From Molecular Glycobiology, Frontier Research
Program, The Institute of Physical and Chemical Research (RIKEN),
Wako-shi, Saitama 351-0198, Japan and the § Department of
Neurology, Division of Neuroscience, Graduate School, Faculty of
Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113, Japan
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ABSTRACT |
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The rabbit H-blood type The H-blood type antigens (Fuc We have extended the analysis of the expression of H determinants in
the mammalian nervous system using anti-fucosyl GM1 antibodies and
U. europaeus agglutinin 1 lectin (10-13). In the human and rabbit nervous systems, H antigens are abundantly expressed in dorsal
root ganglia (DRG), which consist of several types of primary sensory
neurons. The anti-fucosyl GM1 antibodies and U. europaeus agglutinin 1 lectin recognized a subpopulation of neurons in DRG and
the dorsal horn of the spinal cord. The anti-fucosyl GM1 antibodies also bound to the satellite cells surrounding the fucosyl GM1-positive neurons (10, 12). In addition, in rabbits, the anti-fucosyl GM1
antibodies bound to the axons and the myelin of the small myelinated
fibers in the dorsal root and the large neurons in the ventral horn
(13). In rabbit DRG, fucosyl GM1 is readily detectable
immunohistochemically on embryonic day 25, followed by the appearance
of U. europaeus agglutinin 1 lectin-reactive antigens
postnatally (12, 13).
The expression of H antigens in human and rabbit DRG neurons seems to
be under similar control, although little is known about the molecular
basis of their regulated expression. To investigate the mechanisms
underlying the regulation of the biosynthesis of H antigens in DRG
neurons, we recently cloned three types of rabbit PCR Rapid Amplification of 5' and 3' cDNA Ends
(RACE)--
Poly(A)-rich RNAs were extracted from adult rabbit DRG by
the guanidinium isothiocyanate method and purified with Oligotex-dT30 (Takara-Shuzo, Japan). Amplification of the 5'-end of the RFT-I cDNA was performed essentially according to the procedure of
Frothman et al. (17). cDNA was synthesized by reverse
transcription (Superscript II; Life Technologies, Inc.) of 5 µg of
rabbit DRG poly(A)-rich RNA using primer H4B3,
5'-AAGCAAGAAGGCCAGACAGAGCTG-3', which is complementary to nucleotides
+45 to +22 (taking the translational initiation site as +1) of the
RFT-I gene. The excess primer and deoxynucleotide were removed by
passage of the cDNA through a MicroSpin S-400 column (Amersham
Pharmacia Biotech). The cDNA was A-tailed with 0.6 units of
terminal deoxynucleotidyltransferase (Boehringer Mannheim), using 0.05 mM dATP. Two consecutive PCRs were performed with two
nested sets of primers; for the first PCR, the forward primer was
NotI-(dT)18 (Amersham Pharmacia Biotech), and
the reverse primer was H4B3; for the second PCR, the forward primer was
as above but without the T-tail, 5'-AACTGGAAGAATTCGCGGCCGCAGGAA-3', and
the reverse primer was H4B4 (5'-AGAGCTGCCGGCGGCTCGGAGGCCACAT-3'; complementary to nucleotides +28 to +1). The cDNA was amplified for
40 cycles of a step program (95 °C, 30 s; 55 °C, 30 s;
and 72 °C, 30 s). The amplification products were subcloned
into pBluescript II SK(+) (Stratagene) and then sequenced.
For 3'-RACE, cDNA was synthesized by reverse transcription using
(dT)18 (Amersham Pharmacia Biotech). Two consecutive PCRs were performed using forward primer H4A11
(5'-TTTAACCCAGGGGCAGCACAGGGTCT-3'; nucleotides +2082 to +2107) and
reverse primer NotI-(dT)18 and then using the
forward primer H4A12 (5'-TGGAGTCGAGGTCCACACCTCCA-3'; nucleotides +2284
to +2306) and the above reverse primer without the T-tail.
Northern Blot Analysis--
We previously reported two RFT-I
mRNA transcripts, 4.2 and 3.1 kb, the latter of which is major and
specific to DRG (16). To determine the transcription pattern of the
DRG-specific mRNA isoform, we performed Northern blot analysis
using three probes located in the 5'-flanking region relative to the
translation initiation site. Probes A ( Plasmid Construction--
To obtain various lengths of the
5'-flanking region of the RFT-I gene, pH4-SK3.6 was constructed by
sequentially subcloning 2.0-kb XhoI-KpnI
(nucleotides +856 to +2890) and 1.6-kb SacI-XhoI (nucleotides Cell Culture and Promoter Activity Analysis--
Neuro2a and
Chinese hamster ovary (CHO) cells were seeded at 5 × 104 cells/35-mm diameter dish in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum 24 h prior
to transfection, respectively. PC12 cells were seeded in the same
manner in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum and 5% horse serum. The luciferase plasmid (2 µg)
used as a reporter and the pSR-
Plasmid pH4-SK3.6, which contains 5'-untranslated region, the entire
coding region, and 3'-untranslated region with a poly(A) signal, was
linearized with HindIII, combined with pTK-Hyg
(CLONTECH), and then transfected into Neuro2a and
PC12 cells. After culturing for 72 h, the cells were selected with
0.3 µg/ml of hygromycin (Life Technologies, Inc.) and subsequently subcloned.
