From the Verna and Marrs McLean Department of
Biochemistry, Baylor College of Medicine, Houston, Texas 77030 and
the ¶ Department of Biochemistry and Molecular Biology, University
of Texas Health Science Center at Houston, Houston, Texas 77030
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ABSTRACT |
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The maturation of stratified squamous epithelium
of the upper gastrointestinal tract is a highly ordered process of
development and differentiation. Information on the molecular basis of
this process is, however, limited. Here we report the identification of
the first murine forestomach regulatory element using the murine adenosine deaminase (Ada) gene as a model. In the adult
mouse, Ada is highly expressed in the terminally
differentiated epithelial layer of upper gastrointestinal tract
tissues. The data reported here represent the identification and
detailed analysis of a 1.1-kilobase (kb) sequence located 3.4-kb
upstream of the transcription initiation site of the murine
Ada gene, which is sufficient to target cat reporter gene expression to the forestomach in transgenic mice. This
1.1-kb fragment is capable of directing cat reporter gene expression mainly to the forestomach of transgenic mice, with a level
comparable to the endogenous Ada gene. This expression is
localized to the appropriate cell types, confers copy number dependence, and shows the same developmental regulation. Mutational analysis revealed the functional importance of multiple transcription factor-binding sites.
The mucosa of murine upper gastrointestinal tract tissues,
i.e. tongue, esophagus, and forestomach, like the epidermis,
undergoes organized progressive differentiation to form a mature
stratified squamous epithelium, which is comprised almost exclusively
of keratinocytes (1-3). Keratinocyte differentiation is the process whereby a relatively undifferentiated keratinocyte in the basal layer
is converted, via intermediate spinous and granular layers, into a
terminally differentiated corneocyte in the cornified layer (4, 5).
Insights into transcriptional regulation of gene expression in
keratinocytes have been gained mainly through analysis of the
eukaryotic cytokeratin promoters (6-11), and to a lesser extent, the
cornified envelope precursor genes in skin or in epidermal epithelial
cell lines (12-23). Genes expressed in the epidermis appear to be
lineage-specific. For example, keratins 5 and 14 are expressed in the
proliferating basal layers. In the differentiated suprabasal layer,
their expression is down-regulated, as the expression of early
differentiation marker genes keratin 1 and 10 and involucrin are turned
on. Loricrin, filaggrin, transglutaminase, localized in the upper
spinous and granular layers of the epidermis and upper
GI1 tract tissues, are
predominant markers for late epithelial differentiation. Genes
expressed in the squamous epithelium of the upper GI tract, despite
limited research, have been shown to resemble the epidermis. In mouse
forestomach, keratins 5 and 14 are expressed in the basal layers, while
keratins 1 and 10 are expressed in the suprabasal layer where cells are
terminally differentiated (24). Although some progress has been made in
identifying and understanding factors that regulate gene expression in
esophageal epithelium from investigation of the Epstein-Barr DNA virus
(25-28), much work needs to be done. Moreover, while the majority of
research has focused on keratin genes involved in basal cell
proliferation or early differentiation, and the early differentiation
marker involucrin gene, only a few of the several genes involved in the
latest stages of epithelial differentiation have been studied,
including transglutaminase 3, loricrin (20, 23), and small proline-rich
protein 1A (29).
Adenosine deaminase (ADA) is a pivotal purine catabolic enzyme that
converts adenosine and deoxyadenosine to inosine and deoxyinosine, respectively (30). It is ubiquitously distributed among vertebrate tissues, but the level of expression varies markedly among different tissues. In humans, high levels of ADA are found in the proximal small
intestine and thymus. The upper GI tract tissues, especially the
stomach, also express enhanced levels of ADA (31). In adult mice, the
highest levels of ADA also occur in the gastrointestinal tract,
including the absorptive epithelium of the small intestine and the
keratinized squamous epithelium that lines the tongue, esophagus, and
forestomach. In fact, these mucosal layers are the richest naturally
occurring sources of ADA where the enzyme accounts for as much as 20%
of the total soluble protein (32). Immunostaining analysis showed that
ADA is localized predominantly to the granular layer of keratinized
epithelium (32), rendering it another marker of the late stage of
keratinocyte differentiation in the upper GI tract. Given the abundance
of murine ADA and its cellular localization in the upper GI tract,
utilizing the Ada gene as a model for studying gene
expression in the GI tract will likely contribute to our knowledge of
keratinocyte terminal differentiation, and enhance our appreciation for
the function of the vast amount of ADA in the GI epithelium. The work
presented in this paper focuses on defining the
cis-regulatory elements and protein factors that direct this
enhanced level of expression in the GI epithelium, especially the
forestomach epithelium.
