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J. Biol. Chem., Vol. 279, Issue 17, 17449-17458, April 23, 2004

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NF-B Site Interacts with Sp Factors and Up-regulates the NR1 Promoter during Neuronal Differentiation^*

Anguo Liu, Peter W. Hoffman, Weiwei Lu, and Guang Bai, Supported by National Institutes of Health Grant NS38077¶

From the Department of Biomedical Sciences, Dental School, Program in Neuroscience, and Program in Cellular and Molecular Biology, University of Maryland, Baltimore, Maryland 21201 and the Department of Biology, College of Notre Dame Maryland, Baltimore, Maryland 21210

Received for publication, October 13, 2003 , and in revised form, February 2, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The NR1 gene undergoes induction in neurogenesis mainly via promoter de-repression, and up-regulation during neuronal differentiation by undefined mechanism(s). Here, we show that in the distal region the NR1 promoter has an active NF-B site sharing the consensus with the immunoglobulin (Ig)/human immunodeficiency virus NF-B site. Mutation of this site significantly reduced NR1 promoter up-regulation during neuronal differentiation of P19 cells. Electrophoretic mobility shift assays revealed that P19 nuclei constitutively contained p50 and that neuronal differentiation not only increased nuclear p50 but also induced p65 nuclear translocation. Responding to this change was an up-regulation of NF-B-dependent promoter activity. However, inhibition of NF-B nuclear translocation by an IB super-repressor or decoy DNA only moderately inhibited NR1 promoter up-regulation. Interestingly, the NR1 NF-B site strongly interacted with Sp3/Sp1, instead of NF-B factors, in P19 nuclear extracts. This interaction was reduced for Sp3 following neuronal differentiation, accompanied by dynamic expression of Sp factors. Cotransfection of Sp factors (Sp1, 3, or 4) upregulated the NR1 NF-B site dramatically in differentiated neurons, but only moderately in undifferentiated P19 cells. This up-regulation was strong for Sp1 in differentiated cells and for Sp3 in undifferentiated cells. Chromatin-immunoprecipitation assays further demonstrated that Sp1 and Sp3 interacted with the NR1 NF-B site in situ, and Sp3 lost its interaction after neuronal differentiation. We conclude that the NF-B site positively regulates the NR1 promoter during neuronal differentiation via interacting mainly with Sp factors and neuronal differentiation reduces the effect of Sp3 factor on this site.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The N-methyl-D-aspartate (NMDA)¹ subtype of glutamate receptors plays an important role in neuronal differentiation and plasticity. The essential component of the NMDA receptor complexes is encoded by the NR1 gene (1, 2). This gene is induced during neurogenesis and up-regulated in neurons undergoing differentiation (3, 4). In studies of NR1 gene upregulation, a negative mechanism has been proposed: repressor element 1 (RE1) silencing transcription factor (REST)/neuron-restriction silencer factor (NRSF) acts on the RE1 site in the NR1 promoter and suppresses transcription in neural progenitors. After neurogenesis, REST/NRSF is down-regulated, and the suppression of the NR1 gene is removed to let de-repression occur (4). In studies of neuronal differentiation of P19 cells, we observed that there was a time gap between the down-regulation of the REST/NRSF protein and the gradually robust upregulation of the NR1 mRNA/promoter activity during neuronal differentiation (4), suggesting that a positive mechanism is required for NR1 gene transcription after de-repression. The NR1 promoter contains in the proximal region GC-boxes and MEF2 sites that interact with transcription activators sensitive to neuronal differentiation and thus could contribute to this positive mechanism (5–7). However, our recent observation suggests that the distal region of this promoter is required for fully up-regulated transcription (4) and very likely contains one or more important enhancers that positively respond to the signals of neuronal differentiation.

Sequence analysis of the NR1 promoter uncovered a putative nuclear factor-B (NF-B) site in the region distal to the transcription start points (TSPs) (4). NF-B sites interact with dimerized NF-B factors and positively regulates cis-promoter. Five different NF-B factors have been found in mammalian tissues, p65 (Rel-A), p50, p52, C-Rel, and RelB. Homo- or heterodimers of NF-B factors are associated with IB factor in the cytoplasm and remain inactive. Activating signals triggered by environmental cues induce IB dissociation, thus releasing NF-B dimers that then become activated and nuclearly translocated to regulate transcription of target genes (8–10). In the nervous system, NF-B activity is differentially distributed in the developing brain at different stages (11). For example, p50 appears in the early gastrula stage followed by the appearance of p65/c-Rel in neurons during the postnatal period (12). In mature neurons p65 and p50 NF-B factors are constitutively expressed, and other NF-B factors may be induced by selective stimuli, such as neuronal differentiation (11, 13, 14). Although putative NF-B sites were found in several neuronal genes (15–17), their impact on promoter activation during neuronal differentiation is largely undefined. NMDA receptors are also involved in brain development (2), and activation of NMDA receptors provides the major neuronal signal for activation of the NF-B factors (13, 14). Therefore, whether NF-B factors are able to regulate the NMDA receptor genes and thus promote their maturation may be important for retaining long term activation of NF-B factors in neurons undergoing differentiation or in mature neurons.

