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J Biol Chem, Vol. 273, Issue 44, 29022-29031, October 30, 1998
From the Enhancer II (ENII) of hepatitis B virus (HBV) is
one of the essential cis-elements for the transcriptional regulation of
HBV gene expression. Its function is highly liver-specific, suggesting that liver-enriched transcriptional factors play critical roles in
regulating the activity of ENII. In this report, a novel hepatocyte transcription factor, which binds specifically to the B1 region (AACGACCGACCTTGAG) within the major functional unit (B unit) of ENII,
has been cloned from a human liver cDNA library by yeast one-hybrid
screening, and demonstrated to trans-activate ENII via the B1 region.
We named this factor hB1F, for human B1-binding factor. Amino acid
analysis revealed this factor structurally belongs to nuclear receptor
superfamily. Based on the sequence similarities, hB1F is characterized
to be a novel human homolog of the orphan receptor fushi
tarazu factor I (FTZ-F1). Using reverse transcription-polymerase chain reaction, a splicing isoform of hB1F
(hB1F-2) was identified, which has an extra 46 amino acid residues in
the A/B region. Examination of the tissue distribution has revealed an
abundant 5.2-kilobase transcript of hB1F is present specifically in
human pancreas and liver. Interestingly, an additional transcript of
3.8 kilobases was found to be present in hepatoma cells HepG2.
Fluorescent in situ hybridization has mapped the gene locus
of hB1F to the region q31-32.1 of human chromosome 1. Altogether, this
study provides the first report that a novel human homolog of FTZ-F1
binds and regulates ENII of HBV. The potential roles of this FTZ-F1
homolog in tissue-specific gene regulation, in embryonic development,
as well as in liver carcinogenesis are discussed.
Hepatitis B virus (HBV)1
is the major cause of acute and chronic hepatitis, also closely
associated with the development of hepatocellular carcinoma (1). HBV
predominantly infects hepatocytes, which is a prominent characteristic
of hepadnaviruses. The genome of HBV is a small, circular, partially
double-stranded DNA of about 3.2 kb, which contains four partially
overlapping open reading frames (ORF) encoding the surface antigens
(preS/S), the core antigen/e antigen (preC/C), the polymerase (P) and
the X protein (X), respectively (2, 3). These HBV genes express
specifically in liver, controlled by the combinatorial action of the
promoters and enhancers. To date, four promoters (Sp1, Sp2, Cp, and Xp) (4) have been identified to be responsible for the transcription of the
viral mRNAs, and they are regulated by two HBV enhancers. Enhancer
I (ENI) functions in a relatively tissue independent manner (5, 6),
while enhancer II (ENII) shows strong hepatocyte specificity.
Enhancer II of HBV is located within the X ORF, about 600 bp downstream
of ENI (7-9). In our previous study, ENII was mapped in a 148-bp
region from nt 1627 to 1774 (HBV adr1 subtype), partially overlapped
with the core promoter (Cp) (7). According to the functional analyses,
it can be divided into two parts, A and B, with part B as the basal
functional unit, which could be further subdivided into three regions
B1 (nt 1687-1704), B2 (nt 1705-1735), and B3 (nt 1736-1774) (Fig.
1) (7, 10). ENII plays critical roles in
the regulation of the liver-specific viral gene transcription. It is a
major cis element regulating the liver-specific transcription initiated
by Cp, which is critical for viral replication and morphogenesis (11).
ENII also participates in the regulation of other promoters of HBV
genome, and is able to activate heterologous promoters in a highly
liver-specific manner (7, 12).
To elucidate the regulatory mechanism of the liver-specific activity of
ENII, a considerable amount of work has been focused on the study of
trans-acting factors that interact with ENII. As shown in Fig. 1, many
transcription factors have been identified to interact with different
regions of ENII. Among these factors, a set of liver-enriched
transcription factors such as HNF1, C/EBP, HNF3, and HNF4 are believed
to be involved in the determination of hepatocyte specificity of ENII.
Recently, we found the B1 region (nt 1688-AACGACCGACCTTGAG-nt 1703) was
bound specifically by a novel hepatocyte nuclear factor (B1-binding
factor, Fig. 1).2 This binding
activity is present in differentiated hepatoma cell line HepG2 but not
in the nonhepatic cell line HeLa. Mutations in the B1 region not only
aborted the specific binding by this factor, but also significantly
reduced the activity of ENII in co-transfection analysis. In addition,
the disruption of the specific binding was shown to cause a decrease in
viral gene transcription initiated from the core promoter, resulting in
a reduction of the 3.5-kb pregenomic RNA (13, 14). Therefore, this
binding site has an important function in regulating the ENII activity, and consequently, it affects the global HBV gene expression. Our previous efforts to identify the B1-binding factor have suggested it
not to be any members of known liver-enriched factors such as HNF1,
HNF3, HNF4, or C/EBP (13, 15), but a novel hepatocyte transcription
factor uncharacterized before; it was thus considered of great interest
to clone and characterized this factor.
For this purpose, we employed a yeast one-hybrid screen (16-18) to
clone the cDNA of the B1-binding factor. By screening GAL4 activation domain (AD) fused-cDNA library of human adult normal liver, we isolated a cDNA named hB1F (human
B1-binding factor) and investigated both its
interaction with the B1 region in vitro and its
trans-regulatory effects on ENII in vivo. hB1F was further characterized as a novel human homolog of FTZ-F1, an orphan nuclear receptor that was originally identified as a transcriptional factor involved in the regulation of fushi tarazu
(ftz) expression in early Drosophila melanogaster
embryos (19).
cDNA Cloning by Yeast One-hybrid Screen--
The two
oligonucleotides, 5'-gatcAACGACCGACCTTGAG-3' and
3'-TTGCTGGCTGGAACTCctag-5', contained the B1 fragment in capital letters with extra nucleotides in lowercase letters added for cloning
purpose, were chemically synthesized and annealed. Four tandem repeats
of the B1 fragment were placed upstream of the E1b minimal promoter in
HIS3 reporter plasmid (p6012), and the CYC1 minimal promoter
in lacZ reporter plasmid (pCZII), respectively (CLONTECH). These two plasmids were linearized and
sequentially integrated into Saccharomyces cerevisiae YM4271
(MATa, ura3-52, his3-200, ade2-101, lys2-801, leu2-3,
112, trp1-903, tyr1-501) (CLONTECH) to obtain
a reporter yeast strain YMB1-HB. The yeast was then transformed with
human liver MATCHMAKER cDNA library (CLONTECH)
and selected on histidine-deficient (His In Vitro Translation--
EcoRI-digested 2.5-kb
cDNA of the isolated positive clone (named pGAD-16) containing the
complete hB1F coding sequence was inserted downstream of the T7
promoter of the pBS(+) (Stratagene), and used for in vitro
translation in a typical 50-µl reaction volume of TNT coupled
reticulocyte lysate systems (Promega), containing 25 µl of rabbit
reticulocyte lysate, 1 µl of 1 mM amino acids, and 1 µg
of DNA template.
