(Received for publication, November 21, 1996)
From the Departments of Molecular Biology and
Pharmacology, and § Pediatrics, Washington University School
of Medicine, St. Louis, Missouri 63110 and the
Department of
Pathology, University of Cincinnati School of Medicine,
Cincinnati, Ohio 45267
A 35-nucleotide sequence in the liver fatty
acid-binding protein gene (Fabpl) has been identified that
interacts with nuclear proteins present in adult mouse liver, kidney,
stomach, small intestine, and colon. The binding site consists of a
direct heptad repeat (TTCTGNNTT) separated by
five nucleotides. Both heptads are required for formation of stable
complexes with nuclear proteins in gel mobility shift assays. The
in vivo functions mediated by the repeats were determined
by comparing the expression of four Fabpl/human growth
hormone fusion genes in multiple pedigrees of adult transgenic mice.
The transgenes contained (i) nucleotides 596 to +21 of
Fabpl linked to the human growth hormone reporter, (ii) 4 additional copies of the 35-base pair element placed at nucleotide
596 of Fabpl, (iii) 4 additional copies of the sequence placed just upstream of its endogenous site at nucleotide
132, and
(iv) a sequence identical to (iii) but with all heptad repeats mutated
within each of the 4 additional copies of the 35-base pair element.
Transgene expression was defined by RNA blot hybridizations and by
light and electron microscopic immunohistochemistry. The heptad repeat
functions to suppress expression in tubular epithelial cells of the
proximal nephron, in hepatocytes, in the mucus-producing pit cells of
the gastric epithelium, and in absorptive enterocytes located in the
proximal small intestine. There is a gradient of escape from
enterocytic suppression as one moves from the proximal to distal small
intestine. This escape progresses to involve successively less
differentiated cells located closer and closer to the stem cell zone in
crypts of Lieberkühn. The heptad repeat activates gene expression
in the colonic epithelium so that all proliferating and
nonproliferating cells in colonic crypts distributed from the cecum to
the rectum support transgene expression. The heptad has no obvious
sequence similarities to known transcription factor binding sites,
suggesting that mediators of its in vivo activities are
likely to be novel. One candidate factor is a 90-kDa protein identified
in Southwestern blots. The 90-kDa protein also binds to an element in
the matrix metalloproteinase-2 gene that functions as an enhancer in
renal cells, shares sequence homology with the heptad, and generates
similar-sized complexes in gel mobility shift assays as the
Fabpl repeat. The heptad repeat represents a target for
identifying transcription factors that regulate gene expression between
gut and renal epithelia and that also regulate the differentiation
program of the intestine's principal epithelial lineage as a function
of its location along the duodenal-colonic axis. Finally, the
Fabpl regulatory elements described in this report should
be useful for delivering a variety of gene products throughout the
colonic epithelium of transgenic mice.
Epithelial cells that line the intestine and the nephrons of the kidney share many common functions. The molecular mechanisms that regulate gene transcription within and between these epithelia are largely uncharacterized. The "liver" fatty acid-binding protein gene (Fabpl)1 provides a model for investigating these mechanisms.
In the adult mouse and rat, Fabpl is transcribed in hepatocytes and in polarized absorptive enterocytes, the principal epithelial cell lineage of the small intestine (1). Fabpl exhibits a cephalocaudal gradient of expression within the intestine; highest levels of its mRNA and protein products are encountered in differentiated enterocytes that overlie villi located in the middle third of the small intestine; levels diminish as one moves proximally or distally. The gene is silent in the gastric and distal colonic epithelium of both species and in all other epithelia not associated with the gastrointestinal tract.
The contribution of cis-acting suppressors to maintaining this pattern
of expression was revealed from studies in transgenic mice (1). Seven
fusion genes were produced by sequential deletions of the proximal 4000 nucleotides of rat Fabpl's 5-nontranscribed domain and
linkage of each truncated product to nucleotides +3 to +2150 of the
human growth hormone (hGH) gene. The cellular and spatial patterns of
expression of each fusion gene were defined in multiple pedigrees of
adult transgenic mice. Cis-acting suppressors of cecal and colonic
transcription were identified between nucleotides
4000 and
1600.
Suppressors of gastric expression are positioned outside of nucleotides
4000 to +21. Multiple suppressor elements, distributed between
nucleotides
4000 and +21, help determine the intestinal epithelial
cell lineage-specific patterns of Fabpl expression (1).
A suppressor of kidney expression was also found. Although the
endogenous gene remains silent in the mouse and rat kidney throughout
adulthood, analyses of transgenic mice containing nucleotides 132 to
+21 of Fabpl revealed that the steady state concentration of
the hGH reporter mRNA was 10 times higher in kidney than in the
four other tissues where it was expressed (1). Renal expression of hGH
was confined to epithelial cells located in the proximal tubules of
nephrons. The relative levels of hGH mRNA in the other tissues were
small intestine > colon > stomach = liver. Addition of
a 54-bp sequence spanning nucleotides
186 to
133 suppressed kidney
hGH expression 40-fold relative to these four other tissues (1).
In the present report, we have determined that this 54-bp element contains a direct heptad repeat that binds nuclear proteins from kidney, small intestine, colon, and liver. The transcriptional regulatory activities of this repeat in kidney and gut epithelial cell lineages have been defined using transgenic mice. The results indicate that the heptad sequence binds novel transcription factors that modulate gene expression within and between different epithelia.
Preparation of Nuclear Extracts
Nuclear extracts were prepared from tissues harvested from 6- to 20-week-old male or female FVB/N mice. The entire extraction protocol was performed at 4 °C. The distal half of the small intestine was removed immediately after sacrifice, clamped at one end, infused with buffer A (10 mM HEPES, pH 7.6, 15 mM KCl, 2 mM MgCl2) supplemented with protease inhibitors (2 mM Pefabloc SC, 1 µM leupeptin, 1 µM pepstatin, 0.3 µM aprotinin, 130 µM bestatin) until it became distended, then opened with a longitudinal incision, placed in 20 ml of buffer A, and shaken for ~5 s. The washed segment was transferred to fresh buffer A and shaken again. The cycle was repeated three times. Luminal contents were manually extruded from the intact colon prior to preparation of nuclear extracts. All other tissues were placed directly in buffer A.
