From the
A 13-kilobase pair genomic DNA encoding a 78-amino acid
brain-specific calmodulin-binding protein kinase C (PKC) substrate,
neurogranin (Ng/RC3; also known as RC3 or p17), has been sequenced. The
Ng/RC3 gene is composed of four exons and three introns, with the
protein-coding region located in the first and second exons. This gene
was found to have multiple transcriptional start sites clustered within
20 base pairs (bp); it lacks the TATA, GC, and CCAAT boxes in the
proximal upstream region of the start sites. The promoter activity was
characterized by transfection of 293 cells with nested deletion mutants
of the 5`-flanking region fused to the luciferase reporter gene. A
minimal construct containing bp +11 to +256 was nearly as
active as that covering bp
Neurogranin (Ng/RC3),
Ng/RC3 is
expressed in the neurons of the central nervous system and is
especially enriched in neostriatum, neocortex, and hippocampus
(1, 3) . Within neurons, this protein is accumulated in
the cell bodies and dendrites, where it is localized predominantly in
the dendritic shafts and spines
(1, 3, 16) . The
expression of this gene is relatively low in the fetal and newborn rat
brain, and an accelerated expression occurs 2-3 weeks after birth
(1, 3) . The cellular and subcellular localizations and
the pattern of expression of Ng/RC3 during development, in many
respects, resemble those of the PKC-
Previously, we characterized the 5`-flanking
region of the rat PKC-
A homology search of the 2 kb upstream from the
transcriptional initiation site revealed several potential
transcriptional regulatory element sequences for SP1 at nucleotides
The cloned Ng/RC3 genomic DNA fragment encompasses
The Ng/RC3 gene resembles many other protein-coding genes from the
brain, such as PKC-
The expression
of the appropriate neuronal phenotype can be modulated by
developmental, spatial, and local environmental influences
(47, 48, 49) . These extracellular signals
impinge on receptors located at the cell membrane that lead to
transcriptional control involving the assembly of multiprotein
complexes on enhancers and promoters
(50) . Several genes
expressed in the brain, including those encoding dopamine
Phorbol ester is the most potent stimulator of Ng/RC3
gene expression. The stimulatory effects are
The 5`-flanking regions of the Ng/RC3 and
PKC-
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) U22062.
We thank Dr. Joseph B. Watson for providing Ng/RC3
cDNA, Dr. John L. Knopf for the various PKC isozyme cDNAs, and Dr.
Peter Blumberg (NCI) for recombinant PKC-
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1508 to +256, whereas a shorter
one covering bp +40 to +256 had a greatly reduced activity.
Between bp +11 and +40 lies a 12-nucleotide sequence
(CCCCGCCCACCC) containing overlapping binding sites for AP2
(CCGCCCACCC) and SP1 (CCCGCC); this region may be important for
conferring the basal transcriptional activity of the Ng/RC3 gene. The
expression of a Ng/RC3-luciferase fusion construct
(
1508/+256) in transfected 293 cells was stimulated by
phorbol 12-myristate 13-acetate (PMA), but not by cAMP, arachidonic
acid, vitamin D, retinoic acid, or thyroxines T
and
T
. PMA caused a 2-4-fold stimulation of all the
reporter gene constructs ranging from +11/+256 to
1508/+256. The stimulatory effects of PMA could be
magnified by cotransfection with both Ca
-dependent
and -independent phorbol ester-binding PKC-
, -
,
-
, -
, -
, and -
cDNAs, but not by
non-phorbol ester-binding PKC-
cDNA. The Ng/RC3 and PKC-
genes have a similar expression pattern in the brain during
development. These two genes share at least four conserved sequence
segments 1.5 kilobase pair upstream from their transcriptional start
sites and a gross similarity in that they possess several AT-rich
segments within bp
550 to
950. A near homogeneous 20-kDa
DNA-binding protein purified from rat brain was able to bind to these
AT-rich regions of both Ng/RC3 and PKC-
genes with footprints
containing ATTA, ATAA, and AATA sequences.
(
)
also known as RC3
or p17, is a prominent calmodulin-binding protein kinase C (PKC)
substrate specifically expressed in the cerebrum of the adult rat brain
(1, 2, 3) . This 78-amino acid protein resembles
another PKC substrate, neuromodulin (also known as B-50 and GAP-43)
(4, 5, 6, 7, 8) , in its high
affinity binding of calmodulin at low levels of Ca
(2, 9, 10, 11) . Both neurogranin
and neuromodulin are phosphorylated by PKC at a single site located
within a conserved region between these two proteins
(2, 9, 11) . This phosphorylation site domain is
adjacent to the predicted calmodulin-binding region. Phosphorylation of
these two proteins by PKC reduces their affinities for calmodulin. It
has been hypothesized that the PKC-catalyzed phosphorylation of these
two proteins results in the release of calmodulin for other
calmodulin-dependent enzymes
(12) . This signal amplification
step following the activation of PKC has been linked to the regulation
of ion conductance, neuronal plasticity, gene expression, growth, and
differentiation
(13, 14, 15) .
isozyme
(17) ;
however, the Ng/RC3 gene has a more restricted neuronal expression than
PKC-
. The characteristic tissue-specific and developmental
stage-regulated expression of these two proteins is distinctively
different from that of PKC-
and -
(17) . These latter
two PKCs are ubiquitously expressed in a variety of tissues and cell
types; in the brain, the expression of these two genes is progressively
increased from the fetal stage up to 2-3 weeks after birth. The
mechanisms that trigger the delayed expression of both PKC-
and
Ng/RC3 are unknown.
