From the
Intestinal trefoil factor (ITF) is a small peptide bearing the
unique motif of intrachain disulfide bonds characteristic of the
trefoil family. Previous work had localized expression of ITF primarily
within goblet cells in the small and large bowel, making it a candidate
gene for the study of the molecular basis of intestinal and goblet
cell-specific gene expression. In order to study the regulation of ITF
expression, we have cloned the rat ITF gene and sequenced 1.7 kilobases
of the 5`-flanking region. RNase protection analysis demonstrated a
single transcriptional start site. Various lengths of the 5`-flanking
region were linked to the reporter gene luciferase and transfected into
the colon cancer cell lines LS174T and Caco-2, representing,
respectively, cells with and without goblet cell-like phenotype.
Expression in the goblet cell-like LS174T colon cancer cell line was
nearly 10-fold greater than expression in Caco-2 cells which exhibit
columnar enterocyte-like phenotype. The pattern of goblet
cell-associated selective transcription required only 153 base pairs of
the rat ITF 5`-flanking sequence. Transfection of a construct of human
growth hormone under the control of the rat ITF promoter in the N2
subclone of HT-29 cells demonstrated expression of the reporter gene
only in those cells exhibiting a goblet cell phenotype as assessed by
expression of immunoreactive mucin. These initial studies of the
5`-flanking region of the ITF gene demonstrate the presence of
cis-regulatory elements capable of directing goblet cell specific
expression.
Goblet cells are abundant constituents of the surface epithelium
within the small and large intestine. Although these cells have been
long recognized to secrete a complex mixture of mucin glycoprotein onto
the cell surface, their functional importance in gastrointestinal tract
mucosa has not been well defined. Characterization of the molecular
basis of goblet cell differentiation and function has also been quite
limited. Although substantial advances have been made in the cDNA
cloning of genes encoding the protein backbones of mucin glycoproteins
from the gastrointestinal tract and elsewhere, the enormous size and
complexity of these genes have made these of limited utility to define
those elements responsible for goblet cell specific expression
(1, 2, 3, 4, 5, 6, 7) .
Observations in recent years have led to the recognition of a family
of small proteins which are also selectively expressed by
mucin-producing goblet cells in gastrointestinal tract mucosa. This
family of proteins, designated trefoil peptides, are distinguished by a
unique six-cysteine motif (called a ``P'' domain) which
results in the formation of three intrachain loops within these small
peptides due to disulfide bond formation in a 1-5, 2-4, 3-6
configuration
(8, 9) .
Members of the trefoil peptide
family appear to be expressed in a region-specific fashion along the
length of the gastrointestinal tract. Human spasmolytic polypeptide, or
hSP, bears two trefoil motifs and is expressed primarily in the stomach
(10) , although the porcine homologue was originally isolated
from pancreas
(11) . pS2, bearing a single trefoil motif and
initially cloned as the product of an estrogen-responsive gene from a
breast cancer cell line
(12) , is normally expressed only in the
gastric antrum in man
(13) . Cloning of the rodent homologues of
pS2 and hSP confirmed that expression of these distinct, but closely
related peptides is site-specific along the longitudinal axis of the
upper gastrointestinal tract
(14, 15) .
Intestinal
trefoil factor (ITF)
Understanding of the functional role of the trefoil
peptides in gastrointestinal tract biology is now emerging. Recent
studies using an in vitro model have demonstrated that these
peptides may play a key role in facilitating healing at sites of
mucosal injury by enhancing cell migration from the wound edge
(18) . This concept is consistent with observed patterns of
expression of trefoil peptide in association with injury in
gastrointestinal tract disorders. Thus, expression of hSP and pS2,
normally confined to the proximal gastrointestinal tract, as well as
ITF has been observed adjacent to intestinal ulceration in patients
with Crohn's disease and peptic ulcer disease in man
(19) , whereas ITF is expressed in stomach in conjunction with
the other trefoil peptides in an animal model of gastric ulceration
(20) .
The selective expression of ITF in intestinal goblet
cells suggests that characterization of the gene encoding this peptide
may provide insight into regulatory elements responsible for goblet
cell-associated gene expression. Among the trefoil family members, only
the pS2 gene normally expressed in gastric mucosa has been reported
(21, 22) . Although several genes expressed in the
intestine have been cloned and their regulatory elements studied, none
are products of goblet cells. Intestinal fatty acid binding protein,
cloned in rat and human
(23) , human villin
(24) , human
and mouse sucrase-isomaltase
(25, 26) , porcine
aminopeptidase N
(27) , human intestinal alkaline phosphatase
(28) , and human lactase-phlorizin
(29) all represent
proteins expressed in the columnar absorptive enterocyte. It is not
known whether the regulatory elements which direct intestinal-specific
expression of enterocyte products are also important in other
intestinal epithelial cell populations. To investigate the mechanisms
responsible for goblet cell differentiation within the intestine, and
as a basis for further study of the genetic elements responsible for
``ectopic'' expression of ITF in pathologic conditions, we
have cloned the rat ITF gene and its 5`-flanking region. Reporter gene
constructs under the control of the rat ITF promoter were able to
direct expression in a relatively goblet cell-specific fashion.
Phage DNA was prepared from plate lysate by a
modification of methods described in Maniatis et al. (31) .
Phage DNA from each plaque-purified cross-hybridizing phage was
subjected to restriction digestion with a variety of restriction
enzymes and Southern blot was performed with the T3411 probe used to
screen the genomic library. Based upon Southern blot, an appropriate
restriction fragment containing the entire RITF gene was selected for
subcloning into plasmid. The selected restriction fragment was ligated
into appropriately cut pKS+ Bluescript plasmid (Stratagene) and
used to transform competent XL1-Blue E. coli (Stratagene).
