(Received for publication, June 25, 1996, and in revised form, October 30, 1996)
From the Laboratory of Neurochemistry, NINDS,
National Institutes of Health, Bethesda, Maryland 20892 and
§ Department of Anatomy, College of Medicine, Korea
University, Seoul, Korea 136-701
-Conotoxin-sensitive N-type Ca2+
channels, unlike dihydropyridine-sensitive L-type channels, are
exclusively expressed in nervous tissues. To understand the molecular
basis for neuron-specific expression of the N-type channel, we have
isolated genomic clones encoding the human
1B subunit
gene, localized to the long arm of chromosome 9 (9q34) by fluorescence
in situ hybridization, and characterized its 5
-upstream
region. The proximal promoter of the
1B subunit gene
lacks a typical TATA box, is highly GC-rich, and contains several
sequences for transcription factor binding. Primer extension
experiments revealed the presence of two transcription start sites.
In vitro transfection study of the
1B
subunit-luciferase fusion gene showed that the 4.0-kb 5
-flanking
region of the
1B gene functions as an efficient promoter
in neuronal cells but not in glioma or nonneuronal cells, consistent
with the patterns of the endogenous
1B gene expression
in these cells. Deletion analysis of
1B
subunit-luciferase fusion gene constructs further revealed the presence
of several cis-acting regulatory elements, including a potential
repressor located in the distal upstream region (
3992 to
1788) that
may be important for the neuron-specific expression of the N-type
Ca2+ channel
1B subunit gene.
Voltage-sensitive Ca2+ channels
(VSCC)1 found in the plasma membranes of
many excitable cells regulate calcium entry to mediate a wide variety
of physiological functions, encompassing membrane excitability, neurite
outgrowth, enzyme regulation, gene expression in cell bodies, and
neurotransmitter release from nerve terminals (1, 2). A number of
Ca2+ channel types have been described based upon
biochemical, pharmacological, and electrophysiological studies,
including L-, T-, N-, P/Q-, and R-types (3-5). The skeletal muscle
L-type Ca2+ channel, the first one to be defined at the
molecular level, is composed of multiple subunits, 1,
2-
,
, and
(6, 7), and the corresponding
cDNAs have been cloned and sequenced (reviewed in Refs. 8 and 9).
The cloned skeletal muscle
1 subunit (
1S)
exhibits structural features common to voltage-gated cation channel
gene families and is capable of directing expression of
Ca2+ channel activity in heterologous expression systems.
Homology screening resulted in the isolation of five additional
1 subunit cDNAs that encode either dihydropyridine
(DHP)-sensitive L-type channels (
1C and
1D) or DHP-insensitive high voltage-activated Ca2+ channels (
1A,
1B, and
1E). Distinct isoforms of the
1C and
1D subunits are generated by alternative splicing and
are responsible for heterogeneity of DHP-sensitive L-type channel
subtypes present in a variety of excitable and nonexcitable cells
(10-12). Although multiple isoforms also have been reported,
expression of DHP-insensitive Ca2+ channel
1
subunits seems to be restricted to nervous tissues and the cells of
neuronal origin as is the case for the
1B subunit encoding the
-conotoxin GVIA-sensitive N-type channel (13).
Little is known about the molecular mechanisms underlying distinct
cell-type and tissue-specific expression patterns of VSCC subtypes,
despite the progress in our understanding of transcriptional regulation
of other ion channel genes. Neuron-specific expression of the rat brain
type II Na+ channel is regulated by a silencer element
located in the 5-flanking region of the gene (14, 15). The
transcription of a potassium channel gene, Kv1.5, is
regulated by glucocorticoids and cAMP in both GH3 cells and cardiac
myocytes (16-18). The expression of the rat DHP-sensitive L-type
1D subunit gene in NG108-15 cells increases during
differentiation in the presence of prostaglandin E1 or
retinoic acid (19). A recent report provided an initial description of
the transcriptional regulation of the rat
1D subunit gene and identified a novel enhancer that consists of an
(ATG)7 trinucleotide repeat sequence (19). Because of their
differential expression patterns, it is of interest to examine
molecular mechanisms that underlie the regulation of N-type
Ca2+ channel
1B subunit gene expression and
compare it with those involved in the L-type
1D subunit
gene expression. In the present study, we report isolation of the
genomic clones containing the 5
-flanking sequence and its chromosomal
location, and we provide an initial characterization of the
4.0-kilobase pairs (kb) upstream promoter region of the human N-type
Ca2+ channel
1B subunit gene.