A Polytron (Tekmar) was used to disrupt each tissue (five 7-s bursts at full speed). The homogenate was centrifuged for 10 min at 750 × g. The nuclear pellet was resuspended in 20 ml of buffer A plus Nonidet P-40 (0.25%, v/v). Following a 1-min incubation, the suspension was centrifuged as above, and the nuclear pellet was resuspended to a final volume of 10 ml in buffer B (10% glycerol, 50 mM HEPES, pH 7.6, 50 mM KCl, 0.1 mM EDTA, plus the protease inhibitors listed above). Ammonium sulfate was added (1 ml of a 3 M solution). The suspension was shaken for 30 min to lyse nuclei and subsequently spun at 123,000 × g for 60 min to pellet chromatin. An equal volume of 3 M ammonium sulfate was added to the supernatant, and the solution was shaken for 30 min to precipitate nuclear proteins. After centrifugation at 123,000 × g for 30 min, the protein pellet was resuspended in 100 µl of buffer C (10% glycerol, 25 mM HEPES, pH 7.6, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT) and desalted by passage through a Biospin 6GD Sephadex G25 column (Bio-Rad) equilibrated in buffer C. Ten-microliter aliquots of the column effluent were snap-frozen in liquid N2 and stored under liquid N2 until use. The protein content of each extract was determined using the Bio-Rad Protein Assay kit.
The integrity of nuclear proteins was verified using a gel mobility
shift assay (GMSA) with a template consisting of an orphan steroid
hormone receptor binding site. This previously characterized element
(5-TCTCTTGAACTTTGAACTTCCACA-3
) was derived from nucleotides
86 to
63 of the rat intestinal fatty acid-binding protein gene promoter
(Fabpi) (2, 3) and formed complexes with nuclear extracts
from all tissues.
Gel Mobility Shift Assays
Templates for GMSA were made by annealing two complementary
single-stranded synthetic oligodeoxynucleotides (oligos). Oligos were
end-labeled with 33P using polynucleotide kinase
(Boehringer Mannheim). Each gel shift reaction (total volume = 20 µl) contained 12.5 mM HEPES, pH 7.6, 40 mM
KCl, 0.1 mM EDTA, 1 mM DTT, 7.5% glycerol
(w/v), 33P-labeled probe (50 fmol), poly(dI·dC) (6 ng,
Boehringer Mannheim), herring sperm DNA (6 ng, Sigma), and nuclear
extract (50-8000 ng protein). Following a 20-min incubation at room
temperature, the reaction was terminated by subjecting the mixture to
polyacrylamide gel electrophoresis (5% of 29:1
acrylamide:N,N-methylene-bisacrylamide in 45 mM Tris borate buffer, pH 8.0, 1 mM EDTA). All
GMSA assays were performed at least twice and with at least two
independent preparations of tissue nuclear protein extracts.
Southwestern Blots
Nuclear extracts (80 µg of protein/tissue) were fractionated
by electrophoresis through 7% polyacrylamide gels containing 0.1% SDS
according to the protocol described in Ausubel et al. (4)
with the following exceptions: DTT was omitted from the sample loading
buffer and the samples were not heated. Proteins were
electrophoretically transferred to supported nitrocellulose membranes
(Life Technologies, Inc.). Triplicate blots were preincubated for 30 min at 4 °C in 5% Blotto (25 mM HEPES, pH 7.6, 60 mM KCl, 1 mM DTT, 1 mM EDTA, 0.5 µg/ml sheared herring sperm DNA, and 5% nonfat dry milk (w/v))
followed by a 60-min incubation at the same temperature in 0.5%
Blotto. Each blot was probed with one of three concatenated
32P-labeled double-stranded oligos: (i)
5-ACAAACTTCTGCCTTGCCCATTCTGATTTTTATCG-3
(nucleotides
167 to
133
of rat Fabpl); (ii)
5
-ACAAACggagtCCggGCCCAggagtATggTTATCG-3
(identical to (i) but with
mutated bases indicated in lowercase); and (iii)
5
-AGTGGGTTTCTGACAAGTTCAGACTTGCCCAGCAG-3
(nucleotides
1319 to
1285
of the rat matrix metalloproteinase 2 gene; Ref. 5). Concatenation was
accomplished using the Rapid Ligation kit from Boehringer Mannheim and
40 pmol of the double-stranded oligo per 20-µl reaction. The specific
activity of each double-stranded oligo was identical (6000 Ci/mmol).
The entire reaction mixture was added to 5 ml of hybridization buffer
(0.5% Blotto). Following an overnight incubation with the concatenated
probe at 4 °C, blots were washed in 0.5% Blotto for 5 min and then
in a solution containing 25 mM HEPES, pH 7.6, 60 mM KCl, 1 mM DTT, and 1 mM EDTA (3 washes of 5 min, each wash done at 4 °C). DNA-protein complexes were visualized using a storage PhosphorImaging system (Molecular
Dynamics).
Generation of Transgenic Mice
Construction of Fabpl/hGH Fusion GenesThree recombinant
DNAs were produced. Fabpl4× at 596/hGH was
created as follows. A BamHI/EcoRI fragment
spanning nucleotides
596 to +21 of Fabpl was liberated
from pEPLFABP (6) and ligated to BamHI/EcoRI-digested pBluescript II
SK+ (Stratagene), yielding pTS9. Nucleotides +3 to +2150 of
hGH were removed from pBShGH (1) with BamHI and ligated to
BamHI-digested pTS9. pTS10 was identified by diagnostic
restriction digests as a ligation product with the 5
end of the hGH
gene joined to the 3
end of the Fabpl sequence. Two pairs
of complementary synthetic oligos were phosphorylated and annealed to
one another.
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A similar strategy was used to create a second fusion gene,
Fabpl4× at 132/hGH, which contains four
tandem copies of the same sequence as in
Fabpl4× at
596/hGH (i.e.
nucleotides
172 to
133) but inserted at Fabpl nucleotide
132.
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The same strategy used to create
Fabpl4× at 132/hGH was exploited to
generate the third fusion gene,
Fabpl4× mutated at
132/hGH, which contains
nucleotides
596 to +21 of Fabpl with four tandem repeats
added at Fabpl nucleotide
132. These repeats were mutated
copies of nucleotides
172 to
133, where each purine in each heptad
repeat was replaced with a pyrimidine from the opposite base pair and
vice versa.
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DNA fragments containing the three fusion genes were each purified from their vector sequences by agarose gel electrophoresis, extracted from the gel using the Qiaex system (Qiagen), and then used for pronuclear injection into fertilized FVB/N oocytes (7). Injected oocytes were transferred to pseudopregnant Swiss Webster mice using standard techniques (8).