Recent studies revealed that the Sp1 transcription factor that usually binds GC-box (GGGGCGGGC) or GT (GGGTGTGGC) motifs (18) exhibits a high affinity to a subset of NF-B sites. For example, Sp1 activates NF-B sites from the immunoglobulin -chain and P-selectin promoters (19). NF-B factors do not, however, share binding affinity to GC-box sequences. Most recently, it was reported that in the nervous system there is a neuronal B-binding factor that binds the NF-B consensus but is distinct from all identified NF-B factors. This factor was found to be a complex consisting of Sp1, 3, and 4 (20). These factors are activators to most genes except that Sp3 may activate genes having a single GC-box (21, 22) but repress Sp1-mediated transactivation of genes having multiple GC-boxes, possibly via competition for DNA binding sites (23, 24). Protein modification studies also indicated that repressive Sp3 could become an activator after acetylation (25). We have previously demonstrated that Sp factors (Sp1, 3, and 4) are activators to the NR1 promoter via an interaction with the tandem GC-boxes proximal to the TSPs (26). Whether Sp factors also act on cis-element(s) existing in the distal region of the NR1 promoter remains unknown.

In the present study, to search for positive mechanisms underlying the up-regulation of the NR1 gene during neuronal differentiation, we functionally analyzed the putative NR1 NF-B site in P19 cells and cultured cerebrocortical neurons. We collected evidence to show that this putative site activated the NR1 promoter in cells undergoing neuronal differentiation. Additional studies demonstrated that, instead of NF-B factors, this site mainly interacted with the Sp factors to up-regulate the promoter. Furthermore, interaction of this site with Sp factors in situ was demonstrated and, finally, Sp3 binding was shown to be lost following neuronal differentiation, while Sp1 binding became enhanced.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Antibodies were obtained from following resources (followed by their catalogue numbers): Sp1 (07-124) from Upstate Biotechnology Inc. (Waltham, MA); Sp3 (sc13018x and sc644x), Sp4 (sc13019x and sc645x), p65 (sc-109x), p50 (sc-114x), p52 (sc-298x), and c-Rel (sc-272x) from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant proteins of p50 and p49 were purchased from Promega (Madison, WI). Oligonucleotides were synthesized by Genemed Synthesis (South San Francisco, CA).

Construction of Reporter Genes and Expression Constructs—All plasmids used in this study have been described previously (4, 27) with the following exceptions. Mutation of the NF-B site in the rat NR1 promoter was conducted with a Quick Exchange kit (Stratagene, La Jolla, CA) with primers GB402, 5'-TTAATGTCCTGGtaccTCCTGTCTAC, and GB403, 5'-GTAGACAGGAggtaCCAGGACATTAA (lowercased letters represent replaced bases) to form the construct pNRL5.4mtNFB. Plasmid pNRL5.4mtGC carrying mutation of the tandem GC-boxes was generated by exchange of the KpnI-XbaI fragment between pNRL3029mtSp1 x 2 (27) and pNRL5.4 (4). Exchanging KpnI/XhoI fragments between pNRL5.4RE1 (4) and pNRL5.4mtNFB produced pNRL5.4RE1mtNFB bearing the RE1 deletion together with an NF-B mutation.

Preparation of Recombinant Adenovirus—Shuttle plasmid pAdTrack containing the green fluorescent protein (GFP) gene driven by the early gene promoter of human cytomegalovirus (CMV), and adenoviral backbone vector pAdEasy2 were obtained from Dr. Tong-Chuan He (Johns Hopkins University). Recombinant adenovirus rAdGFP expressing GFP was prepared using pAdTrack and AdEasy2 following the protocol described by He et al. (28). Recombinant adenovirus rAdLacZ expressing the bacterial LacZ gene was provided generously by Dr. Christine L. Wilcox (Colorado State University). Recombinant adenovirus Ad5IB encoding an IB super-repressor was a kind gift from Dr. David Brenner (University of Columbia). Amplification and titration of these viruses were accomplished by infecting HEK293 cells with virus following standard protocols (28). Infected HEK293 cells were lysed by a freeze-and-thaw protocol. Cell debris was removed by centrifugation, and the supernatants were saved at -80 °C until use.