Cultures of DRG neurons and cerebellar granule cells were obtained as
follows. DRG were excised from P14 rabbits, trypsinized, treated with
0.01% DNase I and then 10 µg/ml of collagenase I (Sigma), and
finally mechanically dissociated. The cell suspension was plated onto
collagen S (Boehringer Mannheim)-coated coverslips in Dulbecco's
modified Eagle's medium/F-12 medium supplemented with 10% fetal calf
serum and 100 ng/ml of 2.5 S nerve growth factor (NGF; Sigma). Dorsal
roots were also excised and treated as above to obtain fibroblasts and
Schwann cells, but without DRG neurons. Cerebella excised from P5
rabbits were trypsinized and then mechanically dissociated. The cell
suspension was plated onto poly-L-lysine (Sigma)-coated
coverslips at 2-5 × 105 cells/cm2 in
minimum essential medium with 26 mM potassium supplemented with 5% horse serum. After 48 h of plating, the luciferase
plasmid (0.5 µg) and the pSR-
After 48 h of transfection, the cells were washed three times with
PBS and then lysed with cell lysis buffer (PG Electrophoretic Mobility Shift Assay--
Nuclear extracts from
Neuro2a and PC12 cells with and without treatment with NGF (100 ng/ml)
for 90 min were prepared by the method of Masuda et al. (18)
Briefly, 5 × 107 cells were collected by
centrifugation and then sequentially resuspended with buffer A (10 mM HEPES, pH 7.6, 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and
10 µg/ml of leupeptin), buffer A containing 0.2% Nonidet P-40, and
buffer A containing 0.2 M sucrose and centrifugation at
800 × g. The cell pellet was then resuspended with
buffer D (50 mM HEPES, pH 7.9, 400 mM KCl, 10%
glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin)
and centrifuged. Protein concentration of supernatant was determined by
a Bio-Rad protein assay.
The DNA fragments of nucleotides
Binding assays were performed with a labeled probe in the presence of 2 µg of poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech) and 0.4 footprinting units of recombinant human Sp1 (Promega) or 0.5 µg of
nuclear extracts. Binding reactions were carried out for 30 min on ice
in 10 mM HEPES-KOH (pH 7.8), 50 mM KCl, 5 mM MgCl2, 1 mM EDTA, 10% glycerol,
25 mM dithiothreitol, 3.5 mM
phenylmethylsulfonyl fluoride, 5 mM sodium orthovanadate,
10 µg/ml aprotinin, and 10 µg/ml leupeptin. Competitor fragments of
a 20-fold excess amount or anti-Egr-1 polyclonal antibodies (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) were added where indicated. After
incubation, the samples were loaded onto a 4% polyacrylamide gel in
0.5 × TBE. The gel was run in the cold at 200 V and dried, and
then the radioactivity was detected with a BAS 2000 image analyzer
(Fuji Film, Japan).
Determination of the Transcriptional Start Site of the RFT-I
Gene--
We previously showed that two RFT-I mRNA transcripts,
4.2 and 3.1 kb, were expressed in rabbit nervous tissues (16). The 4.2-kb transcript was broadly found in the central and peripheral nervous tissues examined but in low amounts, whereas the 3.1-kb transcript was abundantly and specifically observed in DRG neurons of
small diameter. To isolate the 5'-end of the RFT-I cDNA, we performed RACE-PCR using poly(A) RNA from adult rabbit DRG, and found
two transcriptional start sites, at positions
To analyze the differential use of transcriptional start sites in
rabbit nervous tissues, we next performed Northern analysis using three
kinds of probes (probes A, B, and C in Fig. 1). Probe A, recognizing
3.3- and 4.2-kb mRNAs, gave weak signals at the corresponding bands
in both DRG and brain RNA (Fig. 3). Probe B gave weak signals at 4.2-kb in DRG and brain RNA. Probe C hybridized to a 3.1-kb transcript in DRG RNA strongly enough to be detected on
short exposure. These results showed that the 3.1-kb mRNA of the
RFT-I gene was abundantly and specifically expressed in DRG and that
the 3.3- and 4.2-kb mRNAs were broadly found in DRG and other
nervous tissues but in low amounts.
Promoter Activity Analysis--
To determine the RFT-I gene
promoter activity, we used three types of cells: PC12 rat
pheochromocytoma cells expressing rat H-blood type
pBH4-BP3.0, containing the 3.0-kb 5'-flanking sequence of the RFT-I
gene (
To determine the effect of the differential promoter activity on the
transcriptional pattern, plasmid pH4-SK3.6, containing the 5'- and
3'-flanking regions and the entire coding region of the RFT-I gene, was
stably transfected into Neuro2a and PC12 cells. The transcriptional
pattern was analyzed by reverse transcription-PCR using three pairs of
primers; the H4A5 and H4B4 primers could discriminate the presence of a
3.3-kb mRNA, the H4A5 and H4B5 (5'-GGAGGAGGTCTGGGAAAAGAGGCG-3')
primers could detect a 4.2-kb mRNA, and the H4A4 and H4B4 primers
could detect 3.1- and 4.2-kb mRNAs but could not discriminate them.
