From the Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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A novel cDNA was partially isolated from a
HepG2 cell expression library by screening with the promoter-linked
coupling element (PCE), a site from the -fetoprotein (AFP) gene
promoter. The remainder of the cDNA was cloned from fetal liver RNA
using random amplification of cDNA ends. The cDNA encodes a
239-amino acid peptide with domains closely related to the
Drosophila factor nk-2. The new factor is the
eighth vertebrate factor related to nk-2, hence
nkx-2.8. Northern blot and reverse transcriptase polymerase chain reaction analysis demonstrated mRNA in HepG2, two other AFP-expressing human cell lines, and human fetal liver. Transcripts were not detected in adult liver. Cell-free translation produced DNA
binding activity that gel shifted a PCE oligonucleotide. Cotransfection of nkx-2.8 expression and PCE reporter plasmids into HeLa
cells demonstrated transcriptional activation; NH2-terminal
deletion eliminated this activity. Cotransfection into AFP-producing
hepatocytic cells repressed AFP reporter expression, suggesting that
endogenous activity was already present in these cells. In contrast,
cotransfection into an AFP-negative hepatocytic line produced moderate
activation of the AFP gene. The cardiac developmental factor
nkx-2.5 could substitute for nkx-2.8 in all
transfection assays, whereas another related factor, thyroid
transcription factor 1, showed a more limited range of substitution.
Although the studies have yet to establish definitively that
nkx-2.8 is the AFP gene regulator PCF, the two factors
share a common DNA binding site, gel shift behavior, migration on
SDS-acrylamide gels, and cellular distribution. Moreover, the
nk-2-related genes are developmental regulators, and
nkx-2.8 is the first such factor associated with liver
development.
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INTRODUCTION |
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Developmental processes are frequently associated with expression of specific homeobox transcription factors. Although all share a common form of DNA binding domain, the hundreds of known homeobox factors belong to many subfamilies with a wide variety of secondary domains (1). The Drosophila factor nk-2 is the prototype of a distinct family of homeobox factors. nk-2-related homeodomain factors have been characterized in Drosophila (nk-2, nk-3/bagpipe, and nk-4/tinman/msh2), planarians (Dth1 and Dth2), leeches (lox10), Caenorhabditis (Ceh22), and vertebrates (nkx-2.1 to 2.7) (2). The nk-2-related factors contain a characteristic secondary domain, the "conserved peptide," which has an unknown function and is unrelated to known protein domains.
The three Drosophila homologues have important developmental functions. nk-2 is involved in early neurogenesis, nk-3 is required for visceral muscle formation, and nk-4 is essential for the formation of precardiac mesoderm.
The vertebrate nk-2 factors also regulate development. nkx-2.1, or thyroid transcription factor 1 (TTF-1),1 is a regulator of thyroid-specific gene expression, thyroid development, thyroid cell differentiation, and thyroid cell proliferation (3, 4) and is first expressed several days before thyroid differentiation (5). TTF-1/nkx-2.1 also regulates pulmonary development and gene expression (6, 7).
nkx-2.5/Csx and nkx-2.3 are both expressed in early cardiac primordia and thus replicate the function of tinman (8). nkx-2.6 is also expressed in heart. In Drosophila, ablation of tinman blocks cardiac development, whereas knockout of mouse nkx-2.5 arrests heart development at the looping stage (8). This is a less severe phenotype than the Drosophila knockout and probably reflects functional redundance with nkx-2.3 and nkx-2.6 (9). nkx-2.7, another tinman homologue related to cardiac development, has recently been described in zebrafish (10).
nkx-2.2 is expressed in developing mouse brain, with an onset of expression at about 9 days gestation. The transcripts are found in localized regions that correspond to anatomic boundaries in the developing forebrain. The localization suggests that nkx-2.2 specifies differentiation of the developing diencephalon into its anatomically and functionally defined subregions (11).
