Department of Pediatrics, Yale University, New Haven, Connecticut 06520-8064
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ABSTRACT |
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The
divergent homeobox gene Hex is expressed in both developing
and mature liver. A putative Hex binding site was identified in the promoter region of the liver-specific Na+-bile acid
cotransporter gene (ntcp), and we hypothesized that Hex regulates the ntcp promoter through this
site. Successive 5'-deletions of the ntcp promoter in a
luciferase reporter construct transfected into Hep G2 cells confirmed a
Hex response element (HRE) within the ntcp
promoter (nt 733/
714). Moreover, p-CMHex transactivated a
heterologous promoter construct containing HRE multimers (p4xHRELUC),
whereas a 5-bp mutation of the core HRE eliminated transactivation. A
dominant negative form of Hex (p-Hex-DN) suppressed basal luciferase activity of p-4xHRELUC and inhibited activation of this construct by p-CMHex. Interestingly, p-CMHex transactivated the HRE in Hep G2 cells but not in fibroblast-derived COS cells, suggesting the possibility that Hex protein
requires an additional liver cell-specific factor(s) for full activity. Electrophoretic mobility shift assays confirmed that liver and Hep G2
cells contain a specific nuclear protein that binds the native HRE. We
have demonstrated that the liver-specific ntcp gene promoter
is the first known target of Hex and is a useful tool for
evaluating function of the Hex protein.
transcriptional regulation; liver
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INTRODUCTION |
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HOMEOBOX GENES CONSTITUTE a highly conserved family of transcription factors characterized by the presence of a 60-amino acid motif known as the homeodomain (30, 32, 44). These genes fall into two categories. One group, Hox genes, are tightly clustered on four chromosomes and are identified by similarities in the homeodomain with homologous Drosophila genes involved in determination of body pattern (31, 42). The remaining homeobox genes are referred to as divergent homeobox genes and are scattered throughout the genome. These genes tend to have more limited domains of expression and, where evaluated, have been shown to have tissue-specific function (see, e.g., Refs. 6, 19, and 26).
The divergent homeobox gene, Hex, was originally identified from hematopoietic tissue (3, 22). However, we (4) and others (46, 47) have recently provided evidence that Hex is expressed at high levels in the developing fetal liver, including the epithelium of the bile duct and gallbladder, and in the mature liver. It is also expressed in Hep G2 cells, a human hepatoma cell line (22, 46). This pattern of expression strongly suggests a role for Hex both in hepatobiliary development and in the expression of genes in the mature liver. Tanaka et al. (46) recently reported that a fusion protein consisting of Hex with the DNA binding domain of Gal4 functions in Hep G2 cells as a repressor with a reporter plasmid containing five copies of the Gal4 binding site. Although that group did not test with a putative Hex target, their findings are consistent with Hex functioning as a transcriptional regulator.
Although one report identified a core binding sequence for Hex consisting of 5'-ATTAA-3' (12), no known DNA targets of Hex (and of most other homeobox genes) have been identified. We identified a potential Hex binding site in the promoter of the sodium-dependent bile acid transporter gene (ntcp). The ntcp gene, which is expressed exclusively in hepatocytes, codes for the major transporter of bile acids across the basolateral surface of the hepatocyte (33). Both cDNA (18) and genomic clones (24) for rat ntcp have been reported. Analysis of these clones has greatly facilitated our understanding of the complex molecular mechanisms regulating the enterohepatic circulation of bile acids. However, these mechanisms are still not completely understood.
Bile flow is dependent on the synthesis, transport, and recirculation
of bile acids (reviewed in Refs. 36 and 45). We reported previously
(24) that the minimal promoter of ntcp, from
nucleotide 158 to +47 relative to the transcription start site,
regulated basal and tissue-specific expression of the ntcp gene. Multiple regulatory proteins activate the minimal ntcp
promoter, including hepatocyte nuclear factor (HNF)-1
and an
unidentified factor, footprint B binding protein (24).
