(Received for publication, April 21, 1995; and in revised form, July 12, 1995)
From the
A human cDNA clone for ERM, a member of the ets gene family, has been obtained by polymerase chain reaction with degenerate primers corresponding to highly conserved regions within an Ets DNA binding domain. ERM mRNA is expressed ubiquitously. The gene was mapped to chromosome 3q27. In in vivo transient-expression assays, ERM induced transcription more efficiently from a synthetic element containing both an ets-binding site (EBS) and a cyclic AMP response element (CRE) than from one containing an EBS alone. The activation of a synthetic EBS-CRE site by ERM was likely to involve a leucine zipper protein capable of dimerizing with CRE-BP1 leucine zipper. Indeed, ERM and c-Jun synergistically activated the EBS-CRE without making an apparent ternary complex. The synergy between c-Jun and ERM may be attributed to the enhancing effect of c-Jun on the transcription activity of ERM, because c-Jun increased ERM transcription activity by more than 20-fold in an assay system using a variety of fusion proteins between a Gal4 DNA-binding domain and a portion of ERM. This enhancing effect of c-Jun required the amino-terminal portion of ERM.
The ets oncogene family members are involved in gene
activation through interactions with other gene
products(1, 2, 3, 4) . The first
member of the ets gene family, v-ets oncogene, was
identified as a part of the gag-myb-ets fusion oncogene of the
avian retrovirus E26, which is responsible for leukemic transformation
of chicken erythroblasts and myeloblasts(5, 6) . Since
then, a number of genes related to the v-ets oncogene have
been identified in a variety of cell types from Drosophila to
human(3, 4) . The ets gene family is divided
into subgroups on the basis of amino acid sequence similarity and
overall structure, such as the Ets1/Ets2(7, 8) ,
Elk-1/SAP1(9, 10) , Fli-1/Erg1 (11, 12) , PEA3/ER81(13, 14) ,
PU.1(15) , GABP(16) , and Elf-1/E74
subfamilies(17, 18) . All of these genes have a highly
conserved domain, the Ets domain, which is composed of about 85 amino
acids and located near the carboxyl terminus, except in the Elk-1/SAP1
subfamily and Elf-1. The Ets domain is sufficient for sequence-specific
binding to DNA at a purine-rich core sequence,
GGA(A/T)(19, 20) .
Putative ets-binding
sites (EBS) ()have been identified in a variety of promoters
or enhancers, including the ets-1 promoter(21) ,
interleukin-2 gene enhancer (NFAT-1)(18) , polyomavirus
enhancer (PEA3) (22) , moloney sarcoma virus long terminal
repeat(23) , SV40 enhancer (15) , human T-cell leukemia
virus type I long terminal repeat(24) , human immunodeficiency
virus type 2 long terminal repeat(25) , human herpes simplex
virus ICP4 enhancer(16, 26) , stromelysin
promoter(27) , T-cell receptor
and
enhancers(28, 29, 30) , c-fos promoter(2, 10, 31) , immunoglobulin
3` enhancer(32) , immunoglobulin heavy chain intron
enhancer(33) , and granulocyte macrophage colony-stimulating
factor promoter(34) . The members of the Ets family often show
functional synergism with other transcription factors to achieve
efficient activation of such target genes through an EBS and adjacent
DNA sequences(10, 22, 35) . For instance, a
factor(s) binding to the PEA3 site of polyoma virus enhancer and
collagenase promoter acts synergistically with AP1 to express maximal
transcription activity(22) . Ets-1 and Ets-2 can bind to the
polyoma virus enhancer PEA3 site and cooperate with exogenously
expressed c-Fos and c-Jun(1) . Direct interaction between the
Ets family members and other transcription factors has been
demonstrated in many cases, including Ets-1/Sp1 on the EBSs of human
T-cell leukemia virus type I long terminal repeat(36) ,
GABP
/GABP
on the herpes simplex virus ICP4
enhancer(16, 26) , PU.1/NF-EM5 on the immunoglobulin
3` enhancer (32) , p62 ternary complex factor
(p62
)/serum response factor on the c-fos serum
response element (SRE)(2, 10, 31) , and
Ets-1/CBF on the T-cell receptor
chain
E2 and
E3
enhancers(29) . Thus, Ets family members often require
cooperation with other factors for selective and efficient activation
of their target genes.