Using transgenic mouse techniques, we have identified a 1.1-kb
EagI-HindIII fragment from 5'-flanking region of
the murine Ada gene that is able to direct chloramphenicol
acetyltransferase (CAT) gene expression in the forestomach of
transgenic mice. In situ hybridization shows that the
cat expression directed by the forestomach regulatory
element exhibits the same cellular localization as the endogenous
Ada gene. This cat expression also displays copy
number dependence, shows the same developmental timing and comparable
level as the endogenous Ada gene. Sequence analysis revealed
both general and tissue-specific protein-binding sites within this
1.1-kb sequence. Deletions and site-specific mutations of some
potential protein-binding sites resulted in decreased levels of
reporter gene expression, suggesting the involvement of these factors
in regulating Ada expression in the forestomach.
Plasmid Construction--
The 6.4CAT transgene, described by
Winston et al. (33) as the construct ADACAT, was subcloned
into the BamHI site of Bluescript KS+ II vector
(Stratagene). Deletions were prepared by appropriate restriction
digestion of this parental plasmid. 3.3FCAT and pCAT were generated by
HindIII and XbaI digestion of 6.4CAT,
respectively. 2.0FCAT, 1.1FCAT, 0.9FCAT, 0.6FCAT, and 0.5FCAT were
generated by ligation of the pCAT construct to various restriction
fragments from the 6.4-kb Ada flanking sequence. To
generate AP2FCAT, a pair of complimentary oligonucleotides
CCGTCTACAAAGGTGTGCACTATGGTGTGCACTATGTTTATGGGCTGCCCAAGTAACCCTGGCATCCCTCTACACGGTGAAAGCTTCTAGAGC and
GCTCTAGAAGCTTTCACCGTGTAGAGGGATGCCAGGGTTACTTGGGCAGCCCATAAACATAGTGCACACCTTTGTAGACGG (the nucleotide sequences for AP-2 site are underlined and the italicized letters denote mutations) were synthesized (Life
Technologies, Inc.). After annealing, the double-strand DNA was
digested by AccI and XbaI and subcloned into
1.1FCAT which was also digested by AccI and XbaI.
The AP-2 binding site mutation was later confirmed by nucleotide
sequencing. pCAT contained a murine Ada promoter sequence
from the XbaI site at Transgenic Mice--
Each transgene construct was restriction
digested to remove vector sequence, isolated by agarose gel
electrophoresis, and purified using the QIAquick Gel Extraction Kit
(Qiagen Inc.). DNA was resuspended at 2 ng/µl in 10 mM
Tris-HCl, pH 7.4, 0.1 mM EDTA and was microinjected into
male pronucleus of fertilized FVB/N oocytes. Genomic DNA was collected
from tails of weaning pups and analyzed by Southern blot using a 1.4-kb
cat-SV40 probe to identify transgenic mice (33). Transgene
copy numbers were determined by comparison of radioactivity of
transgene bands with known standards blotted to the same membrane. When
necessary, lines were established by mating with ICR mice.
DNA Hybridizations--
Genomic DNA was isolated from tails at
weaning. For Southern blotting, 10 µg of genomic DNA was digested
with BamHI or NcoI, separated by agarose gel
electrophoresis, transferred to NytranTM Plus membranes
(Schleicher & Schuell), and hybridized according to the manufacturer's
instructions. A 1.4-kb fragment corresponding to the
cat-SV40 sequence was used as a template to generate
32P-labeled probes using a random primer labeling kit (Stratagene).
