The NMDA (N-methyl-D-aspartate) receptors are important in the regulation of neuronal development, synaptic plasticity, learning and memory, and are involved in several brain pathologies. The NR1 subunit is essential for the assembly of functional receptors, as it forms the calcium-permeable ion channel and contains the obligatory co-agonist binding site. Previous studies have shown that NR1 gene (Grin1) expression is up-regulated during neuronal differentiation and its expression is widespread in the central nervous system. We have previously cloned the chicken Grin1 gene and 1.9 kb of the 5′-regulatory region. In the present study, we analysed the molecular mechanisms that regulate chicken Grin1 gene transcription in undifferentiated cells and neurons. By functional analysis of chicken Grin1–luciferase gene 5′-regulatory region constructs, we demonstrate that the basal promoter is delimited within 210 bp upstream from the main transcription initiation site. DNA–protein binding and functional assays revealed that the 5′-UTR (untranslated region) has one consensus NRSE (neuron-restrictive silencing element) that binds NRSF (neuron-restrictive silencing factor), and one SP (stimulating protein transcription factor) element that binds SP3, both repressing Grin1 gene transcription in undifferentiated P19 cells (embryonic terato-carcinoma cells) and PC12 cells (phaeochromocytoma cells). The promoter region lacks a consensus TATA box, but contains one GSG/SP (GSG-like box near a SP-consensus site) that binds SP3 and up-regulates gene transcription in embryonic chicken cortical neurons. Taken together, these results demonstrate a dual role of SP3 in regulating the expression of the Grin1 gene, by repressing transcription in the 5′-UTR in undifferentiated cells as well as acting as a transcription factor, increasing Grin1 gene transcription in neurons.

INTRODUCTION

The NMDARs [NDMA (N-methyl-D-aspartate) receptors] belong to the ionotropic glutamate receptor family, play important roles in neuronal development and synaptic plasticity and are involved in several neurological and psychiatric disorders [1,2]. The NMDARs are calcium-permeable ligand-gated cation channels containing NR1, NR2 and NR3 subunits, encoded by seven genes: Grin1, Grin2ad, Grin3a and Grin3b. [3,4]. The NR1 subunit is essential for the formation of functional NMDARs because it forms the ion channel and contains the glycine co-agonist binding site [4,5].

Changes in NMDAR subunit expression induced by normal and pathological activity, as well as by neurotrophins, have been documented thoroughly [6]. Among the neurotrophins contained in serum, NGF (nerve growth factor) was shown to up-regulate NR1 subunit expression in PC12 cells (phaeochromocytoma cells) [7]. Also, the Grin1 gene is expressed throughout the central nervous system and is up-regulated during neuronal differentiation and brain development [8,9].

The promoter regions of the rat, mouse and human Grin1 genes have been cloned and characterized [1013], and we have cloned the chicken Grin1 gene previously [12]. Several transcription factors have been shown to play an important role in the transcriptional regulation of the rat Grin1 gene. It has been demonstrated that overexpression of SP (stimulating protein transcription factor)1, SP2 and SP4 increases transcriptional activity of the rat Grin1 gene [1416] by directly interacting with GC-rich elements, or through interacting with MEF2C (myocyte enhancer factor 2C), NF-κB (nuclear factor κB) and MAZ (Myc-associated zinc-finger-binding protein) transcription factors [14,16,17]. In contrast, the NRSF (neuron-restrictive silencing factor) represses the transcription of neuronal genes in non-neuronal cells as well as in undifferentiated pluripotent cells [18]. NRSF is known to negatively regulate the transcription of several neuronal genes, including the rat Grin1 [9,13,19], Nav1.2 (voltage-gated sodium channel subunit 1.2) and SCG10 (superior cervical ganglia protein 10) genes [20]. However, the molecular mechanisms controlling Grin1 gene transcription in birds are unknown.

In the present study, we examined the transcriptional regulation of the chicken Grin1 gene in undifferentiated and neuron-differentiated P19 cells (embryonic terato-carcinoma cells) and PC12 cells, and in chicken cortical neurons. The results showed transcriptional up-regulation of the chicken Grin1 gene 5′-regulatory region upon neuronal differentiation of P19 and PC12 cells. By EMSA (electrophoretic mobility-shift assay) and functional analyses, we demonstrated that binding of NRSF and factors to their respective DNA elements in the 5′-UTR (untranslated region) was responsible for the repression of Grin1 gene transcription in neuronal-committed PC12 cells and in undifferentiated P19 cells, and that such repression was attenuated in chicken cortical neurons. In contrast, binding of SP3 to a SP element near a GSG site located in the proximal promoter region activated transcription of the chicken Grin1 gene in cortical neurons. These findings suggest a dual role of SP3 in the transcriptional regulation of the chicken Grin1 gene as a transcriptional repressor together with NRSF by binding to the 5-UTR and probably forming a repressor complex, whereas the interaction of SP3 with a SP-site located in the proximal promoter plays a role as a transcriptional activator of the Grin1 gene in chicken cortical neurons.

EXPERIMENTAL

Cell culture

P19 cells were grown in DMEM (Dulbecco's modified Eagle medium), supplemented with 10% (v/v) FBS (fetal bovine serum). P19 cells were induced to differentiate into neurons by the addition of 0.5 μM retinoic acid for 4 days, and then cells were treated with trypsin and plated into tissue-culture dishes with their growth medium supplemented with 50 ng/ml Ara-C (cytosine arabinoside). PC12 cells were maintained in DMEM supplemented with 10% (v/v) HS (horse serum) and 5% (v/v) FBS. For neuronal differentiation of PC12 cells, cells were plated on poly-D-ornithine-coated tissue-culture dishes. Subsequently, the medium was substituted with DMEM containing 2% (v/v) HS and 50 nM NGF 2.5S (Alomone Labs, Jerusalem, Israel). Primary culture of cortical chicken neurons was performed as described previously [12]. Briefly, forebrains from E7 (embryonic day 7) chicken embryos were digested with 0.5% trypsin in Advanced DMEM (Invitrogen), washed twice with Advanced DMEM supplemented with 20% (v/v) FBS, passed through a nylon mesh, seeded and maintained in Advanced DMEM supplemented with 3% (v/v) FBS and 50 ng/ml Ara-C in poly-D-ornithine-coated culture dishes. All culture media were supplemented with kanamycin (60 mg/l), penicillin G (10 i.u./ml), streptomycin (10 mg/ml), amphotericin B (0.025 mg/ml) and nystatin (10 i.u./ml). The use of chicken embryos was approved by the Ethical Committee of the School of Medicine Universidad Nacionel Autónoma de México.

