Divisions of 1 Developmental Biology and 2 Pediatric Gastroenterology, Department of Pediatrics, University of Cincinnati College of Medicine and Children's Hospital Research Foundation, Cincinnati, Ohio 45229
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ABSTRACT |
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Adenosine deaminase (ADA) is expressed at high levels in the epithelium of proximal small intestine. Transgenic mice were used to characterize the regulatory region governing this activation. A duodenum-specific enhancer is located in intron 2 of the human ADA gene at the central site among a cluster of seven DNase I-hypersensitive sites present in duodenal DNA. Flanking DNA, including the remaining hypersensitive sites, is required for consistent high-level enhancer function. The enhancer activates expression in a pattern identical to endogenous ADA along both the anterior-posterior axis of the small intestine and the crypt-villus differentiation axis of the intestinal epithelium. Timing of activation by the central enhancer mimics endogenous mouse ADA activation, occurring at 2-3 wk of age. However, two upstream DNA segments, one proximal and one distal, collaborate to change enhancer activation to a perinatal time point. Studies with duodenal nuclear extracts identified five distinct DNase I footprints within the enhancer. Protected regions encompass six putative binding sites for the transcription factor PDX-1, as well as proposed CDX, hepatocyte nuclear factor-4, and GATA-type sites.
transgenic mice; PDX-1; temporal regulation; crypt-villus axis; cephalocaudal axis; DNase I footprinting
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INTRODUCTION |
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GENE EXPRESSION IN THE EPITHELIUM of the mammalian small intestine can be envisioned as occurring along three distinct axes (20). The cephalocaudal or anterior-posterior (AP) axis is a linear axis extending along the coiled length of the intestine from the proximal duodenum to the distal ileum. The crypt-villus (CV) axis is a bidirectional axis extending upward and downward from the stem cells anchored in the crypt epithelium. This is the axis of cellular migration and differentiation within the CV structure of the mature intestine's epithelium, which is constantly renewing from the stem cells throughout life. Four distinct cell types are derived from the stem cells. Absorptive enterocytes, mucous-producing goblet cells, and enteroendocrine cells all migrate "upward" along the CV axis out of the crypt onto the villus, whereas Paneth cells migrate "downward" toward the base of the crypt. Each of the four cell lineages completes its differentiation program during this orderly migration that lasts a few days. The third axis considered here is the developmental time axis, along which the structure of the epithelium forms and its function develops. The array of genes expressed in any intestinal epithelial cell (as well as the level of expression) are determined not only by that cell's particular lineage but also by where the cell is along each of the three axes. The conserved pattern of expression for various genes encoding metabolic enzymes and other functional proteins in the cells of the intestinal epithelium show striking similarities, as well as striking differences, along these three axes (50).
For example, a number of different genes are all known to be activated to high levels of expression in rodent small intestinal enterocytes at a similar time along the developmental time axis, the period of the suckling-weanling transition. However, these same genes have significantly different profiles of expression along the AP axis. In a similar manner, a number of genes are all known to be activated at the CV junction during enterocyte migration/differentiation but have very different expression profiles along the AP axis or the developmental time axis (50). The mechanisms and factors regulating these patterns of gene expression and their differences are poorly understood at the molecular level. Detailed analysis of the regulatory programs for a number of these genes is required to understand what is held in common and what the differences are in their control mechanisms that result in the observed patterns. Understanding of both enhancers and repressors of gene activation in specific regions of the intestine may lead to therapeutic strategies for intestinal disease or gut failure, e.g., short bowel syndrome.
Adenosine deaminase (ADA), an enzyme of purine catabolism and salvage, has a highly defined pattern of expression in the mammalian small intestine (15). Along the AP axis, the highest levels of ADA expression are observed in the epithelium of the proximal duodenum of mice and humans, with much lower levels present in the more distal parts of the epithelium (14, 59). Enterocytes of the duodenal epithelium first demonstrate activation of ADA expression to high levels at the CV junction, with expression extending along the CV axis to the villus tip (14, 15, 59). In mice, a significant activation of ADA in duodenal epithelium occurs at 2-3 wk, at the time of the suckling-weanling transition (15, 34). This transition may be linked to the final maturation of the epithelium (39). The equivalent maturation process is thought to occur developmentally much earlier in humans, between 8 and 12 wk of gestation (13). Studies describing detailed characterizations of fetal and newborn expression of ADA in humans have not been reported. However, ADA is known to be expressed at significant levels in newborn human duodenum, so activation of duodenal ADA expression probably occurs in utero.
