From the Immunopathology Section, Laboratory of
Immunobiology, NCI-Frederick Cancer Research and Development Center,
Frederick, Maryland 21702 and § The First Department of
Internal Medicine, Yokohama City University School of Medicine,
Yokohama 236, Japan
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ABSTRACT |
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We previously reported that the transcription of
the human macrophage stimulating protein (MSP) gene was
positively regulated by the binding of NF-Y to the CAATT sequence in
the promoter region of this gene. Here we confirmed our previous
results and further characterized the MSP promoter.
Luciferase assay with deletion constructs showed the importance of the
region, +32 to +39, for the promoter activity in Hep3B cells. Two
nuclear protein-DNA probe (+15 to +40) complexes, C1 and C2, were
detected by electrophoretic mobility shift assay. C2 was specific to
hepatoma cells and contained hepatocyte nuclear factor-4 (HNF-4). DNase
I footprinting with recombinant HNF-4 located another HNF-4-binding
site in the distal region, 89 to
54. Mutations in the CAATT or the
proximal HNF-4-binding site significantly reduced the promoter activity
in Hep3B cells and HNF-4-transfected HeLa cells, whereas mutations in
the distal HNF-4-binding site had no effect. The close proximity
between the CAATT and the proximal HNF-4-binding site suggested that a direct contact between NF-Y and HNF-4 might be important.
Protein-protein interaction between the A-subunit of NF-Y and HNF-4 was
detected by a yeast two-hybrid system. The binding of in
vitro translated HNF-4 to immobilized NF-YA and in
vitro translated NF-YA to immobilized HNF-4 was also detected.
These results suggest the binding of HNF-4 to the proximal
HNF-4-binding site directs the basal transcription of the
MSP gene, and the maximal promoter activity may depend on
the direct association between HNF-4 and NF-Y.
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INTRODUCTION |
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Macrophage stimulating protein (MSP)1 is a hepatocyte-derived serum protein that has high amino acid sequence similarity to hepatocyte growth factor/scatter factor (1-3). MSP was originally purified as a protein that made mouse resident peritoneal macrophages capable of responding to the chemoattractant C5a (4). However, recent study shows that MSP itself is a chemoattractant for mouse resident macrophages (5). The receptor for MSP is a receptor tyrosine kinase, Ron/Stk (6-10). Identification of the MSP receptor revealed that keratinocytes are another target cell of MSP (10), and MSP appears to be involved in the migration of keratinocytes during the healing process after skin injury. We and others (2, 3) previously reported that MSP mRNA was predominantly and constitutively expressed in the liver, probably in hepatic cells.
The transcriptional mechanisms of genes specifically expressed in
hepatic cells, such as albumin, transthyretin, 1-antitrypsin, or
fibrinogen, have been previously investigated (11). In most of the
cases, transcription of these genes is positively regulated by
liver-specific transcriptional factors such as CCAAT/enhancer-binding protein
(C/EBP
) (12), hepatocyte nuclear factor (HNF)-1 (13), HNF-3 (14), and HNF-4 (15). Recently, we cloned the 5'-flanking region
of the human MSP gene and found that the transcription of
the human MSP gene was regulated by positive and negative
regulatory elements (PRE and NRE) (16). The PRE was located in the
region between 34 and +2 and was essential for the maximal
transcription of this gene. This sequence carried the "CCAAT"
sequence and was the binding site for the ubiquitously expressed
trans-activator, MSP-PRE-binding protein-1, and
MSP-PRE-binding protein-2 (identical to NF-Y/CCAAT-binding factor)
(16). The NRE was located in the region between
141 and
34 and
appeared to be responsible for the tissue-specific expression of this
gene.
Waltz et al. (17) also investigated the mechanisms of the
human MSP gene transcription. There were significant
differences between the data obtained by two groups. Waltz et
al. (17) identified putative transcription initiation sites at
75 and
76 (corresponding to
27 and
28 in out study), located on
the CCAAT sequence, by using RNA from HepG2 cells transfected with the
pL5CAT (
1348/+1) chimeric construct, but they could not obtain primer
extension products from RNA isolated from untransfected HepG2 cells.
