From the
Department of Oral & Craniofacial
Biological Sciences, University of Maryland Dental School and Program in
Neuroscience, University of Maryland, Baltimore, Maryland 21201 and the
Department of Biology, College of Notre Dame of
Maryland, Baltimore, Maryland 21210
Received for publication, October 31, 2002 , and in revised form, May 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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The NR2A gene, like other NR genes, is expressed preferentially in neurons and its distribution pattern in the developing and adult brain is similar to that of the NR1 mRNA (7). Several physiological and pathological factors have been shown to regulate its expression at the mRNA level. In neurons of the neonatal brain, NR2A mRNA is progressively increased during development (7). This developmental increase is dependent upon synaptic activity (13) and may be promoted by brain-derived neurotrophic factor (14) or high KCl or NMDA in cultured neurons (15, 16). NR2A mRNA in the developing visual cortex is up-regulated by light and down-regulated by darkness (17). Chronic alcohol treatment delays developmental expression in cultured cerebellar granule neurons (18). Exposure to lead results in specific down-regulation of NR2A mRNA in the developing brain (19), whereas prolonged exposure to nicotine induces an increase of NR2A mRNA in auditory cortex of postnatal rats (20). In the adult brain, NR2A mRNA is up-regulated by treatments of growth hormone (21), substance P (22), and dioxin (23); withdrawal of butorphanol infusion (24); estrogen reduction in acyclic animals (25); ginsenoside Rg1 infusion (26); spinal cord injury (27); and inflammation-induced hyperalgesia (28). NR2A mRNA is down-regulated in the CA1 and CA3 regions of the hippocampus by butorphanol-induced tolerance (24), in the hypothalamus between cycling and acyclic female rats, possibly by estrogen (29), in the pyramidal neurons of the visual cortex by focal thermolesion (30), and in the dentate gyrus granule cells by kindling-induced epileptogenesis (31). Furthermore, abnormal expression of the NR2A gene has been reported in the dorsolateral prefrontal cortex and the occipital cortex of patients with schizophrenia (32) and in entorhinal cortex of Alzheimer's patients (33).
The cellular mRNA level of most genes is predominately controlled by the transcription rate that is in turn determined by promoter activity, although other mechanisms may be involved. The mRNA level of the NR2A gene in neurons is regulated by various factors, as mentioned above, but the mechanisms underlying the regulation remain for the most part unknown. As a first step toward elucidating these mechanisms, we characterized the promoter of the rat NR2A gene. We defined the transcription start points (TSPs) using RNase protection assay (RPA), rapid amplification of 5' cDNA ends (5'-RACE), and in vitro transcription experiments. Then, we tested abilities of the isolated promoter to drive a reporter gene in cultured cells and to interact with nuclear Sp factors, as well as examined the impact of Sp factors on the promoter capability to drive the reporter gene. We further studied the effects of the 5'-untranslated region (5'-UTR) on NR2A gene expression at transcriptional and posttranscriptional levels.
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EXPERIMENTAL PROCEDURES |
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Isolation of Genomic SequenceTo screen the rat genomic
library for the NR2A promoter a probe was generated by PCR with an upstream
primer GB54 (5'-GCTGCGAGCCCTGCTGCAGAG) and a downstream primer GB98
(5'-CAATAACGCCACCACGTTCACATC). The reaction employed rat liver genomic
DNA as a template, and Ex Taq DNA polymerase (TaKaRa/PanVera,
Madison, WI) to amplify DNA for 30 cycles of 30 s at 95 °C and 2 min at 68
°C. The resulting PCR product was treated with Klenow enzyme, cloned into
pGEM3Zf(+) at the HincII site (Promega, Madison, WI), and sequenced
as described previously (34).
The PCR product was confirmed to be NR2A genomic sequence, containing exons
and introns, and named GB5498. Excised DNA probe (GB5498) was labeled in the
presence of [-32P]dATP (ICN Biomedicals, Irvine, CA) with a
random primer extension kit following the manufacturer's instruction (Amersham
Biosciences, Piscataway, NJ). Library screening was carried out as described
previously (34). One positive
clone named NR2A32E was digested completely by EcoRI to produce three
fragments that were subcloned into pGEM3Zf(+) for sequencing. These subclones
were named as NR2A32E32, NR2A32E11, and NR2A32E22. DNA sequencing was
performed as described previously
(34) or by the core facility
of University of Maryland at Baltimore. Sequencing data were analyzed with
Vector NTI software (InforMax, Bethesda, MD). DNA sequence of clone NR2A32E
plus GB5498 was deposited in GenBankTM with an accession number of
AY167029
[GenBank]
.
