(Received for publication, March 27, 1995; and in revised form, May 26, 1995)
From the
Studies using transgenic mice indicate that expression of the human apolipoprotein (apo) A-I gene in the liver and small intestine is controlled by spatially distinct cis-acting DNA elements; hepatic expression is controlled by a domain defined by nucleotides -256 to -1, while small intestinal expression requires elements positioned 9 kilobases 3` to the gene, between nucleotides -1300 and -200 of the convergently transcribed apoC-III gene. In this report we have mapped this enhancer to a 260-base pair (bp) region of the apoC-III promoter spanning nucleotides -780 to -520. The elements contained within this 260-bp apoC-III domain are sufficient to direct a pattern of expression in villus-associated enterocytes distributed along the duodenal-to-ileal axis that resembles that of mouse and human apoA-I. However, the elements produce inappropriate activation of apoA-I expression in proliferating and nonproliferating crypt epithelial cells, and in subpopulations of cholecystokinin- and serotonin-producing enteroendocrine cells. Cis-acting suppressors of these inappropriate patterns of expression are located outside of nucleotides -1300 to -200 of the human apoC-III gene. DNase I protection and gel mobility gel shift assays identified two 21-bp sequences, nucleotides -745 to -725 and -700 to -680 of human apoC-III, which bind nuclear proteins present in a human enterocyte-like cell line (Caco-2). These sequences are conserved in the orthologous mouse apoC-III gene. The 260-bp apoC-III element is the first intestinal enhancer that has been identified in an in vivo system and should provide insights about how cell lineage-specific, differentiation-dependent, and cephalocaudal patterns of gene expression are established and maintained in the perpetually renewing gut epithelium. In addition, novel intestinal transcription factors may bind to the enhancer and regulate its activity.
Apolipoprotein (apo) ()A-I is the major protein
constituent of human high density lipoproteins (HDL). Numerous
epidemiological studies have determined that low levels of plasma HDL
are correlated with increased risk of atherosclerosis and increased
incidence of coronary heart disease (see (1) , and references
therein). Plasma HDL levels are directly related to the plasma
concentration of apoA-I(2) . Transgenic mice that express human
apoA-I in their livers have increased circulating levels of HDL
particles (comparable to human HDL
and
HDL
)(3, 4) . C57Bl/6 transgenic mice that
produce high levels of human apoA-I in their livers have a reduced
incidence of fatty streak formation, compared to their normal
littermates, when exposed to a high fat, atherogenic diet(5) .
In addition, apoE-deficient mice that overexpress a human apoA-I
transgene have decreased atherosclerosis compared to apoE-deficient
mice when fed a chow diet(6) .
ApoA-I is produced in hepatocytes and in the principal epithelial cell lineage of the small intestine: the polarized absorptive enterocytes. Immunocytochemical studies of duodenal/proximal jejunal biopsies obtained from normal adult humans after a 12-h fast or 1 h after a corn oil meal indicated that apoA-I is not detectable in cells that populate the proliferative unit of the intestine: the crypt of Lieberkühn. Rather, apoA-I is first detectable after nonproliferating, differentiating enterocytes exit the crypt. Expression persists as enterocytes complete their rapid upward migration to the apical extrusion zone of an adjacent villus(7) .
The human apoA-I gene is located on chromosome 11, in a cluster that contains the apoC-III and A-IV genes (Fig. 1). Cis-acting DNA elements necessary for apoA-I expression in cultured human hepatoma cells (HepG2) and in the hepatocytes of transgenic mice appear to be located between nucleotides at -256 bp and the start of transcription(3, 8, 9, 10, 11, 12, 13) . However, studies in transgenic mice indicate that a genomic fragment containing up to 5.5 kb of 5` nontranscribed domain of apoA-I, its 4 exons and 3 introns, plus 4 kb of 3` flanking sequence is not expressed in the small intestine. The determinants of intestinal expression appear to reside in a 1.1-kb fragment that extends from nucleotides -200 to -1300 of the adjacent, convergently transcribed human apoC-III gene(14) . This fragment can function in either orientation to direct intestinal expression when it is placed 1.7 kb 3` to apoA-I's terminal exon. Intestinal expression, defined as accumulation of human apoA-I mRNA in total RNA prepared from the proximal third of the small bowel of adult mice, is sustained in the absence of apoC-III exonic and intronic sequences(14) . However, the cell lineage-specific and spatial patterns of apoA-I/C-III transgene expression were not defined in this study.
