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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3210-3221
Systematic Method to Obtain Novel Genes That Are Regulated by
mi Transcription Factor: Impaired Expression of Granzyme B and
Tryptophan Hydroxylase in mi/mi Cultured Mast Cells
By
Akihiko Ito,
Eiichi Morii,
Kazutaka Maeyama,
Tomoko Jippo,
Dae-Ki Kim,
Young-Mi Lee,
Hideki Ogihara,
Koji Hashimoto,
Yukihiko Kitamura, and
Hiroshi Nojima
From the Department of Pathology, Medical School, Osaka University,
Suita, Osaka; the Department of Pharmacology, Ehime University Medical
School, Ehime; and the Department of Molecular Genetics, Research
Institute for Microbial Diseases, Osaka University, Suita, Osaka,
Japan.
 |
ABSTRACT |
The mi locus encodes a member of the
basic-helix-loop-helix-leucine zipper protein family of transcription
factors (hereafter called MITF). We have reported that the expression
of several genes was impaired in cultured mast cells (CMCs) of
mi/mi genotype, and demonstrated the involvement of MITF in the
transcription of these genes. To obtain new genes whose transcription
may be regulated by MITF, we prepared a subtracted cDNA library using +/+ and mi/mi CMCs. We found two clones carrying the
granzyme (Gr) B and tryptophan hydroxylase (TPH) cDNAs in the
subtracted library. The expression of the Gr B and TPH genes decreased
in mi/mi CMCs, and recovered to nearly normal level by the
overexpression of normal (+) MITF but not of mutant (mi)
MITF. The +-MITF bound three and one CANNTG motifs in the Gr B and
TPH promoters, respectively, and transactivated these two genes,
indicating the involvement of +-MITF in their expression. Because TPH
is the rate-limiting enzyme for serotonin synthesis, we examined the
serotonin content of +/+ and mi/mi CMCs. The serotonin
content was significantly smaller in mi/mi CMCs than in +/+
CMCs. The introduction of +-MITF but not of mi-MITF
normalized the serotonin content in mi/mi CMCs.
| |
INTRODUCTION |
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
mi-transcription factor, MITF).1,2 The MITF encoded
by the mutant mi allele deletes 1 of 4 consecutive arginines in
the basic domain (hereafter mi-MITF).1,3,4 The
mi-MITF is defective in the DNA binding activity and the
nuclear localization potential,5,6 and it does not
transactivate target genes.6-10 The mi/mi mice show
microphthalmia, depletion of pigment in both hair and eyes,
osteopetrosis, and decrease in number of mast cells.11-15
In addition to the decrease in number, the phenotype of mast cells is
abnormal in mi/mi mice.16-19 Mast cells in the skin of normal (+/+) mice were stained with berberine sulfate that bound heparin proteoglycan and expressed the mouse mast
cell protease 6 (MMCP-6) and c-kit genes, but mast cells in the
skin of mi/mi mice were berberine sulfate-negative and did not
express MMCP-6 and c-kit genes.17,20-23
We have shown the involvement of +-MITF in the transcription of
c-kit, MMCP-6, MMCP-5, and p75 nerve growth factor (NGF)
receptor gene in cultured mast cells (CMCs).7-10 To obtain
new genes whose transcription may also be regulated by +-MITF, we
elaborated cDNA libraries from +/+ CMCs and mi/mi CMCs and
subtracted the latter from the former. The subtraction process
consisted of the removal of clones carrying inserts complementary to
mi/mi CMC mRNAs from the +/+ CMC cDNA library by hybridization.
After repeating the subtraction process several times, we could yield a
subtracted cDNA library made up of a high frequency of clones that were
expressed in a +/+ CMC-specific manner. Screening 400 clones randomly
selected from this library, we isolated several new genes as potential transcriptional targets of MITF, two of which turned out to be the
granzyme (Gr) B and tryptophan hydroxylase (TPH) genes.
Gr B is a serine protease essential for cell-mediated immunity. It is
most specifically detected in cytotoxic T lymphocytes (CTLs) and
natural killer (NK) cells, but it has also been shown to be expressed
by CMCs and ABFTL1 mastocytoma cells.24 The murine Gr B
gene resides on chromosome 14 and links with a gene complex encoding
the other granzymes (Grs) (Grs C, D, E, F, and G), and the mast
cell-specific serine proteases (MMCP-1, -2, -4, and
-5).25-28 On the other hand, TPH is the rate-limiting
enzyme for the synthesis of serotonin,29-33 a chemical
mediator of immediate hypersensitivity reaction that is preformed and
stored in the basophilic granules of mast cells.34 We
examined whether +-MITF directly transactivated the Gr B and TPH genes.
The +-MITF specifically bound three CANNTG motifs in the Gr B promoter
and one CAGGTG motif in the TPH promoter, and the binding
transactivated the luciferase genes under the control of the Gr B or
TPH promoter.
| |
MATERIALS AND METHODS |
Mice and cells.
The original stock of C57BL/6-mi/+ (mi/+) mice was
purchased from the Jackson Laboratory (Bar Harbor, ME) and was
maintained in our laboratory by consecutive backcrosses with our own
inbred C57BL/6 colony (more than 12 generations at the time of the
experiments described here). Female mi/+ mice were crossed with
male mi/+ mice, and the resulting mi/mi mice were
selected on the basis of their white coat color.11,12
Pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM)
was prepared according to the method described by Nakahata et
al.35 Mice of mi/mi genotype and their normal (+/+)
littermates with an age of 2 to 3 weeks were used to obtain CMCs. Mice
were killed by decapitation after ether anesthesia and the spleens were
removed. Spleen cells derived from mi/mi or +/+ mice were
cultured in  -minimal essential medium (-MEM; ICN Biomedicals,
Costa Mesa, CA) supplemented with 10% PWM-SCM and 10% fetal calf
serum (FCS; Nippon Biosupp Center, Tokyo, Japan). Half of the medium
was replaced every 7 days. Four weeks after initiation of the culture
more than 95% of cells were CMCs.22 CMCs overexpressing
+-MITF or mi-MITF were obtained as described previously and
maintained in -MEM supplemented with 10% PWM-SCM and 10%
FCS.7,8 The NIH/3T3 fibroblast cell line was generously
provided by Dr S.A. Aaronson (National Cancer Institute, Bethesda, MD)
and maintained in Dulbecco's modification of Eagle's medium (ICN
Biomedicals) supplemented with 10% FCS.