Waltz et al. (17) also reported that the binding of HNF-4 to
the region between
135 and
105 (corresponding to
98 and
69 in
our study) activated the transcription of this gene (17). The binding
of HNF-4 to the region was shown by EMSA and supershift.
trans-Activity of HNF-4 was demonstrated with a CAT
construct, pL5CAT5 (
135/
105), constructed by ligating the sequence
between
135 and
105 to the upstream of the herpes simplex virus
thymidine kinase promoter in pBLCAT5. The thymidine kinase promoter
contained TATA box, whereas the wild-type MSP promoter does not contain
TATA box. Thus, the presence of TATA box in pL5CAT 5 (
135/
105)
could influence the outcome of their experiments.
After the publication of our results (16), we further characterized the
PRE and NRE by DNase I protection assay. Two distinct footprints were
detected with Hep3B cell nuclear extracts. One was the region between
33 and
4 that included the CCAAT sequence, and the other was the
region between
80 and
65. In contrast, a broad region between
104
and
4 was protected with HeLa or HOS nuclear extracts. By EMSA and
supershift assay, we confirmed that HNF-4 bound to the region between
80 and
65 as shown by Waltz et al. (17). Co-transfection
of HOS cells with an HNF-4 expression vector and a vector containing 1 kilobase pair of the MSP promoter fused to the
CAT gene dose-dependently increased CAT
activity, suggesting that the binding of HNF-4 to this region was
important for the tissue-specific expression of MSP mRNA. However,
it was surprising that the deletion of the HNF-4 binding sequence from
the pMD1k or mutation on the HNF-4 binding sequence did not affect the
promotor activity of the MSP gene in Hep3B, HepG2, and
HNF-4-transfected HOS cells.2
Thus, although some of our results agreed with that reported by Waltz
et al. (17), we could not explain tissue-specific expression of MSP mRNA simply by the binding of HNF-4 to the previously
reported HNF-4-binding site. There appear to be other mechanisms that
regulate tissue-specific expression of MSP mRNA.
As described above, the 5'-region of the MSP gene does not
contain TATA box. In promoters that do not have TATA box, an initiator (Inr) is functionally analogous to TATA and capable of directing basal
transcription by RNA polymerase II (18, 19). In the promoter region of
the MSP gene, a sequence similar to the weak consensus
sequence for the Inr (3YYCAYYYYY+6) (18) was
found between +23 and +31. In our preliminary study, deletion of the
region between
5 and +49 resulted in a significant decrease (1/20) in
the CAT activity in Hep3B cells. In contrast, deletion of the region
between +37 and +49 had no effect, suggesting that the region between
5 and +36 could be functionally important for the promoter
activity.
In the present study, we first repeated CAT assay to confirm our
previous results that suggested that the transcription of the
MSP gene was regulated by PRE and NRE. After the
confirmation of our previous results, we further analyzed the
MSP promoter located between 5 and +49 and found that the
binding of HNF-4 to the sequence between +32 and +39 was critical for
the basal transcription of this gene, and the maximal promoter activity depended on the cooperation between HNF-4 and NF-Y.
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EXPERIMENTAL PROCEDURES |
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Reagents--
Dulbecco's modified Eagle's medium, minimum
essential medium, RPMI 1640, Dulbecco's phosphate-buffered saline, and
isopropyl -D-thiogalactopyranoside were from Life
Technologies, Inc. Fetal calf serum (FCS) was from HyClone, Logan, UT.
L-[35S]Methionine was from Amersham Pharmacia
Biotech.
-[32P]CTP was from ICN Biomedicals, Inc.,
Costa Mesa, CA. Oligonucleotides were from Operon Technologies, Inc.,
Alameda, CA, and BioServe, Laurel, MD. TnT-coupled rabbit reticulocyte
lysate system and luciferase assay system were from Promega, Madison,
WI. pcDNA3 was from Invitrogen, Carlsbad, CA. pET32a and His·Bind
Resin columns were from Novagen, Madison, WI. Matchmaker Two-Hybrid
System 2 was from CLONTECH, Palo Alto, CA.