Immunocytochemistry

The TUJ1 antibody (provided by Dr Iván Velasco, Instituto de Fisiología Celular, Universidad Autónoma de México, México) was used for the detection of β-tubulin III during neuronal differentiation of P19 and PC12 cells. The cells were induced to differentiate into neurons as described above. Following fixation with 4% (w/v) paraformaldehyde for 10 min, cells were washed in PBS, blocked with 3% (v/v) goat serum for 2 h, and then incubated overnight with the mouse TUJ1 antibody (1:2000 dilution; Chemicon International) at 4°C. After washing with PBS, Alexa Fluor® 488-conjugated anti-mouse secondary antibody (1:500 dilution; Invitrogen) was added for 2 h at room temperature (22°C) and then washed with PBS. Nuclei were dyed with Hoechst staining. The cells were visualized using a confocal Nikon Eclipse TE 2000-U microscope and were analysed with ACT-1 software (Nikon).

Plasmid constructs

We previously cloned a 1.9 kb genomic DNA fragment digested with SstI containing 1817 bp of the 5′-regulatory region from the chicken Grin1 gene and 86 bp of 5′-UTR into the pGL3-Basic plasmid expressing the firefly luciferase gene as a reporter (Promega). [12]. This construct was used to generate five additional DNA restriction enzyme deletion constructs. This plasmid was digested with SmaI to generate a construct containing only 51 bp of the 5′-flanking regulatory region. All of the other constructs were generated by digestion with HindIII and the following enzymes: BamHI (to generate a −210 bp construct); PvuII (to produce a −334 bp construct); RsaI (to generate a −549 bp construct); and NcoI (to generate the −1237 bp construct). All constructs contained the first 86 bp of the 5′-UTR. The obtained DNA fragments were cloned into pGL3-Basic, amplified in Escherichia coli DH5α cells and purified using anion-exchange gravity-flow columns (Qiagen) following the manufacturer's instructions. All constructs were verified by DNA restriction-endonuclease mapping and DNA sequencing. The REST-VP16 plasmid was provided by Dr S. Majumder (Department of Molecular Genetics, M.D. Anderson Cancer Center, University of Texas, Houston, TX, U.S.A.) [21]. The pGL3 promoter containing the basal SV40 (simian virus 40) promoter was used as a positive transfection control.

Mutagenesis

Site-directed mutagenesis of the GSG-like-box, GSG/SP (GSG-like box near a SP-consensus site), S2 and N1 elements was performed using the QuikChange® site-directed mutagenesis kit (Stratagene) with the wild-type −210 bp Grin1–luciferase plasmid construct used as the DNA template and the designed synthetic mutated DNA oligonucleotide primers (see Table 1). Mutations were performed in the presence of 7.5% (v/v) DMSO because of the high Tm (melting temperature of DNA) value of the template, and reactions were performed as follows: 12 cycles of 30 s at 95°C for denaturing, 60 s at the indicated annealing Tm, and 12 min at 68°C for extension. Subsequently, template DNA was eliminated by digestion with DpnI (a methylation-sensitive DNA-restriction enzyme) and the newly synthesized mutated plasmids were transformed into E. coli DH5α competent cells. The mutant plasmids were then amplified and purified using the endo-free plasmid purification kit (Qiagen) following the manufacturer's instructions. The mutations in the obtained plasmids were confirmed by automated DNA sequencing.

Table 1
Oligonucleotides used for EMSAs and site-directed mutagenesis

The consensus core sequences of the elements are underlined and the mutated nucleotides are indicated in bold type. cs, consensus sequence; f, forward; m, mutation; r, reverse.

NameSequence 5′→3′
SP cs f 5′-ATTCGATCGGGGCGGGGCGAGC-3′ 
SP cs r 5′-GCTCGCCCCGCCCCGATCGAAT-3′ 
SPm cs f 5′-ATTCGATCGGTTCGGGGCGAGC-3′ 
SPm cs r 5′-GCTCGCCCCGAACCGATCGAAT-3′ 
S2 f 5′-AGCCGGCGGCGGGCGGAGCGGCGCGG-3′ 
S2 r 5′-CCGCGCCGCTCCGCCCGCCGCCGGCT-3′ 
S2m f 5′-AGCCGGCGGCGTTCGGAGCGGCGCGG-3′ 
S2m r 5′-CCGCGCCGCTCCGAACGCCGCCGGCT-3′ 
GSG/SP f 5′-TCCCGCTGCCGCGGGGGCCGGGGGCGGGCCGGGGGTGGG-3′ 
GSG/SP r 5′-CCCACCCCCGGCCCGCCCCCGGCCCCCGCGGCAGCGGGA-3′ 
GSG/SPm1 f 5′-TCCCGCTGCCGCTTGGGCCGGGGGCGGGCCGGGGGTGGG-3′ 
GSG/SPm1 r 5′-CCCACCCCCGGCCCGCCCCCGGCCCAAGCGGCAGCGGGA-3′ 
GSG/SPm2 f 5′-TCCCGCTGCCGCGGGGGCCGGGTTCGGGCCGGGGGTGGG-3′ 
GSG/SPm2 r 5′-CCCACCCCCGGCCCGAACCCGGCCCCCGCGGCAGCGGGA-3 
NRSE cs f 5′-GCCAAACACGCTTCAGCACCTCGGACAGCATCCGCCGCGC-3′ 
NRSE cs r 5′-GCGCGGCGGATGCTGTCCGAGGTGCTGAAGCGTGTTTGGC-3′ 
NRSEm cs f 5′-GCCAAACACGCTTCGTAACCTCGGACAGCATCCGCCGCGC-3′ 
NRSEm cs r 5′-GCGCGGCGGATGCTGTCCGAGGTTACGAAGCGTGTTTGGC-3′ 
N1 f 5′-GAGCGGGAGGTTCAGCACCAAGGAGAGCTCCCCGCGCCGC-3′ 
N1 r 5′-GCGGCGCGGGGAGCTCTCCTTGGTGCTGAACCTCCCGCTC-3′ 
N1m f 5′-GAGCGGGAGGTTTCGTACCAAGGAGAGCTCTTACGCGTGC-3′ 
N1m r 5′-GCACGCGTAAGAGCTCTCCTTGGTACGAAACCTCCCGCTC-3′ 
N2 f 5′-CGGCGGCGGGCGGAGCGGCGCGGAGCGGAGCGGGAGGTTCA-3′ 
N2 r 5′-TGAACCTCCCGCTCCGCTCCGCGCCGCTCCGCCCGCCGCCG-3′ 
N3 f 5′-CGCTGCGGCAGCGCGGGGCCGCGGAGCGGGAGGAGCCGGCGG-3′ 
N3 r 5′-CCGCCGGCTCCTCCCGCTCCGCCGCCCCGCGCTGCCGCAGCG-3′ 
NameSequence 5′→3′
SP cs f 5′-ATTCGATCGGGGCGGGGCGAGC-3′ 
SP cs r 5′-GCTCGCCCCGCCCCGATCGAAT-3′ 
SPm cs f 5′-ATTCGATCGGTTCGGGGCGAGC-3′ 
SPm cs r 5′-GCTCGCCCCGAACCGATCGAAT-3′ 
S2 f 5′-AGCCGGCGGCGGGCGGAGCGGCGCGG-3′ 
S2 r 5′-CCGCGCCGCTCCGCCCGCCGCCGGCT-3′ 
S2m f 5′-AGCCGGCGGCGTTCGGAGCGGCGCGG-3′ 
S2m r 5′-CCGCGCCGCTCCGAACGCCGCCGGCT-3′ 
GSG/SP f 5′-TCCCGCTGCCGCGGGGGCCGGGGGCGGGCCGGGGGTGGG-3′ 
GSG/SP r 5′-CCCACCCCCGGCCCGCCCCCGGCCCCCGCGGCAGCGGGA-3′ 
GSG/SPm1 f 5′-TCCCGCTGCCGCTTGGGCCGGGGGCGGGCCGGGGGTGGG-3′ 
GSG/SPm1 r 5′-CCCACCCCCGGCCCGCCCCCGGCCCAAGCGGCAGCGGGA-3′ 
GSG/SPm2 f 5′-TCCCGCTGCCGCGGGGGCCGGGTTCGGGCCGGGGGTGGG-3′ 
GSG/SPm2 r 5′-CCCACCCCCGGCCCGAACCCGGCCCCCGCGGCAGCGGGA-3 
NRSE cs f 5′-GCCAAACACGCTTCAGCACCTCGGACAGCATCCGCCGCGC-3′ 
NRSE cs r 5′-GCGCGGCGGATGCTGTCCGAGGTGCTGAAGCGTGTTTGGC-3′ 
NRSEm cs f 5′-GCCAAACACGCTTCGTAACCTCGGACAGCATCCGCCGCGC-3′ 
NRSEm cs r 5′-GCGCGGCGGATGCTGTCCGAGGTTACGAAGCGTGTTTGGC-3′ 
N1 f 5′-GAGCGGGAGGTTCAGCACCAAGGAGAGCTCCCCGCGCCGC-3′ 
N1 r 5′-GCGGCGCGGGGAGCTCTCCTTGGTGCTGAACCTCCCGCTC-3′ 
N1m f 5′-GAGCGGGAGGTTTCGTACCAAGGAGAGCTCTTACGCGTGC-3′ 
N1m r 5′-GCACGCGTAAGAGCTCTCCTTGGTACGAAACCTCCCGCTC-3′ 
N2 f 5′-CGGCGGCGGGCGGAGCGGCGCGGAGCGGAGCGGGAGGTTCA-3′ 
N2 r 5′-TGAACCTCCCGCTCCGCTCCGCGCCGCTCCGCCCGCCGCCG-3′ 
N3 f 5′-CGCTGCGGCAGCGCGGGGCCGCGGAGCGGGAGGAGCCGGCGG-3′ 
N3 r 5′-CCGCCGGCTCCTCCCGCTCCGCCGCCCCGCGCTGCCGCAGCG-3′ 