Large scale preparation of plasmid DNA was performed by CsCl gradient
and by affinity resin purification (Magic Maxiprep; Promega, Madison,
WI). Exons, large portions of introns, and the entire 5`- and
3`-flanking regions were sequenced using Sequenase (U. S. Biochemical
Corp.) and oligonucleotide primers synthesized in a sequential
overlapping manner. Final sequence was determined from both strands.
Reaction products were resolved on denaturing polyacrylamide gels by
standard techniques. Sequence comparisons were made using the BESTFIT
and BLAST programs
(32) . The 5`-flanking region of the RITF
gene was searched for potential enhancer, promoter, and repressor
elements using Signal Scan v.3.01
(33) . The sequences of the
5`-flanking regions of reported genes expressed in intestinal tissue
were retrieved from the GenBank
A construct consisting of
the -1671 RITF promoter driving the human growth hormone gene was
prepared in the following manner: -1671 RITF-Luc was digested to
completion with BamHI and BglII to release the full
length of the 5`-flanking region. The
Deletion constructs of the 5`-flanking region of the
RITF gene driving the luciferase gene were derived from the -1671
RITF-Luc construct taking advantage of convenient restriction sites.
For -980 RITF-Luc, the HincII- XhoI fragment of
the 5`-flanking region was isolated from -1671 RITF-Luc by
electrophoresis on a 1% agarose gel and agarose digestion using GELase
followed by ethanol precipitation in the presence of 3
M ammonium hydroxide. Gel-isolated HincII- XhoI
fragment was ligated into pXP2 digested with SmaI and
XhoI. Plasmid -704 RITF-Luc was derived by digestion of
-1671 RITF-Luc with XbaI followed by Klenow treatment of
the 5` overhang to make it blunt. Plasmid was then cut with
XhoI and the XbaI- XhoI fragment isolated
from an agarose gel as above. The XbaI- XhoI fragment
was ligated into pXP2 digested with SmaI and XhoI.
Further deletion plasmids, -664 RITF-Luc, -331 RITF-Luc,
and -153 RITF-Luc, were constructed by treatment of -1671
RITF-Luc with exonuclease III and mung bean nuclease after creation of
a 5` overhang using BamHI digestion to cut the plasmid at the
5` end of the promoter insert (Erase-a-Base System, Promega). All
plasmids were transformed into DH5
Transfection was
accomplished by the calcium phosphate precipitation method. Sixteen
hours prior to transfection, 8
Co-localization of hGH Expression under Control
of RITF 5`-Flanking Region and Expression of Mucin
Glycoprotein-The N2 subclone of the HT29 cell line obtained
from Dr. Daniel Louvard
(36, 37) was grown in
differentiating and undifferentiating media (as described for H2 cells)
in cell culture flasks with a removable glass slide bottom for
subsequent immunostaining (Lab-Tek Chamber Slide System; Nunc,
Naperville, IL). N2 cells were seeded as 5
Searches for specific known cis-regulatory elements revealed
potential overlapping AP-2 binding sites
(46) from -119
to -105, within the GC-rich region. A CRE consensus sequence
(TGACC)
(47) was found from -1431 to -1427.
Potential CAAT boxes (either CCAAT or GCAAT) located at -1153 on
the sense strand and on the antisense strand starting at -97 and
-1225. Four sites, located at -1431, -1185,
-908, and -369, also fit the consensus sequence of
RG(G/T)TCA which has been reported to promiscuously bind the
transcription factors RAR, RXR, HNF-4, and ARP-1
(48) .
Expression of hGH and mucin glycoprotein in
differentiated N2 cells transfected with -1671 RITF-hGH is
depicted in Fig. 6. Examination of these cells reveals expression
of hGH in virtually all cells expressing mucin glycoprotein. No cells
were observed to express the reporter hGH in the absence of mucin
glycoprotein. In contrast, undifferentiated cells transfected with
-1671 RITF-hGH expressed neither mucin glycoprotein nor hGH. In
differentiated and undifferentiated N2 cells transfected with pTK-GH,
consisting of the human growth hormone gene under control of the
thymidine kinase promoter, hGH was expressed in a nonspecific fashion
among cells and independent of their acquisition of goblet cell
phenotype as assessed by immunostaining for mucin glycoprotein (results
not shown).
Epithelial differentiation in the gastrointestinal tract is a
complex and dynamic process. Not only is tissue-specific phenotype
maintained along the longitudinal axis from esophagus to large bowel,
but in the normal mucosa, vertical differentiation from crypt to villus
is perpetuated as well. Moreover, differentiation into several
region-specific subpopulations is observed within the epithelium along
the length of the gastrointestinal tract. Among the growing list of
cloned genes whose products are intestine-specific, ITF represents the
first within the gastrointestinal tract exclusively expressed by goblet
cells. In this paper we report the cloning of the rat intestinal
trefoil protein gene and the characterization of its 5`-flanking
region.
ITF is only the second gene from among the members of the
family of trefoil proteins to be analyzed. Although the protein
products of the ITF and pS2 genes share the characteristic intrachain
disulfide bonding constituting the trefoil P domain, the regulatory
regions of these otherwise distinct genes appear to share little
homology. Strict regulation of the expression of these two trefoil
proteins along the longitudinal axis of the gastrointestinal tract,
with pS2 normally expressed in the gastric antrum and ITF expressed in
increasing amounts in the small and large bowel, may account for the
relative lack of shared elements in their promoter region.