Identification and Housing of Transgenic AnimalsLive-born
animals were screened at 10 days of age for the presence of transgenes
using the following protocol. DNA was isolated from 1-cm tail snips
after an overnight digestion at 55 °C in 0.4 ml of digestion buffer
(100 mM Tris, pH 8.0, 100 mM NaCl, 10 mM EDTA, 0.5 mg/ml proteinase K (Boehringer Mannheim)).
Following phenol/chloroform extraction and ethanol precipitation, the
DNA pellet was resuspended in 100 µl of water (typical yield of
DNA = 50-100 µg). Polymerase chain reaction was performed in
20-µl incubations containing 50 mM KCl, 20 mM
Tris, pH 8.4, 2 mM MgCl2, 3% glycerol, 10 µM cresol red, dNTPs (200 µM each), 0.5 unit of AmpliTaq polymerase (Perkin-Elmer), 100-500 ng of tail DNA,
and primers that recognize hGH and mouse -actin gene sequences (0.8 pmol of each). The hGH primers (5
-AGGTGGCCTTTGACACCTACCAGG-3
and
5
-TCTGTTGTGTTTCCTCCCTGTTGG-3
) span intron 2 and produce a 360-bp
product. The
-actin primers (5
-CACCACACCTTCTACAATGAGCTG-3
and
5
-TCATCAGGTAGTCAGTGAGGTCGC-3
) span intron 3 and produce a 451-bp
product. The reaction mixture was overlaid with wax (Chill-Out 14, MJ
Research), and subjected to 30 cycles of 1 min at 94 °C, 2 min at
55 °C, and 2 min at 72 °C.
Pedigrees from transgenic founder animals were established and maintained by crosses to non-transgenic FVB/N littermates. Serum hGH levels were measured by radioimmunoassay (Nichols Diagnostic). Pedigrees with high serum levels of hGH (>1 µg/ml) had to be maintained by ovarian transplantation (9). All mice were housed in microisolators on a 12-h light/dark cycle and given an irradiated diet (Pico rodent chow 20, PMI Feeds) ad libitum. Mice were determined to be specific pathogen-free based on the results of screening sentinel animals for hepatitis, minute, lymphocytic choriomeningitis, ectromelia, polyoma, Sendai, pneumonia, and mouse adenoviruses, enteric bacterial pathogens, and parasites.
Analyses of Transgene Expression
Transgenic mice and their normal littermates were sacrificed
between postnatal days 35 (P35) and 42. Some animals received an
intraperitoneal injection of 5-bromo-2-deoxyuridine (BrdUrd, 120 mg/kg
body wt) and 5-fluoro-2
-deoxyuridine (12 mg/kg) 90 min prior to their
death.
Immediately after sacrifice, several tissue samples were recovered. The small intestine was divided into quarters. A 2-cm section was taken from the center of each quarter and rapidly frozen in liquid nitrogen for subsequent RNA isolation. The colon was divided in half and a 2-cm section was taken from the centers of the proximal and distal halves, also for isolation of RNA. The stomach was divided in half along its greater and lesser curvatures. One-half was used for RNA extraction. A wedge of liver and one-half of one kidney were also used to prepare RNA. The remainder of these tissues, plus portions of heart, lung, spleen, pancreas, skin, white adipose tissue, skeletal muscle, and brain were fixed in Bouin's solution at room temperature for 8-18 h, rinsed in ethanol, and embedded in paraffin for immunohistochemical analysis.
Quantitative RNA Hybridization StudiesTotal cellular RNA was isolated from frozen pulverized tissues using RNeasy columns (Qiagen). RNA samples were fractionated by denaturing formaldehyde agarose gel electrophoresis and transferred to Nylon-1 membranes by capillary blotting (Life Technologies, Inc.). The membranes were probed with hGH+3 to +2150 DNA labeled with 33P using random primers (1). Each blot contained reference RNA samples containing known amounts of hGH mRNA. The intensity of the hybridization signal from each mouse tissue RNA sample was quantitated using a storage PhosphorImaging system. Blots were stripped and reprobed with a 33P-labeled human glyceraldehyde-6-phosphate dehydrogenase cDNA (American Tissue Culture Collection). hGH mRNA concentrations in tissue RNAs were calculated by comparing the sample signal to the signals from the RNA standards after normalizing to glyceraldehyde-6-phosphate dehydrogenase mRNA levels.
Single and Multi-label ImmunohistochemistryFive-micron
thick sections prepared from paraffin-embedded samples of stomach,
small intestine, cecum, colon, liver, kidney, pancreas, spleen, white
adipose tissue, skeletal muscle, brain, heart, and lung were incubated
overnight at 4 °C with sheep anti-hGH (Cortex Biochem; diluted
1:10,000 in blocking buffer (1% bovine serum albumin, 0.02% non-fat
dry milk, 0.3% Triton X-100 in PBS)). Bound antibodies were detected
by adding biotinylated donkey anti-sheep immunoglobulins (Ig; Jackson
ImmunoResearch; 1:1000) for 1 h at room temperature, followed by
streptavidin peroxidase conjugate (Vector Laboratories) for 30 min.
Metal-enhanced 3,3
-diaminobenzidine (DAB, Pierce Chemical) was used
as the peroxidase substrate (incubation = 15 s at room
temperature). Sections were counterstained with hematoxylin and
eosin.
Triple label fluorescent visualization of liver fatty acid-binding
protein (L-FABP), hGH, and BrdUrd in the intestinal epithelium was
accomplished using the following multistep protocol: (i) bovine pancreas -chymotrypsin (1 mg/ml in 0.1% CaCl2) for 15 min at 37 °C (antigen unmasking step); (ii) rabbit anti-L-FABP sera
(1:500; Ref. 6) overnight at 4 °C; (iii) normal donkey serum (1:50, Jackson ImmunoResearch) for 1 h at room temperature; (iv)
Cy5-conjugated donkey anti-rabbit immunoglobulin (1:500, Jackson
ImmunoResearch) for 1 h at room temperature; (v) sheep anti-hGH
(1:1000) overnight at 4 °C; (vi) Cy3-donkey anti-sheep Ig (1:500,
Jackson ImmunoResearch) for 1 h at room temperature; (vii) normal
goat serum (1:50, Jackson ImmunoResearch) for 1 h at room
temperature; (viii) goat anti-BrdUrd ((10); 1:1000, biotinylated using
a kit from Boehringer Mannheim) overnight at 4 °C; and (ix)
Cy2-conjugated streptavidin (5 µg/ml, Amersham Corp.) for 1 h at
room temperature. Stained sections prepared from normal and transgenic
mice were then scanned with a Molecular Dynamics Multiprobe 2001 inverted confocal microscope system.