Previously, a fragment of the human ADA gene was identified that is able to recapitulate most of the observed pattern of ADA expression in transgenic mice (15). This 13-kb intragenic fragment was linked in a transgene construction with the human ADA gene promoter and the chloramphenicol acetyltransferase (CAT) reporter gene. From this transgene, CAT expression was consistently activated to high levels specifically in the duodenal epithelium, with very low expression along the remainder of the AP axis of the small intestine, as well as in other tissues. Along the CV axis of the duodenal epithelium, CAT expression was not observed in the crypts but first appeared at the CV junction and extended along the length of the villi. A developmental activation of CAT expression was observed along the temporal axis at or near the time of birth. This is significantly earlier than the activation of the endogenous mouse ADA gene, which occurs 2-3 wk later at the time of weaning. The regulatory region responsible for duodenum-specific activation was further mapped to a 3.4-kb region near intron 2 that was found to contain seven DNase I-hypersensitive sites (HS) in the DNA of duodenal nuclei. These sites were absent from the nuclear DNA of several other tissues, including distal small intestine.
In this study, a duodenum-specific enhancer was mapped to a region of ~300 bp coincident with HS-D, the central HS among the previously identified array. This enhancer is capable of independently specifying a pattern of expression that is almost identical to that of the endogenous mouse ADA gene along all three axes of the small intestine, including developmental activation at or near the time of weaning. Two regions upstream of HS-D, including one associated with HS-C, were found to be required for the precocious temporal activation of the enhancer described previously (15). Footprinting of the enhancer region of ~300 bp revealed five regions protected by proteins in extracts from duodenal nuclei. Potential binding sites for transcription factors were identified within these footprints, and their possible significance is discussed here.
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EXPERIMENTAL PROCEDURES |
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Plasmid constructions.
Construction of the plasmids p5'acba, p5'acba L117, and p5'acba
L1173 has been described previously (15). The plasmid
p5'acba L117 was cut with Sma I, a 4,708-bp fragment was
isolated, and Bcl I linkers (New England BioLabs) were added
and digested with Bcl I. The resulting linkered fragment was
ligated into the plasmid p5'acba, previously cut with BamH
I, to produce the plasmid p5'acba L117
4. The plasmid p5'acba
L117
3 was cut with HinP1 I, and a 4,229-bp fragment was
isolated and ligated into Acc I-cut pGEM4z plasmid. The
resulting plasmid was then cut with Sph I, and the 2,566-bp
fragment from this digest was cloned into the Sph I site of
p5'acba to produce the plasmid p5'acba L117
5. A 3,451-bp
Sph I fragment was isolated from p5'acba L117
3 and
subcloned into the Sph I site of pALTER-1 (Promega) to
produce pALT Sph3.4(
). Site-directed mutagenensis was performed on
pALT Sph3.4(
) as directed by Promega (Altered Sites Protocol) to
change the residue corresponding to human ADA gene residue #20526
(GenBank accession no. M13792) from a C to a G, thereby creating a
Sal I site between HS-B and HS-C. A Sal I
fragment of 1,753 bp was removed from this mutated plasmid and cloned
into the Sal I site in p5'acba to create p5'acba L117
6.