All pairs of primers gave positive bands when RNA from Neuro2a
cell-derived stable transfectants with pH4-SK3.6 was analyzed (data not
shown), indicating the presence of 3.3- and 4.2-kb transcripts of the
RFT-I gene in these cells, although this result did not exclude the
possibility of the presence of a 3.1-kb transcript. On the contrary, a
positive band was amplified only by the H4A4 and H4B4 primers in PC12
cell-derived stable transfectants with pH4-SK3.6 (data not shown),
showing the presence of a 3.1-kb transcript of the RFT-I gene.
Effects of NGF and Mutations on Promoter Activity--
A data base
search for possible binding of transcription factors revealed several
Sp1 binding sites and a N-Myc binding site in the region of nucleotides
We then examined whether or not a possible N-Myc binding site
(nucleotides Electrophoretic Mobility Shift Assay--
To confirm the actual
binding of Sp1 to the putative Sp1 binding site between nucleotides
To determine the transcriptional factors that regulate the specific
promoter activity of this region in PC12 but not in Neuro2a cells, we
next performed the assay using nuclear protein extracts of Neuro2a and
PC12 cells with and without NGF treatment. When nuclear protein
extracts of Neuro2a and PC12 cells were used, the labeled DNA fragment
of nucleotides Promoter Activity Analysis of Cultures DRG Neurons--
DRG
neurons were cultured and transfected with reporter plasmids containing
several lengths of the 5'-flanking region of the RFT-I gene. The
pBH4-SP0.7 construct showed the highest level of promoter activity when
expressed in DRG neurons (Fig. 7), which was consistent with the results for PC12 and Neuro2a cells. However, the pBH4-SSP1.3, pBH4-SSm0.2, and pBH4-NP0.3 constructs showed relatively low promoter activities. The DRG culture contained neurons,
Schwann cells, and fibroblasts, and all of them were transfected with
plasmids and expressed the reporter gene, as revealed by the
transfection of pCMV-EGFP and observation by fluorescence microscopy
(data not shown). To determine the contributions of Schwann cells and
fibroblasts to the promoter activity, we then used a dorsal root
culture, which contained Schwann cells and fibroblasts but no neurons.
All of the constructs showed a relatively low level of promoter
activity (Fig. 7), suggesting that the high promoter activity of the
pBH4-SP0.7 construct could be due to the expression of the reporter
gene in DRG neurons. Next, we determined whether or not the promoter
activity of the pBH4-SP0.7 construct was specific to DRG neurons. The
pBH4-SP0.7 and other constructs showed lower promoter activity when
expressed in cerebellar granule cells, another type of neuron, than in
DRG neurons (Fig. 7).
We previously showed that the expression of RFT-I is strictly
regulated spatially and temporary in the rabbit nervous system and
abundant in adult DRG neurons of small diameter (16). In this study, we
determined the genomic organization and promoter activity that
regulates the DRG neuron-specific expression of the RFT-I gene. Our
results demonstrated that the RFT-I gene used two transcriptional start
sites yielding three types of mRNA and that the minimal promoter
region flanking the translational initiation codon of the RFT-I gene
was sufficient for DRG neuron-specific expression of the gene. DRG
neuron-specific promoter activity has not been detected so far, and
analysis of the proteins binding to the minimal promoter region will
facilitate understanding of the differentiation of DRG neurons.
The RFT-I gene specified three types of mRNA; a 3.1-kb transcript
starting at position The distribution of H antigens in the nervous system is quite similar
in human and rabbit; in both species, small neurons of DRG are positive
for both U. europaeus agglutinin 1 lectin staining and
anti-fucosyl GM1 antibody immunostaining (8, 11, 12). The human H-blood
type In the present study, we detected the promoter activity in the
5'-flanking region (nucleotides The region of nucleotides The GSG element is a consensus binding motif recognized by members of
the early response gene family, such as NGFI-A (32)/Egr-1 (33), Krox-20
(34), Wilms' tumor gene product (30), and NGFI-C (31). Among them,
NGFI-A/Egr-1 and NGFI-C are rapidly and temporally induced in PC12
cells by NGF stimulation. In the present study, an electrophoretic
mobility shift assay involving a nuclear extract of PC12 cells
stimulated with NGF and anti-Egr-1 antibodies revealed that
NGFI-A/Egr-1, at least, actually binds to the Sp1 and GSG-like
overlapping element. It is still possible that other NGF-responsive
transcriptional factors weakly bind to this element and enhance the
promoter activity of this region. Sp1 is a ubiquitous transcriptional
factor that could recruit transcription initiation complexes to the
initiation site in TATA-less promoters (35) and may require other
co-factors for the tissue-specific expression of genes. As for the