Construction of the pAP3neo vector.
The expression vector, pAP3neo, which harbors an SV40 promoter
and allows efficient expression of cDNA inserts in mammalian cells, was
prepared as follows. First, the 0.4-kb DNA fragment (AflIII/Kpn I cut) of the pBluescript II vector
(Stratagene, La Jolla, CA) was replaced by a 0.7-kb DNA fragment
(AflIII/Kpn I cut) of the pcDV1 vector.36
Then, the 0.6-kb DNA fragment (Pst I/HindIII cut) of
the pL1 vector36 carrying the SV40 promoter was inserted
into the Pst I/BssHII site of this plasmid DNA using appropriate adapter/linkers. Subsequently, a pair of chemically synthesized oligonucleotides designed to yield a double-stranded multicloning site was inserted into this plasmid DNA via Pst
I/Kpn I sites. The multicloning site contains the recognition
sequences for the following enzymes: 5 -Sse I(Pst
I)-Sac I-Cpo I-Apa I-Sau I-Mlu
I-T7 RNA polymerase-EcoRI-AatII-Xba
I-BglII-AflIII-Asc I-BstXI-Bal I-SnaBI-BstEII-DraIII-Nhe I-Sce
I-Not I-T3 RNA polymerase-Swa I-Spl
I-Nru I-Pac I-SacII-Kpn I-3. Finally,
a neomycin unit was inserted via the Sfi I site into the middle
of the SV40 promoter, and we named this vector pAP3neo.
Preparation of cDNA libraries carrying directional inserts.
Before the purification of poly (A)+ RNA, total RNA
was extracted by the guanidine thiocyanate/CsTFA method from +/+ and
mi/mi CMCs. The cDNA library was prepared as described by
Gubler and Hoffmann37 with some modifications.
Briefly, cDNA was synthesized from 2 µg of +/+ or mi/mi CMC
poly (A)+ RNA with reverse transcriptase in a reaction
mixture including 5Me-dCTP and 1.6 µg of oligo (dT)
primer carrying an Not I restriction site. RNase H was then
added to the reaction mixture and was followed by DNA polymerase I as
described by Okayama and Berg.36 Then, the cDNA was
blunt-ended with T4 DNA polymerase, and an unphosphorylated BglII-Sma I adapter was ligated to the 5 end. After
digestion with Not I, small DNA fragments of less than 300 bp
were removed by a CHROMA spin 400 column (Clontech, Palo Alto,
CA). The cDNA fragments were directionally inserted
between the Not I (dephosphorylated) and BglII sites of
the pAP3neo vector.
Dephosphorylation of the Not I site of the vector and the
unphosphorylated BglII end of the inserts minimized the
ligation of multiple cDNA inserts in the library. The ligation mixture was pretreated as described before38 and electroporated
into Escherichia coli MC1061A cells. Before propagation of the
cDNA library for preparation of a stock solution, we removed an aliquot and counted the cell number to know how many independent clones the
cDNA library contained (the complexity of a library).
Preparation of the subtracted cDNA library.
The detailed process for the preparation of the subtracted cDNA library
was described previously.39 To prepare single-stranded plasmid DNA, the plasmid DNA prepared from the +/+ CMC cDNA library was
introduced into E coli DH5FIQ cells by electroporation. After 1 hour of culture in rich medium (2× YT),
transformed cells were infected with R408 helper phages. Then,
single-stranded DNA was purified from the supernatant of the 8-hour
culture. To prepare biotinylated RNA drivers, total RNA was extracted
by the guanidine thiocyanate/CsTFA method from mi/mi CMCs, and
poly (A)+ RNA was purified and labeled by photobiotin
(Vector Lab, Burlingame, CA). One microgram of single-stranded DNA
prepared from the +/+ CMC cDNA library was hybridized with 10 µg of
biotinylated RNA at 42°C in 25 µL hybridization buffer containing
40% formamide, 50 mmol/L HEPES (pH 7.5), 1 mmol/L EDTA, 0.1% sodium
dodecyl sulfate (SDS), 0.2 mol/L NaCl, and 1 µg of oligo-poly(rA).
After hybridization for 42 hours, the mixture was transferred to 400 µL of SB (50 mmol/L HEPES [pH 7.5], 2 mmol/L EDTA, 500 mmol/L
NaCl), and 10 µg streptoavidin was subsequently added. The mixture
was incubated at room temperature for 5 minutes and extracted with
phenol/chloroform/isoamyl alcohol (25:24:1). The organic phase was
back-extracted with 100 µL TE (10 mmol/L Tris-HCl [pH 7.5], 1 mmol/L EDTA). The aqueous phases were pooled. Streptoavidin binding and
phenol treatment were repeated once more. The recovered single-stranded
plasmid DNA was subtracted with biotinylated RNA one more time. After repeating the subtraction process, the recovered single-stranded DNA
was converted to double-stranded plasmid DNA by the BcaBEST DNA
polymerase (TaKaRa, Otsu, Japan) reaction at 65°C for 30 minutes. After phenol extraction and ethanol precipitation, the DNA was dissolved in 20 µL TE buffer and 3-µL aliquots were introduced into
E coli MC1061A cells by electroporation.
Screening of +/+ CMC-specific clones by Southern blot
analysis.
Four hundred cDNA clones were prepared from ampicillin-resistant
colonies randomly selected from the subtracted cDNA library by an
automated plasmid purification machine (PI-100; KURABO, Osaka, Japan).