Expression and Purification of Glutathione S-Transferase Fusion
Protein--
The BglII-NdeI fragment of pGAD-16
(nt 158-696) (see Fig. 3B) containing the suspected
DNA-binding domain of hB1F was made blunt-ended and in-frame fused with
glutathione S-transferase by inserting into the
SmaI digested pGEX-3X vector (Amersham Pharmacia Biotech).
The glutathione S-transferase fusion protein was expressed in Eschericha coli. BL21 essentially as described by Smith
and Johnson (22), then was purified with glutathione-Sepharose 4B (Amersham Pharmacia Biotech).
Preparation of Nuclear Extract--
The nuclear extract of HepG2
cells and HeLa cells was prepared according to the method of Andreas
and Faller (23). The detailed procedure has been described previously
(24). Briefly, cells were first suspended in 400 µl of buffer A (10 mM HEPES-KOH pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), kept on ice for 15 min,
and then homogenized in a Dounce homogenizer with a B pestle about
20-25 strokes. After centrifugation, the nuclear pellet was
resuspended in 200 µl of buffer B (20 mM HEPES-KOH, pH
7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl, fluoride, 25% glycerol), and kept on ice for 20 min. The supernatant was collected after centrifugation, immediately
frozen in small aliquots in liquid nitrogen, and stored at
Electrophoresis Mobility Shift Assay
(EMSA)--
Oligonucleotides used in EMSA were the following: B1,
5'-GATCAACGACCGACCTTGAG-3' and 3'-TTGCTGGCTGGAACTCCTAG-5'; B1m (mutant B1, the mutated nucleotides are in lowercase letters),
5'-GATCAACtACaGAtCTcGAG-3' and 3'-TTGaTGtCTaGAgCTCCTAG-5'. EMSA was
performed as described previously (24). In the binding reaction,
32P-end-labeled probe (0.1-1 ng, 10,000 cpm) was mixed
with 1-2 µg of poly(dI-dC) (Amersham Pharmacia Biotech), 2-6 µg
of nuclear extract, or 2 µl of in vitro translation
product in a 20-µl reaction mixture containing 10 mM
Tris-HCl (pH 7.5), 90 mM NaCl, 0.15 mM MgCl2, 0.2 mM EDTA, 0.1 mM
dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and
5% glycerol. After incubation at room temperature for 25 min, the
mixture was resolved on a 5% nondenaturing polyacrylamide gel. In the
competition assay, competitors were added to the reaction mixture and
incubated at 0 °C for 10 min prior to the addition of the probe.
DNase I Footprinting Analysis--
The HBV genome fragment (nt
1634-1816) containing ENII and the core promoter (10) was amplified by
PCR with [ DNA Transfection and CAT Assays--
To construct the CAT
reporter plasmid pENII(B)/CpCAT, the ENII(B)/Cp fragment (nt
1686-1878) containing the basic functional unit of ENII (part B) and
the Cp (10), was amplified by PCR and inserted into the XbaI
and HindIII sites of pBS(+) (Stratagene). The CAT reporter
gene was then cloned downstream of pENII(B)/Cp. Four point mutations of
the B1 region (sequence was listed in an EMSA procedure) were
introduced by PCR to construct mutant reporter plasmid
pENII(B1m)/CpCAT. To construct the eukaryotic expression plasmid of
hB1F, pCMV-hB1F, 2.5-kb hB1F cDNA from pGAD-16 released by
EcoRI digestion was inserted downstream of CMV promoter in
pCMV-poly vector (a gift from E. Lai). The antisense expression plasmid
pAnti-hB1F, which transcribes hB1F in the antisense orientation, was
constructed by inserting the hB1F cDNA in the reverse orientation downstream of CMV promoter in the same vector.
HepG2 and HeLa cells were propagated in Dulbecco's modified Eagle's
medium with 10% fetal bovine serum at 5% CO2 and
37 °C. DNAs were transfected into HepG2 or HeLa cells by the calcium phosphate precipitation method (26). Cells were harvested 48 h
after transfection and the protein extracts were prepared. CAT assay
was performed according to the method of Gorman et al. (27). In the co-transfection assay, different amounts of hB1F expression plasmid (pCMV-hB1F) and 1 µg of the reporter plasmid pENII(B)/CpCAT or pENII(B1m)/CpCAT were used; 1 µg of plasmid pCMV/SEAP (28), which
expresses secreted placental alkaline phosphatase (SEAP), was included
as internal control. The total amount of DNA plasmids used for each
transfection experiment was 15 µg, and the plasmid pBS(+)
(Stratagene) was used to make up the difference. Each transfection experiment was performed at least three times.
