(Received for publication, November 19, 1996, and in revised form, February 18, 1997)
From the Department of Clinical Chemistry and Laboratory Medicine, Kyushu University, Faculty of Medicine, Fukuoka 812-82, Japan and the § Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 227, Japan
Thrombopoietin (TPO), the ligand for c-Mpl, is a
cytokine that regulates megakaryocyte growth and development. We have
cloned the 5-flanking region of the human TPO gene and analyzed its promoter activity. The human TPO gene promoter lacks a TATA box and
directs transcription initiation at multiple sites over a 50-nucleotide
region. Transient expression in a human liver cell line (PLC) of
promoter fragment-luciferase reporter gene constructs containing a
series of 5
-truncated sequences or site-directed mutations identified
a sequence 5
-ACTTCCG-3
from
69 to
63 as a positive cis-acting
element for high level expression of TPO gene. This sequence contains a
core motif (C/A)GGA(A/T) for Ets family proteins in the noncoding
strand. Gel mobility shift assays performed with nuclear protein from
PLC cells identified a DNA binding protein(s) specific for the element.
Anti-E4TF1-60(GABP
) or anti-E4TF1-53/47(GABP
) antibodies
supershifted the complex in gel shift assay. Furthermore, co-expression
of E4TF1-60 and E4TF1-53/47 squelched TPO gene expression in PLC and
HepG2 cells. It is concluded that Ets family transcription factor
E4TF1(GABP
/
), an ubiquitously expressed protein, is required for
high level expression of the TPO gene in liver.
Thrombopoietin (TPO)1 binds to and activates the c-Mpl protein, a member of the hematopoietic cytokine receptor family, and regulates proliferation and maturation of megakaryocytic cells (1-4). Human TPO cDNA has recently been cloned and shown to consist of 353 amino acids containing a 21-amino acid signal peptide (1, 2). The mature form of human TPO has two domains: an amino-terminal half of 153 amino acids with homology to erythropoietin and an unique 179-amino acid carboxyl-terminal domain that contains multiple potential N-glycosylation sites (1, 2). Analysis of truncated forms of TPO showed that the amino-terminal region is sufficient to fully stimulate c-Mpl (1, 2).
Analysis of circulating TPO levels in thrombocytopenic animals revealed
that TPO is rapidly destroyed when the platelet counts are high, but
its turnover becomes slower when the platelet levels are low, raising
the possibility that the TPO level is regulated by platelet mass (5).
More recently, Fiedler et al. (6) reported that the level of
TPO gene expression remains constant, but the level of TPO in
circulation was determined by the rate of Mpl-mediated uptake and
destruction of TPO by platelets in both normal and mutant mice
(c-mpl/
). On the other hand, TPO mRNA levels in bone
marrow cells were inversely related to platelet count while hepatic or
renal TPO message were unaltered (7). Bone marrow cells rather than
liver or kidney were considered to be a more physiologically relevant
source of TPO (7). However, Northern blot and reverse transcriptase-PCR
analysis indicated that TPO was expressed mainly in liver and also
slightly in many tissues such as kidney, smooth muscle, spleen, and
bone marrow. Thus, further work is required to assess the significance
of TPO gene expression in various tissues, including liver, for
regulating the circulating level of TPO.
The human TPO gene locus has been mapped to chromosome 3q26 or q27, a
site where a number of chromosomal abnormalities associated with
elevated platelet counts and increased bone marrow megakaryocytes were
reported in patients with acute nonlymphocytic leukemia (8-11). The
structure of the TPO gene has been reported by four groups (12-15).
Three of them reported that it consists of 6 exons and 5 introns (12,
13, 15), while Chang et al. (14) found 7 exons and 6 introns. Furthermore, these reports suggest different transcription
initiation sites. Thus, the structure of 5-flanking region of the
human TPO gene, especially that of the promoter region, is still
controversial.