Tissue Extracts, CAT and ADA Assays--
Tissue extracts from
the tongue, esophagus, forestomach, hindstomach, small intestine,
liver, spleen, and thymus were prepared, and CAT and ADA enzymatic
activity was measured according to established procedures (33).
In Situ Hybridization--
Forestomach tissues were fixed in 4%
paraformaldehyde, phosphate-buffered saline overnight at 4 °C and
processed for in situ hybridization as described (34). RNA
probes were labeled with [ Sequence Analysis--
The 1.1-kb EagI to
HindIII fragment was subcloned into Bluescript KS+ II vector
for sequencing. Transcription factor consensus sequences were sought
using the Findpatterns program both from the Genetics Computer Group
(GCG) program and Online MatInspector (35).
RNase Protection--
Total RNA from mouse tongue, esophagus,
forestomach, hindstomach, small intestine, and skin was isolated using
TRIzol RNA kit (Life Technologies, Inc.), and its integrity was
verified by formaldehyde-agarose gel electrophoresis. RNase protection was carried out using 30 µg (for Ada and cat
messages) of RNA (33). 32P-Labeled probes were
synthesized by T7 or T3 RNA polymerase from linearized Ada
and cat plasmid templates.
A Forestomach-specific Regulatory Element Resides within a 1.1-kb
Fragment in the 5' Flank of the Murine Ada Gene--
We have shown
previously that a 6.4-kb region immediately upstream of the murine
Ada gene is capable of directing the expression of a
reporter gene to the placenta prenatally and to the forestomach postnatally (33). We sought to identify the forestomach-specific regulatory element by deletion analysis in transgenic mice. Transgenic mice were used because this approach provides a physiological assay
system in which the complete array of necessary regulatory elements can
be defined in a developmental context. To increase the pace of the
analysis, we chose to study founder mice instead of generating
transgenic lines since the same features of cat expression
were observed in both assays. Of five 6.4CAT transgenic lines, four
showed high levels of CAT activity in the forestomach and undetectable
CAT activity in the adjoining hindstomach and small intestine. All
seven 6.4CAT founders showed high levels of forestomach-specific
cat expression. The level of cat expression also
correlated with copy number (Table I).
Thus, the possibility of transgenic mosaicity did not present a
problem, and transgenic founders worked as well as the transgenic lines
to define the Ada forestomach regulatory element.
We tested various 6.4CAT deletion constructs for their ability to
target cat expression in the forestomach of founder adult mice (Fig. 1). Deletion to a
HindIII site (3.3FCAT) disrupted expression, while a 2.0-kb
BamHI-EagI fragment at the 5'-half (2.0FCAT) gave
no detectable expression. The 3' 1.1-kb
EagI-HindIII fragment fused to the cat
reporter cassette (1.1FCAT) restored forestomach expression, and the
expression level was similar to that of the original 6.4-kb fragment.
Other adult tissues where high level Ada expression is
normally found did not show consistent expression of cat,
although the tongue sometimes gave about 100 times lower levels of
cat expression (Fig. 2).
Further deletion from either 5' (0.6FCAT) or 3' (0.5FCAT) ends of this
fragment led to a drastic decrease of cat expression in the
forestomach (Fig. 1). Therefore, the 1.1-kb
EagI-HindIII fragment, as the first recognized
forestomach regulatory element, retains all the information required
for high level forestomach-specific expression of the cat
reporter gene.
The Ada Forestomach Regulatory Element Directs Reporter Gene
Expression to the Same Cellular Localization as the Endogenous Ada
Gene--
To confirm that cat gene expression occurred in
the same cell types as the endogenous Ada gene, the cellular
localization of cat transcripts from the 1.1FCAT transgene
was identified by in situ hybridization. The forestomach
tissue from a transgenic founder containing approximately 14 copies of
the 1.1-kb transgene was sectioned, fixed, and then hybridized with
either cat or Ada sense or antisense
35S-labeled UTP RNA probes. Both cat and
Ada antisense probes produced strong signals in the
keratinized squamous epithelial cell layer. Neither Ada nor
cat mRNA was detected in the adjacent muscle layer of
the forestomach (Fig. 3) or the adjacent
hindstomach tissue (data not shown). Thus, this 1.1-kb
EagI-HindIII fragment contains information
necessary for appropriate cellular localization of cat
reporter gene expression.
