Transcriptional Regulation of Cell Type-specific Expression of
the TATA-less A Subunit Gene for Human Coagulation Factor XIII*
Masafumi
Kida,
Masayoshi
Souri,
Masayuki
Yamamoto
,
Hidehiko
Saito§, and
Akitada
Ichinose¶
From the Department of Molecular Pathological Biochemistry and
Biology, Yamagata University School of Medicine, Yamagata 990-9585, the
Department of Molecular Developmental Biology,
Tsukuba University School of Medicine, Tsukuba 305, and the
§ Department of Medicine I, Nagoya University School of
Medicine, Nagoya 466, Japan
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ABSTRACT |
To study the mechanism of gene regulation for
coagulation factor XIII A subunit (FXIIIA), we characterized its
5'-flanking region using a monocytoid (U937), a megakaryocytoid
(MEG-01), and other cells. Our results confirmed that U937 and MEG-01
contained FXIIIA mRNA. A tentative transcription start site was
determined to be 76 bases upstream from the first exon/intron boundary.
Reporter gene assays revealed that a 5'-fragment (
2331 to +75) was
sufficient to support basal expression in U937 and MEG-01 but not in
the other cells. Deletion analysis confined a minimal promoter sequence from
114 to +75. DNase footprinting, electrophoretic mobility shift,
and reporter gene assays demonstrated that promoter elements for a
myeloid-enriched transcription factor (MZF-1-like protein) and two
ubiquitous transcription factors (NF-1 and SP-1) in this region were
important for the basal FXIII expression. It was also revealed that an
upstream region (
806 to
290) had enhancer activity in MEG-01 but
silencer activity in U937. DNA sequences for binding of
myeloid-enriched factors (GATA-1 and Ets-1) were recognized in this
region, and the GATA-1 element was found to be responsible for the
enhancer activity. These transcription factors play a major role in the
cell type-specific expression of FXIIIA, which differs from
other transglutaminases.
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INTRODUCTION |
Human coagulation factor XIII
(FXIII,1 fibrin-stabilizing
factor) is a plasma transglutaminase (TGase) and circulates in blood as
a heterotetramer consisting of two catalytic A (FXIIIA) and two
noncatalytic B (FXIIIB) subunits (A2B2) (1). It
is a proenzyme that is activated by thrombin that is generated in the
final stage of the blood coagulation cascade. The enzyme promotes clot
stability by forming covalent bonds between fibrin molecules and also
by cross-linking fibrin with other proteins including fibronectin,
2-plasmin inhibitor, and collagen (2, 3). These
reactions lead to an increase in the mechanical strength, elasticity,
and resistance to degradation by plasmin of fibrin clots and promotion of wound healing. Congenital FXIII deficiency is a severe bleeding disorder associated with impaired wound healing and an increased risk
of spontaneous abortion in women (2). In most cases the disorder is due
to FXIIIA deficiency, and only a few cases with FXIIIB deficiency have
been reported (3). The FXIIIA gene is located on chromosome
6p24-25 (4) and spans more than 160 kb of sequence and contains 15 exons (5).
In addition to its presence in plasma, FXIII exists inside monocytes
and macrophages (6-11), megakaryocytes/platelets (9, 12), and in
placenta (13). It is not present in erythrocytes, granulocytes, or
lymphocytes (6). Although the intracellular form of FXIII is a
homodimer of FXIIIA alone without FXIIIB, it has functional
characteristics identical to those of plasma factor XIII. Genetic
studies have confirmed that plasma and intracellular FXIIIA are the
products of the same gene (4, 5, 14). It is evident that FXIIIA is
actively synthesized in circulating monocytes (10, 11), and it may also
be synthesized in megakaryocytes (15). In contrast, the possible
biosynthesis of FXIIIA in human liver has been debated (16-19).
Transplantations of bone marrow have presented indirect evidence that
bone marrow cells are a source of plasma FXIIIA (9).
FXIIIA belongs to a family of TGases, members of which are expressed in
various organs, and is regulated by diverse mechanisms. Both
keratinocyte TGase and seminal plasma TGase are expressed in highly
specialized tissues. The former is expressed prominently in
differentiating keratinocytes (20); the latter is synthesized by
prostatic epithelial cells (21). Tissue TGase is ubiquitously expressed
in many cells and tissues (22). Recent studies revealed transcriptional
regulatory mechanisms for these genes. The expression of tissue TGase
may prove to be regulated by DNA methylation (23), and retinoid
response elements were found in the mouse tissue TGase promoter (24).
In the human TGase 3 gene, SP-1- and Ets-1-like motifs were
required for the function of its promoter (25). Analysis of the
keratinocyte TGase promoter indicated a region containing three
AP-2-like motifs essential for its expression (26). In contrast,
mechanisms regulating the expression of the FXIIIA gene have
not been reported. The mechanism of release of FXIIIA from the
synthesizing cells in vivo also remains unknown to date
(27).
In order to investigate the transcriptional regulatory mechanisms of
the FXIIIA promoter, we have characterized cis-acting sequences and trans-acting factors responsible for the
activation of transcription and for the tissue-specific expression of
the TATA-less FXIIIA gene in the present study.
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EXPERIMENTAL PROCEDURES |
Human Cell Culture--
The megakaryoblastic leukemia cell lines
MEG-01 and MEG-J and the histiocyte lymphoma cell line U937 were
maintained in RPMI 1640 medium with 10% fetal bovine serum and
antibiotics (penicillin, streptomycin, and neomycin) at 37 °C in 5%
CO2. The hepatoma cell line HepG2 and the cervical
carcinoma cell line HeLa were maintained in Dulbecco's modified
Eagle's medium with 10% fetal bovine serum and the antibiotics.
Finally, the transformed embryonic kidney cell line 293 was maintained
in Dulbecco's modified Eagle's medium with 10% heat-inactivated
horse serum and the same antibiotics as mentioned above. HepG2 and HeLa
were obtained from the Japanese Cancer Research Resources Bank and 293 from the Health Science Research Resources Bank. U937 and MEG-J were
kind gifts from Drs. S. Yokoyama and H. Mizoguchi, respectively, and
MEG-01 was provided by H. Saito.