Cell Culture and Luciferase Assay--
HeLa, HOS (osteosarcoma),
and A172 (glioblastoma) cells were cultured in Dulbecco's modified
Eagle's medium containing 10% FCS. Hep3B and HepG2 hepatoma cells
were cultured in minimum essential medium containing 10% FCS. Jurkat
cells were cultured in RPMI 1640 containing 10% FCS. All cell lines
were maintained at 37 °C in 5% CO2. Cells were
transiently transfected with 15 µg of each luciferase construct and 5 µg of pSV- galactosidase plasmid DNA per 100-mm tissue culture
plate by a calcium phosphate precipitation technique (20). After
24 h incubation with each DNA-calcium phosphate precipitate, the
cells were rinsed with phosphate-buffered saline and cultured in fresh
medium for another 48 h. The cells were harvested, and luciferase
activity was measured by the luciferase assay system. The resulting
luciferase activity was corrected based on the
-galactosidase
activity in the same cell extract.
Plasmid Construction--
CAT constructs, pMD5.3k, pMD1.5k,
pMD1k, pMD223, pMD141, pMD34, and pM2, were described previously (16).
To obtain additional 5'-deletion CAT constructs, pMD121, pMD101 and
pMD81, DNA fragments covering from 121 to +49,
101 to +49, or
81
to +49 were amplified by PCR with sense oligonucleotide primers that
corresponded to the region from
121 to
102 (primer
121,
5'-GGCTGCAGTGGGACCTGAGGCCTGGCCC-3'), from
101 to
82 (primer
101,
5'-GGCTGCAGCTCATGGCTCCTGTCACCAG-3'), and from
81 to
62 (primer
81, 5'-GGCTGCAGTCTCAGGTCAGGGTCCAGC-3') with an antisense probe from
+30 to +49 (5'-GGTCTAGACCTTCTGGCTGGAGGCTGCA-3'). PstI or
XbaI linkers were incorporated in the 5'-ends of these primers. The pMD1k was used as the PCR template. The PCR products were
subcloned into the PstI-XbaI site of the pCAT
basic vector after digestion with PstI and
XbaI.
In Vitro Protein-Protein Binding Assay-- Rat NF-YA (22), NF-YB (23), and human TFIIB (24) cDNAs were cloned by reverse transcriptase-PCR, and subcloned into the EcoRI site of the pcDNA3 and pET32a. The coding region of the human HNF-4 cDNA was amplified by PCR from the pMT7HNF-4 (generous gift from Dr. F. M. Sladek, University of California, Riverside, CA) and subcloned into the pcDNA3 (pcHNF4) and pET32a. The murine p65 expression vector CMV-LDp65 and human p50 expression vector CMV-399 (this vector coded amino acids 1 to 399 of p50) were provided by Dr. N. Rice (NCI-FCRDC, Frederick, MD). In vitro coupled transcription and translation were performed with the TnT-coupled rabbit reticulocyte lysate system according to a protocol provided by the supplier. Briefly, 1 µg of each pcDNA3 vector containing a different cDNA and T7 RNA polymerase were added to a 50-µl reaction mix and then the mix was incubated at 30 °C for 2 h. For radiolabeling, L-[35S]methionine was used at final activity of 0.8 µCi/µl.
Recombinant proteins were induced in the BL21(DE3) Escherichia coli strain by addition of 0.3 mM isopropylDNase I Footprinting--
To prepare the coding strand DNA
carrying the MSP promoter region (141 to +49), the pMD141
plasmid (16) was digested with HindIII, and the overhangs
were filled in with [32P]dCTP, followed by a digestion
with EcoRI. The resulting 350-base pair fragment was
purified after gel electrophoresis. To prepare the noncoding strand
DNA, the pGLMD141 was digested with NcoI, and the overhangs
were filled in with [32P]dCTP, followed by a digestion
with PstI. The resulting 243-base pair fragment was purified
after gel electrophoresis. DNase I digestion was performed with 4 ng of
the coding or noncoding strand DNA and bacterially expressed
recombinant HNF-4 protein by the method described by Lefevre et
al. (25), and the digested samples were analyzed on 8%
polyacrylamide gels.