In mice the cardiac mesenchyme forms from the ventral wall of the foregut at 8.5 days gestation, and the hepatic primordium buds from an adjacent area of the foregut at 8.5-9 days gestation (12). There is a strong association between this early phase of cardiac differentiation and nk-2-related factors, but no homeodomain factors of any sort have been associated with initial differentiation of the liver, later differentiation of bile ducts, or regulation of hepatic stem cells. Two homeobox factors, HNF-1 (13, 14) and HNF-6 (15), are associated with the liver, but they regulate only the postdevelopmental phenotype. Another homeobox gene, hlx, has a limited effect on liver development. However, hlx is expressed in hematopoietic cells of fetal liver, not hepatocytes. Mice with targeted disruption of this gene undergo normal early hepatic differentiation, but the failure of hematopoietic cells to colonize the fetal liver results in a small but normally formed liver (16).
Our research has focused on the -fetoprotein (AFP) gene,
characteristically expressed from the earliest stages of liver
development, but silent after birth. A site near the AFP promoter, the
promoter-linked coupling element (PCE), appears to interact with the
main developmental regulators of AFP expression. In HepG2 cells, the
PCE binding activity has been characterized as a distinct transcription
factor, PCF (17, 18). To characterize PCF further, we used a
PCE-containing oligonucleotide for expression cloning, leading to the
isolation of nkx-2.8, which shares numerous properties with
PCF. This is the first demonstration of an association between an
nk-2 homeobox factor and liver gene expression.
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EXPERIMENTAL PROCEDURES |
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Expression Cloning--
A gt11 library of oligo(dT)-primed
cDNA from HepG2 cells (CLONTECH) was screened
for plaques that bound the PCE in the AFP gene promoter (
166 to
155, TGTTCAAGGACA; Ref. 18). This sequence and its complement were
included in a double-stranded oligonucleotide with added TCGA sticky
ends. Probe preparation and screening were modified from previously
described methods (19, 20). The oligonucleotide was labeled with
polynucleotide kinase and [
-32P]ATP and concatenated
with DNA ligase to an average size of 5-10 repeats with a specific
activity of 5 × 108 d/min/µg. Bacteriophage were
plated using Escherichia coli strain Y1090. Plaques were
lifted onto Millipore HATF membranes, air dried, incubated in blocking
buffer (5% nonfat dry milk, 25 mM HEPES, pH 7.9, 5 mM MgCl2, 40 mM KCl, 1 mM dithiothreitol) for 30 min and then probe mix (0.25%
nonfat dry milk, 25 mM HEPES, pH 7.9, 5 mM
MgCl2, 40 mM KCl, 0.1 mM
dithiothreitol, 10 µg/ml sonicated denatured DNA, and 10 ng/ml
labeled concatenated probe) for 12 h. DNA from Micrococcus
lysodeiktikus was chosen for this mixture for its high G+C content
(70%) and provided a very effective background competition that
greatly enhanced the sensitivity of the screen. A screen of 1,000,000 phage plaques produced a single clone with appropriate binding
specificity. A second screen of 2,000,000 plaques from another HepG2
library in
ZAP (Stratagene, La Jolla, CA) provided no additional
positives. Upon sequencing, the positive clone was found to be
truncated at the 5
-end by an in-frame deletion that also removed part
of the
-galactosidase gene. PCR assays (see below) were used to
screen pooled DNA isolated from both libraries but detected only the
single partially deleted clone. The cloned insert was subcloned in
pBluescript KS+ (Stratagene). Because clonal instability was observed,
this and all further subclones were propagated in a DNA repair
and recombination-deficient E. coli host (SURE,
Stratagene).
PCR-based Cloning--
From the sequence of the new clone,
direct and reverse transcriptase PCR assays (below) were used to screen
a variety of RNA sources. A positive detection was obtained from a
commercial cDNA prepared from human fetal liver (Marathon-Ready
cDNA, CLONTECH) which was synthesized using
random amplification of cDNA ends cloning adaptor annealed to the
5-end. Amplification was carried out with primers AP1 and R5A and Tth
DNA polymerase (denaturation at 95 °C for 2 min; 40 cycles of
95 °C for 40 s, 68 °C for 4 min). A weak PCR product was
then reamplified with two combinations of nested primers, using the
same temperature-cycling protocol. Primers AP2 and R13A gave products
of 160 and 340 bp; primers AP2 and R7A gave products of 410 and 590 bp.