In this article, we report the structure and functional
characterization of the human TPO gene promoter. Our data demonstrate that the human TPO gene has multiple transcription initiation sites and
lacks a putative TATA box near the initiation sites. We identified a
strong cis-acting ets motif, ACTTCCG, at 69 to
63 from
the first initiation site for an optimal expression of the human TPO
gene. Furthermore, E4TF1/GABP/NRF-2 (16-18), an ubiquitously expressed
Ets transcription factor, was shown to be involved in the stimulated
expression of the gene. The functional significance of the element and
its trans-acting factor in TPO gene expression is discussed.
The two human hepatocarcinoma cells, PLC and HepG2, and the human cervical carcinoma cell HeLa cell line used in this study were originally obtained from the American Type Culture Collection. These cells were maintained in Dulbecco's modified Iscove medium supplemented with 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37 °C in a 5% CO2 incubator.
Isolation of Human TPO Genomic Clone and Sequencing of the 5A human TPO cDNA from 651 to 1070 nucleotides
(2) was amplified by reverse transcriptase-PCR using a total RNA of
human adult liver with a sense primer (5-AATGCCATCTTCCTGAGCTT-3
) and an antisense primer (5
-CCTGTGTCTGATGTTCCTGA-3
), and used as a probe.
Approximately 1 × 105 plaques of the human genomic
library in the EMBL3 vector, a gift from Dr. Fukumaki in Kyushu
University, were screened using an ECL labeling and detection kit
(Amersham Corp.). One positive clone containing the 5
-flanking region
of TPO gene was obtained, subcloned into the pUC19 plasmid, and
sequenced by the dideoxy sequence method (19) using a SQ-5500 DNA
sequencer (Hitachi).
Poly(A)+ RNAs from PLC,
HepG2, and HeLa cells were obtained using a micro mRNA purification
kit (Pharmacia Biotech Inc.). 10 µg of poly(A)+RNA were
separated on a 1% formaldehyde-agarose gel and transferred to a Hybond
N+ membrane (Amersham Corp.). The membrane was hybridized
with a TPO cDNA probe in 5 × SSC, 1% SDS, 50% formamide at
42 °C overnight. The membrane was washed twice with 1 × SSC,
0.1% SDS at room temperature for 15 min, twice with 0.1 × SSC,
0.1% SDS at 65 °C for 5 min and then subjected to autoradiography
at 80 °C for 24 h. The cDNA fragment from 651 to 1070 was
radiolabeled with [
-32P]dCTP (6000 Ci/mmol), using a
megaprime labeling kit (Amersham Corp.), and was used as a probe.
Poly(A)+ RNAs from human adult liver, PLC
cells, and HepG2 cells were analyzed to determine the transcription
initiation sites using a rapid amplification of 5-cDNA end
(5
-RACE) method and an RNase protection assay. The 5
-RACE analysis
was performed using a Marathon cDNA amplification kit
(CLONTECH). After the first-strand cDNA was
synthesized by incubating 1 µg of mRNA with an oligo(dT) primer
and Moloney murine leukemia virus reverse transcriptase at 45 °C for
60 min, the second-strand cDNA was synthesized with a mixture of
Escherichia coli DNA polymerase I, RNase H, and E. coli DNA ligase (20). After blunt-ending with T4 DNA polymerase,
the double-stranded cDNA was ligated to the Marathon cDNA
adaptor. A PCR reaction was then performed with a gene-specific
internal antisense primer T1 (5
-TGTGAAGGACATGGGAGTCA-3
), which was
complementary to nucleotides 329-348 of the TPO cDNA in exon 3 (1,
13), and the Marathon adaptor sense primer, AP1
(5
-CCATCCTAATACGACTCACTATAGGGC-3
), followed by nested PCR using the
nested gene-specific antisense primer, T2 (5
-TTGCACTTCTGGGCAGAGTA-3
), which was complementary to nucleotides 109-128 of the cDNA in exon
2 (1, 13), and the nested adaptor primer, AP2
(5
-ACTCACTATAGGGCTCGAGCGGC-3
). The amplified products were then
subcloned into the pGEM-T vector (Promega), and a total of 31 independent clones were sequenced. For the RNase protection assay, the
genomic fragment from
239 to +100 was amplified by PCR and subcloned
into the plasmid pGEM4Z (Promega). The plasmid was linearized by
EcoRI digestion, and the antisense cRNA probe was generated
with T7 RNA polymerase using a mixture of 1 mM each ATP,
CTP, and UTP, and 10 µM [
-32P]GTP (3000 Ci/mmol). After 1 µg of cRNA product and 20 µg of poly(A)+ RNAs were mixed, heated at 80 °C for 10 min,
and hybridized in 30 µl of hybridization buffer of 40 mM
PIPES, pH 6.4, 400 mM NaCl, 1 mM EDTA, and 80%
formamide at 45 °C overnight, the mixture was digested by RNase A
(10 µg/ml) and RNase T1 (0.5 µg/ml, Sigma) at 30 °C for 30 min.