A human multiple tissue Northern
blot (Clontech Laboratories, Palo Alto, CA) was prehybridized at
42 °C for 6 h in a hybridization solution (5 × SSPE,
10 × Denhardt's solution, 50% formamide, 2% SDS, and 100 µg/ml salmon sperm DNA) and hybridizied at 42 °C for 24 h
with the probes labeled with 32P by random priming. The
1B probe was a 230-base pair (bp) fragment of the 5
region of the
1B subunit cDNA (corresponding to
nucleotide residue number 263-493 as in GenBankTM
accession no. M94172[GenBank]) which was generated by a polymerase chain
reaction (PCR). The
1D cDNA probe was the 443-bp
cDNA insert isolated from p60Z plasmid (12) (corresponding to
nucleotide residues 2803-3246 as in GenBankTM accession
no. M57682[GenBank]). Following hybridization the blot was rinsed in solution I
(2 × SSC and 0.05% SDS) at room temperature for 15 min twice and
washed in solution II (0.1 × SSC and 0.1% SDS) at 50 °C for
40 min with one change of fresh solution. The blots were exposed to
Biomax MR x-ray film (Eastman Kodak Co.) at
80 °C for 3 days.
To isolate the promoter of the
human 1B subunit gene, approximately 2 × 105 recombinant phages from a human WI-38 lung fibroblast
cell
Fix genomic DNA library (Stratagene, La Jolla, CA) were
screened with both the 230-bp
1B cDNA probe and a
mixture of two oligonucleotide probes (hNAG1 and hNAG2).
Oligonucleotide sequences are: hNAG1, 5
-CCA GCG GGT CCT CTA CAA GCA
ATC GAT CGC GCA GCG CGC GCG GA-3
(265-308, GenBankTM
accession no. M94172[GenBank]); hNAG2, 5
-AGA GCG AGC GGT TGA CGG TGA AGC AGT
TCT GCT TGA CCG GGA TG-3
(328-371, GenBankTM7 accession no. M94172[GenBank]).
Plaques were transferred to nitrocellulose filters, and the filters
were prehybridized in 20% formamide, 5 × SSPE, 1 × Denhardt's solution, 0.1% SDS, and 100 µg/ml salmon sperm DNA for
4 h and hybridized overnight at 42 °C with the
32P-labeled probes at 1 × 106 cpm/ml of
hybridization solution. Filters were washed at room temperature in
2 × SSC, 0.2% SDS for 15 min three times and at 62 °C in
0.1 × SSC, 0.1% SDS for 30 min twice. Autoradiography was
carried out for 48 h at
80 °C with Kodak X-Omat AR film.
The genomic inserts isolated from positive plaques were subcloned into pGEM7Zf(+) plasmid (Promega, Madison, WI). Both strands of the genomic inserts were sequenced by the chain termination sequencing method (20). Sequence analysis and data base searches were performed with the GCG software package.
Primer Extension AnalysisA 21-mer antisense
oligonucleotide primer (NAPE1, as indicated in Fig. 2), complementary
to a portion of the first exon (56-76; GenBankTM accession
no. M94172[GenBank]) of the human N-type calcium channel 1B gene,
was end-labeled with 32P by T4 polynucleotide kinase. The
32P-labeled NAPE1 was annealed to 2 µg of human
neuroblastoma SH-5YSY cell poly(A)+ RNA in 40 mM PIPES (pH 6.8), 1.25 mM EDTA (pH 8.0), 125 mM NaCl, and 75% foramide for 1 h at 42 °C.