Full thickness pieces of gut (each 1 × 6 mm) were cut from the center of each quarter of the small intestine and from the center of each half of the colon. Each piece was immersed for 4 h at 4 °C in phosphate-buffered saline (PBS) containing 2% paraformaldehyde and 0.2% glutaraldehyde. The fixed fragments were washed 3 times with PBS, 3% sucrose, left in fresh buffer overnight at 4 °C, dehydrated with graded ethanols, and embedded in Lowicryl K4M (Polysciences). Sections (50-70 nm) were cut along the plane of the crypt-villus axis and picked up on slotted nickel grids that were coated with Formvar and stabilized with evaporated carbon film (Electron Microscopy Sciences). Grids were floated for 30 min at room temperature on drops of Tris-buffered saline (TBS; 20 mM Tris, 150 mM NaCl, pH 7.4), containing 10% normal mouse serum (Jackson ImmunoResearch) and 0.3% Tween 20. Blocked grids were then transferred to drops of sheep anti-hGH (diluted 1:100 in TBS, 5% normal mouse serum, 0.3% Tween 20). After a 2-h incubation at room temperature, grids were washed with TBS/Tween (3 cycles; 5 min each). Antigen-antibody complexes were visualized with 18 nm colloidal gold conjugated-goat anti-rabbit IgG as described previously (11). Air-dried sections were stained with 4% aqueous uranyl acetate and Reynolds lead citrate (Electron Microscopy Sciences) and viewed under a JOEL Model X100 electron microscope. The specificity of the immunostaining was established by performing two types of control experiments: omitting the hGH antisera or incubating intestinal specimens obtained from normal (nontransgenic) mice with both primary and secondary antibodies.
In Vitro Analyses
Fabpl Contains a Direct Heptad Repeat That Binds Proteins Present in Kidney Nuclear ExtractsGel mobility shift assays (GMSA) were
performed to identify transcription factor binding sites that mediate
suppression of Fabpl in renal proximal tubular epithelial
cells. Nuclear extracts, prepared from adult FVB/N mouse kidneys,
formed a prominent complex with a double-stranded oligo spanning
nucleotides 187 to
123 of rat Fabpl (Fig.
1A plus Fig. 1B, lane 5). A
64-fold molar excess of unlabeled double-stranded template markedly
reduced the amount of labeled complex formed (Fig. 1B, lanes
2-5).
A series of shorter double-stranded oligos, derived from nucleotides
187 to
123, were used as templates and competitors in subsequent
gel mobility shift assays. The results, summarized in Fig. 1,
A-C, revealed that nucleotides
167 to
133 encompassed the nuclear factor binding site(s). This 35-base sequence contains a
direct repeat, 5
-TTCTGNNTT-3
, separated by a 5-nucleotide "spacer":
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Three prominent complexes formed when gel mobility shift assays were
performed with kidney nuclear extracts and nucleotides 167 to
133
(designated A, B, and C in lane
2 of Fig. 2A). Complex formation
was abolished by a 128-fold molar excess of the unlabeled double-stranded template (Fig. 2A, lane 3).
To test whether the heptad repeats contained within nucleotides
161 and
139 were required for binding kidney nuclear proteins, competition assays were performed with mutated oligos. The mutations involved replacement of purines in each heptad with a pyrimidine from
the opposite base pair and vice versa. When all purines in both repeats
were mutated (oligo 13 in Fig. 2B) the ability to effectively compete with the wild type template was lost (Fig. 2A, lane 5). In addition, the "completely" mutated oligo
13 was unable to function as a template for binding, i.e.
neither complex A, B, or C was observed when radiolabeled
double-stranded oligo 13 was incubated with kidney nuclear proteins
(data not shown). An oligo with the TTCTG component of the heptad
NN mutated and the TT dinucleotide left
intact (oligo 14 in Fig. 2B) blocked formation of complex A
more effectively than complex B or C (Fig. 2A, lane 6). In
contrast, an oligo with the TTCTG component left intact and the TT
dinucleotide mutated (sequence 15) blocked formation of complex C more
effectively than complex A or B (Fig. 2A, lane 7). These
findings indicate that the TT dinucleotide in the heptad repeats is
important for generating complex A, and the TTCTG pentanucleotide component of the heptads is important for assembly of the B and C
complexes.
Oligos with either the upstream or the downstream heptad repeat mutated
were not effective competitors for complex formation with the native
167 to
133 template nor were they able, when radiolabeled, to form
detectable complexes with kidney nuclear proteins (data not shown). To
further test the hypothesis that both heptads are necessary for complex
formation under the conditions of the GMSA, we attempted to block
formation of these complexes with a 128-fold molar excess of an oligo
spanning the upstream heptad with five extra bases at both ends
(sequence 16 in Fig. 2B) and an oligo encompassing the
downstream heptad with five extra bases at both ends (sequence 17).
Neither sequence alone nor both oligos together were effective
competitors (lanes 8-10 in Fig. 2A).
Kidney nuclear proteins were fractionated by SDS-polyacrylamide gel
electrophoresis. Nitrocellulose blots of the gels were probed with
double-stranded oligos containing both heptads (nucleotides 167 to
133; sequence 11) or with both heptads mutated (sequence 13). A
prominent ~90-kDa reactive protein was seen when blots were probed
with the wild type sequence (Fig. 3A).
Binding to this protein was markedly reduced with the mutant oligo
(Fig. 3B). This Southwestern blotting result provides
further evidence that kidney nuclear protein(s) specifically bind to
the heptad repeats.
Kidney nuclear extracts were also subjected to Superdex 200 gel filtration chromatography using nondenaturing conditions. Column fractions were analyzed by GMSA employing radiolabeled double-stranded oligo 11 as the template. Complex forming activity was restricted to fractions in two size classes. Nuclear proteins that eluted with a estimated size of 70,000 formed single complexes with mobilities equivalent to that of "C." Proteins that eluted in the size range of 200,000-240,000 formed complexes B and C (data not shown).
An Enhancer Element in the Matrix Metalloproteinase-2 Gene with Homology to the Fabpl Heptad Repeat Forms Similar Complexes with Kidney Nuclear ProteinsThe TRANSFAC data base (12) was used to search
nucleotides 167 to
133 of Fabpl for known transcription
factor binding sites. No significant similarities were detected.