After digestion with both Sma I and Not I to separate the cDNA insert, the plasmid DNA was electrophoresed in an agarose gel
and transferred to nylon membranes. The cDNA probes were synthesized with reverse transcriptase (RT) from +/+ or mi/mi CMC poly
(A)+ RNA in a reaction mixture containing
[-32P]dCTP. Duplicate membranes were hybridized with
the reverse-transcribed cDNA probes for 15 hours. The filters were
washed several times with a final stringency of 0.1× SSC (1×
SSC = 0.15 mmol/L NaCl, 15 mmol/L sodium citrate, pH 7.2) and 0.1%
SDS at 50°C, and autoradiographed at  80°C with an intensifying
screen. Clones that hybridized with higher efficiency to the +/+ cDNA
probe compared to the mi/mi cDNA probe were selected as
candidates for further investigation.
DNA sequencing.
The plasmid DNA was purified individually from the subtracted cDNA
library by PI-100 and directly subjected to DNA sequencing. Dideoxy-chain termination sequencing reactions were performed with T7
dye-labeled primers and thermal cycle sequencing kits purchased from
LI-COR (Lincoln, NE). The reaction products were analyzed by a Model
4000L Automated DNA Sequencer (LI-COR).
Northern blot analysis.
Five micrograms of total RNA prepared from +/+ or mi/mi CMCs
was loaded per lane, fractionated on 1% agarose-formaldehyde gels, and
transferred to nylon membranes by capillary action in 20× SSC. Baked
membranes were prehybridized for 3 hours at 42°C in a buffer
containing 50% formamide, 5× SSC, 5× Denhardt's solution, and
0.1% SDS. The membranes were hybridized with the
[-32P]dCTP-labeled DNA probes at 42°C for 15 hours
in the same buffer. Preparation of the DNA probes was performed
according to the random hexamer labeling method. cDNA inserts of some
clones isolated from the subtracted cDNA library were used as a
template. To prepare MMCP-6, mast cell carboxypeptidase A (MC-CPA),
histidine decarboxylase (HDC), and  -actin probes, cDNA fragments of
approximately 570 bp, 550 bp, 800 bp, and 700 bp were used,
respectively. The MITF probe was prepared from the Xho
I-Pst I cDNA fragment of about 750 bp.1 After
hybridization, the membranes were washed to a final stringency of
0.1× SSC and 0.1% SDS at 50°C, and autoradiographed at 80°C.
To characterize the subtracted cDNA library, 1 µg of the Not
I-digested plasmid DNA prepared from the +/+ CMC, mi/mi CMC, or subtracted cDNA library was used as a template for the T7 RNA polymerase reaction (Stratagene). Two micrograms of synthesized RNA was
loaded per lane, fractionated on 1% agarose-formaldehyde gels, and
transferred to nylon membranes by capillary action in 20× SSC. The
hybridization procedures were the same as described above.
In situ hybridization.
Pellets of +/+ and mi/mi CMCs with or without introduction of
+- or mi-MITF cDNA were fixed with 4% paraformaldehyde in
phosphate buffer (0.1 mol/L, pH 7.2) overnight, embedded in paraffin,
and cut serially to a thickness of 3 µm. Three serial sections were used as follows. The first section was stained with hematoxylin and
eosin (H&E), the second section was used for in situ
hybridization, and the third section was stained with alcian blue to
detect mast cells. Details of the in situ hybridization technique have
been described previously.40 For cRNA probe preparation,
the clones containing Gr B, MC-CPA, TPH, and HDC cDNA inserts were
transcribed with the DIG RNA Labeling Kit (Boehringer Mannheim
Biochemica, Mannheim, Germany). The number of cells
positive for the probes and that of alcian blue-positive cells were
counted in serial sections, and the proportion of the former cells was
calculated.
RT-polymerase chain reaction (PCR) analysis.
Two sets of oligonucleotide primers were synthesized: the Gr B sense
primer 5-GATTACCCATCGTCCCTAGAGCT-3 and antisense primer 5-CATGCCCAGCTCCAATGCAAAC-3 (nucleotides [nt] 884-906 and nt 1097-1118, respectively)25; and the Gr C-G sense primer
5-TCCTGACCCTACTTCTGCCTCT-3 (nt 94-115, nt 106-127, nt 1-22, nt
80-101, and nt 61-82 of Gr C, D, E, F, and G,
respectively)41-43 and antisense primer
5-CTTTTGCCACAGGGATGATCTGC-3 (nt 335-357, nt 359-381, nt 242-264, nt
321-343, and nt 302-324 of Gr C, D, E, F, and G,
respectively).41-43 The Gr C-G sense primer had one
nucleotide mismatch for the Gr F sequence (nt 90; T to A).41 As a positive control, splenocytes of +/+ mice were
activated by culturing in -MEM containing PWM (1:300 dilution) for 2 days before RNA extraction. Various amounts of total RNA (0.5, 0.05, and 0.005 µg) extracted from +/+ and mi/mi CMCs, and +/+
splenocytes were reverse-transcribed in 20 µL of a reaction mixture
containing 200 U of Superscript II (GIBCO-BRL, Grand Island,
NY) and 0.2 µg of oligo (dT) primer. One microliter of
each reaction mixture was amplified in 10 µL of PCR mixture
containing 0.5 U of Taq DNA polymerase (TaKaRa) and 12.5 pmol of one
set of specific primers. The PCR profile consisted of 24 or 30 cycles
of denaturation at 94°C for 30 seconds, reannealing at 63°C for 30 seconds, and polymerization at 72°C for 1 minute, followed by
extension at 72°C for 5 minutes. Half of the respective PCR products
was fractionated on 2% agarose gels and stained with ethidium bromide.
Concentrations of serotonin and histamine.
The concentrations of serotonin and histamine were measured using
high-performance liquid chromatography (HPLC) with electrochemical detection44 and HPLC-fluorometry,45
respectively. Briefly, CMCs were collected, washed with
phosphate-buffered saline (PBS), counted, and sonicated for 20 seconds
in a sonicator (Tomy, Tokyo, Japan) in 1 mL of ice-cold 3% perchloric
acid containing of 5 mmol/L EDTA and 1 mmol/L sodium metabissulfite.
The homogenate was centrifuged at 10,000g for 15 minutes at
4°C and the supernatant was applied directly to the HPLC column. The
concentration of serotonin and histamine per 1.0 × 106
cells was calculated.