gene
(18, 19) . A sequence
of 163 base pairs (bp) upstream from the transcriptional initiation
site was found to possess full promoter activity. This region contains
consensus recognition elements for SP1 and AP2, but without the
canonical TATA and CCAAT boxes at their usual positions upstream from
the transcriptional initiation site. The PKC-
gene contains a
putative repressor-binding site near bp
670 with the ATTA motif
typical of the homeobox-like protein-binding site of the POU family of
factors
(19, 20) . This study was aimed at cloning the
Ng/RC3 gene for the purpose of determining its structure and potential
regulation. In addition, we were interested in determining any
structural similarity between the Ng/RC3 and PKC-
genes. Here, we
describe the structural organization and sequence of the Ng/RC3 gene
and its regulation by phorbol ester and PKC isozymes. The Ng/RC3 and
PKC-
genes are similar in that they lack the TATA and CCAAT boxes
and contain several conserved sequence elements between them. We have
also identified a 20-kDa DNA-binding protein that interacts with the
AT-rich regions of both Ng/RC3 and PKC-
promoters.
Materials
The following materials were obtained
from the indicated sources: a rat genomic library in the Lambda Dash II
vector prepared from male Sprague-Dawley rat testis DNA from
Stratagene; [-
P]ATP (6000 Ci/mmol),
-
S-ATP (1000 Ci/mmol), and
[
-
P]CTP (3000 Ci/mmol) from Amersham Corp.;
restriction enzymes and other DNA-modifying enzymes from New England
Biolabs Inc. and Promega; molecular mass markers, luciferase, and
-galactosidase assay kits from Promega; a Sequenase kit from
United States Biochemical Corp.; TaqI DNA polymerase,
Lipofectin, Lipofectamine, and a DNase I footprinting system from Life
Technologies, Inc.; and a Maxam-Gilbert DNA sequencing kit from DuPont
NEN. Oligonucleotides were synthesized using an Applied Biosystems 380B
DNA synthesizer. PKC-
, -
, -
,
-
, -
, -
, and -
cDNAs were provided by Dr. John L.
Knopf (Genetics Institute), and RC3 cDNA was provided by Dr. Joseph B.
Watson (UCLA).
Isolation of Ng/RC3 Genomic Clone
Approximately
1.5 10
clones were screened by plaque hybridization
with a
P-randomly labeled full-length Ng/RC3 cDNA probe
(1) . Escherichia coli strain LE392 was infected with
recombinant phage and replica-plated on nitrocellulose filters. The
filters were baked at 80 °C for 2 h and then prehybridized at 65
°C with 3
SSC (150 mM NaCl, 15 mM
trisodium citrate, pH 7.0) for 30 min and with 3
SSC plus 1
Denhardt's solution for 2 h. Hybridization with the probe
was carried out in a hybridization solution containing 1 M
NaCl, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0, 1
Denhardt's solution, 100 µg/ml salmon sperm DNA, and
denatured DNA probe at 65 °C overnight. The filters were washed
with 3
SSC for 30 min at room temperature and then twice with 1
SSC containing 0.1% SDS at 50 °C for 30 min. Positive
clones were further hybridized with a variety of oligonucleotide probes
from the 5`-untranslated, translated, and 3`-untranslated regions of
rat Ng/RC3 cDNA to confirm the isolation of a complete genomic clone.
Phage DNA was digested with BamHI, EcoRI,
PstI, and XbaI and subcloned into either the pGEM3Zf
(Promega) or pBluescript IIKS(+) (Stratagene) vector. Each
subclone was hybridized with oligonucleotide probes to determine the
orientation of these genomic fragments and sequenced on both strands by
the dideoxynucleotide chain termination method using
-
S-dATP
(21) . 7-Deaza-dGTP was used in some
sequencing reactions to reduce the effect of GC band compression. To
alleviate the polymerase ``pauses'' due to the secondary
structure, terminal deoxynucleotide transferase was used in the chase
reactions. The genomic DNA sequence was analyzed with the Genetics
Computer Group software program.
Primer Extension Analysis
A 24-base synthetic
oligodeoxynucleotide primer complementary to nucleotides +29 to
+52 of the Ng/RC3 gene was labeled at the 5`-end with
[-
P]ATP by T4 polynucleotide kinase and
purified on a Stratagene NucTrap probe purification column.
P-Labeled oligomer (
10 pmol) was hybridized at 58
°C for 20 min in a reaction mixture containing 10 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 300 mM KCl, and 1 µg
of rat cerebral poly(A
) RNA prepared from adult rat
brain total RNA. The extension reaction was carried out at 41 °C
for 30 min with the addition of 1 unit of avian myeloblastosis virus
reverse transcriptase, unlabeled deoxynucleotide triphosphates, and
sodium pyrophosphate as recommended by the supplier (Promega, primer
extension system). The reaction products were purified by ethanol
precipitation and electrophoresed on a 7 M urea, 6%
polyacrylamide sequencing gel. A single-stranded DNA sequencing product
using the same primer was run in parallel as a reference to determine
the size of the extended products.
