(Received for publication, December 9, 1994; and in revised form, January 16, 1995)
From the
The NMDAR1 receptor subunit is a common subunit of N-methyl-D-aspartate receptors. We have previously characterized 3 kilobases (kb) of 5`-flanking sequence of the NMDAR1 gene and now report on the ability of this region to direct transcription of a reporter gene and on its interaction with nuclear proteins. The sequence 356 base pairs (bp) 5` of the first nucleotide of codon 1 was sufficient to express a luciferase reporter gene in rat PC12 pheochromocytoma cells. Additional sequences upstream of nucleotide -356 influenced the activity approximately 2-fold. A labeled 112-bp fragment (position -356 to -245) formed six complexes (C1A and -B, C2A and -B, and C3A and -B), grouped as three double bands, with nuclear extracts from PC12 cells. Competition with Sp1 oligonucleotides abolished formation of C2A and -B and C3A and -B complexes. Sp1 antibody recognized the C3A complex in supershift experiments. Prior immunoprecipitation of nuclear extracts with Sp1 antibody abolished formation of C2A and -B and C3A and -B complexes. Purified Sp1 protein alone did not form a C3A complex but potentiated its formation when PC12 nuclear extract was added. A GC-rich sequence in this fragment was protected from DNase I digestion by nuclear extract. These results suggest that a 356-bp sequence comprises the NMDAR1 basal promoter, and that NMDAR1 gene expression may be regulated by Sp1-like nuclear factors.
N-Methyl-D-aspartate (NMDA) ()receptors are members of the glutamate family of
ligand-gated ion channels. They play important roles in the central
nervous system and have been implicated in both neurotrophic and
neurotoxic mechanisms. Their activity is important in neuronal
long-term potentiation, a cellular process thought to underlie memory
formation(1, 2, 3) . Overactivity of NMDA
receptors is toxic and results in neuronal death brought about by
excessive intracellular calcium accumulation and a subsequent cascade
of events which may involve activation of intracellular hydrolases or
an apoptotic genetic program(3, 4) .
Recently, two families of NMDA receptor subunits were cloned, and their functional characteristics were delineated. The NMDAR1 gene, the sole member of this family cloned so far, appears to be a subunit common to all NMDA receptors and is capable of forming functional homomeric and heteromeric NMDA receptors(5, 6, 7) . The NMDAR1 gene undergoes alternative splicing to generate several protein isoforms(8, 9, 10) . The NMDAR2 gene family is comprised of four members designated 2A, 2B, 2C, and 2D, which only exhibit channel activity when co-expressed with the NMDAR1 gene(5, 7, 11) . In situ hybridization studies revealed that the NMDAR1 gene is expressed widely in the central nervous system with more prevalent expression in the hippocampus, cerebral cortex, and olfactory bulb(6, 7) . In contrast, the NMDAR2 genes have a more restricted and differential distribution(5, 7, 11) . These expression patterns have been substantiated by immunohistochemical methods with specific antibodies to the various subunits(12, 13) .
The expression of the NMDAR1 gene is neuron-specific and highly regulated under both physiological and pathological conditions(2, 3) . In the developing mammalian central nervous system, there is a progressive increase in NMDAR1 expression until the cessation of cortical neuronal migration(14, 15, 16) . NMDAR1 receptor mRNA is expressed in embryonic carcinoma cells differentiated with retinoic acid into a neuronal phenotype(17) . The levels in cerebral cortex and hippocampus of adult brain are somewhat lower than those found postnatally(14, 15) . Evidence based upon receptor ligand binding studies suggests that NMDA receptors may be diminished during aging and in various neurological diseases(3, 4, 18) . Furthermore, NMDA receptors are down-regulated in hippocampus and cerebral cortex by long-term administration of competitive antagonists(19) , in dentate gyrus granule cells by full kindling-induced epileptogenesis(20) , and in hippocampal CA1 neurons by transient global ischemia(21) . Estrogen replacement in ovariectomized rats significantly up-regulates NMDAR1 mRNA in cerebral cortex(22) . Interestingly, it has recently been shown that NMDAR1 mRNA levels change in a circadian pattern in the suprachiasmatic nucleus of the rat(23) . Taken together, these results suggest that NMDA receptor expression may be highly regulated at the level of gene transcription in both a temporal and cell-specific manner.