Electromobility Shift Assay (EMSA) and Supershift
Assay--
EMSA was carried out as previously reported (16, 20).
Briefly, the probes were prepared by annealing each sense
oligonucleotide with antisense oligonucleotide shown in Table I. The
binding reaction was performed at 20 °C for 30 min in 10 ml of 1×
binding buffer (12 mM HEPES, pH 8.0, 60 mM KCl,
2.5 mM EDTA, 14% glycerol, 2 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride)
containing 10 fmol of each probe end-labeled with
[-32P]ATP, 1 µg of dI-dC, and 3 µg of each nuclear
extract. For the competition assay, 10-, 20-, or 50-fold excess amounts
of appropriate unlabeled probes were added to the binding reaction
mixtures. For supershift assay, nuclear extract was preincubated with 1 µl of normal rabbit serum or antiserum for 20 min before mixing with
the probe. Gel electrophoresis was carried out at 200 V for 2 h
after a prerun at 200 V for 30 min.
Yeast Two-hybrid Assay--
Yeast two-hybrid assay originally
developed by Fields and Song (26) was performed with the Matchmaker
Two-Hybrid System 2 according to the direction supplied from the
manufacturer. Briefly, the full-length cDNAs for NF-YA, NF-YB,
NF-YC, and TFIIB were obtained by reverse transcriptase-PCR. The
cDNAs for HNF-4, NF-YA, NF-YB, and NF-YC were ligated into the
NcoI-EcoRI site, and the TFIIB cDNA was
ligated into the BamHI-EcoRI site of the pAS2-1 vector carrying the GAL4 DNA-binding domain (DNA-BD) (27) or the pACT2
carrying the GAL4 transcriptional activation domain (AD) (28). To
investigate interactions between two transcription factors, a yeast
strain, Y187 (MATa, trp 1, leu 2, URA3::GAL1USA-GAL1TATA-LACZ), was
transformed with various combinations of the pAS2-1 vector coding
different GAL4 DNA-BD fusion proteins and the pACT2 coding different
GAL4 transcriptional AD fusion proteins. -Galactosidase activity in
the yeast was assayed by colony-lift filter assay as qualitative assay
and by liquid culture assay as quantitative assay (29, 30). For
colony-lift assay, yeast colonies were streaked out onto synthetic
dropout selection agar plates without leucine or tryptophan, and the
plates were incubated for 3 days at 30 °C. Sterile VWR filters
presoaked in Z buffer/5-bromo-4-chloro-3-indolyl
-D-galactosidase were placed on the plates. The filters
were lifted carefully and submerged in liquid nitrogen for 10 s.
The filters were thawed at room temperature and placed on presoaked filters and then incubated at 30 °C until blue color appeared. For
liquid culture assay, yeast colonies were inoculated into 5 ml of
synthetic dropout without leucine or tryptophan and cultured overnight
at 30 °C with shaking. Two ml of the overnight cultures were
inoculated into 8 ml of YPD liquid, and the cells were grown at
30 °C for 3-5 h with shaking until the A600
reached 0.5-0.8. One and a half ml of the cultures were transferred
into 1.5-ml microcentrifuge tubes, and the tubes were centrifuged at
14,000 rpm for 30 s. After removal of the supernatants, cells were
washed and finally resuspended in 0.3 ml of Z buffer. One hundred µl of the cell suspensions were transferred to fresh microcentrifuge tubes
and placed in liquid nitrogen until the cells were frozen. The cells
were thawed at 37 °C, and 700 µl of Z buffer containing
-mercaptoethanol were added to the tubes. One hundred sixty µl of
o-nitrophenyl
-D-galactopyranoside (4 mg/ml
in Z buffer) were added to each tube, and the tubes were incubated at
30 °C. When the yellow color developed, the reaction was stopped by
addition of 0.4 ml of 1 M Na2CO3.
The tubes were centrifuged, and the supernatants were subjected to
spectrophotometry at A420.