The
family of trefoil proteins, including ITF, have been implicated in
mucosal healing following injury
(18) . An ulcer-associated cell
lineage has been reported to appear adjacent to areas of
gastrointestinal ulceration, with cells containing EGF-immunostaining
material in the base of the newly budding cell lineage and trefoil
protein-producing cells appearing more distally along the developing
ductule
(49) . Recent reports of enhancement of healing by
trefoil proteins, including ITF, in an in vitro model of
intestinal epithelial injury
(18) and in vivo (50) further supports the notion that the trefoil proteins are
important to the maintenance of the mucosal barrier of the intestine.
Whether the abundantly expressed and luminally secreted trefoil
proteins accomplish this by contributing to the physical properties of
the mucoviscous layer or by specific ligand-receptor interactions
remains unknown. ITF has been observed to be induced in the stomach in
animal models of gastric ulceration
(20) , whereas physiologic
expression is normally restricted to the small and large bowel
(16, 17) (with the reported exception of the mucous
cells of the gastric cardia)
(49) .
The presence of an
EGF-responsive element in the 5`-flanking region of the pS2 gene
(22) has led to speculation about the role of EGF in inducing
expression of trefoil proteins in a response to mucosal injury
(51) . Although scrutiny of the 5`-flanking region of the ITF
gene demonstrates no significant homology to known EGF response
elements, such an element may still be present and could now be
localized using the deletion constructs reported here. In addition, the
signals which lead to the expression of ITF in response to ulceration,
whether a consequence of altered cytoskeletal structure from loss of
contact inhibition or from local release of cytokines or other
mediators, remain obscure and may be more easily unravelled with
isolation of the ITF gene and promoter.
Constructs containing
segments of the 5`-flanking region of the ITF gene were found to be
capable of directing tissue-specific expression to cells with a goblet
cell phenotype, indicating the presence of goblet cell-specific
promoter elements or conversely non-goblet cell repressor elements
within the length of promoter examined. Indeed, relatively high levels
of specific expression with promoters as short as 153 bp of the
5`-flanking region suggests that such an element or elements are
present within close proximity to the transcriptional start site. As a
marker of intestinal goblet cell differentiation, the relatively short
ITF gene may have a distinct advantage over the lengthy mucin genes
(1, 2, 3, 4, 5, 6, 7) for use in transgenic animal experiments or tissue-directed
gene therapy.
A search of the 5`-flanking region of the ITF gene
reveals none of the known regulatory elements which have been
demonstrated to play a role in intestine-specific expression.
Specifically, the SIF-1, -2 and -3 elements of the
sucrase-isomaltase gene
(26) are lacking in the rat ITF
promoter. No areas of significant homology appear to exist between the
promoter of the rat ITF gene and the reported 5`-flanking regions of
the genes for human intestinal alkaline phosphatase, human intestinal
fatty acid-binding protein, porcine aminopeptidase N, human and mouse
sucrase-isomaltase, or human lactase-phlorizin hydrolase. Of interest,
the 5`-flanking region of rat ITF contains a Pit-1 homeodomain
consensus binding site. Although initially described as a
cis-regulatory element specifically directing expression in pituitary
cells
(41) , the presence of this element in a rat intestinal
gene suggests that it might play a role in tissue-specific gene
expression beyond the pituitary, an effect which might be
context-specific, depending upon additional as yet undescribed
regulatory elements. Although lacking a classic Pit-1 homeodomain, the
rat I-FABP gene shares with the rat ITF gene a 14-bp homeodomain-like
element (-429 to -416).
Transient expression of deletion
constructs containing various lengths of RITF gene 5`-flanking region
ligated to a luciferase reporter gene indicate that a length of
5`-flanking region as short as the 153 bp contiguous with the
transcriptional start site is capable of preferentially promoting
expression in cells with a goblet cell-like phenotype. Reporter gene
expression in LS174T cells, a human colon cancer-derived cell line
capable of expressing both mucin glycoprotein and native ITF in a
fashion typical of goblet cells, was nearly 10-fold higher than the
level of expression observed in undifferentiated Caco-2 cells, even
when corrected for differences in transfection efficiency.
Transient
expression experiments comparing undifferentiated and differentiated H2
cells and LS174T cells demonstrate a gradient of expression which
parallels the ability to express native ITF. A small number of H2 cells
grown in conditions not conducive to differentiation nevertheless
spontaneously acquire a goblet cell-like phenotype and secrete mucin,
consistent with the low, but not undetectable, level of luciferase
activity in undifferentiated cells transfected with -1671
RITF-Luc. Even in the presence of differentiation promoting culture
conditions, acquisition of the goblet cell-like phenotype is not
uniform
(44) , accounting for the level of expression which is
nearly 2-fold greater than that observed in undifferentiated H2 cells,
but still less than that observed in LS174T cells, which uniformly
exhibit a goblet cell-like phenotype. Co-localization of mucin
glycoprotein and reporter gene expression under the control of the RITF
promoter in H2 cells, which like N2 cells are capable of achieving a
goblet cell-like phenotype, lends further support to the goblet
cell-specific nature of the RITF promoter. Based on these observations
it is likely that the proximal 5`-flanking region of the rat ITF gene
contains elements which are capable of directing intestine and goblet
cell-specific expression.