However, significant sequence homology was noted between the heptad
repeat and an enhancer element in the rat matrix metalloproteinase-2
gene (MMP-2). MMP-2 is normally expressed at high levels in the
developing mouse kidney and lung (13). In adult animals, expression is
markedly repressed in these tissues. Using cultured glomerular
mesangial and epithelial cells, Harendza et al. (5)
identified an enhancer spanning nucleotides
1322 to
1282 of MMP-2.
Fig. 4A shows the homology between this
enhancer and Fabpl's heptad repeats.
An oligo representing nucleotides 1319 to
1285 of rat MMP-2
competes for complex formation with nucleotides
167 to
133 of rat
Fabpl as effectively as the Fabpl sequence itself
(compare lanes 3 and 4 in Fig. 4B). In
addition, the MMP-2 sequence forms complexes with nuclear proteins
prepared from adult FVB/N kidney (lane 7). These complexes
have mobilities indistinguishable from those formed with nucleotides
167 to
133 of Fabpl (compare lanes 2 and
7 in Fig. 4B). Both the MMP-2 sequence and the
Fabpl sequence compete for complex formation with the
radiolabeled MMP-2 template (lanes 8 and 9).
Finally, the oligo with the mutated heptad repeats (sequence 13 in Fig.
2B) is not as effective a competitor for complex formation
between kidney nuclear proteins and the MMP-2 template as the oligo
with the authentic Fabpl heptad repeats (compare lanes
9 and 10 in Fig. 4B).
The MMP-2 element reacts with two prominent proteins in Southwestern
blots of kidney nuclear proteins: one has the same mobility as the
90-kDa protein that binds nucleotides 167 to
133 of
Fabpl and the other is larger (140 kDa; Fig. 3C).
This result provides further evidence that the heptad repeats in
Fabpl and the homologous MMP-2 enhancer sequence may
interact with similar proteins.
Nuclear extracts were prepared from a
variety of adult FVB/N mouse tissues and assayed for their ability to
form complexes with nucleotides 167 to
133 of Fabpl. The
specificity of complex formation was defined based on the ability of
unlabeled template to compete for complex formation with tissue nuclear
proteins and by the inability of oligo 13 (both heptads mutated) to
function as a competitor. Complexes with similar mobilities to those
obtained with kidney nuclear proteins (lanes 2-4 of Fig.
5) were generated with extracts prepared from liver
(lanes 5-7) and the distal half of the small intestine
(lanes 11-13). Specific complexes also formed with colonic
and stomach nuclear proteins (lanes 8-10 and 14-16, respectively). The major colonic complex co-migrated
with the major complex formed with kidney nuclear extracts (complex C).
However, none of the complexes formed with stomach extracts co-migrated
with those formed with kidney extracts. Thus, all the tissues that
support Fabpl/hGH expression in adult FVB/N transgenic mice
contain nuclear proteins that recognize the direct heptad repeats.
Southwestern blots of nuclear extracts prepared from these tissues indicated that the heptad repeats mediate binding to a protein in liver which has the same size as the reactive protein in kidney. No bands were detectable in small intestinal, colonic, or stomach extracts (Fig. 3, A and B). GMSAs performed using varying amounts of nuclear extracts disclosed that these other tissues had <10% of the complex forming activity/µg of protein as kidney extracts (data not shown).
None of the seven Fabpl/hGH transgenes that had been
analyzed previously are expressed in heart or spleen nor are the
endogenous rat and mouse genes transcribed in these tissues (1).
Nonetheless, cardiac nuclear proteins form specific complexes with
nucleotides 167 to
133 (lanes 17-19 in Fig. 5). The
major complex generated in the GMSA had a mobility similar to the major
complex (C) formed with kidney extracts (compare lanes 2 and
17). Specific complexes also formed with splenic nuclear
extracts (lanes 20-22). The more slowly migrating splenic
complexes had mobilities similar to those generated from kidney, liver,
and distal small intestinal (ileal) nuclear proteins (complexes A and
B). These findings indicate that nuclear factor(s) that bind to the
heptad repeat are present in tissues that do not support expression of
Fabpl or Fabpl/hGH transgenes.
In Vivo Analyses
The in vivo function of the protein binding site(s)
encompassed by Fabpl nucleotides 167 to
133 were
initially tested using two different fusion genes. Four tandem copies
of this sequence plus a 5
spacer of five nucleotides (i.e.
nucleotides
172 to
133) were added to the 5
end of
Fabpl
596 to +21/hGH, creating
Fabpl4x at
596/hGH. In the other construct,
four tandem copies were placed adjacent to nucleotide
132 of
Fabpl in Fabpl
596 to +21/hGH
(i.e. next to the "endogenous" 35-bp sequence spanning
nucleotides
167 to
133). This construct was designated
Fabpl4x at
132/hGH. We reasoned that any
differences in hGH expression between comparably aged FVB/N mice with
Fabpl
596 to +21/hGH,
Fabpl4x at
596/hGH, and
Fabpl4x at
132/hGH transgenes should reveal
the function(s) of the 35-bp element and the positional dependence of
the function(s).
Seven transgenic mice with
Fabpl4x at 596/hGH were identified from 63 live born animals. Six of these seven founder animals expressed the
transgene as determined by the presence of hGH in the serum and hGH
mRNA in various tissues. All of the expressing founder animals were
sterile due to high serum hGH levels (6.0-66.3 µg/ml). Lines were
established by ovarian transplantation from the two female founders
(Go 26 and Go 27).
Blot hybridization analyses were performed using RNAs prepared
from kidney, stomach, each quarter of the small intestine, the proximal
and distal halves of the colon, and liver. Only two differences were
noted between 6-week-old FVB/N
Fabpl4x at 596/hGH mice and comparably aged
Fabpl
596 to +21/hGH animals. Kidney
expression was suppressed 17-fold in mice containing 4 extra copies of
the 35-bp element (reference standard = jejunal hGH mRNA
levels). Colonic expression was slightly augmented (range = 117-314% of jejunal hGH mRNA levels in
Fabpl4x at
596/hGH animals versus
64-81% in Fabpl
596 to +21/hGH mice).
Multilabel immunohistochemical studies of 6-week-old mice from each
pedigree revealed that the cellular patterns of
Fabpl4x at 596/hGH expression in stomach,
small intestine, colon, and liver were indistinguishable from the
extensively characterized patterns of
Fabpl
596 to +21/hGH expression in similarly
aged animals (see Refs. 1, 6, 14-17). Immunoreactive hGH was
undetectable in any cell type in Fabpl4x at
596/hGH kidney using sensitive
immunofluorescence detection methods (data not shown). In addition,
Fabpl4x at
596/hGH was not expressed at
detectable levels in heart, spleen, or any of the other tissues
surveyed (brain, lung, pancreas, skeletal muscle, white adipose, and
skin).