Northern Blot Analysis--
RNA was isolated from a variety of
cells including U937, MEG-01, MEG-J, HepG2, 293, and HeLa by the acid
guanidinium thiocyanate method. Five µg of total RNA was
electrophoresed on a 1% agarose gel containing 6% formaldehyde,
transferred to a nylon membrane (Zeta-Probe, Bio-Rad), and hybridized
with the DIGTM-RNA probe (Boehringer, Mannheim, Germany)
made from the cDNA for FXIIIA (14). Detection was performed using a
DIGTM-nucleic acid detection kit following the
manufacturer's instructions. The membrane was exposed to Kodak XAR
autoradiography film, and the film was subjected to semi-quantitation
of mRNA bands by Densitograph 3.01 Imaging Analyzer (Atto Inc.,
Tokyo, Japan). The fluorescence intensity of the band for FXIIIA was
normalized to that for
-actin.
Rapid Amplification of 5'-cDNA Ends--
To determine the
transcription initiation site of the FXIIIA gene, rapid
amplification of 5'-cDNA ends (5'-RACE) was performed using a
5'-RACE System, version 2.0 (Life Technologies, Inc.), and total RNA
from U937, MEG-01, and HepG2. The cDNA was first synthesized from
500 ng of total RNA using SuperscriptTM II reverse
transcriptase (Life Technologies, Inc.) and an antisense primer
(5'-CACATAGAAAGACTGC-3' designed for exon III of FXIIIA) at 42 °C
for 60 min. The cDNA purified by a GlassMax spin cartridge (Life
Technologies, Inc.) was used in terminal dideoxynucleotide transferase
(TdT) tailing, and PCR was then performed with an internal antisense
primer (5'-ACTGCTCTTCTGCCTCCAAA-3' designed for exon II of FXIIIA) and
a 5'-sense primer termed Abridged Anchor Primer. This was followed by a
nested PCR using a nested gene-specific antisense primer
(5'-GGAAGTGTCGACCATTTTTGACTTTACAAG-3' designed for exon II of FXIIIA)
and a nested adapter primer termed Abridged Universal Amplification
Primer. PCR products were analyzed by 2% agarose gel electrophoresis
and by the dideoxy sequencing method both directly and after subcloning
into pBluescript vectors (Invitrogen, Carlsbad, CA).
The 5'-RACE experiment was repeated by employing a separate antisense
primer for the PCR (5'-GACTTTACAAGGTCCTGCAGG-3' designed for exon II)
and fresh mRNA samples.
S1 Nuclease Mapping--
A chimeric DNA fragment containing the
5'-flanking region, exon I, and exon II of the FXIIIA gene
was prepared by ligation of a PstI fragment (
290 to +74)
of a 5'-genomic clone into a PstI-cleaved cDNA plasmid
for FXIIIA in pBluescript II SK+ (50). A probe for S1 nuclease mapping
was produced by the single-strand PCR method using Taq DNA
polymerase, an antisense primer designed from the sequence in exon II
(GSP2, 5'-GGTCCTGGAAGTTTCTGACAT-3'), and the chimeric DNA plasmid as a
template. Poly(A) mRNAs were purified from MEG-01 and U937 cells
using an oligo(dT)- cellulose column. The probe was hybridized with 2 µg of the mRNA sample in 12.5 mM HEPES, pH 6.4, and
0.4 M NaCl at 60 °C for 8 h and then treated with
500-2000 units S1 nuclease (Life Technologies, Inc.) in 30 mM sodium acetate, pH 4.6, 1 mM zinc acetate,
0.2 M NaCl, and 5% glycerol at 37 °C for 60 min. 10 µg of yeast tRNA was added to the reaction mixture, and then the
protected DNA was recovered by precipitation with ethanol. The samples
were electrophoresed on a 6% polyacrylamide gel containing 8 M urea, together with a sequencing reaction mixture
produced by employing the same antisense primer and template chimeric
DNA as used in the generation of the probe. DNAs were transferred to a
nylon membrane and hybridized with a sense strand digoxigenin
(DIG)-labeled RNA probe, which was generated by 20 units of T7 RNA
polymerase (Boehringer Mannheim) and the chimeric DNA plasmid as a
template. Detection of the DNA was achieved using a DIG nucleic acid
detection kit (Boehringer Mannheim).
Primer Extension--
A DIG-labeled oligonucleotide (GSP2) was
hybridized with 5 µg of poly(A) mRNA at 45 °C for 8 h in
a solution containing 0.4 M NaCl and 12.5 mM
HEPES, pH 6.4. After ethanol precipitation, the oligonucleotide was
extended by Superscript IITM reverse transcriptase using
FXIIIA mRNA as a template. DNA products were subjected to
electrophoresis on a 6% sequencing gel containing 8 M urea
in parallel with dideoxy sequencing ladders. Detection of the DNA was
achieved using the DIG nucleic acid detection kit.
Construction of CAT Plasmids--
Genomic clones of FXIIIA were
obtained in the previous study (5). A 4.5-kb fragment containing the
5'-flanking region of FXIIIA was excised from a phage clone with
HindIII and subcloned into a HindIII site of
promoter-less pCAT-Basic and -Enhancer vectors (Promega, Madison, WI).
A fragment containing
290 to +75 relative to the transcription start
site was isolated from the subcloned vector by digestion with
PstI and inserted into the PstI site of
promoter-less CAT vector (pCAT 0), generating pCAT
290/+75 (Fig.
1). A fragment spanning from
2331 to
115 was obtained by digestion of the first CAT construct containing the 4.5-kb fragment with HindIII and SacI and
inserted into pCAT
290/+75 digested with HindIII and
SacI, generating pCAT
2331/+75. The pCAT
2331/+75 was
digested with SacI and XbaI and blunt-ended and
circularized, generating pCAT
2331/
115. CAT constructs containing fragments spanning from positions
1593,
806,
114 to +75 were obtained by digestion of HindIII and StuI,
SmaI, or SacI of pCAT
2331/+75, respectively,
and religation. The sequences of 5'- and 3'-boundaries of
generated CAT constructs were verified both by digestion with
several restriction enzymes and by dideoxy sequencing. Large
scale plasmid preparation was made using a plasmid purification kit
(Qiagen, Valencia, CA).

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Fig. 1.
Constructs for the 5'-flanking region of
FXIIIA and promoter activity. Left panel, various
lengths of the FXIIIA promoter region (thick solid lines)
were inserted into the promoter-less CAT plasmid (pCAT 0).