Serotonin uptake.
The uptake of serotonin into +/+ or mi/mi CMCs was determined
according to the method described by Miller and Hoffman46 with minor modification. CMCs (1.0 × 105) of +/+ or
mi/mi genotype were preincubated with 0.1 mmol/L iproniazid (Sigma, St Louis, MO) for 30 minutes in the uptake buffer (20 mmol/L
HEPES, 140 mmol/L NaCl, 5 mmol/L KCl, 1.8 mmol/L CaCl2, 0.8 mmol/L MgSO4, 5 mmol/L glucose, and 10 µmol/L pargyline). The buffer was aspirated and fresh uptake buffer containing 200 nmol/L
of [3H] serotonin (30 mCi/µmol; Amersham, Arlington
Heights, IL) was added to the cells. Incubation was continued for 15 minutes at 37°C; uptake was terminated by aspiration of the medium.
After washing three times, the cells were dissolved in 1% SDS/0.2
mol/L NaOH and the radioactivity was measured with a liquid
scintillation counter (Amersham).
Electrophoretic gel mobility shift assay (EGMSA).
The production and purification of GST-MITF fusion protein and anti-GST
antibody were described previously.5 The fusion protein was
used to measure the in vitro binding of MITF to the consensus sequence
in EGMSA. The double-stranded oligonucleotides were labeled by
filling 5-overhangs with Klenow enzyme in the presence of
deoxynucleotides containing [-32P]dCTP. Probes were
separated from unincorporated nucleotides by gel electrophoresis. The
binding reactions were performed at 4°C for 15 minutes with 50 ng of
labeled DNA probe and 3.5 µg of protein in a 20-µL reaction mixture
containing 10 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA, 75 mmol/L KCl, 1 mmol/L dithiothreitol (DTT), 4% Ficoll type 400 (Sigma),
and 50 ng of poly (dI-dC). For competition experiments, unlabeled
double-stranded oligonucleotides at a 100-fold molar excess were added
to the binding reaction mixture before the addition of the protein.
DNA-protein complexes were separated by electrophoresis on a 5%
polyacrylamide gel in 0.25× TBE buffer (1× TBE = 90 mmol/L
Tris-base, 64.6 mmol/L boric acid, and 2.5 mmol/L EDTA, pH 8.3) at 14 V/cm. The gels were dried on Whatman 3MM chromatography
paper (Whatman, Maidstone, UK) and subjected to autoradiography.
Construction of effector and reporter plasmids, and the transient
cotransfection assay.
pEF-BOS expression vector47 was kindly provided by Dr S. Nagata (Osaka University, Osaka, Japan). The expression plasmids containing +-MITF or mi-MITF cDNA was constructed as described previously.7,8 The luciferase gene subcloned into pSP72
(pSPLuc) was generously provided by Dr K. Nakajima (Osaka University
Medical School, Osaka, Japan). To construct reporter plasmids, the
promoter region of the Gr B (nt 910 to +42)48 and TPH
(nt 1052 to +86)31 genes were obtained with PCR and
subcloned into the upstream region of the luciferase gene in pSPLuc.
The deletion of the TPH promoter was produced by PCR using the
appropriate primers. The mutations were introduced by PCR with mismatch
primers. Deleted or mutated products were verified by sequencing.
NIH/3T3 cells (5 × 105) were plated in a 10-cm dish 1 day before the procedure. Cotransfection of 10 µg of a reporter, 5 µg of an effector, and 5 µg of an expression vector containing the
-galactosidase gene was performed by the calcium phosphate
precipitation method. The expression vector containing the
-galactosidase gene was used as an internal control. The cells were
obtained 48 hours after transfection and lysed with 0.1 mol/L potassium
phosphate buffer (pH 7.4) containing 1% Triton X-100 (Nacali, Kyoto,
Japan). Soluble extracts were then assayed for luciferase
activity with a luminometer LB96P (Berthold GmbH, Wildbad, Germany) and
for -galactosidase activity. The luciferase activity was normalized
using the -galactosidase activity and total protein concentration
was estimated according to the method described by Yasumoto et
al.49 The normalized value was divided by the value
obtained after cotransfection with the reporter and pEF-BOS, and was
expressed as the relative luciferase activity.
| |
RESULTS |
Preparation and characterization of a subtracted cDNA library.
The systematic cloning of the genes expressed in a +/+ CMC-specific
manner, including the transcriptional target genes for MITF, was
conducted according to the following strategy. With the aim of
developing a new mammalian expression vector for future experiments, a
plasmid vector (pAP3neo) was constructed by fusing the
Okayama-Berg vector36 with pBluescript II and inserting a
chemically synthesized multicloning site (see Materials and methods).
The introduction of the f1 origin into the vector enabled the
preparation of the single-stranded cDNA library necessary for the
subtraction process. Using this vector, a cDNA library of +/+ CMCs was
prepared by a modified Gubler-Hoffman
method,37 which we refer to as the
Linker-primer method in Materials and Methods. The complexities of the
cDNA libraries used in this study were 1.2 × 106
colony-forming units (CFU) for +/+ CMCs and 1.6 × 106 CFU
for mi/mi CMCs. These numbers indicate that the cDNA libraries contain almost all of the mRNA expressed in these cells without any
leakage. Clones carrying inserts complementary to mRNAs of mi/mi CMCs were subtracted from the +/+ CMC cDNA library after formation of the biotin-avidin complex. Because mi/mi CMCs
contain a reduced proportion of transcripts from MITF-upregulated
genes, the subtraction process should allow enrichment of +/+
CMC-specific cDNA clones and facilitate the cloning of downstream
target genes of MITF.