RT-PCR--
Five primers were synthesized and used in the RT-PCR
for amplification of the complete coding sequence of the hB1F in
HepG2 cells: primer I, 5'-CCCCCAATCTCTTTTTGTTTTGAAAGC-3' (nt 1591-1565 in hB1F); primer II, 5'-GAAAGCAGAGCTCCTAGGGGTTGTAAC-3' (nt 1570-1544); primer III, 5'-GTAACTTATGCTCTTTTGGCATGCAAC-3' (nt 1548-1522); primer
IV, 5'-GAACTGCCTATAATTTCACTAAGAATGTC-3' (nt 32-60); primer V,
5'-CTAAGAATGTCTTCTAATTCAGATACTGG-3' (nt 50-78). 100 ng of
poly(A)+-RNA from HepG2 or HeLa cells were reverse
transcribed with 1 µl (200 units) of SUPERSCRIPT II RNase
H
To examine the distribution of hB1F isoforms with difference within A/B
regions, 1 µg of total RNA isolated separately from fetal liver,
adult liver, HepG2 cells, and HeLa cells by TRIzol reagent (Life
Technologies, Inc.) was reverse transcribed into cDNA with primer
VI (5'-CTTGGATCACCTGAGACATGGCTTCTAGC-3') (nt 521-493). Subsequently,
first round PCR was performed using primer VI and primer IV, and second
round using primer VII (5'-GTCTTTAAAGCACGGACTTACACCTATTG-3') (nt
91-119) and primer VIII (5'-CTTCTAGCTTAAGTCCATTGGCTCGGATG-3') (nt 500-472). The resulting PCR fragments were expected to
include part of the A/B region and the C region, and were analyzed by electrophoresis on a 5% nondenaturing polyacrylamide gel.
Northern Blot Analysis--
Multiple Tissue Northern blots
containing 2 µg of poly(A)+ RNA per lane from multiple
human adult tissues were purchased from CLONTECH.
Blots were hybridized with random radiolabeled 2.5-kb hB1F cDNA
probe (Prime-a-Gene labeling system, Promega) in ExpressHyb hybridization solution (CLONTECH) at 68 °C for
1 h. The filters were washed twice with wash solution I (0.3 M NaCl, 0.03 M sodium citrate (pH 7.0), 0.05%
SDS) for 20 min at room temperature, and twice with wash solution II
(15 mM NaCl, 1.5 mM sodium citrate (pH 7.0),
0.1% SDS) for 30 min at 50 °C. The same filters were hybridized
separately with a
Twenty micrograms of total RNA isolated from HepG2 and HeLa cells by
TRIzol reagent (Life Technologies, Inc.) were electrophoresed in 1.0%
agarose-formaldehyde gels, blotted onto Hybond N+ nylon
membranes (Amersham Pharmacia Biotech), and subjected to Northern blot
hybridization as described above.
Human Chromosomal Localization by Fluorescent in Situ
Hybridization (FISH) Mapping--
A 2.5-kb hB1F cDNA probe was
biotinylated with dATP using the Life Technologies, Inc. BioNick
labeling kit. The FISH detection was performed in See DNA Biotech. Inc.
(Canada), according to Heng and Tsui (29) and Heng et al.
(30).
Nucleotide Sequence Accession Number--
The sequence of the
hB1F cDNA determined in this study has been deposited in the
GenBankTM data base under accession no. HSU80251.
Cloning of cDNA Encoding B1-binding Factor hB1F by Yeast
One-hybrid Screen--
Our previous study has revealed that B1 region
of HBV ENII is bound specifically by a novel liver-enriched nuclear
factor (13-15). As shown in Fig. 2,
using the 16-bp B1 fragment (AACGACCGACCTTGAG) as probe in
EMSA, we detected a specific shift (band s) with the nuclear extract of
the hepatoma HepG2 cell, but not with that of nonhepatic HeLa cell.
When four mutations
(AACtACaGAtCTcGAG) were
introduced into the B1 fragment, the specific binding was no longer
detected. Although HNF4 DNA binding consensus oligonucleotide in large
excess amount seemed to be able to compete the B1 specific binding
(Fig. 2), the possibility of HNF4 binding was ruled out since
HNF4-specific antiserum failed to affect this specific band in a
supershift assay (13). As the interaction of the B1-binding factor with
B1 fragment was of functional significance (13, 14), it was of great
importance to identify this unknown factor.
A yeast one-hybrid screen was employed to clone the B1- binding factor
from human liver MATCHMAKER cDNA library
(CLONTECH). In a total of approximately 4 × 106 transformants, 19 His+ colonies were
selected, of which one colony showed strong
Subsequent sequencing of the 2.5-kb cDNA insert of pGAD-16 revealed
a 1.5-kb ORF. Surprisingly, the ORF is in an orientation reverse to the
GAL4 AD (Fig. 3A). This
cDNA was probably transcribed by a cryptic promoter located around
the ADH1 terminator region of the
vector,3 and similar
observations had been reported previously (31). The complete sequence
of the cDNA insert in pGAD-16 is shown in Fig. 3B. The
presence of an in-frame stop codon TAA upstream of the predicted
initiation codon indicated that the cDNA insert contained the
complete ORF encoding a protein of 495 amino acids. We named it hB1F
for human B1-binding factor. The predicted molecular mass of 54 kDa for
hB1F was verified by in vitro translation (data not
shown).
The amino acid sequence analysis showed that hB1F shared a common
modular structure with the nuclear receptors (32). It consists of
subdomains A to E or F (Fig. 3C), among which the C region
(DNA-binding domain) and the E/F region (ligand-binding domain) are the
two conserved domains of the nuclear receptor superfamily (33, 34). The
homology comparisons suggested hB1F to be a human homolog of orphan
nuclear receptor FTZ-F1 (see below). Since the whole sequences
including the DNA-binding domain and the activation domain have been
encompassed in this factor, it is then not surprising that hB1F could
activate the B1-driven HIS3 and lacZ reporter
genes independent of GAL4 AD in the yeast screening.
hB1F Binds Specifically to B1 Sequence--
To confirm the binding
specificity of the cloned hB1F for the B1 target sequence, in
vitro translated hB1F was incubated with labeled B1 probe and
analyzed by EMSA. As shown in Fig.
4A, the formation of shifted
complex (band s) could be efficiently competed by unlabeled B1
oligonucleotides, but not by B1m, or other nonspecific oligonucletides.
Quite notably, the shifted complex (band s) migrated to a position
similar to that of the complex found with the HepG2 nuclear extract
(Fig. 2), suggesting that the binding fashion of the cloned hB1F to the
B1 sequence was consistent with that of the B1-binding factor observed
in the HepG2 nuclear extract.