Numbers above the constructs indicate positions
relative to the transcription start site (+1). Right panels,
CAT activity of a series of the deletion constructs was presented
relative to that of the pCAT 0. The CAT activity was normalized to
-galactosidase activity. The data are presented as the mean ± S.D. for three independent experiments.
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A series of CAT plasmids harboring either a deletion or nucleotide
substitutions that alter or abolish consensus sequences for
protein-binding sites was also constructed by site-directed mutagenesis
using an overlap extension PCR protocol (28). Two separate PCR
fragments for each half of a final hybrid product were generated
employing mutagenesis primers (Table I)
and outside primers (forward primer, 5'-CTCCTGAAAATCTCGCC-3'; reverse
primer, 5'-CAGGAAACAGCTATGAC-3'). The two products were mixed, and a
second PCR was performed using the two outside primers. The resulting products were digested with HindIII and XbaI and
ligated into HindIII/XbaI sites of a promoterless
CAT plasmid. The inserts were then sequenced to confirm the intended
mutations.
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Table I
Sequences of the double-stranded oligonucleotides used in this study
Lowercase letters indicate the bases mutated. S and AS stand for sense
and antisense, respectively.
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Transient Transfection and CAT Assay--
pCAT constructs (20 µg) and a pSV
-galactosidase vector (10 µg, Promega, Madison, WI)
as an internal control were transfected into cultured cells by
electroporation or the calcium-phosphate precipitation method. Both
pCAT-Basic and -Control vectors were also employed as negative and
positive controls, respectively. After 24 h in culture, the cells
were harvested, and cell lysates were prepared. Each transfection
experiment was repeated at least three times. CAT activity was measured
by the standard method and
-galactosidase activity by the
colorimetric method. CAT expression levels were normalized to
-galactosidase activity to correct for differences in transfection
efficiency and cell number.
Preparation of Nuclear Extracts and DNase I Footprint
Analysis--
Nuclear extracts from culture cells were prepared
according to a modified Dignam's method (29). Final protein
concentrations were determined by the BCA protein assay (Pierce). DNase
I footprinting was performed in a 50-µl reaction mixture containing
25 mM HEPES, pH 7.9, 60 mM KCl, 5 mM MgCl2, 0.25 mM EDTA, 0.75 mM dithiothreitol, 7.5% glycerol, and 3 µg of
poly(dI-dC). Fifty µg of crude nuclear extracts was added and
incubated for 10 min on ice. Then, 32P-end-labeled DNA
fragments
289 to +75 (10 ng, 5 × 104 cpm) were
added, and the incubation was continued for an additional 20 min on
ice. At room temperature, digestion with 1 unit of DNase I (Takara,
Tokyo, Japan) was allowed to proceed for exactly 1 min. To the control
without nuclear extracts, 0.08 or 0.16 units of DNase I was added. The
reaction was terminated by adding a stop solution containing 1.5 M sodium acetate, 20 mM EDTA, and 100 µg/ml
tRNA. The DNA sample was analyzed on a 6% polyacrylamide sequencing
gel. The corresponding FXIIIA sequence generated by the Maxam and
Gilbert method was run alongside the reaction to serve as a marker.
Footprinting assays were performed for both strands of the FXIIIA promoter.
Electrophoretic Mobility Shift Assay (EMSA) and Supershift
Assay--
Complementary single-stranded oligonucleotides were
annealed, end-labeled with 32P, and purified using Sephadex
G-50 quick spin columns (Boehringer Mannheim). Nuclear proteins were
preincubated in a reaction solution containing 20 mM
Tris-HCl, pH 7.9, 2 mM MgCl2, 1 mM
EDTA, 50 mM NaCl, 0.5 mM dithiothreitol, 10%
glycerol, 0.1% Nonidet P-40, and 2 µg of poly(dI-dC). A competitor
DNA was added when needed. After 10 min, the
32P-end-labeled duplex oligonucleotide (2 × 104 cpm) was added, and the reaction was incubated for 20 min on ice.
For supershift experiments, 2 µl of control rabbit IgG or control rat
IgG (Santa Cruz Biotechnology, Santa Cruz, CA) against 1 µl of
NF-1 (a generous gift from Dr. Naoko Tanese),
SP-1,
Est-1 (a
kind gift from Dr. Dennis Watson),
Est-1/Est-2 (Santa Cruz
Biotechnology),
GATA-1 (N6), or
GATA-2 (RC1.1) (provided by M. Yamamoto) antibody was added to the reaction mixture and incubated for
30 min.
 |
RESULTS |
Northern Blot Analysis--
Northern blot analysis employing total
RNAs obtained from cultured cell lines showed a single mRNA band of
about 4.0 kb both in U937 and MEG-01 cells, but the band was not
detectable in MEG-J, HepG2 or 293 cells (Fig.
2), or HeLa cells (data not shown).
Semi-quantitation of the bands by an image analyzer revealed that the
FXIIIA mRNA level in U937 cells was 3-fold that in the MEG-01
cells, when normalized to
-actin mRNA levels.

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Fig. 2.
Northern blot analysis of the mRNA for
FXIIIA in human cultured cell lines. Total RNA from various cell
lines was hybridized to the DIGTM-RNA probe for FXIIIA. A
4.0-kb band, as indicated by an arrow and labeled FXIIIA,
corresponds to the FXIIIA transcript in U937 and MEG-01 cells
(upper panel). -Actin was used as a reference to permit
normalization (lower panel).
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Determination of the Transcription Initiation Site--
Rapid
amplification of the 5'-cDNA ends was performed to identify the
transcription start site of the FXIIIA gene. A single band
was observed in both U937 and MEG-01 cells (data not shown); a PCR
product of the same size was visible in HepG2 cells only after PCR was
performed extensively. Sequencing of 10 subclones of the resulting
transcripts revealed that all 10 clones started at a G nucleotide
located 76 nucleotides upstream from the first exon/intron boundary.
This indicated the presence of a single major transcription initiation
site for the FXIIIA gene (large asterisk in Fig.
3). These results were also confirmed by
direct sequencing of the PCR products for U937 and MEG-01 cells (data not shown). When the 5'-RACE experiment was repeated by employing a new
antisense primer and fresh mRNA samples, two major bands were
observed: one band corresponded to the tentative transcription initiation site (+1), whereas another band fell upon an A nucleotide 218 bases upstream of the tentative initiation site (small
asterisk in Fig. 3).