However, our data indicated that regions upstream of the minimal
ntcp promoter participated in the regulation of
ntcp gene expression.
We hypothesized that Hex would transactivate the
ntcp promoter via a putative Hex response element
(HRE) located in the 5'-upstream region from 733 to
714. The HRE
contains an ATTAA core at
725 to
721. In the studies reported here,
we demonstrate that Hex is indeed a regulator of the
ntcp promoter via Hex binding to the HRE. These
data document the ntcp promoter as the first known target of
Hex and provide a target for evaluation of the
Hex protein.
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EXPERIMENTAL PROCEDURES |
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Materials and supplies.
DNA restriction enzymes, calf intestinal alkaline phosphatase, T4
polynucleotide kinase, Vent DNA polymerase, and T4 DNA ligase were
purchased from New England Biolabs. Taq DNA polymerase and dNTPs were purchased from Boehringer Mannheim. Radiolabeled nucleotides and nylon filters were purchased from Amersham. Tumor necrosis factor- and interleukin-1
were purchased from R&D Systems
(Minneapolis, MN). Cell culture media and fetal calf serum were
purchased from GIBCO-BRL (Gaithersburg, MD). Luciferase and
-galactosidase assay kits were obtained from Promega (Madison, WI).
A lactate dehydrogenase kit was obtained from Sigma (St. Louis, MO).
Routine biochemicals were purchased from Fisher and U. S. Biochemical.
Oligonucleotides.
All oligonucleotides were synthesized by the DNA Synthesis Lab,
Critical Technologies Program, at Yale University on a model 3948 ABI
DNA Synthesizer. The sequences of double-stranded oligonucleotides used
in gel mobility shift assays and single-stranded oligonucleotides for
plasmid construction are shown in Table
1.
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Plasmid construction and sequence analysis.
A full-length mouse Hex cDNA was prepared making use of a
partial restriction map of the Hex gene and a partial
Hex cDNA clone kindly provided by Dr. Leanne Wiedemann
(Leukemia Research Fund Centre, Institute of Cancer Research, London,
UK). Standard screening techniques were used to isolate genomic clones
from a FIX II library (kindly provided by Dr. Adrian Hayday, Yale
University School of Medicine, New Haven, CT). Filters were hybridized
and washed using a labeled 82-bp fragment of the Hex
homeodomain and screened as described previously (5).
Three clones were obtained and plaque purified. A partial sequence
obtained from the 5' end of one clone was used to pick PCR primers that
amplified a single band from genomic DNA. These primers were used to
obtain a P1 clone from Genome Systems. The P1 clone and the partial
cDNA from Dr. Wiedemann were used to complete a full-length
Hex cDNA that was cloned into pcDNA3 (Invitrogen), which
contains a cytomegalovirus promoter, and is referred to as
p-CMHex. A second full-length cDNA clone containing a sequence
for a FLAG epitope tag (Sigma) at the 3' end of the cDNA was prepared
by PCR. The 3' primer contained the FLAG sequence after the
last Hex amino acid and just before a termination codon. The
sequence of this product was confirmed and cloned into pcDNA3; this
clone is referred to as HEXFLAG. Primer sequences are given in Table 1.
Cell lines, nuclear extracts, and electrophoretic mobility shift assays. The procedures for these methods were the same as described previously (24). Briefly, Hep G2 cells (human hepatoblastoma) and COS cells were purchased from American Type Culture Collection and maintained in MEM supplemented with 10% FBS and penicillin-streptomycin-glutamine. Cells were plated in 12-well plates (105 cells/well) for transient transfections and luciferase expression assays, 6-well plates for immunohistochemical localization of Hex by the FLAG epitope tag, or 10-cm plates (106 cells/plate) for isolation of nuclear extracts and RNA. They were grown until they were ~75% confluent before transfection with the calcium phosphate-DNA coprecipitation technique as described previously (24).