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RESULTS |
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Tissue-specific Promoter Activity on the PRE of the MSP
Gene--
As described in the Introduction, our previous results on
the cis-element responsible for the positive regulation of
the MSP gene transcription were significantly different from
that by Waltz et al. (17). Therefore, we first repeated CAT
assay with previously constructed CAT constructs containing different
lengths of the 5'-flanking region of the MSP gene obtained
by progressive deletion of the 5'-end (16) and also with newly created
CAT constructs (Fig. 1B). A
significant CAT activity was detected in the extracts of Hep3B cells
transfected with pMD5.3k. Progressive deletion of the 5'-flanking
region from 5.3k (pMD5.3k) to
34 (pMD34) resulted in a gradual
increase in CAT activity. In contrast, no significant CAT activity was
found in HeLa cells transfected with either pMD5.3k or other constructs
containing the 5'-flanking regions progressively deleted up to
141.
However, when the 5'-flanking region was deleted to
121,
101, or
81, CAT activity was increased 2.5-fold. Further deletion from
81
to
34 resulted in another 2-fold increase in CAT activity. These
results suggested that the region between
141 and
34 might
negatively regulate the transcription of the MSP gene. In
both cells, deletion to +2 (pM2) resulted in a significant decrease in
CAT activity. The region between
34 and +2 carries the CAATT sequence
we previously identified as an NF-Y-binding site. Removal of the
remaining proximal MSP gene sequence (pCAT-basic) resulted
in a total loss of CAT activity, suggesting that the proximal
5'-flanking region between +2 and +49 may also be responsible for the
positive regulation of the MSP gene transcription. Thus,
these results further support our hypothesis that the transcription of
the MSP gene is regulated by PRE and NRE.
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Characterization of Binding Protein to the Proximal PRE-- To characterize nuclear proteins that could bind to the region between +31 and +38, EMSA was performed with a nucleotide probe covering the sequence from +16 to +45. Two specific nuclear protein-DNA complexes, C1 and C2 (Fig. 2A, lane 1), that were competed by addition of excess amounts of unlabeled probe (lanes 2-4), were detected with the nuclear extract from Hep3B cells. Shifted bands that migrated to the region of the C1 were detected with the nuclear extracts from HepG2, HeLa, HOS, A172, and Jurkat cells (Fig. 2B, lanes 2-6), but the C2 was formed only with the nuclear extracts from Hep3B and HepG2 cells.
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Identification of Proximal and Distal HNF-4-binding Sites in the
MSP Promoter Region--
The binding of HNF-4 to the promoter region
of the MSP gene was further investigated by DNase I
protection assay. The experiments were performed with a recombinant
HNF-4 protein expressed in E. coli. As shown in Fig.
3, two regions were protected from DNase I digestion. One was in the proximal region, between +20 and +41 on the
noncoding strand (antisense) and between +14 and +38 on the coding
strand (sense), corresponding to the HNF-4-binding site described
above. The protection of this region was not previously clear with
Hep3B cell nuclear extracts (data not shown). Another region in the
distal region, between 89 and
69 on the coding strand (sense) and
between
54 and
81 on the non-coding strand (antisense), was also
protected from DNase I digestion. This distal HNF-4-binding site is
identical to the one previously reported by Waltz et al.
(17) as an element responsible for the liver-specific expression of
MSP/HGFL. The binding of HNF-4 to the distal HNF-4-binding site was
confirmed by EMSA with Hep3B nuclear extract and also by supershift
assay with an antiserum against HNF4 (data not shown).
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Effects of Mutations in the Proximal HNF-4-binding Site on the Formation of C2 and HNF-4-induced Transcriptional Activity-- To investigate the DNA sequence required for the protein binding to the proximal HNF-4-binding site, mutated oligonucleotide probes were synthesized and tested in EMSA (Fig. 4A). The mutations from "+24TCA+26" to "+24ATC+26" and from "+35CC+36" to "+35GA+36" resulted in significant decreases in the competitive activity (Fig. 4A, M2 and M4). In contrast, the other mutations, M1 (+16TGG+18 to +16CCC+18), M3 (+30TGC+32 to +30CTG+32), and M5 (+40CAG+42 to +40CTG+42), did not affect the HNF-4 binding. These results suggested that the base pairs, +24 to +26 and +35 and +36, were critical for HNF-4 binding.