Figure 1:
Transient
transfection analysis of the intestinal control domain contained in the
human apoC-III gene. Schematic depiction of the human apolipoprotein
A-I/C-III/A-IV gene cluster and recombinant DNAs used for transfection
analysis. The 1.1-kb PstI (P) fragment of the
apoC-III promoter was digested with SacI (S), ApaI (A), BsaI (B), and StuI (St) and subcloned into pA-I-CAT. The element I deletion mutant was created by
digesting the apoC-III -780 to -520 containing plasmid with EcoNI, which removes a 50-bp fragment containing element I,
followed by religation. Recombinant DNAs were transfected into near
confluent, proliferating Caco-2 cells and CAT assays were performed.
Results are presented as the mean activity (± S.D.) relative to
that obtained with pA-I
-CAT alone (n = 3 independent transfections for each
construct).
There is very limited information available about the cis- or trans-acting factors that determine axial, temporal, and cell lineage-specific patterns of transcription in the intestinal epithelium, in part because in vivo assays are required to define these parameters. There are three ``axes'' (two spatial and one temporal) that need to be considered when evaluating endogenous gene or transgene expression in the mouse gut epithelium. The intestine's four principal epithelial cell lineages, enterocytic, goblet, enteroendocrine, and Paneth cell, are all derived from multipotent stem cells located near the base of each crypt(15, 16, 17) . The descendants of these stem cells undergo several rounds of cell division in the midportion of the crypt, creating a transit cell population. Each lineage subsequently completes its differentiation program during an orderly migration. Paneth cells differentiate during a downward migration to the base of the crypt(18) . Enterocytes, goblet cells, and enteroendocrine cells all differentiate as they migrate in vertical coherent bands from each crypt up an adjacent villus to an apical extrusion zone located near the villus tip(19, 20) . The migration-associated differentiation and exfoliation programs of these three lineages are completed in 2-5 days(21, 22, 23, 24) . The differentiation program of a given cell lineage varies along the duodenal-to-ileal axis (25, 26) . Thus, gene expression must be evaluated as a function of cell type, the state of differentiation of that cell (i.e. its position along the crypt-to-villus unit) and its location along the cephalocaudal axis.
We have now used transgenic mice and cultured cell lines to map further the intestinal enhancer contained in the human apoA-I/C-III/A-IV gene cluster, to define its effects on gene expression along the crypt-villus and duodenal-colonic axes, and to characterize nuclear proteins that may modulate its activity.
Cell lines were obtained from the American Type Tissue Collection
and maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum (HepG2 and HeLa) or minimal
essential medium with 20% fetal calf serum (Caco-2). Cells were plated
on 60-mm plastic dishes and incubated for 24 h at 37 °C under an
atmosphere containing 95% air, 5% CO. Duplicate plates were
transfected with 10 µg of each test plasmid, 5 µg of
pCMV-
gal (27) , or 5 µg of pLEN4S (an hepatic nuclear
factor 4, HNF-4, expression plasmid)(28) . Cells were shocked
with 15% glycerol 24 h after transfection and harvested 24 h later.
-Galactosidase and CAT assays were performed as
described(27) . Equivalent amounts of extracted total cell
protein were used in each CAT assay (equivalence was determined by
defining the levels of
-gal expression in cleared cell lysates).
Relative CAT activity was determined by comparing the percentage of
acetylated to unacetylated chloramphenicol obtained with each
apoA-I/C-III-containing recombinant DNA to the percentage obtained with
the pA-I-CAT control that lacked apoC-III sequences.
BamHI linkers were added to a 260-bp ApaI/SacI fragment that contains nucleotides -780 to -520 of the human apoC-III gene. It was subcloned into pH-A-I-14 at its unique BamHI site. A plasmid (pH-A-I-16) was identified containing this fragment in an orientation opposite to the direction of apoA-I gene transcription, just as it is the apoA-I/apoC-III/apoA-IV cluster on chromosome 11 (Fig. 1). BamHI linkers were also added to a 90-bp ApaI/StuI fragment that encompasses nucleotides -780 to -690 of apoC-III. The linker-containing DNA was ligated to BamHI-digested pH-A-I-14 and a recombinant plasmid (pH-A-I-17) was identified where the apoC-III fragment was in an orientation opposite to that of apoA-I.