A plasmid containingImmunohistochemistry. Immunohistochemistry was carried out by plating Hep G2 (or COS) cells in 6-well plates containing a sterile coverslip. Transfections were done with pcDNA3 (control) or HEXFLAG1 as above, after which the cells were fixed with methanol. After rehydration in PBS, nonspecific protein binding was blocked by incubating the fixed cells for 1 h at room temperature in 30% goat serum-70% PBS. Cells were then incubated for 1 h at room temperature in a 1:2,000 dilution of a mouse monoclonal anti-FLAG antibody (Sigma) prepared in PBS containing 10% goat serum and 2% BSA. After five washes at room temperature with PBS, cells were incubated at room temperature for 30 min with a 1:2,000 dilution of a Cy3-tagged goat anti-mouse antibody (Amersham) in the same solution. Cells were again washed in PBS and once in distilled water and mounted on glass slides with Crystal Mount (Biomedia, Foster City, CA). Light and fluorescent micrographs were taken on an Olympus IX70 microscope fitted with a charge-coupled device camera. Light and fluorescent micrographs were combined in Adobe Photoshop. We then printed light micrographs and combined light and fluorescent micrographs.
Statistics.
All statistical comparisons were done by a Student's
t-test. All measurements, including EMSA, were done with an
n 3.
Animals. Adult male Sprague-Dawley rats (Camm Research Institute, Wayne, NJ) weighing 250-275 g were used for these experiments. The Yale Animal Care and Use Committee approved study protocols. Animals received humane care in compliance with the National Research Council's criteria as outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Institutes of Health (NIH Pub. no. 86-23, revised 1985).
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RESULTS |
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Hex transactivates ntcp promoter via an upstream element.
Figure 1A shows a schematic
drawing of the ntcp promoter with known and putative
regulatory regions specified, including the HRE located between
nucleotides 733 and
714. Figure 1B provides the sequence
of the HRE and the 5-bp mutation in the core binding sequence of
Hex producing the mutated HRE, HM5. We used a standard 5'
deletion approach to evaluate the ability of Hex to regulate the ntcp promoter. Three plasmids, p-1237LUC, p-758LUC, and
p-710LUC, were tested. The putative core element of the Hex
binding site from nucleotides
725 to
721 is eliminated from the
shortest construct. Cotransfection of either p-1237LUC (not shown) or
p-758LUC into Hep G2 cells with p-CMHex resulted in an almost twofold
increase in luciferase activity relative to the test plasmids alone
(Fig. 2A). In contrast,
luciferase expression was not different when Hep G2 cells were
transfected with p-710LUC (background) compared with cotransfection
with p-CMHex and p-710LUC. Also, transfection of p-710LUC alone
produced luciferase activity that was only 75% of that found with
p-758LUC (Fig. 2A). This reflects activation of luciferase
from endogenous Hex via binding at a site in p-758LUC that
was deleted in preparing p-710LUC. These data suggest the presence of a
HRE between nucleotides
758 and
710.
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Ntcp promoter sequence from 733 to
714 confers Hex
responsiveness to a heterologous promoter in Hep G2 cells but not in
COS cells.
p-4xHRELUC was used to evaluate whether the HRE was sufficient to
transactivate a heterologous promoter in Hep G2 cells. The parent
vector, pGL3-Promoter, has a minimal SV40 promoter driving expression
of a luciferase reporter gene. Transfection of p-4xHRELUC into Hep G2
cells increased luciferase activity >11-fold compared with vector
alone (Fig. 2B). In contrast, transfection of p-4xHRELUC into COS cells did not significantly increase luciferase activity compared with vector alone (Fig. 2B). These data support the
existence of a liver-enriched factor that can bind and transactivate
via the HRE.
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Hep G2 cells and rat liver contain proteins that bind specifically
to the HRE.
Because liver and Hep G2 cells are known to express Hex
(4, 22, 25, 37,
47), we evaluated liver and Hep G2 cells for the presence
of a protein capable of binding to the HRE. An EMSA was done using the
HRE (double stranded) and HM5 (double stranded) as probes. Nuclear
extracts from both Hep G2 cells and rat liver contained a protein(s)
that specifically bound the labeled HRE (Fig.