Cell Culture, Neuronal Differentiation, Transfection, Infection, and Reporter Assay—Previously described methods were used for culture of P19 cells (4), HEK cells (5), and dissociated rat cerebrocortical neurons (27). Differentiation of P19 cells was induced by retinoic acid (RA) as described previously (4). Transient transfection was accomplished with LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's instruction for cells growing in 12-well plates. Expression constructs were cotransfected with promoter-luciferase genes at a 4:1 ratio in the presence of pCMVgal in an amount one-tenth that of the luciferase construct. Reporter gene assays and normalization of luciferase activity to -galactosidase were carried out as reported previously (4). For infection, selective rAd was added to cells by 100 plaque-forming units per cell for 24 h. Infection efficiency of differentiated P19 cells by rAd was tested with rAdGFP, and more than 80% of cells showed GFP green fluorescence 24 h after infection (data not shown).

The stable cell line of P19 cells harboring the 5.4-kb NR1 promoter (P19NR1–5.4) has been described previously (4). Establishment of the P19 cell line carrying the mutated 5.4-kb NR1 promoter or the NF-B-luciferase gene was accomplished by cotransfection of pcDNA3 with pNRL5.4mtNFB or B-luc as described previously (4) to form P19NR1–5.4mtNFB and P19-NF-B-luc cell lines, respectively. The B-luc DNA was a gift from Dr. D. D. Billadeau (Mayor Clinic) and contains three NF-B sites upstream of the concanavalin-A minimal promoter-luciferase gene (29).

Inactivation of NF-B by decoy DNA was performed following a protocol and utilizing DNA sequences described in the literature for cultured neurons (30). 150 pmol of DNA was transfected by LipofectAMINE 2000 into P19 cells cultured in a 3.5-cm dish. In some experiments, decoy DNA was transfected into P19-NF-B-luc cells for 12 h, and phorbol myristate acetate (PMA, Sigma, St. Louis, MO) at 0.1 µM was used to stimulate the cells for an additional 5 h before reporter gene activity was measured. DNA with a sequence scrambled from the NF-B decoy sequence served as a control to correct for the effect of transfection on the inhibition (30). The same decoy and control DNAs were transfected into P19NR1–5.4 cells differentiated for 6 days, and 12 h later cells were harvested for luciferase assay. The luciferase assay and its normalization to cellular protein contents were accomplished for stable transfectants as described previously (4).

DNA Protein Interaction and Immunoblot Analysis—Electrophoretic mobility shift assays (EMSAs) were performed following a protocol described previously (6). Nuclear proteins were extracted from P19 cells as described in previous studies (4). A probe for the rat NR1 NF-B site (rNR1-NFB) was formed by annealing DNA GB424 (upper strand, 5'-TTAATGTCCTGGGACTTTCCTGT) and GB425 (lower strand, 5'-AGTGTAGACAGGAAAGTCCCAGG) with uneven ends. A probe for the mouse NR1 NF-B site (mNR1-NFB) was produced as above with DNA GB469 (upper strand, 5'-TCTGAGGAGTGGGACTGGCCAGCAT) and GB460 (lower strand, 5'-GCCTAATGCTGGCCAGTCCCACTCC). Both rNR1-NFB and mNR1-NFB were labeled by Klenow enzyme in the presence of [-³²P]dCTP (>3000 mCi/mmol, ICN Biomedicals, Irvine, CA) (4). The NF-B consensus probe of the Ig/HIV promoter was obtained from Promega and 5'-end-labeled with [-³²P]ATP (>6000 Ci/mmol, ICN Biomedicals) by T4 DNA kinase (6). This consensus has a sequence of 5'-AGTTGAGGGGACTTTCCCAGGC with the core sequence underlined. Competitors of the NR1 NF-B sequences were prepared by filling in the uneven ends of the annealed GB424/GB425 and GB469/GB470 with Klenow enzyme individually. Competitors containing consensus of Sp1 GC-box, the Ig/HIV NF-B, or Oct1 were purchased from Promega. Competition and supershift experiments were done as described previously (6). Reaction buffer for NF-B binding was composed of 4% Ficoll (Sigma), 2 µg of poly(dI-dC), 1 µg of BSA, 60 mM KCl, 5 mM dithiothreitol, 20 mM Hepes, pH 8.3 (19). To perform NR1 GC-box EMSA, a probe used was formed by annealing GB59 (5'-GCGTCAGGAAGCGGGGGCGGTGGGAGGGGTAGAACGCGTAGGT) and GB60 (5'-ACCTACGCGTTCTACCCCTCCCACCGCCCCCGCTTCCTGACGC) as described previously (31). Each experiment was repeated at least three times. Immunoblot analysis of Sp1 and Sp3 in nuclear extracts was performed using methods described previously (4, 27).