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Fig. 3.
Nucleotide sequence of the FXIIIA promoter
region. The four protected sites A-D
(boxed) were determined by DNase I footprint analysis (Fig.
4). The tentative transcription initiation site (+1), depicted with a
large asterisk, was determined by the 5'-RACE method.
Another possible transcription initiation site, found by a separate
5'-RACE study, is indicated by a small asterisk. Other
candidates for transcription initiation sites determined by the S1
mapping experiment are shown by open triangles and that
detected by the primer extension assay is indicated by a
circle. Putative regulatory sites are indicated by
horizontal arrows for GATA-1, Ets-1, SP-1, MZF-1 (myeloid
zinc finger-1), PEA-3, and NF-1 (nuclear factor-1). Two upstream and
downstream Ets-1 consensus sequences are designated as Ets-1a and -1b,
respectively.
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An S1 nuclease mapping analysis, employing a DNA fragment containing
the 5'-flanking region, exon I, and exon II of the XIIIA gene, yielded two major bands for both MEG-01 and U937 (data not shown), which corresponded to an A nucleotide 24 bases and a C nucleotide 7 bases upstream from the first exon/intron boundary (open triangles in Fig. 3). A primer extension experiment
was also performed using the total RNA from U937, MEG-01, and HepG2 and
the oligonucleotide GSP2 and yielded a single major fragment (data not
shown) that corresponded to an A nucleotide 40 bases upstream from the
first exon/intron boundary (small circle in Fig. 3).
As described above, the results of the 5'-RACE, S1 mapping, and primer
extension were inconsistent. However, we concluded the tentative +1
nucleotide was a major transcription initiation site for the
FXIIIA gene, because all other candidates detected by S1
mapping and primer extension were rather downstream of the 5'-ends of
our 6 cDNA clones (14) and the clones of other investigators (18).
In addition, it is unlikely that the A nucleotide 218 bases upstream of
the tentative initiation site, which was detected by the second 5'-RACE
trial, is a real one, since no nearby promoters were identified by the
following experiments (see below).
Promoter Activity of FXIIIA--
Fragments of various lengths
containing the transcription initiation site were inserted into a
promoter-less CAT vector, and the constructs were transiently
transfected into various cultured cells. Promoter activities were then
measured by CAT assay after 24 h of culture. A segment of 2.4 kb
that included the 5'-flanking region from
2331 to +75 (transcription
start site numbered as +1) was able to transcribe a CAT reporter gene
in U937 and MEG-01 cells, whereas this expression was as low as
background levels in HepG2, MEG-J, 293 (Fig. 1), and HeLa cells (data
not shown). These results indicated that the expression of FXIIIA is
hematopoietic cell type-specific.
Deletion analysis revealed that a region from
114 to +75 was
essential for the expression (a minimal or core promoter) and that this
region also showed promoter activity in HepG2 cells (Fig. 1). In U937
cells, stepwise deletions from
2331 to
1593,
806, or
290
resulted in progressive increases in CAT activity, whereas a deletion
from
806 to
290 resulted in a decrease in CAT activity in MEG-01
cells. These results indicated that the 5'-upstream region
2331 to
290 contained silencers for the expression in both U937 and MEG-01
cells and that an enhancer specific for MEG-01 cells was present in the
region
806 to
290. A deletion from
290 to
114 resulted in a
slight decrease in CAT activity in U937 cells but an increase in MEG-01
and HepG2 cells. These results indicated that a weak enhancing
element(s) was present in this region for U937 cells, whereas there was
a silencing element(s) for MEG-01 and HepG2 cells.
DNase I Footprint Analysis--
To identify nuclear factor binding
sites in the core promoter and cell type-specific regulatory region,
DNase I footprint assays were performed for the region
290 to +75.
Crude nuclear extracts from U937, MEG-01, HepG2 and 293 cells were used
for the assays, and both sense and antisense strands were employed as
well. Experiments using U937 and MEG-01 nuclear extracts allowed identification of four distinct regions: site A,
39 to
13, and site
B,
92 to
73 (Fig. 4, lanes
5 and 6); sites C and D, from
260 to
223
(lanes 13 and 14). Nuclear proteins from HepG2
cells protected two regions as follows: site B and site C (lanes
7 and 15), whereas 293 nuclear extracts weakly
protected a single region, site B (lanes 8 and
16).

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Fig. 4.
DNase I footprint analysis using sense
(left) and antisense (right) strands
of the FXIIIA promoter region. A DNA fragment spanning 290/+75
of FXIIIA was labeled with 32P at the 3'-end and was
subjected to DNase I digestion in the absence (lanes 3 and
11 and lanes 4 and 12 with 0.08 and
0.16 units of DNase I, respectively) or presence of nuclear extracts
from various cell lines: lanes 5 and 13, U937
nuclear extracts; lanes 6 and 14, MEG-01 nuclear
extracts; lanes 7 and 15, HepG2 nuclear extracts;
and lanes 8 and 16, 293 nuclear extracts.
G (lanes 1 and 9) and C/T
(lanes 2 and 10) sequence markers were obtained
by Maxam-Gilbert sequencing of the end-labeled fragment. Four regions
protected by nuclear extracts are designated as sites A, B, C, and D,
respectively. An asterisk shows the transcription initiation
site (+1). The numbers on the right side of each
autoradiogram indicate the boundaries of the protected sites.
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A search for putative transcription factor binding sites using the
TRANSFAC 3.2 data base revealed that site A contained a PEA-3 (30) and
a partial sequence for NF-1 (31)-binding sites. It was also revealed
that each of sites B, C, and D contained both myeloid zinc finger-1
(MZF-1)- (32) and SP-1 (33)-binding sites, which partially overlap each
other (Fig. 3).
It seemed likely that three other sites downstream of site A and
upstream and downstream of site C/D were also possibly protected (Fig.
4). These regions, however, were excluded from further detailed studies
for the following reasons. Only nonspecific bands were observed for the
three sites when examined by a gel-shift assay (data not shown), and a
computer-assisted search revealed no consensus sequences for
transcription factors in these three regions.
Characterization of Sites A, B, C, and D by EMSA--
To
characterize transcription factors that interact with the binding sites
in the FXIIIA promoter, EMSAs and supershift assays were performed.