6). Although an excess of the nonlabeled
HRE competed successfully with the labeled HRE for binding, this was
not the case for HM5 or for an oligonucleotide containing the unrelated activating protein-1 (AP-1) binding site. Together, these data indicate
that rat liver and Hep G2 cells contain a protein that binds
specifically to the HRE.
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In vitro expressed Hex binds to the HRE in the ntcp promoter.
To determine whether Hex specifically recognized the HRE, a
fusion protein was created between GST and the carboxy-terminal portion
of Hex including the homeodomain starting from amino acid 128. GST and the GST-Hex fusion proteins were isolated from
bacteria using glutathione-coated Sephadex beads (Pharmacia).
Polyacrylamide gel electrophoresis documented that single bands of the
appropriate molecular weights were obtained for both isolates (not
shown). GST alone was unable to bind to HRE and served as a negative
control in the binding assay. In contrast, binding of the
GST-Hex fusion protein to HRE was observed (Fig. 6). A
100-fold excess of nonlabeled HRE competed successfully with labeled
HRE for binding to the fusion protein. The GST-Hex fusion
protein did not bind HM5, nor did the HM5 compete with the fusion
protein for binding to the native HRE. Similarly, the oligonucleotide
containing an AP-1 consensus binding site neither bound to the
GST-Hex fusion protein nor competed with this protein for
binding to HRE. These data indicate that Hex binds
specifically to the sequence from 733 to
714 in the ntcp
promoter that contains the core consensus sequence for Hex
and, along with the reporter gene studies, support a direct effect of
Hex on ntcp expression.
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DISCUSSION |
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The first homeobox gene was cloned from Drosophila about 15 years ago (31). Mutations and misexpression studies on clustered (Hox) and divergent homeobox genes have provided significant information on both early embryonic and mature, tissue-specific functional aspects of these genes (see, e.g., Refs. 1, 2, 8, 13, 14, 16, 19, 21, 26, 27, 34, and 41). However, only a few targets of homeobox genes have been identified (e.g., Refs. 7, 11, 40, and 48). Both mutation/misexpression studies and evaluation of target genes are important for understanding organ development and function.
Because Hex is expressed in the developing hepatobiliary
system and subsequently in the mature liver, we speculated that
Hex is important in liver-specific function and searched for
a potential target for Hex in the liver. The ntcp
gene, which produces a protein integral to maintaining bile flow, was
found to contain a putative Hex binding site upstream of the
minimal promoter (24). We hypothesized that the
ntcp promoter would be regulated by Hex and in
these studies have identified ntcp promoter as the first
known target for Hex. We found a significant but modest
twofold stimulation of ntcp promoter activity via sequences
between nucleotides 733 and
711. This activity was lost when these
nucleotides were deleted, which eliminated a sequence that contained
the in vitro determined consensus Hex binding sequence,
ATTAA (12).
This result did not distinguish between direct binding of
Hex to this small region of DNA and stimulation via a
secondary effect. However, liver and Hep G2 cells, both of which are
known to express Hex mRNA, produce a protein that binds
specifically to the HRE. A fusion protein between GST and the portion
of the Hex protein containing the DNA binding domain binds
to the same site. This is consistent with binding of an endogenous
protein(s) from liver and Hep G2 cells to this region of DNA. A 20-mer
from the ntcp promoter centered on this site drives
expression of a reporter gene under control of a heterologous
promoter in Hep G2 cells when cotransfected with a plasmid expressing a
Hex cDNA. Furthermore, a shortened form of Hex
capable of DNA binding, HEXDN, functioned as a dominant negative when
cotransfected with this plasmid. Finally, an identical reporter
plasmid, p-HM5LUC, except for mutation of the core Hex
binding sequence, is not activated by Hex. Together, these
data allow us to conclude that Hex regulates the
ntcp promoter directly by binding to its consensus sequence between nucleotides 725 and
720. Furthermore, it is possible that
Hex plays a contributing role in overall ntcp
activity in vivo, albeit not as a primary transcriptional activator.