RFT-I gene promoter, NGFI-A/Egr-1 and possibly other members of the
NGF-inducible gene family may be responsible for the PC12 cell-specific
promoter activity in the region of nucleotides The promoter region of nucleotides High promoter activity was detected in the region of nucleotides The pBH4-SSm0.2 and pBH4-NP0.3 constructs showed relatively low
promoter activity as compared with the pBH4-SP0.7 construct, when
transfected into a primary culture of DRG neurons. DRG neurons are
known to express trk (neurotrophin receptor family genes) and to be dependent on neurotrophin-mediated signaling (36, 37). Most
of the small neurons of DRG bearing H antigens on their surface express
TrkA, which binds to NGF, as in the case of PC12 cells. Despite the
fact that the DRG neurons were cultured with 100 ng/ml NGF, the
pBH4-SSm0.2 construct showed a low level of promoter activity in DRG
neurons comparable with that in dorsal roots and cerebellar granule
cells. Some other factors binding to somewhere within the region of
nucleotides 1,2-fucosyltransferase
(RFT-I), gene and its biosynthetic products, H antigens
(Fuc
1,2Gal
), are abundantly expressed in a subset of dorsal root
ganglia (DRG) neurons. To investigate the regulatory mechanisms for the
RFT-I gene expression, we determined the genomic structure and promoter activity of this gene. PCR amplification of the 5' cDNA end
analysis revealed two transcriptional start sites, 498 and 82 nucleotides upstream of the translational initiation codon, the latter
site yielding a major 3.1-kb transcript specifically expressed in DRG, as revealed by Northern blotting. Promoter analysis of the 5'-flanking region of the RFT-I gene using a luciferase gene reporter system demonstrated strong promoter activity in PC12 cells, which express the
rat H-type
1,2-fucosyltransferase gene, and Neuro2a mouse neuroblastoma cells. Deletion analysis revealed the 704-base pair minimal promoter region flanking the translational initiation codon,
for which two distinct promoter activities were detected and
differentially used in PC12 and Neuro2a cells. The minimal promoter
region contained a GC-rich domain (GC content 80%), in which a Sp1
binding sequence and a GSG-like nerve growth factor-responsive element
were found, but lacked TATA- and CAAT-boxes. Promoter analysis with a
primary culture of DRG neurons demonstrated that the minimal promoter
region of the RFT-I gene was sufficient for the expression of a
reporter gene in DRG neurons. We conclude that the TATA-less GC-rich
minimal promoter region of the RFT-I gene controls DRG small
neuron-specific expression of the RFT-I gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
1,2Gal
) are synthesized by
GDP-L-fucose:
-D-galactoside
2-
-L-fucosyltransferase
(
1,2-FT)1 (for a
review, see Ref. 1). The expression of H determinants is strictly
regulated temporally and spatially during vertebrate development
(2-4). The H antigens are rarely detected in adult nervous tissues of
human and other mammals but are present on a subset of neurons.
Analyses with Ulex europaeus agglutinin 1 lectin, which
binds to type 2 H (Fuc
1,2Gal
1,4GlcNAc) determinants, and
anti-H antibodies revealed that the expression of H antigens was
restricted to olfactory bulb and cochlear hair cells in rats (5, 6) and
to primary sensory neurons and their axons in human and other primates
(7-9). Most of the H-positive axons of primary sensory neurons were
unmyelinated and thought to be C-fibers that mediate nociceptive or
thermoceptive inputs or both.
1,2-FT gene,
i.e. one H-type and two Se-type genes, as judged from the
results of kinetic studies (14, 15). Analysis of the expression of
these genes revealed that all three
1,2-FT genes were expressed in
DRG of late embryonic rabbits but that only the H-type
1,2-FT gene,
the RFT-I gene, was expressed postnatally (16). In situ
hybridization demonstrated that abundant RFT-I mRNA was present in
adult rabbit DRG neurons of small diameter. We have shown that the
RFT-I gene specifies the mRNAs of a major 3.1-kb transcript in DRG
and a minor 4.2-kb transcript broadly expressed in rabbit nervous
tissues. These results indicate that the 3.1-kb transcript of the RFT-I
gene is under the control of DRG small neuron-specific promoter
activity. In this study, we determine the genomic structure of the
RFT-I gene and the promoter activity of the 5'-flanking region using a
primary culture of DRG neurons.
EXPERIMENTAL PROCEDURES
481 to
302), B (
294 to
55), and C (
75 to +29) were labeled with
[
-32P]dCTP (NEN Life Science Products), using a pair
of synthetic oligonucleotide primers specific to each probe and Klenow
fragment (Amersham Pharmacia Biotech). Total RNAs (3 µg) from adult
rabbit brain and DRG were fractionated on a denaturing
formaldehyde-agarose gel (1.0%) and then transferred to a nylon
membrane (Nytran; Schleicher & Schuell). Three membranes prepared from
a gel were hybridized with probe A, B, or C.
