Isolation and Characterization of the 5'-Upstream Region of the Human N-type Calcium Channel alpha 1B Subunit Gene
CHROMOSOMAL LOCALIZATION AND PROMOTER ANALYSIS*

(Received for publication, June 25, 1996, and in revised form, October 30, 1996)

Dong S. Kim Dagger , Hyun-Ho Jung Dagger , Sun-Hwa Park § and Hemin Chin Dagger

From the Dagger  Laboratory of Neurochemistry, NINDS, National Institutes of Health, Bethesda, Maryland 20892 and § Department of Anatomy, College of Medicine, Korea University, Seoul, Korea 136-701

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

omega -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 alpha 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 alpha 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 alpha 1B subunit-luciferase fusion gene showed that the 4.0-kb 5'-flanking region of the alpha 1B gene functions as an efficient promoter in neuronal cells but not in glioma or nonneuronal cells, consistent with the patterns of the endogenous alpha 1B gene expression in these cells. Deletion analysis of alpha 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 alpha 1B subunit gene.


INTRODUCTION

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, alpha 1, alpha 2-delta , beta , and gamma  (6, 7), and the corresponding cDNAs have been cloned and sequenced (reviewed in Refs. 8 and 9). The cloned skeletal muscle alpha 1 subunit (alpha 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 alpha 1 subunit cDNAs that encode either dihydropyridine (DHP)-sensitive L-type channels (alpha 1C and alpha 1D) or DHP-insensitive high voltage-activated Ca2+ channels (alpha 1A, alpha 1B, and alpha 1E). Distinct isoforms of the alpha 1C and alpha 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 alpha 1 subunits seems to be restricted to nervous tissues and the cells of neuronal origin as is the case for the alpha 1B subunit encoding the omega -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 alpha 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 alpha 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 alpha 1B subunit gene expression and compare it with those involved in the L-type alpha 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 alpha 1B subunit gene.


MATERIALS AND METHODS

Northern Hybridization

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 alpha 1B probe was a 230-base pair (bp) fragment of the 5' region of the alpha 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 alpha 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.

DNA Cloning and Sequencing

To isolate the promoter of the human alpha 1B subunit gene, approximately 2 × 105 recombinant phages from a human WI-38 lung fibroblast cell lambda Fix genomic DNA library (Stratagene, La Jolla, CA) were screened with both the 230-bp alpha 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 Analysis

A 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 alpha 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.


Fig. 2. Nucleotide sequence of 5'-upstream region through the first intron of human N-type calcium channel alpha 1B gene. Two transcription initiation sites are indicated by bent arrows. Nucleotides are numbered with respect to the 3'-major transcription start site that is indicated as +1. The coding sequence of the first exon is shown as codon triplets, and the intron sequence is depicted in lowercase letters. The 5'-end points of deletion constructs (-3992, -1788, -1559, -1289, -1057, -803, -347, -110), and the common 3'-end point (+86) are indicated by arrowheads and numbers. Putative transcription factor binding sites are marked by boxes with their names indicated below. A homologous sequence to consensus NRSE is underlined. Another common motif found in several neuron-specific genes is double underlined. The NAPE1 oligonucleotide used for primer extension analysis is shown as a dashed line.
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Fluorescence in Situ Hybridization

The plasmid containing a 9.5-kb human alpha 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.

Plasmid Construction

The human alpha 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 alpha 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 pCMVbeta (Stratagene, La Jolla, CA) contain the Rous sarcoma virus (RSV) promoter fused to the luciferase gene and the cytomegalovirus (CMV) promoter fused to the beta -galactosidase gene, respectively.

Cell Culture

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 Assay

Equimolar amounts of the human alpha 1B gene-luciferase plasmids (3 µg were used for the shortest deletion construct -110L) and pCMVbeta 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. beta -Galactosidase was monitored by an assay kit in the same lysate (Promega). The luciferase activity of fusion constructs was normalized to beta -galactosidase activity and expressed as a percentage of the RSV promoter activity of pRSVL.

Reverse Transcription (RT)-PCR Analysis

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 alpha 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.


RESULTS

Differential Expression of alpha 1B and alpha 1D Transcripts

The distribution of alpha 1B and alpha 1D Ca2+ channel transcripts in human tissues was examined by Northern blot analysis. A single 10.5-kb transcript of the alpha 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 alpha 1B mRNA present in human brain is similar to the ~10-kb rat brain transcript (13). The alpha 1D cDNA probe, which is derived from the least conserved intracellular linker of the II and III loop of the alpha 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 alpha 1D probe did not cross hybridize to the L-type alpha 1C and alpha 1S subunit transcripts. The 9.5-, 8.5- and 6.5-kb sizes of the human alpha 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 alpha 1B subunit, unlike the DHP-sensitive L-type channel which shows a broad distribution pattern in various tissues, is limited to nervous tissues.


Fig. 1. Comparison of the tissue distribution of the calcium channel between human alpha 1B and alpha 1D subunit mRNAs. 32P-Labeled alpha 1B (A) and alpha 1D (B) probes were hybridized to the human multiple tissue Northern blots as described under "Materials and Methods." A 230-bp PCR product containing the N-terminal domain of the human alpha 1B cDNA and a 443-bp fragment including the intracellular loop domain II of the rat alpha 1D cDNA were used as hybridization probes. The tissues tested were heart (lane 1), brain (lane 2), placenta (lane 3), lung (lane 4), liver (lane 5), skeletal muscle (lane 6), kidney (lane 7), and pancreas (lane 8). Sizes are indicated in kilobase pairs.
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Cloning and Nucleotide Sequence of the 5'-Upstream Region of Human alpha 1B Subunit Gene

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 alpha 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).