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Synergistic trans-Activity between NF-Y and HNF-4-- Our previous study (16) and the present study identified three cis-elements in the promoter region of the MSP gene, two HNF-4-binding sites and one CCAAT box (binding site for NF-Y). To investigate the roles of these three cis-elements and two trans-activators in the transcription of the MSP gene, we performed luciferase assays by transfecting HeLa cells with HNF-4 expression vector, pMT7HNF-4, and various luciferase constructs shown in Fig. 5. When HeLa cells were co-transfected with pMT7HNF-4 and pGLMD1K containing the wild-type MSP promoter, the luciferase activity was 15-fold increased. Mutation in the proximal HNF-4 site (pmPH) resulted in 75% loss of the luciferase activity in HNF-4-transfected HeLa cells and 71% loss in Hep3B cells. However, mutation in the distal HNF-4-binding site (pmDH) did not affect the activity. Mutation in the CCAAT sequence (pmY) resulted in 60% loss of the activity in HNF-4-transfected HeLa cells and 75% loss in Hep3B cells. When both the CCAAT sequence and the proximal HNF-4 site were mutated (pmYmPH), the luciferase activity was decreased to the basal level (pmDHmYmPH). The luciferase activities obtained with either pmDHmY or pmDHmYmPH were not significantly different from that obtained with pmY or pmYmPH, respectively, in both HNF-4-transfected HeLa cells and Hep3B cells. There was no significant luciferase activity in HeLa cells that was not transfected with the pMT7HNF-4. These results strongly suggest that the binding of NF-Y to the CCAAT sequence and the binding of HNF-4 to the proximal HNF-4-binding site, not to the distal HNF-4-binding site, synergistically activate the transcription of the MSP gene in hepatocytes.
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HNF-4 Contacts with NF-YA and TFIIB in a Y187 Yeast Strain-- Cooperative activity between the CCAAT sequence and the proximal HNF-4-binding site, not the distal HNF-4-binding site, leads to an hypothesis that a direct protein-protein interaction between NF-Y and HNF-4 might be required for the maximal promoter activity of the MSP gene. To test whether HNF-4 interacts with NF-Y or the basal transcriptional complexes, we used a yeast two-hybrid system. HNF-4 was expressed as a fusion protein with GAL4 activating domain (AD) by cloning HNF-4 cDNA into a yeast shuttle vector, pACT2. NF-YA, NF-YB, NF-YC, TFIIB, and TFIID (TATA-binding protein) were expressed as fusion proteins with GAL4-binding domain (BD) by cloning the cDNAs into an yeast shuttle vector, pAS2-1. Interaction between HNF-4 fused to AD and a protein fused to BD reconstitutes GAL4 function in vivo and results in the expression of lacZ gene in Y187 yeast strain. SV40 large T antigen-AD and murine p53-BD were used as positive control (31). The protein-protein interaction between p53 and T antigen was detected as shown in Fig. 6A. When the HNF-4-AD expression vector was co-transfected with pAS2-1 BD expression vector, the HNF-4-AD possessed no intrinsic activation activity (almost same to that of the pAS2-1 vector alone). However, when HNF-4-AD expression vector was co-transfected with NF-YA-BD or TFIIB-BD expression vector, significant levels of galactosidase activities were detected (Fig. 6A). NF-YB-BD, NF-YC-BD, and p53-BD failed to interact with HNF-4-AD. Expression of T antigen-AD, HNF-4-AD, p53-BD, NF-YA-BD, NF-YB-BD, NF-YC-BD, TFIIB-BD, and TFIID-BD in the yeast was confirmed by Western blot analysis (data not shown). The galactosidase activities were also quantified by liquid cultures of the yeast colonies (Fig. 6B). The interaction between HNF-4 and NF-YA was lower than that between p53 and large T antigen but appeared to be stronger than that between HNF-4 and TFIIB.
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Protein-Protein Interaction between HNF-4 and NF-YA in
Vitro--
We also characterized the binding between NF-YA and HNF-4
by using recombinant proteins in vitro. As shown in Fig.
7A, lane 1, in vitro
translated 35S-labeled NF-YA (upper panel) bound
to the column to which bacterially expressed HNF-4 was immobilized and
was eluted from the column (lower panel).