Chromatin Immunoprecipitation—ChIP experiments were performed following the methods described by Baek (32) and Hatzis (33) as well as the instruction described by Upstate Biotechnology Inc. in a ChIP assay kit. Briefly, P19 cells were grown to more than 80% confluence and fixed with 1% formaldehyde in media for 10 min at room temperature to cross-link DNA with proteins. Reaction was terminated by addition of 0.15 M glycine. Nuclei were prepared by incubation of cells in lysis buffer (50 mM Hepes, pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin) on ice for 10 min, and then centrifugation at 6,000 x g for 5 min. Nuclei were dissolved in radioimmunoprecipitation assay buffer plus protease inhibitors (27), and DNAs in lysates were sonicated into fragments between 200 and 1000 bp. Cell debris was removed by centrifugation at 16,000 x g for 10 min at 4 °C, and supernatants were diluted five times with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Hepes, pH 7.9, 167 mM NaCl). After clean up with protein A/G-agarose (Santa Cruz Biotechnology) saturated with 50 µg/ml sheared lambda DNA and 1 mg/ml BSA in TE buffer, the diluted supernatants underwent immunoprecipitation with 5 µg of Sp1 or Sp3 antibody for every 1.5-ml volume at 4 °C overnight. Antibodies were recovered with protein A/G-agarose saturated with sheared lambda DNA/BSA and washed with 1 ml of the following buffers in order: once with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl); once with high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 500 mM NaCl); once with LiCl wash buffer (250 mM LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0); twice with TE buffer, pH 8.0. DNA-protein complexes were eluted from beads twice with 100-µl elution buffer (1% SDS, 100 mM NaHCO₃). The eluted samples were combined and each was treated with 1 µg of RNase A for 15 min at 37 °C. Protein-DNA cross-link was reversed by 300 mM NaCl at 65 °C for 4 h. Eluants were then deproteinized by 10 µg/ml proteinase K at 45 °C for 2 h. Free DNAs were extracted by the conventional phenol/chloroform method. Purified DNA was dissolved in 50 µl of dH₂O. One-tenth of each DNA was amplified for 40 cycles in PCR by employing Ex Taq DNA polymerase (TaKaRa/PanVera, Madison, WI) and primers GB580 (5'-ATATACTGGCCCCATGGTCA) and GB581 (5'-TGGTGACTGGCTGTTCTCTG) to detect the fragment overlapping the mouse NR1 NF-B site. As a positive control (input), sonicated nuclear lysates underwent reverse cross-link and phenol/chloroform extraction and were subject to PCR. For negative control, an antibody against goat IgG was used in immunoprecipitation. PCR products were fractionated and visualized on 1% agarose plus 1% low melt agarose-TAE gel containing ethidium bromide (4).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The NR1 Gene Has a Putative NF-B Site in the Distal 5' Flanking Region—Our previous observations suggest that the distal region of the rat NR1 promoter contains positive regulatory elements (4). A motif search of this region revealed a sequence matching perfectly the 10-bp consensus of the Ig/HIV NF-B site, the most common NF-B site identified previously (10). Its homology to the NF-B consensus, position in the NR1 promoter, and 10 bp of flanking sequences at both ends are presented in Fig. 1. A similar putative site was also found in the distal 5' flanking sequences of the mouse NR1 gene from clone RP23–47P18 on chromosome 2 deposited in the GenBank^TM mouse genome data base (www.ncbi.nlm.gov/gene-bank/mousegenom) (Fig. 1).

View larger version (15K): [in this window] [in a new window]   FIG. 1. Comparison of NF-B consensus with the putative NF-B sites of the rat and mouse NR1 genes. R represents A or G, D is denoted for C, A or T, and Y is for C or T. Sequences matching the consensus are in boldface. Flanking sequences at both ends of the putative sites are shown for 10 bp each side. Vertical dashed lines indicate similarity of sequences. Numbers represent positions of nearby residues relative to the first codon of each gene.

View larger version (15K): [in this window] [in a new window]   FIG. 2. The NF-B site was required for up-regulation of the NR1 promoter during neuronal differentiation. A, schematic of the rat NR1 5' flanking sequences. Locations of the putative NF-B site and other previously analyzed cis-elements are indicated on the bar. B, NR1 promoter activity during neuronal differentiation. P19NR1–5.4 and P19NR1–5.4mtNFB cells were differentiated by RA as described under "Experimental Procedures." At indicated days, luciferase activity in cells was measured and normalized to protein contents. The normalized luciferase activity was used to calculate -fold increase in promoter activity on the basis of the activity in cells before RA treatment (day 0). Mean values ± S.E. from six experiments are shown.

View larger version (62K): [in this window] [in a new window]   FIG. 3. Constitutive presence of NF-B protein (p50) in nuclei of P19 cells. An Ig/HIV NF-B consensus sequence (22 bp) was end-labeled with 32P and used as probe in EMSA experiments. Nuclear proteins were extracted from undifferentiated P19 cells. For supershift experiments, the nuclear proteins were preincubated with antibody specific for p65, p50, C-Rel, p52, or Sp1. Band B was supershifted by p50 antibody to form band C.