Equally interesting and worthy of further study is the fact that in fibroblast-derived COS cells, Hex was not capable of activating a reporter gene in a plasmid containing a minimal viral promoter and four copies of the HRE. This suggests the presence of a factor(s) that interacts with Hex, is necessary for mediating transactivation of target genes, and is found in Hep G2 cells but not in COS cells. Although this factor may well be tissue specific, our data indicate that this factor is not required for nuclear transport of Hex. Cofactors interacting with other homeobox genes and required for their specificity and activity have been described previously (see, e.g., Refs. 9, 15, 17, 23, 28, 35, 39, and 49). The Drosophila protein exd functions as a cofactor with homeobox proteins at different times in development. This is particularly interesting to us because Hex shows a very early pattern of expression, suggesting a role in embryogenesis as well as a later, organ-specific pattern (4, 22, 25, 47). The cofactor required for Hex function in Hep G2 cells may well be required for Hex function early in development. We are currently conducting studies to determine the identity of this cofactor.
Our results differ significantly from a recently reported study suggesting that Hex might function as a transcriptional repressor (46). These authors constructed various chimeric fusion proteins utilizing different portions of Hex. These were tested against an artificial but specially designed reporter construct. In contrast, using a native, liver-specific promoter we clearly show that Hex activates the ntcp promoter in the same cell type. We also tested for Hex activation of the ntcp gene in COS cells and found no evidence of activation (or repression) despite demonstrating that Hex protein translocates to the nucleus in both cell types. The advantage of our assay system is that it relies on binding of the native Hex protein to a native target site in the ntcp promoter. Nevertheless, there are other potential explanations for the differences between our results, including the possibility that Hex functions as a repressor or as an activator depending on the target gene and circumstances. For instance, Zhu and Kuziora (50) found in their analysis of functional domains of the Deformed homeobox protein that mutant Deformed proteins influenced the regulation of other homeotic genes in a manner distinct from that of the native protein. In fact, they found that removal of two transcriptionally active domains, the acidic region and the C tail, converted a chimeric protein from a strong activator to a repressor of the Distal-less element while having little effect on the activation of the empty spiracles element. It is also possible that the Hex-GAL4 fusion protein used by Tanaka et al. (46) does not interact with the liver-enriched cofactor(s) that we have demonstrated is required for target gene activation. Recent reports by Li et al. (29) and Choi et al. (10) provide strong evidence that the interaction of homeodomain proteins with tissue- or cell type-specific cofactors can influence their transcriptional activity, whereby any one homeobox protein can function as either a repressor or activator. Further studies are required to answer this question.
In summary, we have identified the ntcp promoter as the
first known target gene for Hex. Furthermore, we provide
evidence for the existence of an activating cofactor of Hex
present in Hep G2 cells that is absent from COS cells. Hex
joins the small group of liver-enriched transcription factors (e.g.,
HNF-1, HNF-3, HNF-4, HNF-6, and C/EBP) known to regulate
liver-specific gene promoters. These experiments provide an
important tool for evaluation of the Hex protein and a
strong basis for further investigations into the hepatobiliary role of
Hex.
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ACKNOWLEDGEMENTS |
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This work was supported in part by grants from the Charles H. Hood Foundation (H. C. Jacobs), the Yale Liver Center [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) P-3034989, H. C. Jacobs and C. W. Bogue], NIDDK Grant K08-DK-02318 (S. J. Karpen), and National Heart, Lung, and Blood Institute Grant K08-HL-03471 (C. W. Bogue).
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. C. Jacobs, Dept. of Pediatrics, Yale Univ., New Haven, CT 06520-8064 (E-mail: harris.jacobs{at}yale.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 10 December 1999; accepted in final form 18 February 2000.
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