The PCR products were subcloned in pCR-Script SK+ (Stratagene), and
multiple clones were sequenced. All represented the same region, with
predicted size differences based on primer position. The shorter PCR
products were terminated at a high G+C region within the longer one.
Thus all clones represented a single mRNA. Two allelic variants
were recognized. For PCR studies, except where otherwise specified, enzymes were used with buffers and conditions supplied by the manufacturers. The primers are listed in Table
I.
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DNA Sequencing, cDNA, and Genomic Analysis--
Because the
high G+C content caused many compression artifacts, all DNA sequencing
was verified by sequencing with a thermostable DNA polymerase system
(SequiTherm EXCEL, Epicentre Technologies, Madison, WI). The original
clone and four plasmid clones from the PCR amplification of the 5-end
were sequenced fully on both strands. A 241-bp region of overlap
between the bacteriophage and PCR-generated clones was identical in all
clones. All sequences were subjected to BLAST analysis (National Center
for Biotechnology, http://www.ncbi.nih.gov/BLAST/). Related sequences
were downloaded and compared by FASTA alignment routines (21).
mRNA Analysis-- RNA isolation from cell lines and tissues, agarose-urea gel electrophoresis, and Northern blot hybridization were carried out as described previously (22, 23). Poly(A) RNA was purified using a magnetic bead system (Poly(A)Tract, Promega, Madison, WI). For each gel lane, 100 µg of total RNA was processed, 50 µg of E. coli tRNA was added, and the entire sample was ethanol precipitated and redissolved for electrophoresis. For a hybridization probe, the F9:R5 PCR product was cloned in pCR-Script SK+. A riboprobe was generated with T7 RNA polymerase following plasmid linearization with PvuII. For PCRs, 1 µg total RNA was incubated in a single tube with primers R5 and PBDG-B and Superscript II reverse transcriptase (Life Technologies) at 45 °C for 1 h. Amplification of nkx-2.8 transcripts was then carried out on an aliquot using primers F7 and R10A and KlenTaq DNA polymerase (CLONTECH). The sample was denatured at 95 °C for 2 min followed by 7 cycles of 95 °C for 40 s, 72 °C for 4 min; and 40 cycles of 95 °C for 40 s, 68 °C for 4 min. A control PCR for a housekeeping mRNA, porphobilinogen deaminase (PBDG; Ref. 24), was carried out with primers PBDG-A and PBDG-B and Taq DNA polymerase. The sample was denatured at 94 °C for 2 min followed by 40 cycles of 94 °C for 30 s, 55 °C for 20 s, and 72 °C for 30 s. Products were visualized on an ethidium bromide-stained agarose gel or transferred to nitrocellulose and detected by hybridization to a specific probe, the product of an F10:R7 PCR.
Cells, Tissues, and Transfection-- Human cell lines HepG2, HuH7, and HuH1-clone 2 (Clone 2) are derived from hepatocellular carcinomas (25-27); and RPMI 7451 is from a cholangiocarcinoma (28). H4C3 is a rat hepatocellular carcinoma cell line that expresses high levels of albumin and low levels of AFP (23). Human lung carcinoma cell line H441 was used as a source of TTF-1 for gel shifts (29). All were propagated in Williams E medium containing 1-5% fetal calf serum, penicillin-streptomycin, and glutamine. Specimens of human fetal and adult liver were provided by Dr. Stephen Strom (University of Pittsburgh).