View larger version (68K): [in this window] [in a new window]   FIG. 4. Effect of neuronal differentiation on nuclear NF-B factors in P19 cells. A, Change of NF-B binding in P19 cells undergoing neuronal differentiation. Nuclear proteins were extracted from P19 cells differentiated by RA for days as indicated. EMSA experiments were performed for the radiolabeled Ig/HIV NF-B probe. B, neuronal differentiation increased nuclear translocation of p50 and translocated p65 to nuclei. EMSAs were performed with nuclear proteins of P19 cells differentiated for 9 days. Nuclear proteins were preincubated with indicated antibody individually before the probe was added. Others are the same as A.

View larger version (20K): [in this window] [in a new window]   FIG. 5. Inhibition of nuclear translocation of NF-B factors moderately decreased activity of the NR1 promoter in P19 cells undergoing neuronal differentiation. A, inhibition of the NR1 promoter activity by IB super-repressor in P19 cells. P19NR1–5.4 cells were differentiated by RA for 5 and 7 days and then infected with Ad5IBorrAdLacZ (control) for additional 24 h. Luciferase activity and protein contents in cell lysates were assayed. Luciferase activity was normalized to protein contents to produce relative luciferase activity. B, effect of NF-B decoy on the NR1 promoter activity in P19 cells. P19NR1–5.4 cells were differentiated for 6 days and transfected with NF-B decoy or control DNA for 12 h. Relative luciferase activity was obtained in the same way as A. Mean values ± S.E. from six experiments are presented. Student's t test was used to assess the difference between treated cells and controls at the time of differentiation.

View larger version (57K): [in this window] [in a new window]   FIG. 6. The putative NF-B site of the rat NR1 promoter formed a specific complex with nuclear proteins extracted from P19 cells. EMSA experiments were performed for interaction between nuclear proteins extracted from undifferentiated P19 cells (P19 N.P.) and a radiolabeled probe rNR1-NFB. An NF-B binding buffer was employed. As indicated, before probe addition, nuclear proteins were preincubated with 50x competitive DNAs of the rat NR1 NF-B site (rNFB, 30 bp), the mouse NR1 NF-B site (mNFB, 30 bp), Sp1 consensus (Sp1, 22 bp), NF-B consensus (NFB, 22 bp), or Oct1 consensus (Oct1, 22 bp).

View larger version (60K): [in this window] [in a new window]   FIG. 7. Identification of nuclear proteins in the NR1 NF-B nuclear complex. A, NF-B factors were not involved in the complex formation. EMSA experiments were performed as described in Fig. 2 with radiolabeled rNR1-NFkB. Nuclear proteins were preincubated with antibodies against individual NF-B factors as indicated before the probe was added. B, the NF-B site of the rat NR1 promoter interacted with an Sp3-like protein in P19 cells. EMSA experiments were performed as described above. Nuclear proteins were preincubated with antibody specific for Sp1, Sp3, or Sp4 as indicated. C, the mouse NR1 NF-B site interacted with Sp proteins in P19 cells. EMSA experiments were performed for radiolabeled mNR1-NFB with nuclear proteins extracted from undifferentiated P19 cells. Nuclear proteins were preincubated with indicated antibody before probe addition. Bands 1 and 2 were interrupted by Sp3 antibody, whereas band 3 was removed by Sp1 antibody.

View larger version (57K): [in this window] [in a new window]   FIG. 8. Effect of neuronal differentiation on Sp protein and DNA binding in P19 cells. A, modulation of binding to the NF-B site of the rat NR1 promoter. EMSA experiments were performed with radiolabeled rNR1-NFB and nuclear proteins extracted from P19 cells at indicated days after RA treatment. B, expression of Sp1 protein in P19 cells undergoing neuronal differentiation. Immunoblot of Sp1 was conducted with nuclear proteins used in A. C, protein level of Sp3 in P19 cells undergoing neuronal differentiation. Immunoblot analysis was performed with an Sp3 antibody as in B. D, effect of neuronal differentiation on binding to the NR1 GC-box. EMSA was performed with a GC-box probe of GB59/60 and nuclear proteins used in A.