The protected RNA fragments were recovered, resolved on a 6%
sequencing gel, and subjected to autoradiography at
80 °C for
24 h.
Human TPO
promoter-luciferase reporter plasmids were constructed by cloning
various lengths of the 5-flanking sequence of the TPO gene into the
multicloning sites of pLUC-Basic (Promega). The plasmid pUC19-T5500,
containing a 5.5-kb fragment of the 5
-flanking region, was used as a
template for PCR to generate fragments containing from
1318 to +100,
from
107 to +100, from
88 to +100, and from
58 to +100 of the TPO
gene, respectively. Briefly, the required fragments of the human TPO
gene were amplified by using primer sets containing KpnI and
BglII sites at each end, and the resulting fragments were
subcloned into KpnI and BglII sites of the
pLUC-Basic to obtain pLUC-T1318, pLUC-T107, pLUC-T88, and pLUC-T58,
respectively. pLUC-T39 containing
39 to +100 fragment was obtained by
subcloning SacI/BglII fragment of pUC19-T5500
into the SacI/BglII sites of pLUC-Basic. For the
plasmids obtained by using PCR, the sequence of each clone was
confirmed. The plasmid pLUC-T735 was obtained after self-ligation of
the large fragment generated by digestion of pLUC-T1318 with
SmaI and PvuII, and pLUC-T397 was obtained by
self-ligation of the large fragment after pLUC-T1318 was digested with
SmaI and AvrII and treated by the Klenow fragment
to make the AvrII site blunt-ended. pLUC-T158 was
constructed by subcloning a 258-bp fragment of BamHI and
HindIII digestion of pLUC-T1318 into
BglII/HindIII sites of pLUC-Basic. Plasmid
pLUC-T4063 was constructed as follows. First, a 5500-bp
HindIII/EcoRI fragment from pUC19-T5500 was
subcloned into the HindIII and EcoRI sites of
pLUC-Basic, and the resulting pLUC-T5500 was completely digested by
EcoRI and then partially digested by AvrII to
obtain a 8800-bp fragment. pLUC-T735 described above was also digested
with EcoRI and AvrII to obtain a 800-bp fragment.
Finally, both 8800- and 800-bp fragments were ligated to produce
pLUC-T4063.
Plasmids containing the mutations, as defined in the figure legends, were constructed using the plasmid pLUC-T158 as the matrix by an overlap extension PCR protocol (21). Briefly, two separate PCR products, one for each half of the hybrid product, were generated with either an antisense- or a sense-mutated oligonucleotide and one outside primer. The two products were mixed, and a second PCR was then performed using the two outside primers. The product was digested with BamHI and HindIII and ligated into BglII/HindIII sites of pLUC-Basic. The inserts were sequenced to confirm the intended mutations.
Transient Expression of Human TPO-Luciferase Gene and Assay of Luciferase andAll the plasmids of
pLUC-TPO fusion genes and pCI--galactosidase gene that was used for
internal control of transfection were purified using the QIAGEN plasmid
purification kit (QIAGEN, Inc.) and at least two different batches of
plasmids for each construct were tested for transfections. Cells were
plated on a 35-mm dish at a density adjusted so that they reached
70-80% confluence (approximately 3 × 105 cells)
prior to transfection. 4 µg of each plasmid DNA in 100 µl of 0.3 M NaCl and 20 µg of cationic liposome (Promega) in 100 µl of H2O were mixed and added to the cells. After 60 min
of incubation at 37 °C, the transfection medium was replaced by 2 ml
of complete medium containing 10% fetal calf serum. After an
additional 40-h culture, the cells were lysed by 150 µl of Picagene
reporter lysis buffer LC
(Toyoinki) and centrifuged at 10,000 × g for 10 min to remove cell debris. The supernatants were
assayed for luciferase and
-galactosidase activities using the
luciferase assay and
-galactosidase assay systems (Promega),
respectively. The luciferase activities were normalized to those of
-galactosidase and expressed as the percentage of that of a plasmid
as described in the figure legends.