CaPO4 transfection of cell lines HepG2, Clone 2, and HeLa was carried out as described previously (30). H4C3 cells were transfected with LipofectAMINE (Life Technologies, Inc.); 10-cm culture plates were inoculated with 1 × 106 cells and transfected after 2 days, using 10 µg of DNA and 60 µl of LipofectAMINE/plate, according to the supplier's protocols. Individual transfection experiments always consisted of a series of identical plates transfected simultaneously under identical conditions. Each determination was the average of two transfections. All described results were reproducible in at least two separate experiments.Reporter and Expression Plasmids-- A PCF reporter plasmid, pPCE4-HIV-CAT, was constructed from plasmid pAPF1-HIV-CAT (31) by cutting with SalI and PstI to remove the HNF4 binding sites and substituting an 80-bp oligonucleotide array consisting of four copies of the PCE from the AFP promoter. The oligonucleotide contained PCF binding sites in the same orientation spaced at 20-bp intervals. A control plasmid containing no binding sites, pHIV-CAT, was produced by blunt ligating an SalI-PstI-digested plasmid. AFP and albumin gene expression plasmids have been described previously (18, 30, 32).
nkx-2.8 expression plasmids were constructed in pCI (Promega), which contains a CMV early enhancer/promoter and SV40 splicing and polyadenylation signals. The vector also contains a multicloning site with an adjacent T7 RNA polymerase promoter. Full-length pCMV-Nkx2.8 and 5Gel Shift Analysis--
pCMV-Nkx2.8 and pCMV-Nkx2.8 DNAs were
linearized with BamHI, transcribed with T7 RNA polymerase,
and translated using a TNT® Coupled Wheat Germ Extract
System (Promega) according to the supplier's protocols. Labeling with
[14C]leucine was also described in these protocols. Cell
extracts and gel shift procedures were previously described (18).
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RESULTS |
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cDNA Cloning and Sequence-- Using a combination of approaches, overlapping products have been cloned which encode a novel transcription factor (Fig. 1). BLAST analysis of translated sequences showed two characteristic domains, a homeobox (Fig. 2A) and a conserved peptide (Fig. 2B). Both domains established a relationship to the Drosophila factor nk-2. These comparisons also demonstrated that the encoded factor is new, the shortest member of the family described so far. The new factor is the eighth vertebrate nk-2-related factor, hence nkx-2.8.
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Gene Structure-- The predicted cDNA structure was confirmed with a series of PCR studies of fetal liver cDNA, and parallel studies were carried out on genomic DNA. This analysis verified the unique cDNA and also localized an intron. The following primer combinations were studied: F7:R10A, F7:R7A, F7:R12A, F7:R13A, F12:R13A, and F5A:R16 (Table I). In the cDNA analysis, each combination gave a single strong product, suggesting that the mRNA has no splicing variants. Amplification with F7:R7A and F7:R10A gave genomic DNA products ~750 bp larger than cDNA products, whereas genomic DNA did not amplify with F7:R12A. All other products were identical for both cDNA and genomic DNA. These results suggested a ~750-bp intron at the position of primer R12A. A genomic PCR product was cloned and partially sequenced (Fig. 3), confirming an intron within the codon located 31 amino acids from the NH2-terminal side of the homeobox. This is similar to the structure of the human TTF-1 gene, which has a single 966-bp intron within a codon 36 residues proximal to the homeobox (29).
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mRNA Analysis-- Standard Northern blot analysis of total RNA was attempted with several probes, but because of the high G+C content, specific signals were obscured by nonspecific hybridization to rRNA. To solve this problem, a riboprobe plasmid was constructed containing the F9:R5 amplimer, a region within the homeobox which had lower G+C content (Fig. 4A). In blots of total RNA, this probe detected a transcript with an apparent molecular mass of 1.6 kb but still showed cross-hybridization to rRNA even under stringent conditions (not illustrated). However, when poly(A) RNA was isolated for blots, the hybridization was predominately to this single 1.6-kb transcript, which was clearly detected in RNA from three hepatocarcinoma cell lines, HepG2, HuH7, and Clone 2 (Fig. 4B). The cholangiocarcinoma cell line, R7451, and adult tissues including liver, appeared to lack this transcript, although very weak signals near 1.6 kb and at higher molecular masses could represent either low levels of transcript or cross-hybridizing species. mRNA was also isolated from three fetal liver specimens and compared with three additional adult liver specimens. A weak signal at 1.6 kb was suggested in at least two of the fetal liver mRNAs. The Northern blot studies can only be considered semiquantitative because poly(A) RNA preparations vary significantly.