RNA MappingRPAs were carried out as described previously (34). Templates of riboprobes were generated by subcloning restriction fragments of clone 2A32E22 or PCR products of the clone or its subcloned fragments into pGEM3Zf(+) or pGEM-T vector (Promega). Primers used in PCR to generate templates are as follows in pairs: GB339 (5'-CACCCTCTCCGCAGTCCTGCTC) as downstream primer plus M13 reverse primer for riboprobe 4 and 5; GB461 (5'-AAGCAGCAAGTGTGTATG) as an upstream primer and GB462 (5'-CTGCTCCACGCAGGGCAC) as a downstream primer for riboprobe 11. NR2A genomic sequences covered by each riboprobe are illustrated in Fig. 1. Sequences contributed by vectors are as follows: 29 nucleotides (nt) for riboprobes 13, 92 nt for riboprobe 4, 59 for riboprobe 5, 38 for riboprobes 68, 31 for riboprobe 9, 38 for riboprobe 10, and 46 for riboprobe 11. Correction of RNA migration to a DNA sequencing ladder was done as described previously (34). 5'-RACE was performed with a Marathon cDNA Amplification kit (Clontech) or a GeneRacer kit (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Total RNA was extracted from the adult rat brain and liver (Sprague-Dawley) with a guanidinium isothiocyanate/CsCl method as described previously (35). Rat brain poly(A)+ RNA was purchased from Clontech or prepared as described previously (35). DNA contamination in RNA extracts was monitored by directly running 1 µg of RNA samples in PCR with primers designed for the rat NR2A cDNA (see below). Any samples showing DNA contamination were not used. Primers used for 5'-RACE with a Marathon cDNA Amplification kit (Clontech) were GB97 (5'-GGCAATACCAGCAAGGTCCAGTAG) and GB98 (5'-CAATAACGCCACCACGTTCACATC), and those for RNA ligase-mediated (RLM) 5'-RACE with a GeneRacer kit were GB390 (5'-GCGTTCGGTTCCTCACTTGGTCAG), GB391 (5'-TGTGAGTGAGTGAGTGTGGGAGT), and GB462 (5'-CTGCTCCACGCAGGGCAC).
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In Vitro TranscriptionIn vitro transcription was performed
on the basis of a protocol described by Schwartz et al. with
modifications (36). Briefly,
nuclei were isolated from adult rat brain (Sprague-Dawley) by centrifugation
on a sucrose phase and resuspended in nuclear buffer (10 mM Hepes,
pH 7.9, 3 mM MgCl2, 0.1 M EDTA, 1
mM DTT, 0.1 M phenylmethylsulfonyl fluoride, 15%
glycerol, 10 µg/ml each of bestatin, leupeptin, and aprotinin). Crude
nuclear proteins were obtained by addition of an equal volume of nuclear
buffer supplemented with 0.7 M KCl, rotation for 15 min at 4
°C, and centrifugation at 125,000 x g for 20 min. Crude
nuclear proteins underwent ammonium sulfate precipitation and dialysis against
a buffer consisting of 25 mM Hepes, pH 7.9, 100 mM KCl,
15% glycerol, 0.25 mM EDTA, 1 mM DTT, and 0.5
mM phenylmethylsulfonyl fluoride. Protein concentration was
determined by the Brandt method (Bio-Rad). For in vitro
transcription, 0.1 pmol of NR2A promoter-luciferase constructs were mixed with
50 µg of nuclear proteins in a transcription buffer (40 mM
Hepes, pH 7.9, 1 mM MgCl2, 1 mM DTT).
Reactions were pre-run at 30 °C for 30 min. Then, 10 mM rNTP
and 40 units of RNasin (Promega) per reaction were added for a further
incubation at 30 °C for 30 min. Then, 1 µl of RNasin (40 units,
Promega) and 2 µl of RNase-free DNase (2 units, Promega) were added for
incubation at 37 °C for 20 min to remove DNA templates. The reaction was
quenched by addition of a stopping buffer (40 mM Hepes, pH 7.9, 20
mM EDTA, 25 µg/ml yeast tRNA, 0.2 M sodium acetate,
and 1% SDS). RNA in the reactions was then extracted with a conventional
phenol/chloroform method and subjected to RNase protection assay with
riboprobe 8, 9, or 11 as described above. RNase A and T1 were used to digest
single-stranded RNA. Protected RNAs were denatured and fractionated on 6%
PAGE-7 M urea-TBE gel. 32P-Labeled X174 DNA
digested by HinfIII was used as a DNA ladder. Autoradiography was
recorded with Kodak BioMax x-ray film.
Construction of NR2A-reporter PlasmidsA 2896-bp fragment
consisting of 1548-bp 5'-flanking sequence and 1348 bp of the 1349-bp
5'-untranslated region (5'-UTR) of the rat NR2A gene was prepared
by combining an EcoRI-XbaI fragment of NR2A32E22 with an
XbaI/filledin-StyI fragment of a 5'-RACE product. The
2896-bp fragment was cloned into pGEM3Zf(+) at EcoRI/HincII
sites to form pG2A2896. This plasmid was digested with EcoRI and
HindIII and the resulting NR2A fragment was cloned into pGL2basic
(Promega) at the XhoI/HindIII sites by a
ligation-fill-in-ligation strategy to form pNR2A28971.
pNR2A2897
338 was prepared by subcloning the
EcoRI-XbaI region of the 2896-bp fragment into pGL2basic.
Digesting pNR2A2897
338 with ApaI-HindIII or
BglII-HindIII followed by fill-in-ligation of the vector
produced pNR2A2897
576 and pNR2A2897
1071, respectively. To create
pNR2A2897
822 or pNR2A2897
1703, a SacI or NheI
fragment of pNR2A2897
338 was inserted into pGL2Basic.
