(Received for publication, May 5, 1997)
From the Department of Biochemistry I, National Defense Medical College, Namiki 3-2, Tokorozawa, Saitama 359, Japan
ERK2 (extracellular-signal regulated kinase 2, also
known as p42 mitogen-activated protein kinase) is an integral member of the mitogen-activated protein kinase cascade that is crucial for many
cellular events such as proliferation and differentiation. Here, we
determined the genomic organization of the Erk2 gene and
characterized its promoter. The Erk2 gene spans over 60 kilobases, and the coding region is split into eight exons. In the
coding region, exon-intron organization was exactly conserved between the two mouse genes for ERK2 and ERK1 except one junction shifted by
one nucleotide. Primer extension and S1 nuclease analyses identified two major transcription start sites located at 219 and
223 relative to the translation start site. The 5
-flanking sequence lacked TATA box
but contained a CCAAT box located approximately 60 base pairs upstream
of transcription start sites. Sequencing of the 5
-flanking region also
revealed potential cis-acting elements for multiple
transcriptional regulatory factors including Sp1, zif268,
Ets, CREB, and PuF sites. The promoter activity of the 5
-flanking
region was examined using chloramphenicol acetyltransferase as a
reporter gene. Transient transfection experiments using Chinese hamster
ovary cells defined a maximal promoter activity in a 371-base pair
region immediately upstream of the translation start site. Furthermore,
we demonstrated, using mouse P19 embryonal carcinoma cells, that this
371-base pair sequence is likely to be sufficient to confer the
transcriptional activation of the ERK2 promoter during the retinoic
acid-induced differentiation of P19 cells.
The mitogen-activated protein (MAP)1 kinase cascade is activated by a myriad of proliferation- and differentiation-inducing stimuli (1-6). The MAP kinase pathway is utilized by broad organisms, including slime mold, yeast, fly, plants, and mammals, in a well conserved fashion. Following the activation of receptor tyrosine kinases and G-protein-coupled receptors, the signal is transmitted sequentially through an array of proteins including Ras, MAP kinase kinase kinases (Raf, MEK kinase, Mos), and MAP kinase kinase (MEK). MAP kinases are then phosphorylated on the tyrosine and threonine residues by dual-specificity kinase MEK. The activated MAP kinases translocate to the nucleus where the signal is converted to change transcriptional activity. MAP kinase activation also leads to phosphorylation of various proteins including membrane receptors, other protein kinases, cytoskeletal proteins, and regulatory enzymes.
ERK1 (extracellular signal-regulated kinase 1) and ERK2, known as 44- and 42 kDa-protein, respectively, were the first vertebrate MAP kinases to be described (7-9) and are the most closely related isoforms among the increasing MAP kinase multigene family (10) sharing approximately 90% identity in the amino acid sequence (9). These two kinases are similar in many aspects, such as ubiquitous tissue distribution (9, 11, 12), sensitivity to activation by MEK (13), and substrate specificity (2). Although ERK1 and ERK2 could be functionally redundant, it seems more likely that distinct patterns of gene expression impart specific biological role(s) to each ERK isoform in accordance with selective activation of the ERK isoforms; various stimuli selectively activate ERK2 in different cell contexts (14-20). Distinct regional distribution of ERK1 and ERK2 in brain structures (9, 21, 22) appears to result from unique transcriptional regulation on each gene.
Studies examining the specificity of growth factor signaling have suggested that the duration of the MAPK cascade activation makes cells decide about proliferation versus differentiation (4). Thus, proper regulation of Erk gene expression may be required for determining cell response by restricting the potential for activation of the corresponding cascade. However, little is known about the transcriptional regulation of ERKs. Early works have demonstrated an increased expression of ERK2 mRNA in the developing brain with concomitant change in the protein level (9). It was also shown that neuronal differentiation of P19 mouse embryonal carcinoma cells is associated with an increase in ERK2 mRNA (9). Analysis in cultured kidney mesangial cells has suggested that the regulation of the Erk2 gene expression resembles that of an immediate early gene (23).