The plasmid pALT Sph3.4(
) was also separately subjected to
site-directed mutagenesis to create BsiW I sites flanking
HS-C of ADA. The affected residues are those corresponding to human ADA
gene residues #20664 (A to G), #20667 (A to C), #21041 (G to T), and
#21043 (G to C). The mutated plasmid, pALT 3.4(
)Cmut, was digested
with BsiW I, and the resulting 8,755-bp fragment was ligated
to form pALT 3.4
C. In an analogous manner, site-directed mutagenesis
in pALT Sph3.4(
) of residues corresponding to human ADA gene residues
#21041 (G to T), #21043 (G to C), #21360 (G to T), and #21362 (G to C)
created BsiW I sites flanking HS-D. The mutated plasmid,
pALT 3.4(
)Dmut, was digested with BsiW I, and the
resulting 8,812-bp fragment was ligated to produce pALT 3.4(
)
D. An
11,290-bp Sph I fragment from p5'acba L117 was isolated and
inserted into the Sph I site of pALTER-1 to create pALT
Sph11(
). pALT Sph11(
) was then digested with Bbs I, and
an 8,244-bp fragment was isolated and ligated to a Bbs I
partial digest fragment from either pALT 3.4(
)
C (a 4,745-bp
fragment) or pALT 3.4(
)
D (a 4,802-bp fragment). The resulting
plasmids were then digested with Afl III, and 9,146-bp
(
C) or 9,203-bp (
D) fragments were isolated and ligated to a
7,448-bp Afl III fragment from pALT Sph11(
) to create pALT
Sph11(
)
C and pALT Sph11(
)
D. These plasmids were then each
digested with Sph I, and 10,914-bp (
C) or 10,971-bp
(
D) fragments were isolated and ligated to a 9,876-bp Sph
I fragment isolated from p5'acba L117. Plasmid clones with both
orientations were obtained and named p5'acba L117
C(+), p5'acba L117
C(
), p5'acba L117
D(+), and p5'acba L117
D(
). The
plasmid pADACAT 211 (16) was digested with Sal
I, filled with Klenow and dNTPs, and ligated to eliminate the
Sal I site and replace it with a Pvu I site. The
resulting plasmid was then digested with Hind III, and the
ends were filled with Klenow and dNTPs. This linearized plasmid was
digested with Nco I, and a 913-bp fragment was isolated and
ligated to a 4,750-bp fragment isolated by digesting p5'acba with
Bgl II, filling with dNTPs and Klenow, and cutting again
with Nco I. The resulting plasmid was cut with Sal I, and the 13-kb Sal I fragment from
ADA117 (56) was inserted to create p5'acba L117
prom.
Plasmid DNAs for transgene isolation were prepared as described
previously (5).
Fragment isolation and transgenic mouse production.
Transgenes V-VIII have been described previously (15).
Transgene V-A is a 15,132-bp Pvu I fragment isolated from
the plasmid p5'acba L117prom. Transgene IX is a 10,179-bp
Nde I-Pvu I fragment isolated from the plasmid
p5'acba L117
4. Transgene X is a 8,030-bp Nde I-Pvu
I fragment isolated from the plasmid p5'acba L117
5. Transgene
XI is a 7,217-bp Nde I-Pvu I fragment isolated
from the plasmid p5'acba L117
6. Transgene XII is an 18,095-bp
Nde I-Pvu I fragment from p5'acba L117
C(+).
Transgene XIII is an 18,152-bp Nde I-Pvu I
fragment from p5'acba L117
D(+). Transgene XII(
) is an 18,095-bp
Nde I-Pvu I fragment from p5'acba L117
C(
). Transgene XIII(
) is an 18,152-bp Nde I-Pvu I
fragment from p5'acba L117
D(
). Transgene fragments were routinely
isolated from low-melting-point agarose by digestion with
-agarase,
phenol extraction, precipitation, and purification over an Elutip-D
column (Schleicher & Schuell). Transgenic mice were produced from FVB/N
mice. Tail DNA samples from F0 mice were digested with
EcoR I and BamH I. DNA was electrophoresed, Southern blotted onto MAGNA NT membranes (MSI), and probed with a
radiolabeled 1.4-kb EcoR I fragment from pBLCAT6
(10) encompassing most of the CAT coding sequence. Mice
that possessed a hybridizing band of the appropriate size (1.4 kb) were
mated with nontransgenic FVB/N mice to establish lines from each
founder. Offspring were analyzed by both PCR and/or Southern blot for
transmission of the transgene.
Analysis of transgenic mice. CAT reporter gene expression was routinely analyzed in F1 transgenic mice between 4 and 7 wk of age. Small intestine segments were isolated from duodenum (2-cm section adjacent to pyloric sphincter), jejunum (2-cm section from the center of the small intestine), and ileum (2-cm section proximal to the cecum). For expression profiles along the AP axis of the small intestine, sequential 2-cm segments were harvested along the small intestine, starting at the pyloric sphincter. Other tissues routinely assayed included liver, thymus, spleen, tongue, esophagus, colon, and stomach. A more extended panel of tissues, which were assayed once for each transgene, included quadriceps muscle, lung, heart, ovary/testes, kidney, bone marrow, and brain. For the duodenal temporal expression studies, mice were harvested as early as embryonic day 16, in addition to a number of subsequent prenatal and postnatal time points. Methods of tissue extraction, CAT activity determination, and protein concentration determination have been described previously (4). Transgene copy number determination was carried out as described previously (15).