Hybrids were ethanol-precipitated and extended by avian myeloblastosis
virus (AMV) reverse transcriptase in a mixture containing 0.06 µg of
actinomycin D. Extension products were analyzed on 8%
ployacrylamide-urea sequencing gels.
Fluorescence in Situ Hybridization
The plasmid containing a
9.5-kb human 1B subunit genomic insert (pNAG Sac2-2) was
labeled with biotin-dUTP by nick translation. The labeled probe was
combined with human Cot-1 DNA and hybridized to a normal metaphase
chromosome from phytohemagglutinin-stimulated peripheral blood
lymphocytes in a solution containing 50% formamide, 10% dextran
sulfate, and 2 × SSC. Specific hybridization signals were
detected by incubating the hybridized slides in fluoresceine conjugated
avidin. The slides were counterstained with propidium iodide and
analyzed.
The human 1B
gene-luciferase fusion plasmids were constructed by subcloning into the
polylinker region of pGL2-Basic vector (Promega) with the following
restriction fragments from the human
1B genomic clone
pNAG Sac2-2: a 4.0-kb BamHI/BssHII fragment (
3992L), a 1.7-kb XhoI/BssHII fragment
(
1788L), and 0.1-kb NotI/BssHII fragment
(
110L). Another deletion constructs were generated by Discrete-Delete
ExoIII/mung bean nuclease deletion kit (Epicentre Technologies,
Madison, WI). All plasmids were sequenced to determine the deletion end
points and to exclude the possibility of recombination in host
Escherichia coli. The control plasmids pRSVL (a gift from Dr. Sung O Huh; Sloan-Kettering Institute, New York) and pCMV
(Stratagene, La Jolla, CA) contain the Rous sarcoma virus (RSV) promoter fused to the luciferase gene and the cytomegalovirus (CMV)
promoter fused to the
-galactosidase gene, respectively.
Human neuroblastoma SH-5YSY and BE(2)-C cells, which were provided by Dr. June Biedler (Sloan-Kettering Institute, New York), were grown in 1:1 Eagle's minimal essential medium and Ham's nutrient medium F12 supplemented with 10% fetal bovine serum (FBS). Two mouse neuroblastoma X rat glioma hybrid cell lines, NG108-15 and 140-3, were grown in Dulbecco's modified Eagle's medium with 10% FBS, 100 µM hypoxanthine, 1 µM aminopterin, and 16 µM thymidine. Murine neuroblastoma NS20Y and human glioma U251 cells were cultured in Dulbecco's modified Eagle's medium with 10% FBS. PC12 cells were maintained in Dulbecco's modified Eagle's medium containing 10% FBS and 5% horse serum. HeLa and HepG2 cells were grown in Eagle's minimal essential medium with 10% FBS. All culture media were supplemented with penicillin G (100 units/ml) and streptomycin (100 µg/ml).
Transient Transfection and Luciferase AssayEquimolar
amounts of the human 1B gene-luciferase plasmids (3 µg
were used for the shortest deletion construct
110L) and pCMV
were
cotransfected into subconfluent cells in 60-mm culture dishes using
LipofectAMINE (Life Technologies, Inc.). Cells were harvested 24 h
after transfection and lysed in 1 × cell culture lysis reagent,
and activities were assayed using luciferase assay reagent (Promega).
The light emitted was integrated over a 15-s interval on a Monolight
2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA)
and expressed as light units.
-Galactosidase was monitored by an
assay kit in the same lysate (Promega). The luciferase activity of
fusion constructs was normalized to
-galactosidase activity and
expressed as a percentage of the RSV promoter activity of pRSVL.