The Ada Forestomach Regulatory Element Directs Reporter Gene
Expression with the Appropriate Developmental Timing--
In
gastrointestinal tissues, the level of ADA protein is subject to
pronounced developmental control, being low at birth and achieving
enormous levels within the first 5 weeks of postnatal life. To
determine whether the developmental timing of cat expression coincided with forestomach Ada gene expression during
epithelial layer formation, the early onset of cat
expression was compared with that of ada expression in the
epithelial layer. CAT activity was measured in the forestomach of
transgenic mice at birth, at 10 days, and at approximately 4 months.
CAT activity was low at birth, but by weaning, CAT was highly expressed
in the forestomach, and significant expression appeared by 4 months
(about 120 days) of age (Fig. 4).
In situ hybridization also showed that the expression is
localized only in the epithelial layer of the forestomach (data not
shown). From these data, we conclude that this 1.1-kb
EagI-HindIII fragment contains sufficient genetic
information for reproducing the endogenous developmental pattern of
Ada gene expression in the forestomach.
The Forestomach Enhancer Directs Reporter Gene Expression in a Copy
Number-dependent Manner--
To examine whether this
1.1-kb EagI-HindIII fragment allows a
position-independent, copy number-dependent expression of
the reporter gene, the level of cat expression in the
forestomach of each transgenic founder mouse was compared with its copy
number. This comparison revealed a linear relationship between the
level of cat expression in each transgenic mouse and its
transgene copy number (Fig. 5). The
forestomach regulatory element therefore functions in an integration
site independent and copy number-dependent manner.
The Forestomach Enhancer Is Capable of Directing cat Expression in
the Transgenic Forestomach in a Level Similar to the Endogenous Ada
Gene--
To determine whether the quantity of cat
expression in the transgenic forestomach was comparable to that of
endogenous Ada expression, the respective levels of each
steady state message were compared in gastrointestinal tissues,
including tongue, esophagus, forestomach, hindstomach, and small
intestine. RNA was isolated from these tissues, and the ratio of
cat message to Ada message in the forestomach was
determined by RNase protection assay using 32P-labeled cDNA probes for both
Ada and cat genes. While Ada message was observed in tongue, esophagus, forestomach, and small intestine, consistent with the observed pattern of enzyme activity, cat
message was detectable only in the forestomach. When adjusted for gene dosage, the magnitude of cat expression per transgene in the
forestomach was similar to the endogenous Ada gene. Thus,
the forestomach regulatory element is capable of delivering reporter
gene expression to the transgenic forestomach at a high level, being
comparable to endogenous Ada genes (Fig.
6A).
Because cat transcripts and endogenous Ada
transcripts may differ in their stability, a forestomach
enhancer-driven Ada minigene (1.1FADA) was constructed and
transgenic mice were generated. This minigene contained the endogenous
last intron, poly(A) signal and 3'-untranslated region to ensure
similar processing and stability as the endogenous gene (36). A 36-bp
deletion at the 5'-untranslated region was engineered to allow the
transgene mRNA to be easily distinguished from the endogenous
mRNA (Fig. 6B). Total RNA was extracted from tissues of
gastrointestinal tracts in different F0 mice carrying the
1.1FADA. RNase protection assays were performed on these tissues using
the same Ada probe for the 1.1FCAT mice. The protected band
for endogenous Ada was 310-bp, while that for minigene is
275-bp (Fig. 6B). The ratios in several transgenic founder
mice were close to 1 when adjusted to transgene copy numbers (Table
II). It thus indicates that this
forestomach enhancer is capable of directing high level expression of
the Ada minigene as well as the cat reporter gene
in the forestomach, indicating that it may contain most, if not all,
the genetic information required for the expression of Ada
gene in the forestomach.