Transfection and luciferase assays

The P19 and PC12 stable-transfected cell lines were generated by co-transfecting the pGL3-Grin1 1817 bp construct and pCDNA3, which contains the Geneticin-resistance gene. Briefly, P19 and PC12 cells were grown for 2 weeks in the appropriate growth medium supplemented with 250 μg/ml and 500 μg/ml Geneticin respectively. Three different unique clone Geneticin-resistant colonies were selected from each cell line and tested for firefly luciferase activity. The stably transfected cell lines were induced to differentiate as described above, and firefly luciferase activity was measured at 24 h intervals. The results were normalized against total protein content and expressed as the ratio of firefly luciferase activity per mg of protein.

For transient-expression studies, co-transfections were performed using Lipofectamine™ 2000 reagent (Invitrogen) following the manufacturer's protocol in serum-free DMEM, with 1 μg of the chicken pGL3-Grin1 constructs and 0.1 μg of pRL-CMV (Promega) or REST-VP16 plasmid. Transfections were performed in undifferentiated P19 and PC12 cells and in primary culture cortical chicken neurons and then incubated for 3 h at 37°C. Subsequently, the transfection medium was replaced with the appropriate growth medium and cells were incubated at 37°C and 5% CO2 for 24 h. Cells were lysed in passive lysis buffer (Promega) for 30 min and firefly and Renilla luciferase activities assays were performed using the dual-luciferase assay reagent kit (Promega) in a multiwell plate luminometer (Wallac Victor2, PerkinElmer). Firefly luciferase activity was normalized using Renilla luciferase activity or total protein in the samples as indicated. Pharmacological treatments were performed after 24 h of transient transfection. Briefly, growth medium was supplemented with 100 nM mithramycin A and cells were maintained in supplemented medium for 16 h until the luciferase assay was performed as stated above.

EMSA

The DNA oligonucleotide sequences employed for the EMSAs are listed in Table 1. Nuclear extracts were prepared from undifferentiated and neuron-differentiated P19 and PC12 cells, as well as from primary cultures of E7 cortical chicken neurons as described previously [22]. Briefly, cells were lysed in a hypo-osmotic buffer containing 20 mM Hepes, 20% (w/v) glycerol, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT (dithiothreitol) and 1 mM PMSF (pH 7.9) and incubated at 4°C for 10 min. Then nuclei were pelleted and lysed in a hyper-osmotic buffer containing 20 mM Hepes, 20% (w/v) glycerol, 0.5 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 1 mM PMSF and 3.5 fmol dsDNA (double-strand DNA) oligonucleotides 5′-end labelled by T4 polynucleotide kinase and 1 μl of [γ-32P]ATP (6000 Ci/mmol; PerkinElmer) (pH 8.0). The DNA–protein binding reaction was performed in a buffer containing 50 mM Tris/HCl (pH 7.6) 20% (w/v) glycerol, 5 mM MgCl2, 2.5 mM EDTA, 1 mM DTT and 1 mM PMSF, with 150 mM NaCl for NRSE (neuron-restrictive silencing element) oligonucleotides or with 300 mM NaCl for the other oligonucleotides. Briefly, 10 μg of nuclear extract, 1 μg of poly-dI/dC and a 100-fold excess of unlabelled dsDNA oligonucleotides were added for the DNA-competition assay as indicated, and 1 μl of [32P]-labelled dsDNA oligonucleotides were incubated at room temperature to allow DNA–protein binding. The mixture was incubated with 2 μg of super-shift antibodies for NRSF (H-290), SP1 (PEP2 and H-225), TFIID (transcription factor IID; N-12) and EGR-1 (early growth-response gene product 1; C-20) (all from Santa Cruz Biotechnology). Reaction products were resolved by non-denaturing PAGE (5% gels) containing 3% (w/v) glycerol for 4 h at 20 mA. Gels were dried and exposed to X-Omat film (Kodak) at −80°C for 12–72 h.