Since hB1F shares the common structure with nuclear receptors (Fig. 3,
B and C), a partial hB1F cDNA (nt 158-696,
encoding amino acids 35-215) containing the putative DNA-binding
domain was fused with glutathione S-transferase and
expressed in E. coli. The purified protein glutathione
S-transferase-hB1F(DBD) was able to bind to the B1 target
region specifically (data not shown). To localize further the hB1F
specific binding site(s) in HBV ENII and core promoter region, we
performed a DNase I footprinting analysis, using the labeled fragment
of HBV DNA (nt 1634-1816) as target. After incubation with the
purified glutathione S-transferase-hB1F(DBD), one region of
ENII (nt 1689-ACGACCGACCTTGAGGCA-nt 1706) was shown to be completely
protected from DNase I digestion (Fig. 4B). It agrees well
with the B1 region of ENII (nt 1688-AACGACCGACCTTGAG-nt 1703). No other
binding site of hB1F was found in ENII and Cp region.
The above results provided strong evidence of the specific binding of
hB1F to the B1 sequence. Together with the results obtained from a
yeast one-hybrid screen, we have demonstrated that hB1F is the human
cellular factor binding specifically to the B1 region of enhancer II of
HBV.
hB1F Activates the Enhancer II of HBV--
To investigate the
functional importance of hB1F to the activity of enhancer II, we
performed co-transfection analysis in nonhepatic cells (HeLa) which do
not express hB1F protein (see the results of Northern blot analyses,
Fig. 8B). The CAT activity of the reporter plasmid
pENII(B)/CpCAT is very low in HeLa cells due to the liver specificity
of ENII (Fig. 5A, lane
1) (24). When eukaryotic expression plasmid pCMV-hB1F, which
contains hB1F cDNA under the control of CMV promoter was
co-transfected, the CAT level of pENII(B)/CpCAT could be stimulated
with the increasing amount of pCMV-hB1F (Fig. 5A). However,
if mutations were introduced into the B1 region, the activity of ENII
could no longer be stimulated by hB1F (Fig. 5B). These
results indicated that hB1F activates ENII via the B1 site. The
trans-activating effect of hB1F to ENII was also confirmed in the
experiment of co-transfecting pENII(B)/CpCAT with the antisense
expression plasmid pAnti-hB1F into HepG2 cells which express endogenous
hB1F. In that experiment, the antisense hB1F mRNA was shown to be
able to suppress the activity of ENII (data not shown).
hB1F Is a Human Homolog of FTZ-F1--
Homology comparison
revealed that hB1F was most closely related to members of the FTZ-F1
subfamily of orphan nuclear receptors. Since the identification of
FTZ-F1 in D. melanogaster (19), its homologs or close
relatives have been isolated in insects (35) and in vertebrate species
(36-43), and these factors constitute a distinct FTZ-F1 subfamily of
nuclear receptors. In Fig. 6, the results
of homology comparisons of hB1F with some representative vertebrate
FTZ-F1 factors and Drosophila DmFTZ-F1 are summarized.
The two zinc finger structural motifs present in the DBD of hB1F are
95% identical with that of mLRH-1, xFF1rA, and zFF1A; 89% with hSF-1,
mSF-1, and bAd4BP; and 82% with DmFTZ-F1 (Fig. 6). At the
carboxyl-terminal end of DBD, hB1F shares a typical 30-amino acid
FTZ-F1-box (Fig. 3B) with all members of the FTZ-F1 subfamily, which is implicated in determining the specificity of
monomeric binding of FTZ-F1 with its target DNA sequence
(5'-PyCAAGGPyCPu-3') (37). As shown in Fig. 6, the FTZ-F1-box of hB1F
matches almost exactly with the sequences of vertebrate FTZ-F1
factors.
Three subregions of the ligand binding domain (E/F region) of hB1F,
region II, region III (44), and activation function-2, all share a high
degree of identity with mLRH-1, xFF1rA, and zFF1A, but a considerably
lower one with hSF-1, mSF-1, and bAd4BP (Fig. 6).
From the above sequence analysis, we categorized hB1F as a member of
FTZ-F1 subfamily of nuclear receptors. It is a novel human homolog of
FTZ-F1, distinct from the factor hSF-1 which was identified as a human
FTZ-F1 homolog before (43). Based on the sequence similarities among
the FTZ-F1 subfamily, we suggested that hB1F, together with its close
relatives such as mLRH-1, rFTF, xFF1rA/xFF1rB, and zFF1A/zFF1B
constituted one subgroup of vertebrate FTZ-F1; while hSF-1, bAD4BP and
mSF-1/mELP, which have been also reported to share high homology with
each other (45), composed the second subgroup.
An hB1F Isoform hB1F-2 Is Identified--
Since the specific B1
binding activity was first identified in HepG2 cells and was found
absent in HeLa cells, we used RT-PCR to examine the existence of this
factor in these two cell lines. As expected, hB1F could be amplified
from HepG2 cells but not from HeLa cells (data not shown). The
amplified fragments from HepG2 cells were cloned into pcDNA3 vector
and sequenced. We found two forms of amplified sequences, one of them
matched with the cloned hB1F, while the second form had extra 138-bp
nucleotides encoding 46 amino acid residues in the A/B region (Fig.
7A) with the rest of the
coding sequence identical to hB1F. This factor was designated as
hB1F-2. The 5' and 3' ends of the 138 bp nucleotides follows the
"GT-AG" exon-intron junction rule (Fig. 7A), suggesting that hB1F-2 and hB1F might be two isoforms resulting from different splicing.
A panel of different pairs of primers was designed to conduct RT-PCR to
investigate whether the isoforms with different A/B regions are also
present in normal liver tissues. As indicated in Fig. 7B,
these two forms of A/B region (with or without the 138 bp) were present
not only in HepG2 cells, but also in human adult liver and fetal
liver.
hB1F mRNA Is Restricted Mainly to Pancreas and Liver--
The
tissue distribution of hB1F gene expression was examined by Northern
blot analysis of poly(A)+ RNA isolated from a variety of
human adult tissues (multiple tissue Northern blot I and II,
CLONTECH) with a labeled 2.5-kb hB1F cDNA
probe. Among 16 tissues examined, a transcript of 5.2 kb was detected
in an abundant amount in pancreas, less in liver, and very little in
lung (Fig. 8A). No signal was
detected in any other tissues tested, including heart, brain, placenta,
skeletal muscle, kidney, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocytes (Fig. 8A
and data not shown).