Synthetic oligonucleotides used in EMSAs (Table I) were designed from
the regions protected by nuclear proteins and from known consensus
sequences for transcription factors (30-36). EMSA of site A showed a
single complex with nuclear proteins from U937 and MEG-01 cells, but
the band was hardly detected with nuclear proteins from HepG2 and 293 cells (Fig. 5A) and HeLa and
MEG-J cells (data not shown). This complex was competed and eliminated by a 20- or 200-fold molar excess of the unlabeled site A
oligonucleotide termed self-competitor (Fig. 5A,
left and middle, lanes 2, 3, 11, and
12), as well as by a NF-1 consensus oligonucleotide (Fig. 5A, left and middle, lanes 6, 7, 15, and 16). The shifted band was not abolished by a PEA-3
consensus oligonucleotide (Fig. 5A, left and
middle, lanes 4, 5, 13, and 14). An
oligonucleotide with mutations at the NF-1 sequence in site A (Table I)
formed only a faint complex (Fig. 5A right, lanes 20, 22, 24, and 26). These results suggested that site A is an
NF-1-binding site.

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Fig. 5.
EMSAs for sites A, B, C, and D. Nuclear
extracts were prepared from U937, MEG-01, HepG2, and 293 cells, as
indicated at the bottom of the panels. Specific DNA-protein
complexes are indicated by asterisks. NS depicts
a nonspecific complex, and F shows the position of free
oligonucleotide. Indicated amounts of molar excess of cold
self-competitors and consensus oligonucleotides were added. Nucleotide
sequences of the probes and competitors are shown in Table I.
A, an oligonucleotide spanning 42 to 10 of FXIIIA (site
A wild type) was labeled and incubated with 5 µg of nuclear extracts
from various cell lines, as indicated at the bottom of the
panels (lanes 1-19, 21, 23, and 25). Experiments
with a mutant probe (site A mutant in Table I) were also performed
(lanes 20, 22, 24, and 26). Lanes 1, 8, 10, 17, and 19-26 were samples with no competitor (termed
0). For competition analysis, an unlabeled oligonucleotide
for site A (wild type) was added as a cold competitor (termed
Self) in 20, 200, and 100 × molar excess (lanes
2 and 11, 3 and 12, and
9 and 18, respectively). Lanes 4 and
13 and 5 and 14 contained samples with
an unlabeled PEA-3 oligonucleotide (consensus PEA-3) as a competitor in
20 and 200 × molar excess, respectively. Lanes 6 and
15 and 7 and 16 contained samples with
an unlabeled NF-1 oligonucleotide (consensus NF-1) as a competitor in
20 and 200 × molar excess, respectively. B,
competition analysis of site B. The probe used was a follows: site B
wild type in lanes 1-27, 29, 31, and 33; and
site B mutant in lanes 28, 30, 32, and 34. The
competing oligonucleotide used was as follows: none in lanes 1, 10, 14, 23, and 27-34; site B wild type in lane
2 (20 × molar excess), lane 3 (200 ×),
lane 15 (20 ×), and lane 16 (200 ×); consensus
SP-1 in lane 4 (20 ×), lane 5 (200 ×),
lane 11 (100 ×), lane 17 (20 ×), lane
18 (200 ×), and lane 24 (100 ×); consensus ZN1-4 in
lane 6 (20 ×), lane 7 (200 ×), lane
12 (100 ×), lane 19 (20 ×), lane 20 (200 ×), and lane 25 (100 ×); and consensus ZN5-13 in
lane 8 (20 ×), lane 9 (200 ×), lane
13 (100 ×), lane 21 (20 ×), lane 22 (200 ×), and lane 26 (100 ×). C, competition
analysis of site C. The probe used in lanes 1-15, 18, 21,
and 23 was as follows: site C wild type; mutant 1 of site C
in lanes 16 and 19; mutant 2 in lanes
17 and 20; and mutant 3 in lanes 22 and
24. The competing oligonucleotide of 100-fold molar excess
was as follows: none in lanes 1, 6, 11, 13, and
15-24; site C wild type in lanes 2, 7, 12, and
14; consensus SP-1 in lanes 3 and 8;
consensus ZN1-4 in lanes 4 and 9; and consensus
ZN5-13 in lanes 5 and 10. D,
competition analysis of site D. The probe was site D wild type in
lanes 1-16 and 18 and site D mutant in
lanes 17 and 19. The competing oligonucleotide of
100-fold molar excess was as follows: none in lanes 1, 2, 7, 12, 14, and 16-19; site D wild type in lanes 3, 8, 13, and 15; consensus SP-1 in lanes 4 and
9; consensus ZN1-4 in lanes 5 and 10;
and consensus ZN5-13 in lanes 6 and 11.
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|
Site B formed two distinct complexes. The slower migrating complex was
found with MEG-01 nuclear extracts (Fig. 5B,
middle), whereas only the faster one was detected for U937,
HepG2, and 293 cells (Fig. 5B, left and middle).
Competition experiments showed that both complexes were completely
abolished by a 200-fold molar excess of an unlabeled
self-oligonucleotide for U937 and MEG-01 cells (Fig. 5B,
left and middle, lanes 3 and
16). Two oligonucleotides for the consensus MZF-1 (32),
ZN1-4 and ZN5-13, specifically competed with the faster migrating
complex (Fig. 5B, lanes 6-9, 12, 13, 19-22, 25, and
26), suggesting that this site binds to an MZF-1-like
protein. The slower migrating complex of MEG-01 was eliminated even by
a 20-fold molar excess of unlabeled SP-1 oligonucleotide (Fig.
5B, middle, lane 17), suggesting that this site
also binds an SP-1 protein. An oligonucleotide with a mutated MZF-1
sequence in site B failed to form either complex (Fig. 5B, right, lanes 28, 30, 32, and 34).
EMSAs for site C demonstrated similar results to those for site B. The
faster migrating complex of site C (Fig. 5C), however, was
not completely abolished by specific MZF-1 oligonucleotides even in a
500-fold molar excess (data not shown), whereas the faster migrating
complexes of sites B and D were abolished by the same MZF-1
oligonucleotides in a 200-fold molar excess.
In EMSAs using mutated oligonucleotides as a probe for site C, mutant 1 with four base changes at the MZF-1 site failed to form a complex (Fig.
