From the Department of Molecular Glycobiology, Frontier Research
Program, The Institute of Physical and Chemical Research (RIKEN),
Wako, Saitama 351-01, Japan
The mouse gene encoding ST8Sia IV/PST, one of two
polysialic acid synthases, was isolated and characterized. The mST8Sia
IV/PST gene was found to comprise over 60 kilobases and to be composed of five exons. Primer extension analysis revealed that transcription started from 333 nucleotides upstream of the translational initiation site. Transfection with nested deletion mutants of the 5'-flanking region fused to the luciferase reporter gene revealed that the promoter
activity of the
107/+145 region was correlated with the gene
expression of mST8Sia IV/PST in embryonal carcinoma P19 and
neuroblastoma F11 cells. This proximal promoter region lacks an
apparent TATA box but has putative binding sites for transcription factors Sp1 and NF-Y (CCAAT binding protein) at nucleotide positions
66/
57 and
47/
37, respectively. Individual deletions and
mutations of the inverted Sp1 binding site or inverted NF-Y binding
site caused significant reduction of the promoter activity, indicating that each binding site was involved in essential transcription control.
Mobility shift assaying also revealed that Sp1 and NF-Y in a nuclear
extract of P19 cells bind to the promoter region of the mST8Sia IV/PST
gene. Deletion of the region from
60 to
40, which contains parts of
both the Sp1 and NF-Y binding sites, completely abolished the promoter
activity, suggesting that both Sp1 and NF-Y are synergetically involved
in transcription regulation of the mST8Sia IV/PST gene in P19 and F11
cells. Although the overall structures of the two polysialic acid
synthase genes (ST8Sia II/STX and IV/PST) are very similar, there is no
extensive sequence homology between the 5'-flanking regions of the
ST8Sia II/STX and IV/PST genes, suggesting that these two genes are
expressed under different regulatory systems.
 |
INTRODUCTION |
Polysialic acid (PSA)1
is a linear homopolymer of
2,8-sialic acid residues mainly
associated with the neural cell adhesion molecule (N-CAM) in mammalian
cells and is implicated in the reduction of N-CAM adhesion through its
large negative charge (1). In the late embryonic and early postnatal
stages, neurons mainly express the highly polysialylated form of N-CAM
(2, 3). However, in the course of neural development, the content of
PSA associated with N-CAM decreases, resulting in an increase in the adhesive ability of the N-CAM itself (3-5). Recent data imply important functions of PSA in the pathfinding and targeting in the
innervation of axons, migration of neuronal cells and tumor cells, and
spatial learning and memory (6-8).
In 1995, Eckhardt et al. (9) cloned the cDNA of a
sialyltransferase, which is the key enzyme for PSA expression in
Chinese hamster ovary cells, and named the enzyme
polysialyltransferase-1 (PST-1). We independently cloned a mouse
cDNA encoding an
2,8-sialyltransferase, ST8Sia IV, whose amino
acid sequence exhibits 99.8% identity to that of hamster PST-1, and
showed that ST8Sia IV is a PSA synthase (10). On the other hand, we
demonstrated that ST8Sia II/STX is another PSA synthase (12-17). In
the mouse, the amino acid sequences of the two types of PSA synthases,
ST8Sia II/STX and IV/PST, exhibit 56% identity, which is the highest
score among the sialyltransferases cloned so far. Northern blot
analysis indicated that expression of the mST8Sia II/STX gene was
restricted to the brain and testis, whereas the mST8Sia IV/PST gene was
expressed strongly in the lung, spleen, and heart, rather than the
brain (10, 12). Expression of the mST8Sia II/STX gene in the brain was
strictly regulated during development (12). Expression of the mST8Sia
IV/PST gene was also higher in fetal than adult brain but was less
regulated during brain development as compared with that of the mST8Sia II/STX gene (10). Our recent results indicated that mST8Sia II/STX and
IV/PST synthesize PSA of different sizes in vitro and in vivo (17, 18). However, it is not clear why two types of PSA synthases exist and how they are differently expressed.
To elucidate the mechanisms underlying the differential expression of
the mST8Sia II/STX and IV/PST genes, it is important to know the
structures and activities of their promoters. We recently reported the
entire genomic organization and the promoter structure of the mST8Sia
II/STX gene (19). We demonstrated that the minimal promoter region of
the mST8Sia II/STX gene conferred the cell type-specific expression in
the reporter gene. The minimal promoter was embedded in a GC-rich
domain (GC content, 74%), in which two Sp1 binding motifs as well as a
long purine-rich region were found, but it lacked TATA and CAAT boxes.
In the present study, we describe the genomic structure of the mST8Sia
IV/PST gene and its promoter sequence involved in the regulation of
transcription activity.
 |
EXPERIMENTAL PROCEDURES |
Isolation of Genomic and cDNA Clones Encoding mST8Sia
IV/PST--
A mouse genomic cosmid library was constructed and
screened as described previously (20). The locations of the exons of the mST8Sia IV/PST gene were determined by PCR (GeneAmp XL PCR Kit,
Perkin-Elmer) with specific oligonucleotide primers or by hybridizing
radiolabeled sialyltransferase cDNA to the same blots.
The 3'-untranslated region of the mST8Sia IV/PST cDNA was isolated
from a 3-day-old mouse brain cDNA library (20) by PCR using primers
O5-3A, O5-3B, and O5-3C (Table I).