To further elucidate the mechanisms that underlie these regulatable
aspects of the Erk2 gene, we cloned the Erk2 gene
and identified the promoter region. Determining the organization and promoter sequence of the Erk2 gene will enable a comparative
analysis with those of the previously isolated mouse Erk1
gene (24). It would also help to investigate the possible occurrence of
alternatively spliced transcripts that could give rise to different
forms of ERK2 protein observed in human tissues (12). A comparison of structural features between the genes for ERK1 and ERK2 might provide
insights into evolution of these closely related kinase genes after
gene duplication. In the present study, we determined the structure and
5-flanking sequence of the mouse Erk2 gene and
characterized its promoter.
Restriction and modifying enzymes were purchased from Life Technologies, Inc., New England Biolabs, Inc., Promega Corp., and Boehringer Mannheim. Radiolabeled nucleotides and [14C]chloramphenicol were from NEN Life Science Products. Nested deletion kit was from Pharmacia Biotech Inc. Cell culturing media were from Life Technologies, Inc. Bovine serum and fetal bovine serum were from JRH Biosciences.
Northern Blot Hybridization AnalysisFor Northern blot
analysis, 5 µg of poly(A)+-rich RNA from mouse brain and
lung were fractionated on a formaldehyde, 1% agarose denaturing gel
and transferred to Hybond-N+ nylon membrane (Amersham Corp.). The blot
was prehybridized in 50% formamide, 6 × SSPE, 1% SDS, 2 × Denhardt's solution and 100 µg/ml denatured herring sperm DNA at
42 °C for 3 h. Hybridization was performed at 42 °C for
10 h with 2 × 106 cpm of labeled probe/ml in the
same solution as prehybridization except for omitting Denhardt's
solution. Washing was carried out 3 times in 1 × SSC and 0.1%
SDS at room temperature for 10 min and then in 0.1 × SSC and
0.1% SDS at 65 °C for 45 min. The probe used was mouse ERK2
cDNA fragment (consisting of 60 bp of the 5-untranslated region
and 5
485 bp of the coding region; Ref. 25) labeled with
[
-32P]dCTP by the random primer method (26); the N
terminus coding region showed the least sequence homology between ERK1
and ERK2. Preliminary dot blot hybridization analysis confirmed that
under this condition, the ERK2 cDNA probe hybridized with more than 1,000-fold selectivity toward ERK2 cDNA sequence over ERK1 cDNA sequence. Autoradiography was done at
80 °C with an intensifying screen for 3 days.
A P1 clone isolated
from a c129 mouse embryonic stem cell library was obtained from Genome
Systems (St. Louis, MO). The PCR primers used for screening were PS-1U
(5-ACAAAGTTCGAGTTGCTATCA-3
) and PS-1D (5
-ATTGATGCCAATGATGTTCTC-3
)
which amplify a 122-bp product between nucleotides 152 and 273 (translation start site as +1) of the mouse ERK2 cDNA sequence
(25). To obtain a higher yield of P1 plasmid DNA, the P1 plasmid in
Escherichia coli NS3529 was transferred into the strain
NS3516 by production of a P1 transducing phage (27). P1 plasmid was
purified by the plasmid preparation method 1 suggested by the
supplier.
The clone 7278 P1 plasmid was subjected to restriction site mapping. A
4.5-kb EcoRI fragment containing the Erk2 gene
5-flanking region was identified by probing the EcoRI
digests with 32P-labeled oligonucleotide probe U3
(5
-CGCGAAGCGTCGAACCGAAC-3
) which represents the 5
extremity of the
known mouse ERK2 cDNA sequence. Plasmid pBS-E4.5 was made by
subcloning this fragment into pBluescript (Stratagene). To analyze the
gene structure, blots of restriction digests of clone 7278 were
hybridized either with end-labeled oligonucleotide probes designed
according to the ERK2 cDNA sequence or with ERK2 cDNA fragments
labeled by the random primer method. The exon-containing fragments were
identified and subcloned for sequencing.
The sizes of introns G and H were determined by PCR using U7
(5-CATACCTGGAGCAGTATTATGAC-3
)-D1 (5
-CCACAATGCACACGACCGTC-3
) and U2 (5
-CCAGCCAGGATACAGATCT-3
)-D4
(5
-GCTGCTACTACCAGAAACTGC-3
) primer combinations, respectively.