Deletion of 200-bp 5' Region and the Mutation of a Putative AP-2
Factor-binding Site Results in Decreased CAT Expression in the
Transgenic Forestomach--
A data base search was performed to
identify potential transcription factor-binding sites within this
1.1-kb forestomach regulatory element. The sequence analysis revealed
multiple candidates (Fig. 7) which have
been shown to be of importance in regulating gene expression in
epidermal as well as esophageal epithelium. Among these are a cluster
of AP-1, C/EBP, and CACCC-like binding motifs within the 5' 200-bp of
the forestomach element, and an AP-2 factor consensus site at the 3'
end. Deletion of the 200-bp 5' region resulted in a drastic decrease of
the expression level of cat reporter gene, indicating the
functional importance of this region (Fig.
8). Similarly, a two-base mutation of CC
to TT at the AP-2 site led to almost total diminution of activity,
strongly indicating the importance of this site in the enhancement of
forestomach expression (Fig. 8). Therefore, mutational analysis
suggests that multiple factors in the forestomach epithelium may act
through the forestomach regulatory element.
In addition to its low level of ubiquitous expression in almost
all tissues, the murine Ada gene is expressed at
substantially elevated levels in a small collection of diverse tissues,
especially in those of the gastrointestinal tract. The expression of
Ada in the GI tract displays a cell type- and
lineage-specificity, confined to the epithelial cells lining the mucosa
of the tongue, esophagus, forestomach as well as small intestine (32).
The relative level of ADA protein is extremely high, comprising
approximately 20% of soluble protein in the mucosal layer (32). To
facilitate a greater understanding of the physiological significance of
ADA in the gastrointestinal tract and the regulation of gene expression in the upper GI tract, we wish to identify signaling pathways that
govern the temporal and cellular expression of the murine Ada gene during GI development using transgenic mouse
assays. The data reported here represent the identification and
detailed analysis of a 1.1-kb sequence located 3.4 kb upstream of the
transcription initiation site of the murine Ada gene, which
is sufficient to target cat reporter gene expression to the
forestomach in transgenic mice. This expression is localized to the
appropriate cell types, confers copy number dependence, and shows the
same developmental regulation. These facts indicate that this
forestomach-specific regulatory element contains all the
cis-acting genetic information that is necessary to
reproduce the endogenous pattern of murine Ada expression in
the forestomach.
Both immunofluorescence staining from previous work in our laboratory
(32) and in situ hybridization in the present study showed
that murine Ada message is localized predominantly to the keratinized stratified squamous epithelium, especially to the late
differentiated keratinocytes. Ada is undetectable in any non-squamous cells in the lamina propria or muscularis mucosae in the
mucosa, submucosa, external muscularis mucosae, or serosa. The
identified forestomach enhancer is capable of directing cat reporter gene expression mainly to the terminally differentiated cells.
Detailed analysis of this 1.1-kb regulatory element in the forestomach
epithelium is therefore of importance, since it will give us insights
into the molecular basis of gene regulation on the terminally
differentiated cells in the upper GI epithelium.
Although tongue and esophagus are histologically similar to
forestomach, CAT assays as well as RNase protection assays showed that
this forestomach regulatory element does not direct enhanced cat expression in those tissues, at least not at expected
high levels as compared with those of the endogenous Ada
expression. These interesting observations, along with a number of
previous studies both in human and mouse, indicate that the regulation of murine Ada in each tissue is complex and involves
different regulatory elements for different or even closely related
tissues or cell types, in this case, all the upper GI tract tissues. In fact, we and others have shown that distinct regulatory modules govern
expression in both the murine thymus (37) and placenta (38) as well as
the human thymus (39) and small intestine (40). In addition, a locus
control region has also been identified in intron 1 region of the human
ADA gene (39, 41), and similarly, an element responsible for ubiquitous
expression in intron 1 of the murine Ada gene (37).