Construction of Ng/RC3-Luciferase Fusion Genes
A
2.8-kb EcoRI/ BamHI genomic fragment encompassing the
5`-flanking region, the first exon, and part of the first intron was
subcloned into pGEM3Zf(+), and deletion mutants were generated
from this clone by polymerase chain reaction using specific primers for
each construct. SacI and BglII site-containing
primers were used at the 5`-ends for Ng/RC3(1508/+256) and
Ng/RC3(
940/+256) constructs, respectively. A KpnI
linker was used for the 5`-ends of all other deletion mutants. The
3`-ends of all the deletion mutant constructs contained a
HindIII linker. Polymerase chain reaction products digested
with SacI/ HindIII for Ng/RC3(
1508/+256),
BglII/ HindIII for Ng/RC3(
940/+256), and
KpnI/ HindIII for the rest of the constructs were
subcloned in the sense orientation upstream from the luciferase gene in
the pGL2-Basic vector (Promega). All these constructs do not contain
the neurogranin translational initiation codon (at nucleotide
+260) and thus were not expected to interfere with the expression
of luciferase. Various constructs were transformed into E. coli DH5
, and plasmid DNA was purified with a QIAGEN plasmid kit.
Prior to transfection, samples of all plasmids were digested with the
appropriate restriction endonucleases and resolved by agarose gel
electrophoresis to ensure the quality of the preparations. Orientation
of the insert was confirmed by sequencing.
Cell Transfection and Luciferase and
Transformed human embryonic kidney 293 cells (American
Type Culture Collection) were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum and 25
mM HEPES in a humidified atmosphere at 37 °C containing 5%
CO-Galactosidase
Assays
. Subconfluent cultures (
40-60% confluent,
24-48 h after plating) in 35-mm culture dishes were washed twice
with Dulbecco's modified Eagle's medium containing 25
mM HEPES, pH 7.5, and incubated with DNA-Lipofectin or
DNA-Lipofectamine complexes containing 1.5-2.5 µg of Ng/RC3
construct plus 0.3-0.6 µg of pSV-
-galactosidase control
vector or PKC cDNA in duplicate or triplicate. Transfection was carried
out for 6 h, and cells were washed with fresh Dulbecco's modified
Eagle's medium containing 25 mM HEPES and fed the same
medium supplemented with 10% fetal bovine serum. After 42-48 h,
cells were rinsed twice with phosphate-buffered saline and lysed with
25 mM Tris phosphate, pH 7.8, containing 2 mM
dithiothreitol, 2 mM CDTA, 10% glycerol, and 1% Triton X-100
at room temperature for 10-15 min. Cells were scraped from the
dish and transferred to microcentrifuge tubes for centrifugation to
remove cell debris. Luciferase and
-galactosidase activities in
the supernatant fluid were measured with Promega assay systems for
these two enzymes. Protein concentrations were determined by the
Coomassie Blue dye binding method
(22) .
Isolation of Rat Brain AT-rich DNA-binding
Protein
A heat-stable nuclear protein was found to bind an
AT-rich ApaI/ PstI fragment (997/
454) of
the Ng/RC3 gene. The binding activity of this protein was also stable
to 2% perchloric acid treatment, similar to several PKC substrates such
as Ng/RC3, neuromodulin, and MARCKS
(23) . For large-scale
preparation, 500 g of frozen rat brain were thawed and homogenized with
a Polytron homogenizer in 2500 ml of buffer A (20 mM Tris-HCl,
pH 7.5, containing 1 mM dithiothreitol, 0.5 mM EDTA,
0.5 mM EGTA, and 10% glycerol) containing 0.5 mM
phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 10,000
g for 30 min, filtered through glass wool, and
precipitated with 2.2% HClO
, and the supernatant was
neutralized with KOH. Following removal of KClO
by
centrifugation, the supernatant was concentrated by ultrafiltration
(Amicon YM-5 membrane) and dialyzed with buffer A by repetitive
dilution/concentration inside the ultrafiltration chamber. The
concentrated solution was applied to a DEAE-cellulose column (1.6
10 cm, packed in a Pharmacia Biotech HR 16/10 column)
equilibrated with buffer A, and the flow-through fractions were
collected and further concentrated by ultrafiltration. These fractions
were purified by high pressure liquid chromatography using a Vydac
C
reversed-phase column (214TP510, 10
250 mm)
eluted with a 0.1% trifluoroacetic acid/0.1% trifluoroacetic acid
+ 100% acetonitrile gradient. The DNA-binding protein was eluted
between 20 and 25% acetonitrile and exhibited a molecular mass of 20
kDa as determined by SDS-polyacrylamide gel electrophoresis.
Homogeneous protein was prepared by additional chromatography on a Mono
S column (Pharmacia Biotech HR 5/5) eluted with a buffer A/buffer A
+ 1.0 M KCl gradient. The DNA-binding protein was eluted
at 0.4 M KCl.