In situ hybridization. The in situ hybridization procedures were carried out as described previously (15). In brief, probes for in situ hybridization were prepared from a pGEM-4Z plasmid containing a 550-bp Hind III-Nco I fragment from pSV0-CAT (21) that encompasses the 5' end of the CAT reporter gene sequence. Sense and antisense 33P-labeled probes were synthesized from linear templates with T7 or SP6 polymerases using an in vitro transcription system (Promega). Duodenal samples from transgenic mice were isolated, rinsed in 1× PBS, and fixed in 4% paraformaldehyde in 1× PBS at 4°C overnight, followed by overnight incubation at 4°C in 30% sucrose, 1× PBS. Tissues were frozen in M1 embedding matrix (Lipshaw) and cut into 10-µm sections. Tissue sections were then fixed, acetylated, prehybridized, and hybridized (22, 23) with a solution containing ~5 × 105 cpm/µl 33P-labeled CAT riboprobe. After overnight hybridization at 48°C, sections were washed at high stringency and treated with RNase A (50 µg/µl; Worthington Biochemical) and RNase T1 (50 U/ml; Life Technologies) at 37°C for 30 min. Sections were dehydrated and exposed to Kodak NTB-2 emulsion for varying times (normally ~1 wk) at 4°C. Histological staining of sections was performed with hematoxylin and eosin.
Nuclei isolation and extract preparation.
Duodenum from organ transplant donors was obtained at the time of organ
retrieval under an Institutional Review Board-approved protocol. The
intestine was flushed with cold saline, blotted to remove mucus,
wrapped in aluminum foil, and frozen at 80°C. To prepare nuclear
proteins, ~1 cm3 of adult human duodenum was
freeze-fractured. Pieces were thawed in lysis buffer, and nuclei were
isolated over sucrose gradients as described previously (5,
16), with the following exceptions. Polyamine lysis buffer was
supplemented with additional protease inhibitors as follows (in
µg/ml): 40 bestatin, 1 aprotinin, 1 leupeptin, and 1 pepstatin A. Protease inhibitors were included in all buffers except dialysis
buffer. Proteins in the crude nuclear extract were precipitated with
ammonium sulfate and resuspended in Dignam Buffer D (20 mM HEPES-KOH,
pH 7.9, 20% vol/vol glycerol, 0.1 M KCl, and 0.2 mM EDTA).
Footprinting. DNase I footprinting was carried out essentially as described previously (5). A 659-bp BamH I-Pst I fragment containing HS-C and HS-D was subcloned into pUC12 between the BamH I and Pst I sites. The resulting plasmid, pUC 660, was used to generate probe for both strands of DNA. For the upper strand footprinting reactions, pUC 660 was digested with Hind III and filled using Klenow, [32P]dATP, and cold free nucleotides. Labeled DNA was then digested with Nco I, and the 312-bp fragment was isolated and purified. For the lower strand footprint, pUC 660 was digested with Nco I and filled using Klenow, [32P]dCTP, and cold free nucleotides. Labeled plasmid was then digested with Hind III, and the 312-bp segment was isolated and purified. DNase I footprinting was performed on each fragment using 0-25 µg of duodenal nuclear extract.
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RESULTS |
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A duodenum-specific regulatory region.
Transgenic mice were used previously to identify a regulatory region in
the human ADA gene that activates high-level expression of a linked CAT
reporter gene in the epithelium of the small intestine (15). Along the AP axis of the intestine, this high-level
expression was limited specifically to the epithelium of duodenum.
Ability to drive this pattern of transgene expression was originally
associated with a large fragment of ~13 kb from the middle of the
human ADA gene called fragment c (see Fig.
1). It was proposed that fragment c
contains a duodenum-specific enhancer, responsible for most, if
not all, of the pattern of ADA expression observed in the small intestine. Studies with additional transgenes further localized the
putative enhancer to a 3.4-kb fragment called fragment g
(15). This fragment (shown in Fig. 1A) was also
found to contain sites of altered chromatin structure in duodenal
nuclear DNA (but not liver nuclear DNA) from transgenic mice. These
sites were observed in the form of seven distinct DNase I HSs (HS-A to
HS-G). Since regulatory regions are often associated with regions of
altered chromatin, it was proposed that the putative duodenum-specific enhancer was associated with one or more of these HSs.