Sp Factors Up-regulate the NR1 Promoter by Acting on Both the NF-B Site and GC-boxes—Results obtained above suggest that Sp factors may regulate the NR1 promoter by interacting with a new binding site in addition to the previously identified GC-boxes (6). To further evaluate the functional outcome of this new interaction, we cotransfected the 5.4-kb NR1 promoter-reporter fusion gene with various Sp expression constructs into undifferentiated P19 cells. As shown in Fig. 9A, NR1 promoter activity was enhanced by overexpressed Sp factors even in the presence of the RE1 site in the promoter. It has been known that this RE1 site interacts with a transcription suppressor, REST/NRSF, which is abundant in undifferentiated P19 cells (4). Sp1 increased promoter activity 3-fold, whereas others factors increased activity 1.5-fold. The NR1 promoter with mutation of the NF-B site or GC-boxes lost responsiveness to Sp factors and showed activity comparable to that seen in cells cotransfected with vector (Fig. 9A). We assumed that the high level of REST/NRSF in P19 cells (4) might negatively block the effects of Sp factors on the promoter. To test this assumption, we mutated the RE1 site from the promoter and examined the effect of individual Sp factors on the promoter again. As indicated in Fig. 9B, RE1 mutation allowed the promoter to be regulated by Sp1 factor more strongly, up to more than 5-fold. Interestingly, cotransfection of Sp4 also produced a more than 4-fold increase, whereas Sp3 produced an increase of only slightly above a 2-fold. Taken together, results above suggest that undifferentiated cells lack sufficient Sp factors to overcome the repression caused by REST/NRSF or the latter is sufficient to inhibit the promoter regardless the presence of Sp factors in the cells.

View larger version (31K): [in this window] [in a new window]   FIG. 9. Effects of Sp factors and their binding sites on the NR1 promoter in P19 cells. A, Sp factors act on NF-B and GC-box and positively regulate the NR1 promoter in P19 cells. P19 cells were transfected with NR1 promoter-luciferase constructs as indicated. Luciferase activity was normalized to cotransfected -galactosidase activity to produce the relative luciferase activity. B, effect of RE1 mutation on Sp factor-regulated activity of the NR1 promoter. NR1 promoter constructs with mutations were cotransfected into P19 cells with Sp expression constructs as indicated. Others are the same as A. Mean values ± S.E. from six experiments are shown.

View larger version (31K): [in this window] [in a new window]   FIG. 10. Effects of Sp factors and their binding sites on the NR1 promoter in neurons. A, Sp factors positively regulate the NR1 promoter in neuronally differentiated P19 cells by acting on both distal NF-B site and the proximal GC-boxes. Sp expression constructs were cotransfected with wild-type (wt) or mutated NR1 promoter (5.4 kb) into P19 cells differentiated by RA for 5 days and reporter gene activities were measured 1 day after transfection. Luciferase activity normalized to -galactosidase activity was used to calculate -fold increase over those cotransfected with vector (pCDNA3). B, Sp factors positively regulate the NR1 promoter by acting on both distal NF-B site and the proximal GC-boxes. Sp expression constructs were cotransfected with wild-type (wt) and mutated NR1 promoter (5.4 kb) as indicated into cerebrocortical neurons cultured in vitro for 7 days. The -fold increase in promoter activity was calculated as in A. Mean values ± S.E. from six experiments are presented.

View larger version (14K): [in this window] [in a new window]   FIG. 11. Interaction of Sp factors with the NF-B site of the NR1 gene in living cells. ChIP experiments were performed with antibody against Sp1 or Sp3 in P19 cells treated with RA-induced differentiation for indicated time. Coprecipitated DNA was examined for the NF-B site of the NR1 gene by a PCR protocol as described under "Experimental Procedures." DNA products generated by specific antibody or genomic DNA (input) had an expected 180-bp size on the agarose gel as indicated, whereas nonspecific antibody served as a negative control and produced no DNA band (contl). Results shown represent one of three repeated experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It is well known that transcription factors specifically bind selective DNA sequences that have a limited variation following a consensus. In certain cases, one type of consensus can be bound by different groups of transcription factors (41), and one group of transcription factors may interact with a different consensus (42). For example, Sp factors may bind GC-boxes, GT motifs, or basic transcription elements (18). NF-B factors and Sp factors belong to different families of transcription factors based on distinct DNA consensuses and protein structures. It was reported previously that NF-B and Sp cooperatively activated the human immunodeficiency virus type 1 gene when their cognate binding sites are close together on the promoter (43). In our studies, however, Sp factors selectively bind to the NR1 NF-B site. This selection is clearly not due to a lack of the NF-B factors in the nucleus, because the Ig/HIV NF-B consensus was bound by the NF-B factors, p50 and p65, from the same nuclear preparation (Fig. 3). Therefore, affinity may be the mechanism underlying the diversity seen between these two NF-B sites. Hirano et al. (19) reported previously that Sp factors in nuclear proteins extracted from HeLa cells interacted with a subgroup of NF-B sites, but in their studies bindings of NF-B and Sp factors coexisted to the same probe, suggesting similar affinities of these factors to the DNA. Interestingly, in the present studies, sequence comparison indicates that these two sequences used in EMSA (Fig. 1) share exactly the same 10-bp consensus, but have distinct flanking sequences. Therefore, the flanking sequence may determine selectivity of transcription factors. It was reported previously that nucleotide residues directly flanking the 10-bp NF-B consensus had an impact on its affinity to NF-B factors (44). For example, replacement of the first G residue 5' of the 10-bp NF-B consensus reduces its affinity to p50 homodimer, but not p50-p65 heterodimer (44). Recently, we also observed that the flanking sequence is required for REST/NRSF binding to the RE1 site in the NR1 promoter (4). However, in this study, we observed a new phenomenon that flanking sequence may determine which protein the NF-B consensus binds, NF-B factors or Sp factors. This selection may result from the large difference of the affinities of these transcription factors to the DNA. The NR1 NF-B site was able to bind recombinant NF-B proteins but interacted almost exclusively with Sp factors when the DNA probe was incubated with crude nuclear extracts in which both families of Sp and NF-B factors exist. This possibility is further supported by an observation that neither a GC-box consensus nor the Ig/HIV NF-B site competed interaction between Sp factors and the NR1 NF-B site (Fig. 6). We cannot, however, exclude the possibility that this selection may depend on the components in nuclear extracts or status of protein modification. For example, many transcription factors interact with each other and then regulate the cis-promoter. A great body of data has revealed that Sp factors interact with other transcription factors, including NF-B factors (45). Again, posttranslation modification may modulate these interactions and thereby increase the complexity of gene expression as well as link gene transcription to signal transduction. This possibility is supported by the fact that both NF-B factors and Sp factors can be modified via phosphorylation (46) or acetylation (47), thus changing their relationship with DNA. Our previous studies have indicated that signaling of nerve growth factor, which is involved in neuronal differentiation, causes phosphorylation of Sp1 and thus modifies its action on the NR1 promoter (26). The impact of protein modification of other Sp factors on their regulation of the NR1 promoter during neuronal differentiation is currently under investigation in our laboratory.