5C, right, lanes 16 and 19), as did mutant 3 with
one base mutation at the same MZF-1 site (Fig. 5C, right, lanes 22 and 24). The mutation in mutant 3 is a G to A polymorphism found in the 5'-flanking region of two
unrelated cases with FXIII deficiency (37, 38). EMSAs employing mutant
2, which has a typical SP-1-binding site generated by a T to C change,
resulted in an increase in the slower migrating complex and an
elimination of the faster migrating complex (Fig. 5C, right,
lanes 17 and 20), suggesting that the slower migrating
complex was formed with SP-1. The intensity of the slower migrating
complex of U937 was much less than that of MEG-01, which is consistent
with the fact that Western blotting detected a lesser amount of SP-1 in
U937 than in MEG-01 (data not shown).
EMSAs for site D demonstrated essentially the same results as those for
site B. There was a nonspecific complex in U937 cells (Fig. 5D,
lane 2), whereas two specific complexes appeared to form with an
MZF-1-like protein and an SP-1 protein in MEG-01 cells (Fig. 5D,
lane 7). The slower migrating complex was prominently recognized
in MEG-01 cells. This complex was competed out with unlabeled self,
SP-1, and ZN5-13 oligonucleotides (Fig. 5D, left, lanes 8, 9 and 11), whereas it was not completely eliminated by ZN1-4 (Fig. 5D, left, lane 10). A mutant oligonucleotide
with four bases changes at the MZF-1 site eliminated both complexes in
MEG-01 (Fig. 5D, right, lane 19).
All the above EMSA experiments for sites A, B, C, and D were confirmed
by cross-competitions, employing consensus sequences for transcription
factors as probes and wild type oligonucleotides for four sites as
competitors (Table I).
Finally, supershift assays were performed in order to identify a
nuclear protein(s) for each of the above transcription factor-binding sites. When an antibody against NF-1 was added to the binding reaction,
a supershifted band appeared for both U937 and MEG-01 cells (Fig.
6 left), indicating that the
protein bound to site A was NF-1. An
SP-1 antibody competed away
only the slower migrating complexes of sites B, C, and D (Fig. 6,
middle), strongly suggesting that these complexes were made
by SP-1. Since no antibody against human MZF-1 suitable for supershift
assays is available at present, a protein forming the faster migrating
complexes could not be determined in this study.

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Fig. 6.
Supershift assays in EMSAs. Control IgG
or a specific antibody against NF-1 ( NF-1), SP-1 ( SP-1), GATA-1
( GATA-1), or GATA-2 ( GATA-2) was added to the reaction mixture of
EMSA and incubated for 30 min. An asterisk alone,
arrow with F, arrow with
NS, and arrow with S indicate a
specific complex, a free probe, a nonspecific band, and a supershifted
complex, respectively. An arrow with C depicts a
complex that is competed by an antibody. When the assay for SP-1
(middle) was repeated in the presence of the control rabbit
IgG, the complex shown by an arrow with C
remained unchanged (data not shown).
|
|
Functional Analysis of Regulatory Sequences by Site-directed
Mutagenesis--
In order to determine the functional significance of
the protein-binding sites detected by footprinting and EMSAs, we
constructed a series of substitution mutants and a deletion mutant that
alter or abolish the consensus sequences by site-directed mutagenesis (Table I). The CAT activity of the mutants was compared with that of
the wild type in transfection assays in U937 and MEG-01 cells (Fig.
7, middle four
constructs). Mutations in either site B or C resulted in drastic
decreases in CAT expression, whereas a mutation in site D had no
significant effect in either cell. These results suggest that the
MZF-1/SP-1 sequences in sites B and C are important for FXIIIA
expression. In particular, the former sequence must be essential for
FXIIIA expression, since the mutation in site B reduced the CAT
activity of the minimal promoter to virtually background levels in both
U937 and MEG-01 cells (Fig. 7, bottom).

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Fig. 7.
Functional analysis of mutated
regulatory sequences. CAT activity of a series of the mutated
constructs, generated by site-directed mutagenesis using primers
in Table I, is presented relative to that of the pCAT 0. The CAT
activity was normalized to -galactosidase activity. The data are
presented as the mean ± S.D. for three independent
experiments.
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|
A mutation in site A in the minimal promoter, as well as its complete
deletion, also resulted in moderate and drastic reductions in activity,
respectively, indicating that the NF-1 site is important for the FXIIIA
expression, as well.
Characterization of GATA-1 and Ets-1 Sites in the Enhancer Region
Specific for MEG-01 Cells--
Deletion analysis in the CAT assay
revealed the presence of an enhancer region specific for MEG-01 which
spans from
806 to
290 (Fig. 1). A computer-assisted search in the
TRANSFAC 3.2 data base revealed that this region contained one GATA-1
and two Ets-1 consensus sequences (Fig. 3), which were reported as
enhancers in the 5'-flanking regions of several megakaryocyte-specific
genes (34, 39-42). These sites were examined first by EMSAs. For the GATA-1 site, there was one shifted band in U937 cells (Fig. 8 left, lane 1) and
two in MEG-01 cells (lane 6). These complexes were competed
away by an excess self-competitor (lanes 2 and
7). The faster migrating complex was competed by a mutant
oligonucleotide for the GATA-1 site (lanes 3 and
8), and the slower migrating complex was abolished by a
GATA-1 consensus oligonucleotide (lanes 4 and 9),
although the labeled mutant oligonucleotide failed to form any complex
(lanes 5 and 10). Thus, it was suggested that the
slower migrating complex was formed by a GATA-1-like protein(s), and
the faster one by a nonspecific protein. This assumption is confirmed
by the fact that both antibodies against GATA-1 and -2 supershifted
only the slower migrating complex (Fig. 6, right).

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Fig. 8.
EMSAs of a GATA-1 and two Ets-1 sites.
Left, an oligonucleotide spanning from 762 to 748
containing the GATA-1 site (GATA-1 wild type) was labeled and incubated
with 5 µg of nuclear extracts from various cell lines, as indicated
at the bottom of the panels (lanes 1-4 and
6-9). Experiments with a mutant probe (GATA-1
mutant) were also performed (lanes 5 and
10). Specific DNA-protein complexes are indicated by
asterisks. NS with arrow shows
nonspecific complexes. The position of the free probe is marked as
F. The competing oligonucleotide of 100-fold molar excess
was as follows: GATA-1 wild type in lanes 2 and
7; GATA-1 mutant in lanes 3 and 8;
and, consensus GATA-1 in lanes 4 and 9 (Table I).
