©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure and Regulation of the Gene Encoding the Neuron-specific Protein Kinase C Substrate Neurogranin (RC3 Protein) (*)

Takayuki Sato , Dian-Mo Xiao , Hua Li , Freesia L. Huang , Kuo-Ping Huang (§)

From the (1) Section on Metabolic Regulation, Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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 Tand 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.


INTRODUCTION

Neurogranin (Ng/RC3),() 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) .

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- 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.

Previously, we characterized the 5`-flanking region of the rat PKC- 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.


EXPERIMENTAL PROCEDURES

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 10clones 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 -Galactosidase Assays

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. 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 KClOby 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 Creversed-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.


RESULTS

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, AGAGAGAGAGAGAGAGAG. 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 (Aat nucleotide +1811, Aat nucleotide +3944, ACACAat nucleotide +4198, and ACAat nucleotide +6494) and T tracts (Tat nucleotide +5462 and Tat 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).

A homology search of the 2 kb upstream from the transcriptional initiation site revealed several potential transcriptional regulatory element sequences for SP1 at nucleotides 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 (GT17)-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- Genes

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 Nachannel, 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.




DISCUSSION

The cloned Ng/RC3 genomic DNA fragment encompasses 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.

The Ng/RC3 gene resembles many other protein-coding genes from the brain, such as PKC- (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 Nachannel, 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) .

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 -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 Tand 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 Tto 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) .

Phorbol ester is the most potent stimulator of Ng/RC3 gene expression. The stimulatory effects are 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.

The 5`-flanking regions of the Ng/RC3 and PKC- 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



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank/EMBL Data Bank with accession number(s) U22062.

§
To whom correspondence should be addressed: NIH, Bldg. 49, Rm. 6A 36, 49 Convent Dr. MSC 4510, Bethesda, MD 20892-4510. Tel.: 301-496-7827; Fax: 301-496-7434.

The abbreviations used are: Ng/RC3, neurogranin; PKC, protein kinase C; bp, base pair(s); kb, kilobase pair(s); CDTA, 1,2-diaminocyclohexane- N, N, N`, N`-tetraacetic acid; MES, 4-morpholineethanesulfonic acid; PMA, phorbol 12-myristate 13-acetate.

D.-M. Xiao, T. Sato, and K.-P. Huang, unpublished results.


ACKNOWLEDGEMENTS

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-.


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