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Mapping a duodenum-specific enhancer.
Additional transgenes have been produced and analyzed to further map
and characterize the identified duodenum-specific regulatory region.
These transgenes were prepared from several human ADA intragenic
fragments that correspond to truncations of fragment g or
deletions in fragment c (Fig. 1A). The intragenic
fragments were included in transgene constructions whose basic design
was that used previously (Fig. 1B; Ref. 15). The
results from transgenic mouse lines established with these transgenes
are summarized in Table 1.
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Extended ADA promoter sequences are not required for duodenal
enhancer function.
The structures of a number of transgenes from this and the previous
study (15) are summarized in Fig. 2,
along with those of two transgenes (Transgenes V-A and IX) that have
not been discussed previously. Transgene IX contains fragment
h, a fragment that contains HS-B and -A segments and several
kilobases of DNA upstream of those regions. The single line of mice
examined from this transgene displayed no CAT expression in duodenum.
This is not surprising since this transgene does not contain the HS-D
enhancer region. Transgene V-A is identical in structure to Transgene
V, except that the ADA promoter used was truncated from 3.7 kb to a
segment of ~200 bp previously defined as the basal promoter
(16). Three lines of mice from Transgene V-A showed
high-level expression (55,000, 45,000, and 93,000 U/copy number)
specifically in duodenum, demonstrating that only the basal ADA
promoter is required to interact with the duodenal enhancer. Upstream
ADA promoter sequences are not necessary for transgene activation in
the duodenum. One line of mice from Transgene V-A, with a single copy
of the transgene, showed no expression in the duodenum, indicating that
there are some transgene insertion sites that are completely refractory to the enhancer function of fragment c.
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Expression patterns along the AP and CV axes are maintained.
CAT expression profiles were examined along the various axes of the
small intestine in transgenic mice derived from a number of the
different transgenes shown in Fig. 2. Previously, the profile of CAT
expression along the AP axis of the small intestine was determined for
a mouse from Transgene V, line 10 (15). It was found to be localized almost exclusively to the first 4-6 cm, a
pattern that is virtually superimposable on the profile of endogenous ADA expression. Similar studies were carried out for a number of other
transgenes, and the results with Transgenes VIII, X, XI, and V-A are
shown in Fig. 3. The profiles of CAT
expression along the AP axis are all very similar and almost identical
to that observed previously. Results with Transgene XII, line 3 (not shown) are also almost identical to these. Together, these
results demonstrate that the enhancer mapped to HS-D independently
specifies this pattern of activation along the AP axis and that it is
duodenum specific in nature.
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Upstream sequences modify the timing of enhancer activation.
Previous studies showed that Transgenes IV and V, which both contain
the full-length fragment c, showed perinatal activation of
transgene expression in the duodenal epithelium ~2 wk before activation of the endogenous ADA gene in mouse duodenum
(15). This endogenous activation occurs at the time of the
suckling-weanling transition. The temporal pattern of activation along
the developmental time axis was examined for various transgenes in this
study (Fig. 5). Transgenes V-A and VII
displayed activation profiles very similar to those seen previously
with Transgenes IV and V, with high-level CAT expression being
activated in duodenum at about the time of birth. However, Transgene VI
had a dramatically different profile of transgene activation, with
high-level CAT expression not appearing in duodenum until between 2 and
3 wk. This profile is almost identical to that previously described for
the endogenous mouse gene. The initial interpretation of this
observation is that the presence of sequences upstream of the duodenal
enhancer result in precocious activation of its function in the
perinatal period. These sequences map to the region upstream of exon 2 in fragments c and e. Results with Transgenes
VIII, X, and XI confirm these observations, since all lack this region
and all show delayed activation.
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Multiple proteins bind in the duodenal enhancer region.
To begin to characterize the factors responsible for duodenal enhancer
function, DNase I footprinting studies were carried out on the HS-D
region. Nuclear extracts were prepared from human, mouse, and rat
duodenum, and these extracts were used separately to footprint the
enhancer region. The pattern of protection was very similar with all
three extracts and on both DNA strands. The footprints observed with
the human duodenal nuclear extract on the lower and upper strand of the
enhancer DNA are shown in Fig. 6,
A and B, and are summarized graphically in Fig.
6C. Five separate and distinct footprints were identified.