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Analysis of Translated nkx-2.8 Protein--
The full-length and
deleted nkx-2.8 expression plasmids were translated in
vitro and the products analyzed by oligonucleotide gel shift. Gel
shifts (Fig. 5A) were carried
out with a PCE oligonucleotide (tcgaTGTTCAAGGACA). Full-length
nkx-2.8 protein produced a band shift that comigrated with
the PCF band shift of HepG2 cells but was distinctly lower than the
band shift of the related factor, TTF-1. As expected, the 5-deleted
protein produced a band shift lower than that of intact
nkx-2.8. The analysis demonstrated that the expression
plasmids containing the cDNAs are functional and encode peptides
that bind the PCE.
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nkx-2.8 Function in a Nonhepatocytic Cell Line-- HeLa cells, expected to be free of endogenous expression of nkx-2.8 and other hepatocytic transcription factors, were used for the initial evaluation of transcriptional activity. nkx-2.8 was analyzed directly and compared with the related factors nkx-2.5 and TTF-1 (Fig. 6). For this analysis, a reporter plasmid, pPCE4-HIV-CAT, was constructed in which four PCE sites were placed in tandem near a basal promoter. In HeLa cells, the reporter showed clear activation over unstimulated levels. Maximum 9-fold stimulation occurred at 1 µg. Higher levels had somewhat lower activity but still stimulated the reporter. nkx-2.5 also showed comparable (14-fold) activation. The deleted form of nkx-2.8 showed no transcriptional stimulation, nor did TTF-1, even though the latter showed clear binding to a PCF oligonucleotide (Fig. 5).2 Thus nkx-2.8 is a functional transcriptional activator, can act through a PCE site, and requires the NH2-terminal 31 amino acids for activation. nkx-2.5 has comparable function, although another nk-2-related factor, TTF-1, cannot activate the same reporter plasmid in HeLa.
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nkx-2.8 Function in Hepatocytic Cell Lines--
For evaluation of
nkx-2.8 in hepatocytic cells, we attempted to study
differentiated hepatocytic cell lines with both fetal (AFP+, albumin+) and adult (AFP,
albumin+) phenotypes (Fig.
7A). In general,
hepatocellular carcinoma cell lines either have a fetal phenotype like
HepG2 or are undifferentiated. Cell lines reported to have an adult
hepatocytic phenotype are infrequent and problematic, although we
utilized two such lines, Clone 2 and H4C3. Morinaga et al.
(27) characterized gene expression in the HuH1 variant, Clone 2. HuH1
has a fetal phenotype, but Clone 2 was reported to have selectively
lost AFP expression. However, our analysis of Clone 2 showed reduced
but still significant AFP expression. Moreover, we also observed clear
nkx-2.8 expression in Clone 2 (Fig. 4). Reuber hepatoma cell
lines like H4C3, common models for the adult hepatocyte phenotype, also
have problematic features. Because H4C3 is rat-derived, we did not
analyze for nkx-2.8 transcripts. However, we previously
detected low AFP mRNA expression (23) and a weak PCF gel shift from
these cells (18).2
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DISCUSSION |
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An Unusual Gene and Transcript-- nkx-2.8 mRNA has an unusual DNA sequence. The overall G+C content is 66%, whereas the coding region is 71%. Moreover, the exons contain 129 CG dinucleotides, potential DNA methylation sites. Such CG islands are found upstream of some genes but are rarely incorporated into coding regions. A high G+C content is also a feature of the mammalian TTF-1 (71% G+C), nkx-2.2 (65%), and nkx-2.5 (70%) genes (11, 29, 36). Interestingly, when examples of these genes have been sequenced from lower vertebrates, the high G+C content is not conserved (10, 37). The Xenopus nkx-2.5 gene, for example, is only 48% G+C.
