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HEMATOPOIESIS
From the Department of Pathology, Osaka University
Medical School, Suita, Japan; the Research Function of Biotechnology,
Frontier Collaborative Research Center, Tokyo Institute of Technology,
Yokohama, Japan; and the Division of Cellular and Molecular Medicine,
Glycobiology Program, University of California, San Diego, CA.
The mi transcription factor (MITF) is a
basic-helix-loop-helix leucine zipper (bHLH-Zip) transcription factor
that is important for the development of mast cells. Mast cells of
mi/mi genotype express normal amounts of abnormal MITF
(mi-MITF), whereas mast cells of tg/tg genotype
do not express any MITFs. The synthesis of heparin is abnormal in the
skin mast cells of mi/mi mice. Because N-deacetylase/N-sulfotransferase 2 (NDST-2) is essential for the synthesis of heparin, the amount of NDST-2 messenger RNA (mRNA) was compared among cultured mast cells (CMCs) of +/+,
mi/mi, and tg/tg genotypes. The NDST-2 mRNA was
detected by in situ hybridization in the skin mast cells of +/+ and
tg/tg mice, but not in the skin mast cells of
mi/mi mice. The amount of NDST-2 mRNA decreased significantly in CMCs derived from mi/mi mice when compared
to the values of +/+ and tg/tg mice, suggesting that the
defective form of MITF inhibited the expression of the NDST-2
transcript. The expression of NDST-2 transcript was mediated by the
GGAA motif located in the 5'-untranslated region. GA binding protein
(GABP) bound the GGAA motif and increased the amount of NDST-2
transcript. The mi-MITF appeared to inhibit the ability of
GABP to express NDST-2 transcript by disturbing its nuclear
localization. This is the first study to show that expression of an
abnormal form of a bHLH-Zip transcription factor can dramatically alter
the intracellular location of another DNA/RNA binding factor, which in
turn brings about profound and unexpected consequences on transcript expression.
(Blood. 2001;97:3032-3039) The mi locus of mice encodes a member of
the basic-helix-loop-helix leucine zipper (bHLH-Zip) protein family of
transcription factors (hereafter called MITF).1,2 The
mi/mi mutant mice show depletion of pigment in both hair and
eyes, microphthalmia, osteopetrosis, and deficient natural killer
activity.3,4 In addition, the development of mast cells is
defective in mi/mi mice. The skin mast cells of
mi/mi mice decrease in number and reduce the amount of mouse
mast cell protease-4 (MMCP-4), MMCP-6, and c-kit messenger
RNAs (mRNAs).5-11 Cultured mast cells (CMCs) derived from
the spleen of mi/mi mice are deficient in the expression of
various genes, such as MMCP-4,11 MMCP-5,12
MMCP-6,13 c-kit,14 p75 nerve
growth factor receptor,15 granzyme B (Gr B),16 tryptophan hydroxylase (TPH),16
integrin- MITF encoded by the mi mutant allele (mi-MITF)
deletes 1 of 4 consecutive arginines in the basic
domain.1,19,20 The mi-MITF is defective in the
DNA binding ability and the nuclear localization potential.21,22 The mi-MITF does not appear to
transactivate target genes due to these
abnormalities.11-18,22 The tg is another mutant
allele of the mi locus.1,23 The
tg/tg mice possess the insertional mutation at the promoter
region of mi gene and do not express any
MITFs.1,24 The tg/tg and mi/mi mice
share several phenotypic features, but the phenotypic abnormality of tg/tg mice is apparently mild when compared to that of
mi/mi mice. The transcription of c-kit, Gr
B, and TPH genes as significantly reduced in
mi/mi CMCs, but the reduction was moderate in
tg/tg CMCs.25 The presence of
mi-MITF caused more severe abnormalities than the absence of
normal (+) MITF. In addition to the loss of transactivation ability,
the mi-MITF possesses an inhibitory effect on the
transcription of some particular genes in mast cells.25
Heparin is a highly sulfated proteoglycan that is abundantly contained
by mast cells in the skin of mice.26-28 Because berberine sulfate is a dye that binds heparin, most of the skin mast cells of +/+
mice are stained with berberine sulfate.29-31 In the skin of mi/mi mice, the content of heparin decreases, and few
mast cells are stained with berberine sulfate.9 In