To determine if successful subtraction had been achieved, we examined
the subtracted cDNA library by the following experiments. Firstly, the
sense RNAs of the cDNA inserts were synthesized by the T7 RNA
polymerase reaction using the Not I-digested plasmid DNA of
either the +/+ CMC, mi/mi CMC, or subtracted cDNA libraries as
templates. They were then fixed to nylon membranes to perform Northern
blot analysis (we refer to them as library-Northern blots hereafter)
(Fig 1). Both the MC-CPA and -actin
genes have been shown to be expressed equally in +/+ and mi/mi
CMCs, and the MMCP-6 gene to be expressed only in +/+
CMCs.15 When hybridized with the MC-CPA and -actin cDNA,
the hybridized bands were easily detected with equal intensity in both
the RNAs from the +/+ and mi/mi CMC cDNA libraries as well, but
were rarely detectable in the RNA from the subtracted cDNA library. On
the other hand, when MMCP-6 was used as a probe, the hybridized bands
were easily detectable in the RNA from the +/+ CMC cDNA library but no
such band was observed in the RNA from the mi/mi CMC cDNA
library. In the RNA from the subtracted cDNA library, the intensity of
the hybridized band was much stronger than that in the RNA from the +/+
CMC cDNA library. The results of library-Northern blot analysis
obtained with the RNAs from +/+ and mi/mi CMC cDNA libraries
were consistent with those obtained with the total RNAs of +/+ and
mi/mi CMCs, indicating that RNA synthesis from these libraries
had been performed without bias. Thus, we concluded that compared with
the original +/+ cDNA library, the subtracted cDNA library contained
much smaller numbers of cDNA clones from transcripts present at the
same level in +/+ and mi/mi CMCs, such as the MC-CPA and
-actin gene transcripts, and much larger numbers of cDNA clones from
transcripts present in +/+ CMCs but absent from mi/mi CMCs,
such as the MMCP-6 gene transcript.
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| Fig 1.
Characterization of the subtracted cDNA library by
Northern blot analysis. Sense RNAs were synthesized by the T7 RNA
polymerase reaction using the Not I-digested plasmid DNA of
the +/+ CMC, mi/mi CMC, and subtracted cDNA libraries as
templates. Two micrograms of synthesized RNA was loaded per lane,
electrophoresed, transferred, and fixed onto nylon membranes. Probes
were prepared from the cDNAs for MMCP-6, MC-CPA, -actin, or Gr B
using the random hexamer labeling method.
|
|
Secondly, 400 cDNA clones were isolated from the subtracted library,
and the cDNA insert of each clone was analyzed by Southern blotting
using cDNA reverse-transcribed from +/+ or mi/mi CMC poly
(A)+ RNA as probes (Fig 2).After adjustment of the two -actin-specific signals to equal
intensity, it may be expected that the clones which hybridize more
efficiently with the +/+ cDNA probe than with the mi/mi cDNA
probe carry cDNA inserts transcribed specifically in +/+ CMCs.
Effectively, the subtracted cDNA library contained such clones (clones
no. 5, 11, 232, 239, and 382 in Fig 2) at high frequency (approximately
15%). These two experiments showed that we had achieved successful
subtraction and that it was possible to obtain a subtracted cDNA
library of sufficient quality to allow an effective search for +/+
CMC-specific cDNA clones.
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| Fig 2.
Screening of +/+ CMC-specific clones by Southern blot
analysis. After digestion with both Sma I and Not I to
separate the cDNA insert, plasmid DNAs of 400 clones randomly selected
from the subtracted cDNA library were electrophoresed in agarose gel and bound to nylon membranes. Duplicate membranes were hybridized with
32P-labeled cDNAs synthesized from poly(A)+
RNA of +/+ (upper) or mi/mi (lower) CMCs. In the rightmost
lanes, an equal amount of the -actin cDNA fragment was loaded as a
control, and we graphically equalized the intensity of their bands for two filters to normalize the intensity of other sets of the bands. The
clones which hybridized to a greater degree with +/+ CMC-cDNAs than
with mi/mi CMC-cDNAs, such as clones no. 5, 11, 232, 239, and
382, were selected, and subjected to DNA sequencing and a computer-assisted homology search. The identity of these clones is also
shown by the clone number.
|
|
The subtracted cDNA library had a complexity of 1.0 × 106
CFU, as judged by counting the cell number at the final electroporation step (see Materials and Methods). This number indicates that the subtraction process caused almost no leakage of the cDNA species present in the original cDNA library. To know how many independent cDNA
clones the subtracted cDNA library contained, 100 clones were randomly
selected from the library. A mixture of the cDNA inserts prepared from
20 of these 100 clones by Sma I/Not I digestion were
labeled with [-32P]dCTP. After stripping from the
membranes the reverse-transcribed cDNA probes described above, the
membranes were rehybridized with this new cDNA probe, and the number of
clones showing hybridization signals was counted (data not shown). Five
of the 400 clones proved to have cDNA inserts identical to those of the
selected 20 clones. The same procedures were performed using the
mixture of cDNA inserts from the remaining 80 clones. Out of 400 clones, 20 proved to have identical cDNA inserts to those of the 80 clones. These results indicate that 20 and 80 clones correspond to
5/400 (1.25%) and 20/400 (5%) of the total clone number present in
the library, respectively, and that the library contains approximately
20/0.0125 or 80/0.05 clones, ie, approximately 1.6 × 103
independent cDNA clones. Considering that the subtracted cDNA library
contained +/+-specific clones with a frequency of 15%, we estimated
that the number of species of genes transcriptionally upregulated by
MITF is, at most, 240 genes.
Identification of the genes transcribed in a +/+
CMC-specific manner.
By screening the cDNA inserts of the subtracted library by Southern
blot analysis, 40 clones were picked up and subjected to further
analyses. About a 1-kb region from the 5-end of these cloned cDNAs was
sequenced and analyzed with a computer-assisted homology search. Half
(16 clones) of them showed no homology to any published sequences.
Among the rest of the clones, we repeatedly found cDNAs encoding MMCP-5
(two clones) or MMCP-6 (two clones), both of which are known to be
transcribed in a +/+ CMC-specific manner.5,8,9 We also
identified cDNAs encoding Gr B (four clones), tryptophan hydroxylase
(TPH) (clone no. 232), gp49B50 (clone no. 239), and ST2L
(clone no. 382), suggesting that these genes are novel transcriptional
targets of MITF.