View this table:
[in this window]
[in a new window]
|
Table I
Primers used in this study
Primers O5-3A, O5-3B, and O5-3C were used for isolation of the
3'-untranslated region of the mST8Sia IV/PST cDNA, and primers O5-EX2 and O5-N5 were used for 5'-RACE. Primer O5-EX2 was used for
primer extension analysis. Primers O5-ATGNco, O5-530X, O5-440X, O5-350N, O5-310X, and O5-150H include a restriction enzyme
recognition site (underlined, Nco, NcoI; X, XhoI;
N, NheI; H, HindIII), respectively, and were used
for construction of the plasmids. Primers Sp*-N, Sp*-C, FSp*-N, and
BSp*-C were used for construction of an Sp1 binding site-replaced
mutant and contain EcoRI sites. Primers Ap*-N, Ap*-C, and
FAp*-N were used for construction of an AP-2 site-replaced mutant and
contain BglII sites. The lowercase letters denote mutated
nucleotides.
|
|
PCR Amplification of the 5'-cDNA End
(RACE)--
Amplification of the 5' end of mST8Sia IV/PST cDNA was
performed as described previously (20). cDNA was synthesized by
reverse transcription of 5 µg of mouse brain poly(A) RNA using primer O5-EX2, a 32-mer oligonucleotide complementary to mST8Sia IV mRNA (nucleotide positions +325 to +293, Table I). After the cDNA had
been A-tailed, two consecutive PCRs were performed with two nested sets
of primers. For pair 1, the forward primer was
NotI-(dT)18 (Pharmacia Biotech Inc.), and the
reverse primer was O5-EX2. For pair 2, the forward primer was as above
but without the T-tail, 5'-AACTGGAAGAATTCGCGGCCGCAGGAA-3', and the
reverse primer was O5-N5 (Table I). The cDNA was amplified for 35 cycles of a step program (94 °C, 40 s; 55 °C, 40 s; and
72 °C, 60 s). The amplified products were subcloned into pUC119
and then sequenced.
Primer Extension Analysis--
pO5-22E1.5 was constructed by
subcloning a 1.5-kb EcoRI fragment from COS O5-22, which
contains the 1030-bp 5'-flanking region of the mST8Sia IV/PST gene,
into the pUC118 plasmid. The O5-EX2 primer was end-labeled with
[32P]ATP using T4 polynucleotide kinase. The radiolabeled
O5-EX2 was hybridized with 5 µg of poly(A) RNA prepared from mouse
brain or 5 µg of yeast tRNA, as a control to extension specificity, and then extended with Superscript II (Life Technologies, Inc.) as
described previously (19). The primer extension products were separated
on a 6% sequencing gel along with dideoxy-mediated a chain termination
sequencing reaction of pO5-22E1.5, using O5-EX2 as the primer.
Analysis of Promoter Activity--
To obtain various lengths of
the 5'-flanking region of the mST8Sia IV gene, PCR with Tth DNA
polymerase (GeneAmp XL PCR Kit, Perkin-Elmer) was performed using two
primers, O5-ATGNco (Table I) and a reverse sequencing primer, with
pO5-22E1.5 as the template. The mST8Sia IV/PST-luciferase fusion gene
expression plasmids were constructed by subcloning the following
restriction fragments from the PCR products into pPicaGene-Basic II
(pPGBII, Toyo-ink, Japan): pBO5-BN0.87 carries a 0.87-kb
BamHI-NcoI fragment and pBO5-SaN0.16 carries a
0.16-kb SacI-NcoI fragment, respectively. Other
series of deletion plasmids were constructed by subcloning the
restriction enzyme-digested PCR products amplified using the primer set
of O5-ATGNco and the restriction enzyme site introducing mutagenic
primers into pPGBII. The primers and template plasmids used were as
follows: pBO5-XN0.53 carries a 0.53-kb XhoI-NcoI fragment amplified by using the primer set of O5-530X/O5-ATGNco; pBO5-XN0.44 carries a 0.44-kb XhoI-NcoI fragment
amplified by using the primer set of O5-440X/O5-ATGNco; pBO5-NhN0.35
carries a 0.35-kb NheI-NcoI fragment amplified by
using the primer set of O5-350Nh/O5-ATGNco; pBO5-XN0.31 carries a
0.31-kb XhoI-NcoI fragment amplified by using the
primer set of O5-310X/O5-ATGNco; and pBO5-XH0.25 carries a
0.25-kb XhoI-HindIII fragment amplified by using
the primer set of O5-440X/O5-150H, respectively, with pO5-22E1.5 as the
template (Table I). For the pBO5-NhN3.5 construct, a 2.8-kb
NheI-XhoI fragment from COS O5-22 was subcloned
into pBO5-XN0.67 digested with the same restriction enzymes. As
controls, plasmids pBSV, containing the luciferase gene driven by the
SV40 promoter, and pPGBII, containing the promoterless luciferase gene, were transfected into parallel cultures of each cell line. The luciferase activity due to each luciferase reporter plasmid was normalized to the
-galactosidase activity by cotransfecting an internal control plasmid, pSR
-gal, carrying a
-galactosidase gene
under the control of the SR
promoter. In all the cell lines tested,
pPGBII was inactive to the expression of luciferase activity, whereas
pBSV caused a high level of expression.
NIH3T3, F11, undifferentiated-P19, Neuro2a, F9, B16, LL/2, C2C12, and
NMuMG cells were seeded at 5 × 104 cells per 60-mm
diameter dish in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum 24 h prior to transfection, respectively.
For the neural differentiation of P19 cells into neuronal cells, the
cells were seeded into and aggregated in bacteriological grade dishes
in the presence of 1 mM retinoic acid at the cell density
of 1 × 105/ml. After 3 days, the aggregates
were trypsinized, and then approximately 1 × 105
cells per 60-mm diameter dish (tissue culture grade dishes) were plated
in Dulbecco's modified Eagle's medium, 10% fetal calf serum 24 h prior to transfection.
