary, we have defined a 12.4-kb genomic region of the
villin gene that is capable of driving transgene expression in all
cells of the crypt and tip and along the entire large and small
intestines. This genomic region also contains LCR-like sequences that suppress position effect variegation. We have
already used this regulatory fragment to derive mice that express Cre recombinase efficiently in the intestinal compartment, a tool that will be extremely valuable for the creation of
conditional genetic mutations. Our identification of multiple
33283
GENES: STRUCTURE AND
REGULATION:
cis Elements of the Villin Gene Control
Expression in Restricted Domains of the
Vertical (Crypt) and Horizontal
(Duodenum, Cecum) Axes of the Intestine
Blair B. Madison, Laura Dunbar, Xiaotan T.
Qiao, Katherine Braunstein, Evan Braunstein
and Deborah L. Gumucio
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This article cites 25 references, 13 of which can be accessed free at
http://www.jbc.org/content/277/36/33275.full.html#ref-list-1
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J. Biol. Chem. 2002, 277:33275-33283.
doi: 10.1074/jbc.M204935200 originally published online June 13, 2002