PCR was performed using 50 ng of P1 phage DNA as template with 30 cycles of 1-min denaturation at 94 °C, 45-s annealing at 60 °C,
and 3-min extension at 72 °C. The PCR products were sequenced to
verify the expected products.
By a combination of subcloning at known restriction sites and the generation of a series of nested deletion clones (28), genomic DNA fragments were sequenced by the dideoxy chain termination method using a PRISM sequencing kit and the model 373A automated sequencer (Applied Biosystems).
Isolation of RNATotal RNA was isolated from BALB/c mouse brain and lung using TRIzol LS RNA extraction reagent (Life Technologies, Inc.). Poly(A)+-rich RNA was prepared by one round of purification through an oligo(dT)-cellulose spin column (Pharmacia).
Primer Extension and S1 Nuclease AnalysesPrimer extension
and S1 nuclease analyses were performed based on the protocol described
by Sambrook et al. (29). For primer extension,
oligonucleotides PE-1 (5-GCCGCCGCCGCCGCGTTCGGTTCGACGCT-3
) and PE-2
(5
-GATCGGGAACGAGGAAGGAGGACAACACAG-3
), with their 3
termini
located 53 and 158 nucleotides upstream from the translation start
site, respectively, were end-labeled with [
-32P]ATP.
Both of the primers (1 × 105 cpm of PE-1 or 2 × 105 cpm of PE-2) were hybridized with 5 µg of
poly(A)+ RNA in 30 µl of hybridization buffer at 30 °C
for 16 h. Primer extension was carried out in a total volume of 20 µl in 50 mM Tris/Cl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM dNTPs, and 0.5 units/ml cloned ribonuclease inhibitor
(Life Technologies, Inc.) with 200 units of Superscript II reverse
transcriptase (Life Technologies, Inc.) at 42 °C for 2 h. S1
analysis was performed with single-stranded DNA probe prepared as
follows. Oligonucleotide PE-2 was end-labeled with
[
-32P]ATP and extended on sense strand DNA of plasmid
pBS-E4.5 with Klenow enzyme. The synthesized double-stranded DNA was
digested with XbaI and fractionated on a 5% acrylamide, 8.3 M urea denaturing gel. The desired probe fragment was
eluted from the gel in 0.5 M NH4OAc, 10 mM Mg(OAc)2, 1 mM EDTA, 0.1% SDS,
and 10 µg/ml tRNA at 37 °C for 12 h, followed by extraction
with phenol/chloroform and ethanol precipitation. Five micrograms of
poly(A)+ RNA was co-precipitated and hybridized with 4 × 104 cpm (approximately 2 fmol) of single-stranded DNA
probe at 30 °C for 16 h. The hybrid was digested with 1,000 or
3,300 units/ml S1 nuclease (Boehringer Mannheim) at 37 °C for 30 min. After stopping the reaction, samples were precipitated with
ethanol.
The primer-extended and S1 nuclease-resistant fragments were resolved
on a 5% acrylamide, 8.3 M urea gel concurrently with a
sequencing ladder that was generated with the same end-labeled oligonucleotide (PE-1 or PE-2) as primer to locate the 3 end of the
products. Sequencing reaction was carried out using Sequenase version
2.0 (U. S. Biochemical Corp.) with sense strand DNA of pBS-E4.5 as
template.
To evaluate promoter
activity of the 5-flanking sequence of the mouse Erk2 gene,
a series of reporter plasmids were constructed in pCAT-Basic (Promega).