We generated five FVB/N
Fabpl4x at 132/hGH founders that expressed
the transgene. Members of the three pedigrees established by ovarian transplantation exhibited identical patterns of transgene expression. Renal suppressor activity is retained when four extra copies of the
35-bp element are placed adjacent to the endogenous copy at
132.
However, the suppression is less than when the copies are placed at
596: 3-fold versus >17-fold. Immunohistochemical studies indicated that renal suppression in
Fabpl4x at
132/hGH mice is not associated
with a change in cell type specificity; hGH is confined to proximal
tubular epithelial cells (Fig. 6A), just as
it is in Fabpl
596 to +21/hGH animals.
Light microscopic immunohistochemical studies
of the effect of the heptad repeat on cellular patterns of transgene
expression. A, section of kidney from a P42
Fabpl4× at 132/hGH mouse was incubated with
sheep anti-hGH sera. Antigen-antibody complexes were visualized with
peroxidase-conjugated donkey anti-sheep immunoglobulins and
metal-enhanced DAB. The section was counterstained with hematoxylin and
eosin. hGH present in epithelial cells of the proximal nephron appears
red-brown. The arrows point to a glomerulus composed of hGH-negative cells. B, confocal
micrograph of a section of crypt-villus units from the proximal jejunum
of a P42 Fabpl
596 to +21/hGH mouse. The
section was incubated with rabbit anti-liver fatty acid binding protein
(L-FABP; visualized with Cy5-conjugated donkey anti-rabbit Ig), sheep
anti-hGH (detected with Cy3-donkey anti-sheep Ig), and biotinylated
goat anti-BrdUrd (visualized with Cy2-streptavidin). Rapidly dividing
crypt cells are marked in S-phase by incorporation of BrdUrd and appear
blue. The crypt-villus junction is indicated by open
arrows. L-FABP (red-orange) is confined to post-mitotic villus-associated enterocytes and is not expressed in dividing or
non-dividing crypt epithelial cells. hGH is detected in the supranuclear Golgi apparatus of villus enterocytes (yellow,
e.g. closed arrows) and in BrdUrd-positive and -negative
crypt cells (where hGH is seen as green staining
material). C, high power confocal view of one of the
crypts shown in B demonstrating expression of hGH (green) throughout the
crypt in both proliferating (blue) and nonproliferating
epithelial cells. The open arrows point to the crypt-villus
junction. D-I, the 35-bp element functions as a suppressor
of gene expression in proliferating and differentiating enterocytes
depending upon their location along the duodenal-ileal axis of a P42
Fabpl4× at
132/hGH mouse. D,
crypt-villus units from the proximal duodenum (first 3 cm of the small
intestine) stained with sheep anti-hGH (detected with
peroxidase-conjugated donkey anti-sheep Ig and DAB). The section was
counterstained with hematoxylin and eosin. Placement of four copies of
the 35-bp element at nucleotide
132 silences hGH expression in all
but a few scattered villus epithelial cells located in the upper
two-thirds of these duodenal villi (e.g. open arrow). The
closed arrowheads point to the crypt-villus junction. The
closed arrow in the upper right corner of the
panel points to a cluster of hGH-positive cells in the upper third of a
villus sectioned perpendicular to its crypt-villus axis. The
hGH-positive cells were identified as enterocytes and goblet cells by
EM immunohistochemistry (see Fig. 8, B and F).
Although not detectable with light microscopic methods, EM
immunohistochemistry established that hGH is also present in Paneth
cells located at the base of all proximal duodenal crypts (see Fig.
8G). E, high power view of two proximal duodenal crypts stained with sheep anti-hGH, Cy3-donkey anti-sheep Ig, biotinylated goat anti-BrdUrd, and Cy2-streptavidin. hGH is not detectable in proliferating BrdUrd-positive (green)
epithelial cells located in the mid-portion of the crypt or in
nonproliferating/differentiating cells located in the upper crypt and
lower half of the villus. Scattered hGH-positive villus epithelial
cells (red; arrows) are limited to the upper half
of villi. F, crypt-villus unit in the proximal jejunum
(distal end of the proximal quarter of the small intestine) stained as
in D. The percentage of villus epithelial cells that express
the transgene is increased compared with the proximal duodenum.
G and H, crypt-villus units located at the mid-point of the small intestine. The section in G was
processed as in D. H is a high power view of the
crypts shown in G and was stained as in E. At the
mid-point along the duodenal-ileal axis, all villus enterocytes and
goblet cells contain hGH (G) as do nonproliferating cells in
the uppermost portion of crypts (G and H). The
arrow in G points to hGH-positive cells in the
upper crypt. The arrows in H indicate the
location of the crypt-villus junction. I, crypt positioned
95% of the distance from the gastroduodenal to the ileal-cecal
junctions. hGH (brown-orange) is detectable in all crypt
cells including Paneth cells located at the crypt base (closed
arrow) and in the putative stem cell zone positioned just above
Paneth cells. J, crypts from the proximal quarter of the
colon stained with sheep anti-hGH (visualized as red with Cy3-donkey anti-sheep Ig) and goat-anti-BrdUrd (visualized with Cy2-streptavidin). Fabpl4× at
132/hGH is
ubiquitously expressed in proliferating (yellow) and
nonproliferating crypt epithelial cells as well as in fully
differentiated cells located at the surface epithelial cuff surrounding
the orifice of each crypt (one such cuff is indicated by the
closed arrowheads). The mucin-containing globules of goblet
cells appear black (e.g. open arrow).
Closed arrows point to the bases of crypts. K and L, sections of crypts from the distal quarter of the colon
(K) and rectal glands (L), stained as in
J. Expression of
Fabpl4× at
132/hGH is sustained in the most
distal portions of the colonic epithelium. M and
N, mutation of the heptad repeats abolishes the suppressor and activator functions of the 35-bp element when placed at
132. M, proximal duodenal crypt from a P40 F1
Fabpl4× mutated at
132/hGH mouse stained
as in D. Unlike
Fabpl4× at
132/hGH which is silent in
proximal duodenal crypts (see D and E), Fabpl4× mutated at
132/hGH is actively
expressed in proliferating and nonproliferating crypt epithelial cells.