pNR2A2897
1223 was produced by replacing the proximal 2301-bp sequence
in pNR2A2897
1 with fragment (2302/1223) at the
PstI sites. pNR2A2897
1234, pNR2A2897
1352, and
pNR2A2897
1380 were generated with PCR using GB492
(5'-TTAAAATCAAAAACATATATAC), GB493 (5'-TCTTCTCTAAGTTGTTCCAGAC),
and GB494 (5'-GGAGCCCTGTGGTTGGCACT), respectively, with M13 20
primer on the pG2A2896 and replacing the XhoI-HindIII region
of pNR2A2897
338 with the PCR products. 5' deletions of the
2897-bp fragment were conducted to produce constructs lacking the 338 or 822
bp of the proximal 5'-UTR. NheI digestion and vector
self-ligation of pNR2A2897
338 and pNR2A2897
822 created
pNR2A1706
338 and pNR2A1706
822, respectively. pNR2A1229
822
was produced by inserting the HincII-SacI fragment of a
pG2A2896 subclone into pGL2Basic at the Smal/SacI sites. A
BglII/SacI fragment of pNR2A1229
822 was ligated to
pGL2basic to form pNR2A1077
822. pNR2A1352
822 was derived from
riboprobe 11. The SacI-HindIII fragment of
pNR2A2897
338 was inserted into pNR2A1352
822 to produce
pNR2A1352
338, into pNR2A1229
822 to form pNR2A1229
338, and
into pGL2basic to create pNR2A822
338. Construct pNR2A1072
338 was
formed by ligation of a BglII-HindIII fragment of
pNR2A2897
338 to pGL2basic. Vector self-ligation of
ApaI-Smal-treated pNR2A822
338 produced
pNR2A580
338. pNR2A1700
1223 was formed by subcloning an
NheI-HindIII fragment of pNR2A2897
1223 to pGL2Basic.
pNR2A1228
1071 was produced by vector self-ligation of
BglII-digested pNR2A1228
338. Subcloning a
BamHI/XbaI fragment of pNR2A2897
576 to pGL2Basic at
the XhoI-XbaI sites generated pNR2A822
576.
Pfu-turbo DNA polymerase (Stratagene, La Jolla, CA) was used in all
PCRs above, and the fidelity of PCR products was examined by DNA
sequencing.
Cell Culture, Transfection, and Reporter Gene AssayPC12,
HeLa, HEK293, and P19 cells were maintained and transfected as described
previously (37,
38) except that
LipofectAMIN2000 (Invitrogen) was used according to the manufacturer's
instruction. Co-transfection of luciferase and -galactosidase constructs
and assay of reporters were done as described previously
(37,
38). In some experiments,
cells were used for RNA extraction 2 days after co-transfection.
Co-transfection of cDNA expression constructs was conducted with a 4:1 ratio
to luciferase constructs in addition to a CMV-LacZ construct (pCMV
gal)
at one-tenth of total DNA, and pDNA3 was used as vector DNA for
co-transfection. Luciferase activity was normalized to
-galactosidase
activity. Sp factor constructs were described in previous studies
(39).
Neuron-rich primary culture was prepared as described by Plesnila et
al. (40). Briefly, cells
were dissociated from the cerebral cortex of rat embryos at gastrulation day
15 as described previously
(38) and plated at
105/cm2 on poly-L-lysine-coated surface with
Neurobasal media plus 2% B27 serum-free supplement (Invitrogen). At day 8 of
culture in vitro, more than 95% of cells were neurons proved by both
morphology and microtubule-associated protein 2 immunostaining. At the same
time, cells were transfected with DNA using LipofectAMIN2000. LipofectAMIN2000
was mixed with DNA at a ratio of 2 µl:1 µg, and 2.5 µg of DNA was
used for cells cultured in a 10-cm2 surface. DNA constructs of the
NR2A promoter luciferase gene and pCMVgal (9:1) were transfected to
cells. Two days after transfection, cells were lysed for reporter gene assays
as described previously (39).
Transfection efficiency was about 5% determined by green fluorescent cells
after transfection of pEGFP (Invitrogen). Glial cells were isolated from the
cerebrocortex of rats at postnatal day 1 and cultured for 8 days before
transfection as described by Plesnila et al.
(40). Transfection and
reporter gene assays were performed as described for neurons.
Reverse Transcription-PCRTotal RNA was extracted from rat
and mouse brains as described above and from cultured cells with TRIzol
(Invitrogen) following the manufacturer's instructions. Human brain RNA was
obtained from Dr. John W. Kusiak at the National Institutes of Health. RNA was
treated with RNase-free DNase (Promega) and recovered with an RNA clean kit
(Zymo Research, Orange, CA). 2 µg of total RNA was annealed to a poly(dT)
primer (Promega) at 65 °C and reversibly transcribed into cDNA by
SuperScript II reverse transcriptase (Invitrogen) in 20 µl at 42 °C for
30 min. Three µl of the reaction of cell lines, 2 µl of cultured
neurons, or 1 µl of brain tissues was used in a PCR to detect NR2A mRNA
with human primers, GB509 (5'-TGATGAGGGAACCTCTTTGG)/GB510
(5'-GGTTGCTCTTCTCCATCAGC), rat primers, GB508
(5'-TGTGAAGAAATGCTGCAAGG)/GB511 (5'-GAACGCTCCTCATTGATGGT), or
mouse primers, GB512 (5'-CCATCAGCAGGGGCATCTACAG)/GB513
(5'-ACTGTGTTGGGGTTGGACTCAT). Primers of GB450
(5'-ACCACAGTCCATGCCATCAC) and GB449 (5'-TCCACCACCCTGTTGCTGTA) were
used to detect the glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA from
human, rat, and mouse. To detect reporter gene mRNAs, primers GB149
(5'-GCGGTTGCAAAACGCTTCCATCTTCCAGGGATAC)/GB148
(5'-AAATAGGATCTCTGGCATGCGAGAATCTGACGCAG) were used for luciferase
transcripts and GB122 (5'-ATGTGCGGCGAGTTGCGTGACTACCTA)/GB123
(5'-ATTCATTGGCACCATGCCGTGGGTTTC) for Escherichia coli
-galactosidase. PCR was performed with Ex Taq DNA polymerase
and cycled with annealing temperatures between 50 and 60 °C and extension
at 72 °C for 45 s. 40 cycles were used for NR2A mRNA, 30 cycles for
luciferase mRNA, and 26 cycles for
-galactosidase. DNA products were
visualized with UV illumination on agarose gel containing 10 µg/ml ethidium
bromide. Images were taken on a Kodak Image Station 440CF and analyzed with 1D
Image Analysis software as stated previously
(39).