PLC and HeLa cell nuclear extracts
were prepared from 1 × 108 cells according to the
method of Dignam et al. (22). In vitro translated
proteins were prepared by using 1 µg of pET3d plasmids containing
each cDNA for E4TF1-60, E4TF1-53, or E4TF1-47 (23) in the TNT
T7-coupled reticulocyte lysate system (Promega). For gel mobility shift
assay, oligonucleotides were labeled with [-32P]ATP
(6000 Ci/mmol) and T4 polynucleotide kinase. Approximately 1 ng of the
labeled oligonucleotide (10000 cpm) was mixed with 2 µl (5 µg of
protein) of nuclear extract or 1 µl of in vitro translated
proteins in a total of 20 µl of binding buffer (15 mM
HEPES, pH 7.9, 50 mM KCl, 15 mM NaCl, 4 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 0.5 mM
phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 10 units/ml
Trasylol) containing 1 µg of poly(dI·dC). After incubation at room
temperature for 20 min, the reaction mixture was separated on a 5%
nondenaturing polyacrylamide gel with 0.5 × TBE buffer (40 mM Tris borate, 1 mM EDTA). The gel was dried
and subjected to autoradiography at
80 °C for 24 h. For
supershift experiments, 1 µl of control antibody or 0.5 µl of
polyclonal rabbit antibody against either E4TF1-60 or E4TF1-53/47 was
added to the reaction mixture 20 min after the addition of probe, and
the mixture was further incubated for 20 min at room temperature.
The human TPO genomic clone EMBL3-T1 was isolated as
described under "Materials and Methods." The clone contained a
15-kb insert containing 5 kb of the 5-flanking region (Fig.
1A). Southern blot analysis of human genomic
DNA with a DNA fragment from this clone revealed that restriction
fragments obtained by HindIII and BamHI digests
were identical in size to those in the clone, indicating that no major
DNA rearrangements occurred during isolation of the clone (data not
shown). The 4876 nucleotides of the 5
-flanking region and the first
exon were sequenced on both strands, and a portion of the sequence that
encompasses transcription initiation site(s) is shown in Fig.
1B (see also Fig. 2).
Northern Blot Analysis
Northern blot analysis of 5 µg of poly(A)+ RNA from PLC, HepG2, and HeLa cells showed the presence of a 1.8-kb single band in both PLC and HepG2 mRNAs, but no band was detectable in HeLa mRNA, even after a 3-fold longer exposure (data not shown).
Determination of the Transcription Initiation SiteTo
determine the transcription initiation sites of the human TPO gene,
both 5-RACE and RNase protection assays were performed as described
under "Materials and Methods." As shown in Fig. 2, A and
C, 5
-RACE assay of human adult liver cDNA revealed a
total of six initiation sites from +1 to +51 nucleotides relative to the first initiation site. Among 31 clones analyzed, five clones began
at +1, one clone at +8, two clones at +11, 14 clones at +24, seven
clones at +25, and the other two clones at +51. Thus, the initiation
sites are clustered between at +1 and +51, with the major sites at +24
and +25. RNase protection assays were also performed using a cRNA probe
from
239 to +100 nucleotides of the human TPO gene. The results
showed three major RNase-protected bands at +24, +25, and +51 (Fig. 2,
B and C), and their positions matched those of
the most prominent sites determined by 5
-RACE assay. Similar results
were obtained when mRNAs from HepG2 or PLC cells were analyzed,
indicating that the same transcription initiation sites as in adult
liver are employed in these two cell lines (data not shown). A putative
TATA box was not observed in any promoter region of the
multi-initiation sites (see Fig. 1B). Chang et
al. (14) recently reported the existence of exon 0 at a more
5
-upstream region than shown in Fig. 1. If that were the case, a
protected band at
189 must be detected in our RNase protection assay,
since their exon 2, which is from
189 to +114, should be protected by
our cRNA probe of
239 to +100. However, as shown in Fig.