DNase I Footprinting
Analysis
Ng/RC3(1508/+256) plasmid DNA was digested
with ApaI/ PstI and BstEII/ PstI and
PKC-
(
1612/+243)
(18) with
AvrII/ AatII, gel-isolated, dephosphorylated with calf
intestinal alkaline phosphatase, and end-labeled with
[
-
P]ATP and T4 polynucleotide kinase. The
labeled DNA was precipitated with ethanol and dissolved in TE buffer
(10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The labeled
ApaI/ PstI fragment from Ng/RC3 was digested with
BstEII to produce a shorter single terminus-labeled probe of
the ApaI/ BstEII fragment (
997/
707). The
end-labeled Ng/RC3 BstEII/ PstI
(
706/
454) and PKC-
AvrII/ AatII
(
979/
691) probes were used directly. Footprinting was
performed using the DNase I footprinting system of Life Technologies,
Inc. Reaction mixtures (50 µl) containing 10 mM Tris-HCl,
pH 7.5, 50 mM NaCl, 1 mM EDTA, 5% glycerol, 20
µg/ml poly(dI-dC), and 0.3-300 ng of purified protein were
incubated at room temperature for 20 min. After the addition of 1 ng of
labeled DNA probe, the mixture was further incubated at room
temperature for 30 min. DNase I digestion was done at room temperature
for 1 min following the addition of 50 µl of DNase I buffer (10
mM HEPES, pH 7.8, containing 5 mM MgCl
, 1
mM CaCl
, and 25 mM NaCl) and 40-60
ng of DNase I. The reaction was terminated by the addition of 10 µl
of stop buffer (100 mM MES, pH 6.0, containing 150 mM
EDTA, 5% SDS, and 250 µg/ml herring sperm DNA), and the products
extracted with phenol/chloroform/isoamyl alcohol (25:24:1),
precipitated with ethanol, and run on an 8% polyacrylamide gel
alongside a Maxam-Gilbert sequencing reaction
(62) of the same
DNA.
Cloning and Characterization of Ng/RC3 Genomic
DNA
Three positive phage recombinants were identified from a
Stratagene rat genomic library with a P-labeled
full-length Ng/RC3 cDNA as a probe. All these phage clones contained an
11-kb EcoRI fragment hybridized positively with the probe. One
of them, Ng/RC3-T3, was chosen for further characterization by
restriction enzyme mapping and Southern blotting. Digestion of the
phage clone with BamHI generated a 4.7-kb fragment hybridized
with a 330-bp restriction fragment ( BamHI/ XbaI)
derived from the 5`-end of Ng/RC3 cDNA. Both 11-kb EcoRI and
4.7-kb BamHI fragments were gel-isolated, subcloned into
pGEM3Zf, and characterized by restriction enzyme mapping. Hybridization
of the various restriction fragments with oligonucleotide probes
derived from the various sequence regions of rat Ng/RC3 cDNA indicated
that this genomic clone contained the entire rat Ng/RC3-coding region.
The entire 13-kb genomic fragment was sequenced; a restriction map of
this genomic clone is shown in Fig. 1(see Fig. 2for
nucleotide sequence).
Figure 1:
Organization and restriction map of rat
Ng/RC3 gene. The genomic clone is 12,728 nucleotides in length and
consists of four exons, represented by filled boxes.
The protein-coding region in cDNA, represented by hatched boxes, is derived from exons 1 (15 nucleotides) and 2
(219 nucleotides), and the 5`- and 3`-untranslated regions are
represented by open boxes. B,
BamHI; E, EcoRI; P, PstI;
X, XbaI.
Figure 2:
Nucleotide sequence of rat Ng/RC3 gene.
Nucleotide 1 is the assigned transcriptional start site. The
transcribed sequence is in upper-case letters, and the
nontranscribed sequence is in lower-case letters. Predicted
transcription factor-binding sites for SP1, AP1, and AP2 between
nucleotides 2000 and +40 and polyadenylation signals TTTAAT
(at nucleotide +8152) and AATAAA (at nucleotide +8199) are
underlined. Other unique features of the Ng/RC3 gene, such as
the A tract, the T tract, the GT box, and the 38-nucleotide
( nt) repeat, are also shown.
The exon/intron boundaries were determined by
comparison with rat Ng/RC3 cDNA sequence
(1) . This gene is
composed of four exons and three introns (Fig. 1). The boundaries
of the exon/intron junctions conform the consensus 5`-splice donor site
of GT and the 3`-splice acceptor site of AG (). The splice
acceptor dinucleotide AG is preceded by a CT-rich sequence at the
3`-end of introns 1 and 3, which both contain the sequence TCCTCAG. The
splice donor sites of introns 1 and 2 contain the sequence GTGAG. The
first exon (274 bp) contains the entire 5`-untranslated region and
those coding for the N-terminal 5 amino acids. The second exon (227 bp)
contains the region coding for the remaining 73 amino acids and a short
tail of 3`-untranslated region. The third exon contains only 18 bp of
the 3`-untranslated region, and the fourth exon contains the remaining
3`-untranslated region, which contains two polyadenylation signals
(TTTAAT at nucleotide +8152 and AATAAA at nucleotide +8199)
located near the end and a long purine tract,
AGA
GA
GA
GA
GA
GA
GA
GA
G.
Two prominent transcripts of 1.0 and 1.5 kb that arise by the use of
two polyadenylation sites of this gene have been identified from adult
rat brain poly(A
) RNA
(1) . The second and
third introns are relatively short as compared with the first one (6.25
kb). This large first intron contains several long A tracts (A
at nucleotide +1811, A
at nucleotide
+3944, A
CA
CA
at nucleotide
+4198, and A
CA
at nucleotide +6494)
and T tracts (T
at nucleotide +5462 and T
at nucleotide +5660) and a 38-nucleotide repeat (nucleotides
+4847 to +4884 and nucleotides +5808 to +5845) with
one mismatch. In addition, there is a GT-rich segment containing a GTG
repeat 20 times within 89 nucleotides (nucleotides +4553 to
+4641).