contrast, most of the skin mast cells of tg/tg mice are
stained with berberine sulfate as in the case of +/+ skin mast
cells,32 indicating that the content of heparin decreases
in mi/mi skin mast cells but not in tg/tg skin
mast cells. There is a possibility that mi-MITF may possess
the inhibitory effect on the synthesis of heparin as in the case of
serotonin.25 In fact, serotonin content of
mi/mi CMCs decreases due to the poor transcription of
TPH gene.16,25 N-deacetylase/N-sulfotransferase
2 (NDST-2) is essential for the synthesis of heparin, and the
disruption of the NDST-2 gene causes the depletion of
heparin in mast cells.33,34 In the present study, we
compared the amount of NDST-2 mRNA among mast cells of +/+,
mi/mi, and tg/tg genotypes and found the
inhibitory effect of mi-MITF on the expression of NDST-2
transcript in mast cells.
Mice
In situ hybridization
Cells Pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) was prepared according to the method described by Nakahata and coworkers.38 Mice of mi/mi, tg/tg, and control +/+ genotype were used at 2 to 3 weeks of age to obtain CMCs. Mice were killed by decapitation after ether anesthesia and spleens were removed. Spleen cells were cultured in -minimal essential medium (-MEM; ICN Biomedicals, Costa Mesa, CA)
supplemented with 10% PWM-SCM and 10% fetal calf serum (FCS; Nippon
Bio-supp Center, Tokyo, Japan). Half of the medium was replaced every 5 days. Cells derived from the spleen of each mutant genotype reached to
1 × 107 in number within 4 weeks. More than 95% of
cells contained alcian blue-positive granules and were considered to
be CMCs 4 weeks after initiation of the culture. The MST cells, a
heparin-producing cell line derived from the Furth murine mastocytoma,
were maintained in -MEM supplemented with 10% FCS.39
The NIH/3T3 cells and COS-7 cells were maintained in Dulbecco modified
Eagle medium (DMEM, Flow Laboratories, Irvine, United Kingdom)
supplemented with 10% FCS.
Northern blot analysis Each RNA sample was prepared from 1.0 × 107 of CMCs or MST cells by the lithium chloride-urea method.36 Northern blot analysis was performed using NDST-237 and GAPDH cDNAs40 labeled with-[32P]-dCTP
(DuPont/NEN Research Products, Boston, MA; 10 mCi/mL) by random
oligonucleotide priming. The NDST-2 cDNA used was same as that used for
in situ hybridization. After hybridization at 42°C, blots were washed
to a final stringency of 0.2 × standard sodium citrate (SSC; 1 ×
SSC is 150 mM NaCl and 15 mM trisodium citrate, pH 7.4), and subjected
to autoradiography.
Determination of 5'-end of cDNA The 5'-rapid amplification of cDNA end (5'-RACE) was performed with the SMART RACE cDNA Amplification Kit (Clontech Laboratories, Palo Alto, CA) according to the manufacturer's instructions. The primer for the first PCR was 5'-AGATTCCCATCCTGTGTCTGCCAATGAG (corresponding to the region between nt 66 and nt 38)37 and the primer for the nested PCR was 5'-CTGGTGGGCTCTCGATAACAAGTGGATG (corresponding to the region between nt 30 and nt 3).37 The total RNA obtained from MST cells was used as the template.Promoter region of the NDST-2 gene The DNA fragment containing the promoter region of the NDST-2 gene was isolated with the Mouse Genome Walker Kit (Clontech Laboratories) according to the manufacturer's instructions. The primers used were same as those used in 5'-RACE. The isolated fragment was cloned into pBS and sequenced. The isolated fragment contained the most upstream point of the 5'-RACE product, and the remaining upstream sequence was considered to be the promoter region.Construction of reporter plasmids The luciferase gene subcloned into pSP72 (pSPLuc) was generously provided by Dr K. Nakajima (Osaka City University Medical School, Osaka, Japan).41 To construct reporter plasmids, a DNA fragment containing a promoter and 5'-untranslated region (5'-UTR) of the NDST-2 gene ( 1300 to +668, +1 shows the
transcription initiation site determined by 5'-RACE) was cloned into
the upstream of luciferase gene in pSPLuc. The deletion of
the promoter or 5'-UTR of the NDST-2 gene was produced by
PCR. The mutation was also introduced by PCR. All of the PCR products
were verified by sequencing.