The luciferase plasmid (5 µg) used as the reporter and the pSR
-gal
plasmid (0.5 µg) used as an internal control for transfection efficiency were transfected into the cells by means of LipofectAMINE (Life Technologies, Inc.). After 48 h transfection, the cells were
washed three times with phosphate-buffered saline and then lysed with
cell lysis buffer (PG
-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 quadruplicate, averaged, and then normalized to the
-galactosidase activity to correct for the transfection efficiency.
-Galactosidase activity was measured using a Luminescent
-Galactosidase Detection Kit II (CLONTECH).
Site-directed Mutagenesis and Deletion of the Sp1, NF-Y, and AP-2
Binding Sites--
The primers used for the construction of
site-replaced mutants and site-directed deletion mutants of the Sp1,
NF-Y, and AP-2 binding sites are shown in Table I. All PCRs were
performed using pO5-22E1.5 as the template. The Sp1 binding
site-replaced mutant, pBO5-XN0.53(Sp1*), was constructed by subcloning
into XhoI-NcoI-digested pPGBII an
XhoI-EcoRI fragment amplified with the primer set
of O5-530X/Sp*-N and an EcoRI-NcoI fragment with
the primer set of Sp*-C/O5-ATGNco. The Sp1 binding site-deleted
mutants, pBO5-XN0.53 (Sp1*
75/
56) and pBO5-XN0.53 (Sp1*
60/
40),
were constructed as follows. pBO5-XN0.53 (Sp1*
75/
56) was
constructed by subcloning into pPGBII an
XhoI-EcoRI fragment amplified with the primer set of O5-530X/FSp*-N and an EcoRI-NcoI fragment with
the primer set of Sp*
C/O5-ATGNco. pBO5-XN0.53 (Sp1*
60/
40) was
constructed by subcloning into pPGBII an
XhoI-EcoRI fragment amplified with the primer set
of O5-530X/Sp*-N, and an EcoRI-NcoI fragment with the primer set of BSp*-C/O5-ATGNco. A series of NF-Y binding
site-deleted mutants, pBO5-XN0.53 (
53/
51), pBO5-XN0.53
(
50/
48), pBO5-XN0.53 (
47/
45), pBO5-XN0.53 (
44/
42),
pBO5-XN0.53 (
41/
39), and pBO5-XN0.53 (
39/
37), was
constructed by subcloning the NcoI-XhoI-digested PCR products into pPGBII. For the construction of
pBO5-XN0.53(
53/
51), PCR (94 °C, 40 s; 50 °C, 40 s; and 72 °C, 90 s) was performed using two fragments amplified
with the primer sets of O5-530X/A-N and A-C/O5-ATGNco, respectively, as
templates, without primers for the first 5 cycles, followed by
amplification in the presence of the primer set of O5-530X/O5-ATGNco
for 30 cycles. pBO5-XN0.53(
50/
48), pBO5-XN0.53(
47/
45),
pBO5-XN0.53(
44/
42), pBO5-XN0.53(
41/
39), and
pBO5-XN0.53(
39/
37) were constructed as above using the corresponding primer sets. The AP-2 binding site-replaced mutant, pBO5-XN0.53 (Ap2*), was constructed by subcloning into
XhoI-NcoI-digested pPGBII an
XhoI-BglII fragment amplified with the primer set
of O5-530X/Ap*-N and an EcoRI-NcoI fragment with
the primer set of Ap*-C/O5-ATGNco. The AP-2 binding site-deleted
mutant, pBO5-XN0.53(
+42/+87), was constructed by subcloning into
pPGBII an XhoI-BglII fragment amplified with the
primer set of O5-530X/FAp*-N and a BglII-NcoI fragment with the primer set of Ap*-C/O5-ATGNco. All plasmids were
verified by restriction mapping and sequencing.
Cloning of the NF-Y Gene--
The NF-YA gene was
cloned from P19 cells by reverse transcriptase-PCR using primers
5'-GAAGCTTCAGGACTCTTAAC-3' and 5'-TGACTGATCAGCTCTGCCACC-3' (22). PCR
products were cloned into pBluescriptII SK+ and then sequenced. The
NF-YB gene (22) was cloned from an adult mouse brain
cDNA library by plaque hybridization and then sequenced.
In Vitro Transcription and Translation--
In vitro
transcription of the NF-YA and NF-YB genes was
performed using an mCAP mRNA capping kit (Stratagene) according to the manufacturer's instructions. The resulting mRNA samples (2 µg) were applied to a rabbit reticulocyte lysate system (Amersham Corp.) for in vitro translation.
Gel Shift Assay--
The DNA fragment from
107 to
16 was
prepared from pBO5-XN0.44 by digestion with XhoI and
PstI and then end-labeled with [32P]dCTP using
Klenow polymerase. Binding assays were performed with a labeled probe
(10-20 k cpm) in the presence of 2 µg of poly(dI-dC)·poly(dI-dC)
(Pharmacia) and 2 µg of a nuclear protein extract or an appropriate
volume of the products translated in vitro. Binding
reactions were carried out for 30 min at 0 °C in 25 mM
HEPES-KOH (pH 7.9), 0.5 mM EDTA, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and
10% glycerol. Competitor fragments or anti-Sp1 antibodies (Santa Cruz
Biotechnology) were included where indicated. The DNA fragment from
339 to
221, which was prepared from pBO5-XN0.63 by digestion with
XhoI and BlnI, and the DNA fragment from
107 to
16 were used as nonspecific and specific competitors, respectively.