We thank Drs. Timothy Wang, Loyal Tillotson, and Mark
Babyatsky for helpful suggestions in carrying out transfection studies.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
is a third member of the
trefoil peptide family identified in man
(16) and in rat
(17) which contains a single P domain. In contrast to pS2 and
hSP, ITF is normally selectively expressed in the normal small and
large intestinal mucosa complementing the pattern of expression of the
other members of the family in the normal gastrointestinal tract
(16, 17) . More specifically, ITF expression is normally
confined to the goblet cell population with the intestinal epithelium
(17) .
Genomic Library Screening, Mapping, Subcloning, and
Sequencing
A rat genomic library constructed by a partial digest
of DNA from an adult male Sprague-Dawley rat with Sau3A cloned
into EMBL3/SP6/T7 phage was obtained from Clontech Laboratories,
Inc. (Palo Alto, CA). This library contains 2.3
10
independent clones with an average insert size of 16 kb (range:
8-20 kb). Phage were plated out on 150-mm plates at a density of
5
10
plaque-forming units per plate in a bacterial
lawn of competent Escherichia coli NM538. After 8 h of growth
at 37 °C duplicate nitrocellulose filter lifts were made. After
phage denaturation and cross-linking by standard methods, membranes
were prehybridized in a solution containing 50% formamide, 5
SSC (SSC: 0.15
M NaCl, 0.05
M sodium citrate), 20
m
M Tris-Cl, pH 7.5, 5
Denhardt's solution, 0.1%
sodium dodecyl sulfate, 100 µg/ml of salmon sperm DNA at 42 °C
for 2 h. Membranes were then placed in a solution identical to the one
used for prehybridization substituting 10% dextran sulfate for SDS and
using 3
10
cpm/ml of
P-labeled T3411
probe. T3411, the rat intestinal trefoil factor cDNA described
previously
(17) , was labeled by random priming using Klenow DNA
polymerase (U. S. Biochemical Corp.)
(30) . Push columns
(Primerase columns; Stratagene, La Jolla, CA) were used to remove
unincorporated nucleotides. Hybridization was performed for 48 h at 42
°C, followed by three 10-min washes at room temperature in 2
SSC, 0.1% SDS, and then two 20-min washes at 37 °C in 0.2
SSC, 0.1% SDS. Membranes were blotted and exposed overnight to
Kodak XAR film with intensifying screens at -70 °C. Positive
plaques were picked and phage eluted into
diluent before
secondary screening on 100-mm plates. Conditions for secondary and
tertiary screening were performed in a manner identical to primary
screening.
(
)
Plate lysate was spun at
10,000
g for 10 min at 4 °C and the supernatant
collected and subjected to 72,000
g for 3 h at 4
°C. Pellets were resuspended in 500 µl of
diluent, two
samples pooled, spun briefly to pellet contaminants, and the cleared
supernatant layered over a CsCl gradient with SM (SM: 100 m
M NaCl, 10 m
M MgSO
, 50 m
M Tris-HCl, pH
7.5, 0.01% gelatin) as the diluent, set up as 1 ml with
=
1.7, 1 ml with
= 1.5, and 2 ml with
= 1.45 in
a 13
51-mm centrifuge tube. Gradients were subjected to 120,000
g at 20 °C for 1 h. Intact bacteriophage was
collected from the interface of
= 1.5 and 1.45 and
dialyzed twice for 1 h against 100 m
M NaCl, 50 m
M Tris-HCl, pH 8, 10 m
M MgCl
. Phage solution
was digested with 50 µg/ml proteinase K at 65 °C for 4-12
h and phage DNA extracted with phenol once, phenol/chloroform once, and
chloroform once, then ethanol precipitated and resuspended in 50 µl
of TE buffer (10 m
M Tris-Cl, pH 8, 1 m
M EDTA).
data base and compared with the
sequence of the 5`-flanking region of the rat ITF gene using the local
and global homology search capabilities of the ALIGN PLUS program
(Scientific & Educational Software, State Line, PA).
RNase Protection Assay
The transcriptional start
site was established by RNase protection assay based upon the sequence
established from the subcloned gene and its 5`-flanking region. A
315-bp fragment straddling the junction of the 5`-flanking region and
the first exon was amplified by a polymerase chain reaction primed with
synthetic oligonucleotides designed as a sense strand 23-mer starting
230 bp upstream of the translational start site and an antisense 24-mer
61 bp 3` to the translational start site. The RITF gene subcloned into
pKS+ plasmid was used as template, and 35 cycles of 94 °C for
1 min, 64 °C for 1.5 min, 72 °C for 2 min were performed,
followed by 72 °C for 8 min to complete extension. PCR product was
polished with Klenow fragment and subcloned into the SrfI site
of the plasmid vector PCR Script (Stratagene), and competent XL1-Blue
cells were transformed. Plasmid DNA was prepared with plasmid affinity
columns (Wizard Miniprep; Promega). Orientation and sequence was
confirmed by sequencing with universal primers as described above. The
plasmid was linearized by digestion with NotI to provide a 5`
overhang, and a 315-bp P-labeled riboprobe was generated
by in vitro transcription with priming at the T7 site of the
PCR Script vector using the MAXIscript in vitro transcription
kit (Ambion; Austin, TX). Probe was purified on a 5% acrylamide, 8
M urea gel prior to hybridization with total RNA from rat
colon and RNase digestion using the RPA II Kit (Ambion; Austin, TX).
Products of digestion were resolved by electrophoresis on a 5% Long
Ranger, 8
M urea gel (AT Biochem; Malvern, PA) and exposed to
Kodak XAR film at -70 °C between two intensifier screens.