Middle, an oligonucleotide spanning 563 to 539
containing the upstream Ets-1 site (Ets-1a wild type) was used in EMSA
as a probe (lanes 1-4 and 6-9). Experiments
with a mutant probe (Ets-1a mutant) were also performed (lanes
5 and 10). The competing oligonucleotide of 100-fold
molar excess was as follows: Ets-1a wild type in lanes 2 and
7; Ets-1a mutant in lanes 3 and 8; and
consensus Ets-1 in lanes 4 and 9.
Right, an oligonucleotides spanning 490 to 466
containing the downstream Ets-1 site (Ets-1b wild type) was used in
EMSA as a probe (lanes 1-4 and 6-9).
Experiments with a mutant probe (Ets-1b mutant) were also performed
(lanes 5 and 10). The competing oligonucleotides
of 100-fold molar excess were Ets-1b wild type in lanes 2 and 7, Ets-1b mutant in lanes 3 and 8,
and consensus Ets-1 in lanes 4 and 9.
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Fig. 8 (middle panel) shows the results of EMSAs for the
upstream Ets-1 site (termed Ets-1a). A labeled Ets-1a oligonucleotide formed three complexes with nuclear extracts from U937 (lane
1) and MEG-01 (lane 6) cells. All complexes were
competed away by an excess self-competitor (lanes 2 and
7), whereas a mutant oligonucleotide for the Ets-1 site
competed only the slowest migrating complex (lanes 3 and
8). Two faster migrating complexes were abolished by an
Ets-1a consensus oligonucleotide (lanes 4 and 9),
and these complexes disappeared when the mutant probe was employed
(lanes 5 and 10). Accordingly, it was suggested
that the two faster migrating complexes were formed by an Ets-1 like
protein(s) and the slowest one by a nonspecific protein.
EMSAs for the putative Ets-1b site showed the same results as those for
Ets-1a (Fig. 8, right). These results suggested that GATA-1
and Ets-1 might contribute to the expression of FXIIIA in MEG-01 cells.
In order to determine the functional significance of these
protein-binding sites detected by EMSAs and the supershift assays, we
performed functional analyses by employing substitution mutants that
alter the consensus sequences by site-directed mutagenesis (Table I). A
mutation of the GATA-1 site led to a great reduction in the CAT
activity in MEG-01 cells, whereas only a mild decrease was observed in
U937 cells (Fig. 7, top four). In contrast, mutations of
both Ets-1a and -1b sites had no significant effect on the CAT
activity. Thus, these results indicate that the GATA-1 site is
responsible for the enhancer activity of this region for FXIIIA expression in MEG-01 cells, but the Est sites are not. Moreover, both
Est-1 and
Est-1/Est-2 antibodies failed to supershift or compete
away the complexes of Ets-1a and Ets-1b sites (data not shown),
suggesting that these complexes might be formed with Ets-like proteins
other than Est-1 and Est-2.
 |
DISCUSSION |
FXIIIA is present inside certain cells originating from the bone
marrow including monocytes/macrophages (6-11) and
megakaryocytes/platelets (9, 15), whereas FXIIIB is apparently produced
in hepatocytes (43, 44). It was previously demonstrated that peripheral
monocytes and U937 cells contained the mRNA for FXIIIA (10, 12). In the present study, we detected the FXIIIA mRNA not only in U937 cells but also in a megakaryocytoid cell line (MEG-01) by Northern blot
analysis. These results are consistent with the microscopic and
biochemical observations as mentioned in the Introduction.
In the present study, we have tentatively defined the transcription
start site of FXIIIA by 5'-RACE using total RNAs obtained from both
U937 and MEG-01 cells. Since no intronic sequence was found in the
transcripts of these cell lines, it was concluded that there are no
alternative promoter regions for the FXIIIA gene. The
transcription initiation site is located 76 nucleotides upstream from
the exon I/intron A boundary and only 13 nucleotides upstream from the
longest cDNA (14). Thus, exon I codes for only the 5'-untranslated
region in the FXIIIA gene (5).
This study revealed several cis-elements and
trans-acting factors involved in the constitutive and cell
type-specific expression of FXIIIA. These include hematopoietic- or
myeloid-specific elements such as MZF-1, GATA-1, and Ets-1 as well as
general elements such as NF-1 and SP-1.
Core Promoter of the FXIIIA Gene--
It is common in many
TATA-less promoters that transcription initiates from a cluster of
nuclear protein-binding sites. Protein binding to the NF-1 element at
site A was abundant in U937 and MEG-01 cells expressing the FXIIIA
mRNA but essentially absent in HepG2, 293, HeLa, and MEG-J cells.
These results are consistent with the fact that the latter four cell
lines did not show any transcript in the Northern blotting analysis.
The mutation of site A led to a moderate loss of the promoter activity
and a complete loss of the NF-1 binding to site A. Accordingly, the
NF-1 element in site A is important for the expression of FXIIIA in
U937 and MEG-01 cells. The NF-1 element at site A alone, however, is
not sufficient to drive FXIIIA expression, since a DNA fragment
containing site A alone did not demonstrate any CAT activity. The
removal of site A (27 bases,
13 to
39) led to a nearly complete
loss of the promoter activity, suggesting that proper space arrangement around the transcription start site may be required for
FXIIIA gene expression.
The MZF-1/SP-1 element at site B (
92 to
73) appeared to be most
essential for the expression of FXIIIA, since the removal or mutation
of this site completely abolished the promoter activity in all cell
lines. Both SP-1 and MZF-1-like proteins bind site B in MEG-01 cells,
whereas only MZF-1-like protein binds site B in U937 cells. MZF-1
protein is preferentially expressed in myeloid leukemia cell lines and
in myeloid progenitor cells from normal marrow (45). Accordingly, the
MZF-1 element at site B may play a central role in FXIIIA transcription.
It is of note that the DNA-protein complex formed by SP-1 was present
in MEG-01 but absent or in trace amounts only in U937 cells for sites C
and D as well as for site B. The SP-1 protein may modulate but may not
be indispensable for FXIIIA expression, since U937 cells contain a
large amount of FXIIIA mRNA and demonstrate strong promoter
activity despite the fact that U937 cells contain only a small amount
of SP-1 protein.