N, crypts from the distal quarter of the colon of the same
mouse, stained with the same reagents used in M. Unlike
Fabpl4× at
132/hGH which is active in
proliferating and nonproliferating cells of distal colonic crypts,
Fabpl4× mutated at
132/hGH is silent.
Bars = 25 µm.
The 35-Base Pair Element Can Function as a Suppressor of Gene Expression in Liver and Stomach
Moving the four tandem repeats of
the 35-bp element from nucleotide 596 to
132 results in the
acquisition of two "new" suppressor activities. Reporter expression
is abolished in hepatocytes as assayed by measurements of hGH mRNA
in liver RNA (Fig. 7) or by immunohistochemistry (data
not shown). Expression is also silenced in the stomach: mucus-producing
pit cells support expression of a variety of
Fabpl
596 to +21/reporter transgenes (17,
18), but immunoreactive hGH is not detectable in the pit cell or any
other gastric epithelial lineage of
Fabpl4x at
132/hGH animals (data not shown;
cf. Fig. 7).
The 35-Base Pair Element Can Function as a Suppressor of Gene Expression in Proliferating and Differentiating Enterocytes Depending upon Their Location Along the Duodenal-Ileal Axis of the Small Intestine
The adult mouse small intestinal epithelium undergoes continuous renewal (reviewed in Ref. 19). Cellular proliferation is confined to ~1 million flask-shaped mucosal invaginations known as crypts of Lieberkühn, several of which surround the base of each villus. Each crypt contains multipotent stem cells, located near its base. This stem cell gives rise to the four principal epithelial cell lineages of the small intestine. Enterocytes (comprising >80% of the epithelial cells), goblet cells, and enteroendocrine cells differentiate as they undergo an orderly upward migration from a crypt to the tip of an adjacent villus. Cells are removed at the villus tip by apoptosis and/or exfoliation (20). The cycle is completed every 2-5 days (21-25). In contrast, Paneth cells, which elaborate anti-microbial peptides and growth factors, differentiate as they migrate to the base of the crypt where they reside for ~20 days (26, 27).
Throughout the length of the small intestine,
Fabpl596 to +21/hGH and
Fabpl4x at
596/hGH are active in
proliferating and nonproliferating crypt epithelial cells (Fig.
6, B and C). Moving the 4 tandem copies of the
35-bp sequence to nucleotide
132 of Fabpl results in
suppression of hGH expression in members of the enterocytic lineage.
Light and electron microscopic immunohistochemical studies of
6-week-old Fabpl4x at
132/hGH mice indicated
that in the very proximal portion of the small intestine
(i.e. within 3 cm of the gastroduodenal junction), hGH is
not detectable in proliferating and nonproliferating cells located in
the upper half of the crypt or in most differentiated enterocytes, save
a small subpopulation of scattered cells in the upper three-quarters of
the villus (Figs. 6, D and E, and 8B).
In contrast, hGH is expressed in each of the three other epithelial
lineages: Paneth cells (Fig. 8G), rare
enteroendocrine cells (Fig. 8C), and members of the goblet
cell lineage. The latter include oligomucus (Fig. 8D),
granule goblet, and common goblet cells (Fig. 8F) as well as
"intermediate cells" (Fig. 8E) that have morphologic
features intermediate between those of granule goblet and Paneth
cells.2
There is gradient of escape from this enterocyte-specific suppression
along the duodenal-ileal axis. Proceeding distally through the next
25% of the small intestine of
Fabpl4x at 132/hGH mice, the percentage of
villus enterocytes that are hGH-positive increases. In addition, their
distribution expands to include the lower quarter of the villus (Fig.
6F). By the mid-point of the small intestine, hGH production
has generalized to all villus enterocytes as well as to
nonproliferating cells in the uppermost portion of the crypt (Fig. 6,
G and H). EM immunohistochemical studies
disclosed that the transgene remains active in the goblet and Paneth
cell lineages, just as it is in the proximal small intestine (data not
shown).
In the distal half of the small intestine, there is a progressive loss of suppression of hGH expression in proliferating and nonproliferating cells located in the crypt. The more distal the location of a crypt, the deeper the cellular location of hGH expression within that crypt. In the terminal 10% of the small intestine, hGH is expressed in the putative stem cell zone positioned five cell layers above the crypt base (Fig. 8H) and in differentiating and differentiated members of all four lineages distributed along the length of the crypt-villus axis (Fig. 6I).
The 35-Base Pair Element Can Function as an Activator of Gene Expression throughout the Colonic EpitheliumThe colon lacks villi and Paneth cells. Multipotent stem cells, thought to be located at the crypt base (29), give rise to three epithelial cell types, enterocytes (colonocytes), goblet cells, and enteroendocrine cells. Differentiation occurs as cells migrate up each colonic crypt and onto a hexagonal-shaped surface epithelial cuff that surrounds each crypt orifice (30, 31).
Fabpl596 to +21/hGH and
Fabpl4x at
596/hGH are active in
proliferating and nonproliferating crypt epithelial cells in the cecum
and proximal half of the colon of P35-P42 mice. The transgenes are
silent in the distal quarter of the colon. In the middle portion of the colon, only scattered crypts support hGH expression.
Moving the 4 tandem repeats of the 35-bp sequence to 132 does not
change the pattern of hGH expression in the cecum or proximal half of
the colon. The reporter is present in all crypt epithelial cells, as
judged by light or EM immunohistochemistry (e.g. Fig. 6J). However, moving the elements to
132 produces a marked
activation of transgene expression from the mid-colon to the rectum.
Greater than 99% of crypts in the mid- and distal thirds of the colon are hGH-positive (Fig. 6K). These hGH-positive crypts extend
to the rectum (Fig. 6L). All proliferating and
nonproliferating cells in distal colonic crypts appear to support
transgene expression, whether judged by light or EM
immunohistochemistry. This includes enterocytes (colonocytes) and
goblet cells, as well as cells located at the presumptive stem cell
zone at the crypt base. Expression is sustained as cells migrate upward
to the surface epithelial cuff surrounding the crypt orifice.
RNA hybridization studies established that steady state levels of hGH
mRNA were 2-3-fold higher in the distal compared with proximal
colon of Fabpl4x at 132/hGH mice (Fig.
7).