To examine the distribution of hB1F in hepatoma cells and nonhepatic
cells, total RNAs isolated from HepG2 and HeLa cells were hybridized
with the cDNA probe of hB1F. As expected, no signal was detected in
HeLa cells. Interestingly, however, there are two different sizes of
hB1F transcripts in HepG2 cells. Besides the 5.2-kb transcript
consistent with that seen in liver and pancreas, there was an
additional transcript of 3.8 kb (Fig. 8B).
hB1F Gene Is Located at Human Chromosome 1 q31-32.1--
The human
chromosomal localization of the hB1F gene was mapped by FISH, using the
biotinylated 2.5-kb hB1F cDNA as a probe. Among 100 checked mitotic
cell spreads, 81 showed signals on one part of the chromosomes (Fig.
9A), suggesting the
hybridization efficiency was >80%. Using 4,6-diamidino-2-phenylindole
banding to identify the specific chromosome, we were able to assign the signals to the long arm of chromosome 1 (Fig. 9B). The
detailed position was further determined based on the summary from 10 best photographs (Fig. 9C). No other locus was picked by
FISH detection under the condition used. Therefore, the hB1F gene is
located at human chromosome 1, region q31-32.1.
Enhancer II is a critical cis-element for the liver-specific
transcriptional regulation of HBV gene expression. In order to elucidate the regulatory mechanism of liver-specific activity of ENII,
it is of great importance to identify cellular factors interacting with
this cis-element, especially the liver-specific or liver-enriched
transcription factors. In this report, we described the cloning and
identification of a novel hepatocyte nuclear factor that interacts with
the ENII B1 region (nt 1688-AACGACCGACCTTGAG-nt 1703). Using the B1
sequence as a bait in yeast one-hybrid screening, the cloned factor
named hB1F from human liver MATCHMAKER cDNA library has been proved
by EMSA and DNase I footprinting to be specifically bound to the B1
sequence within ENII. The co-transfection assay demonstrated that hB1F
trans-activated ENII via its direct interaction with the B1 site. The
results with a survey of tissue specificity of its expression showed
that it was restricted to liver and pancreas for normal tissues,
present only in differentiated hepatoma cell line HepG2, but not in the
nonhepatic HeLa cells. It is thus clear that we have successfully
cloned the hepatocyte transcription factor which binds specifically to
the B1 region and activates the enhancer II of hepatitis B virus.
It has been known that the strong hepatocyte specificity of ENII is due
to the requirement of hepatocyte transcription factors. So far, several
liver-enrich factors have been found to interact with ENII. These
include HNF1 (15), C/EBP (46), HNF3 (24, 47), and HNF4 (48, 49). Now we
can add hB1F to the list of liver-enriched transcription factors
interacting with ENII. It should be noted that none of these
liver-enriched factors is exclusively expressed in the liver, but the
liver is the only tissue that expresses all of these factors.
Therefore, it is probably the combination of these different
liver-enriched factors that determines the hepatocyte specificity of
ENII, and consequently direct the restricted hepatocyte expression of
HBV genes.
hB1F is a novel nuclear receptor. Structurally, it belongs to the
FTZ-F1 subfamily that binds target DNA as monomers. The binding
sequence of hB1F within the B1 region (nt
1688-AACGACCGACCTTGAG-nt 1703) agrees well with the
consensus FTZ-F1 response element (5'-PyCAAGGPyCPu-3') derived from the
DNA-binding sequence of known FTZ-F1 factors (19, 37, 40, 42, 50-52).
This binding site contains a single hexad half-site (5'-AGGPyCPu-3')
(53) and three 5'-flanking bases (5'-PyCA-3') critical for the specific
recognition of FTZ-F1 factors. In addition to the B1 region, two other
binding sites of nuclear receptors within the ENII/Cp region have been
previously described. One is located in the A unit of ENII, which binds
HNF4 (48, 49), while the other is located in the B3 region, which was
recently found to bind HNF4, RXR, PPAR, COUPTF1, or ARP1 (49, 54).
Unlike the binding site of hB1F, both of these two sites are homologous
to the nuclear receptor DR-1 element containing direct repeat of the
hexad half-site (RGG/TTCA) (49), and the nuclear receptors interact
with the target sites as homo- (i.e. HNF4) or hetero-dimers
(i.e. RXR/PPAR). As shown in DNase I footprinting assay
(Fig. 4B), hB1F does not bind to those DR1 elements in A and
B3 regions of ENII with high affinity. However, considering its binding
sequence covers a half-site of the DR element, it is not surprising
that the HNF4 binding consensus oligonucleotide in an excess amount was
able to compete with the binding of hB1F in EMSA (Fig. 2).
Since a number of trans-acting factors (Fig. 1) have been identified to
be required for the optimized function of ENII, an important question
arises as to how these factors modulate the activity of ENII. First,
the optimum activity of ENII probably requires cooperative interactions
among these transcription factors. In the preliminary study, we have
observed a synergism between hB1F and HNF1 in activating ENII (data not
shown). Potential interaction of these two factors and with other
factors is now under investigation. Second, it is conceivable that
these factors may respond to different microenvironmental cues either
within the cell or from extracellular stimuli, or may function at
different stages of HBV transcription. Obviously, a clearer picture of
the complex network of interaction and cross-talk of these factors will
be needed to understand better the mechanism of transcriptional
regulation of HBV ENII.