To determine if these cDNAs are actually transcribed exclusively in +/+
CMCs, we performed Northern blot analysis using total RNA obtained from
+/+ and mi/mi CMCs. As shown in Fig
3, the Gr B and TPH mRNA expression was
easily detected in +/+ CMCs but rarely detectable in mi/mi
CMCs. The gp49B and ST2L genes were also transcribed preferentially in
+/+ CMCs. From the clones displaying no homology to any published
sequences, clone no. 5 was randomly selected and shown to be expressed
in a +/+ CMC-specific manner. These results indicated that the data
obtained from Southern and Northern blotting were consistent. To
confirm the success of the subtraction process again, the radiolabeled
cDNA probe of clone no. 11 insert (Gr B cDNA) was hybridized with the
library-Northern blots (Fig 1). As in the case of MMCP-6, the
subtracted cDNA library contained much larger numbers of cDNA clones
carrying the Gr B cDNA than the original +/+ CMC cDNA library,
confirming the efficiency of the subtraction process. Hereafter, we
focused on the poor expression of Gr B and TPH mRNA in mi/mi
CMCs.
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| Fig 3.
Expression of the Gr B, gp49B, ST2L, TPH, and HDC genes
in +/+ and mi/mi CMCs. Five micrograms of total RNA
prepared from +/+ or mi/mi CMCs was loaded in each lane and
fixed onto nylon membranes by capillary action. The membranes were
hybridized with specific DNA probes. To prepare the Gr B, gp49B, ST2L,
TPH probes, the cDNA inserts of clones no. 11, 232, 239, and 382 were
used. Expression of MMCP-6 and MC-CPA mRNA were shown as controls.
Arrows indicate the specific signals which are accompanied by their
molecular size. Arrowheads indicate the position of 18S and 28S in each panel. Reprobing with the -actin probe allowed verification that an
equal amount of mRNA was loaded per lane.
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Recovery of Gr B and TPH expression by the introduction of
+-MITF cDNA into mi/mi CMCs.
Previously, we were able to successfully introduce +- or
mi-MITF cDNA into mi/mi CMCs.7,8 Using
these cells, we examined by in situ hybridization whether
+-MITF-overexpression in mi/mi CMCs allowed the recovery of Gr
B and TPH expression. Approximately 90% and 70% of +/+ CMCs expressed
Gr B and TPH mRNA, respectively, whereas only a few mi/mi CMCs
were able to do so (Table 1). The introduction of +-MITF cDNA, but not mi-MITF cDNA, increased
the proportions of Gr B and TPH mRNA-expressing CMCs up to a level equivalent to those of +/+ CMCs (Table 1). To demonstrate the restoration of Gr B and TPH expression in mi/mi CMCs more
quantitatively, total RNAs were extracted from +/+ CMCs, mi/mi
CMCs, and mi/mi CMCs containing +-MITF cDNA, and subjected to
Northern blot analysis. As shown in Fig 4,mi/mi CMCs containing +-MITF cDNA overexpressed the MITF mRNA
and recovered the Gr B and TPH expression to the nearly normal levels.
After longer exposure, no difference in endogenous MITF gene expression
was observed between the original +/+ and the mi/mi CMCs (data
not shown).
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| Fig 4.
Recovery of Gr B and TPH expression after the
introduction of +-MITF cDNA into mi/mi CMCs. Five micrograms
of total RNA from +/+ CMCs, mi/mi CMCs, and mi/mi
CMCs overexpressing +-MITF were fractionated on 1% agarose gels and
subjected to hybridization with the MITF, Gr B, and TPH probes. The
+-MITF cDNA introduced into mi/mi CMCs appeared to be
transcribed abundantly. Reprobing with the -actin and MC-CPA probes
showed that equal loading had been achieved.
|
|
Gr B shows a distinct expression profile among multiple granzymes.
The cDNA sequences of granzymes (Grs) B-G are highly similar to one
another. Because the Gr B probe used in the Northern blot analysis
(Figs 3 and 4) was a full-length Gr B cDNA (clone no. 11), it will
crossreact with transcripts from all of the Gr genes. To discriminate
Gr B transcripts from those of other Grs, we performed RT-PCR analysis
(Fig 5A). We designed two sets of
oligonucleotide primers: one set was Gr B-specific and was expected to
amplify the 235-bp cDNA fragment derived from the Gr B 3 untranslated region (UTR); the other set was common to Grs C-G and was expected to
amplify the 264-bp (Grs C, E, F, and G) and/or 276-bp (Gr D) cDNA fragment derived from the Grs C-G open reading frame. A series of
diluted total RNAs (0.5, 0.05, and 0.005 µg) from +/+ CMCs, mi/mi CMCs, and +/+ PWM-stimulated splenocytes were
reverse-transcribed, and the resulting cDNAs were PCR-amplified with
the Gr B- or Gr C-G-specific primers. Half of the respective PCR
products were analyzed on agarose gels. The specificity of the reaction
was demonstrated by the presence of a single band of the expected size
among the PCR products and by DNA sequencing. The intensity of the
bands corresponding to Gr B or Grs C-G increased proportionally with
the amount of template RNA. This showed that PCR amplification was
semiquantitative throughout the entire PCR reaction. After only 24 PCR
cycles, amplification of the Gr B-specific cDNA was easily detectable
on agarose gels, and Gr B expression in mi/mi CMCs appeared to
be less than one tenth of that seen in +/+ CMCs (Fig 5A). This
observation was compatible with the result of Northern blot analysis
shown in Fig 3. On the other hand, amplification of the bands
corresponding to Grs C-G was hardly detectable in either +/+ or
mi/mi CMCs after 24 cycles using a set of the primers common to
Grs C-G. At an increased PCR cycle number of 30, bands of about 270 bp
in size became visible for both types of cells (Fig 5A). Judging from
the band intensity, Gr C-G expression was slightly higher in
mi/mi CMCs than in +/+ CMCs. Because the Gr B 3UTR sequence,
which was PCR-amplified with Gr B-specific primers, lacks homology to
the cDNA sequences of other Grs, a fragment containing this sequence is
a useful probe for the specific detection of the Gr B transcript. When
we reassessed Gr B expression in CMCs by Northern blot analysis using
this Gr B-specific probe, we obtained a result (Fig 5B) similar to
that shown in Fig 3, where full-length Gr B cDNA was used as a probe.