As a first step to explore this regulation, we previously isolated and characterized a 3-kb genomic fragment encompassing the 5`-flanking sequence of the NMDAR1 gene and mapped the transcriptional start sites (24) . Our results suggested that the NMDAR1 gene promoter has the characteristics of a housekeeping gene in that there are multiple start sites and it contains a proximal GC-rich region with no TATA or CAAT box motifs.
In the present study we studied the ability of rat pheochromocytoma cells (PC12) to correctly transcribe NMDAR1 mRNA and evaluated the ability of the 3-kb promoter fragment to direct the expression of a reporter gene construct in transient transfection assays. We also investigated DNA-protein interactions by gel mobility shift assays and DNA footprinting assays with promoter fragments thought to be important in the expression of the NMDAR1 gene. Our results suggest that the NMDAR1 gene proximal promoter region is sufficient for gene expression and that this promoter may be regulated by immediate early genes.
Figure 1: 5` Non-overlapping deletions of the NMDAR1 gene promoter. The 3029-bp 5`-flanking sequence of NMDAR1 gene is shown with an open bar. Progressive 5` deletions of this fragment were derived as described in detail under ``Experimental Procedures.'' Their 5` ends are indicated with numbers, which also represent their size. The locations of the putative motifs are indicated on the top of the bar. The arrow and surrounding tick marks represent the cluster of multiple transcription start sites. For other details about the 3029-bp sequence, please see (24) .
A Pharmacia kit was utilized for DNA footprinting. After titrating the DNase I concentration to create the best ladder, enough extract was applied under the conditions in gel mobility shift assay to saturate the probe. For the control ladder, the same amount of bovine serum albumin was added. At the end of the incubation, the probe was nicked by DNase I digestion for 1 min at room temperature. The digestion was stopped by adding 120 µl of stop buffer and extracted once with phenol/chloroform. Precipitated DNA was denatured at 95 °C for 5 min in loading buffer from the Sequenase II kit and fractionated on a 10% sequencing gel. DNA ladders of chemically cleaved G and G + A were also prepared and run on the gel(26) .
In some experiments, the density of specific bands in autoradiographs was analyzed with an LKB Ultroscan XL Enhanced Laser Densitometer.
Figure 2: PC12 cells transcribe the NMDAR1 gene from the same sites as in the brain. The 5` ends of NMDAR1 mRNA were mapped by RNase protection with riboprobe 2 as described under ``Experimental Procedures.'' Ten µg of total cellular RNA were hybridized to probe, and the protected bands were fractionated on an 8% DNA sequencing gel. Lane 1, riboprobe 1 (253 nucleotides) that was used to correct for the migration of RNA; lane 2, rat brain RNA; lane 3, PC12 cell RNA; lane 4, C6 cell RNA; lane 5, HeLa cell RNA; lane 6, yeast RNA. This is an autoradiograph exposed for 3 days at -80 °C with one intensifying screen. A 2-week exposure showed the same results.
Figure 3:
The activity of NMDAR1 promoter in
transiently transfected cells. Cells were transfected with chimeric
NMDAR1 promoter constructs, and the reporter gene assays were performed
as described under ``Experimental Procedures.'' Relative
luciferase activity is expressed after correcting for the transfection
efficiency with co-transfected pCMV/-galactosidase. A luciferase
gene driven by SV40 early gene promoter was used as positive control,
and the luciferase vector pGL-2Basic was used as a promoterless gene
control. All values are presented as the mean ± S.E. from at
least three separate experiments. The results were from PC12 cells (A and D), C6 cells (B), and HeLa cells (C). The results from construct pNRL356
242 with the
deletion at the 3` end of exon 1 are shown in D.
To determine whether the NMDAR1 core
promoter only contains nonspecific basal transcription activity,
additional transfection studies were carried out in non-neuronal C6
glioma and HeLa cells. Results in Fig. 3, B and C, showed that this NMDAR1 promoter construct in these two
cell lines had low activity compared to the SV40 construct. The changes
in activity among the other NMDAR1 constructs are small and vary less
than 2-fold in HeLa and 7-fold in C6 cells. In HeLa cells, since the
cytomegalovirus promoter has high -galactosidase activity and
luciferase activity overall was low, the relative luciferase activity
is lower than that in either PC12 or C6 cells. In view of these
results, this proximal sequence may have a role in basal, cell
type-specific NMDAR1 gene expression.
Figure 4:
Gel mobility shift analysis of the
interactions of NMDAR1 promoter with nuclear factors. A 112-bp fragment
(position -356 to -245) of NMDAR1 promoter was labeled
either on the sense or antisense strand by Klenow enzyme. Gel mobility
shift experiments were done as described under ``Experimental
Procedures.'' A, binding of different nuclear extracts.