707 to +856) fragments from RG11 into the pUC118 plasmid. A 0.7-kb SacI-PstI fragment was
subcloned into pPicaGene-Basic II (pPGBII; Toyo-ink, Japan) to obtain
pBH4-SP0.7. A 0.6-kb SacI fragment was subcloned into the
SacI site of pBH4-SP0.7 to obtain pBH4-SSP1.3. A 1.6-kb
BamHI-SacI fragment was subcloned into the upper
SacI site of pBH4-SSP1.3 to obtain pBH4-BP3.0. Series of deletion plasmids were constructed by subcloning the restriction enzyme-digested DNA fragments. SacI-SmaI (169 bp), SmaI (325 bp), and NcoI-PstI
(291 bp) fragments were subcloned into pPGBII to obtain pBH4-SSm0.2,
pBH4-Sma0.3, and pBH4-NP0.3, respectively. The correct constructs were
verified by DNA sequencing using the fluorescein isothiocyanate-labeled
BII primer (5'-AGTGCAAGTGCAGGTGCCAGAA-3') and an A.L.F. DNA sequencer
(Amersham Pharmacia Biotech). The pBH4-SacB8 (containing nucleotides
707 to
626) and pBH4-SacB8m (containing the same region with
mutations) constructs were made by integrating the DNA fragments
amplified by PCR using pBH4-SSm0.2 as a template and a pair of primers,
the forward BII primer and reverse primer H4B8
(5'-TCCGGGCTCCCAGCCCCCGCCCCCAGGG-3') or H4B8m (5'-TCCGGGCTCCCAGCCCCTGTCCCCAGGG-3',
mutations underlined), into pPGBII, respectively. The pBH4-A6Pst
(containing nucleotides
257 to
4) and pBH4-A6mPst (containing the
same region with mutations) constructs were made by integrating the DNA
fragments amplified by PCR using pH4-SK3.6 as a template and a pair of
primers, forward primer H4A6 (5'-GAGCCGGTGGGCTTGGCACGTGGGGAGG-3') or
H4A6m (5'-GAGCCGGTGGGCTTGGCAAATGGGGAGG-3', mutations
underlined) and reverse primer H4B4, into pPGBII, respectively. The DNA
fragments containing the SV40 early promoter cut from pPicaGene-Control
Vector II (Toyo-ink) were subcloned into pPGBII to obtain pBII-SV40.
The DNA fragments containing CMV promoter cut from pRc-CMV (Invitrogen)
were subcloned into pEGFP-1 (CLONTECH) to obtain
pCMV-EGFP.
-gal plasmid (0.2 µg) used as an
internal control for transfection efficiency were transfected into the
cells by means of Lipofectoamine (Life Technologies, Inc.). As negative and positive controls, the pPGBII and pBII-SV40 plasmids, respectively, were also transfected.
-gal plasmid (0.05 µg) were
transfected into the cells by means of FuGene 6 (Boehringer Mannheim).
To verify the viability of cells and actual transfection of the
plasmids into DRG neurons or cerebellar granule cells, pCMV-EGFP (0.5 µg) was also transfected in another coverslip culture.
-50; Toyo-ink, Japan).
Luciferase activity was measured using a PicaGene Luciferase assay
system (Toyo-ink) and a Luminescencer AB-2000 (Atto, Japan). Light
activity measurements were performed in duplicate, averaged, and then
normalized relative to
-galactosidase activity in order to correct
for the transfection efficiency.
-Galactosidase activity was
measured using a Luminescent
-galactosidase detection kit (CLONTECH).
707 to
626 with and without
mutations in the Sp1 and GSG overlapping elements were cut out with
SacI and HindIII from pBH4-SacB8m and pBH4-SacB8.
The synthetic DNA primers, H4A8
(5'-GGCCCTGGGGGCGGGGGCTGGGAGCCCGGA-3') and H4B8, and H4A8m
(5'-GGCCCTGGGGACAGGGGCTGGGAGCCCGGA-3',
mutations underlined) and H4B8m, were annealed to yield A8B8 and A8mB8m fragments, respectively. The DNA fragments were end-labeled with [32P]dCTP using Klenow polymerase.
RESULTS
498 and
82 (Figs.
1 and 2).
The RACE-PCR results revealed that there are three types of mRNA
transcripts; one transcript (3.1-kb mRNA in Fig. 1) started at
position
82 and contained no intron, the second one (3.3-kb mRNA
in Fig. 1) started at position
98 and skipped the intron of
nucleotides
265 to
3, yielding an additional 3.3-kb transcript, and
the last one (4.2-kb mRNA in Fig. 1) also started at position
498
but without splicing out the 3.3-kb mRNA intron sequence. We also
performed 3' RACE-PCR and found a poly(A) signal at position +2579
without an intron.
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Fig. 1.
Restriction map of RG11 and structure of the
RFT-I gene. The exons of RFT-I are indicated as
rectangles and the shaded box
represents a coding region. The probes used for Northern blotting
analysis are indicated by thick lines. Shown
below is the splicing pattern for each transcript of the
RFT-I gene. B, BamHI; S,
SacI; K, KpnI; X,
XhoI. Numbers above the restriction
enzyme sites indicate the nucleotide positions taking the translational
initiation site as +1.
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Fig. 2.
Nucleotide sequence of the 5'-flanking region
of the RFT-I gene. The sequence of nucleotides 750 to +45 is
shown. The exons used in the 3.1- or 3.3-kb transcript are indicated as
uppercase letters. The exon-intron junctions of
the 3.3-kb transcript are TACACAGgtacgcc ... cctgcagCCATGTG. The
primers used in this study and some of the putative transcriptional
factor-binding elements are also shown.