2B, no such protected band at the
189 position (indicated
by an arrow) was observed. Thus, the significance of exon 0 described by Chang et al. could not be assessed in our assay.
To
localize the region essential for human TPO gene expression, a series
of 5- to 3
-deletion mutants were generated and transiently expressed
in PLC cells (Fig. 3A). Since PLC cells expressed the TPO gene and employed the same transcription initiation sites as adult human liver (Fig. 2), we used the PLC cell line to assay
the promoter activity. Furthermore, it was demonstrated that the
transcription of the reporter gene also initiated from several start
sites that were essentially the same as in endogenous TPO gene (data
not shown). As shown in Fig. 3B, all the plasmids containing
5
deletions of various lengths from
4063 to
88 promoted high level
expression of luciferase activity that was approximately 14-25 times
higher than the background of pLUC-Basic. This indicated the motifs
observed in the region from
4063 to
88 had no significant regulatory effect, while pLUC-T158 reproducibly showed a 1.3-2-fold higher activity than the other constructs. This might suggest the
existence of an inhibitory element(s) from
397 to
158 and an
enhancing element(s) from
158 to
107. In contrast, a further deletion to
58 resulted in an extreme reduction of activity to about
13% of that of pLUC-T88. The results clearly indicate that a positive
regulatory element(s) is located from
88 to
58, and this region is
essential for optimal transcription of the TPO gene. The pLUC-T39
plasmid still showed as much activity as pLUC-T58, 2.6 times higher
than pLUC-Basic. We next transfected the deletion mutants into HeLa
cells, which do not express the TPO gene. Fig. 3C shows that
HeLa cells did express the pLUC-T58 reporter gene to an equivalent
level to that in PLC cells, indicating that the 3
-sequence from
58
functions as a minimal promoter in both PLC and HeLa cells. However,
the proximal cis-element(s) from
88 to
58 increased the expression
only 1.8-fold in HeLa cells compared with a significant activation in
PLC cells (6.2-fold). This result suggests that the promoter of the TPO
gene can function in HeLa cells but that the cis-element from
88 to
58 supports tissue-specific activation only in PLC cells.
Mutational Analysis of the Proximal Cis-regulatory Region of the TPO Gene
We next introduced clustered mutations into the plasmid
pLUC-T158. As shown in Fig. 4, the activities of
mutants, M5, 6, 8, 9, and 10, all showed 75-80% reduction as compared
with that of pLUC-T158, and M3, 7, and 11 exhibited small deviations or
changes from the control, while M4 showed a slightly enhanced activity (50%). The M5 mutations introduced into the longer 5 sequence, pLUC-T1318-M5 and pLUC-T4063-M5, also reduced the activities. The data
clearly indicate that the sequence ACTTCCG from
69 to
63 functions
as a major positive cis-element for the efficient expression of the TPO
gene. It was also noted that ACTTCCG contains a core sequence of
ets motif (C/A)GGA(A/T) in the noncoding strand (24, 25).
Other mutations, M1, M2, and M14, that were directed toward a GGGTG
(CACCC) element at
123 to
119 (26), a GGGAAG (ets-like)
element at
85 to
80 (24, 25), and a ATTGG(CCAAT) element at
26 to
22 (27), respectively, exhibited almost comparable activity to that
of the wild type. It is indicated that these elements do not play
significant regulatory roles in TPO gene expression.