Construction of expression plasmids The pBS containing the whole coding region of +-MITF or mi-MITF was constructed in our laboratory (hereafter called pBS-+-MITF and pBS-mi-MITF, respectively). The SmaI-HincII fragment of pBS-+-MITF, or pBS-mi-MITF was introduced into the blunted Xba I site of pEF-BOS expression vector kindly provided by Dr S. Nagata (Osaka University, Osaka, Japan).42 To generate the Myc-tagged MITF construct, we subcloned the SmaI-HincII fragment of pBS-+-MITF or pBS-mi-MITF into the StuI site of the CS2+MT expression vector that provides 6 copies of the Myc epitope tag at the N-terminal end of the protein (a gift from Dr I. Matsumura, Osaka University, Osaka, Japan).43 The resultant chimeric gene was subcloned into pEF-BOS expression vector. The dominant negative form of GABP cloned in the pET3d expression vector (Novagen, Madison, WI) was digested with the BamHI and BglII. The BamHI-BglII fragment of the pET3d expression plasmid was introduced into the BglII site of pCAGGS expression vector.Transfection and luciferase assay The transfection to MST cells was performed by electroporation. The transfection to NIH/3T3 cells or COS-7 cells was performed with the TransFast Transfection Reagent (Promega, Madison, WI) according to the manufacturer's instructions. In luciferase assays, 10 µg of a reporter and 3 µg of an expression vector containing -galactosidase gene were cotransfected. The expression
vector containing -galactosidase gene was used as an
internal control. In some experiments, 10 µg of an expression plasmid
containing effector cDNA was cotransfected with 10 µg of reporter
plasmids. The cells were harvested 48 hours after the transfection and
lysed with 0.1 M potassium phosphate buffer (pH 7.4) containing 1%
Triton X-100. Soluble extracts were then assayed for luciferase
activity with a luminometer LB96P (Berthold, Wildbad, Germany) and for -galactosidase activity. The luciferase activity was normalized by
the -galactosidase activity and total protein concentration according to the method described by Yasumoto and
coworkers.44 The normalized value was expressed as the
relative luciferase activity.
Electrophoretic gel mobility shift assay Nuclear extract of MST cells was obtained as described before.22 The sequence of oligonucleotide used as a probe was 5'-GGAGAAGCGGAAGGGGAAGGGA (the GGAA motifs are underlined). The oligonucleotide was labeled with-[32P]-dCTP by filling 5'-overhangs and used as probes
of EGMSA. DNA-binding assays were performed in a 20 µL reaction
mixture containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 75 mM KCl, 1 mM
dithiothreitol (DTT), 4% Ficoll type 400, 50 ng poly (dI-dC), and 25 ng labeled DNA probe. After the incubation at 4°C for 15 minutes, the
reaction mixture was subjected to electrophoresis at 14 V/cm on a 5%
polyacrylamide gel in 0.25 × TBE buffer (1 × TBE is 90 mM Tris-HCl,
64.6 mM boric acid, and 2.5 mM EDTA, pH 8.3). In the competition
experiment, 10-fold molar excess amount of unlabeled oligonucleotide
was added. In the supershift experiment, the antibody (Ab) against
GABP, GABP, or Ets-1 (Santa Cruz Biotechnology, Santa Cruz, CA)
was added. The polyacrylamide gels were dried on Whatman 3MM
chromatography paper and subjected to autoradiography.