The synthetic DNA fragments, 5'-CGCCCCCTCAGCACGGTGATTGGCTGG-3'
(nucleotide positions from
62 to
36) and
5'-AGGCCAGCCAATCACCGTGCTGAGGGGG-3' (complementary to nucleotide
positions from
60 to
33), were used as competitors after the two
synthetic DNAs had been annealed. After incubation, the samples were
loaded onto a 4% polyacrylamide gel (acrylamide:bisacrylamide, 19:1)
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).
 |
RESULTS |
Isolation of mST8Sia IV/PST Genomic Clones--
The screening of
an NIH3T3 cell cosmid library with mST8Sia IV/PST cDNA resulted in
the isolation of three independent genomic clones. A restriction map of
the approximately 100-kb region containing the mST8Sia IV/PST gene is
shown in Fig. 1. The locations of the mST8Sia IV/PST exons were determined by PCR and Southern blot hybridization using a variety of oligonucleotides designed according to
the known mST8Sia IV/PST cDNA sequence. Since cross-hybridization experiments indicated that CosO5-12 and CosO5-19 did not overlap, further screening of 5 × 105 genomic clones (inserts
ranging from 30 to 40 kb in size) was performed with a 2-kb fragment of
CosO5-12 including exon 4 and a 1.3-kb fragment of the T3 primer
end of CosO5-19 as probes. However, no overlapping clones were
obtained. Southern blot analyses of the NIH3T3 genomic DNA using the
above two probes showed that the distance between the termini of
CosO5
12 and
19 was over 12 kb (data not shown).

View larger version (7K):
[in this window]
[in a new window]
|
Fig. 1.
Structure of the mST8Sia IV/PST gene.
A, schematic representation of the mST8Sia IV/PST gene
locus. The shaded bars denote the three genomic clones
(CosO5-22, CosO5-12, and CosO5-19) isolated from a cosmid library. The
five exons of the mST8Sia IV/PST gene are shown as filled
rectangles and the 5'- and 3'-untranslated regions as open
rectangles. The intronic sequences are indicated by the
solid lines between the exons. B, domain
structure of mouse brain ST8Sia IV/PST. The boxes indicate
translated sequences and horizontal bars indicate
untranslated sequences. A hydrophobic signal anchor sequence
(transmembrane domain) and sialyl motifs L and S are denoted as
TM, SM-L, and SM-S, respectively. The
splicing sites are indicated by vertical arrows.
|
|
We sequenced the exons to determine their exact sizes and the
intron/exon junctions (Table II). The
sequences of all the intron/exon splice junctions conformed to the
GT-AG rule (23). The mST8Sia IV/PST cDNA was divided into 5 exons,
ranging from 132 to 5656 bp, with intron sizes of 8-24 kb, and
spanning more than 60 kb of genomic DNA (Fig. 1). Exon 1 contained the
entire 5'-untranslated region and the beginning of the coding region to
amino acid residue 37, containing a cytoplasmic domain, a short
hydrophobic signal anchor sequence, and a part of the stem domain.
Exons 3-5 encoded the putative active domain of the enzyme, and exon 5 contained a large 3'-untranslated region. We previously reported
mST8Sia IV/PST cDNA sequences lacking the whole 3'-untranslated
region. Therefore, to determine the 3' end of the 5-kb mST8Sia IV/PST transcript, the mouse brain cDNA library was screened by PCR using primers distributed along the 3' part of the gene. Sequence analyses of
the PCR products revealed that the size of the transcribed RNA was 6786 bp, thus it included a large 3'-untranslated region of 5372 bp. Poly(A)
addition occurred 23 nucleotides downstream of the sequence at the T
residue of the polyadenylation signal (AATAAA).
View this table:
[in this window]
[in a new window]
|
Table II
Exon/intron junctions of the mST8Sia IV/PST gene
The nucleotide sequences at the intron (lowercase letters) and exon
(uppercase letters) junctions are shown. The derived amino acid
sequence is displayed below the nucleotide sequence. Exons are numbered
from the 5' end, as described in Fig. 1. The exon and intron sizes are
indicated in bp. The numbering starts, position +1, at the adenosine of
the initiator methionine.
|
|
Mapping of the Transcription Initiation Site--
The
transcription initiation site was determined by primer extension with
RNA recovered from 1-day-old mouse brain, in which the mST8Sia IV/PST
gene was expressed (Fig. 2). Northern
blot analysis indicated that the mST8Sia IV/PST gene gave a single transcript, whose size was about 5 kb. The primer extension products obtained with primer O5-EX2 were analyzed on a 6% sequencing gel. The
end points of the extension were determined by comparison with a
sequence ladder derived from the same genomic DNA template and the
original primer, O5-EX2. The end point was determined to be a guanine
(+1), which corresponded to a position 333 nucleotides upstream from
the initiation codon, ATG. Moreover, we performed 5'-RACE-PCR on
newborn mouse brain poly(A) RNA to identify the 5' end of the mST8Sia
IV/PST gene, and the longest RACE-PCR product corresponded to the
transcription initiation site determined in the primer extension
experiments. Therefore, mST8Sia IV/PST mRNA was transcribed at a
single position 333 nucleotides upstream from the initiation codon at
least in mouse brain and gave a single transcript.

View larger version (82K):
[in this window]
[in a new window]
|
Fig. 2.