Increasing amounts of crude nuclear extracts, 2.25, 4.5, and 9 µg
for PC12 and HeLa, 4.5 and 9 µg for C6, were added to reaction
mixtures. Six major complexes and free probe are indicated. The
smearing in the lane with the highest nuclear extract appeared due to
overloading. Overexposure of autoradiography showed that C6 had
multiple bands with similar density including duplexes C1 to C3. The left-hand lane is a control with probe alone. B,
competition by consensus oligonucleotides. 4.5 µg of crude PC12
nuclear extract was preincubated with or without 100-fold excess of
consensus oligonucleotides as indicated in the figure before adding
labeled probe. All oligonucleotides used for competition were from
Promega or Stratagene. The sequences of consensus oligonucleotides are
as follows: AP1(c-jun), CGCTTGATGAGTCAGCCGGAA; AP2, GATCGAACTGACCGCCCGCGGCCCGT; AP3,
CTAGTGGGACTTTCCACAGATC; CREB, AGAGATTGCCTGACGTCAGAGAGCTAG; CTF/NF1, CCTTTGGCATGCTGCCAATATG; GRE,
TCGACTGTACAGGATGTTCTAGCTACT; NF /B,
AGTTGAGGGGACTTTCCCAGGC; Oct1, TGTCGAATGCAAATCACTAGAA; Sp1, ATTCGATCGGGGCGGGGCGAGC; TFIID,
GCAGAGCATATAAGGTGAGGTAGGA. C, supershift of nuclear factor-DNA
complex by Sp1 antibody. Ten minutes after addition of probe to 4.5
µg of PC12 nuclear extract, increasing amounts of Sp1 antibody,
3.12, 6.25, 12.5, 25, and 50 ng were added to the mixture and incubated
for a further 20 min. In duplex C3, band A was further retarded in the
presence of Sp1 antibody. D, the binding of purified Sp1
protein. Human Sp1 protein purified from Sp1 cDNA-transfected HeLa
cells was added to labeled 112-bp probe in the absence or the presence
of PC12 extract (2.25 µg) represented with the solid bar.
Increasing amounts of Sp1 protein, from 0.0625, 0.125, 0.25, and 0.5
footprinting unit, were added to probe with PC12 cell extracts and 2
units of Sp1 protein to probe without extract (left lane).
Addition of 0.0625 unit of Sp1 increased the intensity of band A
approximately 8-fold based upon densitometric scanning of the
autoradiograph. E, effect of heating on potentiation of C3A
formation by Sp1 protein. Labeled 112-bp probe was incubated with
buffer (lane 1), 2.25 µg of PC12 extract (lane
2), or 4.5 µg of boiled PC12 extract (lanes 3 and 4). Sp1 protein (0.125 unit) was added to the reaction in lane 4. The band appearing in lane 4 migrated at the
same position as C3A in lane 2 and is 53.13% of C3A in lane 2 based upon densitometric scanning of the
autoradiograph. F, effect of Sp1 protein removal on the
formation of protein-DNA complexes in NMDAR1 promoter. Sp1 protein in
PC12 extract was precipitated by antibody as described under
``Experimental Procedures.'' Labeled probe was exposed to
PC12 extract (lane 1, 4.5 µg; lane 2, 2.25
µg) or Sp1-deficient extract (lane 3, 9 µg; lane
4, 18 µg).
As the first step to identify the nuclear
factors, competition experiments with a series of double-stranded
oligonucleotides, which contain specific motifs including many GC-rich
consensus sequences, were performed. As can be seen in Fig. 4B, C2 and C3 doublets were completely competed by
preincubation with a 100-fold excess of Sp1 oligonucleotide. Other
oligonucleotides did not compete with the binding. We then tested the
effect of an Sp1 specific antibody on the reaction mixture (Fig. 4C). In PC12 extracts, only one band, C3A, was
further retarded by the polyclonal Sp1 antibody which is capable of
specifically binding Sp1 proteins in human, rat, and mouse tissues.