Identification of the Transcription Initiation Site

Primer extension analysis was performed in order to determine the transcription initiation site of the human alpha 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.


Fig. 3. Mapping of the transcription initiation site of the human N-type calcium channel alpha 1B gene. Primer extension using 2 µg of poly(A)+ RNA of human neuroblastoma SH-5YSY cells (lane 1) or 2 µg of E. coli tRNA was carried out as described under "Materials and Methods." The arrowheads indicate two transcription initiation sites. On the left is shown a sequencing ladder which was used as a size marker.
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Analysis of the sequence immediately upstream of the transcription initiation sites reveals that the alpha 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<UNL>G</UNL>AGGC) and minor transcription initiation sites (GTC<UNL>G</UNL>GGTG) are different from the initiator sequence (CTC<UNL>A</UNL>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).

Chromosomal Localization of alpha 1B Gene

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 alpha 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 alpha 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.


Fig. 4. Fluorescence in situ hybridization of the human alpha 1B gene to human metaphase chromosome. The chromosome location of the human alpha 1B gene was determined by fluorescent in situ hybridization as described under "Materials and Methods." A, alpha 1B gene probe alone. B, alpha 1B gene probe was cohybridized with a 9-centromere-associated satellite probe. The arrows indicate the fluorescent signal of the alpha 1B subunit gene.
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Cell Type-specific Expression by 5'-Flanking Region of the alpha 1B Gene Promoter

To address whether the 5'-flanking sequence of the human alpha 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 alpha 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 alpha 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).


Fig. 5. Cell type-specific expression of the human alpha 1B subunit gene. A, cell type-specific expression directed by the 5'-flanking sequence of the human alpha 1B gene. The alpha 1B-luciferase fusion gene -3992L was cotransfected with internal control plasmid pCMVbeta . The luciferase activity was normalized to beta -galactosidase activity and expressed as a percentage of the RSV promoter activity in that cell. The histograms show the mean ± S.E. of three independent experiments, each of which was performed in triplicate. B, RT-PCR analysis of endogenous alpha 1B gene expression. The first-strand cDNA was reverse-transcribed using total RNA from each cell and amplified with a specific alpha 1B gene primer and a GAPDH primer for 30 cycles. The PCR products were electrophoresed on an 1.5% agarose gel.
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RT-PCR, which was carried out to detect the endogenous alpha 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 alpha 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 alpha 1B-luciferase fusion gene construct and endogenous alpha 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 alpha 1B gene in a neuron-specific manner.

The Distal Upstream Region of the alpha 1B Gene Promoter for Neuron-specific Expression

To locate a cis-acting regulatory element for neuron-specific expression of the alpha 1B gene, a series of alpha 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 alpha 1B subunit.


Fig. 6. Deletion analysis of the human alpha 1B gene promoter. A series of alpha 1B gene fragments containing progressive 5' deletions and a common 3' end at +86 nucleotide were inserted upstream of promoterless luciferase gene pGL2-Basic. The promoter activity of each alpha 1B-luciferase fusion gene in NS20Y and HeLa cells was determined and expressed as a percentage of the RSV promoter activity (A) or as a fold of repression (B). Results are the mean ± S.E. from at least three separate transfection experiments. A repression fold was defined as a ratio of luciferase activity in NS20Y cells to that of HeLa cells expressed as a percentage of luciferase activity of the reference construct pRSVL. Statistical significance was calculated using Student's t test. *p < 0.01
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DISCUSSION

In this study we report the cloning, chromosomal localization, and molecular analysis of the 5'-flanking region of the human N-type Ca2+ channel alpha 1B subunit gene. A single 10.5-kb alpha 1B mRNA transcript was detected only in the brain among the human tissues examined, whereas L-type alpha 1D mRNAs were detected in a variety of tissues (Fig. 1). The alpha 1B transcripts were generated from the single alpha 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 alpha 1B gene contained a promoter which was capable of directing expression of the alpha 1B transcript in neuronal cells and repressing its expression in nonneuronal cells (Fig. 5). Deletion analysis of alpha 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 alpha 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 alpha 1B gene in the central nervous system and wide distribution of L-type alpha 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 alpha 1D gene. Furthermore, the transcription of the rat alpha 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 alpha 1B gene, however, revealed the nucleotide sequence (NRSE-alpha 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-alpha 1B fragment showed a 80% sequence identity to that of the NRSE. We have subcloned NRSE-alpha 1B into the 5' upstream of SV40 promoter linked to luciferase reporter gene to test whether or not an NRSE-alpha 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 alpha 1B gene in various cell lines was in excellent agreement with RT-PCR analysis of the endogenous alpha 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 alpha 1B gene. In vitro transient transfection of truncated alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 1 subunit genes in the nervous systems.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U76666[GenBank].


   To whom correspondence should be addressed: Laboratory of Neurochemistry, Bldg. 36, Rm. 4D25, NINDS, NIH, Bethesda, MD 20892. Tel.: 301-496-9917; Fax: 301-496-1339; E-mail: hemin{at}codon.nih.gov.
1    The abbreviations used are: VSCC, voltage-sensitive calcium channel; DHP, dihydropyridine; FBS, fetal bovine serum; RT, reverse transcription; PCR, polymerase chain reaction; NRSE, neural-restrictive silence element; NRSF, neural-restrictive silencer element-binding factor; SV40, simian virus 40; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CMV, cytomegalovirus; RSV, Rous sarcoma virus; AMV, avian myeloblastosis virus; PIPES, 1,4-piperazinediethanesulfonic acid; nt, nucleotides, bp, base pair(s); kb, kilobase pair(s).

Acknowledgments

We thank Dr. Harold Gainer for encouragement and support, and Jim Nagle of NINDS DNA Sequencing Facility for sequencing.


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