Total RNAs were
prepared by a guanidium thiocyanate-phenol extraction method (21).
First strand cDNA was synthesized using 1 µg of total RNA, which
had been treated with RNase-free DNase, by using SuperScript
preamplification system (Life Technologies, Inc.). Following the first
strand cDNA synthesis, PCRs were done in a 50-µl reaction mixture
containing 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 100 µM of each dNTP,
1.25 units of Taq polymerase (Perkin-Elmer), and 50 pmol
each of primer pairs for 1B subunit gene and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers used were
as follows (the nucleotide residue number and accession nos. are in
parentheses): CaCh SphI, 5
-ACG CCA TCA TCG GCA TGC ACG
TTT-3
(4838-4861, M92905); CaCh AvaI, 5
-CCT AGG ATG GAA
GAA TCC CGC GT-3
(5143-5165, M92905); GAPDH-S, 5
-GGA CAT TGT TGC CAT
CAA CGA C-3
(108-129, M17701); GAPDH-AS, 5
-ATG AGC CCT TCC ACG ATG
CCA AAG-3
(525-548, M17701). The PCR was performed for 30 cycles (1 min at 94 °C, 2 min at 60 °C and 2 min at 72 °C). The final
extension was carried out at 72 °C for 5 min at the end of cycling.
The amplified products were separated on 1.5% agarose gels and
visualized by ethidium bromide staining.
The distribution of 1B and
1D Ca2+ channel transcripts in human tissues
was examined by Northern blot analysis. A single 10.5-kb transcript of
the
1B gene was present in the brain, but there was no
hybridized band detected in other human tissues examined (Fig.
1A). The size of the
1B
mRNA present in human brain is similar to the ~10-kb rat brain
transcript (13). The
1D cDNA probe, which is derived
from the least conserved intracellular linker of the II and III loop of
the
1D subunit (10), strongly hybridized to the
transcripts of two sizes, 9.5 and 8.5 kb, in the brain, placenta, lung,
liver, kidney, and pancreas. An additional band of 6.5 kb in size was
detected in the kidney and pancreas (Fig. 1B). No bands were
detected in the heart and skeletal muscle, indicating that the
1D probe did not cross hybridize to the L-type
1C and
1S subunit transcripts. The 9.5-, 8.5- and 6.5-kb sizes of the human
1D transcripts are
similar to those present in rat brain (10, 22), but smaller than the
11-kb transcript found in pancreatic islets (23). Thus, expression of
N-type Ca2+ channel
1B subunit, unlike the
DHP-sensitive L-type channel which shows a broad distribution pattern
in various tissues, is limited to nervous tissues.
Cloning and Nucleotide Sequence of the 5
Screening a human lung fibroblast
genomic library yielded two overlapping clones which, taken together,
contained 6.5 kb of the 5-upstream region, the first exon, and 2.5 kb
of the first intervening sequences. A major portion, approximately 4.7 kb, of the human
1B genomic clone was sequenced and is
shown in Fig. 2. The 4670-nt sequence contains 3991 nt
of the 5
-flanking region, 430 nt of the first exon (148 nt of
5
-untranslated region and 282 nt of coding sequence), and the 249-nt
part of the first intron. The exon 1 sequence is identical to that of
the human N-type Ca2+ channel cDNA reported by Williams
et al. (24).
Multiple GC boxes (GGGCGG), Sp1-binding sites (25), are found in the
proximal 5-flanking region between
388 and
75. Two of the GC boxes
overlap with the NGFIA-binding site (GCGGGGGCG) (26) located at
nucleotides
118 to
110. Two potential AP1-binding sites (TGAGTCAG)
(27) are located at
3014 and
972. In addition, three AP2-binding
sites (CCCCAGGC) (28) are present at
3786,
3548, and
603, and an
AP4-binding site (CAGCTGTGG) (29) at
354. Interestingly, nine copies
of a 39-bp direct repeat are found in the region between
2880 and
2530.