This antibody showed high titer since 25 ng of antibody was able to
shift almost all C3A complex from 4.5 µg of extracts to a new,
slower migrating band. Increasing the amount of antibody to 50 ng did
not significantly change the other three bands. This suggests that the
C3A complex may contain Sp1-like proteins. Then we attempted to verify
this by adding purified Sp1 protein to the labeled 112-bp DNA. However,
even though we put 2 footprinting units in a single reaction, we did
not see any binding (Fig. 4D, left lane). Sp1
protein belongs to a family of zinc finger proteins and requires zinc
ions as cofactor. Although the storage buffer of Sp1 protein contains 5
µM ZnSO, we did not supplement any buffers
with zinc during nuclear extraction. Metal ions, such as
ZnCl
, ZnSO
, CaCl
, or MgCl
up to 1 mM, were added in the assay, but no binding was
seen (data not shown). These same results were observed with three
different lots of Sp1 proteins. Only when we added nuclear extract,
even as small an amount as 2.5 µg, did a band appear from 0.0625
unit of Sp1 protein migrating at the same position as C3A. This
suggests that an Sp1-like protein probably contributes to the C3A
binding complex.
Since Sp1 bound the 112-bp DNA fragment only in the presence of nuclear extract and did not bind in the presence of added zinc, we tested the heat sensitivity of the extract for Sp1 potentiation. Boiling the PC12 extracts dramatically decreased the formation of all complexes on the probe, since, compared to half as much native extract (lane 2, Fig. 4E), 4.5 µg of boiled extract (lane 3, Fig. 4E) showed much weaker, but the same bands only in an overexposed autoradiograph (data not shown). A densitometric analysis of the results in Fig. 4E indicates that, in 4.5 µg of boiled extract, the C3A complex potentiated by Sp1 protein (0.125 unit) is only equal to 53.13% of the C3A in half as much native extract, while 0.0625 unit of Sp1 protein is able to intensify the C3A in 2.25 µg of nuclear extracts 8-fold (Fig. 4D). This suggests that the binding of Sp1 protein to the 112-bp probe required the presence of heat-sensitive factors in nuclear extracts.
To clarify the relationship of this Sp1-like protein with the C2 and C3 doublets which were both competed by Sp1 oligonucleotides, we precipitated the Sp1-like protein from nuclear extracts with Sp1 antibody before testing extracts in gel shift experiments. As seen in Fig. 4F (lanes 3 and 4), prior precipitation of nuclear extracts with anti-Sp1 antibody prevented formation of both C2 and C3 duplexes. This result is similar to the Sp1 oligonucleotide competition experiments. This may suggest that Sp1-like protein and other factors access the 112-bp DNA in a mutually dependent way, or Sp1-like protein forms a complex with the other factors and they were co-precipitated by antibody-protein A-Sepharose.
Sequence analysis of the 112-bp fragment indicates that there are several GC-rich motifs: a GSG sequence which is recognized by immediate early gene family members including NGFI-A (32, 33, 34) and two successive Sp1 sites. One Sp1 site has a GGCGGG core sequence and overlaps the 3` end of GSG motif, and the other Sp1 site has a GGAGGG sequence (Fig. 5). To examine whether these motifs are the targets recognized by proteins involved in the DNA interactions as seen in gel shift experiments, we labeled either the sense or the antisense strand of the 112-bp fragment and examined the sequence protected from digestion with DNase I by PC12 nuclear extracts. The cluster of GC-rich motifs spanning almost 28 bp, on the antisense strand, was protected (Fig. 6). This evidence supports the idea that Sp1 factors are involved in the interaction with the NMDAR1 promoter. Since Sp1 protein is reported to protect a short sequence less than 10 bp (35, 36) , other factors must join this interaction which is consistent with the multiple retarded bands appearing in gel shift results.
Figure 5: Sequence of DNA probe used in gel mobility shift. The putative motifs are indicated, and the distal transcription start site is represented with an arrow.
Figure 6:
DNA footprinting analysis of the 112-bp
NMDAR1 promoter fragment. The end of the antisense strand was filled in
by Klenow enzyme with [-
P]dCTP. After
incubating the fragment with 35 µg of PC12 extracts or bovine serum
albumin as a control, increasing amounts of DNase I from 0.04 to 0.06
unit for PC12 extract and 0.1 to 0.4 for control were added to the
reactions. The solid bar represents the PC12 extracts, and the open triangle represents the amount of DNase I. A G + A
ladder of the probe was fractionated on the same gel and is shown in
the left lane. The next three lanes are the bovine
serum albumin control lanes.