Identification of the transcription
initiation sites of the mST8Sia IV/PST gene of mouse brain. The
transcription initiation site has been mapped by means of primer
extension analysis. For the primer extension reaction, the O5-EX2
primer was [ -32P]ATP-end-labeled, hybridized with 5 µg of poly(A) RNA from 1-day-old mouse brain or 5 µg of yeast tRNA,
and then reverse-transcribed. The primer extended products were run on
a sequencing gel along with a sequencing reaction of pO5-22E1.5, using
O5-EX2 as the primer. Lane 1, primer extension with yeast
tRNA; lane 2, with 1-day-old mouse brain mRNA. The
arrow indicates the position of the transcription initiation
site.
|
|
Analysis of the 5'-Flanking Region--
Analysis of the sequence
immediately upstream of the transcription initiation site revealed that
the mST8Sia IV/PST gene promoter consists of a G + C-rich sequence
lacking a canonical TATA box (Fig. 3). In
this promoter, an apparent G + C-rich region extends from
100 to +150
(GC content, 64%). The G + C-rich region of the mST8Sia IV/PST gene
promoter is shorter and its GC content is lower than those of the
mST8Sia II/STX gene promoter (nt
175 to +168, 74%) (18). The
TATA-less mST8Sia IV/PST gene promoter contains an inverted Sp1 binding
site at positions
66 to
57 (8 of 10 matching), an inverted NF-Y
(CCAAT binding protein) binding site at positions
47 to
37 (9 of 11 matching), and an AP-2 binding site, CC(G/C)C(A/G)GGC, at
positions +84 to +91 (7 of 8 matching). The 1-kb 5'-flanking sequence
of the mST8Sia IV/PST gene does not exhibit extensive homology with the
upstream region of the mST8Sia II/STX gene.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 3.
Nucleotide sequence of the 5'-flanking region
of the mST8Sia IV/PST gene. The transcription initiation site (+1)
is indicated by a vertical arrow. The sequence of the first
exon is shown in capital letters and those of the
untranscribed regions in lowercase letters. The coding
sequence of the first exon is shown as codon triplets. The
putative binding sites for several transcription factors are indicated.
The O5-EX2 primer is indicated by arrows. For the detection
of promoter activity, the start point of each construction is indicated
by an arrowhead.
|
|
Demonstration of Promoter Activity--
To characterize the
regions regulating the transcription activity of the gene, we
constructed a series of chimeric plasmids containing different lengths
of the 5'-flanking region of the mST8Sia IV/PST gene fused to the
promoterless luciferase gene in pPGBII (Fig.
4). One of the constructs, pBO5-NhN3.5,
was assayed for promoter activity by transient transfection into
several cell lines at first (Table III).
Of these cell lines, embryonal carcinoma P19 cells showed the highest
promoter activity, neuroblastoma F11 cells showed a moderate level of
promoter activity, and NIH3T3 fibroblast cells showed a very low level
of promoter activity. The level of endogenous mST8Sia IV/PST gene
expression in P19 cells was similar to that in F11 cells, but NIH3T3
cells do not express the mST8Sia IV/PST gene at all (data not shown).
Thus, we decided to use P19, F11, and NIH3T3 cells for the following study for comparison of the promoter activities.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
mST8Sia IV/PST gene promoter activity and
identification of the regulatory regions. A schematic
representation of DNA constructions containing various lengths of the
mST8Sia IV/PST promoter linked to the luciferase gene is presented.
Each DNA fragment subcloned into the luciferase reporter plasmid is
defined to its position in the mST8Sia IV/PST gene promoter relative to the transcription initiation site (+1). 5 µg of each construct was
transfected into NIH3T3, F11 (F11), undifferentiated P19
(P19), or neural-differentiated P19 (P19(Dif))
cells. Luciferase activity was normalized to the -galactosidase
activity of a cotransfected internal control plasmid, pSR -Gal, and
expressed as a percentage of the SV40 promoter activity in that cell
type. Each datum is the average of four or five experiments.
|
|
View this table:
[in this window]
[in a new window]
|
Table III
Relative promoter activities of several types of cells
pBO5-NhN3.5 was used to assay the promoter activity as luciferase
activity. Luciferase activity was normalized as to the
-galactosidase activity of a cotransfected internal control plasmid,
pSR -Gal, and expressed as a percentage of the SV40 promoter activity
in that cell type.
|
|
Sequential deletions of the region between nucleotide positions
3140
and
541 had little effect on the luciferase activity. Further
deletions from nucleotide positions
541 to
200 increased the
promoter activity in differentiated P19 cells as well as in undifferentiated P19 and F11 cells. Although further deletions from
nucleotide positions
200 to
107 (pBO5-XN0.44) had little effect on
the activity, deletions from nucleotide positions
200 to
15
(pBO5-NhN0.35) caused a drastic decrease in the promoter activity from
one-fourth to one-fifth that of pBO5-XN0.44 in the examined cells. On
further truncation beyond the transcriptional initiation site to
nucleotide position +26, the promoter activity of pBO5-XN0.31 was
reduced to the basal level. Deletion to nucleotide position +173
reduced the promoter activity to the level seen with the promoterless
control vector, pPGBII. On the other hand, truncation from nucleotide
positions +333 to +145 (pBO5-XH0.25) had little effect. All the
constructs exhibited barely detectable activity in NIH3T3 cells. Taken
together, we concluded that the region between
107 and +145 was
responsible for high levels of expression in mST8Sia IV/PST
mRNA-expressing cells. We have shown that P19 cells express the
endogenous mST8Sia IV/PST gene and that the level of the gene
expression slightly increases during neural differentiation on retinoic
acid treatment (24). It should be noted that the promoter activities
due to most of the constructs were approximately 1.5-fold higher than
those in undifferentiated P19 cells. This observation correlates with
the mST8Sia IV/PST mRNA expression in P19 cells during neural
differentiation.