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Fig. 3.
Northern blotting of RFT-I. The
differential expression of three types of RFT-I mRNA was analyzed
using three probes. Probe A hybridized to 4.2- and 3.3-kb transcripts
in adult rabbit DRG and brain, probe B hybridized to a 4.2-kb
transcript in both tissues, and probe C hybridized to a 3.1-kb
transcript in DRG only. The results for probe C were obtained with a
shorter exposure (overnight) than for probe A or B (three overnight
exposures).
1,2-FT, a
counterpart of RFT-I; Neuro2a mouse neuroblastoma cells originating
from the neural crest but not expressing
1,2-FT; and nonneuronal CHO
cells. A series of reporter plasmids containing progressive deletions
of the 5'-flanking region of the RFT-I gene fused to the promoterless
luciferase gene were mixed with an internal control plasmid,
pSR-
-Gal, carrying a
-galactosidase gene under the control of the
SR
promoter, and then transfected into cells. The luciferase
activity due to each luciferase reporter plasmid was normalized as to
the
-galactosidase activity. The promoter activity was calculated
relative to the SV40 promoter activity taken as 100%.
2970 to
4), showed high levels of promoter activity when
expressed in PC12 and Neuro2a cells but not in CHO cells (Fig.
4). The pBH4-SP0.7 reporter plasmid also
showed high promoter activity in PC12 and Neuro2a cells, although the
activity was lower than that of pBH4-BP3.0. The 0.7-kb 5'-flanking
region was divided into three portions, and each fragment was ligated
into a pPGBII plasmid. The pBH4-SSm0.2 plasmid, containing the 0.2-kb sequence (
707 to
539), showed a substantial level of promoter activity when expressed in PC12 but not in Neuro2a cells, whereas the
pBH4-NP0.3 plasmid, containing the 0.3-kb sequence (
294 to
4),
showed a level of promoter activity comparable with pBH4-SP0.7 in
Neuro2a cells but not in PC12 cells. pBH4-Sma0.3 showed little promoter
activity in both types of cells. These results suggested that the
promoter activity was differentially regulated in PC12 and Neuro2a
cells.
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Fig. 4.
RFT-I gene promoter activity analysis.
Shown is a schematic representation of DNA constructs containing
various lengths of the 5'-flanking region of the RFT-I gene. Each
construct and the pSR- -gal plasmid were transfected into PC12,
Neuro2a, or CHO cells. Luciferase activity was normalized as to
-galactosidase activity, and the relative promoter activity was
calculated relative to SV40 early promoter activity taken as 100%.
Each experiment was performed in duplicate, and the results are the
averages of four experiments.
707 to
4. Among possible Sp1 binding sites, one (nucleotides
650
to
638) showed the highest homology to the consensus sequence and
overlapped a GSG (GCGGGGCG)-like motif (nucleotides
645 to
637). To
determine whether or not these elements were functional, we constructed
plasmids pBH4-SacB8 (containing nucleotides
707 to
626) and
pBH4-SacB8m (the same region with mutations) and transfected them into
PC12 cells. PC12 cells transfected with pBH4-SacB8 and treated with 100 ng/ml of NGF for 48 h showed 2-fold higher luciferase activity
than those without NGF (Fig.
5A). The mutations in the Sp1
and GSG overlapping domain decreased the luciferase activity and
abolished the effect of NGF treatment.
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Fig. 5.
Effects of NGF and mutations on the promoter
activity. A, PC12 cells were transfected with the
pBH4-SSm0.2, pBH4-SacB8, and pBH4-SacB8m constructs and then cultured
with and without 100 ng/ml of NGF for 48 h. The wild type
(pBH4-SSm0.2 and pBH4-SacB8) and mutational (pBH4-SacB8m) sequences of
the Sp1 binding site and the overlapping GSG-like (8/9 consensus)
elements are shown. B, Neuro2a cells were transfected with
the pBH4-A6Pst and pBH4-A6mPst constructs, and then cultured for
48 h. The wild type (pBH4-A6Pst) and mutational (pBH4-A6mPst)
sequences of the inverted N-Myc binding site are shown. Luciferase
activity was normalized relative to -galactosidase activity, and the
relative promoter activities were calculated with SV40 early promoter
activity taken as 100%. Each experiment was performed in duplicate,
and the data are the means ± S.E. for three experiments.
244 to
233) found within the pBH4-NP0.3 construct could be demonstrated functionally. We constructed plasmids pBH4-A6Pst (containing nucleotides
264 to
4) and pBH4-A6mPst (the same region
with mutations), and transfected them into Neuro2a cells. The
pBH4-A6Pst and pBH4-A6mPst constructs showed comparable promoter activities when transfected into Neuro2a cells (Fig. 5B),
suggesting that the N-Myc was not a major factor for the activity.
650 and
638, we performed an electrophoretic mobility shift assay.