Sequence and statistical analyses

The nucleotide sequence of the 1.9 kb 5′-regulatory region of the chicken Grin1–luciferase gene construct has been reported previously [12] (see GenBank® Nucleotide Sequence Database accession number AY663367). In silico DNA-sequence analyses were performed using BLAST (National Center for Biotechnology Information), MacVector (Accelrys Software), MathInspector (Genomatix), Transfac (BioBase) and EMBOSS CpG Plot programs and algorithms. The results are means±S.E.M. (n≥3) and were analysed by one-way ANOVA and Dunnett's test using the Prism 4.0 program (GraphPad). P<0.05 was considered to be statistically significant.

RESULTS

Grin1 gene transcription increases along neuronal differentiation

To analyse the pattern of transcriptional activity of the chicken Grin1 gene during neuronal differentiation, we used the pluripotent P19 cells [23] and the neuron-committed PC12 cells [24] as two cellular models of neuronal differentiation. We generated stable transfectants of both cell lines by expressing a chimaeric Grin1–luciferase gene construct containing 1.9 kb of the chicken Grin1 gene 5′-regulatory region subcloned into pGL3-Basic [12]. To confirm that the two neuronal-differentiation models were working properly, we assessed the expression of β-tubulin III in P19 and PC12 cells in the undifferentiated and neuronal-differentiated stages using immunocytochemistry. The results showed that although undifferentiated P19 cells did not express β-tubulin III, following the induction of differentiation by retinoic acid, a progressive increase in the expression of the protein was observed from day 5 to day 7 (Figure 1A). In contrast, β-tubulin III expression was observed in undifferentiated PC12 cells, and increased progressively up to the sixth day following NGF-induced neuronal differentiation (Figure 1C).

The P19 stable-transfectant cells showed low transcriptional activity in the undifferentiated stage, which increased starting on the fifth day after retinoic-acid-induced neuronal differentiation, reaching an 8-fold increase in stimulation by the eighth day of treatment (Figure 1B). In PC12 stable-transfectant cells, NGF-induced differentiation promoted a 4- to 5-fold increase in transcriptional activity after 2 days and then reaching a steady state from between 3 and 6 days (Figure 1D).

Transcription of the chicken Grin1 gene is up-regulated during neuronal differentiation of P19 and PC12 cells

Figure 1
Transcription of the chicken Grin1 gene is up-regulated during neuronal differentiation of P19 and PC12 cells

(A) Immunocytochemistry of β-tubulin III expression in P19 cells during 0, 2, 4, 5 and 7 days of retinoic-acid-induced neuronal differentiation. A β-tubulin III antibody (TUJ1) labelled with FITC and Hoechst stain to dye the nuclei was used. (B) Stable transfectants in P19 cells containing 1.9 kb of the chicken Grin1 gene 5′-regulatory region driving the expression of the firefly luciferase gene were induced to differentiate into neurons by treatment with retinoic acid for 8 days. Luciferase activity and total protein amount was measured every 24 h. The results are expressed as the ratio of luciferase activity/total protein. **P<0.01. (C) Immunocytochemistry of β-tubulin III expression in PC12 cells during 0, 2, 4 and 6 days of NGF-induced neuronal differentiation. A β-tubulin III antibody (TUJ1) labelled with FITC and Hoechst stain to dye the nuclei was used. (D) Stable-transfected PC12 cells containing the 1.9 kb of the chicken Grin1 gene 5′-regulatory region were induced to differentiate into neurons by treatment with NGF for 6 days. Luciferase activity and total protein amount were measured every 24 h. The results are expressed as the ratio of luciferase activity/total protein. **P<0.01.

Figure 1
Transcription of the chicken Grin1 gene is up-regulated during neuronal differentiation of P19 and PC12 cells

(A) Immunocytochemistry of β-tubulin III expression in P19 cells during 0, 2, 4, 5 and 7 days of retinoic-acid-induced neuronal differentiation. A β-tubulin III antibody (TUJ1) labelled with FITC and Hoechst stain to dye the nuclei was used. (B) Stable transfectants in P19 cells containing 1.9 kb of the chicken Grin1 gene 5′-regulatory region driving the expression of the firefly luciferase gene were induced to differentiate into neurons by treatment with retinoic acid for 8 days. Luciferase activity and total protein amount was measured every 24 h. The results are expressed as the ratio of luciferase activity/total protein. **P<0.01. (C) Immunocytochemistry of β-tubulin III expression in PC12 cells during 0, 2, 4 and 6 days of NGF-induced neuronal differentiation. A β-tubulin III antibody (TUJ1) labelled with FITC and Hoechst stain to dye the nuclei was used. (D) Stable-transfected PC12 cells containing the 1.9 kb of the chicken Grin1 gene 5′-regulatory region were induced to differentiate into neurons by treatment with NGF for 6 days. Luciferase activity and total protein amount were measured every 24 h. The results are expressed as the ratio of luciferase activity/total protein. **P<0.01.

Functional analysis of the chicken Grin1 gene 5′-regulatory region

The functional analysis of the chicken Grin1 gene 5′-regulatory region was performed by transient transfection of pGL3-Grin1 deletion constructs into primary cultures of chicken cortical neurons. The constructs contained 51 bp, 210 bp, 334 bp, 549 bp, 1237 bp and 1817 bp of the upstream 5′-regulatory region, as well as 86 bp of the 5′-UTR fused to a reporter firefly luciferase gene. To define the basal chicken Grin1 gene promoter, we analysed the transcriptional activity of the constructs in chicken cortical neurons at E7. Figure 2(A) shows that the Grin1–luciferase construct containing only 51 bp upstream from the transcription initiation site did not exhibit transcriptional activity, whereas the construct containing 210 bp showed significant transcriptional activity in cortical neurons. The 334 bp and 549 bp constructs showed 2–2.5-fold higher transcriptional activity compared with the 210 bp Grin1–luciferase construct. In contrast, the 1237 bp Grin1–luciferase construct showed a decrease in transcriptional activity by 30% compared with the 549 bp construct, whereas the 1815 bp construct induced a 2-fold increase in activity similar to that induced by the 549 bp Grin1–luciferase construct (Figure 2A).

Functional and in silico analysis of the chicken Grin1 gene 5′-regulatory region

Figure 2
Functional and in silico analysis of the chicken Grin1 gene 5′-regulatory region

(A) The chimaeric chicken 5′-flanking region Grin1–luciferase constructs that contain 51 bp, 210 bp, 334 bp, 549 bp, 1237 bp and 1817 bp of the 5′-regulatory region and 86 bp of the 5′-UTR were transiently transfected into E7 primary cortical neurons in culture. The results are expressed as the ratio of firefly/Renilla luciferase activities. Statistical significance was determined relative to the −210 bp Grin1–luciferase construct and ANOVA with Dunnett's analysis was performed (**P<0.01). †, statistically significant relative to the 210 bp construct. (B) Nucleotide comparison of the Grin1 gene regulatory region among human, rat, mouse and chicken shows a conserved pattern of transcription-factor-binding sites among species. The chicken Grin1 gene nucleotide sequence shown has been previously reported (GenBank® nucleotide database sequence accession number AY663367) [12]. Nucleotide similarity among chicken, human, rat and mouse is indicated by asterisks (*). The putative SP sites and GSG-like-box are boxed, and the putative NRSEs are shaded grey. The transcription-initiation sites described previously for the Grin1 genes are circled [1013]. Position +1 in the sequences corresponds to the translation initiation site. (C) The complete Grin1 gene and regulatory-region sequence were analysed for the presence of a CpG dinucleotide island using EMBOSS CpG Plot and MacVector. The CpG dinucleotide distribution within the sequence is plotted and reveals the presence of a CpG island spanning the chicken Grin1 gene 5′-flanking sequence. The structure of the genomic region is represented below the plot.