The central footprints, FP-2 and -3, were the strongest with all three
nuclear extracts used. The flanking footprints, FP-1, -4, and -5, generally appeared weaker and were much more easily seen on one strand
than the other. This is probably due, at least to some extent, to the length of the probes used and the experimental conditions employed. FP-3 is clearly bipartite, with FP-3b evident at lower extract concentrations than FP-3a. This difference is even more obvious with
the rodent nuclear extracts, where FP-3b is much stronger than FP-3a,
which was difficult to see in some experiments.
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DISCUSSION |
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ADA is a critical enzyme of purine metabolism in mammals, deaminating adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. These products can then be either catabolized further and excreted or salvaged back for reutilization. Since ADA sits at this important metabolic "crossroad," the amount of this enzyme in a particular cell is a critical factor in determining the balance in pools of purine nucleosides and nucleotides present. Aberrant pools of purines, caused by defects in ADA expression, can have dramatic consequences. The absence of this enzyme in humans leads to a severe combined immunodeficiency characterized by an absence of both B and T cells, as well as less dramatic changes in other tissues (31). ADA-deficient mice, produced by embryonic stem cell gene-targeting experiments, die at birth with defects of the liver, lungs, and small intestine (36, 55). Overexpression of ADA in red blood cells leads to a hereditary form of severe hemolytic anemia (54). Thus regulated expression of ADA is critical in many, if not all, cell types. Although ADA is expressed ubiquitously in mammalian cells, the amount of enzyme present is highly variable and highly regulated.
Regulation of the ADA gene has been studied in both mice and humans, in which ADA expression was found to be governed principally at the level of transcription (8, 33, 59). The basal promoters for the human and mouse ADA genes are comprised principally of multiple Sp1-type binding sites, which are required for activation of promoter function (1, 16, 27, 56). However, these promoters alone are not sufficient to produce the observed tissue-specific pattern of ADA expression (4). Control of transcription is exerted to a great extent by a system comprised of a number of discrete regulatory modules present in and near the ADA gene. These modules activate ADA gene transcription in specific tissues or sets of tissues. A thymus-specific regulatory module is located in the first intron of both the human and mouse ADA genes (4, 5, 8, 11, 57). This module contains a classic transcription enhancer (5). In the human gene, sequences flanking the enhancer (called facilitators) were identified that impart consistent function to the enhancer in transgenic mouse thymus. Transient transfection studies in T cells indicated that the promoter Sp1 sites are required to mediate this enhancer function (16). Two distinct regulatory modules have been identified in the mouse ADA gene that activate ADA expression specifically in the placenta (45, 46) and in the forestomach (60). These are both located in the distal promoter region, 5.4 kb and 3.4 kb upstream of the transcription start sites, respectively. A regulatory region distinct from the thymic enhancer that activates in a ubiquitous manner was also identified in mouse ADA gene intron 1 (58). In transgenic mice, it was found that this region was required for activation of ubiquitous expression and for generation of a strong hypersensitive site at the promoter. Evidence for a similar segment from the human ADA gene that activates expression in a ubiquitous manner was also seen in transgenic mouse studies (4, 5). The duodenum-specific regulatory region, identified previously (15) and characterized here, represents a distinct new member of this group of regulatory modules that together specify the tissue-specific pattern of ADA expression. An equivalent regulatory module within the mouse ADA gene is almost certain to exist, but at present the location of such a module is unknown (12). In addition, it is likely that there are other unidentified regulatory modules that govern expression in tissues with high-level ADA expression, such as mouse tongue and esophagus.
Perhaps the best characterized of the ADA regulatory modules is the human thymic module (3-5, 17, 24). The present studies with the duodenum-specific regulatory module suggest that a number of similarities exist between it and the thymus-specific module. Both regulatory modules, when intact in transgenic constructions, are capable of specifying reproducible and consistent high-level expression of the transgene from line to line in the functional tissue, either thymus or duodenum. Both are centered on a classic transcription enhancer that is independent of distance and orientation in its function. Both of these enhancers are comprised of regions with multiple factor binding sites, and both lie within tissue-specific DNase I HSs, indicating regions of open chromatin. In both cases, regions flanking the enhancer are required for consistent high-level function in vivo in transgenic mice, even though the flanking regions themselves have no inherent enhancer capability alone. Removal of the flanking sequences causes a dramatic general reduction in transgene expression and much greater variation in expression from line to line for the same transgenic construction, indicating a much greater influence of insertion site. It has been proposed for the thymic regulatory module that the role of the flanking sequences is to control or modulate regional chromatin to allow formation and function of the enhancer complex (3, 5). The same may well be true for the duodenum-specific control module, although direct experimental support for this hypothesis is currently lacking.