The position independence, copy number dependence associated with
transgenes carrying the forestomach regulatory region, and the strong
enhancer activity observed in the transgenic forestomach qualify this
element as locus control region (LCR), a regulatory element acting as a
dominant activator in establishing the transcriptional competency of a
complete gene locus/chromatin domain (42). Since their initial
description in the human To fully understand the molecular aspect of the regulation of
this forestomach enhancer, we conducted mutational analysis of this
enhancer to identify important transcription factor-binding sites. In
the process of determining protein factors involved in regulating this
forestomach enhancer using footprinting and gel mobility shift assays,
we encountered some difficulties. Since the keratinized stratified
squamous epithelium where Ada is highly expressed in the
forestomach is terminally differentiated and enucleated, it is very
difficult to prepare nuclear extract from the forestomach mucosa.
However, sequence analysis of this enhancer revealed several potential
transcription factor-binding sites that have been shown to be
functionally important on other keratinocyte-specific promoters or
enhancers, among which is an AP-2 factor-binding site located at the 3'
end of this regulatory element. AP-2 consensus binding motifs have been
recognized in many keratin promoters and enhancers and other
epidermis-specific or squamous epithelium-specific genes (46, 47).
Despite this, it has been shown that AP-2 determines, in
vitro, the level of transcription rather than functioning as a
sole determinant of the epithelial specificity of gene expression (48).
Similar to their findings, the cat reporter gene expression in our AP-2 mutant construct (AP2FCAT) decreased significantly, however, forestomach restricted expression remained (data not shown).
This result indicates that the role of the AP-2-like factor in squamous
epithelium gene expression is quantitative rather than qualitative.
That is, rather than determining epithelial specificity, this AP-2-like
factor modulates, in vivo, the level of expression of
epithelial genes. AP-2 contains three isoforms AP-2 Within a 200-bp region at the 5' end of the forestomach enhancer, there
is a cluster of transcriptional binding motifs that have been shown to
regulate the expression of other squamous epithelium-specific genes.
These motifs include six AP-1 sites, two CCAAT/enhancer-binding proteins (C/EBP) binding sites and a CACCC-like motif. AP-1 binding motifs are found in the keratin promoters (11, 54) and other early or
late differentiation stage genes (19, 21, 55), and are important
determinants of the keratinocyte stage of differentiation (23). C/EBP
proteins, members of the bZIP family of DNA-binding proteins/transcription factors, play a fundamental role both in the
differentiation of preadipocytes to adipocytes and in the regulation of
the expression of many different genes encoding cytokines in several
cell types (56-60). High level expression of C/EBP has recently been
shown to be associated with squamous differentiation in epidermis,
suggesting that it may play an important role in regulating one or more
aspects of the squamous epithelium differentiation program (61, 62).
The importance of the CACCC-like element in the transactivation of
genes expressed in the upper GI tract has been demonstrated in its
interaction with gut-enriched Krüppel-like factor and activating
the Epstein-Barr virus EL-2D promoter (26, 27). Gut-enriched
Krüppel-like factor is localized to suprabasal cells in skin,
tongue, esophageal, and intestinal squamous epithelial cells (63),
rendering it a good candidate for regulating the suprabasal-layer
expressed Ada gene. Although the role of each possible
transcription factor has not been assessed, the functional importance
of this 200-bp region is evident since deletion of this sequence
abolished reporter gene activity in the transgenic forestomach.
Additional experiments are required to fully assess the importance of
the above mentioned transcription factors.
The evidence that the Ada forestomach enhancer consists of a
collection of distinct genetic regulatory motifs suggests that a
combination of multiple transcription factors are required for the
forestomach epithelium-specific expression of murine Ada, even though many of these factors, such as AP1 and AP-2, also appear in
nonepithelial cells. It is possible that appropriate combinations of
these potential factors, including AP-2 factor, AP1 factors, C/EBP
factors, gut-enriched Krüppel-like factor, and other ubiquitously
as well as epithelium-specific factors, may provide a mechanism for the
determination of epithelium specificity (64). Future experiments will
provide more insight into this aspect of the molecular mechanism of
gene regulation in the squamous epithelium.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSUION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSUION
REFERENCES
750 bp to the NcoI site
at +90 bp ligated to the cat cDNA followed by the SV40
small T antigen intron and the early polyadenylation site.