Gel Mobility Shift Assay
The nuclear protein factor(s)
specific for binding to the cis-element from 69 to
63 was analyzed
by gel mobility shift assay using wild type or mutated oligonucleotides
(Fig. 5A). As shown in Fig. 5B,
lane 2, the nuclear proteins from PLC cells produced three
bands, a prominent band I and two faint complexes, II and III, of
higher mobilities. Band I was shown to contain two bands of close
mobilities on short exposure of the gel. The specificity of these bands
for the sequence was shown by a competition experiment in which a 50 times molar excess of cold specific oligonucleotide completely
abolished the formation of band III (lane 3) while partially
preventing the formation of bands I and II. In contrast, the same molar
excess of mutated oligonucleotides failed to compete (lanes
6-8). Among a series of mutated oligonucleotides, M3, 4, 11, and
13, which had little effect on expression, efficiently competed with
the wild type sequence (lanes 4, 5, 9,
and 10), while M11 competed less (lane 9). In
contrast, M5, 9, and 10, which markedly reduced expression, had little
effect on complex formation (lanes 6-8). HeLa cell nuclear
proteins also yielded three bands that showed similar mobilities and
sequence specificities to those in PLC cells (lanes 11-19),
indicating that the factor(s) is also present in cells that do not
express TPO. Since the cis-element of the TPO gene contains a core
sequence, -CGGAA-, recognized by Ets domains, oligonucleotides from the
stromelysin gene promoter (
209/
187, STROM
209/
187) (24, 28) and
cytochrome oxidase subunit IV gene promoter (+13/+36, RCO4 +13/+36)
(25, 29) corresponding to DNA-binding sites for Ets-2 and E4TF1/GABP,
respectively, were tested for competition. Both STROM and RCO4
sequences strongly inhibited formation of bands I, II, and III, while
inhibition by RCO4 was more marked than by STROM under conditions where
the control sequence had no effect (Fig. 6). Taken
together, these data indicate that (i) the DNA-binding protein(s) that
recognizes the cis-element of the TPO gene is present in liver as well
as HeLa cells and (ii) shares DNA sequence specificity with E4TF1/GABP or Ets-2, putative Ets family proteins (18, 24, 25, 28-31).
E4TF1 Binds to the ets Consensus Site of the TPO Gene Promoter
The effect of antibodies against two subunits of E4TF1,
E4TF1-60, or E4TF1-53/47, on complex formation was investigated. Fig. 7A showed that an antibody against E4TF1-60
sharply reduced the formation of bands I, II, and III, and generated a
supershifted complex (shown by an arrow) in both PLC and
HeLa cell nuclear extracts (lanes 2 and 6).
Anti-albumin antibody, used as a control, had no effect on complex
formation under these conditions (lanes 4 and 8).
In addition, anti-E4TF1-60 antibody yielded no band when incubated
with the probe in the absence of nuclear extract (lane 9).
Thus, the effect of anti-E4TF1-60 antibody was specific, and bands I,
II, and III all contained E4TF1-60 protein as a component. Anti-E4TF1-53/47 antibody, in contrast, slightly reduced the intensity of band I with generation of a band of lower mobility (indicated by an
arrowhead in lanes 3 and 7). The
intensities of bands II and III remained almost unchanged (lanes
3 and 7). As shown in lane 10, incubation of
the antibody with the probe formed no band, indicating that the
antibody did not react with the probe DNA. These results suggested that
the band generated by anti-53/47 antibody in lanes 3 and
7 represented a specific supershifted complex of band I
containing E4TF1-53/47 protein. This view was supported by the
experiment shown in Fig. 7B. In this experiment, we tested
in vitro translated E4TF1-60, E4TF1-53, and E4TF1-47 for
binding to the ets motif of the TPO gene promoter (Fig.
7B). E4TF1-60 produced a complex c (lane 2),
while neither E4TF1-53 nor E4TF1-47 alone bound the oligonucleotide W
(lanes 3 and 4). In contrast, when mixtures of
E4TF1-60 and E4TF1-53, or E4TF1-47 were tested, a prominent band a
or b of lower mobilities was produced along with abolishment or
reduction of band c (lanes 5-7). The mobilities of complex
c, or a and b, were similar or very close to that of band III or I
produced by PLC nuclear proteins, respectively (compare lanes
2, 5, 6, and 7 with lane
8). From these data, it is evident that bands III or I formed by
nuclear extract represented the complex by monomeric E4TF1-60 or
heteromer of E4TF1-60 and E4TF1-53/47, respectively. Band II could
not be reproduced by in vitro translated subunits of E4TF1,
suggesting that it might represent a complex of DNA and E4TF1-60 that
has been modified in vivo.