Mice with an additional transgene
(Fabpl4× mutated at 132/hGH) were
generated to determine whether the position-dependent
suppressor and activator activities of the 35-bp element were mediated
by its heptad repeats. Fabpl4× mutated at
132/hGH is analogous to
Fabpl4× at
132/hGH, except each of its 4 copies of the 35-bp element contains the seven nucleotide substitutions
per heptad that abolished complex formation in the gel mobility shift
assay (see sequence 13 in Fig. 2B). Seven founder
Fabpl4× mutated at
132/hGH transgenic mice that
contained
50 ng of hGH/ml serum were identified among the 96 live
born animals screened. All of these founders plus offspring derived
from two of them were analyzed. Immunohistochemical studies revealed
that all animals had similar phenotypes.
Mutation of the heptad repeat abolished the effects of placing 4 copies
of the 35-bp element at 132. Suppression in hepatocytes and in the
pit cell lineage of the stomach was lost; P35-P42 mice with
Fabpl4× mutated at
132/hGH had readily
detectable levels of immunoreactive hGH in these cell types (data not
shown). Suppression of transgene expression was also lost in crypts
located in the proximal half of the small intestine; proliferating and
nonproliferating epithelial cells distributed throughout the crypt were
hGH-positive (Fig. 6M). Finally, transgene expression was no
longer detectable in crypts located in the distal half of the colon
(Fig. 6N).
As with Fabpl4× at 132/hGH,
Fabpl4× mutated at
132/hGH is not
expressed in heart, spleen, brain, lung, pancreas, skeletal muscle,
white adipose, or skin.
Fig. 9 summarizes the effects of manipulating the
position and sequence of the heptad repeat on transgene expression. The pleiotropic activities mediated by the heptad repeat were determined by
comparing the pattern of expression of three Fabpl/hGH
transgenes containing (i) nucleotides 596 to +21 of Fabpl;
(ii) nucleotides
596 to +21 but with four additional copies of its
nucleotides
172 to
133 (containing the direct heptad repeats)
placed at
132; and (iii) a sequence identical to (ii) but with all
heptad repeats mutated within each of the four additional copies of
nucleotides
172 to
133 (the "endogenous" copy was not mutated).
The suppressor and activator activities were revealed when four
additional copies of nucleotides
172 to
133 were added at
nucleotide
132 of Fabpl
596 to +21. When
the heptad repeats in these four additional copies were mutated, the
pattern of expression was indistinguishable from that of the
"parental" Fabpl
596 to +21 sequence
without any inserts. These results allowed us to conclude that the
suppressor and activator activities were dependent upon the
interactions of trans-acting factors with the heptad repeat, rather
than to a change in the relative spacing of cis-acting elements within
596 to +21 due to insertion of an additional 160 bp of DNA.
Comparison of the pattern of expression of another transgene containing
four additional copies of nucleotides 172 to
133 just 5
to
nucleotides
596 to +21 (Fabpl4× at
596)
with the pattern of expression produced by
Fabpl4× at
132 allowed us to conclude that
renal suppressor and colonic activation activities can be still be
expressed even when these elements are moved >400 bp upstream.
The function of the heptad repeat illustrates a central theme of gene regulation within the rapidly self-renewing small intestinal epithelium: controlling expression in a given epithelial lineage as a function of its state of differentiation and location along the duodenal-ileal axis. The endogenous Fabpl gene is turned on just as members of the enterocytic lineage exit the crypt. Expression is sustained as cells complete their migration up the villus to the apical extrusion zone. The heptad repeat in Fabpl acts as an enterocyte lineage-specific suppressor. Remarkably, this suppressor activity is expressed at different points in the lineage's differentiation program depending upon the location of enterocytes along the duodenal-ileal axis. In the proximal small intestine, the heptad suppresses expression in proliferating undifferentiated, differentiating, and fully differentiated enterocytes. As one travels distally toward the ileum, there is a smooth gradient of escape from this suppression that progresses from mature enterocytes in the upper villus to involve successively less differentiated cells located closer and closer to the stem cell zone in the crypt.
The function of the heptad repeat is distinctive among previously characterized cis-acting elements that regulate gene expression in the small intestinal epithelium. For example, functional mapping studies of cis-acting sequences in other members of the Fabpl gene family conducted in transgenic mice have disclosed domains that regulate cell lineage-specific, differentiation-dependent, and cephalocaudal patterns of transcription (11, 32, 33). Like the heptad repeat, a 20-bp element in the homologous intestinal fatty acid-binding protein gene (Fabpi) functions as a differentiation-dependent suppressor of gene expression in the enterocytic lineage. However, the interaction of the 20-bp element with transcription factors causes uniform suppression of expression throughout a crypt, regardless of whether the crypt is located in the proximal, mid, or distal portions of the small intestine. This 20-bp Fabpi element has no obvious sequence similarities to the Fabpl heptad repeat (or to other known transcription factor binding sites). Thus, the heptad repeat represents a novel target for identifying transcription factors that affect the interrelationship between differentiation and axial position in the intestinal epithelium. Although a number of transcription factors have been identified in the intestine (reviewed in Ref. 34), none have been assigned an in vivo role in regulating this interrelationship.
The ability of the heptad repeat to mediate suppression in the stomach, liver, and kidney, and activation in the colon, suggests that it also represents a target for identifying transcription factors that regulate gene expression between different epithelia. One candidate factor is a 90-kDa protein identified in Southwestern blotting experiments. Evidence supporting its candidacy includes the following: (i) an oligo containing the heptad repeats binds to a 90-kDa protein, whereas binding is markedly reduced when the heptad sequences are mutated; (ii) the 90-kDa protein also binds to an element in the matrix metalloproteinase-2 gene that functions as an enhancer in renal cells, shares sequence homology with the heptad, and generates similar-sized complexes in gel mobility shift assays as the Fabpl heptads; and (iii) among the nuclear extracts prepared from tissues where the heptad repeat affects gene expression, the 90-kDa protein is most abundant in kidney nuclear extracts which also have the highest specific activity for complex formation in the GMSA. The heptad repeat bears no obvious sequence similarities to known transcription factor binding sites. Thus, the mediators of its in vivo activities are likely to be novel.
Finally, the various combinations of transcriptional regulatory
elements present in the Fabpl/hGH transgenes described above can now be used to deliver other proteins to different lineages and
locations within the intestine. In particular,
Fabpl4× at 132 can be exploited to deliver
gene products to the entire colonic epithelium of transgenic animals.
This provides an opportunity to design gain-of-function or
loss-of-function experiments that test the effects of various factors
on colonic epithelial biology or that create mouse models of human
colonic pathology.
We are indebted to David O'Donnell and Maria Karlsson for their help in generating and maintaining transgenic mice, to Lisa Roberts for her participation in the EM immunohistochemical studies, and Sean McCaul for assistance with the Southwestern blotting experiments.