DNA-Protein Interaction AnalysisCrude nuclear extractions
were prepared from cells as previously described
(37). DNA sequences used as
probes in electrophoretic mobility shift assays (EMSA) are as follows:
GC-box-a, 5'-GCCTCCGGGGTGGGCGACTGC; GC-box-b,
5'-CCGGGGAGCGGGCGGAGAGCGTGGT; GC-Box-c,
5'-GCATCCTGGGCGGGTGTGTGC. Probes were labeled by T4 DNA kinase in the
presence of [-32P]ATP (ICN Biomedicals), and EMSA was
conducted in a method reported previously
(37). Competition and
supershift of EMSA were performed as described previously with antibodies
against Sp1, Sp3, or Sp4 (37).
A consensus of GC-Box (Sp1 site) from Promega was used as a competitor.
Results of EMSA were analyzed and quantified with a PhosphorImager as
described in our previous studies
(41).
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RESULTS |
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Identification of TSP(s) or 5' End(s) of NR2A mRNANext, we searched for the TSP(s) by mapping the 5'-end(s) of NR2A mRNA. First we performed 5'-RACE experiments on rat brain poly(A)+ RNA using an NR2A gene-specific primer, either GB97 or GB98. After two rounds of PCR, we obtained fragments that contained at most an additional 236-bp sequence beyond the 222-bp 5'-UTR of the NR2A cDNA. Sequence analysis indicated that all of the newly obtained sequences are extensions of NR2A cDNA in the clone NR2A32E22 and thus belong to the same exon that encodes the 5' sequence of the cDNA. These results suggest that the NR2A gene has multiple TSPs.
Considering that the reverse transcriptase is sensitive to the secondary structure of mRNA and very often generates truncated products (43), the cDNAs in our 5'-RACE experiments may have resulted from incomplete reverse transcription. To confirm results obtained from the 5'-RACE above and search for possible upstream TSPs, we performed an RPA to map the 5'-end(s) of the NR2A mRNA. In addition, RPA is also able to determine TSP strength by quantifying the relevant transcripts (44). We designed 11 complementary riboprobes for use in the RPA based on the NR2A32E22 sequence (Fig. 1). RPAs with riboprobes 48 and rat brain or cerebral cortex RNA each resulted in a major protected band or clusters representing full protection of the NR2A sequence in the probes. In addition, each probe also yielded a number of small, weak protected bands representing minor TSPs (summarized in Table I). Riboprobes 13, however, repeatedly produced multiple packed bands indicating an interference of RNA secondary structure of the probes (data not shown). As a NR2A mRNA-free control, RNA extracted from the rat liver did not show any protection. Fig. 2 (A and B) shows representative results obtained from riboprobes 5 and 6 after digestion by RNase I. The same results were obtained from RNase A/T1 digestion (not shown). As shown in the Fig. 2 (A and B), RNA extracted from the adult rat brain generated strong single clusters with bands that varied by 24 nt, and these clusters represent the full sequence of the NR2A gene in the probes. A number of weak bands followed these clusters and delineated minor TSPs. These results suggest that the major TSP(s) must be upstream of the 5'-end of riboprobe 8 (1073, Fig. 1). To uncover the major TSP(s), we employed riboprobes 9 and 10, both of which end at 1230 (Fig. 1). As shown in Fig. 2C, riboprobe 9 revealed a strong band that is 7 bp less than the NR2A sequences in the riboprobe and corresponds to a protected sequence ending at 1222/1223. Riboprobe 10 produced the same results (data not shown). RPAs with riboprobe 11, which covers further upstream sequences, resulted in multiple protected bands (Fig. 2D). Among them, there are two groups of strong bands delineating major TSPs at positions 1233/1234 and 1222/1223. Both upstream and downstream of these strong bands are several weak bands indicating minor TSPs (Figs. 1 and 2). Density analysis of these protected bands indicated that the utilization difference between the major and minor points was more than 3.55 times in pair comparison. Detection of major TSPs at 1222/1223 by riboprobes 911 strongly supports their utilization by the NR2A gene in the rat brain. The largest protected band of riboprobe 11 represents a minor TSP at 1343 (Figs. 1 and 2D and Table I). On the basis of RPA results, we designed new primers of GB390, GB391, and GB462 (see "Experimental Procedures" for sequences) complementary to NR2A mRNA and performed a highly specific RLM-5'-RACE, which reacts only with 5'-caped mRNA. This experiment revealed multiple 5'-ends of the NR2A mRNA from rat brain poly(A)+ RNA. All newly obtained sequences are extensions of the previous 5'-RACE products in NR2A32E22. Most of these sites exist in the 1243/726 region (Table I), with the most distal position at 1349 relative to the first codon of open-reading frame (ORF). This position is 7 bp upstream of the site revealed by RPA (Figs. 1 and 2D) and may represent a very weak TSP that could be uncovered only by 5'-RACE. As a result of combination of these two methods, many of the minor TSPs were revealed by both 5'-RACE and RPA or more than two different riboprobes in RPAs, and are indicated by underlining in Table I. Taken together, our data suggest that the NR2A gene starts transcription at multiple sites with differential strength in exon 1 and that the NR2A transcript has a 5'-UTR with a maximum of 1349 nt encoded by a combination of exon 1 (1140 bp), exon 2 (191 bp), and part of exon 3 (18 bp).