Figure 2
Functional and in silico analysis of the chicken Grin1 gene 5′-regulatory region

(A) The chimaeric chicken 5′-flanking region Grin1–luciferase constructs that contain 51 bp, 210 bp, 334 bp, 549 bp, 1237 bp and 1817 bp of the 5′-regulatory region and 86 bp of the 5′-UTR were transiently transfected into E7 primary cortical neurons in culture. The results are expressed as the ratio of firefly/Renilla luciferase activities. Statistical significance was determined relative to the −210 bp Grin1–luciferase construct and ANOVA with Dunnett's analysis was performed (**P<0.01). †, statistically significant relative to the 210 bp construct. (B) Nucleotide comparison of the Grin1 gene regulatory region among human, rat, mouse and chicken shows a conserved pattern of transcription-factor-binding sites among species. The chicken Grin1 gene nucleotide sequence shown has been previously reported (GenBank® nucleotide database sequence accession number AY663367) [12]. Nucleotide similarity among chicken, human, rat and mouse is indicated by asterisks (*). The putative SP sites and GSG-like-box are boxed, and the putative NRSEs are shaded grey. The transcription-initiation sites described previously for the Grin1 genes are circled [1013]. Position +1 in the sequences corresponds to the translation initiation site. (C) The complete Grin1 gene and regulatory-region sequence were analysed for the presence of a CpG dinucleotide island using EMBOSS CpG Plot and MacVector. The CpG dinucleotide distribution within the sequence is plotted and reveals the presence of a CpG island spanning the chicken Grin1 gene 5′-flanking sequence. The structure of the genomic region is represented below the plot.

In silico analysis of the chicken Grin1 gene 5′-regulatory region

To understand the role played by transcription factors in the transcriptional activity of the chicken Grin1 gene promoter, we performed in silico comparative analyses of the proximal chicken Grin1 gene 5′-regulatory region sequence with their counterparts in human, rat and mouse. Figure 2(B) shows the nucleotide alignment of the DNA sequence of the proximal 210 bp of the 5′-regulatory region and 86 bp of 5′-UTR of the chicken Grin1 gene aligned with the 5′-regulatory region of the rat, mouse and human genes. It is revealed that there is a low overall nucleotide-sequence similarity (31%) among species (Figure 2B). The DNA-sequence analysis shows that the proximal 5′-flanking region of the chicken Grin1 promoter is highly GC-rich (90%) and contains a CpG-dinucleotide island that spans 397 bp of the 5′-flanking genomic region, including the proximal promoter and the 5′-UTR (Figure 2C). Interestingly, although nucleotide similarity among species is low, conservation of the consensus DNA-binding sites for related transcription factors in the 5′-flanking region between mammals and birds is evident. As in the case of mammalian Grin1 gene promoters, the chicken Grin1 gene 5′-regulatory region does not possess a consensus TATA box, it instead has a putative GSG-like-box and one SP site at −14 bp to −30 bp upstream from the main transcription-initiation site (Figure 2B). It has been proposed that these sites play an equivalent role in recruiting basal transcription factors for RNA-polymerase-II-mediated transcription [25,26]. Analysis of the 5′-UTR of the Grin1 genes revealed the presence of putative binding elements for NRSF and SP transcription factors (Figures 2B and 2C). The chicken Grin1 gene constructs contain two consensus SP-binding sites (named S1 and S2) and three putative NRSEs (named N1, N2 and N3) in the 5′-UTR.

DNA–protein interaction of the NRSE and SP elements located in the 5′-UTR

To determine whether the putative NRSE and SP elements have DNA–protein-binding properties we performed EMSAs using wild-type and mutated synthetic dsDNA oligonucleotides designed for these sites (Table 1). We found that of the three putative NRSE sites (N1, N2 and N3), only the N1 element located in the 5′-UTR bound NRSF specifically (results not shown). Figure 3(A) shows the competition assays using the N1-oligonucleotide probe and nuclear extracts from undifferentiated P19 cells. As a positive control, we used an NRSE-consensus sequence from the rat Grin1 gene [9] (see Table 1). Two high-molecular-mass DNA–protein complexes were observed with both probes, and were specifically competed by a 100-fold molar excess of the unlabelled dsDNA consensus and N1 probes. These complexes were competed when a NRSF-specific antibody was added to the binding reaction. In contrast, when we used nuclear extracts from neuron-differentiated PC12 and P19 cells, and primary cultures of E7 chicken cortical neurons, no DNA–protein complexes were observed (Figure 3), probably as a result of a low abundance of NRSF in the nuclear extracts.

NRSF binds to the NRSE (N1) site present in the 5′-UTR of the chicken Grin1 gene

Figure 3
NRSF binds to the NRSE (N1) site present in the 5′-UTR of the chicken Grin1 gene

(A and B) Nuclear extracts obtained from undifferentiated P19 cells were incubated with a 32P-labelled NRSE consensus dsDNA oligonucleotide probe from the rat Grin1 gene [9], and the NRSE site (N1) of the chicken Grin1 gene. Two slow-migrating DNA–protein complexes were observed with both probes and specifically competed with a 100-fold molar excess of the N1-unlabelled probe (NRSE cs), but not with the N1m probe (NRSE mcs). The DNA–protein complexes were competed with the NRSF antibody (NRSF Ab). The two high-molecular-mass complexes were not observed with nuclear extracts from differentiated P19 cells (C), E7 chicken cortical neurons (D) or undifferentiated PC12 cells (E).

Figure 3
NRSF binds to the NRSE (N1) site present in the 5′-UTR of the chicken Grin1 gene

(A and B) Nuclear extracts obtained from undifferentiated P19 cells were incubated with a 32P-labelled NRSE consensus dsDNA oligonucleotide probe from the rat Grin1 gene [9], and the NRSE site (N1) of the chicken Grin1 gene. Two slow-migrating DNA–protein complexes were observed with both probes and specifically competed with a 100-fold molar excess of the N1-unlabelled probe (NRSE cs), but not with the N1m probe (NRSE mcs). The DNA–protein complexes were competed with the NRSF antibody (NRSF Ab). The two high-molecular-mass complexes were not observed with nuclear extracts from differentiated P19 cells (C), E7 chicken cortical neurons (D) or undifferentiated PC12 cells (E).