Computer analysis of RNA folding predicts an unusually high degree of secondary structure for the nkx-2.8 mRNA. The combination of high G+C content and secondary structure has led to technical problems in the cloning, purification, and quantification of the mRNA. The abundance of nkx-2.8 mRNA may have been underestimated because of these technical problems. Nevertheless, a low level of mRNA is characteristic of the nkx-2 gene family except for TTF-1 and may typify genes that have limited regulatory function. The studies of this paper provide a technical paradigm for a difficult class of mRNAs.Relationship of Binding Sites-- It is not surprising that there is a close relationship between the PCE and TTF-1 binding sites (Fig. 8) because the nkx-2.8 homeobox is extremely similar to the TTF-1 homeobox and to those of other members of the family. The sites share a common motif, TCAAG, which is also the central motif of the Drosophila nk-2 binding site (38). nkx-2.5 also binds strongly to a TTF-1 site (33). The structure and sequence of these nk-2-family homeodomains differ considerably from the more common Antennapedia class homeodomains. The latter bind to a TAAT central motif that corresponds to CAAG in the nk-2 family sites (39).
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nkx-2.8 and PCF-- nkx-2.8 was obtained through efforts to clone the AFP gene developmental regulator activity we characterized as PCF (18). There are marked similarities between PCF and nkx-2.8. Both bind the PCE and activate from that site, and both are expressed in the same cells, along with AFP. However, the exact peptide composition of PCF is not fully resolved. Photoaffinity labeling demonstrated a broad PCF band migrating at 34 kDa, and partially purified PCF showed multiple bands in the 32-34-kDa range. nkx-2.8 is basic, and PCF also purifies as a basic protein(s). Although only 26 kDa, nkx-2.8 has anomalous migration on SDS-acrylamide gels which causes it to migrate near 34 kDa, the position of PCF components. The multiple PCF bands might represent isoforms, post-translational modifications, related factors, coactivators, or even unrelated peptides that copurify. It appears likely, however, that nkx-2.8 is a component of PCF.
There is a more significant problem in the relationship between PCF and nkx-2.8. Surprisingly, transfection of nkx-2.8 into AFP-positive lines HepG2 and Clone 2 caused transcriptional repression of AFP gene reporters, whereas transfection in H4C3 cells led only to weak AFP gene activation. In contrast, PCF has been characterized as a strong activator in HepG2 cells. Although other explanations are possible, repression may indicate squelching of an activity already present in HepG2 and Clone 2. The weak activation in H4C3 cells (with stronger activation by other nk-2-related factors) does indicate that nkx-2.2 and related factors can act as AFP gene activators in a hepatocytic cell setting. The weak effects are disappointing, but this may reflect the limitations of available cell lines as experimental models. H4C3 cells may lack other components necessary for full activation of AFP or even, according to standard models, actively repress the gene (40, 41). It will be necessary to examine other cell systems, potential coactivators, and possibly transgenic systems before the role of nkx-2.8 is fully resolved. Galarneau et al. (42) have used transfection experiments to demonstrate that another factor, FTF, can activate AFP transcription on binding to the PCE. FTF is also known as LRH and PHR and is related to Drosophila FTZ-TF1. However, PCF from HepG2 cells is a distinctive activity unrelated to FTF, and FTF is undetectable in HepG2 cells (18, 42). Moreover, the levels of FTF are highest in adult liver, where the AFP gene is silent. Recent studies in Drosophila have shown an important relationship that may explain interaction of PCF, nkx-2.8, and FTF. In Drosophila, FTZ-TF1 and the homeodomain protein ftz are mutually dependent cofactors. The two factors interact to activate important low affinity sites (43, 44). Similar interaction between nkx-2.8 and FTF, or other members of each family, might account for the complex regulation that is observed at the