An inverted sequence of the core motif CCAGGAG (14) shared by several
neuron-specific genes is found at 190 to
184. This consensus
element or its inverted sequence occurs in the 5
-flanking region of
the genes encoding the rat type II Na+ channel (14), the
rat peripherin (30), the mouse neurofilament (31), the rat SCG 10 (32),
rat GAP-43 (33), and the mouse synapsin II (34).
Primer
extension analysis was performed in order to determine the
transcription initiation site of the human 1B gene.
32P-Labeled oligonucleotide primer NAPE1 was annealed to
poly(A)+ RNA of human neuroblastoma SH-5YSY cells, extended
by AMV reverse transcriptase, and the extended products were separated
by polyacrylamide-urea gel electrophoresis. A predominant band of 79 nt
and a weaker band of 83 nt were detected on an autoradiogram (Fig.
3, lane 1). No extended products were
observed when extension reaction was performed with E. coli
tRNA templates which was used as a control for specificity of
hybridization (Fig. 3, lane 2). From the size of the
extended products and the location of the NAPE1 oligonucleotde primer,
we were able to place major and minor initiation sites at 148 and 152 nt upstream of the translation start site, respectively.
Analysis of the sequence immediately upstream of the transcription
initiation sites reveals that the 1B subunit gene
promoter contains a CCAAT box (
59 in antisense orientation) but lacks a typical TATA consensus motif. The sequences surrounding the major
(GGT
AGGC) and minor transcription initiation sites
(GTC
GGTG) are different from the initiator sequence
(CTC
NTCT) present in the promoter region of TATA-less
genes, including the synapsin I gene (35) (the underlined nucleotide
represents the transcription initiation site). In addition, this
promoter is highly rich in G + C content and 72 CpG and 85 GpC
dinucleotides are located in a region of 500 bp (positions
400 to
+100).
Fluorescent in situ hybridization using the
biotin-labeled probe resulted in specific labeling of the distal end of
long arm of chromosome 9 (Fig. 4A). A second
experiment was carried out in which a chromosome 9 centromere-associated satellite probe was cohybridized with the human
1B genomic probe to confirm the identity of the
specially labeled chromosome. This experiment showed the specific
labeling of the centromeric heterochromatin and the distal long arm of
chromosome 9 (Fig. 4B). Measurement of 10 specifically
hybridized chromosome 9s demonstrated that the human Ca2+
channel
1B gene is located at a position which is 97%
of the distance from the centromere to the telomere of chromosome arm 9q, an area that corresponds to band 9q34. A total of 80 metaphase cells were examined with 65 exhibiting specific signals.
Cell Type-specific Expression by 5
To address whether the
5-flanking sequence of the human
1B subunit gene
contains the regulatory sequences utilized in a cell type-specific
manner we made a fusion gene construct
3992L, containing a 4.0-kb
5
-flanking sequence of the
1B gene (
3992 to +86)
linked to the promoterless luciferase reporter vector pGL2-Basic. This
plasmid was transfected into a variety of neuronal and nonneuronal cell
lines and assayed for luciferase activity. As controls, the plasmids
pGL2-Basic and pRSVL were transfected into parallel cultures of each
cell line. In all the cell lines tested, the pGL2-Basic plasmid was
ineffective in driving expression of luciferase activity, while
transfection of the pRSVL resulted in high levels of expression. The
results of such an analysis are shown in Fig.
5A. In neuronal cells such as SH-5YSY,
BE(2)-C, NS20Y, NG108-15, and PC12 cells, luciferase activities from
the
1B fusion gene construct
3992L, were approximately
40-60% of those from the RSV promoter. In contrast, reporter gene
expression was very low, maximally 5% of RSV activity, in the glioma
cell line U251 as well as in the nonneuronal HeLa and HepG2 cells. Interestingly, the luciferase gene was poorly expressed in one of the
mouse neuroblastoma-rat glioma hybrid cell lines, 140-3 cells,
consistent with our recent electrophysiological studies showing that
140-3 cells do not expressed any of high voltage-activated currents
(36).