The two putative SP-factor-binding sites present in the 5′-UTR of the −210 bp construct were also examined. Figure 4(A) shows that the SP-consensus oligonucleotide and the S2 oligonucleotide formed three main DNA–protein complexes (a, b and c) with nuclear extracts from undifferentiated P19 and PC12 cells and from E7 chicken cortical neurons (Figures 4B–4D), and they were specifically competed by a 100-fold molar excess of the SP consensus and S2-unlabelled dsDNA probes. The two lower-molecular-mass DNA–protein complexes (b and c) were super-shifted when the SP3 antibody was added to the binding reaction (d), and were not super-shifted upon the addition of the SP1 or SP4 antibodies (Figure 4). When nuclear extracts from E7 chicken cortical neurons were tested, addition of the SP3 antibody specifically competed against the complex (Figure 4B). Addition of the SP1 antibody to the binding reactions did not compete the DNA–protein complexes formed using nuclear extracts from undifferentiated P19 and PC12 cells, but partially competed the upper complexes (a and b) formed when using nuclear extracts from chicken cortical neurons.

SP3 factor binds to the SP element (S2) present in the 5′-UTR of the chicken Grin1 gene

Figure 4
SP3 factor binds to the SP element (S2) present in the 5′-UTR of the chicken Grin1 gene

(A) EMSA analysis of the 32P-labelled SP1 consensus dsDNA oligonucleotide probe (see Table 1) when incubated with nuclear extracts from undifferentiated P19 cells. Three major complexes were observed and competed with 100-fold molar excess of the unlabelled SP1-consensus probe (SP1cs), but were not competed with the SP1m probe (SP1csm). The two lower-molecular-mass complexes were super-shifted when 1 μg of the SP3 antibody was added to the binding reaction, but not with the SP1 and SP4 antibodies. The chicken Grin1 gene SP1 dsDNA oligonucleotide (S2) was incubated with nuclear extracts from undifferentiated P19 cells (B), undifferentiated PC12 cells (C) or E7 chicken cortical neurons (D). With all cell extracts, the three upper complexes observed (a, b and c) were competed with 100-fold molar excess of the S2-unlabelled probe, but were not competed by the S2m probe. The complexes a and b were super-shifted by the SP3 antibody, but not with the SP1 antibody in undifferentiated P19 and PC12 cells. With E7 chicken cortical neuron extracts, SP1 and SP3 antibodies competed with the complex (D). Ab, antibody.

Figure 4
SP3 factor binds to the SP element (S2) present in the 5′-UTR of the chicken Grin1 gene

(A) EMSA analysis of the 32P-labelled SP1 consensus dsDNA oligonucleotide probe (see Table 1) when incubated with nuclear extracts from undifferentiated P19 cells. Three major complexes were observed and competed with 100-fold molar excess of the unlabelled SP1-consensus probe (SP1cs), but were not competed with the SP1m probe (SP1csm). The two lower-molecular-mass complexes were super-shifted when 1 μg of the SP3 antibody was added to the binding reaction, but not with the SP1 and SP4 antibodies. The chicken Grin1 gene SP1 dsDNA oligonucleotide (S2) was incubated with nuclear extracts from undifferentiated P19 cells (B), undifferentiated PC12 cells (C) or E7 chicken cortical neurons (D). With all cell extracts, the three upper complexes observed (a, b and c) were competed with 100-fold molar excess of the S2-unlabelled probe, but were not competed by the S2m probe. The complexes a and b were super-shifted by the SP3 antibody, but not with the SP1 antibody in undifferentiated P19 and PC12 cells. With E7 chicken cortical neuron extracts, SP1 and SP3 antibodies competed with the complex (D). Ab, antibody.

Mutations of the N1 and S2 elements release transcriptional repression in undifferentiated P19 and PC12 cells

We demonstrated by EMSA that NRSF interacts with a NRSE element present in the 5′-UTR and in the proximal chicken Grin1 gene promoter. Because the NRSF has been shown to play an important role in the transcriptional regulation of different neuronal gene promoters, including the rat Grin1 gene, we generated mutant constructs by site-directed mutagenesis to test their functional activity in vivo. To test the functionality of the N1 site, the −210 bp construct with a N1m (mutated N1 site) was transiently transfected into undifferentiated P19 cells. Figure 5 shows that the transcriptional activity of the N1m construct was 4-fold higher compared with the wild-type −210 bp construct. In addition, the transcriptional activity of the N1m construct in neuronal-lineage-committed PC12 cells that lack the NRSF was only slightly increased (30%) compared with the wild-type construct. Finally, in E7 chicken cortical neurons, the N1m construct activity was 2-fold higher compared with the −210 bp control construct.

Mutagenic analysis of the functional SP and NRSE sites

Figure 5
Mutagenic analysis of the functional SP and NRSE sites

Site-directed mutagenesis of the SP site S2 (S2m) and N1 (N1m) sites was performed as described in the Experimental section. The mutated Grin1 gene chimaeric constructs were transiently transfected into undifferentiated P19 cells (A), undifferentiated PC12 cells (B) and E7 chicken cortical neurons (C). The white bars represent the transcriptional activity observed when the wild-type −210 bp Grin1–luciferase construct was co-transfected with the REST-VP16 plasmid and Dual-luciferase assays were performed as described in the Experimental section. The results are expressed as a ratio of the firefly/Renilla luciferase activities. Statistical significance is relative to the −210 bp Grin1–luciferase wild-type construct and was used as a control. ANOVA with Dunnett's test was performed (*P<0.05 and **P<0.01). (D) Undifferentiated P19 cells were transiently transfected with the −210 bp Grin1–luciferase construct as described in the Experimental section. The cells were maintained with medium supplemented with 100 nM mithramycin A for 16 h. Luciferase assays were then performed on cell extracts and the total protein amount was measured. The results are the ratio of luciferase activity/total protein. Statistical significance is relative relative to the control (wild-type) and ANOVA with Dunnett's test was performed (**P<0.01).

Figure 5
Mutagenic analysis of the functional SP and NRSE sites

Site-directed mutagenesis of the SP site S2 (S2m) and N1 (N1m) sites was performed as described in the Experimental section. The mutated Grin1 gene chimaeric constructs were transiently transfected into undifferentiated P19 cells (A), undifferentiated PC12 cells (B) and E7 chicken cortical neurons (C). The white bars represent the transcriptional activity observed when the wild-type −210 bp Grin1–luciferase construct was co-transfected with the REST-VP16 plasmid and Dual-luciferase assays were performed as described in the Experimental section. The results are expressed as a ratio of the firefly/Renilla luciferase activities. Statistical significance is relative to the −210 bp Grin1–luciferase wild-type construct and was used as a control. ANOVA with Dunnett's test was performed (*P<0.05 and **P<0.01). (D) Undifferentiated P19 cells were transiently transfected with the −210 bp Grin1–luciferase construct as described in the Experimental section. The cells were maintained with medium supplemented with 100 nM mithramycin A for 16 h. Luciferase assays were then performed on cell extracts and the total protein amount was measured. The results are the ratio of luciferase activity/total protein. Statistical significance is relative relative to the control (wild-type) and ANOVA with Dunnett's test was performed (**P<0.01).