The duodenum-specific enhancer that maps to a region of ~300 bp in HS-D activates transgenic CAT expression in a pattern that is virtually identical to that of the endogenous mouse ADA gene along all three important axes in the intestine: the AP axis, the CV axis, and the developmental time axis. This indicates that the information controlling that pattern is contained principally or entirely within this small enhancer region. What role each of the various factors that are proposed to bind elements in the enhancer may play in establishing the various aspects of this pattern is an area of intense interest and current investigation in our laboratory. It seems likely that the homeodomain factor PDX-1 is involved in the limitation of high-level expression along the AP axis of the small intestine to duodenal epithelium. In adult rodents, PDX-1 (previously known also as IDX-1, STF-1, and IPF-1) is expressed principally in cells of the pancreas and in duodenal epithelium (35, 37, 41, 42). Disruption of the Pdx1 gene by homologous recombination in mice produces mice that not only are apancreatic but also have aberrant epithelial structure in the rostral duodenum (28, 40). It has recently been proposed that PDX-1 plays a role in specifying the duodenal expression of the human calbindin-D9k (CaBP9k) gene by binding a site in the proximal promoter (7). Preliminary studies in our laboratory indicate that all of the putative sites identified can specifically bind PDX-1 (unpublished results). It is, however, not likely that PDX-1 is the only transcriptional activator responsible for enhancer function in the duodenum, since very low ADA expression and little or no CAT transgene expression is observed in pancreatic cells in which PDX-1 is present at high levels. Most enhancers rely on the combinatorial effect of multiple factors for their function (6, 49). Therefore, it is likely that several factors would be involved in forming the active enhancer complex, perhaps including CDX or GATA factors, for which putative binding sites were also identified within the enhancer footprints. In studies of some genes expressed in the pancreas, PDX-1 has been shown to require other homeodomain proteins as partners (such as Pbx1 or Pax6) bound at contiguous sites to specify function in some regulatory contexts (2, 43). A recent study indicates that the factors PDX-1 and CDX-2 can physically interact and bind each other (26). Whether such heterodimers or interactions are involved in the ADA enhancer function remains to be investigated.
Activation of expression in enterocytes near the CV junction along the CV axis of migration and differentiation is a common observation for a number of genes besides ADA, including the genes for sucrase-isomaltase (SI), lactase-phlorizin hydrolase (LPH), intestinal and liver fatty acid binding proteins, and aminopeptidase N (50). Transcriptional induction of gene expression is thought to be intimately associated with the cessation of proliferation of cells in the upper third of the crypt (50). The molecular basis of activation in enterocytes at this point in migration/differentiation is not well understood. In addition, genes that demonstrate this activation at the CV junction often have radically different patterns of expression along the AP axis and/or the developmental time axis. These complex patterns are thought to be the combinatorial result of a variety of transcription factors and, at least in some cases, a variety of distinct regulatory regions that are involved in controlling gene expression (50). Comparison and contrast of the factors and regulatory regions involved in the control of various genes will allow us to begin to assign them specific functions.