In vitro binding assay The 35S-labeled +-MITF or mi-MITF protein was synthesized using the reticulocyte lysate system (TNT system, Promega). The +-MITF or mi-MITF cDNA in pBluescript was transcribed with T7 RNA polymerase and translated in the presence of 35S-methionine. For the binding assays, the 35S-labeled +-MITF or mi-MITF protein was incubated for 1 hour at room temperature with GST-GABP, GST-GABP,
or GST alone immobilized on glutathione-agarose beads. The beads were
washed 4 times. Proteins retained on the beads were subsequently
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and autoradiography.
Immunoprecipitation The Myc-tagged +-MITF or Myc-tagged mi-MITF was coexpressed with GABP or GABP in COS-7 cells. The whole cell
extract was obtained by the method as described before.22
The whole cell extract was incubated with LIP buffer (10 mM HEPES, 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride) and protein G Sepharose (Amersham-Pharmacia Boitec, Bucks,
United Kingdom) for 1 hour with gentle rocking and centrifuged at 3000 rpm for 3 minutes. The supernatants were transferred into new tubes and
incubated with protein G Sepharose and anti-GABP or anti-GABP Ab
for 1 hour in LIP buffer. Immunecomplexes were washed 4 times with LIP
buffer, resuspended in loading buffer, boiled, and analyzed by
immunoblot with anti-Myc monoclonal Ab (9E10, Pharmingen, San
Diego, CA).
Immunocytochemistry The GABP cDNA or GABP cDNA was transfected alone, or both
GABP and GABP cDNAs were cotransfected to NIH/3T3 cells. The cells were fixed with 100% methanol and permialized by treatment with
0.2% Triton X-100 in PBS. The cells transfected with GABP cDNA were
incubated with mouse anti-GABP Ab, and the cells transfected with
GABP cDNA were incubated with mouse anti-GABP Ab, respectively. The cells transfected with both GABP and GABP cDNAs were
incubated with mouse anti-GABP Ab, or with mouse anti-GABP Ab,
independently. Immunoreacted cells were detected with antimouse IgG Ab
conjugated with fluorescein isothiocyanate (FITC) (MBL, Nagoya, Japan).
In a separate experiment, GABP cDNA was cotransfected to NIH/3T3 cells with +-MITF cDNA. Because the anti-GABP Ab was the mouse Ab47 and the anti-MITF Ab was the rabbit Ab,22
we detected the cells expressing both GABP and +-MITF using the
double-staining method. The transfected cells were incubated with the
mixture of anti-GABP Ab and anti-MITF Ab, and then with the mixture
of the above-mentioned antimouse IgG Ab conjugated with FITC and the
antirabbit IgG Ab conjugated with rhodamine (MBL). The cells expressing
GABP were detected with the green filter and the cells expressing
+-MITF were detected with the red filter of the fluorescence microscope
(Olympus BX-50, Tokyo, Japan). We observed the cells possessing both
the green and red signals. The GABP cDNA was cotransfected with
mi-MITF cDNA. The GABP cDNA was also cotransfected with
either +-MITF cDNA or mi-MITF cDNA. The cells expressing both GABP and mi-MITF, the cells expressing both GABP
and +-MITF, or the cells expressing both GABP and mi-MITF
were detected with the double-staining method, as described above.
The expression of the NDST-2 gene was analyzed in skin
mast cells of +/+, mi/mi, and tg/tg mice by in
situ hybridization. Signals for NDST-2 mRNA were detected in skin mast
cells of +/+ and tg/tg mice but not in skin mast cells of
mi/mi mice (Figure 1). We
compared the amount of NDST-2 mRNA among CMCs derived from +/+,
mi/mi, or tg/tg mice using Northern blot. The
amount of NDST-2 mRNA reduced significantly in mi/mi CMCs
when compared to that of +/+ or tg/tg CMCs (Figure
2).