To examine in more detail the role of NRSF in the transcription of the chicken Grin1 gene, we performed co-transfection of the −210 bp construct and a plasmid expressing REST-VP16, which is a negative competitor of endogenous NRSF [21,27]. The results showed a strong transactivation of the chicken −210 bp Grin1 construct in undifferentiated P19 cells (8-fold), PC12 cells (20-fold) and in E7 chicken cortical neurons (16-fold) (Figures 5A–5C).

The function of the chicken Grin1 gene S2 element was also evaluated. Surprisingly, Figure 5 shows that transient transfection of the S2m (S2 mutated construct) into undifferentiated P19 cells increased the promoter activity 6-fold, whereas in PC12 cells, transcriptional activity was increased 2-fold over the control wild-type plasmid and, in E7 chicken neurons, S2m activity was 4-fold higher than observed for the control plasmid.

To complement the results observed by transient transfection using the −210 bp S2m, we used mithramycin A, a drug that interferes with binding of transcription factors to GC-rich elements. The results confirm that treatment of undifferentiated P19 cells with 100 nM mithramycin A for 16 h increased 2-fold the transcriptional activity of the −210 bp Grin1–luciferase gene construct (Figure 5D).

The proximal promoter SP site activates transcription of the chicken Grin1 gene

The chicken Grin1 gene proximal promoter region contains a GSG/SP at position −14 to −30 bp instead of a classical TATA box (see Figure 2 and Table 1). Therefore we investigated the role of the GSG-like-box and the adjacent SP site. First, we explored the protein-binding capabilities of the GSG/SP site. EMSAs performed using the GSG/SP probe showed the formation of a specific DNA–protein complex that was competed with a 100-fold molar excess of the unlabelled GSG/SP probe, but not with the GSG/SPm2 (GSG/SP mutated in the SP site) probe (Figure 6A). In addition, the complex was also competed by the GSG/SPm1 (GSG/SP mutated in the putative core GSG site) probe (results not shown). The GSG/SP complex observed was super-shifted with the SP3 antibody, but not by the SP1, SP4, TFIID and EGR-1 antibodies (Figure 6A). The complex was not formed with nuclear extracts of undifferentiated P19 or PC12 cells (Figures 6B and 6C).

The SP3 factor binds to the GSG/SP site present in the promoter

Figure 6
The SP3 factor binds to the GSG/SP site present in the promoter

Nuclear extracts from E7 chicken cortical neurons (A), undifferentiated P19 cells (B) and undifferentiated PC12 cells (C) were incubated with 32P-labelled GSG/SP probe. In neuronal nuclear extracts, one complex was competed with 100-fold molar excess of the unlabelled wild-type GSG/SP probe, but was not competed by the GSG/SPm2 probe. The DNA–protein complex was super-shifted when the SP3 antibody was added to the binding reaction, but not when the SP1, SP4, TFIID and EGR-1 (Egr1/2) antibodies were added to the reaction. With undifferentiated P19 and PC12 cells, no complexes were formed. Ab, antibody.

Figure 6
The SP3 factor binds to the GSG/SP site present in the promoter

Nuclear extracts from E7 chicken cortical neurons (A), undifferentiated P19 cells (B) and undifferentiated PC12 cells (C) were incubated with 32P-labelled GSG/SP probe. In neuronal nuclear extracts, one complex was competed with 100-fold molar excess of the unlabelled wild-type GSG/SP probe, but was not competed by the GSG/SPm2 probe. The DNA–protein complex was super-shifted when the SP3 antibody was added to the binding reaction, but not when the SP1, SP4, TFIID and EGR-1 (Egr1/2) antibodies were added to the reaction. With undifferentiated P19 and PC12 cells, no complexes were formed. Ab, antibody.

To assess the functionality of the GSG/SP site, we generated two mutants of these sites using the −210 bp Grin1–luciferase constructs GSG/SPm1 and GSG/SPm2. The constructs were transiently transfected into undifferentiated P19 cells and PC12 cells, and into E7 chicken cortical neurons. The GSG/SPm1 construct did not significantly change the transcriptional activity compared with the wild-type construct (Figure 7). The transcriptional activity of GSG/SPm2 did not change in undifferentiated P19 and PC12 cells, but was decreased by 50% compared with the wild-type construct in cortical neurons (Figure 7). These results are in agreement with the lack of binding activity of the GSG/SP DNA probe using nuclear extracts of undifferentiated P19 and PC12 cells.

Mutagenic analysis of the GSG/SP site

Figure 7
Mutagenic analysis of the GSG/SP site

Site-directed mutagenesis of the GSG site (GSG/SPm1) and the SP site near the GSG element (GSG/SPm2) sites was performed as described in the Experimental section. The mutated Grin1 gene chimaeric constructs were transiently transfected into undifferentiated P19 cells, undifferentiated PC12 cells and E7 chicken cortical neurons. Dual-luciferase assays were performed. The results are expressed as the ratio of firefly/Renilla luciferase activity. Statistical significance was determined relative to the −210 bp Grin1–luciferase wild-type construct and was used as a control. ANOVA with Dunnett's test was performed (**P<0.01).

Figure 7
Mutagenic analysis of the GSG/SP site

Site-directed mutagenesis of the GSG site (GSG/SPm1) and the SP site near the GSG element (GSG/SPm2) sites was performed as described in the Experimental section. The mutated Grin1 gene chimaeric constructs were transiently transfected into undifferentiated P19 cells, undifferentiated PC12 cells and E7 chicken cortical neurons. Dual-luciferase assays were performed. The results are expressed as the ratio of firefly/Renilla luciferase activity. Statistical significance was determined relative to the −210 bp Grin1–luciferase wild-type construct and was used as a control. ANOVA with Dunnett's test was performed (**P<0.01).

DISCUSSION

The understanding of the molecular mechanisms that control the expression of NMDA receptors is essential for determining the role played by glutamate in normal neuronal processes and under pathological conditions. Furthermore, the identification of the conserved transcriptional mechanisms among mammals and birds could provide more insight into the regulation of gene expression among species [28]. However, to date, there are no studies on the transcriptional regulation of glutamate receptor genes in birds. Thus the aim of the present study was to investigate, for the first time, the mechanisms involved in the transcriptional regulation of the chicken Grin1 gene.

In agreement with previous findings using the rat Grin1 gene promoter constructs [9], the 1.9 kb construct displayed a low transcriptional activity in undifferentiated P19 cells, and this was significantly increased during neuronal differentiation after 8 days of retinoic acid treatment. Similarly, the transcriptional activity of the chimaeric Grin1–luciferase gene in PC12 cells was up-regulated during NGF-induced neuronal differentiation. These results support the hypothesis that Grin1 gene expression is repressed in undifferentiated cells and suggest that transcriptional activation of the Grin1 gene during neuronal differentiation occurs by removing such repression.