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Sequence analysis of the proximal 5'-flanking region revealed a long polypyrimidine track, a direct repeat sequence, and an exonic TATA box upstream of the major TSPs (Fig. 1). Although the flanking sequence is TA-rich, exon 1 is over 50% GC in the region close to the 5'-end. There is no DNA sequence with more than 65% homology to the initiator (Inr) consensus, YYANWYY, because the initiator may direct transcription initiation for most genes (45). We also did not find an RE1/NRSE site using the same searching criteria to the consensus, TTCAGMACCDYGSASAGHRCC (46), considering that the RE1/NRSE DNA cis-element plays an important role in neuronal expression (41).
The Promoter of the NR2A GeneTo test whether the isolated 5'-flanking sequence has promoter activity, we utilized reporter gene constructs, because they are highly sensitive and capable of testing cell-type specificity (47). First, using an RTPCR technique, we examined NR2A mRNA in cultured rat cerebrocortical neurons to determine whether an endogenous mechanism for NR2A transcription was active. Our results showed that NR2A mRNA increased about 2.5-fold from 2 days in vitro (DIV) to 8 DIV (Fig. 3A). These cultures retained more than 95% neurons by cell morphology and MAP-2 immunostaining (data not shown). We also employed RT-PCR to examine several cell lines for expression of NR2A mRNA. We designed our PCR reactions to include an exon-exon junction in the product and thus were able to distinguish cDNA from products generated by contaminating genomic DNA. The fidelity of the PCR products was confirmed by DNA sequencing. As reported by others (48, 49), the mouse embryonic cell line, P19, in the non-differentiated state, and the rat pheochromacytoma cell line, PC12, express NR2A mRNA, albeit at a level lower than brain tissues (Fig. 3B). In contrast, RNA from human HEK293 or HeLa cells showed no NR2A transcript even after 40 PCR cycles but exhibited robust and stable GAPDH mRNA after 30 cycles (data not shown).
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Next, considering that the promoter of a given gene transcribed by RNA
polymerase II usually includes a core promoter, approximately between
nucleotides 35/+35 relative to the TSP and 50200 bp of
flanking sequences
(5052),
we fused a 1548-bp 5'-flanking sequence from clone NR2AE22 with various
exonic sequences from the 5'-UTR (1349 bp in total) of the NR2A gene as
illustrated schematically in Fig.
4A. We then tested the ability of these constructs to
drive transcription of a luciferase reporter gene in neural cells
(Fig. 4B) and cell
lines (Fig. 4C). The
largest fragment (pNR2A2897
1) exhibited significantly higher promoter
activity in neuronal culture or PC12 cells than that in glial or HEK293 cells.
The same results were obtained from differentiated P19 (neuronal) and HeLa
(non-neuronal) cells (data not shown). Removal of 338 bp of proximal sequence,
which comprises part of exon 1, the entire exon 2, and part of exon 3,
significantly increased promoter activity in all cells tested. Further removal
of the 3' 1072-bp exonic sequence encoding 5'-UTR
(pNR2A2897
1072), while preserving all major TSPs, decreased promoter
activity to a level comparable to that of pNR2A2897
1 in all types of
cells (Fig. 4, B and
C). Promoter activity was totally lost when all exonic
sequences, bases 1350/1 (pNR2A2897
1350), were deleted or
DNA fragments tested were in the reverse orientation (e.g.
pNR2A2897
822, 3'
5'). In comparison to the SV40 early
gene promoter, the NR2A promoter showed relatively higher activity in neurons
but lower activity in cell lines and comparable activity in glial cells. These
results indicated that the sequences studied here demonstrate promoter
activity preferentially in neurons or neuronal cell lines and that this
activity requires both major TSPs and their downstream sequences of exon
1.
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From results above (Fig. 4, B
and C), it can bee seen that the maximal promoter
activity was given in neuronal cells by NR2A fragments consisting of the
1548-bp 5'-flanking sequence plus exons lacking 338822 bp
proximal to the 5'-UTR. On the basis of these constructs
(pNRL2897338 and pNRL2897
822), we deleted the 5'-flanking
sequence to different sizes and examined the contribution of the
5'-flanking sequence to the NR2A promoter activity in cultured neurons
and PC12 cells. Results obtained from deletions of the parent construct
pNRL2897
338 are shown in Fig.
4D. Surprisingly, deletion of the entire
5'-flanking sequence and 5' 121-bp sequence of exon 1
(pNRL1228
338) did not cause any significant loss of promoter activity.