The inserts from pH-A-I-16
and pH-A-I-17 were released with EcoRI/SalI,
gel-purified, and microinjected into fertilized oocytes obtained from
(C57Bl/6J CBA/J)F1 females that had been mated to (C57Bl/6J
CBA/J)F1 males. Founders containing each construct were
identified by probing Southern blots of PstI-digested tail DNA
with a radiolabeled 0.7-kb DNA fragment that extends from the SacI site in exon 4 of the human apoA-I gene to a PstI site 300 bp 3` to this terminal exon(14) .
Pedigrees were established and maintained by crosses to nontransgenic
littermates. Animals were caged in microisolators under a strict light
cycle (lights on at 0600 and off at 1800) and fed a standard chow diet ad libitum (no. 5001; Ralston Purina, St. Louis, MO).
Control experiments revealed that (i) none of the labeled secondary antibodies bound to tissue sections in the absence of primary antibody or in the presence of preimmune sera, (ii) none of the normal littermate tissues gave a detectable signal when incubated with the anti-human apoA-I sera followed by a fluoroprobe-tagged secondary antibody, and (iii) overnight incubation of a 1:1000 dilution of anti-human apoA-I sera with purified human apoA-I (Sigma; 10 µg/ml antisera) completely blocked its ability to react with gut epithelial cell lineages in mice that expressed a human apoA-I/C-III transgene (data not shown).
Sections were also
incubated with FITC-labeled soybean agglutinin (Glycine max;
final concentration = 5 µg/ml; obtained from Sigma;
carbohydrate specificity = GalNAc3GalNAc;
GalNAc
/
3/4Gal; cell specificity = goblet and Paneth
cells plus enterocytes)(26) . The protocol for staining with
soybean agglutinin was similar to that described above for antibodies,
except that powdered skim milk was omitted from PBS-blocking buffer and
the sections were incubated with the lectin for 16 h at 4 °C. After
three washes in PBS, followed by one wash with PBS, coverslips were
mounted on the sections with PBS/glycerol (1:1, v/v) containing 5%
1,4-diazabicyclo-[2.2.2]octane (Sigma).
Figure 5:
DNase I protection analysis of nucleotides
-780 to -520 of the human apoC-III gene. PanelA, a 260-bp SacI/ApaI apoC-III fragment
was end-labeled and incubated with bovine serum albumin (lane1) or nuclear protein extracts prepared from
proliferating HepG2 (lane2) and Caco-2 (lane3) cells; panelB, comparison of the 5`
nontranscribed domains of the orthologous human and mouse apoC-III
genes. Boxed areas correspond to footprinted elements I and
II. The positions of G C substitutions made in each hexamer of
elements I or II are shown (see text and Fig. 6C).
Figure 6: Gel mobility shift analysis using elements I and II contained in the apoC-III gene promoter. PanelA, double-stranded oligonucleotides corresponding to elements I, II, and I-II were synthesized, radiolabeled, and incubated with no protein (1) or nuclear proteins prepared from near-confluent, proliferating HeLa (2), HepG2 (3), and Caco-2 (4) cells. b represents a complex formed with HepG2 and Caco-2 extracts, while complex a is Caco-2-specific. Other bands are not specific. Panel B, a 100-fold molar excess of unlabeled oligonucleotide was used as competitor in assays containing Caco-2 nuclear extracts. Competitors used were elements I, II, I-II, and a nonspecific oligonucleotide (ns). Panel C, a 100-fold molar excess of unlabeled mutant competitor oligonucleotide (see Fig. 5B and ``Experimental Procedures'') were mixed with Caco-2 nuclear extracts as described in panelB. Mutants are designated Im1 and 2 (mutations in the first and second putative hexamers of element I, respectively) and IIm1 and 2 (mutations in the first and second hexamers of element II, respectively). PanelD, nuclear extracts prepared from Caco-2 cells were incubated with double-stranded oligonucleotides corresponding to element I. HNF-4-specific (lane2) and ARP-1 specific (lane3) antibodies were then added and incubation continued. Complex b is supershifted by the HNF-4 antibody (ss).