RT-PCR, which was carried out to detect the endogenous
1B mRNA expression in the same cell lines used in
transfection studies, yielded the amplified product corresponding to
the predicted size of 355 bp in SH-5YSY, BE2(C), NS20Y, NG108-15 and
PC12 cells but not in 140-3, U251, HeLa, and HepG2 cells (Fig.
5B). The level of endogenous
1B gene
expression in NS20Y, NG108-15, and PC12 cells, as judged by the
intensity of the amplified bands on agarose gels, seemed to be higher
than in SH-5YSY and BE(2)-C cells, suggesting that there is a good
correlation between reporter gene expression from the
1B-luciferase fusion gene construct and endogenous
1B gene expression. Taken together these results, we
conclude that the 4.0-kb 5
-flanking sequence contains the
cis-regulatory elements important for directing expression of the
1B gene in a neuron-specific manner.
To locate a cis-acting regulatory
element for neuron-specific expression of the 1B gene, a
series of
1B-luciferase fusion plasmids were constructed
and transfected into NS20Y cells and HeLa cells. Progressive 5
deletions between nucleotides
3992 and
110 were made from the
3992L construct using either specific restriction enzymes or
exonuclease III digestion protocols. As shown in Fig.
6A, a deletion from
3992 to
1788 resulted
in an approximately 10-fold increase in luciferase activity in HeLa cells, but no change in NS20Y cells, indicating the presence of a
repressor element between
3992 and
1788 that inhibits the reporter
gene expression in HeLa cells. Further deletions of the region between
1788 and
1289 had little effect on luciferase activity in both cell
lines (Fig. 6A). However, removal of the region from
1289
to
1057 resulted in a small but significant 2.0-fold increase only in
HeLa cells, suggesting that this region may contain another weak
repressor element. Extension of 5
deletions to nucleotide
110
gradually reduced the luciferase activity in NS20Y and HeLa cells (Fig.
6, A and B). On the basis of these results, it is
likely that at least two negative regulatory elements with different
strengths, distal (
3992 and
1788) and proximal (
1289 and
1057)
region, may play critical roles in the neuron-specific regulation of
the N-type Ca2+ channel
1B subunit.
In this study we report the cloning, chromosomal localization, and
molecular analysis of the 5-flanking region of the human N-type
Ca2+ channel
1B subunit gene. A single
10.5-kb
1B mRNA transcript was detected only in the
brain among the human tissues examined, whereas L-type
1D mRNAs were detected in a variety of tissues (Fig.
1). The
1B transcripts were generated from the single
1B gene, which was mapped to the distal end of the long
arm of human chromosome 9 (Fig. 4), utilizing the major transcription
start site located at 148 nt and the minor start site located at 152 nt
5
-upstream from the ATG translation start site (Fig. 3). The 4.0-kb
5
-flanking sequence of the
1B gene contained a promoter which was capable of directing expression of the
1B
transcript in neuronal cells and repressing its expression in
nonneuronal cells (Fig. 5). Deletion analysis of
1B
subunit-luciferase fusion gene constructs indicated the presence of
cis-acting regulatory elements located in the distal upstream region
(
3992 to
1788) that may be critical for the neuron-specific
expression of the
1B subunit gene (Fig. 6).
Over the past decade, considerable progress has been made in
elucidating molecular mechanisms for the transcriptional activation of
tissue- and cell type-specific expression of genes in nonneuronal cell
types, such as erythrocytes, lymphocytes, and hepatocytes. More
recently, the molecular bases for neuron-specific gene expression has
also been examined (reviewed in Refs. 37 and 38). The transcription
factor, termed neural-restrictive silencer element (NRSE)-binding
factor (NRSF) (39-41) binds a 21-bp NRSE sequence present in the
5-upstream region of neural-specific genes to selectively repress the
transcription of these genes in nonneuronal cells.