However, deletion of the most 5' region of exon 1 (pNRL580
338)
resulted in a reduction of, but did not abolish, promoter activity in cultured
neurons. In non-neuronal cells (glial, HEK293, and HeLa cells), similar
results were obtained (data not shown), although the activity remained lower
than that in neuronal cells, consistent with results obtained from 3'
deletion studies (Fig. 4, B and
C). In PC12 cells, an exonic fragment from bases
1228 to 339 (pNRL1228
338) generated higher activity than
any other fragments tested. This fragment contains the proximal major TSPs and
most of the internal exon 1 sequence but not the TATA-box. Taken together,
these results suggest that exon 1 of the NR2A gene contains basal promoter
activity that is neuron-preferential and independent of the TATA-box.
Furthermore, the 5'-flanking sequence up to 1548 bp may slightly enhance
the promoter (Fig.
4B), and the exonic sequence encoding the 3'
portion of 5'-UTR may play a negative role.
In view of exon 1 containing multiple TSPs
(Table I) and exhibiting
promoter activity of the NR2A gene (Fig.
4D), we next searched the core promoter within the exon 1
sequence. We generated small, sequential fragments of the exonic sequence that
encodes the 5'-UTR of the NR2A gene (left panel,
Fig. 5A) and tested
their ability to drive the reporter gene transcription in cultured cells.
These fragments vary from 157 to 342 bp in length, and most of them were
derived from exon 1. Interestingly, all fragments independently showed more
promoter activity in neurons or neuronal cells line (PC12) than that in
dissociated glial cells or non-neuronal cell line (HEK293)
(Fig. 5A). The high
activity remains within three fragments spanning from positions 1077 to
339, but is lower than that in a fragment consisting of the most
sequence of exon 1 seen in Fig.
4D (pNRL1228338) that contains major TSPs. These
data suggest that the core promoter resides in exon 1 and requires major TSPs
as well as downstream sequences for full activity. As negative controls,
fragments in reverse orientation, e.g. 1077
822 or
827
528 in front of the luciferase gene, failed to show significant
promoter activity (data not shown).
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To further confirm whether fragments tested above are able to directly transcribe RNA, we performed in vitro transcription experiments. We used nuclear proteins prepared from adult rat brains to transcribe RNA from varied constructs as listed in Table I, and then detected nascent RNA products using RPA. Based on the size of protected RNA probes, we estimated the TSP locations in these fragments. Interestingly, all tested constructs that had promoter activity transcribed nascent RNAs. Fig. 5B schematically illustrates positions of the fragments tested and TSPs revealed by RPA. Fig. 5C is representative of in intro transcripts analyzed by RPA. As can be seen, nascent RNAs were transcribed not only from large fragments showing maximal promoter activity above but also from exonic fragments exhibiting partial promoter activity. As summarized in Table I, all TSPs revealed from these internal fragments repeated or were close to the results revealed by 5'-RACE or RPA above (Table I and Fig. 5C). Major TSPs showed again the strongest activity (Fig. 5C, lanes 6 and 7, and Table I). These data further confirmed that the core promoter along with multiple TSPs resides in exon 1 of the NR2A gene.
Regulation of NR2A Promoter Activity by Sp FactorsSequence analysis of bases 1227/577 revealed three putative GC-boxes (Fig. 1). GC-boxes in a promoter region have been shown to interact with Sp1, Sp3, and Sp4 transcription factors (39) as well as other transcription factors, such as Egr1 (53). To test their role in forming the NR2A promoter, we co-transfected Sp cDNAs with NR2A promoter/luciferase gene constructs into PC12 cells. As shown in Fig. 6A, promoter activity was increased by overexpression of Sp1 or Sp4 but not by Sp3.
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We then examined the binding capacity of these cis-elements to endogenous nuclear proteins in EMSA experiments. Results in Fig. 6B show that all three GC-boxes formed three bands with nuclear proteins extracted from P19, PC12, or HeLa cells. Similar binding patterns were formed by all experimental probes as well as by a consensus GC-box oligonucleotide, although there are varying binding intensities among different cell lines. To identify proteins involved in producing these bands, we preincubated nuclear extracts with unlabeled oligonucleotide probes and found that the binding was diminished only by specific probes (lane 3, Fig. 6C) but not by nonspecific probes, such as Oct 1 (lane 4, Fig. 6C). Pretreatment of nuclear proteins with antibodies specific to each of the Sp factors showed that bands 1 and 3 were disrupted by an Sp1 antibody (lane 5, Fig. 6C), whereas band 2 was disrupted by an Sp4 antibody (lane 6, Fig. 6C), suggesting that these Sp factors are involved in specific band formation.
As shown in Fig. 6B, there are differential binding intensities among the three GC-boxes, even within the same cell line. To more accurately evaluate their binding capacity, we used equal amounts of each probe to react with the equal amounts of P19 cell nuclear proteins in EMSA and quantified the signal using a PhosphorImager. We combined signals from free probe, band 1 and band 2 as the total signal of the probe, and then calculated the signal shifted by each band as a percentage of the total signal in cognate lanes. As indicated in Fig. 6D, GC-box-c generated the largest percentage shift, whereas the GC-box-a generated the least, suggesting that the GC-box-c has the best binding capacity to Sp proteins.
Role of 5'-UTR in Regulation of NR2A Gene
ExpressionAs shown in Fig.
4, the construct containing the full-length 5'-UTR had
significantly lower reporter activity than those lacking the 338-bp proximal
sequence of the 5'-UTR. Sequence analysis revealed that the 338-bp
5'-UTR has five small ORFs (Fig.