Addition of
nucleotides -1300 to -520 of human apoC-III to
apoA-ICAT produces a 4-fold increase
in reporter expression in near confluent Caco-2 cells (Fig. 1).
A 90-bp fragment spanning nucleotides -780 to -690 produce
a comparable increase in CAT levels (6-fold). Nucleotides -760 to
-710 appear necessary for this enhancement, since their deletion
eliminates the effect (Fig. 1). The positive effects produced by
nucleotides -780 to -690 are cell-specific; no increase in
CAT activity was detectable in HepG2 or HeLa cells over that produced
by apoA-I
CAT alone (data not shown).
Figure 2: RNase protection analysis of transgene expression in adult mouse liver, intestine, and brain. A, schematic representation of the recombinant DNAs used to produce mouse lines 427 (transgene expression confined to the liver), A14 (transgene expression in liver and small intestine), A16, and A17. B, a human apoA-I specific riboprobe was hybridized to yeast tRNA (tRNA), total cellular RNA isolated from 1-day post-confluent HepG2 cells (HG2), and total cellular RNA isolated from the liver (L), proximal third of the small intestine (I), and brain (B) from four lines of adult transgenic mice. Human apoA-I mRNA yields a protected fragment of 230 bases (arrow).
Adult mice belonging to all four pedigrees produce human apoA-I mRNA in their livers (Fig. 2B and Table 1). The steady state levels of human apoA-I mRNA in liver RNA prepared from adult members of each pedigree are similar (data not shown). This is not the case in the proximal third of the small intestine, where the steady state levels of human apoA-I mRNA vary between comparably aged members of the four lines. Nonetheless, the range of mRNA concentrations in duodenum/proximal jejunum is similar to that observed in adult transgenic mice with nucleotides -200 to -1300 of apoC-III (compare lines A14 and A16 in Fig. 2B).
Deletion of nucleotides -689 to -520 from the 260-bp apoC-III sequence that supports small intestinal expression abolishes human apoA-I expression in the duodenum and proximal jejunum but has little effect on steady state human apoA-I mRNA levels in liver (Table 1). This reduction is not an insertion site effect since it was observed in adult members of two transgenic pedigrees (data not shown). No apoA-I expression was detectable in the brains of any transgenic mice from any of these lines (Fig. 2B).
Single and multilabel
immunocytochemical surveys were conducted to compare the cell
lineage-specific, differentiation-dependent, and regional patterns of
expression of the various transgenes with the pattern of expression of
the endogenous mouse apoA-I gene. Adult, nontransgenic littermates
contain immunoreactive apoA-I in villus-associated enterocytes. No
immunoreactive protein is detectable in proliferating and
nonproliferating crypt epithelial cells (Fig. 3A).
Multilabel studies with antibodies directed against mouse apoA-I and a
Paneth cell-specific marker (lysozyme) or a lectin (soybean agglutinin)
that reacts with glycoconjugates produced in the majority of adult
(C57Bl/6J CBA/J)F1 small intestinal goblet cells indicated that
neither of these lineages contains detectable levels of the
apolipoprotein (data not shown).
Figure 3:
Multilabel immunocytochemical analysis
reveals that nucleotides -1300 to -200 and nucleotides
-780 to -520 of the human apoC-III gene direct human apoA-I
expression to proliferating and nonproliferating jejunal crypt
epithelial cells as well as villus-associated enterocytes. The patterns
of apoA-I expression along the crypt-to-villus axis of adult mice
belonging to pedigrees A14 (containing nucleotides -1300 to
-200 of the apoC-III gene) and A16 (containing nucleotides
-780 to -520 of the apoC-III gene) are indistinguishable. Panels A-C, a section of the proximal jejunum of a 10
month-old mouse from pedigree A14 (apoC-III). The section was incubated with rabbit anti-mouse and
goat anti-human apoA-I sera. Antigen-antibody complexes were visualized
with FITC-conjugated donkey anti-rabbit and CY3-conjugated donkey
anti-goat sera. Panel A, mouse apoA-I is confined to
villus-associated enterocytes (solidarrowheads).