Primer Extension
Confirmation of the predicted
transcriptional start site was carried out by primer extension using a
primer designated RITF-C (5`-caggaggacgtttcgggtcctt-3` within exon I of
the RITF gene (predicted nucleotides +86 to +107). After
labeling of the primer 5` end with [-
P]ATP
by standard techniques, primer extension was carried out by the method
of Sambrook et al. (31) after initial precipitation of
the primer with 150 µg of total RNA prepared from rat colonic
mucosa in 100 µl by the addition of 3
M sodium acetate (10
µl) and absolute ethanol (250 µl) at -70 °C. Products
of primer extension were evaluated on an 8% Long Ranger gel run side by
side with a sequence ladder produced with the RITF 20C plasmid, using
the RITF-C oligonucleotide as primer (1 pmol/m),
S-dATP
(DuPont NEN and the Sequenase version one sequencing kit (U. S.
Biochemical Corp.).
Construction of Luciferase Plasmids
The
promoterless luciferase gene construct pXP2
(34) was a gift
from Dr. Lee Kaplan. Starting with pRITF20C, an 8.7-kb EcoRI
fragment of one cross-hybridizing phage containing the entire RITF gene
as well as 1.7 and 1.8 kb of 5`- and 3`-flanking regions,
respectively, was subcloned into pKS+ Bluescript and the plasmid
cut at the NcoI site, a single base pair upstream of the start
ATG codon. The 5` overhang was polished by mung bean nuclease digestion
followed by ligation with phosphorylated XhoI linker using T4
DNA ligase. The plasmid was then digested with BamHI to
completion, and the resulting 1.7-kb fragment, containing only the
5`-flanking region, was resolved on a 1% agarose gel and isolated by
digestion with GELase (Epicenter Technologies; Madison, WI) according
to the manufacturer's instructions and ethanol precipitation. The
multiple cloning site of pXP2 was digested with BamHI and
XhoI to completion and double-cut vector was isolated from a
1% agarose gel with Geneclean (BIO 101; La Jolla, CA) by the
manufacturer's instructions. The 1.7-kb
BamHI- XhoI fragment was ligated into prepared vector
to form the construct -1671 RITF-Luc and transformed into
competent E. coli DH5
cells (Clontech Laboratories; Palo
Alto, CA). To confirm correct orientation and preservation of the start
codon of luciferase, plasmid DNA was subjected to restriction mapping
and sequencing of the insertion junctions.
1.7-kb
BamHI- BglII fragment was isolated electrophoretically
on a 1% agarose gel and isolated by GELase as above. p
GH (Nichols
Institute; San Juan Capistrano, CA), a plasmid containing a polycloning
site preceding the human growth hormone gene, was digested with
BamHI and dephosphorylated with calf intestinal alkaline
phosphatase. To increase the efficiency of cloning, BamHI-cut
and dephosphorylated p
GH was ligated with T4 DNA ligase, and
unligated plasmid was electrophoretically separated from undigested and
religated plasmid on a 1% agarose gel and isolated using Geneclean (BIO
101). The BamHI- BglII fragment of -1671
RITF-Luc and BamHI-cut p
GH were ligated using T4 DNA
ligase, taking advantage of the compatibility of BamHI and
BglII cohesive ends. DH5
E. coli were
transformed and minipreps of plasmid DNA digested with EcoRI
to screen for clones with correct insert orientation. The new plasmid,
-1671 RITF-hGH, was sequenced at both insert junctions and
through the translational start site of the growth hormone gene to
confirm the proper sequence of the insert and the initiation site of
translation.
E. coli, grown in LB
media, and plated out. Positive colonies were selected by the method of
Grunstein
(35) using a 240-bp probe of the 3`-most end of the
5`-flanking region of RITF generated by PCR. Synthetic 19-mer primers
for PCR were selected based on the known sequence of the 5`-flanking
region of the RITF gene and subjected to denaturing at 95 °C,
annealing at 42 °C, and extension at 72 °C for 30 cycles,
followed by a terminal extension at 72 °C for 8 min. Correct size
of probe was confirmed on agarose gel and identity confirmed by mapping
with restriction digestions. Probe was radiolabeled by nick translation
with [
-
P]dCTP and
[
-
P]dATP. Unincorporated nucleotide was
eliminated chromatographically using a Primerase column (Stratagene).
All deletion constructs were mapped by restriction digestion and the
insert-vector junctions confirmed by sequencing as above. Plasmid for
transfections was prepared by alkaline lysis and resin column
purification (Qiagen; Chatsworth, CA). Plasmid preparation purity was
confirmed by A
/ A
of
>1.6, and supercoiling of DNA was established by the appearance on
agarose gel electrophoresis prior to use in transfection experiments.
Cell Culture, Transfections, and Reporter Gene
Assays
Caco-2 cells, an intestinal cell line capable of
enterocyte-like differentiation obtained from the ATCC, were grown in
Dulbecco's modified Eagle's medium supplemented with 4.5
g/liter
D-glucose, 25 m
M HEPES, 10% fetal calf serum,
4 m
M
L-glutamine, 50 units/ml penicillin, and 50
µg/ml of streptomycin. Caco-2 cells were grown at confluence for at
least 2 weeks prior to use in transfections. LS174T cell line also
obtained from the ATCC was maintained in Eagle's minimum
essential medium with Eagle's balanced salt solution,
non-essential amino acids, sodium pyruvate,
L-glutamine,
penicillin, and streptomycin. The H2 subclone of the HT-29 cell line
obtained as a gift from Dr. Daniel Louvard was grown in either
Dulbecco's modified Eagle's medium with glucose
(nondifferentiating conditions) or in the presence of galactose as the
sole source of carbohydrate to induce goblet cell-like differentiation
as described previously
(36, 37) . All cell lines were
grown in 5% COat 37 °C.