As a positive control, pRSVCAT (30) was used. Plasmid
pCAT-B-(
2425/+100) was constructed by inserting a 2523-bp
SphI/NaeI fragment of pBS-E4.5 between the
SphI/PstI sites (with PstI site
blunted) of the pCAT-Basic polylinker. To construct pCAT-B-(
2425),
which contains 2.6 kb of the 5
-flanking region immediately upstream of
the translation start site, we generated a 570-bp PCR fragment
encompassing nucleotides
338 to +223 with an additional
SalI site at its 3
end. Primers used were as follows:
5
-primer, 5
-CTCAGGGGTCCCGTGGTTGGTGTG-3
; and 3
-primer,
5
-AGAGTCGACGTTGGCTGCACAGCCGCC-3
. Plasmid pBS-fl0.57 was made by
subcloning this fragment into pBluescript, and the nucleotide sequence
of the insert was confirmed by dideoxy sequencing. Then
pCAT-B-(
2425/+100) was digested with Eco47III and
SalI and inserted with a 398-bp fragment, which was derived
from the digestion of pBS-fl0.57 at the corresponding
Eco47III site and the SalI site to generate
pCAT-B-(
2425) (number in parentheses with a construct represents the
position of the 5
end of the insert relative to the upstream major
transcription start site). A series of 5
-deleted reporter plasmids was
constructed using convenient restriction sites. Plasmid pCAT-B-(
2425)
was cut with HindIII and filled with Klenow and then cut
with AvaI, BstEII, SacI,
DraIII, and NaeI to make pCAT-B-(
1156),
pCAT-B-(
781), pCAT-B-(
542), pCAT-B-(
366), and pCAT-B-(+99),
respectively. The linearized plasmid blunted the protruding end and was
self-ligated. Plasmid pCAT-B-(
1235) was created by self-ligation of
HindIII-cut pCAT-B-(
2425). Other 5
-deleted plasmids
contain EcoO109I/SalI (pCAT-B-(
219)), Eco47III/SalI (pCAT-B-(
174)),
AatII/SalI (pCAT-B-(
147)),
HinfI/SalI (pCAT-B-(
42)), and
BglI/SalI (pCAT-B-(+20)) fragments derived from
pBS-f10.57 (with their 5
ends blunted) between the HindIII (blunted) and SalI sites of the pCAT-basic polylinker. All
constructs were sequenced to verify the orientation and cloning
junction of the insert.
CHO-K1 cell was maintained
in Ham's F-12 medium supplemented with 10% fetal bovine serum.
Reporter constructs were transfected by the lipofection method using
pFx-4 lipid (Invitrogen) according to the protocol recommended by the
supplier. Briefly, 3 × 105 cells were seeded on 60-mm
dishes 1 day before transfection. Using 15 µg of pFx-4, 4 µg of
reporter construct was cotransfected with 1 µg of pCH110 (Pharmacia).
The internal standard plasmid, pCH110, which bears the lacZ
gene under the control of SV40 early promoter, was included to
normalize transfection efficiencies. Transfection was carried out for
4 h, and the medium was changed to the complete medium. After a
48-h incubation, the cells were harvested, and cell extracts were
prepared in 1 × reporter lysis buffer (Promega). The cell
extracts were assayed for CAT activity using a phase extraction method
(31), and -galactosidase activity of CHO cell extracts was
determined using the colorimetric method as described (29).
P19 mouse embryonal carcinoma (EC) cells were cultured in -minimal
essential medium supplemented with 7.5% bovine serum and 2.5% fetal
bovine serum (32). Activities of transfected reporter genes in
undifferentiated and differentiated P19 cells were evaluated as
follows. One day before transfection, 7 × 105
undifferentiated P19 cells were seeded on 60-mm dishes. Transfection was carried out in serum-free medium (Opti-MEM, Life Technologies, Inc.) using 10 µl of LipofectAMINE (Life Technologies, Inc.) to introduce 1.8 µg of reporter construct and 0.6 µg of pCH110. After transfection for 6 h, cells were trypsinized and filtered through a 40-µm nylon mesh (Falcon 2340, Becton Dickinson) to remove
undispersed cells. For undifferentiated cells, one-third of the cells
was replated on 6-well culture plates and cultured without retinoic acid (RA). For induction of neural differentiation, the remainder of
the cells was divided into two 35-mm bacterial grade Petri dishes
(Falcon 1008, Becton Dickinson) and cultured in the presence of 1 µM all-trans-RA (Nacalai Tesque, Tokyo,
Japan). After 24 h, the media of the undifferentiated and
differentiated cultures were changed to fresh medium without or with
RA, respectively. The undifferentiated cells were harvested 3 days
after replating, and the differentiated cells were harvested 2 or 3 days after replating. The
-galactosidase activity of the P19 cell
extracts was measured using the Galacto-Light Plus chemiluminescent
assay system (Tropix) in a Berthold LB 9505 C luminometer. All
determinations of enzymatic activities were performed within a linear
range of the assay system.