Immunoreactive protein is seen associated with the Golgi-apparatus of
these cells and in the lamina propria (openarrow),
reflecting basolateral secretion of the apolipoprotein. Panel
B, the same section as in panelA, showing the
distribution of human apoA-I in villus-associated enterocytes, the
lamina propria, as well as the crypt. PanelC, a
double exposure of the same section as shown in A and B, demonstrating co-expression of mouse and human apoA-I in
villus-associated enterocytes (yellow). However, only human
apoA-I is apparent in crypt epithelial cells (red-orange, closedarrows). Panels D-F, a
10-month-old mouse from pedigree A16 (apoC-III
) received an intraperitoneal injection of BrdUrd 2 h
before sacrifice. A section of the proximal jejunum was incubated with
goat anti-human apoA-I and biotinylated goat anti-BrdUrd sera.
Antigen-antibody complexes were visualized with CY3-conjugated donkey
anti-goat sera and FITC-conjugated streptavidin. Panel D,
proliferating S-phase cells in jejunal crypts detected with the
anti-BrdUrd sera (green nuclear staining, openarrows). Panel E, same section as in D but stained with anti-human apoA-I sera. Note that the
distribution of human apoA-I along the crypt-to-villus axis is
identical to that observed in comparably aged mice belonging to
pedigree A14 (see panelB). PanelF, a double exposure directly demonstrating human apoA-I (orange) in proliferating (green, solidarrows) and nonproliferating crypt epithelial cells. Bar = 25 µm.
Multilabel immunocytochemical studies of enteroendocrine cells in the proximal small intestine of adult mice (20, 25) suggest that a committed enteroendocrine cell progenitor yields descendants that follow one of three principal differentiation pathways during their migration from the crypt to the villus tip. One pathway yields cells that initially express substance P alone, followed by substance P and serotonin, followed by secretin alone or serotonin alone. Another pathway produces cells that sequentially express gastrin plus CCK, followed by gastrin plus CCK plus glucagon-like peptide-1 (GLP-1), followed by gastrin plus CCK or secretin alone. A third pathway yields cells that only express gastric inhibitory peptide. Serotonin- and CCK-producing cells are the most abundant enteroendocrine cell subpopulations in the adult mouse duodenum(25) . Multilabel studies using anti-serotonin or anti-CCK sera revealed that enteroendocrine cells in the first two pathways do not contain detectable levels of mouse apoA-I (data not shown).
Immunocytochemical analyses indicated that the expression
domain of the mouse apoA-I gene in nontransgenic adult (C57Bl/6J
CBA/J)F1 mice extends from villus-associated enterocytes in the
proximal boundary of the duodenum to the distal jejunum (duodenum,
jejunum, and ileum are operationally defined in this study as the
proximal, middle, and distal thirds of the small intestine).
Comparably aged adult transgenic littermates containing the 260-bp
apoC-III element (nucleotides -780 to -520) also express
human apoA-I in their villus-associated enterocytes. Moreover, the
regional pattern of human apoA-I expression along the duodenal-to-ileal
axis mimics that of mouse apoA-I. Unlike the intact endogenous mouse
apoA-I gene, the transgene is expressed in proliferating and
nonproliferating duodenal and jejunal crypt epithelial cells (Fig. 3, D-F). Multilabel light microscopic
studies using lysozyme and the soybean agglutinin failed to detect
human apoA-I in duodenal, jejunal, or proximal ileal Paneth cells or in
lectin-positive goblet cells (data not shown). However, 20% of
CCK-producing and 0.1% of serotonin-producing enteroendocrine
cells co-express the transgene (Fig. 4, A-C). These cells do not produce detectable
amounts of mouse apoA-I in transgenic mice or, as noted above, in their
normal littermates.