Effects of Overexpression of E4TF1-60 and -53
Since our
results indicated that the ets motif of the human TPO gene
promoter is recognized by E4TF1/GABP/NRF-2, the effect of
overexpression of two subunits of E4TF1, E4TF1-60 or -53 (32), on TPO
promoter activity was investigated. Transfection of increasing amounts
of plasmid encoding E4TF1-60, from 0.1 to 2.0 µg, into PLC cells
suppressed TPO gene promoter activity in a dose-dependent manner. In contrast, transfection of 0.1 or 0.5 µg of plasmid for
E4TF1-53 enhanced TPO promoter activity 1.1- or 1.4-fold, respectively, while higher amounts of the plasmid suppressed the activity. Co-transfection of both plasmids for E4TF1-60 and -53 suppressed the activity as did E4TF1. These data suggest that the
subunits of E4TF1 can sequester some factor or factors needed for
transactivation of the human TPO gene and are consistent with the
possibility that both E4TF1 subunits are involved in transactivation of
human the TPO gene, and the amount of E4TF1-53 might be limiting in
cells compared with that of E4TF1-60. Similar results were obtained
using HepG2 cells (white columns in Fig. 8).
Squelching by overexpressed Ets protein has also been observed in
insulin-mediated prolactin gene expression (33).
The present analysis of the 5-flanking region of the human TPO
gene has clarified several features of the mechanisms that regulate its
expression. Our analysis of the transcription initiation site of the
TPO gene in human adult liver revealed multiple initiation sites with
the majority at +24 and +25, rather than at
20 or +48 as previously
reported. The sites were essentially identical in both 5
-RACE and
RNase protection assays and were found to be also utilized in the liver
cell lines PLC and HepG2. More recently, Chang et al. (14)
reported the existence of one more exon (exon 0) from
1498 to
1397
with a transcription initiation site at
1498. Although their
initiation site could not be detected in our RNase protection analysis,
reverse transcriptase-PCR of liver mRNA, performed using primers
flanking the region from exon 0 and exon 3, did amplify a band of the
expected length. But that amount was significantly lower than that of
the product amplified using exon 1 primers (data not shown). Thus, exon
0 detected in the study of Chang et al. might be utilized in
the adult liver as one of several initiation sites but considerably
less frequently than the other sites reported in this study.
Alternatively, it might reflect different mechanisms of TPO gene
expression during liver development, because we used human adult liver
whereas they used fetal liver. However, this possibility awaits further
investigation.
The analysis of 5-flanking regions of the TPO gene clearly
demonstrated that the 7-bp sequence -ACTTCCG- from
69 to
63, was
essential for the high level expression of human TPO gene in liver
cells. The sequence contains a core (C/A)GGA(A/T) motif for an Ets
family of transcription factors in its noncoding strand. It has been
reported that Ets proteins recognize a purine-rich (C/A)GGA(A/T) motif
in the middle of 10-bp nucleotides, and the flanking sequences are
considered to be also involved in determining sequence specificity of
each Ets protein (23-25, 34, 35). Since mutations in the flanking
region of the ets consensus sequence of TPO gene, M3, 4, and
11, also exerted a marginal effect, that is slight reduction in M3 and
M4 and slight stimulation in M5, respectively, a total 10-bp sequence
might be involved in the specific binding of a factor to the human TPO
ets sequence.
The present study further demonstrated that a specific DNA binding protein(s) for ACTTCCG motif of the human TPO gene is highly likely to be E4TF1/GABP. This is supported by the following results. (i) The DNA-protein complex detected in gel shift assays was effectively abolished by an ets sequence for E4TF1/GABP from an RCO4 gene promoter that matches 8 out of 10 nucleotides of that of the TPO gene (24, 25, 29). Furthermore, there is a complete match of sequence observed between that of the human TPO gene and of the MCO5b site D, another site for E4TF1/GABP/NRF-2 binding (29). (ii) Antibodies against subunits of E4TF1, E4TF1-60 or E4TF1-53/47, specifically supershifted the complex. (iii) The combination of in vitro translated E4TF1-60 and E4TF1-53/47 produced a complex with essentially the same mobility as that produced by a PLC nuclear extract. (iv) Overexpression of E4TF1-60 and -53/47 squelched TPO promoter activity in PLC and HepG2 cells.