Based on the sequence similarity, hB1F is categorized as a novel human
homolog of FTZ-F1. Another human FTZ-F1 homolog, hSF-1, has been
identified recently (43). Although they share close sequence homology,
hB1F and hSF-1 represent two subtypes of human FTZ-F1, which might
evolve from one ancestor FTZ-F1 (55). They are encoded by two distinct
genes, show different tissue distributions, and serve different roles.
hSF-1, encoded by a gene at human chromosome 9 q33 (56), is expressed
in all primary steroidogenic tissues and acts as a crucial
transcription factor of enzyme involved in steroid production. It has
been also delineated to be essential in the regulation of
hypothalamic-pituitary-steroidogenic organ axis (reviewed in Parker and
Schimmer (45)). In contrast, the gene of hB1F is located at human
chromosome 1 q31-32.1. It is expressed specifically in pancreas and
liver that do not express hSF-1. The function analysis has suggested
hB1F is an important trans-activator accounting for liver-specific
activity of enhancer II of HBV. As a hepatocyte transcription factor,
hB1F is likely to be involved in regulating other liver-specific genes.
This is supported by the report that the rat FTZ-F1 homolog rFTF,
belonging to the same subgroup with hB1F, was able to activate the
We identified two isoforms, hB1F and hB1F-2, from HepG2 cells. They
differ only in the A/B region. The A/B region of hB1F-2 is 46 amino
acids longer than that of hB1F. These two isoforms might be derived
from differential splicing, a common feature found in the nuclear
receptor superfamily. It has been well known that an activation domain,
activation function-1 is located in the highly varied A/B region of
nuclear receptor, which usually synergizes with activation function-2
domain of nuclear receptors, and functions in a cell type- and
promoter-specific manner. Isoforms of many nuclear receptors differing
in their A/B region have different function properties (59), suggesting
a similar possibility in the case of hB1F and hB1F-2. In our RT-PCR
experiments, both isoforms seem to be present in adult liver, fetal
liver, and HepG2 cells. But whether they are expressed at different
levels or they have different functions needs to be further
investigated.
Finally, it is noteworthy that two different sizes of hB1F transcripts
were revealed by Northern blot in HepG2 hepatoma cells (Fig.
8B). The transcript of 5.2 kb is consistent with that
detected in human adult normal liver and pancreas, whereas the other
short transcript of 3.8 kb is specific for HepG2 cells. Compared with normal liver, there might be a different regulation of hB1F expression in HepG2 cells at the transcription level or post-transcription process, such as differential splicing of mRNA, and/or the usage of
different promoters and/or different poly(A)+ sites. At the
present time, the difference of these two hB1F transcripts in HepG2
cells and their respective function and biological significant are
still unknown. However, the detection of an hB1F 3.8-kb transcript in
HepG2 cells may allow us to study the variation of hB1F expression in
light of liver carcinogenesis.
We thank S. Z. Ao and Y. Luo for
technical advice on yeast one-hybrid screening and Y. D. Zhang for
critical reading of the manuscript. We also thank E. Lai for
providing plasmid pCMV-poly.
*
This work was supported by the Chinese Academy of Sciences
and the National Natural Science Foundation of China.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) HSU80251.
§
Present address: Molecular and Cellular Biology Dept., Roswell Park
Cancer Institute, Elm and Calton Sts., Buffalo, NY 14263.
¶
Present address: Division of Oncology M228, Dept. of Medicine,
Stanford University, School of Medicine, Stanford, CA 74305.
**
To whom correspondence should be addressed: Shanghai Institute of
Biochemistry, Chinese Academy of Sciences, 320 Yue Yang Rd., Shanghai
200031, Peoples Republic of China. Tel.: 0086-21-64374430; Fax:
0086-21-64338357; E-mail: wangyuan{at}server.shcnc.ac.cn.
The abbreviations used are:
HBV, hepatitis B
virus; ENII, enhancer II; ENI, enhancer I; Cp, core promoter; hB1F, human B1-binding factor; ORF, open reading frame; GAL4AD, GAL4
activation domain; EMSA, electrophoresis mobility shift assay; CAT, chloramphenicol acetyltransferase; FISH, fluorescent in situ
hybridization; FTZ-F1, fushi tarazu factor 1; mSF-1, murine
steroidogenic factor 1; mELP, murine embryonal long terminal repeat
binding protein; bAD4BP, bovine adrenal AD4-binding protein; hSF-1, human steroidogenic factor 1; zFF1A/zFF1B, zebrafish FTZ-F1 A and B; xFF1rA/xFF1rB, Xenopus FTZ-F1-related A and B; mLRH-1, murine liver receptor homolog 1; rFTF, rat fetoprotein transcription
factor; DmFTZ-F1, D. melanogaster FTZ-F1; B1m, mutant B1; AD, activation domain; CMV, cytomegalovirus; PCR, polymerase chain
reaction; RT, reverse transcriptase; DBD, DNA-binding domain; nt, nucleotide; bp, base pair(s); kb, kilobase pair(s); SEAP, secreted
placental alkaline phosphatase.
3
M. Li and Y. Wang, unpublished data.
2
Y. Xie, M. Li, Y. Wang, P. H. Hofschneider, and
L. Weiss, submitted for publication.
Cloning and Characterization of a Novel Human Hepatocyte
Transcription Factor, hB1F, Which Binds and Activates Enhancer II
of Hepatitis B Virus*
,§,,¶,
, and**
Shanghai Institute of Biochemistry, Chinese
Academy of Sciences, Shanghai 200031, Peoples Republic of China and the
Molecular Biology Division, Clontech Laboratories, Inc.,
Palo Alto, California 94303
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (27K):
[in a new window]
Fig. 1.
Nucleotide sequence of the ENII/Cp region of
HBV (subtype adr1) and the identified trans-acting factors binding
sites. ENII (nt 1627-1774) can be divided into two parts, A and
B, and part B could be further divided into B1 (nt 1687-1704), B2 (nt
1705-1735), and B3 (nt 1736-1774) according to functional analysis
(7, 10). The underlined sequences represent the Sp1
recognition sequences (60), the C/EBP recognition sequences (46), the
HNF4 recognition sequences (48, 49), the HNF3 recognition sequences
(24, 47), the HNF1 recognition sequences (15), and the recognition
sequences of members of nuclear receptors (49). The double
underlined sequence represents the specific binding site of a
novel hepatocyte nuclear factor (B1-binding factor).
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
) plates
containing 3-aminotriazole (45 mM was shown optimal for suppressing HIS3 background growth). Large colonies
(His+) were transferred onto Hybond N filters (Amersham
Pharmacia Biotech) and further screened for 
-galactosidase activity
(20). After being placed in liquid nitrogen for 30 s, the filters
were incubated in a buffer containing 0.8 mM
6-bromo-4-chloro-3-indolyl--D-galactosidase at 30 °C.