In contrast, the probe prepared from a PCR product common to the other
Grs did not detect any transcript after the same exposure time (5 hours). These results showed that the expression profile of Gr B is
distinct from those of other Grs, and that Gr B gene expression in
mi/mi CMCs is drastically reduced.
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| Fig 5.
Expression profile of the Gr B gene and the other Grs of
the murine Gr B locus in +/+ and mi/mi CMCs. (A) Serially
diluted total RNA (0.5, 0.05, and 0.005 µg) from +/+ and
mi/mi CMCs, and splenocytes of +/+ mice were
reverse-transcribed and PCR-amplified with the Gr B-specific
(uppermost panel) or Gr C-G-specific (lower two panels) primers. The
PCR was stopped after the indicated number of cycles, loaded on 2%
agarose gels, and stained with ethidium bromide. As a positive control,
+/+ splenocytes were used after PWM-stimulation. Molecular size
standards are shown on the right. (B) Five micrograms of total RNA from
+/+ and mi/mi CMCs, and +/+ splenocytes stimulated with
PWM were fractionated on 1% agarose gels and fixed onto nylon
membranes. The PCR-amplified DNA fragment seen in (A) was used as a
probe to detect the Gr B-specific (upper panel) and the Gr
C-G-specific (lower panel) transcripts. Reprobing with the -actin
probe showed that equal loading had been achieved.
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Impaired expression of TPH but not of HDC in mi/mi CMCs.
TPH and HDC are the rate-limiting enzyme of the serotonin and histamine
synthesis, respectively29,51; both chemical mediators were
stored in the basophilic granules of mast cells.34 Although
the TPH gene was transcriptionally downregulated in mi/mi CMCs,
the HDC gene was equally transcribed between +/+ and mi/mi CMCs
(Fig 3). The proportion of the HDC mRNA+ CMCs was
comparable among +/+ CMCs, mi/mi CMCs, mi/mi CMCs
overexpressing +-MITF, and mi/mi CMCs overexpressing
mi-MITF (Table 1). The serotonin content was significantly
smaller in mi/mi CMCs than in +/+ CMCs (Table
2). In contrast, the histamine content was comparable between +/+ and mi/mi CMCs (Table 2). The
introduction of +-MITF cDNA to mi/mi CMCs increased the
serotonin content to nearly normal levels, but the introduction of
mi-MITF cDNA to mi/mi CMCs did not. The introduction of
+- or mi-MITF cDNA to mi/mi CMCs did not affect the
histamine content (Table 2). Since mast cells take up serotonin from
media,52,53 we next evaluated the serotonin uptake by +/+
and mi/mi CMCs. The amount of serotonin that was taken up from
the media was comparable between +/+ and mi/mi CMCs (Table
3).
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|
Table 2.
Concentration of Serotonin and Histamine in +/+
CMCs, mi/mi CMCs, and mi/mi CMCs Overexpressing +- or
mi-MITF
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Binding of +-MITF to CANNTG motifs in the 5-flanking sequences
of the Gr B and TPH genes.
Bleackley et al54,55 reported that two positive regulatory
elements (nt 682 to 427 and nt 243 to 112)48
were present in the Gr B 5-flanking region. Since the basic domain of
the bHLH-Zip family recognizes the CANNTG motif,56,57 we
searched for this motif in the two positive elements. We found three
CANNTG motifs in the 5 element (nt 682 to 427)48
and examined whether the +-MITF protein bound each CANNTG motif by
EGMSA. As shown in Fig 6A,the +-MITF protein bound all of the three CANNTG motifs. The binding
was inhibited by the excess amount of oligo 6 containing a CACATG
motif, but not of oligonucleotides containing mutated motifs
(CAGATG between nt 563 and 558 to
CTGAAG, CACGTG between nt
530 and 525 to CTCGAG, and
CATTTG between nt 521 and 516 to
CTTTAG)48 (Fig 6A).
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| Fig 6.
EGMSA using GST-+-MITF fusion protein. (A)
Six kinds of oligonucleotides (oligo 1 to 6) were synthesized: oligo 1 to 5 were derived from the Gr B gene promoter and oligo 6 from the
gp49B gene promoter.50 In vitro binding of oligo 6 to
+-MITF was confirmed in the other experiment (data not shown). CANNTG
motifs are boxed and the mutations introduced into the motifs are shown
by underlines. The labeled oligonucleotide containing the CAGATG motif
(oligo 1), the CACGTG motif (oligo 3), or the CATTTG motif (oligo 4) was used as a probe. The probes were incubated with purified
GST-+-MITF fusion protein in the presence or absence of unlabeled
oligonucleotide competitors. To compete the in vitro binding between
the three CANNTG motifs (oligo 1, 3, and 4) and GST-MITF fusion
protein, excess amount of unlabeled oligo 2, oligo 5, or oligo 6 was
added. The arrowhead indicates the complex obtained with the labeled oligonucleotides and GST-MITF fusion protein. (B) Five kinds of oligonucleotides (oligo 7 to 11) were synthesized based on the promoter
sequence of the TPH gene. The labeled oligonucleotide containing the
CAAGTG motif (oligo 7), the CAGATG motif (oligo 8), the CAACTG motif
(oligo 9), or the CAGGTG motif (oligo 10) was used as a probe.
Competition for the binding of GST-+-MITF to the labeled oligo 10 was
also examined. The excess amount of a nonlabeled oligo 6 or an
oligonucleotide mutated at the CAGGTG motif (to
CTGGAG, oligo 11) was added.