Figure 4:
Confocal microscopic studies disclose
anomalous expression of human apoA-I in serotonin-producing
enteroendocrine cells of adult transgenic mice. A section of the
proximal intestine of a 10-month-old transgenic mouse containing
apoA-I/apoC-III
was incubated with goat anti-human apoA-I and rabbit
anti-serotonin sera. Antigen-antibody complexes were visualized with
CY3-conjugated donkey anti-goat and FITC-conjugated donkey anti-rabbit
sera. Panel A, human apoA-I is evident in the Golgi
apparatus of enterocytes (closedarrowheads) and in
the lamina propria (closedarrow). Diffuse
cytoplasmic staining is also evident in a single cell (openarrowheads). Panel B, view of
the same section at the same focal plane as shown in panelA but stained with anti-serotonin sera. A single
enteroendocrine cell with diffuse cytoplasmic staining is evident. Panel C, overlaid images demonstrate co-expression of
human apoA-I and serotonin in the enteroendocrine cell (yellow-green). Bar = 1
µm.
Precocious expression of human apoA-I in crypt epithelial cells and inappropriate expression in CCK- and serotonin-producing enteroendocrine cells is also evident in transgenic mice that contain the construct with nucleotides -1300 to -200 of the human apoC-III gene. The distribution of human apoA-I along the crypt-to-villus axis and the fractional representation of apoA-I-positive CCK and serotonin cells in adult mice belonging to these pedigrees are indistinguishable from the patterns observed in mice that only contain nucleotides -780 to -520 (Fig. 3, B, C, E, and F, plus data not shown). Light microscopic surveys also indicated that adult mice with the 1.2-kb apoC-III fragment do not produce human apoA-I in members of the Paneth or goblet cell lineages (data not shown).
Together, these findings allow us to conclude that nucleotides -780 to -520 of the human apoC-III gene contain cis-acting sequences that are sufficient to direct a duodenal-to-jejunal pattern of expression of the upstream human apoA-I gene in villus-associated enterocytes of adult transgenic mice which mimics that of the intact endogenous mouse apoA-I gene. However, nucleotides -780 to -520 lack cis-acting sequences that suppress precocious expression of human apoA-I in proliferating and nonproliferating crypt epithelial cells and in subpopulations of CCK- and serotonin-producing enteroendocrine cells. Moreover, these suppressors of crypt and enteroendocrine cell expression are not represented in the region encompassed by nucleotides -1300 to -200.
The orthologous human and mouse apoC-III genes show a high degree of sequence similarity within, but not outside of, these two protected sites (Fig. 5B). The sequences of elements I and II were used to search the transcription factor binding site data base (fastsa.tfd; (35) ). Element I (GGGTCCAGAGGGCAAAATAGGG) shows significant similarity to the consensus recognition site of the nuclear hormone receptor superfamily(36) . The consensus sequence is a direct repeat (DR) of the hexamer AGGTCA with a variable number of intervening nucleotides from 0 to 5. The sequence of element I most closely resembles a DR-2 motif with the half-sites GGGTCC and AGGGCA and two spacer nucleotides (Fig. 5B). Element II has no obvious sequence similarity to known transcription factor binding sites.
Double-stranded oligonucleotides corresponding to elements I, II, and I plus II (I-II) were then synthesized, radiolabeled, and incubated with nuclear extracts from HeLa, HepG2, and Caco-2 cells. When element I was used as the template DNA, one complex forms after incubation with HepG2 and Caco-2 extracts (labeled b in Fig. 6A). Another complex (a in Fig. 6A) is Caco-2- specific. One Caco-2-specific complex forms with element II (nucleotides -700 to -680) (Fig. 6A, a). A similar complex forms when Element I-II is used (Fig. 6A). Element II blocks assembly of the radiolabeled complex a formed between Caco-2 nuclear proteins and element I. Conversely, element I blocks formation of complex a that forms when element II binds Caco-2 nuclear proteins (Fig. 6B).
Mutational analysis revealed that two of the direct repeat half-sites are important for complex a formation. Oligos were synthesized where the second G in each hexamer of element I or II was changed to a C (see Fig. 5B). When nucleotides -740 or -689 were mutated, complex a was not formed (Fig. 6C).