1). A previous study with a limited 282-bp sequence indicated that
the mouse NR2A 5'-UTR interfered with the translation rate of that mRNA,
even though the complete upstream 5'-UTR was not available, and thus its
impact on this interference could not be fully determined
(54). Because reporter
activity really represents activity of protein products that are regulated by
both transcription and translation, we explored the mechanism by which
inclusion of the 338-bp proximal 5'-UTR might inhibit reporter activity
by reducing translation of the message RNA. We transfected cells with
pNR2A28971 or pNR2A2897
338 and then extracted RNA for RT-PCR of
luciferase and
-galactosidase messages. To avoid interference by the
transfected plasmids, we pretreated the RNA with RNase-free DNase. Our
results, as summarized in Fig.
7 indicated that inclusion of the 337-bp sequence slightly reduced
luciferase mRNA level (<50%). These reductions were much smaller than those
seen in reporter gene activity: a 4.3-fold reduction in PC12 cells and a
2-fold in P19 cells (Fig.
4C). These results suggest that sequences of exons 2 and
3 may not only negatively regulate transcription but also attenuate NR2A gene
expression by a different mechanism, very likely at the translational level,
after transcribed into mRNA.
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DISCUSSION |
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The core promoter in eukaryotic cells includes 35/+35 bp sequences
relative to the TSP and contains three common elements: 1) an upstream TATA
motif plus a TFIIB recognition element in some cases, 2) one or more short
initiator (Inr) sequences, and 3) a downstream promoter element (DPE) or other
elements (45,
50,
52,
55). Among these elements, the
DPE is well conserved from Drosophila to human and may replace the
TATA-box by interacting with several basal transcription factors in the
transcription initiation of TATA-less genes
(56). Sequence analysis of the
NR2A exon 1 revealed only one TATA-box consensus located among multiple TSPs,
and this site seems unimportant for promoter activity (Figs.
4 and
5). However, there is no
consensus for either an Inr or a DPE in the exon 1. Currently, the Inr
consensus remains loose or partially unidentified yet
(45). Additionally, Kutach and
Kadonaga (55) searched 205
promoters in a Drosophila core promoter data base and found that
31% of the promoters did not have either a TATA motif or a DPE. They
proposed that these genes might utilize other adjacent cis-elements,
such as GC-boxes, to direct basal transcription. In the sequence of rat NR2A
exon 1, we uncovered three putative GC-boxes, a putative AP-2 site, and a
putative NF
B site. We further demonstrated that these GC-boxes are able
to actively regulate promote transcription (Figs.
1 and
7). In addition, a sequence of
CA repeats was found in exon 1 (Fig.
1). Although the significance of CA repeats has yet to be fully
elucidated, it was reported that the GAPDH gene in P. anserine is
positively regulated by a CA repeat sequence
(57), and therefore this
sequence may also function as a regulatory element for the NR2A gene. All of
these elements may act as part of the core or proximal promoter of the NR2A
gene by supporting transcription initiation, however, this hypothesis needs to
be explored. Although several lines of evidence obtained from rat brain RPA,
reporter gene assay, and in vitro transcription demonstrate that
transcription of the rat NR2A gene can be started from multiple points
internal to exon 1, only two clusters of major TSPs can be protected by brain
RNA and are required for full activity of the promoter. Therefore, we believe
that the NR2A core promoter resides in a signal exon.
We also noted that the functional fragments of the exon 1 sequence of the NR2A gene exhibit high activity in cultured neurons, suggesting a cell-type preference. NR2A is an important neuronal protein, and its mRNA is found only in neuronal cells (3) or at a very low level in limited non-neuronal tissues (58, 59). Our studies further confirmed that the NR2A promoter is active only in neuronal tissue or cell lines (Fig. 3). The core promoter plus adjacent regulatory cis-element(s) located within 250/+250 bp regions to the TSP constitutes the proximal promoter (50). Some of these regulatory elements may be gene-specific and participate in tissue-specific or combinatorial regulation of the promoter (51). Previous studies indicate that, for some neuronal genes, the proximal promoter exhibits neuron-preferential activity (60). Very often these short promoters contain crucial cis-element(s) near the TSPs. For example, a 356-bp NR1 promoter, including an RE1/NRSE site downstream of multiple TSPs, showed cell-type specificity in PC12 cells (37). The RE1/NRSE site is believed to specifically suppress neuronal promoters in non-neuronal cells and thus restrict promoter activity in neurons (61). Additionally, a 386-bp genomic sequence of the growth-associated protein 43 gene encompassing multiple TSPs confers high promoter activity in cultured neurons (62). A silencer sequence found in this 386-bp promoter restricts promoter activity in neurons. It is possible that the short fragments of the NR2A gene studied here may use the sequence in front of the TSPs as proximal 5'-flanking sequence to harbor gene-specific regulatory element(s) and form individual proximal promoters (50). Then, these proximal promoters, instead of the core promoters, confer upon the NR2A gene a neuron-preferential expression, although the regulatory elements involved remain elusive. It also should be noted that the involvement of the core promoter in tissue-specific expression has been recently proposed (51). Whether neuron-preferential expression of the NR2A gene is controlled by the core promoter or the proximal promoter and which regulatory sequences are involved are interesting questions, currently under further investigation in our laboratory.
In addition, our results showed that deletion of the entire 5'-flanking sequence did not significantly alter rat NR2A promoter activity (Fig. 4). This may be caused by a number of mechanisms. First, the most upstream TSP is a very weak site that can be revealed only by a highly sensitive RLM-5'-RACE method (Fig. 1). Therefore, it is possible that the contribution from this site and its 5'-flanking sequence to promoter activity is too weak to be detected by the reporter gene technique. Second, the sequences proximal to the TSPs within exon 1 are sufficient to drive NR2A promoter activity in neurons. Third, other regulatory cis-elements for the rat NR2A gene reside either further upstream or downstream regions and are not in the sequences studied here. Using sequences already cloned in our laboratory, we are currently investigating this possibility.