35S-Labeled HNF-4 also bound to the column (lane
2). Homodimerization of HNF-4 was previously characterized by
others (11). 35S-Labeled NF-B p50 and
35S-labeled RelA failed to bind to the column (lanes
3 and 4). Conversely, 35S-labeled HNF-4
bound to the columns to which bacterially expressed NF-YA or HNF-4 was
immobilized (Fig. 7B, lanes 2 and 4)
but not to the column to which bacterially expressed NF-YB was
immobilized (Fig. 7B, lane 3).
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DISCUSSION |
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In the present study, we identified two HNF-4 binding elements in
the 5'-flanking region of the human MSP gene. The direct repeat 1 (DR1) "(AGGTCA)N(AGGTCA)" is the consensus DNA sequence for HNF-4 binding (32). This sequence is highly conserved in the
sequence of the distal HNF-4-binding site
(83AGGTCT)C(AGGTCA
71) and weakly conserved
in the sequence of the proximal HNF-4 binding site
(+21GGGTCA)C(AGTGCA+33). The mutations from
+24TCA+26 to +24ATC+26
(M2) and +34GCC+36 to
+34GGA+36 (M4) resulted in a loss of HNF-4
binding activity and a decrease in the HNF-4-induced enhancer activity.
On the other hand, the mutations from
"+19TGG+21" to
"+19CCC+21" (M1) and
"+30TGC+32" to
"+30CTG+32" (M3) did not affect either
HNF-4 binding or the enhancer activity. Waltz et al. (17)
investigated the effects of mutated bases on the binding activity of
the distal HNF-4-binding site. Oligoprobes containing mutations between
78 and
70 were not able to compete with the wild-type oligoprobe
for HNF-4 binding. Thus, the sequences responsible for HNF-4 binding
appear to be different between the distal and proximal HNF-4-binding
sites.
We previously detected four different transcription initiation sites, T (+1), G (+7), G (+20), and A (+26), by primer extension assay (16). One of the transcription initiation sites, A (+26), is located in the proximal HNF-4-binding site. As we previously reported, the promoter region of the MSP gene does not contain TATA box. In the genes that lack TATA box, an element called an initiator (Inr) is functionally analogous to TATA (18, 19). The Inr overlaps the transcription initiation site of numerous mammalian protein-coding genes. The Inr has a weak consensus sequence (5'-PyPyCAPyPyPyPyPy-3' or 5'-PyPyAN(T/A)PyPy-3', where Py is pyrimidine) and the functional Inr-binding protein would interact with a relatively low affinity (19). The binding affinity of HNF4 to the proximal HNF-4-binding site was also low compared with that between HNF-4 and the distal HNF-4-binding site (data not shown). Thus, the characteristics of the proximal HNF-4-binding site are similar to that of the Inr.
Transcription of genes is initiated by RNA polymerase II with other basal transcription factors including TFIIA, -B, -D, -E, -F, -G/J, -H, and -I. Before RNA synthesis begins, RNA polymerase II and basal factors bind to a promoter in an ordered series of steps and form a preinitiation complex (PIC) (33). Since HNF-4 binds very closely to the transcription initiation sites, we speculated a possible protein-protein interaction between HNF-4 and PIC. Recently, a protein-protein interaction between HNF-4 and TFIIB was reported (34). TFIIB is a member of the PIC and directly associated with TFIID that binds to TATA box (also known as TATA-binding protein). TFIIB plays a critical role in determining the transcription start site (35). We confirmed a direct interaction between TFIIB and HNF-4 by a two-hybrid system in yeast (Fig. 6, A and B).
We speculated another possible protein-protein interaction between HNF-4 and NF-Y. NF-Y consists of three subunits, NF-YA, NF-YB, and NF-YC (22, 23). NF-Y binds to the CCAAT sequence in the 5'-flanking sequences of many genes and regulates the transcription of those genes (22). The CCAAT sequence was found near upstream of the TATA sequence of some genes. The binding of NF-Y to the CCAAT sequence was also essential for the transcription of some TATA-less genes (36). These reports suggested a direct association of NF-Y to the PIC. In the present study, we have shown that HNF-4 is capable of directly binding to the A-subunit of NF-Y, suggesting that the binding of HNF-4 to both TFIIB and NF-Y is important for the positioning of PIC and the initiation of the transcription of the MSP gene.