NMDA receptors play many important roles in neurons of the central nervous system. Their neuronal location has been substantiated by ligand autoradiography (37, 38) and most recently confirmed by in situ detection of their subunit mRNAs and immunohistochemical detection of their proteins(5, 6, 7, 12, 13) . Cell type-specific and developmentally regulated expression of genes is controlled mainly at the transcriptional level(39, 40) . It was somewhat surprising therefore when our initial characterization of the NMDAR1 gene promoter showed that it had characteristics of housekeeping genes, that is, the proximal region was GC-rich and had no TATA or CAAT box motifs. This type of promoter is characteristic of many genes that are constitutively expressed in a nonspecific manner(41) . However, it has been shown more recently that several genes which lack TATA and CAAT boxes in their promoters have limited tissue distribution and their expression may be regulated(42, 43, 44, 45) . The NMDAR1 gene is in this latter category in that its expression is limited to neuronal cells, it is differentially regulated during development, and its expression is subject to pharmacological manipulation(14, 15, 19, 22) . In order to understand how the NMDAR1 gene promoter controls expression of this gene, we have previously cloned and characterized the 5` region of this gene(24) . In this report, we describe the generation and testing of reporter gene constructs which delineate the promoter region required for cell type-specific expression and attempt to define sequence motifs important in this expression.
It has recently been reported that PC12 cells contain messages for NMDAR1 and NMDAR2 family members(46, 55, 56, 57) . We have confirmed this result and have shown that PC12 cells utilize the same transcription start sites on the NMDAR1 gene as does rat brain. This suggests that PC12 cells have transcription machinery comparable to rat brain neurons, and, therefore, the regulation of the NMDAR1 gene may be similar.
It is interesting to note the differential utilization of
the two major transcription start sites of the NMDAR1 gene. In PC12
cells, the distal one is primarily used while in rat brain the proximal
one is favored. This may be explained by the fact that mRNA from PC12
cells is representative of a more discrete cell lineage with a
characteristic transcription system while mRNA from rat brain
represents the sum total of many different neuronal cells, each
possibly containing different cohorts of transcriptional proteins.
Recently, using both Northern blotting and in situ immunohistochemistry, several investigators have observed that
NMDAR1 message is developmentally and postnatally up-regulated in most
brain regions suggesting that differentiation of neurons is accompanied
by expression of the NMDAR1 gene(14, 15) . We and
others did not see any significant change in endogenous NMDAR1 message
after treatment of PC12 cells with nerve growth factor for up to 9
days(46) . ()These observations do not exclude the
possibility that other neurotrophic factors may play a role in the
up-regulation of NMDAR1 gene message, an area we are currently
exploring.
Using reporter gene technology, we showed that a basal promoter activity of the NMDAR1 gene is associated with a proximal fragment (from nucleotides -356 to -1 relative to the first nucleotide of the start codon) of the NMDAR1 gene. The observed activity in PC12 cells was slightly greater than with the SV40 promoter, while this activity in either HeLa or C6 glioma cells was at least 10-fold less than the SV40 promoter, suggesting a predominant expression in the PC12 cell line. In particular, a 112-bp 5` portion of the 356-bp fragment (-356/-245) seemed to contain sequences important for activity. When this 112 bp was included in the reporter construct, activity was 35 times greater than the next shorter deletion construct (pNRL356 versus pNRL239). Our knowledge of factors controlling neuronal-specific gene expression is still limited. Relatively few transcription factors have been identified which are either exclusively or preferentially expressed in neuronal cells to regulate neuronal genes (47, 48) or are expressed in non-neuronal cells to suppress neuronal gene expression(49) . In scanning the NMDAR1 promoter region for consensus motifs of transcription factors, we found the sequence TATTTATAGA (-804/-795) which is close to the consensus binding site for myocyte enhancer factor 2C (MEF 2C)(50) . This gene expresses specific splice variants in the central nervous system(51) .
In the 5` portion of the 356-bp fragment, we
previously identified a GSG binding motif which is the consensus for a
family of immediate early genes (24 and Fig. 5). Members of this
family can be induced in neurons by neurotrophic factors like nerve
growth factor or neurotransmitters like glutamate and may therefore
participate in central nervous system gene
regulation(32, 33, 34) . In addition, there
are two Sp1 sites, the 5` one overlapping the 3` end of the GSG motif
and the 3` one spanning -288/-283 with the sequence GGGAGG.