Previous experiments
analyzing ERK2 mRNA transcripts have been performed using RNA
isolated from rat (9) and human (12) tissues. To characterize the mouse
gene for ERK2, we examined an expression pattern of ERK2 mRNA by
Northern hybridization analysis using poly(A)+ RNA from
mouse tissues. Hybridization and washing of the blot were carried out
under stringent conditions to avoid cross-hybridization of the ERK2
cDNA probe to the ERK1 transcripts. As has been reported by Boulton
et al. (9), who used rat total RNAs, mouse ERK2 mRNA was
expressed at much higher levels in brain than in the peripheral lung
tissue (Fig. 1). In rat tissues, three
distinct ERK2 transcripts were identified at different ratios among
tissues. Similarly, multiple ERK2 mRNA transcripts were expressed
in the human (12). As shown in Fig. 1, mouse ERK2 transcripts displayed several isoforms as do rat and human ERK2 transcripts. Four ERK2 transcripts, 8.1, 5.3, 3.1, and 2.3 kb in size, were found in brain.
The lung transcripts showed a distinct pattern. The 8.1- and 5.3-kb
transcripts were expressed at barely detectable amounts. In contrast,
discrete transcripts of 3.6 and 3.0 kb were observed in the lung at
higher levels. The 1.9-kb transcript was detected only in lung. These
findings confirm that the characteristics of ERK2 mRNA transcripts,
namely the abundance in brain and the tissue heterogeneity, are also
observed in mouse tissues.
Structure of the Erk2 Gene
We analyzed one P1 clone, which
was isolated from a mouse embryonic stem cell P1 library by screening
with PCR, and found it contained an 80-kb insert and covered all of the
exons encoding the ERK2 protein sequence. Through a combination of
restriction mapping, Southern blot, and sequencing analyses, the
exon-intron organization of the Erk2 gene was elucidated as shown in
Fig. 2 and Table I. The
entire protein-coding sequence was identical to the reported cDNA
sequence (25). The gene spanned over 60 kb, and the protein-coding
region was divided into eight exons. Exon 1 consisted of approximately
220 bp of the 5-untranslated region and 113 bp of the coding sequence.
Exon 8 contained the 3
-coding and untranslated region. The nucleotide
sequences of the exon-intron boundaries (Table I) conformed to the
consensus sequence for splice donor and acceptor sites (GT-AG rule;
Ref. 33). Internal exons ranged in size from 110 to 190 bp. Introns ranged from 0.2 kb (intron E) to the large 32 kb (intron A). In the
coding sequence, positions of the splice junctions were in good
agreement with those of the previously cloned Erk1 gene
(24), except the intron D splicing site. The 5
splice site of intron D
was shifted by one nucleotide toward intron D. A 4.5-kb
EcoRI genomic fragment containing the 5
-flanking region of
the Erk2 gene was identified by Southern analysis and
subcloned into pBluescript as described under "Experimental
Procedures." The plasmid, pBS-E4.5, was used for further analysis of
the 5
-flanking region.
|
To determine the
transcription start site(s) of the Erk2 gene, primer
extension analysis and S1 nuclease protection assay were performed in
combination. For primer extension analysis, two radiolabeled
oligonucleotides were used in the extension reaction. First extension
analysis used oligonucleotide PE-1, which is proximal to the
translation start site (complementary to bases 25 to
53, the
adenine residue of the ATG start codon is referred to as +1), as a
primer. When extension was carried out with poly(A)+ RNA
from mouse brain, three major products were observed at bases
219,
223, and
330, with minor products at
205,
208,
226, and
278
(Fig. 3A). In the reaction using
lung poly(A)+ RNA, the product at
330 was not observed.