10
cells were plated
out in triplicate in 35-mm wells of a six-well cell culture plate.
Complete media was refreshed 2 h prior to transfection. Efficiency of
transfection was standardized by co-precipitation of the construct of
interest with pTK-GH, consisting of the minimal thymidine kinase
promoter driving the human growth hormone gene as a reporter gene
(38) , and adjusting for the amount of human growth hormone
expressed, as determined by a commercially available radioimmunoassay
(hGH Allegro Kit; Nichols Institute Diagnostics). Calcium
phosphate-precipitated plasmid DNA was added to each well and incubated
at constant 5% CO
for 4 h before a 2-min exposure to 15%
glycerol. Cells were subsequently cultured for 48 h prior to assay for
reporter gene expression. For luciferase activity, cells were lysed and
assayed immediately for light production in the presence of luciferase
substrate using a commercial luciferase assay system (Promega) measured
in a luminometer (Analytical Luminescence Laboratory, Monolite 2010).
Luciferase activity was expressed as relative light units. All
luciferase activity was adjusted for transfection efficiency reflected
in the level of growth hormone, expressed as nanograms of hGH/ml of
medium. Where noted, promoter activity was expressed as a percentage of
the expression of the maximal promoter construct RSV-Luc (a gift from
Dr. Loyal Tillotson), consisting of the RSV promoter joined to the
luciferase gene.
10
cells/flask 16 h prior to transfection. Complete medium was
replaced with serum-free medium and 2 µg/flask plasmid DNA, either
-1671RITF-hGH or pTKGH, was combined with 1.5 µl of cationic
liposome (Transfectam; Promega) and added to the cells in culture.
Sixteen hours later, complete medium was returned to the cells. After
48 h of further culture, cells were fixed and dried. Slides were
blocked with 1.5% normal goat serum for 30 min at 25 °C and then
incubated with guinea pig-anti-human growth hormone (Arnel; New York,
NY) at 1:20,000 in L4-1-3 supernatant (a mouse monoclonal anti-human
mucin previously described; Ref. 39) for 1 h at 25 °C. After
washing with PBS, slides were further incubated with
rhodamine-conjugated goat-anti-guinea pig IgG (Cappel; Durham, NC) 0.12
mg/ml and fluorescein-conjugated goat-anti-mouse IgG (Cappel) 0.24
mg/ml at 25 °C for 1 h, followed by washing with PBS, drying, and
mounting with glycerol:PBS (6:1). Samples were examined using a Bio-Rad
MRC 600 scanning confocal imaging system attached to a Zeiss Axiovert
35 inverted microscope (Zeiss; New York). The fluorescence of
fluorescein-anti-mouse IgG and rhodamine-anti-guinea pig IgG was
excited using the 488- and 568-nm lines, respectively, of the
argon-krypton mixed gas laser in the confocal microscope. The confocal
parameters of scan rate, aperture, gain, black level, and frames
accumulated were the same for all samples.
Cloning of the Rat ITF Gene
The rat genomic
library was screened with the full-length cDNA probe of rat ITF. This
probe has been shown previously to specifically hybridize with ITF mRNA
in Northern blot analysis and in situ hybridization
(17) . Screening of 500,000 plaques yielded four independent
phage picks. Analysis by Southern blot hybridization revealed that all
four phage picks contained the entire ITF gene within an 8.3-kilobase
EcoRI fragment. This fragment was subcloned into the
EcoRI site of pKS+ Bluescript, and the new plasmid was
named pRITF20C. The subcloned fragment contained approximately 1.7 kb
of the 5`-flanking region and 1.8 kb of the 3`-flanking region. The
gene was observed to be divided into three exons. Approximate
exon-intron borders were noted by divergence of the genomic sequence
from the cDNA sequence. As depicted in Fig. 1, Exon I extended
119 bp, Exon II extended 144 bp, and Exon III extended 184 bp, whereas
Introns A and B were approximately 2.3 and 2.0 kb, respectively.
Figure 1:
Structure of
RITF20 phage clone of the RITF gene. An 8.3-kb EcoRI
fragment of the genomic clone was inserted into pBluescript KS+
and designated pRITF20C. The relative locations of each of the three
exons are indicated, along with restriction sites utilized for
subcloning and creation of deletion constructs. The entire 5`- and
3`-flanking regions ( FR) and exon-intron borders were
sequenced using a strategy of sequential overlapping primers. S, SfiI; E, EcoRI; H,
HincII; X, XbaI; N, NcoI;
B, BamHI.
Identification of Transcriptional Start Site
The
transcriptional start site of the ITF gene was first established by
ribonuclease protection assay using total RNA isolated from rat colon
hybridized to a 315-bp probe spanning an interval from 224 bp 5` to the
translational start site to 91 bp 3` to the first nucleotide of the
start codon. As demonstrated in Fig. 2 A, optimized
digestion with ribonuclease yielded a single protected fragment,
corresponding to a transcriptional start site located 34 bp 5` to the
translational start site and 27 bp 3` to the presumed TATA box. The
putative single start site was confirmed by primer extension using RNA
obtained from rat colonic mucosa and a complementary primer from
nucleotide +86 to +107. As demonstrated in
Fig. 2B, a single product was identified of the precise
predicted size, confirming the putative transcriptional start site.