The latter Sp1 site has been shown to be a low affinity
site(52) . In many TATA box-less genes, Sp1 sites exist in the
vicinity of the core promoter and function like general transcriptional cis element factors assisting in the formation of a
preinitiation complex(39, 41) . In the present study,
putative, functional Sp1 sites in the NMDAR1 gene promoter were
confirmed by both gel shift and DNA footprinting experiments. However,
Sp1 binding activity was detected in HeLa and C6 glioma cell extracts,
but only PC12 cells had high levels of reporter gene activity. A
similar situation occurs with the myeloid-specific CD11b promoter which
contains an Sp1 and a myeloid-specific factor PU.1 (53) .
Although an Sp1 site was recognized by HeLa cell extracts in
vitro, in vivo footprinting showed that only myeloid
cells bind Sp1 suggesting that Sp1 factors may interact with PU.1 to
control cell-type expression. Another example is that of the
neuronal/muscle-specific expression of rat Na/K-ATPase 2 subunit
gene which is controlled by the interaction of one E-box binding factor
and an Sp1 factor(52) .
In the NMDAR1 promoter, an Sp1-like protein may cooperate with GSG binding proteins to control gene expression. This is supported by the following evidence. The construct pNRL356 produced about 35-fold more activity in PC12 cells than the next shorter construct pNRL239 (Fig. 3). The additional 112 bp of sequence (-356/-245) in pNRL356 contains a GSG and two Sp1 sites which are protected from DNase digestion by nuclear extracts (Fig. 6). Gel mobility shift experiments with the labeled 112-bp fragment revealed four complexes, C2A + B and C3A + B, which were specifically competed by Sp1 consensus oligonucleotides and were abolished by prior immunoprecipitation of nuclear extracts with Sp1 antibody. One of these complexes (C3A) could be supershifted by Sp1 antibody (Fig. 4, B and C). An explanation for this may rest on protein-protein interactions in complex formation and the time of antibody addition in each experiment. Prior removal of Sp1-like proteins by immunoprecipitation of nuclear extracts before initiation of complex formation may prevent formation of these four complexes. However, an Sp1-like protein may be accessible to antibody only in the C3A complex and either masked by other proteins or in an altered conformation in the remaining complexes. In the latter gel mobility shift assays, the Sp1 antibody is added after initiation of complex formation, and, therefore, potential interaction of transcription factors with each other and DNA has already occurred. Interestingly, purified Sp1 protein did not form any complex with this fragment but did so only in the presence of PC12 nuclear extract (Fig. 4D). Increasing amounts of purified Sp1 protein potentiated the formation of the same C3A complex which is supershifted by Sp1 antibody. The C3B complex also may increase slightly in these experiments. However, this increase may be due to shadowing from the more predominant C3A band or aberrant retardation of C3A complex in the C3B location. These results suggest that Sp1 binding is crucial for the formation of C2 and C3 complexes, and Sp1 protein may require the presence or binding of other transcription factors before it can interact with the promoter. We are presently investigating the protein composition of the other complexes. Pecorino et al.(54) observed that a neuronal GC box binding factor from murine brain enhances the expression of plasminogen activator in vitro. This activity shares the same recognition sites as Sp1 in the proximal promoter but cannot be retarded by Sp1 antibody in gel shift experiments. Thus, Sp1-like factors or GC box binding factors may have a role in neuronal-specific gene expression. We cannot rule out the possibility that neuronal-specific factors may recognize general transcription factors like Sp1 and control expression indirectly.
Using DNA footprinting, we confirmed that in the NMDAR1 basal promoter a previously identified sequence, CGCCCCCGC, was bound by nuclear proteins (24 and Fig. 4A). This sequence matches perfectly the consensus of GSG or Egr motifs which are recognized by most members in a zinc finger protein family including NGFI-A (also named Egr-1, krox 24, Zif/268, TIS 8), NGFI-C, krox-20/Egr-2, and ERG-3(32, 33, 34) . The variations in reporter activity caused by additional sequences 5` of nucleotide -356 suggest the existence of some other regulatory elements. For example, near the 5` end of pNRL919, which has maximal reporter activity, is a MEF2C site (-804/-795). Originally described by Leifer et al.(51) , MEF2C is a muscle-specific transcription factor and is highly expressed during development. However, it is also expressed in the central nervous system.
The characteristics of the promoter we have described for the NMDAR1 gene may be important in conferring a more general expression throughout the brain albeit in a neuronal-specific pattern. The possibility of a requirement for several transcription factors (Sp1-like and neuronal-specific?) interacting to control widespread neuronal expression of NMDAR1 gene is an attractive hypothesis which will require more testing.