The positive interaction was determined by the appearance of blue
colonies. The LacZ+ colonies were selected and plasmids
were recovered (21). The candidate plasmids isolated from the positive
clones were transformed into YMB1-HB to retest for His+
phenotype and -galactosidase activity. Those that could reproduce the positive phenotypes were called true positive clones and their cDNA inserts were sequenced and further characterized.
70 °C.
-32P]ATP-end-labeled 5'-primer (nt
1634-CCAGGTCTAGACCAAGGTC-nt 1651) and unlabeled 3'-primer (nt
1816-GGTGCTGGTTAACAGACCA-nt 1798), then used as a target DNA. The HBV
sequence was derived from plasmid pADR-1, which contains an entire copy
of the HBV genome of the subtype adr1 (25). For DNase I footprinting
analysis, 2.5-7.5 µg of purified glutathione
S-transferase fusion protein was incubated with 5 µg of
poly(dI-dC) and the probe in a 50-µl reaction mixture containing 10 mM Tris-HCl (pH 7.5), 90 mM NaCl, 0.15 mM MgCl2, 0.2 mM EDTA, 0.1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 5% glycerol for 25 min at room temperature. The DNase I
digestion was then carried out as described in SureTrack footprinting
kit (Amersham Pharmacia Biotech).
reverse transcriptase (Life Technologies, Inc.), using
2 pmol of primer I. The first round PCR was performed with primers I and IV, and the second round with primers II and V. To increase the
specificity of the final PCR product, a third round PCR was performed
with primers III and V. The resulting fragment was subcloned into
pcDNA3 (Invitrogen) and subsequently sequenced.
-actin cDNA control probe.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (61K):
[in a new window]
Fig. 2.
EMSA of B1 probe with HepG2 and HeLa nuclear
extracts. Lanes 1-5, labeled B1 probe with 6 µg of
HepG2 nuclear extract. Band s indicates the B1-specific
binding band. Lane 1, no competitor was added. Lane
2, unlabeled B1 was used as competitor (40 molar excess).
Lanes 3 and 4, unlabeled HNF4 DNA binding
consensus oligonucleotide (61) was used as competitor (lane
3, 100 molar excess; lane 4, 250 molar excess).
Lane 5, unlabeled nonspecific competitor (mixture of
HaeIII-digested pBS fragments, 250 molar excess). Lane
6, the labeled B1m probe with 6 µg of HepG2 nuclear extract.
Lane 7, the B1 probe with 6 µg of HeLa nuclear
extract.
-galactosidase activity
(LacZ+). The plasmid of this dual positive colony was
recovered from the yeast, and named pGAD-16. When retransformed into
the reporter yeast strain, pGAD-16 restored His and LacZ activity,
indicating it was a true positive clone.
View larger version (57K):
[in a new window]
Fig. 3.
Sequence analysis of hB1F cDNA.
A, diagramatic map of the cDNA insert of pGAD-16,
showing the ORF of hB1F cDNA is in reverse orientation to GAL4AD.
PADH1, the promoter of ADH1;
TADH1, the terminator of ADH1. B,
nucleotide sequence of hB1F cDNA and deduced amino acid sequence of
hB1F protein. In-frame stop codon TAA in the 5'-untranslated region is
underlined. Stretches of white amino acids on
black background are the conserved C region (DBD);
region II, region III and AF-2
(activation function-2) within the E/F region (ligand-binding domain).
P-box and D-box of DBD are underlined. A 30-amino acid
FTZ-F1-box which is located at the carboxyl-terminal of DBD is
double-underlined. Nucleotide and amino acid
positions are shown on the right. C, schematic
subdomain structure of hB1F, showing that it shares a common modular
structure of nuclear receptor superfamily, which is composed of five to
six subdomains (from A to E or
F).
View larger version (29K):
[in a new window]
Fig. 4.
In vitro DNA protein-binding assay to
verify the specific binding of hB1F to the B1 target site.
A, EMSA of B1 probe with in vitro translated
hB1F. Lane 1, labeled B1 probe alone. Lane 2, B1
probe with 2 µl of in vitro translation product of
negative control. Lanes 3-11, B1 probe with 2 µl of
in vitro translated hB1F. Lane 3, no competitor
was added. Lanes 4-6, unlabeled B1 was used as competitor
(lane 4, 10 molar excess; lane 5, 40 molar
excess; lane 6, 100 molar excess). Lanes 7-9,
unlabeled B1m was used as competitor (lane 7, 10 molar
excess; lane 8, 40 molar excess; lane 9, 100 molar excess). Lanes 10 and 11, unlabeled
nonspecific competitor (mixture of HaeIII-digested pBS
fragments. Lane 10, 40 molar excess; lane 11, 100 molar excess). Band s indicates the specific binding band.
B, DNase I footprinting analysis of the ENII/Cp region of
HBV by expressed glutathione S-transferase-hB1F(DBD). The
labeled fragment (nt 1634-1816) containing HBV ENII and Cp region was
used as target DNA. Lanes 1 and 2, G reaction.
Lanes 3 and 4, G + A reaction. Lane 5,
no protein was added. Lanes 6-8, different amounts of
purified glutathione S-transferase-hB1F(DBD) was added
(lane 6, 2.5 µg; lane 7, 5 µg; lane
8, 7.5 µg). The nucleotide sequence of the protected region is
shown on the right.
View larger version (24K):
[in a new window]
Fig. 5.
hB1F trans-activates the ENII of HBV in HeLa
cells. A, the eukaryotic expression plasmid pCMV-hB1F
was co-transfected with reporter plasmid pENII(B)/CpCAT, in which
ENII(B)/Cp fragment (containing the basic functional unit B of ENII and
core promoter) was placed upstream of CAT gene. B, pCMV-hB1F
was co-transfected with reporter plasmid pENII(B1 m)/CpCAT which
contains mutant B1. For both figures, in lane 1 with no
pCMV-hB1F expression plasmid. In lanes 2-5, increasing
amount of pCMV-hB1F expression plasmids were added. 1 µg of
pENII(B)/CpCAT or pENII(B1 m)/CpCAT, and different amount of pCMV-hB1F
were used. 1 µg of pCMV/SEAP (28) was included as internal control
for transfection efficiency. The means of the CAT activity of three
independent experiments are shown on the top of the figure.