As mentioned above, both SI and LPH show activation in enterocytes at the CV junction. In addition, both demonstrate their highest levels in the jejunal area of the small intestine, with declining expression detected both proximally and distally along the AP axis. However, in most mammals they have a reciprocal relationship of expression along the developmental time axis, which is regulated transcriptionally (29, 51). In rodents, LPH is high at birth and gradually declines, whereas SI is low at birth with a sharp increase occurring about the time of the suckling-weanling transition. A small evolutionarily conserved segment of ~200 bp from the proximal promoter of the mouse SI gene has been shown to direct intestine-specific gene transcription in the enterocytes of transgenic mice (53). The pattern of this expression is very similar to the endogenous SI gene along both the temporal and CV axes. Binding of CDX-2 to a caudal-type site within this short promoter segment has been shown to be critical to its function (47). Transgenic mouse studies have suggested that a 2-kb proximal promoter segment from the rat LPH gene can direct correct tissue, cell, and CV expression but may not contain the elements for appropriate temporal or AP control (30). This promoter segment also contains a binding site for CDX-2 through which this factor can activate transcription (52). The CaBP9k gene is expressed in a steep gradient along the rat small intestine, with highest levels in the proximal duodenum. This intestinal pattern, which is very reminiscent of that for ADA, can be recapitulated with a transgene containing an extended rat CaBP9k promoter segment of 4.4 kb that contains binding sites for CDX-2 (32, 44). In addition to the studies cited above, a large body of experimental evidence has implicated both the caudal family members CDX-1 and CDX-2 in control of gene expression during cell differentiation in intestinal epithelium, as well as intestinal development and cell proliferation (18, 48). It has been suggested that in the process they may play a key role in determining positional expression of genes along the AP axis, as well as along the CV differentiation axis (18). Consequently, it will be important to confirm the binding of CDX-type factors to putative sites in the ADA duodenal enhancer and to investigate their role in enhancer control of gene expression along the various axes. The same will be true for the putative binding sites for GATA factors, as well as the other identified sites. GATA-4, -5, and -6 are known to have specific patterns of expression in the intestinal epithelium (19), but little functional information has been determined for them in the small intestine to date (19, 61).
The identification of upstream "temporal" elements that are distinct from the core enhancer and have no inherent capability to independently activate transcription of the linked reporter gene is an intriguing result. Although the core ADA enhancer has some similarities in temporal regulation to the 200-bp promoter of the SI gene, as described above, the ADA upstream temporal segments seem to represent a novel type of temporal element. The transgenic fragments used in this study are derived from the human ADA gene. Since the core enhancer itself activates CAT transcription near the suckling-weanling transition like the endogenous mouse ADA gene, the upstream segments that modify timing of enhancer function may be a specific adaptation in the human ADA gene. In contrast to rodents, development of the human intestine is largely completed in utero by the end of the first trimester, well before birth (38). Although direct comparison of two such different species is subjective at best, this would appear to be at an earlier relative time in human development than in mouse development. If the temporal elements do represent a human adaptation, they are capable of responding to mouse developmental cues in some fashion that mimics the earlier activation of ADA in the human system. Alternatively, the precocious activation associated with some of the specific transgenes may represent an artifact of the transgenic system or reflect the absence of additional sequences that are normally present in the intact ADA gene. A clearer interpretation of the significance of these temporal segments might be obtained by identification and characterization of the analogous duodenal regulatory region in the mouse ADA gene. Regardless of the interpretation of the in vivo significance of the temporal segments, it is clear that the enhancer can be activated to full adult levels perinatally. This seems to indicate that all of the factors necessary to form the enhancer complex are present at this earlier time point and that the key to timing of enhancer activation may be accessibility to enhancer binding sites within the chromatin. Determination of the time course of formation of DNase I HSs associated with the enhancer (especially HS-D) in transgenes present in duodenal nuclear DNA from mice derived from the various transgenic constructs will shed significant light on the question.
A wide variety of studies in recent years have begun to demonstrate that modular regulatory systems are a common theme in the regulation of metazoan genes, in which distinct modules are used to regulate a gene's expression in specific tissues or subsets of tissues. This study adds to our knowledge of the complex modular regulatory system that regulates the tissue-specific expression pattern of the ADA gene. In addition, it adds ADA to the growing list of genes for which regulation in the intestinal epithelium is beginning to be understood. Future studies with the ADA gene's duodenum-specific enhancer and other intestine-specific regulatory regions will help us unravel the factors and mechanisms involved in specifying positional expression along the AP axis, in regulating gene expression along the CV differentiation axis, and in controlling genes during development of the intestinal epithelium.
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ACKNOWLEDGEMENTS |
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We thank Todd French, Lara Picard, and Patrick Ryan for technical assistance and work with the mouse colony. We also thank Mary Beth Thomas for help with the in situ hybridization methods.
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FOOTNOTES |
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This work is supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-52343) to D. A. Wiginton. S. Y. Lowe was supported by a National Heart, Lung, and Blood Institute training grant (T32 HL-07752) and by a Patricia Roberts Harris Fellowship. M. B. Cohen was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-47318).
Address for reprint requests and other correspondence: D. A. Wiginton, Division of Developmental Biology, Dept. of Pediatrics, Children's Hospital Research Foundation, Cincinnati, OH 45229 (E-mail: dan.wiginton{at}chmcc.org).
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.
Received 8 March 2000; accepted in final form 2 June 2000.
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