208 (GGGCGT),
216 (GGGCGG), and
1544 (CCCAGCCTC);
for AP1 at nucleotides
1229 (CTAGTCA),
1393 (TGAATCA),
and
1423 (GTGACTAA); and for AP2 at nucleotides
725
(GGGAGGGG),
951 (CCCCACCC),
955 (CCCACCCC), and
1358 (GGGATGGG). The 5`-untranslated region contains an SP1 at
nucleotide +22 (CCCGCC) that overlaps with an AP2 at nucleotide
+23 (CCGCCCACCC). The proximal promoter region
(
300/+1) lacks a TATA box; several homologous ones
(24) are found within nucleotides
1330 and
410
(TATAAA at nucleotide
1324, TAAATA at nucleotides
1322
and
1274, TATATA at nucleotides
936 and
417,
TATTTA at nucleotide
691, TTTAAA at nucleotides
689 and
608, and AATAAA at nucleotides
816 and
768). These
AT-rich segments appear at a higher frequency between nucleotides
940 and
600. The Ng/RC3 promoter lacks the sequence
elements for binding of steroid hormone receptors including
glucocorticoid, estrogen, thyroid, and retinoic acid.
Determination of Ng/RC3 Gene Transcriptional Initiation
Site
Primer extension using a 5`-P-labeled
synthetic deoxyoligonucleotide primer complementary to a region near
the 5`-end of Ng/RC3 cDNA (nucleotides +29 to +52) was
extended with reverse transcriptase on rat cerebral
poly(A
) RNA (Fig. 3). For direct comparison, the
same primer was used for single-stranded plasmid sequencing of a
PstI/ BamHI genomic fragment (
453/+513) as
a marker. A cluster of extension products and several distinct ones of
both higher and lower molecular sizes were detected. These findings
confirm the previous results of Watson et al. (1) , who
demonstrated that Ng/RC3 transcription begins at multiple sites. We
have designated the site corresponding to one of the prominent
extension products upstream from the clustered ones as the initiation
site, which is 10 nucleotides upstream from the 5`-end of Ng/RC3 cDNA.
This start site is 259 bp upstream from the translational start site.
Figure 3:
Mapping of transcriptional start site of
rat Ng/RC3 gene by primer extension. A P-end-labeled
oligonucleotide corresponding to nucleotides +29 to +52 in
the antisense orientation was hybridized with 0.5 µg ( lane 1) and 1 µg ( lane 2) of adult rat
cerebral poly(A
) RNA and extended by reverse
transcriptase. Products were separated on a 7% polyacrylamide gel
containing 7 M urea. Primer-extended products, detected by
autoradiography, are indicated by arrows. A single-stranded
DNA sequencing product is represented by lanes G,
A, C, and T.
Promoter Activity of Ng/RC3 Genomic
Fragment
Several deletion constructs upstream from the
translational initiation site fused with a luciferase-carrying reporter
plasmid (pGL2) were transfected into 293 cells to characterize the
function of the Ng/RC3 promoter. Efforts to identify Ng/RC3-expressing
cells in several neuroblastoma (NS20Y, NG108-15, and N1E-115)-,
pituitary (T3)-, and hypothalamus (GT1
7)-derived cell lines
and in PC12 cells were unsuccessful. Human embryonic kidney 293 cells
was chosen for the in vitro assay of the promoter activity.
This cell line has previously been used for the expression of the rat
PKC-
gene
(18) . pGL2-Basic and pGL2-SV40/enhancer
constructs were used as negative and positive controls, respectively.
The plasmid DNAs were introduced into the cells by the Lipofectin or
Lipofectamine delivery method, and luciferase activity was determined
42-48 h after transfection. The promoter activity of
Ng/RC3(
1508/+256) was
50-60% of the pGL2 control
containing the SV40 promoter and enhancer. The nested deletion mutants
upstream from nucleotide +11 exhibited promoter activities that
were 80-160% of Ng/RC3(
1508/+256) (Fig. 4). The
Ng/RC3(+11/+256) construct was
90% as effective as the
Ng/RC3(
1508/+256) construct, whereas
Ng/RC3(+40/+256) was only 14% as effective and other deletion
constructs farther downstream were inactive. These results suggest that
the 29 bp between nucleotides +11 and +40 contain an
important sequence element for the promoter activity. This region
contains two potential regulatory protein-binding sites,
+22/+27 (CCCGCC) for SP1 and +23/+32 (CCGCCCACCC)
for AP-2. The binding sites for SP1 and AP2 overlap.
Ng/RC3(
23/+256) was the most active among the various
constructs tested; the downstream Ng/RC3(+11/+256) construct
and the two upstream Ng/RC3(
41/+256) and
Ng/RC3(
141/+256) constructs were considerably less active.
These results suggest that region
23/+11 may contain an
enhancer element and region
141/
23 a suppressor element
to regulate the promoter activity. A more detailed mutational analysis
of these regions with successive microdeletions as well as base
substitutions will be needed to define the sequence element important
for transcriptional regulation. The other constructs containing
upstream sequence, Ng/RC3(
940/+256) and
Ng/RC3(
541/+256), were comparable to
Ng/RC3(
1508/+256).