PCE.nkx-2.8 and Other nk-2-related Factors-- The nk-2-related factors are defined by distinctive homeoboxes, and the studies in this paper demonstrate that nkx-2.8, nkx-2.5, and TTF-1 all bind the PCE. nkx-2.5 substituted for nkx-2.8 in all of the transfection assays, suggesting very similar function for these two factors, whereas TTF-1 showed more limited ability to substitute. Like many activation domains, the nkx-2.8 NH2 terminus is rich in proline and acid residues, and the deleted 31-amino acid region shares this composition. Direct NH2-terminal deletion eliminated activation function. In contrast, nkx-2.5 and TTF-1 have terminal regulatory domains with more internal activation domains because short NH2-terminal deletions increased transcriptional activation before more extensive NH2-terminal deletion removed the apparent activation domain (4, 33). nkx-2.8 has a shorter NH2-terminal region (86 amino acids) than either nkx2.5 (136 amino acids) or murine TTF-1 (159 amino acids). nkx-2.8 is thus more compact than other members of the family and may lack an NH2-terminal regulatory region, but it nevertheless retains activation function in its shortened NH2 terminus. Moreover, the unique NH2-terminal domain of nkx-2.8 might be subject to specific regulation in hepatocytic cells which is evaded by nkx-2.5 and TTF-1.
The present studies have established that nkx-2.8 gene expression is associated with AFP expression in fetal but not adult liver and in hepatocellular carcinoma. AFP is expressed not only in fetal liver but also in primitive endoderm and yolk sac. A common target in the AFP gene could be regulated by different members of the nk-2 gene family in these settings, just as cardiac development involves the partially redundant expression of several different nk-2-related genes (2, 8-10). Future areas of investigation include the role of nkx-2.8 overexpression in neoplasia and the identification of additional nkx-2 genes related to endodermal development. Our findings that associate nkx-2.8 with liver development remain preliminary, limited by our initial choice of a human cell model, because the human is not a suitable organism for the detailed study of development. For characterization of animal development, we recently cloned segments of the rat and mouse nkx-2.8 genes and found that they are extremely similar to their human counterpart in both NH2-terminal and homeobox regions.3 Such strong conservation suggests a gene with important function. ![]() |
ACKNOWLEDGEMENTS |
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We are grateful to Steven Strom for providing specimens of human fetal and adult liver and to Roberto Di Lauro and Robert J. Schwartz for providing TTF-1 and nkx-2.5 expression plasmids.
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FOOTNOTES |
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* This study was supported by National Institutes of Health Grant CA68440 and American Cancer Society Grant NP-82227.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF000295, AF000296, and AF000297.
Present address: Institute for Transcriptional Informatics, 220 Wilson Blvd., Suite 245, Arlington, VA 22201-3324.
§ Present address: Pfizer-Central Research, Box 943, Eastern Point Rd., Groton, CT 06320.
¶ Present address: Dept. of Pathology, University Hospitals of Cleveland, 11000 Euclid Ave., Cleveland OH 44106.
To whom correspondence should be addressed: Dept. of
Pathology, University of Pittsburgh, School of Medicine, BST S405,
Pittsburgh, PA 15261. Tel.: 412-648-8253; Fax: 412-383-7969;
E-mail: loc{at}med.pitt.edu.
1
The abbreviations used are: TTF-1, thyroid
transcription factor 1; AFP, -fetoprotein; PCE, AFP gene
promoter-linked coupling element; PCF, HepG2 transcription factor that
binds PCE; PCR, polymerase chain reaction; bp, base pair(s); PBDG,
porphobilinogen deaminase; HIV, human immunodeficiency virus; CAT,
chloramphenicol acetyltransferase; CMV, cytomegalovirus; kb,
kilobase(s); FTF, fetal transcription factor.
2 N. Crawford and J. Locker, unpublished results.
3 G. Apergis and J. Locker, unpublished results.
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REFERENCES |
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