Figure 2:A, RNase protection assay of RITF
gene. The transcriptional start site of the RITF gene was first
established using an RNase protection assay. A 315-bp fragment spanning
-196 to +119 was amplified by polymerase chain reaction,
polished with Klenow fragment, subcloned into the SrfI site of
PCRScript plasmid. Plasmid linearized with NotI was primed at
the T7 site of the PCR Script vector, and a 315-bp antisense riboprobe
was generated using T7 RNA polymerase with incorporation of
[-
P]UTP (800 Ci/mmol; DuPont NEN). Probe
purification, hybridization, digestion, and electrophoresis were
performed as described under ``Materials and Methods.'' Probe
was hybridized with 10 µg of total RNA isolated from rat colon.
Sequencing ladders for A, C, G, and T, utilizing the antisense PCR
primer used to generate probe and pRITF20C as template, are seen to the
left. Right, a single undigested band is observed,
corresponding with the initiation site of transcription denoted by the
arrow along the sequence displayed at the far left. + and - indicate the sense and antisense strands.
B, Primer extension. RITF transcriptional start site was
confirmed by primer extension using a primer oligonucleotide from exon
I (putative nucleotides +86 to -107). Primer was
precipitated with 150 µg of total RNA isolated from rat colonic
mucosa. Hybridization and primer extension were carried out at 30
°C using the method of Maniatis (31). Precipitated products were
suspended in 9 µl of TE + 1 µl of 6
loading
buffer, heated at 85 °C for 10 min and then run in duplicate on an
8% polyacrylamide gel (90 watts
2 h). A sequence ladder using
RITF 20C plasmid DNA and
S-dATP was run simultaneously as
displayed in the figure. A single product was observed
( arrow) corresponding to the predicted start site identified
by consensus sequence and RNase protection.
Analysis of Rat ITF 5`-Flanking Region
The start
codon ATG was found to be at +34, embedded within a canonical
Kozak consensus sequence
(40) . Examination of the roughly 1.7
kb of cloned 5`-flanking region revealed a number of potential
transcriptionally active elements (Fig. 3). Over the roughly 280
bp spanning from -657 to -376, an AT-rich region was
observed, approaching 90% for these two nucleotides. A smaller region
of roughly 75% GC content was observed between -150 and
-105. The dinucleotide repeat ``AC'' was found repeated
20 times from -776 to -737. The sequence of the RITF
5`-flanking region was compared with the reported sequences of
5`-flanking regions of genes expressed in intestinal tissues, including
the human and mouse sucrase-isomaltase genes, the rat lactase-phlorizin
gene, the human and porcine aminopeptidase genes, and the human and rat
intestinal fatty acid binding protein genes
(23, 24, 25, 26, 27, 28, 29) .
Using the local and global search functions of the ALIGN program, no
areas of high homology were found, except for a 100-bp region of ITF
and rat intestinal fatty acid binding protein, which had 85% identity
between the two sequences. This corresponds to an Alu-like element of
the rat I-FABP gene
(23) . In addition, a cannonical Pit-1
homeodomain
(41) is present at -571 to -561. A
14-bp homeodomain-like element consisting of the nucleotides
ATTAAAATACATTT is found at -429, with the identical sequence
found at -615 of the rat I-FABP gene.
Figure 3:
Sequence of RITF gene. The entire
5`-flanking region contained within pRITF20C, as well as the exonic
sequences with presumed exon-intron borders, are displayed. Translated
regions of exons I, II, and III are indicated by bold italic
letters. Outlined letters represent corrections of the originally
published cDNA sequence of rat ITF. Stippled underlining denotes a 100-bp region with 85% identity between the rat ITF and
rat intestinal fatty acid-binding protein (rIFABP) genes, with an
Alu-like sequence. Vertical hatch underlining indicates a
Pit-1 homeodomain binding domain consensus sequence, whereas
diagonal hatch underlining denotes a 14-bp homeodomain-like
sequence found also in the 5`-flanking region of the rIFABP gene. An
open box underlines a region with 75% GC content, whereas
the solid box lies over overlapping potential AP-2 sites
contained within this region. The TATA box is underlined. Also
observed are 20 repetitions of the dinucleotide CA between -776
and -737 and a roughly 280-bp span between -657 and
-376 with
90% AT content.
Comparison with the
5`-flanking region of pS2, the only other trefoil protein for which a
genomic clone has been reported
(21, 22) , revealed no
striking homology. Specifically, the 13-base pair imperfect palindrome
considered to be the estrogen-responsive element of the pS2 promoter
(42) was not found in the 1.7 kb of RITF 5`-flanking region, nor
was a canonical estrogen-responsive element discovered. In addition, no
strong homology was found with the epidermal growth factor
(EGF)-responsive region of pS2
(22) or with other reported
EGF-responsive elements
(43, 44, 45) .
Expression of the Reporter Gene Luciferase under the
Control of Deletions of Rat ITF 5`-Flanking Region in Intestinal Cell
Lines
Constructs in which the reporter gene luciferase was
ligated to various lengths of the 5`-flanking region of the RITF gene
were transfected into LS174T cells, representing a goblet cell-like
intestinal cell line, and Caco-2 cells, representing a columnar
absorptive-like enterocyte phenotype. The level of expression of the
various deletion constructs containing from -1671 to -153
of the 5`-flanking region was between 8 and 18% of the expression
observed for the maximal promoter-reporter construct, RSV-Luc, in the
LS174T cells (Fig. 4). In contrast, the level of expression in
Caco-2 cells was consistently less than 2% that of RSV-Luc. The
relatively high level of expression in LS174T cells was observed even
in cells transfected with the construct containing only 153 bp of RITF
5`-flanking sequence (-153 RITF-Luc).