Several members of the superfamily of nuclear hormone receptor transcription factors have been shown to be involved in regulating apolipoprotein gene expression(10, 37, 38) . Gel mobility shift assays were performed using antibodies to two of these factors, hepatic nuclear factor 4 (HNF-4) and apolipoprotein regulatory protein 1 (ARP-1), Caco-2 nuclear extracts, and radiolabeled element I to determine whether complex a or b was formed by interactions between this element and either of these proteins. Complex b, which forms with both Caco-2 or HepG2 nuclear extracts, is supershifted by anti-HNF-4 (Fig. 6D, lane2), indicating the presence of HNF-4 or an HNF-4-like protein. The Caco-2-specific complex a is not affected by addition of this antibody preparation. Moreover, none of the complexes were supershifted after addition of anti-ARP-1 (lane3). Comparable studies were also performed with element II. Neither anti-HNF-4 nor anti-ARP-1 produced a supershift of complex a formed when element II was incubated with HepG2 or Caco-2 extracts (data not shown).
We performed transient transfection
assays to examine the effects of HNF-4 on apoA-I expression in
near-confluent, proliferating Caco-2 cells. Introduction of an HNF-4
expression vector into cells containing pA-I-CAT is associated with a 3-fold increase in CAT activity (Fig. 7). This increase reflects an interaction with a
previously described HNF-4 binding site located at nucleotides
-214 to -192 of the human apoA-I gene(39) . When
the HNF-4 expression plasmid was co-transfected into cells containing
either (i) the 260-bp apoC-III fragment encompassing elements I and II
linked to apoA-I
-CAT or (ii) the 90-bp
apoC-III fragment containing element I and only half of element II
linked to apoA-I
-CAT, CAT activity
increased 20-26-fold compared to apoA-I
-CAT (Fig. 7). The increase produced by the 260-bp
apoC-III fragment was not significantly different from that produced by
the 90-bp fragment. When element I was deleted from the 260-bp
construct, HNF-4 responsiveness was not observed (Fig. 7). The
effect was cell-specific, i.e. overexpression of HNF-4 had no
effect on CAT activity in proliferating HepG2 or HeLa cells containing
either of the two apoC-III/apoA-I-CAT DNAs (data not shown). In
addition, when the 260- and 90-bp apoC-III fragments were linked to an
adenovirus major late promoter-CAT reporter construct and transient
transfection assays were performed as described above, CAT activity was
greatly increased when cotransfection of HNF-4 expression plasmids
occurred (data not shown).
Figure 7:
Effect of HNF-4 on expression of human
apoA-I in near confluent Caco-2 cells. HNF-4 expression plasmids were
co-transfected (+) with plasmids containing human apoC-III
nucleotides -780 to -520 (I+II), -780
to -690 (I), or an apoC-III deletion mutant (see Fig. 1), linked to apoA-I-CAT.
Results are presented as mean activity (± S.D.) relative to that
obtained with pA-I-CAT alone (n = 3 independent
transfections for each construct).
Together, these data suggest that HNF-4 binding to element I (nucleotides -745 to -725) increases apoA-I transcription in Caco-2 cells and may be one interaction that contributes to the ability of nucleotides -780 to -520 of apoC-III to direct human apoA-I gene expression in the small intestine of transgenic mice. It is apparently not the only interaction required given the inability of nucleotides -780 to -690 to perform the same function in vivo. Element II may also be required for intestinal expression in vivo. Although it does not appear to bind HNF-4, element II's ability to compete with element I for binding to Caco-2-specific nuclear proteins suggests that the two sites may either be recognized by a common or similar transcription factor(s) and/or that factor binding to one site may influence binding at the other site.
The experimental results presented in this report indicate
that intestinal expression of the apoA-I gene is dependent upon the
presence of an enhancer element located 9.2 kb 3` to its promoter.
The human apoC-III gene is expressed predominantly in hepatocytes and
villus-associated enterocytes, whereas apoA-IV is expressed exclusively
in villus-associated
enterocytes(40, 41, 42) . Preliminary studies
conducted in transgenic mice indicate that intestinal expression of
human apoC-III and A-IV also requires the presence of sequences located
in the 5` nontranscribed domain of the apoC-III gene(43) .
Thus, the enhancer or enhancers positioned in this region may operate
to effect intestinal expression of the entire 17-kb A-I/C-III/A-IV
locus, i.e. they represent a locus control region. It will be
particularly important to assess the ability of either nucleotides
-1300 to -200 or -780 to -520 of human apoC-III
to direct appropriate lineage-specific, differentiation-dependent, and
cephalocaudal patterns of human apoC-III and apoA-IV gene expression in
transgenic mouse gut. Specifically, do the 260- or 1100-bp sequences
produce precocious activation of apoC-III or apoA-IV synthesis in
proliferating crypt epithelial cells and/or support expression in cell
lineages other than enterocytic?