E4TF1/NRF-2 has been cloned from HeLa cells and shown to bind to ets elements in the cytochrome c oxidase subunit IV gene (16) or adenovirus early region 4 gene (17). GABP, a rat homologue of E4TF1/NRF-2, binds to an ets element in the rat HSV IE gene (18). E4TF1 contains three subunits, E4TF1-60, -53, and -47, among which E4TF1-60 specifically binds to DNA through the Ets domain in its carboxyl terminus (16, 17). E4TF1-53 and -47, homologues of Drosophila Notch and Caenorhabditis elegans Glp-1 and Lin-1, respectively, associate with E4TF1-60 through four tandemly repeated Notch-ankyrin motifs in the amino terminus and mediate transcriptional activation through the COOH terminus (16, 17). The factor has been proposed to maximally activate the promoter by binding to two or more ets sites in a tetrameric form (17, 18). A construct containing one more ets sequence just upstream of the wild type ets motif of the TPO gene exhibited enhanced expression (data not shown), consistent with the idea that two ets sequence synergistically activate the TPO gene. We speculate that the human TPO gene is unique in containing only one ets element and is thus incapable of forming a more competent DNA-protein complex. This might explain why the level of TPO expression is low, even in the liver.
Although the present study clearly indicated that an ubiquitously expressed E4TF1/GABP is involved in stimulated expression of TPO gene in liver, it remains to be defined why the TPO gene is not expressed in HeLa cells that contained an equivalent level of E4TF1/GABP to that in liver (Figs. 5B and 7A). We speculate that E4TF1/GABP is necessary but not sufficient for liver-specific expression of the TPO gene, and there might be a liver-specific mechanism of gene activation, since the element transactivated the TPO gene in liver more strongly than in HeLa cells (Fig. 3C). These could include (i) liver-specific post-translational modification of E4TF1/GABP protein, although in our in vitro footprinting experiment to determine ets site occupancy, no apparent differences were observed between liver and HeLa cells (data not shown); (ii) the presence of liver-specific mediator(s) or co-activator(s) that intervenes between E4TF1/GABP and general transcription machinery. This might be one of the likely mechanism since the TPO reporter gene containing ets motif could not be transactivated in transient expression study of HeLa cells; (iii) the difference of higher order chromatin structure of the TPO gene that is open in liver but closed in HeLa cells for the factor(s) to be recruited into the gene promoter. Alternatively, there still remains the possibility that other liver-specific as yet undefined Ets transcription factor(s) that is closely related to but different from E4TF1/GABP is involved in the liver-specific expression of TPO gene.
Finally, our results clearly demonstrated that the ets motif
ACTTCCG at 69 to
63, which is recognized by E4TF1/GABP, an ubiquitously expressed Ets protein, is essential for the high level
expression of the human TPO gene in liver cells. This work provides us
the first clues toward understanding the molecular mechanism of
expression of the human TPO gene. More research into this question,
especially the functional role of E4TF1/GABP in liver-stimulated
expression and regulation of circulating TPO level in vivo,
is necessary, and such an investigation is now in progress.
The nucleotide sequence data reported in this article will appear in the DDBJ, EMBL, and GenBankTM nucleotide sequence data bases with accession no. AB000528.
We thank Dr. H. Sumimoto (Kyushu University, Faculty of Medicine, Japan) and Dr. Tanner (University of Bristol, UK) for critical reading of the manuscript, and Dr. J. A. Conaway (Oklahoma Medical Research Foundation, Oklahoma City) for critical reading and editing the revised manuscript. We also thank Dr. Y. Fukumaki (Kyushu University, Research Laboratory for Genetic Information, Japan) for his generous gift of the human genomic library.