Figure 4:
Deletion analysis of 5`-flanking region of
Ng/RC3 gene by transient transfection. Ng/RC3 promoter constructs with
varying degrees of 5`-deletions linked to the luciferase reporter were
transfected into 293 cells. Aliquots of cell extracts were assayed for
luciferase (10-15 µg of protein) and -galactosidase
(
50 µg of protein) activities. The relative activity of the
various constructs was normalized with the
-galactosidase
activity. The activity of the Ng/RC3(
1508/+256) construct
was taken as 100%. The data represent the average of at least three
independent experiments of duplicate or triplicate
measurements.
Stimulation of Ng/RC3 Promoter Activity by Phorbol Ester
and by Cotransfection with PKC cDNAs
Several potential
modulators were tested for their effects on the promoter activity using
293 cells transfected with Ng/RC3(1508/+256). After
transfection, the cells were incubated with arachidonic acid (0.01,
0.1, and 1 mM), 8-Br-cAMP (0.02, 0.2, and 2 mM),
retinoic acid (0.01, 0.1, and 1 mM), vitamin D (0.001, 0.01,
and 0.1 mM), thyroxines T
(0.5, 5, and 50
µM) and T
(0.01, 0.1, and 1 mM), or
PMA (0.002, 0.02, and 0.2 µM) for 42-48 h. With the
exception of PMA, the chemicals had insignificant effects on the
reporter gene activity. Stimulation of the
Ng/RC3(
1508/+256) construct by PMA was dose-dependent, and
-PMA was inactive. Maximal stimulation was observed at 100
nM PMA. A 2-4-fold stimulation by PMA was seen with all
the fusion constructs containing the Ng/RC3 gene upstream from
nucleotide +11 (Fig. 5). Within the Ng/RC3 promoter region
(from nucleotides
1508 to +40), there are three AP1 sites
(nucleotide
1423, GTGACTAA; nucleotide
1393, TGAATCA; and
nucleotide
1229, CTAGTCA) and five AP2 sites (nucleotide
1358, GGGATGGG; nucleotide
955, CCCACCCC; nucleotide
951, CCCCACCC; nucleotide
725, GGGAGGGG; and nucleotide
+23, CCGCCCACCC). The stimulatory effects of PMA on the various
Ng/RC3 constructs were not positively correlated with the number of
these potential phorbol ester-responsive elements, indicating no
additive effect among these potential sites. The AP2-binding site has
been shown also to respond to cAMP and retinoic acid
(25, 26) ; these two effectors, however, had no effect
on the transcriptional activity of the various Ng/RC3 constructs.
Figure 5:
Effect of PMA on promoter activity of
various Ng/RC3 constructs. 293 cells were transfected with the various
Ng/RC3-luciferase constructs for 6 h, followed by treatment with 100
nM PMA for 42 h (see ``Experimental Procedures'' for
detail). Cell extracts were used for measurements of luciferase and
-galactosidase activities. The normalized luciferase activity of
each construct without PMA was taken as 100%, and -fold stimulation by
PMA was determined. The data represent the average of three independent
experiments of duplicate or triplicate
measurements.
The PMA-mediated responses are believed to be due to stimulation of
PKCs. We examined the role of each PKC subspecies in the control of
Ng/RC3 gene expression by cotransfection of the various PKC cDNAs with
the Ng/RC3(23/+256) construct. Cotransfection of this
construct with PKC-
, -
, -
,
-
, -
, and -
cDNAs caused a 20-40-fold stimulation
over the control without PKC cDNA or cotransfected with the same amount
of
-galactosidase cDNA (Fig. 6). Cotransfection of the same
promoter construct with PKC-
cDNA had an insignificant effect on
the promoter activity. The lack of a stimulatory effect of
cotransfection with PKC-
cDNA was not due to a failure to express
this kinase in the 293 cells; in the cotransfected cells, PKC-
was
expressed at least 20 times more than the control judging from
immunoblot analysis. These results indicate that both
Ca
-dependent (PKC-
, -
,
-
, and -
) and Ca
-independent
(PKC-
and -
) phorbol ester-binding PKCs, but not the
non-phorbol ester-binding kinase (PKC-
), are active in the
stimulation of the Ng/RC3 gene promoter activity. From several
experiments, we consistently observed a higher stimulatory potency for
PKC-
and a lower stimulatory potency for PKC-
and -
when compared with PKC-
, -
, and
-
; the cause for these differences is unknown.
Figure 6:
Effects of various PKC isozymes on Ng/RC3
promoter activity. The Ng/RC3(23/+256) construct (1.6
µg/35-mm dish) was cotransfected with PKC-
,
-
, -
, -
, -
, -
, and
-
cDNAs or with pGL2-
-galactosidase (0.4 µg/35-mm dish)
into 293 cells for 48 h. The stimulatory effects were estimated using
Ng/RC3(
23/+256) + pGL2-
-galactosidase without PKC
cDNA as a standard. The data represent the average of three
measurements. The inset shows an immunoblot of extracts
derived from cells cotransfected with ( lane 1) and
without ( lane 2) PKC-
cDNA and with purified
recombinant PKC-
( lane 3) obtained from
transfected Sf9 cells. STD,
standard.