The restricted expression of the N-type 1B gene in the
central nervous system and wide distribution of L-type
1D transcripts provide us with an excellent opportunity
to examine and compare molecular bases governing Ca2+
channel gene expression. Consistent with the broad mRNA expression within and outside of the central nervous system, we did not find any
sequences with similarity to the NRSE sequence in the 5
-upstream region of the rat
1D gene. Furthermore, the
transcription of the rat
1D gene is regulated by both
cis-acting positive and negative elements in the 5
promoter region and
by an enhancer that consists of (ATG)7 trinucleotide
repeats (19). Inspection of 5
-flanking sequence of the human
1B gene, however, revealed the nucleotide sequence
(NRSE-
1B) homologous to the NRSE (nucleotide
810 to
789 as shown in Fig. 2). Although overall sequence identity to the
21-bp NRSE consensus sequence is 57%, the 5
half of 10-bp NRSE-
1B fragment showed a 80% sequence identity to that
of the NRSE. We have subcloned NRSE-
1B into the 5
upstream of SV40 promoter linked to luciferase reporter gene to test
whether or not an NRSE-
1B could function as a repressor
element in nonneuronal cells. Luciferase activity assay showed that one
or two copies of this putative motif did not affect the SV40 promoter
activity, whereas one copy of the NRSE from SCG 10 gene was sufficient
to repress its activity to 30% of the control in HeLa cells (data not
shown). Since the promoter activity of 4.0-kb 5
flanking region of the
human
1B gene in various cell lines was in excellent agreement with RT-PCR analysis of the endogenous
1B
mRNA expression (Fig. 5), we used the two of these lines, NS20Y and
HeLa, to search for the cis-acting regulatory elements further 5
upstream of the
1B gene. In vitro transient
transfection of truncated
1B-luciferase fusion gene
indicated that the region between
3992 and
1788 contains a
repressor element(s) responsible for the neuron-specific expression of
the N-type
1B subunit gene (Fig. 6). Since sequence analysis did not indicate the presence of any sequence with similarity to the NRSE, a repressor functional in the N-type
1B
subunit gene may be distinct from the ones already identified. Further studies are required to establish whether this region contains a unique
neuron-specific element that is capable of binding a NRSF.
Our results in the present study suggest that selective repression by
negative cis-regulatory elements is responsible for neuron-specific
expression of the human Ca2+ channel 1B
subunit gene as is the case for the type II Na+ channel and
other genes exclusively expressed in the nervous system. In addition to
the NRSE that is the primary determinant for the tissue specificity,
the core promoters are also important for conferring substantial
neuronal specificity to several genes such as synapsin I, II, and
myelin basic protein (34, 42, 43). In contrast, the activity of the
human
1B subunit gene core promoter (
110L plasmid) was
apparently similar in NS20Y and HeLa cells (Fig. 6A),
indicating that the core promoter itself does not confer the neuron
specificity to the
1B gene. However, we cannot rule out
the possibility of concerted interactions between cell type-specific
distal upstream repressor element(s) and the general minimal
promoter.
In summary, we have presented an initial characterization of the human
N-type Ca2+ channel 1B subunit gene and
identified a region in the 5
-upstream of the gene (
3992 and
1788)
that contains negatively acting cis-regulatory elements responsible for
neuron-specific expression of the
1B gene. Further
deletion analyses of the region between
3992 and
1788 and the
studies of the DNA-protein interactions between transcription factors
and the putative repressor elements should help to elucidate the
molecular mechanisms of transcriptional regulation underlying
spatiotemporal expression of VSCC
1 subunit genes in the
nervous systems.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U76666[GenBank].
We thank Dr. Harold Gainer for encouragement and support, and Jim Nagle of NINDS DNA Sequencing Facility for sequencing.