In the mobility shift experiments involving the DNA fragments of
nucleotides
707 to
626, recombinant Sp1 bound to the DNA fragments
(Fig. 6A, lane
2). The shifted band completely disappeared in the presence
of the nonlabeled specific competitor (DNA fragments from
707 to
626, the same as the labeled probe; Fig. 6A,
lane 3) and partially disappeared in the presence of the same region of the DNA fragments with mutations (lane
4). Recombinant Sp1 did not bind to the labeled probe of the
DNA fragments from
707 to
626 with mutations.
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Fig. 6.
Electrophoretic mobility shift assay.
A, the labeled probes of the DNA fragments from 707 to
626 without (SacB8, lanes 1-4) or with
(SacB8m, lanes 5-8) mutations in the Sp1 binding
site were incubated with 0.4 footprinting units of recombinant human
Sp1 either alone (lanes 2 and 6) or
with the nonlabeled wild-type (lanes 3 and
7) and mutant (lanes 4 and
8) competitors in a 20-fold molar excess. The
arrow indicates the binding of recombinant Sp1 to the
labeled SacB8 probe, which completely disappeared in the presence of
the nonlabeled SacB8 DNA fragment and partially disappeared in the
presence of the nonlabeled SacB8m DNA fragment. B, the
labeled probes of the DNA fragments from
655 to
626 without (A8B8,
lanes 1-7) or with (A8mB8m, lanes
8-11) mutations in the Sp1 and GSG overlapping domain were
incubated with 0.5 µg of nuclear extract from Neuro2a
(lanes 2 and 9) and PC12 cells with
(lanes 4-7 and 11) and without
(lanes 3 and 10) NGF treatment. The
nonlabeled wild-type (lane 5) and mutant
(lane 6) competitors in a 20-fold molar excess or
anti-Egr-1 polyclonal antibodies (lane 7) were
also added.
655 to
626 (A8B8) appeared as two shifted bands with
apparently the same electromobility (Fig. 6B,
lanes 2-4, bands a and
c). An additional band (band b) was
observed when using a nuclear extract of PC12 cells treated with NGF
for 90 min, and this shifted band disappeared upon the addition of the
anti-Egr-1 polyclonal antibodies (Fig. 6B, lane 7). All shifted bands completely disappeared in the presence
of the nonlabeled specific competitor (A8B8, lane
5) but were not abolished by the mutant competitor (A8mB8m,
the same region of the DNA fragments with mutations; lane
6). No shifted band was found when using the labeled DNA
fragment of nucleotide
655 to
626 with mutations (A8mB8m) and
nuclear protein extracts of Neuro2a and PC12 cells.
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Fig. 7.
RFT-I gene promoter activity in DRG
neurons. Primary cultures of DRG, DR, and cerebellar tissue were
transfected with reporter plasmids, pBH4-SSP1.3, pBH4-SP0.7,
pBH4-SSm0.2, and pBH4-NP0.3 and then cultured for 48 h. The
viability of cells and actual transfection of the reporter plasmids
were verified by transfection of the pCMV-EGFP construct to another
primary culture preparation and observation by fluorescence microscopy.
Luciferase activity was normalized relative to -galactosidase
activity, and the relative promoter activity was calculated with SV40
early promoter activity taken as 100%. Each experiment was performed
in duplicate, and the results are the averages of three
experiments.
DISCUSSION
82 was abundantly and exclusively expressed in
DRG neurons, and 3.3- and 4.2-kb transcripts both starting at position
498 were broadly found in the rabbit nervous system but in low
amounts. Recently, evidence of multiple transcriptional start sites in
such glycosyltransferase genes as the rat and human
2,6-sialyltransferase genes (19-21), murine
1,4-galactosyltransferase gene (22), human
1,6-N-acetylglucosaminyltransferase V gene (23), human
1,4-N-acetylglucosaminyltransferase gene (24), and human
1,3- and
1,2-fucosyltransferase genes (25, 26) has been
accumulated. Similar to our results for the RFT-I gene, the murine
1,4-galactosyltransferase gene uses at least two transcriptional start sites yielding 4.1- and 3.9-kb mRNAs; the former is
ubiquitously expressed in all tissues, and the latter is abundantly
expressed in lactating mammary glands (22). The differential use of
multiple transcriptional start sites and associated promoters of the
glycosyltransferase gene could regulate the tissue- and stage-specific
expression of glycosyltransferase genes and subsequent glycosylation patterns.
1,2-FT FUT1 gene is known to control the H antigens
on erythrocytes, whereas the expression of the FUT1 gene in
human nervous tissues remains undetermined. The H antigens on DRG small
neurons in humans seem to be synthesized by FUT1, because
FUT1 preferentially uses type 2 precursor glycochain as an
acceptor to yield type 2 H, which the U. europaeus
agglutinin 1 lectin recognizes, rather than FUT2 (27-29).
The exon-intron organization and splicing patterns of FUT1,
which are different from those of the RFT-I gene, have been determined
in human bone marrow cells, HEL human erythroleukemic cells, and MCAS
human ovarian cancer cells (26). It would be interesting to analyze the
transcriptional pattern of FUT1 in human DRG neurons and to compare it with that of the RFT-I gene.