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The 5-flanking sequence of the mouse TPH gene was reported by Stoll
and Goldman.31 We isolated the fragment between nt 1054
and +86 (+1 shows the transcription initiation site),31 which contained four CANNTG motifs. The +-MITF bound one of four CANNTG
motifs (CAGGTG between nt 322 and nt 317),31 whereas the +-MITF did not bind the remaining three CANNTG motifs (CAAGTG between nt 1041 and 1036, CAGATG between nt 977 and 972,
CAACTG between nt 552 and 547)31 (Fig 6B). The
excess amount of oligo 6 containing a CACATG motif inhibited the
binding of +-MITF, whereas the excess amount of the oligonucleotide
that had the mutation at the CAGGTG motif (CAGGTG
to CTGGAG) did not inhibit the binding of +-MITF
(Fig 6B). The +-MITF did bind to oligo 1 but did not bind to oligo 8 (Fig 6A and B); nevertheless, both possessed the identical core
sequence, CAGATG. Presumably flanking sequences affect the binding.
+-MITF directly transactivated the Gr B and TPH promoters.
The binding specificity of +-MITF protein for three CANNTG motifs of
the Gr B promoter and for one of four motifs of the TPH promoter
suggested that these motifs may mediate the transactivation effect of
+-MITF. We elucidated this possibility by the transient cotransfection
assay. The 5-flanking sequences of the Gr B (nt 910 to +42, +1 is
the transcription start site)48 were cloned upstream of
the luciferase gene. This luciferase construct was cotransfected into
NIH/3T3 fibroblasts along with the expression plasmid containing +-MITF
or mi-MITF cDNA. The coexpression of +-MITF increased the
luciferase activity approximately fivefold (Fig
7A). In contrast, coexpression of
mi-MITF did not increase luciferase activity at all. Then, we
introduced mutations into the three CANNTG motifs as is the case of
oligonucleotides used in EGMSA. Of the seven kinds of mutated reporter
plasmids, three kinds of plasmids bearing mutations at either CACGTG
motif, at both CACGTG and CATTTG motifs, or at both CAGATG and CATTTG
motifs, showed significant reductions in luciferase activity. When
mutations were introduced into all of the three motifs, the
transactivation effect of +-MITF was abolished. When mi-MITF
was expressed, no transactivation effect was detected using any mutated
reporter, either. These results indicate that +-MITF transcriptionally
activates the Gr B gene by recognizing three CANNTG motifs, of which
the CACGTG motif appears to be particularly important.
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| Fig 7.
(A) The effect of coexpression of +-MITF or
mi-MITF cDNA on the luciferase activity under the control of
the normal or mutated Gr B promoter. Three square black boxes represent
CANNTG motifs between nt 910 and +42. The boxes with X have
mutated motifs: CAGATG (nt 563 to 558) motif is mutated to
CTGAAG, CACGTG (nt 530 to 525) to CTCGAG, CATTTG (nt 521 to
516) to CTTTTG. The open and filled bars represent the mean ± SE
of the relative luciferase activities obtained by three independent
experiments. (B) The effect of coexpression of +-MITF or
mi-MITF cDNA on the luciferase activity under the control of
the normal, deleted, or mutated TPH promoter. Four square black boxes
represent CANNTG motifs between nt 1054 and +86. The boxes with X
have a mutated motif: CAGGTG (nt 322 to 317) to CTGGAG. The data
represent the mean ± SE of three experiments. In some cases, the SE
was too small to be shown by the bars.
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To examine the transactivation effect of +-MITF for the TPH gene, the
promoter region and a part of the first exon of the TPH gene (nt
1054 to +86)31 was cloned upstream of the luciferase gene. We also constructed the deleted reporter plasmids containing the
TPH promoter starting from nt 990, 565, 335, or 330. The coexpression of +-MITF with the reporter plasmid containing the CAGGTG
motif increased the luciferase activity (Fig 7B). In contrast, the
coexpression of +-MITF and the reporter plasmid without the CAGGTG
motif did not increase the luciferase activity. The mutation of the
CAGGTG motif to CTGGAG
completely abolished the transactivation activity of +-MITF. The
mi-MITF did not transactivate any reporter plasmids (Fig 7B).
| |
DISCUSSION |
In the present study we attempted to isolate as many target genes as
possible that are transcriptionally upregulated by MITF. For this
purpose we prepared a subtracted cDNA library of high quality using +/+
and mi/mi CMCs. From the +/+ CMC cDNA library, the clones
carrying inserts complementary to mi/mi CMC mRNAs were removed
by hybridization. When we characterized the resulting subtracted cDNA library by application of Southern and Northern analyses, we found that it might contain +/+ CMC-specific clones at
high frequencies. When 400 clones were randomly selected from the
subtracted cDNA library and analyzed, 22 genes were found to be
transcribed preferentially in +/+ CMCs: 6 corresponded to Gr B, TPH,
ST2L, gp49B, MMCP-5, and -6, and 16 were unknown genes. MMCP-5 and -6 genes have already been described as +/+-specific genes and targets of
MITF.5,8,9 This result enabled us to judge that the
subtraction method described here was successful enough to answer our
purpose.
By +-MITF introduction, EGMSA and transient cotransfection assay, we
showed that MITF directly involved in transcriptional activation of the
Gr B and TPH genes through recognition of the CANNTG motifs. The CAGGTG
motif located between nt 322 and 31731 was essential
for the activation of the TPH promoter. Reed et al58
analyzed the mouse TPH promoter in the TPH gene-expressing tumor mast
cell line P815-HTR. Because P-815-HTR cells expressed +-MITF (Morii E,
unpublished data, May 1997), the result that the deletion
between nt 343 and nt 25531 containing the CAGGTG
motif decreased the promoter activity in P815-HTR cells is consistent
with the present result. Pham et al59 determined the
genomic structure of the murine Gr B locus on chromosome 14, where Gr B
is located at the 5 end and followed by the genes for Grs C, F, G, D,
and E, cathepsin G, and MMCP-2. They also showed that all of the Grs in
this locus seem to share a common regulatory pattern in natural killer
(NK) cells. From these observations they proposed a model to explain
the regulatory mechanism of multiple Gr gene expression. The model
comprised a regulatory element, located upstream of the Gr gene
cluster, and a responsive sequence for this element within each of the Gr promoters. Interaction between the two would guarantee balanced expression of the genes making up the Gr locus. They presumed this
common regulatory element to be another example of a locus control |
Abstract
Introduction
Abstract