Mapping of the Functional Regions in mST8Sia IV/PST
Transcription--
Figs. 3 and 4 indicate the presence of a putative
Sp1 binding site (inverted form, nt
66 to
57), a putative NF-Y
binding site (inverted form, nt
47 to
37), and a putative AP-2 site (nt +84 to +91) within pBO5-XN0.53, which gave the maximum
transcriptional activity. To examine the involvement of the putative
Sp1, NF-Y, and AP-2 binding sites in mST8Sia IV/PST mRNA
transcription, we first constructed two internal deletion mutants of
pBO5-XN0.53, one of which lacked the inverted Sp1 binding site and its
upstream sequence (nt
75 to
56; pBO5-XN0.53 (
75/
56)), and
the other lacked the Sp1 binding site and its downstream sequence (nt
60 to
40; pBO5-XN0.53 (
60/
40), in which the inverted NF-Y
binding site was also deleted) (Fig. 5).
In addition, we introduced a mutation into the inverted Sp1 binding
site by replacing four nucleotides (pBO5-XN0.53 (Sp1*); cctccgcccc
changed to cctccgaatt), which failed to bind to recombinant
Sp1 (data not shown). The deletion upstream of the inverted Sp1 binding
site reduced the promoter activity to 30-40% (construct pBO5-XN0.53
(
75/
56)) as compared with that of the wild-type construct. The
promoter activity of the Sp1-mutated construct (pBO5-XN0.53 (Sp1*)) was also reduced to 40-50% (Fig. 5). In contrast, the deletion downstream of the inverted Sp1 binding site (construct pBO5-XN0.53 (
60/
40)) caused a drastic decrease in the promoter activity to the basal level
in all cells examined (Fig. 5). A mobility shift experiment involving a
nuclear protein extract of P19 cells revealed that only one shifted
band (Fig. 6, band C) disappeared in the presence of the
synthetic DNA fragment corresponding to nucleotide positions
62 to
33, suggesting the nuclear protein of P19 cells bound in this region
(Fig. 6, lane 5).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 5.
mST8Sia IV/PST gene promoter activity on
mutant analysis. A schematic representation of pO5-XN0.53 mutants
with deletion or mutation of the Sp1, NF-Y, and AP-2 binding sites in
the mST8Sia IV/PST promoter, respectively. The sequences of the
putative inverted Sp1 binding site (Sp1; nt 66 to 57), the putative
inverted NF-Y binding site (NF-Y; nt 47 to 37), and the putative
AP-2 binding site (AP-2; nt +84 to +91) are shown in the figure. The
deleted parts are indicated by the notch marks. The
mutational sequences are shown as underlined italics. The
relative promoter activity was measured as luciferase activity, which
was normalized to the -galactosidase activity of a cotransfected
internal control plasmid, pSR -Gal. The values are presented as
percentages of the promoter activity due to pO5-XN0.53, from which was
subtracted the basal activity due to pO5-XN0.31. The activities of the
promoter of pO5-XN0.53 relative to those of pBSV in F11,
undifferentiated P19, and differentiated P19 cells were 93.6 ± 13.6, 109.1 ± 13.4, and 126.3 ± 10.9%, respectively. The
basal activities due to pO5-XN0.31 relative to those of pBSV in F11,
undifferentiated P19, and differentiated P19 cells were 15.0 ± 3.7, 21.4 ± 1.4, and 19.5 ± 5.2%, respectively.
|
|

View larger version (61K):
[in this window]
[in a new window]
|
Fig. 6.
Gel shift assays of the mST8Sia IV/PST
proximal promoter region with a nuclear extract of P19 cells. The
5'-end-labeled DNA fragment from 107 to 16 (lane 1) was
incubated with the nuclear extract of P19 cells either alone
(lane 2) or with 25 times the amount of the non-labeled
specific competitor (DNA fragment from 107 to 16, lane
3), 25 times the amount of the non-labeled nonspecific competitor
(DNA fragment from 339 to 221, lane 4), and 16 pmol of
the non-labeled specific competitor (synthetic DNA fragment from 62
to 33, lane 5), the anti-Sp1 antibodies (lane
6), and then subjected to the gel shift assay. Lane 7 shows the results of a gel shift assay involving 0.4 footprinting units of recombinant Sp1 instead of the nuclear extract of P19 cells.
|
|
To clarify the involvement downstream of the Sp1 binding site, we
constructed a series of internal deletion mutants of pBO5-XN0.53 and
analyzed their promoter activities. Each 3-base deletion from nucleotide positions
53 to
45 of pBO5-XN0.53 (constructs
pBO5-XN0.53 (
53/
51), pBO5-XN0.53 (
50/
48), and pBO5-XN0.53
(
47/
45)) had little effect on the promoter activity (Fig. 5). In
contrast, the deletion of the sites from
41 to
39 and from
39 to
37 (constructs pBO5-XN0.53 (
41/
39) and pBO5-XN0.53
(
39/
37)) led to a reduction of the promoter activity to 42% (in
the case of F11 cells), 15% (in the case of undifferentiated P19
cells), and 20% (in the case of differentiated P19 cells) as compared with that of the wild-type construct (Fig. 5). Deletion of the site
from
44 to
42 moderately reduced the promoter activity to 50-70%
that of the wild-type construct. Therefore, the site from
44 to
37,
corresponding to the inverted NF-Y binding site, was required for the
maximal promoter activity in both differentiated and undifferentiated
P19 cells and F11 cells. Since the deletion of the
60/
40 site
reduced the promoter activity to the basal level, but the deletion of
the
75/
56,
41/
39, or
39/
37 site reduced it only partly,
both the inverted Sp1 binding site at
65/
57 and the inverted NF-Y
binding site at
44/
37 are critical for the function of the promoter
in these cells.