In the second extension reaction employing the distal primer PE-2
(complementary to bases
129 to
158), the extended products
terminated at two major sites,
219 and
223, and three minor sites,
205,
208, and
226 (Fig. 3B). The reactions with brain
and lung poly(A)+ RNA gave the same products, and the sites
coincided with those of the reaction using PE-1. The two longer
products obtained with PE-1 as primer (at
278 and
330) were not
observed in the extension reaction with PE-2. They might reflect
transcripts with different 5
ends that were generated from alternative
splicing occurring within the 100-bp stretch between PE-1 and PE-2. We
cannot exclude the possibility that another 5
-untranslated exon lies
in the 5
-upstream region.
S1 nuclease assay was also performed to confirm the results obtained
with the primer extension analyses. A 284-nucleotide single-stranded
DNA probe was hybridized to brain and lung poly(A)+ RNA and
digested with S1 nuclease. The protected fragments analyzed on a
sequencing gel gave the consistent pattern observed in the primer
extension using PE-2, with two major protected fragments at 220 and
223 and three minor fragments at
205,
209, and
226 (Fig.
3B). The minor signals at
205 and
209 were weak and only
visible after longer exposure (data not shown). The two
nuclease-resistant fragments (at
209 and
220) that are longer than
the corresponding primer-extended products seemed to have 3
ends
relatively resistant to S1 nuclease digestion.
Taken together, these findings revealed that the Erk2 gene
is transcribed from multiple start sites with the major sites being mapped at bases 219 and
223. The context of the major start site at
223 agrees well with the eukaryotic initiator consensus sequence
(YYA+1NWYY; Ref. 34), although the preceding C residue is
utilized as the initiation site instead of the authentic A residue
(Fig. 3C).
To identify sequence elements that may be involved in
transcriptional regulation, we determined the nucleotide sequence of the 5-flanking region contained in pBS-E4.5 (Fig.
4). The 400-bp stretch, with the major
transcription start sites set on center, had high GC content (68%). No
canonical TATA box was found around the transcription start sites. A
CCAAT box was evident at 60 nucleotides upstream from the major
transcription start site. Six Sp1 (35) binding core sequences were
distributed around the transcription start sites; two of the Sp1 sites
were located upstream of the start site (at
86 and
39 nucleotides
relative to the upstream major start site), and four were at positions
+11, +98, +104, and +116. Two of the downstream Sp1 sites comprised a
part of the zif268 (also known as Krox-24, Egr-1, NGFI-A;
Ref. 36) site (GCGGGGGCG; Fig. 4).
A computer-assisted search revealed multiple sequence elements having
homology to known binding sequences for transcription factors. These
included two AP-2 sites (consensus sequence: 5-CCSCRGGC, Ref. 37) at
180 and +259, two Ets sites (MGGAAG, Ref. 38) at
107 and +24, CREB
site (TGACGTCA, Ref. 35) at
153, PuF site (GGGTGGG, Ref. 39) at
236, Antennapedia site (ANNNNCATTA, Ref. 40) at
461,
bicoid site (TCTAATCCC, Ref. 41) at
503, MyoD site (CANNTG, Ref. 42)
at
779, and ELP site (YCAAGGYCR, Ref. 43) at
786. A comparison by
dot matrix analysis of the 5
-flanking sequences between the
Erk1 and Erk2 genes revealed no apparent overall
homology throughout the 5
-flanking region (data not shown).
To assess promoter activity of the cloned 5-flanking region
by its ability to drive CAT gene expression, we first made a construct by inserting a 2.6-kb fragment, which extends immediately upstream of the translation start site, in front of the CAT
gene (pCAT-B-(
2425)). Transient transfection assay was performed
using CHO cells, which express high levels of ERK2 protein (11), and the 2.6-kb fragment showed a significant promoter activity (Fig. 5). The CAT activity of pCAT-B-(
2425) was
27% of that driven by the Rous sarcoma virus long terminal repeat
(pRSVCAT), indicating that the Erk2 gene promoter could
direct high level transcription. We subsequently examined the effect of
a series of 5
deletions of the 2.6-kb fragment on the promoter
activity. As shown in Fig. 5, deletion to base
781 had little effect
on CAT activity, whereas progressive deletions to bases
542 to
148
resulted in a gradual increase. The increases indicated the existence
of multiple repressive elements in this region. The maximal activity
was marked by a pCAT-B-(
148) construct with a 2.5-fold increase over
that of pCAT-B-(
2425). Truncation of a 106-bp sequence from
pCAT-B-(
148) resulted in more than 85% loss of CAT activity. These
results suggested that the 371-bp region between
148 and +223 could
direct maximal transcription and that the putative
cis-acting elements between
148 and +223, such as Sp1
sites and CCAAT box, are likely essential in basal transcriptional
activity of the Erk2 gene promoter.