- 35S]UTP (1000 Ci/mmol,
Amersham). The Ada probes were generated from a 400-bp
fragment from the 5' end of Ada cDNA. The cat
probes were generated from the first 280-bp of the cat
coding sequence. Samples were hybridized overnight at 60 °C and
treated as described (34). Slides were dipped in Kodak NTB-2 emulsion
and exposed overnight at 4 °C. After development, the slides were
stained with Hoechst 33258 to identify nuclei. Sections were viewed
using an Olympus BX60 fluorescent microscope equipped with dark-field optics with a red filter and photographed using a SPOT digital camera (Diagnostics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSUION
REFERENCES
Comparison of CAT specific activities in adult forestomachs of 6.4 CAT
transgenic lines and founders
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Fig. 1.
Transgenic constructs and their expression in
transgenic forestomachs. The 6.4CAT deletion constructs were made
using the restriction sites of the 6.4-kb 5'-flanking sequence shown as
6.4ADA. The arrow indicates the transcription initiation
site. 5 weeks after birth, transgenic mice, identified by Southern
analysis of tail DNA, were sacrificed and tissue homogenates were
analyzed for the presence of CAT activity (stars) in the
forestomachs. The number of transgenic founders examined is indicated
as n or by the number of asterisks (*). The bar
represents the average of CAT activities. ND (not detected)
represents undetectable CAT activity (<1 pmol/min/mg/copy) in
transgenic founder mice. Ln (line) indicates that the
transgenic data were from F1 transgenic lines.
F0 denotes data from founder mice. B,
BamHI; H, HindIII; Ea,
EagI; Bs, BstEII.
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Fig. 2.
The expression of CAT activity driven by the
forestomach regulatory element in various adult tissues. CAT
activity was measured in extracts from two 5-week-old founders carrying
construct 1.1FCAT (numbers 3091 and 3081). To, tongue;
Fs, forestomach; Hs, hindstomach; SI,
proximal small intestine; Lv, liver; Sp, spleen;
Th, thymus. Arrowheads denote the acetylated
products.
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Fig. 3.
In situ hybridization to
cat reporter gene transcripts in transgenic
forestomach epithelium. Transverse sections through a 6-week-old
1.1FCAT founder mouse of 14 copies were hybridized to either
Ada antisense and sense (panels A and
B), or cat antisense and sense (panels
C and D) 35S-labeled riboprobes.
L, lumen; M, muscle layer; Ep,
epithelium layer; Bl, basal layer; Gl, granular
layer. The bar is 100 µm.
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Fig. 4.
Comparison of developmental regulation
between CAT and ADA enzymatic activities in transgenic
forestomachs. A FVB male 1.1FCAT founder of 4 copies was mated
with ICR females, and forestomachs of transgenic progenies were
isolated and their ADA (shaded bars) and CAT (white
bars) activities were measured at different postnatal time points.
Each star denotes the value of ADA or CAT activities from a
F1 mouse, while each bar represents the mean value.
d, day, age of F1 mice.
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Fig. 5.
The copy number dependence of cat
expression. Forestomachs were isolated from 5-week-old
transgenic founders. CAT activities were measured and plotted against
their respective copy numbers as determined by Southern analysis.
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Fig. 6.
Comparison of the level of cat
expression in construct 1.1FCAT and a forestomach enhancer driven
Ada minigene with that of the endogenous
Ada gene. A, comparison of
cat expression with endogenous Ada gene
expression. 1.1FCAT transgene construct is shown above and includes the
1.1-kb forestomach enhancer (dotted box), 800-bp murine Ada
promoter (shaded oval), cat cDNA (white
box), SV40 small antigen intron region and polyadenylation site
(hatched box). 30 µg of total RNA isolated from tissues of
a 1.1FCAT founder was incubated with a mixture of uniformly radiolabled
280-bp cat and 410-bp Ada probes. The
cat probe protects a 270-bp fragment, while the
Ada probe protects a 310-bp fragments. B,
comparison of expression of the Ada minigene driven by the
forestomach element with that of the endogenous Ada gene.