Comparison of 5`-Flanking Sequences of Ng/RC3 and
PKC-
Computer analysis of the promoter sequences of
these two genes 1.7 kb upstream from their transcriptional start sites
by the Genetics Computer Group Bestfit program revealed four highly
conserved boxes with a similarity of >85% within a sequence of 10 bp
or greater (). These conserved sequence segments do not
contain any known regulatory protein-binding site. These two genes also
do not contain a consensus sequence for the neuron-restrictive silence
element found in SCG10, type II Na Genes
channel, and
synapsin genes
(27, 28, 29) or the TATA and
CCAAT boxes in the upstream region proximal to their transcriptional
initiation sites. Previously, we have identified a potential
developmental stage-regulated suppressor-binding site within the
PKC-
promoter (nucleotide
669, GAATTAATAGG)
(19) ;
this site is not conserved in the Ng/RC3 gene. Although both PKC-
and Ng/RC3 promoter regions lack the TATA box, these two genes contain
several AT-rich sequence elements within nucleotides
670 to
920 of the PKC-
promoter and nucleotides
510 to
940 of the Ng/RC3 promoter (Fig. 7).
Figure 7:
Comparison of AT-rich sequence regions of
Ng/RC3 and PKC genes. The AT-containing sequences are
double-underlined.
Identification of Upstream AT-rich DNA-binding
Protein
The AT-rich region of the Ng/RC3 promoter (nucleotides
510 to
940) contains several elements of TATA and TATA
box variants (TATATA, ATTATA, AATAA, and TTTAAA) and polyadenylation
signals (AATAAA and ATTAAA). A 20-kDa heat- and acid-stable nuclear
protein purified from rat brain (Fig. 8 A) was found to
bind to a
P-5`-end-labeled ApaI/ PstI
fragment (
997/
454) encompassing the AT-rich region.
Further digestion of this fragment with BstEII indicated that
the protein-binding site was located in the
BstEII/ PstI fragment (
707/
454), but not
in the ApaI/ BstEII fragment (
997/
708).
The prominent footprint sites were mapped at regions
677/
688 (AAATAGGTCA) and
665/
649
(GATTAAAACAATAATTATG) (Fig. 8 B). This protein also
protected an AvrII/ AatII fragment
(
979/
691) of the PKC-
promoter at region
920/
910 (ATATTCCTCCA) from digestion by DNase I
(Fig. 8 C). The binding of this protein to these regions
was competitively inhibited by each of their respective unlabeled DNAs,
but not by poly(dI-dC). The common features of these protein
recognition sites are that they are rich in AT and contain ATTA and
ATAA (TATT) or its palindrome, AATA. A search of the transcription
factor-binding sites data bank revealed that the footprint site in the
PKC-
promoter contained a Pu box (TTCCTC)
(30) and that
the Ng/RC3 site within region
665/
649 contained an
enhancer protein-binding site found in the promoter of c- mos (TTAAAAC)
(31) . The DNA-binding proteins for these latter
two sites have not been purified, and their identities to the purified
20-kDa protein have yet to be established.
Figure 8:
Identification of footprint sites of
20-kDa DNA-binding protein within AT-rich regions of Ng/RC3 and
PKC- promoters. A, SDS-polyacrylamide gel electrophoresis
(10-20% polyacrylamide gradient gel) of a purified preparation of
the DNA-binding protein (1 µg) ( lane 2) alongside
of molecular mass markers ( lane 1). B,
prominent footprint sites of the 20-kDa DNA-binding protein on the
P-labeled BstEII/ PstI fragment
(
707/
454) of the Ng/RC3 gene. C, footprint site
of the 20-kDa DNA-binding protein on the
P-labeled
AvrII/ AatII fragment (
979/
691) of the
PKC-
gene. In B and C, samples were resolved on
an 8 M urea, 8% polyacrylamide gel alongside a Maxam-Gilbert
sequencing reaction with each of the labeled
probes.
13
kb. It contains four exons coding for a 78-amino acid protein. This
gene is expressed at a high level in adult rat cerebral neurons;
however, it is undetectable in PC12 cells, several cultured
neuroblastoma cell lines, and pituitary- and hypothalamus-derived cell
lines, perhaps due to a loss of a certain differentiated phenotype of
these cells. In comparison, neuromodulin, another brain-specific PKC
substrate homologous to Ng/RC3, is expressed at high levels in fetal
and newborn rats and is also expressed in several neuroblastoma cells.
The sequence information derived from this study will be used for
identifying the regulatory elements and their binding proteins that
confer the spatial and temporal expression pattern in vivo.
(18) , synapsin I
(32, 33, 34) , amyloid precursor protein
(35, 36) , PEP19
(37) , aldolase C
(38) ,
the membrane protein Thy-1
(39) , and
-enolase
(40, 41) , in lacking the TATA and CCAAT boxes proximal
to the transcriptional initiation site. The Ng/RC3 gene is transcribed
at multiple initiation sites, as frequently observed in these TATA-less
promoters
(42) . The most prominent start sites of these genes
are clustered at a (G + C)-rich region referred to as the
``TATA-less (G + C)-rich promoters'' often found in the
housekeeping genes
(43) . The Ng/RC3 gene, however, does not
have the ``(G + C)-rich'' feature 40-80 bp
upstream from the start site. Downstream from the cluster of major
start sites lies an SP1 consensus site at nucleotide +22, which
overlaps with an AP2 site. Deletion of this region silences the
reporter gene activity, suggesting that this region is important for
conferring the basal transcriptional activity. In spite of the
restricted neuronal expression pattern, the Ng/RC3 gene, similar to the
neuromodulin/GAP-43 gene
(44, 45) , does not contain the
consensus sequence for the neuron-restrictive silence element found in
SCG10, type II Na
channel, and synapsin I genes
(27, 28, 29) . It seems that the determinants
for the neuronal specificity of the Ng/RC3 and neuromodulin/GAP-43
genes are different from those of these three genes. A 386-bp
neuromodulin/GAP-43 promoter fragment that contains TATA and CCAAT box
consensus sequences can direct neural specific gene expression
(45) . In comparison, the largest Ng/RC3 reporter gene
construct, Ng/RC3(
1508/+256), is still expressed at a high
level in non-neuronal cells, suggesting that the neural restrictive
determinant is located elsewhere. The regulatory mechanisms of the
neuromodulin/GAP-43 and Ng/RC3 genes are likely distinct as the former
is expressed at high levels in immature neurons and in the
``growth state'' of mature neurons during axon extension and
synaptogenesis
(46) , whereas Ng/RC3 is expressed in selective
populations of mature neurons
(1, 3) .