Activation of the Erk2 Gene Promoter during Differentiation of P19 EC Cells
The earlier study showed an increase in ERK2 mRNA
levels during differentiation of mouse P19 EC cells (9). We, therefore, examined whether the isolated promoter sequence is responsible for the
transcriptional activation during differentiation of P19 EC cells.
Undifferentiated P19 cells were transiently transfected with either
pCAT-B-(2425) or pCAT-B-(
148). The transfected cells were divided
into aliquots and left undifferentiated or induced to differentiate in
the presence of 1 µM RA (see "Experimental Procedures"). This concentration of RA has been shown to be enough for inducing the neuronal differentiation of P19 cells, and 2-day exposure to RA induces P19 cells into an irreversibly differentiated state (44). The CAT activity was normalized for transfection efficiency
with reference to the
-galactosidase activity derived from the
cotransfected internal control plasmid. Concerning the normalization,
it would be pertinent to note that, as judged from the
-galactosidase activity, expression of the reporter plasmids in the
differentiated cells on day 3 was lowered to 16-66% that on day 2. In
contrast, both
-galactosidase activity and CAT activities of the
undifferentiated cells remained almost constant during days 2 and 3 (data not shown).
As seen in Fig. 6, differentiation of P19
cells was accompanied by a significant increase in transcriptional
activity of the ERK2 promoter. After 3 days of RA treatment, the CAT
activity of differentiated P19 cells transfected with pCAT-B-(148)
was elevated 3.5-fold over that of the corresponding undifferentiated cells. Similar to the results obtained in CHO cells, pCAT-B-(
148) showed higher activity than pCAT-B-(
2425) in both undifferentiated and differentiated cells. These findings indicated that the 371-bp region between bases
148 and +223 is sufficient to confer the transcriptional activation of the Erk2 gene during the
RA-induced differentiation of P19 cells.
In this study, we determined the genomic structure of the mouse
Erk2 gene and identified its promoter. The Erk2
gene is distributed over 60 kb and has 8 exons. The size of the gene is
much larger than that of the previously cloned mouse Erk1
gene (24), about 8 kb in length, because of the expansion of the
corresponding introns. The exon-intron organization of the coding
region is highly conserved between two genes for ERK2 and ERK1. The
only difference was seen in intron D with its 5 splice site shifted by
only one nucleotide. All of the protein kinase conserved subdomains (45) are distributed throughout the exons in the same manner as the
Erk1 gene; in contrast to the other kinase genes (46, 47) in
which some of the kinase subdomains are interrupted by the introns, all
of the kinase subdomains of the Erk2 gene are retained in a
single exon. Primer extension and S1 nuclease analyses identified two
major transcription start sites 219 and 223 bp upstream of the
translation start site. Sequence analysis of the 5
-flanking region
revealed multiple potential cis-acting elements including
Sp1, zif268, Ets, CREB, and PuF sites. TATA box was not
found, whereas a CCAAT box was found located about 60 bp upstream of
the two major transcription sites. A comparison between the 5
-flanking
sequences of the Erk2 and Erk1 genes showed no
apparent homology in contrast to the highly conserved gene structure.
The divergence in the regulatory sequence is likely to enable a unique expression pattern of each Erk gene by employing distinct
combinational assortments of transcription factors.
Since ERKs occupy a pivotal position in MAPK cascades, any change in
expression levels of ERK isoform could be reflected in the amplitude
and duration of signaling of the corresponding MAPK module.