The forestomach enhancer driven Ada minigene is shown above
and includes the murine Ada cDNA (hatched boxes),
endogenous polyadenylation sequence (ATAA), intron 11, and 2 kb of 3' flank from the murine Ada gene (white
boxes), 800-bp Ada promoter containing a 36-base pair
deletion ( ) in the 5'-untranslated region (shaded oval)
and forestomach enhancer (dotted box). 30 µg of total RNA
isolated from tissues of several transgenic founders carrying the
forestomach enhancer driven Ada minigene was incubated with
the Ada probe. The protected band for the endogenous
Ada is 310-bp, while that for the minigene is 275-bp.
To, tongue; Eso, esophagus; Fs,
forestomach; Hs, hindstomach; SI, proximal
small intestine; Ada, murine Ada probe.
Ratios of endogenous Ada to Ada minigene mRNA in forestomachs of
transgenic mice
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Fig. 7.
Sequence of the Ada
forestomach regulatory element. The sequence of the 1.1-kb
EagI to HindIII fragment is shown with key
restriction sites marked by lowercase letters. The sequence
motifs for AP-1, C/EBP, CACCC-like, and AP-2 are shown
boldfaced and underlined.
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Fig. 8.
Functional analysis of binding sites for
ubiquitous transcription factors and AP-2 factors. Two different
1.1FCAT mutant constructs, as depicted in the inset, were
generated and microinjected into FVB/N zygotes. 5 weeks after birth,
CAT activities in the forestomach (shown as *) of the resulting
transgenic founder mice were measured. The bars represent
the average level of CAT activities in the forestomachs.
DISCUSSUION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSUION
REFERENCES
-globin locus, LCR elements have been
recognized in several other genes, including the human ADA T-cell
enhancer/LCR and duodenal enhancer (40, 41). Unlike human T-cell/LCR
and duodenal enhancer, the identified forestomach regulatory element
neither drives ubiquitous low level expression, nor requires additional
sequences for full activation. It, in fact, seems to contain all the
information to ensure integration site independent, copy number
dependent expression of transgenes. LCRs have been suggested to exert
their functions by recruiting protein factors that lead to
hyperacetylation of histones and subsequently to the unfolding of the
chromatin (42-44). The forestomach regulatory element presumably
contains multiple binding sites for sequence-specific transcriptional
activators for its function both as an enhancer and as an LCR, and it
may exert its LCR function by recruiting histone acetyltransferase,
such as CBP/p300 (45), to the Ada promoter. The
hyperacetylation of histones and subsequent unfolding of the chromatin
may eventually result in activation of the Ada promoter.
Further analysis of this element would give us more insight regarding
how LCR elements function.
, AP-2
, and
A-2
, all of which have distinct but partially overlapping patterns
of expression (49-53). Our RNase protection assays (data not shown)
and data from other laboratories (51-53) confirmed the presence of all
three AP-2 isoforms in the upper GI tract tissues, which suggest that
AP-2
, AP-2
, and A-2
may all be involved in the regulation of
this forestomach enhancer. Further transfection analysis is needed to
delineate the definite role of each AP-2 gene in regulating this
enhancer and, moreover, squamous epithelium genes in general.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Michael Blackburn for the Ada minigene construct. We also thank Drs. John Schwartz, Michael Blackburn, Jeffrey Lawton, and Daqing Shi for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK46207.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U73107.
§ Present address: Dept. of Internal Medicine, University of Texas Medical Branch at Galveston, Galveston, TX 77555.
To whom correspondence should be addressed. Tel.:
713-500-6124; Fax: 713-500-0652; E-mail:
rkellems{at}bmb.med.uth.tmc.edu.
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ABBREVIATIONS |
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The abbreviations used are: GI, gastrointestinal; ADA, adenosine deaminase; CAT, chloramphenicol actyltransferase; kb, kilobase(s); bp, base pair(s); LCR, locus control region.
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