We attempted to isolate the promoter region and 5'-UTR of the
NDST-2 gene. The sequence of 512 base pair (bp) upstream of the translation initiation site has been reported by Orellana and
colleagues, but further upstream regions have not been
cloned.37 First, we carried out 5'-RACE to determine the
transcription initiation site of the NDST-2 gene. MST cells
containing abundant NDST-2 transcript were used as the source of mRNA.
The 5'-RACE product of the NDST-2 cDNA started from an adenine, which
was located 1013 bp upstream of the 5'-end reported by Orellana and
coworkers (Figure 3A).37 The
5'-UTR of the NDST-2 gene was composed of 1525 bp (512 bp
for the previously reported part + 1013 bp for the isolated part).
Then, we isolated the DNA fragment containing the promoter region. The
isolated fragment contained the above-mentioned 5'-RACE product (1013 bp) and the remaining upstream sequence (1300 bp) (Figure 3B, the
transcription initiation site was indicated as +1).
To determine the motif(s) that mediate the transactivation, we
constructed the reporter plasmid containing the promoter region and
5'-UTR of the NDST-2 gene (
We examined the region between +649 and +668 in detail. Between +649
and +668, no CANNTG motif that is recognized and bound by MITF was
present. On the other hand, the region contained 2 GGAA motifs, which
are the consensus sequences recognized by GA binding protein (GABP)
(Figure 4B, named 5'-GGAA and 3'-GGAA, respectively). GABP is a member
of ets-family transcription factors, and is composed of 2 subunits,
GABP The EGMSA was done using the nuclear extract of MST cells. The
oligonucleotide containing the 2 GGAA motifs was used as a probe.
Multiple bands were detected by mixing the probe and nuclear extract,
but the addition of excess amount of nonlabeled oligonucleotide with
the same sequence as the probe reduced the intensity of 2 bands (Figure
5). The intensity of these 2 bands was
not reduced by the addition of the nonlabeled oligonucleotide mutated
at the 5'-GGAA motif, but were reduced by the addition of the
nonlabeled oligonucleotide mutated at the 3'-GGAA motif (Figure 5).
This indicated that the protein in the nuclear extract of MST cells specifically bound the 5'-GGAA motif but not the 3'-GGAA motif. To
characterize the protein that specifically bound the 5'-GGAA motif, we
performed super-shift assay with the Ab against the GABP
To examine whether the endogenous GABP increased the amount of NDST-2
transcript through the 5'-GGAA motif, we used a dominant negative form
of GABP. The dominant negative form of GABP is a mutant of
We examined the effect of MITF on the expression of NDST-2 transcript
by GABP. We cotransfected the reporter plasmid to MST cells with the
effector plasmid containing +-MITF or mi-MITF cDNA. The
coexpression of +-MITF did not affect the luciferase activity, but the
coexpression of mi-MITF reduced the luciferase activity in a
dose-dependent manner (Figure 8).