Figure 4:
Transient expression of RITF
promoter-luciferase constructs in intestinal cell lines. Results are
expressed as a percentage of luciferase activity of the RSV promoter
driving luciferase gene. Luciferase activity was measured as relative
light units adjusted for efficiency of transfection standardized by
co-transfection of pTK-GH, the human growth hormone gene under the
influence of the thymidine kinase promoter, measured as human growth
hormone immunoreactivity in the media of the transfected well using a
commercially available radioimmunoassay as described under
``Materials and Methods.'' Deletion constructs are numbered
according to the length of the 5`-flanking region relative to the
transcriptional start site as determined by RNase protection assay
depicted in Fig. 2. pXP2 is a promoterless luciferase
construct. Plasmid constructs were transfected by the calcium phosphate
precipitate method as described. CACO, human
intestinal cell line; LS174T, human intestinal cell line with
goblet cell-like phenotype. n = number of independent
transfections. Results are expressed as the mean ±
S.E.
In an experiment
assessing the level of expression of -1671 RITF-Luc in H2 cells
grown under differentiating and nondifferentiating conditions, as well
as LS174T cells, expression was found to be greatest in the LS174T cell
line (Fig. 5). More importantly, H2 cells grown under
differentiating conditions were found to express the reporter gene
after transfection by the method of calcium phosphate precipitate at
levels approximately 2-fold higher than that observed for H2 cells
grown in nondifferentiating conditions. Enhanced expression of the
RITF-luciferase in the H2 cells in association with differentiating
conditions was also observed when cells were transfected by an
alternative method using cationic liposome (data not shown). The levels
of luciferase activity of these three cell lines were proportional to
the concentration of ITF produced in their respective media as assessed
by Western blot. Thus, undifferentiated H2 cells produced minimal
amounts of ITF, whereas differentiated H2 cells produced greater
concentrations and LS174T cells produced nearly 10-fold
higher.(
)
Figure 5:
Transient expression of RITF-Luc in cell
lines with a goblet cell-like phenotype. Transfections of the
full-length construct -1671 RITF-Luc were performed by the
calcium phosphate method in the LS174T intestinal cell line and in the
H2 subclone of the HT29 intestinal cell line grown in
galactose-containing media to induce a goblet cell-like phenotype or in
glucose to maintain an undifferentiated phenotype. A representative
experiment is shown. Results are displayed as the mean ± S.E. of
relative light units standardized for transfection efficiency by
co-transfection with pTK-GH and human growth hormone activity as
nanograms/ml of medium. Asterisks denote p 0.01
by a two-sided Student's t test comparing the level of
expression to that observed in H2 cells grown in
glucose.
Immunofluorescent Localization of hGH
Expression Directed by the 5`-Flanking Region of RITF and
Co-localization with Mucin Glycoprotein Expression
To further
evaluate the ability of the 5`-flanking region of the RITF gene to
direct goblet cell-specific expression, we sought to determine the
relationship of reporter gene expression directed by the RITF promoter
to that of mucin glycoprotein characteristic of goblet cells in the N2
subclone of the HT29 colon cancer cell line. N2 cells grown under
differentiating and nondifferentiating conditions were transfected with
a construct consisting of the human growth hormone gene under the
control of -1671 RITF 5`-flanking region. Double
immunofluorescent staining using a commercially available guinea pig
anti-human growth hormone antibody and a monoclonal mouse anti-human
mucin glycoprotein, previously described, were used as the primary
antibodies. Use of rhodamine-labeled anti-guinea pig IgG and
fluorescein isothiocyanate-labeled anti-mouse IgG permitted
simultaneous co-localization by laser confocal microscopy of expressed
human growth hormone in N2 cells transfected with -1671 RITF-hGH
by cationic liposome.
Figure 6:
Co-localization of the expression of human
growth hormone under control of the RITF promoter with native mucin
expression in N2 cells. N2 cells, a subclone of the HT29 intestinal
cell line, were grown in galactose-containing media in glass culture
dishes to induce goblet cell differentiation before being seeded onto
glass slide flasks. The -1671 RITF-hGH construct was transfected
by a method of cationic liposome as described under ``Methods and
Materials.'' Slides were fixed 62 h after transfection and
incubated first with polyclonal guinea pig anti-human growth hormone
and monoclonal mouse IgG anti-human mucin glycoprotein as primary
antibodies, followed by rhodamine-conjugated goat anti-guinea pig IgG
and fluorescein-conjugated goat anti-mouse IgG as developing
antibodies. Laser confocal microscopy was performed as described to
permit co-localization of growth hormone and mucin. Specificity of
fluorescent staining was confirmed by absence of staining with omission
of primary antibody (not shown). A-C depict
representative fields of the goblet cell differentiated cells
transfected with -1671 RITF-LGH constituent. A,
laser-stimulated fluorescence of rhodamine appearing as areas of
red, indicating expression of ITF-human growth hormone.
B, laser-stimulated fluorescence of fluorescein appearing as
areas of green, localizing native mucin expression.
C, computer-assisted superimposition of images from A and B display cells co-expressing both human growth
hormone and mucin as yellow or orange. Expression of RITF-hGH was not
observed in transfected control N2 cells grown in nondifferentiating
(glucose-containing) medium ( D), although nearly all of the
nondifferentiated cells did express growth hormone after transfected
with a construct (pTK-GH) containing the human growth hormone under the
control of the nonspecific thymidine kinase promoter ( E). The
pTK-GH was similarly expressed in nearly all N2 cells maintained in
galactose-free media, including those which failed to exhibit goblet
cell phenotype. The appearance of N2 cells by conventional phase
contrast is depicted in F.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.