In this study, although we did not detect apparent binding activity of NF-B factors to the NR1 NF-B site from P19 cells, inhibition of nuclear translocation of NF-B factors indeed reduced, even by a small scale, the NR1 promoter activity. Furthermore, the NR1 NF-B site is able to bind purified recombinant NF-B factors. Therefore, it is still possible that NF-B factors may increase their binding affinity to this site and thus regulate the NR1 gene transcription in a competition with Sp factors. Several lines of studies suggest that this possibility may be true. For example, Qin et al. reported that an endogenous NMDA agonist, quinolinic acid, induced nuclear translocation of NF-B factors in the rat striatal neurons (48) and a down-regulation of NMDA receptor binding followed (49). This may be relevant to NR1 regulation, because we observed a down-regulation of NR1 mRNA following the time of the nuclear translocation of NF-B factors in quinolinic acid-injected striatum.² Because activation of glutamate receptors induces the nuclear translocation of NF-B factors (13, 14), it is also possible that NF-B factors are involved in a negative autoregulation of the NR1 transcription. For example, Mao et al. (20) reported that glutamate stimulation down-regulated NR1 mRNA in cultured neocortical neurons. The detailed mechanism regarding the role of NF-B factors in the NR1 transcription needs further investigation.

In this study, we observed that Sp factors were able to up-regulate the NR1 promoter even in undifferentiated P19 cells. Taken together with our previous report that deletion of the negative cis-element RE1 releases NR1 promoter activity robustly (4), one might assume that undifferentiated cells have enough activators for the NR1 gene transcription and the derepression is the only key factor in NR1 promoter activation. However, in P19 cells, REST/NRSE is down-regulated 4 days after RA treatment (4). At the same time, NR1 promoter activity remains at a very low level and only begins to increase 6 days after RA treatment. Furthermore, this increase lasts more than 2 days (Fig. 2). Therefore, a change of transactivators is still required for the NR1 promoter. Sp factors very likely contribute to this effect. This likelihood is supported by the following facts: 1) cotransfection of Sp factors with an NR1 promoter lacking the RE1 site into undifferentiated P19 cells gained much more activation of the promoter (Fig. 9B), suggesting a shortage of Sp factors in the cells; 2) differentiated P19 neurons or cerebrocortical neurons activated the NR1 promoter a much high level than undifferentiated P19 cells and overexpression of Sp factors resulted in less up-regulation of the NR1 promoter, indicating that neurons already have a high level activators like Sp factors; and 3) Sp1 binding to the NR1 GC-box undergoes dynamic changes during neuronal differentiation; it is down-regulated at the early stage and up-regulated robustly in the late stage (Fig. 8D). Meanwhile, the nuclear Sp1 level is moderately increased, whereas the nuclear Sp3 level increased in the early stage and robustly decreases in the late stage of neuronal differentiation (Fig. 8B).