We identified consensus DNA sequences for regulatory elements that may drive Grin1 gene expression within the first 210 bp of the 5′-regulatory region. In silico analysis of the proximal 5′-regulatory region of the cloned Grin1 genes showed that the organization pattern of transcription-binding sites within this region is conserved, despite the overall low DNA sequence similarity among species (31%). The 5′-regulatory regions of human, mouse and rat Grin1 have been shown to include putative cis-elements, such as NRSE, SP and a GSG-like-box in a similar distribution, although in distinct locations [29]. In addition, these elements are also present in other glutamate receptor genes [30], such as Grin2a [9], Grik5 [31] and Grm1 [32]. Consistent with these findings, we demonstrated that the Grin1 gene proximal 5′-regulatory region that includes the 5′-UTR exhibits a pattern of cis-elements encompassing putative binding sites for NRSF- and SP-transcription factors, as well as a GSG-like-box that allows the formation of the pre-initiation complex required for Grin1 gene transcription in the absence of a TATA box [25].

Because SP factors are expressed in a diversity of cell types, silencing of Grin1 gene expression by transcriptional repression in undifferentiated and non-neuronal cells is required to maintain their cell-type specific phenotype. It has been shown previously that NRSF represses transcription of the rat Grin1 gene in undifferentiated P19 cells [9]. Moreover, NRSF can repress SP1 transcriptional activation by recruitment of the TFIID C-terminal region, which leads to inhibition of transcription by preventing the assembly of the pre-initiation transcriptional complex [33,34]. NRSF has been shown to repress neuron-specific gene expression [18] in both non-neuronal and undifferentiated cells by recruitment of HDAC (histone deacetylase complex) by mSin3A/B to the N-terminal and by CoREST (co-repressor-element-silencing transcription factor) to C-terminal [35,36]. However, transcriptional repression may also be achieved through the inhibition of SP1, direct inhibition of the TATA-binding protein [33], or by the recruitment of small CTD (C-terminal domain) phosphatases that inhibit transcription driven by RNA polymerase II [37]. In the present study, the results clearly show that NRSF-mediated transcriptional repression of the chicken Grin1 gene depends on the cell type as well on the stage of neuronal differentiation. The Grin1 gene transcriptional repression in pluripotent undifferentiated P19 cells requires NRSF repression, but also requires other repressor mechanisms. The neuronal-lineage-committed PC12 cell line lacks expression of NRSF and thus needs NRSF-independent mechanisms of gene repression.

It has been described that some SP factors can function either as activators or as repressors of gene transcription [38,39], playing a main role in transcriptional regulation [40]. SP-mediated transcriptional repression might proceed by titration of one or more promoter-specific transacting factors [41], by recruitment of HDACs [42] or by interaction with a methylation-specific binding protein [43]. Additionally, SP-mediated repression might also depend on the number of SP-binding sites present on the promoter [44]. In the present study, we show that repression of the chicken Grin1 gene is also mediated by binding of SP3 to the S2 element, and that repression depends on the cell type and on the stage of neuronal differentiation. In agreement with recent studies showing that the interaction of SP3 with NRSF results in transcriptional repression [45], our results suggest that SP3 and NRSF could form a repressor complex in the chicken Grin1 gene 5′-UTR, blocking the initiation of transcription.

We used EMSA and functional analyses of wild-type and mutated constructs to show that SP3 activates the transcriptional activity of a proximal promoter chicken Grin1 gene construct by binding to GSG/SP located in the 5′-proximal promoter region. These results are in agreement with the finding that the SP family of transcription factors positively regulate rat Grin1 gene promoter transcription by the direct interaction of SP factors with GC-rich elements [14,16,17]. In the present study, we found that the SP3 transcription factor plays a dual role in transcriptional regulation of the chicken Grin1 gene. Previous reports have demonstrated that post-translational modification of SP factors, such as SUMOylation [46], acetylation [47] and phosphorylation [48], could modify the function of these factors in the transcriptional regulation of genes, supporting the dual role of SP3 in the regulation of gene expression. However, it is not known whether these post-translational modifications regulate the function of SP1 and SP3 in the process of neuronal differentiation, and this requires further study.

In conclusion, results from the present study demonstrate that the chicken Grin1 gene transcription is up-regulated during neuronal differentiation, and is repressed in undifferentiated P19 and PC12 cells by binding of NRSF and SP transcription factors to the proximal promoter and 5′-UTR. We speculate that these factors are probably assembled as a strong repressor complex, which inhibits the transcription of the Grin1 gene in the undifferentiated cells. During the neuronal differentiation process NRSF transcriptional repression ends, resulting in the proximal promoter GSG/SP site binding to SP3, up-regulating the transcription of this gene. However, further studies are required for a better understanding of the precise epigenetic and transcriptional mechanisms involved in the transcriptional repression of the Grin1 gene in non-neuronal and pluripotent cells.

Abbreviations

     
  • Ara-C

    cytosine arabinoside

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • dsDNA

    double-strand DNA

  •  
  • DTT

    dithiothreitol

  •  
  • E7

    embryonic day 7

  •  
  • EGR-1

    early growth-response gene product 1

  •  
  • EMSA

    electrophoretic mobility-shift assay

  •  
  • FBS

    fetal bovine serum

  •  
  • HDAC

    histone deacetylase complex

  •  
  • HS

    horse serum

  •  
  • MAZ

    Myc-associated zinc-finger-binding protein

  •  
  • N1m

    mutated N1 site

  •  
  • NGF

    nerve growth factor

  •  
  • NMDA

    N-methyl-D-aspartate, NMDAR, NMDA receptor

  •  
  • NRSE

    neuron-restrictive silencing element

  •  
  • NRSF

    neuron-restrictive silencing factor

  •  
  • PC12 cell

    phaeochromocytoma cell

  •  
  • P19 cell

    embryonic terato-carcinoma cell

  •  
  • S2m

    mutated S2 construct

  •  
  • SP

    stimulating protein transcription factor, GSG/SP, GSG-like box near a SP-consensus site

  •  
  • GSG/SPm1

    GSG/SP mutated in the putative core GSG site

  •  
  • GSG/SPm2

    GSG/SP mutated in the SP site

  •  
  • TFIID

    transcription factor IID

  •  
  • Tm

    melting temperature of DNA

  •  
  • UTR

    untranslated region

This work was supported by a grant from the Programa de Apoyo para la Investigación y la Inovación Tecnológica, Universidad Nacional Autónoma de México (No. IN206706 to A. Z.-H.) and by grants from the Secretaría de Educación Pública, Consejo Nacional de Ciencia y Tecnología (No. 42801 to A. Z.-H. and No. 42640-Q to A. L.-C.). We thank Dr S. Majumder (Department of Molecular Genetics, M.D. Anderson Cancer Center, University of Texas, Houston, TX 77030, U.S.A.) for the REST-VP16 plasmid and Dr Iván Velasco (Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Distrito Federal, 04510, México) for the β-tubulin III antibody.

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