Upstream Regulatory Sequences--
cis-Acting elements
located upstream from the promoter regions can also modulate the
transcriptional activity. The MZF-1/SP-1 elements at sites C and D were
present side by side in a region that acted as a weak enhancer in U937
cells, but as a silencer in MEG-01 cells. The MZF-1-like protein and
SP-1 bound to these sites in MEG-01 cells, whereas the complex formed
by SP-1 was not observed in U937 cells. Thus, it is possible that SP-1
may act as a negative trans-activator or repressor by
interacting with the MZF-1-like protein at sites C and D in MEG-01
cells. MZF-1-like protein per se, in turn, is likely to be a
weak trans-activator of basal expression in U937 cells.
The nuclear protein that bound to site C showed low affinity for MZF-1
consensus oligonucleotides despite the fact that the MZF-1 sequence of
site C is exactly the same as that of site D (Fig. 3). Therefore, it is
suggested that this MZF-1-like protein bound to site C may differ from
that bound to sites B and D and could be a novel MZF-1 protein.
It is of interest that a G to A polymorphism was found in the
5'-flanking region of two patients with FXIII deficiency (37, 38). This
polymorphism falls exactly upon site C and eliminates the complexes of
site B; moreover, the site C mutant 3 did not form complexes with
either SP-1 or the MZF-1-like protein. Accordingly, the replacement of
G by A nucleotide may affect FXIII expression and its plasma levels in
the population.
It has been reported that GATA-1 and Ets proteins play important roles
in the expression of many genes in the megakaryocytic lineage (34-36,
40-42). A GATA-1 and two Ets-1 motifs were found in an enhancer region
of FXIIIA, and nuclear proteins bound to these sites. The mutation of
the GATA-1, but not those of Ets elements, resulted in a great
reduction in the promoter activity in MEG-01 cells. Accordingly, it was
concluded that the GATA-1 motif is involved in the megakaryocytic
expression of the FXIIIA gene.
It is of interest that there is also a short tandem repeat (STR)
polymorphism in the 5'-flanking region of FXIIIA (46). Since the GATA-1
site and STR polymorphism are adjacent to each other, the STR
polymorphism may have implications in activation of expression by the
GATA proteins and/or their binding affinity to DNA.
Possible interactions between these proteins (MZF-1-like protein, SP-1,
and GATA-1 and GATA-2) and with FXIIIA promoter elements likely
modulate FXIIIA expression, resulting in a complex pattern of tissue-
and cell type-specific gene regulation. A precedent for this is found
in the fact that MZF-1 represses the gene expression of CD34, a
hematopoietic stem cell antigen, in non-hematopoietic cell lines such
as NIH3T3 and 293, whereas it activates gene expression in
hematopoietic cell lines such as K562 and Jurkat (47).
Gene Regulation of TGase Family--
There are many other TGases
(48). The genes for these TGases share significant similarity in their
organization and have evolved from a common ancestral gene. Their
nucleotide sequences in the 5'-flanking regions, however, are not
homologous to each other, and mechanisms for their gene regulation seem
to be diverse as well (25, 26, 49). For example, the FXIIIA
gene has exon I encoding only a 5'-untranslated region, and its
translation start site ATG is present in exon II, whereas other TGase
genes have their translation start sites inside exon I adjacent to the transcription start sites (Fig. 9). The
genes for keratinocyte and tissue TGases are regulated by TATA-like
(CATA) and TATA promoters, whereas that for FXIIIA is not. Accordingly,
these genes do not share common transcription regulatory elements (Fig.
9). Thus, the tissue-specific transcriptional regulation of each gene
is controlled by differential interactions between positive and
negative cis-acting elements and their corresponding
transcription factors. In conclusion, the TATA-less promoter of FXIIIA
is controlled by transcription factors expressed with tissue
specificity, preferentially in myeloid cells, which distinguishes
FXIIIA from other members of the TGase family.

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Fig. 9.
Schematic diagram of 5'-flanking regulatory
sequences in FXIIIA and other TGases. The transcription and
translation initiation sites were indicated by +1 and ATG,
respectively. *, the sequences of transcription factors in the
5'-flanking region of tissue TGase have not been experimentally
determined by protein-binding experiments.
|
|
Further investigations are required to examine possible effects on the
promoter activity of the polymorphisms including the nucleotide
substitution and STR polymorphism in the 5'-flanking region of FXIIIA.
In addition, the possibility of cooperative regulation in the
transcription of the FXIIIA and FXIIIB genes will
be studied in the future, since plasma levels of both subunits are
partially determined by the presence and absence of their counterparts
(2, 3). Since U937 and MEG-01 cells are known to be stimulated and
differentiated by phorbol ester, granulocyte/macrophage colony-stimulating factor, and retinoic acid, possible effects of these
agents on the transcriptional activity of FXIIIA need to be examined,
as well.
 |
ACKNOWLEDGEMENTS |
We thank Drs. S. Yokoyama and H. Mizoguchi
for providing U937 and MEG-J cell lines, respectively; Drs. Naoko
Tanese and Dennis Watson for providing an
NF-1 and
Est-1
antibodies, respectively; Drs. T. Izumi and T. Yamazaki for helpful
discussions; and L. Boba for help in preparation of the manuscript.
 |
FOOTNOTES |
*
This study was supported in part by Research Grant 08457271 from the Ministry of Education, Science and Culture of Japan.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) AB021744.
¶
To whom correspondence should be addressed: Dept. of Molecular
Pathological Biochemistry and Biology, Yamagata University School of
Medicine, Iida-Nishi 2-2-2, Yamagata 990-9585, Japan. Tel.:
81-23-628-5275; Fax: 81-23-628-5280; E-mail:
aichinos{at}med.id.yamagata-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
FXIII, coagulation
factor XIII;
FXIIIA, factor XIII A subunit;
FXIIIB, factor XIII B
subunit;
CAT, chloramphenicol acetyltransferase;
EMSA, electrophoretic
mobility shift assay;
MZF-1, myeloid zinc finger-1;
5'-RACE, rapid
amplification of 5'-cDNA ends;
STR, short tandem repeat;
TGase, transglutaminase;
kb, kilobase pairs;
PCR, polymerase chain reaction;
DIG, digoxigenin.
 |
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