View larger version (36K):
[in a new window]
Fig. 6.
Comparison of the functional domains of the
hB1F with selected vertebrate FTZ-F1 homologs and D. melanogaster
FTZ-F1. The percentage of amino acid identity in conserved
regions (including DBD, FTZ-F1-box, region II, region III, and
activation function-2) relative to hB1F is indicated. The numbers above
each bar represent the first and last amino acids of functional
domains. Data sources were: mSF-1 (39); mELP (40); bAD4BP (42); hSF-1
(43); zFF1A/zFF1B (36); xFF1rA/xFF1rB (37); mLRH-1 (38); rFTF (41);
DmFTZ-F1 (19).
View larger version (31K):
[in a new window]
Fig. 7.
hB1F-2 is an isoform of hB1F.
A, comparison of the nucleotide sequence and amino acid
sequence within the A/B region of hB1F and hB1F-2, showing the extra
138-bp nucleotides in hB1F-2 cDNA and the extra 46 amino acids in
hB1F-2 protein. B, RT-PCR results showing that two isoforms
with different lengths of the A/B region are both present in HepG2
cells, adult liver tissue, and fetal liver tissue. Lane 1,
1-kb DNA ladder; lane 2, fetal liver; lane 3,
adult liver; lane 4, HepG2 cells; lane 5, HeLa
cells; lane 6, plasmid pCMV-B1F was amplified by PCR as a
control, showing the 410-bp fragment without the extra 138-bp
nucleotides in the A/B region; lane 7, plasmid
pcDNA3-hB1F-2 (containing hB1F-2 complete coding region in
pcDNA3 vector) was amplified by PCR as a control, showing the
550-bp fragment with the extra 138-bp nucleotides in the A/B
region.
View larger version (38K):
[in a new window]
Fig. 8.
Northern blot analyses of (A)
human tissue panel and (B) HepG2 and HeLa cells.
Radiolabeled hB1F cDNA (2.5 kb) was used as a probe. A -actin
cDNA probe was hybridized to the same filters as a control
(bottom). A, poly(A)+ RNA blot
contains 2 µg of poly(A)+ RNA per lane from multiple
human adult tissues (human multiple tissue Northern blot I,
CLONTECH). The arrowhead indicates a
5.2-kb hB1F transcript band. B, 20 µg of total RNA
isolated from HepG2 and HeLa cells was separated in 1.0% agarose
formaldehyde gels and blotted onto nylon membrane. The
arrowheads indicate 5.2- and 3.8-kb hB1F transcripts.
View larger version (32K):
[in a new window]
Fig. 9.
Mapping hB1F gene to human chromosome 1 q31-32.1 by FISH. The 2.5-kb hB1F cDNA was biotinylated and
used as a probe. A, the FISH signals on the spread mitotic
chromosome. The site of hybridization is indicated by the
arrow. B, the same mitotic figure stained with
the chromatin-binding fluorescent dye 4,6-diamidino-2-phenylindole to
identify chromosome 1. C, diagram of FISH mapping results
for the hB1F probe on human chromosome 1. Each dot
represents the double FISH signals counted.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-fetoprotein gene (41). On the other hand, since hB1F is most
abundantly expressed in the pancreas, it might also contribute to the
regulation of pancreas-specific genes. In addition, with respect to
members of FTZ-F1 subfamily are highly conserved and some of them have been revealed to be crucial in embryonic development (57), it could be
predicted that hB1F plays important roles in human embryonic development. As hB1F is expressed in liver and pancreas, two
coderivatives of the gut endoderm (58), it is reasonable to propose
this factor be involved in the early development of embryonic gut
endoderm and tissue differentiation, which needs to be addressed by
in vivo functional analyses.
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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H. Ishida, K. Ueda, K. Ohkawa, Y. Kanazawa, A. Hosui, F. Nakanishi, E. Mita, A. Kasahara, Y. Sasaki, M. Hori, et al. Identification of Multiple Transcription Factors, HLF, FTF, and E4BP4, Controlling Hepatitis B Virus Enhancer II J. Virol., February 1, 2000; 74(3): 1241 - 1251. [Abstract] [Full Text]
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V. J. Lannoy, A. Rodolosse, C. E. Pierreux, G. G. Rousseau, and F. P. Lemaigre Transcriptional Stimulation by Hepatocyte Nuclear Factor-6. TARGET-SPECIFIC RECRUITMENT OF EITHER CREB-BINDING PROTEIN (CBP) or p300/CBP-ASSOCIATED FACTOR (p/CAF) J. Biol. Chem., July 14, 2000; 275(29): 22098 - 22103. [Abstract] [Full Text] [PDF]
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J.-F. Pare, S. Roy, L. Galarneau, and L. Belanger The Mouse Fetoprotein Transcription Factor (FTF) Gene Promoter Is Regulated by Three GATA Elements with Tandem E Box and Nkx Motifs, and FTF in Turn Activates the Hnf3beta , Hnf4alpha , and Hnf1alpha Gene Promoters J. Biol. Chem., April 13, 2001; 276(16): 13136 - 13144. [Abstract] [Full Text] [PDF]
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C.-T. Sun, W.-Y. Lo, I.-H. Wang, Y.-H. Lo, S.-R. Shiou, C.-K. Lai, and L.-P. Ting Transcription Repression of Human Hepatitis B Virus Genes by Negative Regulatory Element-binding Protein/SON J. Biol. Chem., June 22, 2001; 276(26): 24059 - 24067. [Abstract] [Full Text] [PDF]
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Y.-K. Lee and D. D. Moore Dual Mechanisms for Repression of the Monomeric Orphan Receptor Liver Receptor Homologous Protein-1 by the Orphan Small Heterodimer Partner J. Biol. Chem., January 18, 2002; 277(4): 2463 - 2467. [Abstract] [Full Text] [PDF]
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