Two features of the
``inappropriate'' pattern of apoA-I gene expression noted in
the small intestine of apoA-I/C-III transgenic mice have been
encountered during the course of functional mapping studies of
transcriptional regulatory elements in other nonhomologous genes that
are expressed in villus-associated enterocytes, lending additional
support to several evolving concepts about regulation of gene
expression in this epithelial lineage. First, distinct
elements appear to control expression in enterocytes distributed along
the crypt-to-villus and duodenal-to-ileal axes. This is true for the
rat intestinal fatty acid binding protein gene (Fabpi; (32) ), the rat liver fatty acid-binding protein gene (Fabpl; (44) ), the mouse ileal lipid-binding protein
gene (Ilbp; (45) ), and the human sucrase-isomaltase
gene(46) . Correct cephalocaudal patterns of expression appear
to be determined by cis-acting activators as well as spatial
suppressors (Fabpi, Fabpl, Ilbp).
Prohibition of precocious activation of expression in proliferating and
nonproliferating crypt epithelial cells requires the presence of
cis-acting suppressors (Fabpi, Fabpl,
sucrase-isomaltase). Second, precocious activation of
transgene expression in the crypt is often associated with
inappropriate expression in other cell lineages. There is an apparent
correlation between the cell stratum where transgene expression is
first initiated and the extent of inappropriate expression in these
lineages: the closer to the stem cell zone (postulated to be at stratum
5; (47) and (48) ), the greater the number of
lineages, other than the enterocytic, that support expression (32, 44) . This may reflect differing degrees of
commitment of the cells that support initiation of transgene
expression; inappropriate expression in nonenterocytic lineages could
arise due to the absence of cis-acting elements in the various
transgenes that normally bind lineage-specific repressors produced by
cells after they are allocated to enteroendocrine, goblet, and Paneth
cell lineages. Transgenes offer a set of instruments for measuring
where and defining how lineage allocation occurs in the crypt. By
crossing mice containing the human apoA-I/C-III fusion gene, described in this study, with mice that
contain nucleotides -596 to +21 of rat Fabpl linked
to a human growth hormone reporter (a transgene which is expressed
deeper in the crypt and in all four lineages; cf. (44) and (49) ), it may be possible to determine which
crypt epithelial cell populations support expression of one or both
transgenes using double label electron microscopic immunocytochemistry (50) . The results could be correlated with previously
published transmission electron microscopic studies of the morphologic
differentiation of the multipotent stem cells'
descendants(18, 21, 22, 23, 24) .
Moreover, these bi-transgenic mice could be used for isolation of gut
stem cells, their early descendants, and/or members of specific
lineages if the reporters encode gene products that allow for positive
and negative selection (e.g. by fluorescence-activated cell
sorting) ex vivo.
Finally, it is important to emphasize the difficulty in using established enterocyte-like cell lines such as Caco-2 to predict the activity of specific cis-acting transcriptional regulatory sequences in vivo, or the functional importance of trans-acting factors that bind to these sites. For example, the similarities in activities of the 90- and 260-bp apoC-III elements in Caco-2 cells contrasts with their markedly different capacities to support transgene expression in the mouse gut epithelium. Defining the state of proliferation and differentiation of Caco-2 cells (and whether they support expression of the intact endogenous copy of the gene being studied) is certainly important when designing and analyzing experiments conducted in this line. Nonetheless, a previous study that correlated the pattern of expression of various Fabpi reporter transgenes along the crypt-to-villus axis of mice with their relative activities in proliferating/preconfluent and ``differentiated''/postconfluent Caco-2 cells suggested that ``differentiated''/postconfluent cells have a transcriptional regulatory environment which resembles that of enterocytes positioned in the upper crypt rather than more differentiated descendants located on the villus(51) . It appears that transgenic mice will remain a requisite final audit of the significance (relevance) of transient transfection studies conducted in cell lines derived from the gut epithelium.