707 to
4) of the RFT-I gene when
reporter plasmids were transfected into PC12 and Neuro2a cells. PC12
(rat pheochromocytoma) and Neuro2a (murine neuroblastoma) cells share a
common developmental origin, the neural crest, with DRG neurons.
Although the species of PC12 and Neuro2a cells and rabbit DRG neurons
are all different, common mechanisms and highly conserved factors might
function to control the expression of H-type
1,2-FT or other neural
crest-originating neuron-specific genes. Indeed, the pBH4-SP0.7
construct containing the region of nucleotides
707 to
4 showed high
promoter activity in PC12 and Neuro2a cells. In this region, at least
two promoter domains are thought to exist; the pBH4-SSm0.2 (nucleotides
707 to
539) construct showed the promoter activity when expressed
in PC12 cells, and the pBH4-NP0.3 (nucleotides
294 to
4) construct
showed the promoter activity when expressed in Neuro2a cells.
707 to
539 included a GC-rich domain of
80% GC content, in which several Sp1 binding sites were found in a
data base search for transcription factors. One of the Sp1 binding
sites showing the highest homology with the consensus sequence
overlapped a GSG-like (8/9 consensus) NGF-responsive element (30, 31).
Promoter analysis involving pBH4-SSm0.2 with and without mutations
demonstrated the reduction of the promoter activity and the abolition
of NGF responsiveness upon the insertion of mutations, suggesting that
these elements are functional. An electrophoretic mobility shift assay
involving recombinant Sp1 and the labeled DNA fragment (nucleotides
707 to
626) with and without mutations confirmed the actual binding
of Sp1 to the putative Sp1 binding site of nucleotides
650 to
638.
The partial disappearance of signals upon the addition of an excess
amount of unlabeled DNA fragments with mutations could be due to the
weak binding of Sp1 to another Sp1 binding site (nucleotides
658 to
646) exhibiting lower homology with the consensus sequence.
707 to
539.
707 to
539 seemed to control the
expression of a 3.1-kb DRG-specific transcript, because PC12
cell-derived stable transfectants with pH4-SK3.6 expressed a transcript
starting at position
82. On the other hand, Neuro2a cell-derived
stable transfectants with pH4-SK3.6 expressed mRNAs starting at
position
498 corresponding to 3.3- and 4.2-kb transcripts, although
reverse transcription-PCR analysis of these transfectants did not
exclude the presence of a transcript starting at position
82. The
promoter region of nucleotides
294 to
4 might control the
expression of the 3.3- and 4.2-kb mRNAs of the RFT-I gene. A N-Myc
binding site found in this region was not thought to be functional,
because the pBH4-A6Pst and pBH4-A6mPst (containing mutations at the
N-Myc binding site) constructs showed comparable promoter activity when
transfected into Neuro2a cells.
707
to
4, when the pBH4-SP0.7 construct was transfected into a primary
culture of DRG neurons. This activity was thought to be due to the
expression of the reporter gene in DRG neurons, because the same
construct showed far lower promoter activity in a primary dorsal root
culture. The difference between DRG and dorsal root cultures was the
presence or absence of neurons. Moreover, the pBH4-SP0.7 construct
showed low promoter activity in a primary cerebellum culture, which
contained another type of neuron, cerebellar granule cells. This
observation supports the notion that the region of nucleotides
707 to
4 of the RFT-I gene involves the promoter sequences sufficient for
the DRG neuron-specific gene expression.
538 to
4 might be necessary for sufficient promoter
activity. It is still possible that the Sp1 binding site and the
overlapping GSG-like element could act in a stage-specific manner
during the late embryonic period when DRG neurons are critically
dependent on NGF for their survival.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Yoshitaka Nagai, Director of the Glycobiology Research Group, and Dr. Tomoya Ogawa, Coordinator of the Research Group (Frontier Research Program of RIKEN), for continued support and encouragement of this research.
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FOOTNOTES |
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* This work was supported by a Frontier Research Program Grant from RIKEN and by Grants-in-aid for Scientific Research on Priority Areas 10152263 and 10178104, Grant-in-aid for Scientific Research (C) 09680639, and Grant-in-aid for Encouragement of Young Scientists 10780486 (to S. H.) from the Ministry of Education, Science, Sports 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) Y18013.
¶ Special Postdoctoral Researcher of the RIKEN.
To whom correspondence should be addressed: Molecular
Glycobiology, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan. Tel.: 81-48-467-9615; Fax: 81-48-462-4692.
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ABBREVIATIONS |
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The abbreviations used are:
1, 2-FT,
GDP-L-fucose:
-D-galactoside
2-
-L-fucosyltransferase;
RFT-I, rabbit H-blood type
1,2-FT;
DRG, dorsal root ganglia;
Se-type, secretor-type;
PCR, polymerase chain reaction;
RACE, rapid amplification of cDNA ends;
PCR, polymerase chain reaction;
CHO, Chinese hamster ovary;
NGF, nerve
growth factor;
bp, base pair(s);
kb, kilobase pair(s);
CMV, cytomegalovirus;
NGFI, nerve growth factor-induced gene. The
nomenclature for gangliosides and glycolipids follows the system of
Svennerholm (38)..
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REFERENCES |
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