The mutation of the AP-2 binding site (pBO5-XN0.53 (Ap2*)) had little
effect on the promoter activity. On the other hand, the deletion of the
region between nucleotide positions +42 and +87 increased the promoter
activity (Fig. 5).
Involvement of Sp1 and NF-Y in Transcription of the mST8Sia IV/PST
Gene--
To determine whether or not the inverted Sp1 binding site
between nucleotide positions
65 and
57 is recognized by Sp1, we performed a mobility shift assay. In the mobility shift experiments involving the DNA fragment from
107 to
16, recombinant Sp1 bound to
the DNA fragment (Fig. 6, lane 7). When a nuclear protein
extract of undifferentiated P19 cells was used for the experiment, the labeled DNA fragment appeared as several shifted bands (Fig. 6, bands A-D). These shifted bands were not abolished by the
nonspecific competitor (DNA fragment from
339 to
221) but
completely disappeared in the presence of the non-labeled specific
competitor (DNA fragment from
107 to
16, the same as the labeled
probe), indicating that the nuclear extract of P19 cells contained some
proteins that specifically bind to this fragment. The corresponding
band, which was observed in the presence of recombinant Sp1, appeared
(lane 2) in the presence of the nuclear protein extract of
P19 cells (band B). This shifted band disappeared on the
addition of the anti-Sp1 polyclonal antibodies (Fig. 6, lane
6). The same results were obtained with nuclear protein extracts
of differentiated P19 cells and F11 cells. Thus, the inverted Sp1 site
at
65/
57 was functional in the examined cells.
Since the inverted CCAAT motif, which corresponds to the NF-Y binding
site, was included in the site from
44 to
37, we analyzed whether
or not this motif was recognized by NF-Y. In the mobility shift
experiment involving the DNA fragment from
107 to
16, NF-Y
translated in vitro bound to the DNA fragment (Fig.
7, lane 3), whose mobility
corresponded to that of band C observed when a nuclear
extract of P19 cells was used (Fig. 6, lane 2, and Fig. 7,
lane 2). The shifted band was not abolished by the
nonspecific competitor (DNA fragment from
339 to
221) but
completely disappeared in the presence of the non-labeled specific
competitor (synthetic DNA fragment from
62 to
33; Fig. 6,
lane 5, and Fig. 7, lane 5). Thus, the inverted
CCAAT motif at
44/
37 was recognized by NF-Y. These results
suggested that Sp1 and NF-Y are involved in the transcription of
mST8Sia IV/PST mRNA.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 7.
Gel shift assays of the mST8Sia IV/PST
proximal promoter region with NF-Y translated in
vitro. The 5'-end-labeled DNA fragment from 107 to 16
(lane 1) was incubated with the nuclear extract of P19 cells
(lane 2). The labeled DNA fragment was also incubated with
NF-Y translated in vitro either alone (lane 3), with 25 times the amount of the non-labeled nonspecific competitor (DNA
fragment from 339 to 221, lane 4), or with 20 pmol of
the non-labeled specific competitor (synthetic DNA fragment from 62 to 33, lane 5) and then subjected to the gel shift
assay.
|
|
 |
DISCUSSION |
We recently reported the genomic organization and promoter
activity of the mST8Sia II/STX gene, a PSA synthase gene, whose expression is highly regulated during brain development (19). In the
present study, we showed that the genomic organization of the mST8Sia
IV/PST gene, another PSA synthase gene, is highly similar to that of
the mST8Sia II/STX gene, whereas the sequence of the 5'-flanking region
of the mST8Sia IV/PST gene does not exhibit extensive homology with the
upstream region of the mST8Sia II/STX gene. We showed that the proximal
promoter region of the mST8Sia IV/PST gene has the ability to express
the transcriptional activity, which correlated with the endogenous
mST8Sia IV/PST gene expression in several cell lines.
So far, the genomic organizations of six other sialyltransferase genes
have been reported (19, 20, 25-29). Among them, the genomic structures
of the mST8Sia II/STX gene is fairly similar to that of the mST8Sia
IV/PST gene. In particular, three introns are inserted into the regions
coding for the putative active domains of the enzymes, mST8Sia II/STX
and IV/PST (Fig. 8A). The
entire amino acid sequence of mST8Sia IV/PST shows 56% identity with that of mST8Sia II/STX, and its putative active domain exhibits higher
similarity to that of mST8Sia II/STX (Fig. 8B). However, the
amino acid sequences of exons 1 and 2 in mST8Sia IV/PST are not
conserved in the corresponding exons of mST8Sia II/STX. On the other
hand, both the genomic organization and the amino acid sequence of the
mST8Sia IV/PST gene showed no similarity, except in the sialyl motifs,
to other known
2,3- and
2,6-sialyltransferase genes. These
observations suggest that the mST8Sia II/STX and IV/PST genes are
evolutionarily related and distant from other sialyltransferase
genes.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 8.