Accordingly, expression of Erk genes should be strictly regulated to maintain cell function. Thus, to elucidate the
transcriptional machinery that modulates ERK2 expression, we analyzed
promoter activity of the 5-flanking region of the gene. The 2.6-kb
fragment of the 5
-flanking region showed marked promoter activity when transiently transfected in CHO cells with cat as a reporter
gene. Since the sequence analysis of this 2.6-kb region had revealed multiple potential cis-acting elements that are implicated
in regulation of developmental- and tissue-specific gene expression, we
constructed a series of deletion constructs and examined their promoter
activity. Deletion to base
781 had little effect on CAT activity,
whereas further progressive deletions to
148 resulted in a gradual
increase. This suggests that some of the putative cis-acting
elements between bases
781 and
148, namely MyoD, bicoid,
Antennapedia, PuF, AP-2, CREB and Ets sites, could mediate a
repressive effect on the Erk2 gene expression in CHO cells. The highest CAT activity of pCAT-B-(
148) was dramatically reduced by
removing of a 5
106-bp sequence, suggesting the importance of two Sp1
sites and CCAAT box included between
148 and
42.
The marked increase in the ERK2 promoter activity during the RA-induced differentiation of P19 EC cells agreed well with the results obtained by the Northern hybridization experiment (9). The proximal 371-bp region was sufficient to confer the responsiveness, and the promoter activity had been elevated significantly by day 2. Since the neural differentiation of P19 cells was shown to be induced irreversibly by 2-day treatment with RA (44), it is highly likely that the increased expression of ERK2 is critical for the process. Supporting this idea is a current model that the neuronal differentiation of PC12 pheochromocytoma cells is determined by the sustained activation of the ERK pathway (4). In the nervous system, expression of ERKs shows developmentally regulated and restricted patterns (9, 21, 22). A role of ERK2 for modulation of neural activity has been suggested in hippocampal long term potentiation (20) which is associated with the induction of a set of immediate early genes. Taking these observations together, it seems that the transcriptional control of the Erk2 gene is essential for development and function of the nervous system.
The previously isolated kinase genes mostly lack both the TATA and
CCAAT boxes or have them at a nonstandard position (47-50). Together
with other common features such as multiple transcription start sites,
GC-enriched 5-flanking sequence, and multiple Sp1 sites, these kinase
genes are considered as housekeeping types. The Erk2 gene
promoter is unusual in that it has characteristics of both housekeeping
genes and regulated genes. While this promoter has the latter three
features common to housekeeping genes, it also contains a canonical
CCAAT box and potential cis-acting elements that act in
various developmental- and tissue-specific contexts. Indeed, the
Erk2 gene promoter is inducible as demonstrated in our
experiment using P19 cells. A similar promoter has been described in
the aspartate aminotransferase gene (51) which is expressed not only
constitutively but also in a hormonally regulated manner. In the
aspartate aminotransferase gene, two CCAAT boxes, which accounted for
the basal activity of the promoter, interact with a different set of
CCAAT box-binding proteins depending on the tissue examined. It seems
that constitutive activity of the promoter may correlate with the
ubiquitous occurrence of the different CCAAT box-binding proteins
including the CCAAT/enhancer-binding protein (C/EBP) family (52),
CTF/NF-I (35), and NF-Y/CP1/CBF-related proteins (53). On the other
hand, differences in combinations of these transcription factors may be
reflected in tissue-specific or regulated activity of the promoter.
Likewise, the unique property of the Erk2 gene promoter
might make it possible to compromise different regulatory requirements
for widespread expression and responsiveness for various stimuli; the
transcriptional regulation of the Erk2 gene might involve
multiple CCAAT box-binding proteins as in the aspartate
aminotransferase gene. Further experiments are needed to precisely
define the cis-acting elements and transcription factors
involved in regulating of the Erk2 gene expression.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D87264-D87271.
We thank Toshiyuki Suganuma for helpful advice, Minoru Sugawara for kindly providing plasmid pRSVCAT, and Yuko Nakamura and Ryoko Seita for technical and secretarial assistance.