The physical interaction between MITF and GABP was examined by an in
vitro binding experiment. The 35S-labeled +-MITF or
35S-labeled mi-MITF was subjected to
coprecipitation with GST, GST-GABP
We carried out coimmunoprecipitation studies to confirm the interaction
between MITF and GABP. The Myc-tagged +-MITF or Myc-tagged mi-MITF was coexpressed with GABP in COS-7 cells, and their
whole cell lysate was analyzed. The immnoprecipitated product with
anti-GABP
The effect of MITF on nuclear translocation of GABP was examined by
immunocytochemistry. First, we examined the subcellular localization of
GABP
NDST-2 catalyzes the initial sulfation step of heparin
synthesis.37,48 Recently, 2 groups reported the phenotype
of the mice whose NDST-2 genes were disrupted
(NDST-2 In mast cells of NDST-2 To examine the inhibitory effect of mi-MITF, we cloned the promoter region and 5'-UTR of the NDST-2 gene and found that the 5'-UTR between +649 and +668 was important for the transactivation of the NDST-2 gene. Unexpectedly, no CANNTG motifs, which were recognized and bound by +-MITF, were present between +649 and +668. Instead, a GGAA motif, which was recognized and bound by GABP, was observed. The expression of dominant negative GABP reduced the luciferase activity of the reporter plasmid containing the GGAA motif, indicating that GABP increased the amount of NDST-2 transcript through the GGAA motif. The mi-MITF reduced the luciferase activity of the reporter plasmid containing the GGAA motif in a dose-dependent manner. This suggested that the mi-MITF possessed the inhibitory effect on the ability of GABP to express NDST-2 transcript. The GGAA motif was located in the 5'-UTR of the NDST-2 gene. The protein that binds to the motif in 5'-UTR regulates the expression of genes in the following 2 ways.49-51 One is that the protein binds to the transcribed RNA and stabilizes it, in which the protein regulates the expression of genes in a post-transcription level.49,50 Another is that the protein binds to the DNA and regulates the expression in a transcription level, as in the case of usual transcription factors.51-55 Further studies including nuclear run-on analysis will clarify whether the GABP regulated the expression of NDST-2 transcript as an RNA-binding factor or a DNA-binding factor. GABP is a heterodimer and is composed of GABP When coexpressed with mi-MITF, either GABP The +-MITF did not affect the luciferase activity of the reporter plasmid under the control of the 5'-UTR of the NDST-2 gene containing the GGAA motif. The +-MITF might not be necessary for the expression of NDST-2 transcript, because the NDST-2 gene was normally expressed in tg/tg mast cells that lacked any MITFs. Besides the NDST-2 transcript, the expression of c-kit, Gr B, and TPH transcripts were negatively affected by the presence of mi-MITF.25 The level of expression of c-kit, Gr B, and TPH transcripts did decrease in tg/tg CMCs25 but that of NDST-2 transcript did not decrease in tg/tg CMCs. In other words, the +-MITF may enhance the expression of c-kit, Gr B, and TPH transcripts but not the expression of NDST-2 transcript. NDST-2 is the first gene whose expression was affected negatively by mi-MITF but whose expression was not affected positively by +-MITF. In vitro binding assay and immunoprecipitation assay showed that GABP
and MITF physically interacted with each other. The physical
interactions between the ets family transcription factors and the
bHLH-Zip family transcription factors have been
reported.57 The present result may be another example of
such interactions. GABP Heparin plays an important role for the storage of mediators in mast
cell granules, because amounts of various mediators, such as MMCP-4,
MMCP-5, and MMCP-6, decrease in NDST-2 Heparin is synthesized with multiple steps. Besides NDST-2, various enzymes, such as copolymerase EXT-1 and EXT-2, are involved in heparin synthesis in mast cells.60 Further studies will reveal the importance of such enzymes. Taken together, the mi-MITF possessed the inhibitory effect on the expression of NDST-2 transcript by disturbing the nuclear localization of GABP. The decreased heparin content in mi/mi skin mast cells may be attributable to the deficient expression of the NDST-2 transcript.
The authors thank Dr H. Arnheiter of National Institutes of Health for VGA9-tg/tg mice, Dr K. Nakajima of Osaka City University for pSP-Luc, Dr S. Nagata of Osaka University for pEF-BOS, and Dr I. Matsumura of Osaka University for CS2+MT.
Submitted September 8, 2000; accepted January 17, 2001.
Supported by grants from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare, the Organization of Pharmaceutical Safety and Research, the Welfide Medicinal Research Foundation, and the Uehara Memorial Foundation. GenBank accession number AF293452.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Eiichi Morii, Dept of Pathology, Osaka University Medical School, Yamada-oka 2-2, Suita 565-0871, Japan; e-mail: morii{at}patho.med.osaka-u.ac.jp.
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Abstract
Introduction
/