Sp factors share similar C-terminal zinc-finger DNA binding domain, and Sp1, 3, and 4 bind DNA with a similar affinity, even though they may differentially regulate the transcription (42). For example, Sp3 represses several promoters that positively respond to Sp1 (23), whereas Sp3 and Sp1 both activate some promoters (21, 22, 27), including the GC-boxes of the NR1 promoter (26). From data of the current study, we believe that Sp1, 3, and 4 are activators of the NR1 promoter via interaction with the NF-B site. Interestingly, we observed differential effects of Sp factors on the NR1 promoter. Sp3 activated the NR1 NF-B site with the least strength but retained the highest binding affinity in vitro with P19 nuclear proteins. In comparison, Sp1 demonstrated the strongest activation of the NR1 promoter, while having a much lower binding affinity than Sp3. However, our ChIP data demonstrated that the interaction of this site in situ in well differentiated P19 cells favors Sp1 over Sp3 (Fig. 11). Therefore, we hypothesize that that Sp3 occupies the enhancer sequence and inefficiently regulates the promoter in undifferentiated cells or cells at the early stage of neuronal differentiation. Following neuronal differentiation, Sp1 may gain high binding affinity and replace Sp3 to up-regulate NR1 transcription, because Sp1 cotransfection showed the strongest effect in differentiated P19 cells. This hypothesis is further supported by evidence from the present study. First, application of Sp1 or Sp4 antibody in EMSA increased Sp3 binding, indicating a competition among these factors. Second, neuronal differentiation robustly increased Sp1 binding to the NR1 GC box as well as nuclear Sp1 protein level (Fig. 8). Last, the NR1 NF-B site becomes nonpermissive to Sp3 after neuronal differentiation (Fig. 11). Our hypothesis is also supported by the previous reports. For example, studies of mouse L cells demonstrated that Sp3 protein competes with Sp1 for the binding site but does not synergize the promoter via interaction with other cis-elements like Sp1 does (24). Synergistic effects of different DNA cis-elements via transcription factors may be important for robust up-regulation of the NR1 gene during neuronal differentiation. Our previous studies also revealed that Sp1 is interacting with Mef2C to up-regulate the NR1 promoter synergistically in neurons (5). Because Sp3 is unable to produce such synergistic effect, differentiating neurons may generate a mechanism to let the promoter be nonpermissive to this factor, but still permissive to Sp1. This mechanism may underlie the missing of Sp3 interaction with the NR1 NF-B site in differentiated P19 cells. Earlier studies also indicated that Sp3 repressed Sp1-mediated promoter activation (23). In addition, Sp factors may undergo covalent modifications and thus change their functionality. For example, the Sp1 protein may be modified by multiple kinases and gain different DNA binding affinities (18, 26, 50). Sp3 may be acetylated and switch from a repressor to an activator (25). Whether and how these changes occur after neuronal differentiation is a very interesting question and needs further investigation.

The function of Sp factors in cultured embryonic neurons could be slightly different, because Sp3 displayed the strongest effect on the promoter (Fig. 10B). This difference may be caused by the high heterogeneity of the cell population. Mao et al. also reported that nuclear proteins extracted from cultured neurons formed with the NF-B consensus a single complex that could be disrupted only by mixed antibodies against Sp1, Sp3, and Sp4, but not by any antibody against NF-B factors. In contrast, we saw Sp3 and Sp1 separately interacted with the NR1 NF-B site. Sp4 is distributed mainly in the central nervous system (51), and we found that it exerts a strong effect on the NR1 promoter in neurons. In summary, we believe that all Sp factors are transactivators of the NR1 promoter and act on the NF-B site as well as the GC-boxes. Neuronal differentiation may differentially modify the interaction between individual Sp factors and these two cis-elements using undefined mechanism(s) and, thus, up-regulate the transcription of the NR1 gene.

	FOOTNOTES

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

^¶ To whom correspondence should be addressed: Dept. of Biomedical Sciences, University of Maryland Dental School, 666 W. Baltimore St., Baltimore, MD 21201. Tel.: 410-706-2082; Fax: 410-706-0193; E-mail address: GNB001{at}dental.umaryland.edu.

¹ The abbreviations used are: NMDA, N-methyl-D-aspartate; NR1, NMDA receptor 1 subunit; TSP, transcription start point; rAd, recombinant adenovirus; RE1, repressor element 1; REST, RE1-silencing transcription factor; NRSF, neuron-restriction silencer factor; CMV, cytomegalovirus; GFP, green fluorescent protein; RA, retinoic acid; PMA, phorbol 12-myristate 13-acetate; EMSA, electrophoretic mobility shift assay; BSA, bovine serum albumin; ChIP, chromatin immunoprecipitation.

² Y. Chai and G. Bai, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Dr. T.-C. He for AdEasy system, Dr. C. L. Wilcox for rAdLacZ, Dr. D. Brenner for Ad5IB, and Dr. D. D. Billadeau for B-luc DNA.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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