-hydroxylase
(51) , tyrosine hydroxylase
(52, 53) , proenkephalin
(54, 55) , and
vasoactive intestinal peptide
(56, 57) , are responsive
to stimulation by both cAMP/protein kinase A- and PMA/PKC-dependent
pathways. The expression of the Ng/RC3 reporter gene construct
Ng/RC3(
1508/+256) in transfected 293 cells was stimulated
by PMA, but not by cAMP, retinoic acid, thyroxines T
and
T
, or vitamin D. These effectors were chosen because of
their known effects on cellular growth and differentiation. A survey of
the 5`-flanking region of the Ng/RC3 gene failed to identify the
consensus sequence-binding sites for ATF (cAMP-responsive
element-binding protein, TGACGC/TC/AG/A)
(58) , retinoic acid
and vitamin D receptors (AGGTCATGACCT)
(58) , and the thyroid
hormone receptor ((AGGTCA)
)
(58) . These results,
however, should be considered tentative until a cell line expressing
this gene is identified. Recently, Iniguez et al. (59) reported that administration of thyroxine T
to
hypothyroid rats increased Ng/RC3 mRNA levels without affecting the
developmental timing of expression. Thyroid hormone was thought to
enhance the transcriptional activity and/or to stabilize the mRNA
(59) .
2-4-fold for
the various deletion mutants ranging from
1508/+256 to
+11/+256 at an optimal concentration of 100 nM.
Within this region, there are several consensus sites for AP1 and AP2;
the potential functions of these sites are unclear as the deletion
mutant Ng/RC3(+11/+256), without any of these upstream
elements, was also effectively stimulated by PMA. The
Ng/RC3(+11/+256) mutant contains an overlapping site for SP1
(+22/+27, CCCGCC) and AP2 (+23/+32, CCGCCCACCC),
which may be responsive to the phorbol ester. To confirm that the
PMA-mediated effects were due to the activation of PKC, we
cotransfected PKC cDNAs with the Ng/RC3(
23/+256) reporter
gene construct. Those cells transfected with PKC-
,
-
, -
, -
, -
, and -
caused a 20-40-fold stimulation over the control, whereas cells
transfected with PKC-
were poorly stimulated. The latter PKC is
not stimulated by PMA and thus is not expected to be effective. The
extent of stimulation by cotransfection with PKC cDNAs was nearly an
order of magnitude greater than with PMA. This may be due to
down-regulation of PKC following prolonged treatment of cells with PMA,
which reduces the steady-state level of the kinase. The current results
strongly suggest that stimulation of PKC will enhance the expression of
its substrate, Ng/RC3. Since stimulation of PKC will also enhance the
phosphorylation of Ng/RC3, the signaling pathway involving this
substrate could be greatly amplified due to both increased synthesis
and phosphorylation.
genes share at least four conserved sequence elements
upstream from their start sites; two of these pairs contain a GGAGAG
sequence (, boxes 1 and 3). Future study will be directed
to identify the binding proteins for these sequence elements.
Previously, we have identified a protein-binding site (GAATTAATAGG) in
PKC-
as a putative silencer during development as the binding
activity is high in the newborn rat brain and reduced in the adult rat
brain when active synthesis occurs
(19) . This sequence element,
however, is not conserved in the Ng/RC3 gene. Thus, the developmental
signal that triggers the transcription of these two genes may not be
identical. Both PKC-
and Ng/RC3 genes also exhibit gross
similarities in that they contain AT-rich sequences at nucleotides
600 to
900. We have identified a 20-kDa DNA-binding
protein that protects AT-rich sequences, with ATTA and ATAA (TATT) and
its palindrome, from DNase I digestion. Although there are several
other segments within these AT-rich regions that also contain these
sequence features, they were not protected by this DNA-binding protein
from DNase I digestion. These results indicate that the surrounding
sequences are also important in defining the binding sites for this
protein. A preliminary study indicates that this protein is a substrate
of PKC and that the binding to DNA is attenuated by
phosphorylation,
(
)
an observation similar to that
found for CCAAT enhancer-binding protein
(60) . This AT-rich
DNA-binding protein is different from the TATA-box binding protein,
which has a molecular mass of 35-38 kDa
(61) . The
identification of other DNA-binding proteins for the Ng/RC3 and
PKC-
genes is essential to elucidate the regulatory mechanism of
these two neuron-specific genes.
Table: Exon/intron junction sequences of the rat
Ng/RC3 gene
Table: Conserved sequence segments in the
5`-flanking regions of the Ng/RC3 and PKC- genes
.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.