The distal HNF-4-binding site resides in the NRE located between 141
and
34 (16). When the NRE was deleted from the wild-type MSP
promoter, NF-Y-induced CAT activity could be detected in HeLa and HOS
cells. Therefore, we previously speculated that the binding of some
repressor proteins to the NRE negatively regulated the transcription of
the MSP gene in non-hepatic cells. In the present study,
several additional CAT plasmids were constructed, and CAT functional
assay was performed with HeLa cells. The previously reported NRE
appears to be divided into two NREs. One is located between
141 and
121 (NRE1), and another is between
81 and
34 (NRE2). Since HNF-4
binds to the 5'-end of the NRE2, HNF-4 could inhibit the binding of
putative repressors to the region. We observed a wide area including
the NRE protected from DNase Idigestion with the nuclear
extracts from HeLa and HOS cells. However, we were unable to
demonstrate the binding of repressor proteins to this region by EMSA
(data not shown). Further study is necessary to understand the
mechanisms involved in tissue-specific transcription of the
MSP gene.
We detected two different nuclear protein-DNA complexes by EMSA with an oligonucleotide carrying the proximal HNF-4-binding site sequence and the nuclear extracts of Hep3B and HepG2 cells (Fig. 2A, C1 and C2). The C2 was specific to Hep3B and HepG2 cells and contained HNF-4, whereas shifted bands that migrated to the region of the C1 were also detected with the nuclear extracts of nonhepatic cells. This ubiquitously expressed protein could bind to the several mutated probes M1, M3, and M5, but not to M2 and M4, showing the same binding pattern as HNF-4. The binding of a ubiquitously expressed protein to an HNF-4-binding site on the apolipoprotein CIII promoter (termed element B) was previously reported. The binding of HNF-4 and CIIIB1 to this element activates the apoCIII promoter (37). Conversely, the binding of chicken ovalbumin upstream promoter-transcriptional factor 1 (38) or apoAI regulatory protein 1 (also known as chicken ovalbumin upstream promoter-transcriptional factor TFII) (39) to the element negatively regulates the promoter activities. These factors might be involved in the formation of the C1. As we reported previously (16) and also in the present article, CAT activities were increased even in HeLa cells after removal of the NRE from the promoter region of the MSP gene. There appear to be other proteins that bind to the proximal HNF-4-binding site and function as a trans-activator for the MSP gene transcription.
In summary, we identified two HNF-4-binding sites in the promoter region of the MSP gene. The proximal HNF-4-binding site, not the distal HNF-4-binding site, and the CCAAT sequence that we previously characterized as an NF-Y-binding site cooperatively regulated the transcription of the MSP gene. Finally, we presented evidence that indicated direct association between NF-YA and HNF-4.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Frances M. Sladek and Nancy Rice for generous gifts of the pMT7HNF-4, rat HNF-4 expression vector, and an antiserum against HNF-4, and the murine p65 expression vector CMV-LDp65 and human p50 expression vector CMV-399, respectively. We are also grateful to Dr. Jun Fukushima for helpful discussion.
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FOOTNOTES |
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* 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.
¶ To whom correspondence should be addressed: Bldg. 560, Rm. 12-71, NCI-FCRDC, Frederick, MD 21702. Tel.: 301-846-1560; Fax: 301-846-6145; E-mail:yoshimur{at}mail.ncifcrf.gov.
1 The abbreviations used are: MSP, macrophage stimulating protein; C/EBP, CCAAT/enhancer-binding protein; HNF, hepatocyte nuclear factor; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; NRE, negative regulatory element; PRE, positive regulatory element; PAGE, polyacrylamide gel electrophoresis; FCS, fetal calf serum; PCR, polymerase chain reaction; PIC, preinitiation complex; Inr, initiator; BD, binding domain; AD, activation domain.
2 A. Ueda and T. Yoshimura, unpublished data.
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REFERENCES |
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