Recent studies have shown that exonic regions of many genes contain various
types of cis-acting regulatory elements. Among them, GC-boxes seem
universal (37,
63). In this study of the NR2A
promoter, three functional GC-boxes were found to interact with Sp1 and Sp4
factors and to be important for the activity of the core promoter. Sp factors
are known to interact with GC-boxes located in proximal promoter regions of
most neuronal genes and participate in the regulation of these genes. Sp
proteins, including Sp1, Sp3, and Sp4, generally share the same binding
element, although whether the mechanism is in coordination or competition
remains unknown. Sp1 may function by interacting with other nuclear factors to
form enhancersomes or may recruit the basal transcriptional machinery
independently (64). For
example, the interaction of Sp1 with TAFII130 participates in the regulation
of the dopamine D2 receptor. In Huntington's patients, huntingtin interferes
with this interaction, resulting in a deficit of neuronal gene expression,
including the dopamine D2 gene
(65). Sp1 factor is also
involved in the regulation of promoters of other glutamate receptor genes. For
example, Sp1 interacts with MEF2c and up-regulates the NR1 promoter in neurons
(38). However, whether Sp1 and
Sp4 further regulate the NR2A promoter via protein-protein interactions with
other transcription factors is unknown. Putative GCG and NFB elements
are also found in the exonic region of the NR2A gene. Their functional impact
on the promoter needs to be further investigated.
Structurally, the exonic organization of the NR2A gene shows a certain similarity to other members of the NR2 family by starting the ORF in an internal exon. For example, mNR2B and 2C begin their ORF in exon 4 (66). In addition, the promoters of NR2B and NR2C genes also lack TATA-box and contain multiple TSPs and GC-rich sequences. These structures are common features of neuronal genes. However, the NR2A gene has a large initiative exon that contains several unusual structures, including five small ORFs, a CA-rich region, and an internal GC-rich region that have been discussed above. Wood et al. (54) previously reported that small ORFs in a limited fragment of the NR2A 5'-UTR interfered with the rate of translation. Our results, obtained from reporter gene transcripts and protein activity assays, further support this concept. In the present study, the full-length 5'-UTR has been tested, and therefore, a possible counteractive effect from the upstream 5'-UTR can be ruled out. More interestingly, the mouse NR2B gene also has multiple ATGs in the 5'-UTR (66). This similarity suggests a common evolutionary origin of NR2 subunit genes and supports their classification as a gene family. It also suggests the possibility of a common regulatory mechanism operating at the post-transcriptional level. Sequence comparisons among human, rat, and mouse NR2A genes deposited in GenBankTM indicate that they do share high homology in exonic region but display low homology in the 5'-flanking regions.
During preparation and review of this manuscript, two studies regarding regulatory sequence of the NR2A gene were reported. Richard et al. (67) mapped the 5'-most ends of the rat NR2A gene at 591 nt relative to the first codon in ORF, whereas Desai et al. (68) estimated that TSPs are located in two regions, 1133/1078 and 486/447 relative to the first codon of the mouse NR2A ORF. Consistent with our observation, studies of Desai et al. indicate that the NR2A neuron-preferential promoter activity resides in the sequence between bases 1253/210. Our studies indicate that exon 1 of the rat NR2A gene covers all of these sequences (Figs. 1 and 2). Using three overlapping deletion fragments, they claimed that a 40-bp sequence between bases 486 and 447 determines neuronal specificity. However, this 40-bp sequence was shown to have promoter activity only after it was linked with a 133-bp upstream or with 237-bp downstream sequences. Therefore, whether this 40-bp sequence initiates transcription for both fragments above or regulates TSPs that are located in either upstream or downstream sequences remains to be demonstrated.
In our studies, we demonstrate that the rat NR2A gene has multiple TSPs in a single, large initiative exon. Two clusters of these TSPs play major roles in initiation of mRNA in rat brain and in control of promoter activity preferential in neuronal cells. Our studies also reveal that three GC-boxes located in these small regions positively regulate promoter activity and interact specifically with Sp1 and Sp4 transcriptional factors. Finally, we confirm that the 5'-UTR sequence may participate in post-transcriptional regulation of the NR2A gene independently upon any upstream sequence either at transcriptional level or mRNA level.
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FOOTNOTES |
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* A part of the preliminary results of this study was reported at the 27th
Annual Meeting of the Society for Neuroscience, New Orleans, LA (1997). 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.
¶ Supported by National Institutes of Health Grant NS38077. To whom correspondence should be addressed: The Dept. of Oral & Craniofacial Biological 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;
NR2A, NMDA receptor 2A subunit; TSP, transcription start points; 5'-UTR,
5'-untranslated region; RACE, rapid amplification of cDNA ends; RPA,
RNase protection assay; nt, nucleotide(s); RLM, RNA ligase-mediated; DTT,
dithiothreitol; CMV, cytomegalovirus; RT, reverse transcription; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift
assay; ORF, open reading frame; DIV, days in vitro; Inr, initiator;
DPE, downstream promoter element; RE1/NRSE, repressor element
1/neuron-restriction silencer element.
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REFERENCES |
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