Comparison of the genomic structures of the
mST8Sia IV/PST and II/STX genes. A, intron/exon structures
of the mST8Sia IV/PST and II/STX genes. The protein domain structure is
represented schematically by a rectangle, which is
subdivided to show the major structural elements of the protein. Sialyl
motifs L and S are underlined. The nucleotide sequences of
the exons at identical positions in the exon/intron junctions of the
mST8Sia IV/PST and II/STX genes are shown. The derived amino acids are
indicated. B, comparison of the deduced amino acid sequences
of the five exons of the mST8Sia IV/PST gene with those of the
corresponding exons of the mST8Sia II/STX gene.
|
|
In this study, we mapped a highly active promoter region of the mST8Sia
IV/PST gene by transient transfection of a series of deleted promoter
sequences in P19 and F11 cells (Fig. 4). Deletion analyses demonstrated
that the promoter sequence from
107 to +15 is critical for the
function of the promoter, because its removal effectively reduced the
reporter gene expression. The promoter activity of the pBO5-XN0.53
construct in F11, P19, and NIH3T3 cells was correlated with the
endogenous gene expression in each cell line. The proximal promoter
region of the mST8Sia IV/PST gene may be capable of directing specific
expression of the gene. By using an in vitro neural
differentiation model system with P19 cells, we previously revealed
that the promoter activity of the mST8Sia II/STX gene (nt
158 to
+167) was low in undifferentiated P19 cells but that it increased about
10 times during neuronal differentiation. However, in the case of the
promoter region of the mST8Sia IV/PST gene, a drastic increase in the
activity was not observed during the differentiation. Therefore the
proximal promoter regions of both mST8Sia II/STX and IV/PST are
differently regulated during at least P19 cell differentiation. In
fact, mST8Sia II/STX mRNA expression increased 20-fold during
differentiation, but mST8Sia IV/PST mRNA expression increased only
a few times (24). Therefore, the proximal promoter regions may possess
specific regulatory elements.
The proximal promoter region of the mST8Sia IV/PST gene contained an
inverted Sp1 binding site (nt
64 to
57) and an inverted CCAAT motif
(NF-Y binding site, nt
47 to
37). A mobility shift assay showed
that the inverted Sp1 binding site was functional in the examined cells
(Fig. 6). The results of deletion and mutation of the Sp1 binding site
suggested that the Sp1 binding site is partly involved in transcription
regulation in P19 and F11 cells (Fig. 5). We also showed the
involvement of NF-Y in the transcriptional regulation, NF-Y binding to
the inverted CCAAT motif located in the region from
44 to
37 in
mobility shift assays (Fig. 7). It should be noted that deletion of the
60 to
40 region (pBO5-XN0.53(
60/
40)) abolished the promoter
activity almost completely, whereas deletion of either the inverted Sp1
site (
64/
57) or the inverted NF-Y site (
44/
37) reduced the
promoter activity only partly (about 40% as compared with the wild
type), and deletion of the site from
53 to
45 had little effect on
the promoter activity. Thus, the two different sites, the inverted Sp1
site and the inverted NF-Y binding site, are required for the promoter
activity in P19 and F11 cells. Probably, the synergetic effect of Sp1
and NF-Y is essential for the transcription of mST8Sia IV/PST mRNA.
Sp1 and NF-Y are thought to be ubiquitous transcription factors. In fact, NIH3T3 cells express Sp1 and NF-Y at almost the same levels to
P19 and F11 cells (data not shown), although the promoter activity in
NIH3T3 cells is very low. Therefore, the minimal promoter region identified in this study seems to be an essential transcription unit,
and some other transcription factors may be involved in the specific
promoter activity in P19 and F11 cells. The mobility shift experiment
indicated the occurrence of other nuclear proteins that specifically
bind to the proximal promoter region of mST8Sia IV/PST. This may
suggest the existence of other sites that are required for the
transcriptional regulation of the mST8Sia IV/PST gene in the proximal
promoter region. However, we could not identify such additional sites
at this stage. Identification of such sites is required.
We recently demonstrated that the minimal promoter region of the
mST8Sia II/PST gene conferred cell type-specific expression in the
reporter gene. The minimal promoter was embedded in a GC-rich region
(GC content, 74%), in which two Sp1 binding motifs as well as a long
purine-rich region were found, but it lacked TATA and CAAT boxes.
Comparison of the promoter regions of the mST8Sia II/STX and IV/PST
genes revealed no extensive sequence homology (Fig.
9). However, both the promoters of these
two genes have functional Sp1 binding site(s) but lack canonical TATA
boxes. This type of promoter is usually associated with housekeeping genes but has also been found in a number of tissue-specific genes, including the neural cell-specific promoters of the neuron-specific enolase, type II sodium channel, syanpsins I and II, and
D1A dopamine receptor genes (30). Although Sp1 binding
sites are found in the proximal promoter regions of the mST8Sia II/STX
and IV/PST genes, NF-Y binding sites are not found in the proximal
promoter region of the mST8Sia II/STX gene (
158/+167). In addition to the difference that NF-Y is involved in the regulation of the expression of the mST8Sia IV/PST gene, but not in that of the mST8Sia
II/STX gene, identification of other factors that interact with the
proximal promoter regions of the mST8Sia II/STX and IV/PST genes may
facilitate understanding of the differential regulation of the two
genes. For example, there is a putative cAMP-responsive element-binding
protein binding site in the proximal promoter regions of both genes.
Now, we are trying to identify regulatory factors, including
cAMP-responsive element-binding protein, that interact with the
proximal promoter regions of the mST8Sia II/STX and IV/PST genes.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 9.
Comparison of the 5'-flanking regions of the
mST8Sia IV/PST and II/STX genes. The putative binding sites for
several transcription factors are shown schematically. The minimal
promoter regions of the mST8Sia IV/PST and II/STX genes are shown by
bold lines. CREB, cAMP-responsive element-binding
protein.
|
|
We are grateful to Dr. Yoshitaka Nagai,
Director of the Glycobiology Research Group, and Dr. Tomoya Ogawa,
Coordinator of the Group, Frontier Research Program of the Institute of
Physical and Chemical Research (RIKEN), for their continued support and encouragement regarding our research.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X86000 and Y09483-8.