The interleukin-6 response element in the junB promoter, named JRE-IL6, contains an ets-binding site (JEBS) and a CRE-like site(37) . There are constitutive (37) and interleukin-6-inducible JEBS-binding proteins(38) . Recently we showed that the major component of the interleukin-6-inducible proteins was Stat3/acute-phase response factor (APRF) in both a human hepatoma cell line, HepG2, and rat liver(38) . During the effort to clone a constitutive JEBS-binding protein, we recloned a member of the ets gene family, ERM(39) , belonging to the PEA3 subfamily. In this report, we show that ERM and c-Jun synergistically activate transcription from a synthetic element containing an EBS and a CRE without apparent cooperative binding to the corresponding site. Moreover, we show that c-Jun enhances the transcriptional activity of ERM. This functional property is specific to ERM and is not observed with a closely related Ets protein, ER81. Thus ERM is the first ets gene family member that acts synergistically with c-Jun and whose transcriptional activity is enhanced by c-Jun.
Synthetic response elements are as
follows: 3 EBS, 5`-TCGAGCAGGAAGTCAGACTTCCTGCGCAGGAAGT-3`;
3
mEBS, 5`-TCGAGCAGCTAGTCAGACTAGCTGCGCAGCTAGT-3`; EBS-AP1,
5`-CGCGGAAGTTATAAAGCATGACTCAG-3`; EBS-CRE,
5`-CGCGGAAGTTATAAAGCATGACGTCAG-3`. The structure of the EBS-AP1 was
equivalent to the PEA3-AP1 motif in the collagenase promoter with a
nine-base pair spacing between the two binding sites(35) . The
typical AP1 site was replaced by a somatostatin CRE (42) for
the EBS-CRE. Complementary oligonucleotides were made so that there was
an appropriate restriction site at each end. One copy each of 3
EBS and 3
mEBS oligonucleotides was inserted into the SalI site of pBSB luc to make 3
EBS-pBSB luc and
3
mEBS-pBSB luc, respectively. To make 3
EBS-AP1 luc and
3
EBS-CRE luc constructs, the oligonucleotides containing the
EBS-AP1 or EBS-CRE with an ApaI and a SalI
restriction site at each end were concatamerized and subcloned upstream
of the minimal junB promoter at the ApaI and SalI sites of pBSB luc. To make a Gal4 luciferase reporter
construct, p5GBS luc, a KpnI-HindIII fragment of
G5BCAT (a gift from Dr. M. R. Green) containing five copies of Gal4
binding sites and a TATA box of the adenovirus E1b gene promoter was
subcloned into the KpnI-HindIII site of pBS luc.
The expression vectors encoding Gal4(1-147) fusion proteins were as follows. GAL4-ERM (pGAL510(1-510)) containing a complete coding sequence of ERM was constructed by fusing a HindIII (Klenow-filled) fragment of pBS-ERM to the BamHI site (Klenow-filled) of pSG424 (a gift from Dr. M. Ptashne). pGAL326 was generated by deleting the NdeI-SalI fragment from pGAL510. pGAL276 was constructed by subcloning the SmaI fragment of pGAL510 into the SmaI site of pSG424. The HindIII (Klenow-filled)-PvuII fragment of pBS-ERM and the NdeI-SalI fragment (Klenow-filled) of pGAL510 were used to make pGAL165 and pGAL327-510, respectively. pGAL166-326 was constructed by subcloning the PvuII-NdeI fragment of ERM, to which had been added an EcoRI linker, to the EcoRI site of pSG424. pGAL-ER81 containing a complete coding sequence of ER81 was constructed by fusing an EcoRI-XbaI fragment of the ER81 cDNA to the EcoRI-XbaI site of pSG424.
Figure 1:
ERM transactivation potential. A, ERM-activated transcription through the ets-binding site. NIH 3T3 cells were transfected with 1.2
µg of either the control reporter plasmid, pBSB luc (Control), the indicated reporter constructs in combination
with the ERM expression plasmid pEF-ERM (shadedbar),
or the vector control, pEF-BOS (blackbar). The
reporter constructs used were as follows: 3 EBS-pBSB luc
containing three copies of the wild-type EBS (indicated as 3
EBS) and 3
mEBS-pBSB luc containing three copies of mutant
EBS (3
mEBS). Transfected cells were maintained in
-minimal essential medium containing 0.5% fetal calf serum. At 48
h posttransfection, cells were harvested, and extracts were analyzed
for luciferase expression. The pEF-lacZ reference plasmid was included
to normalize the differences in transfection efficiency. Luciferase
values were normalized for
-galactosidase and expressed relative
to the activity observed with the control reporter plasmid in the
absence of the ERM expression plasmid. Values represent the means of
three different experiments, and the standard deviations of the mean
values are indicated by errorbars. B,
ERM-activated transcription efficiently from the 3
EBS-CRE
reporter construct and from the 3
EBS-AP1 construct. NIH 3T3
cells were transfected as above with 1.2 µg of either the reporter
constructs 3
EBS-CRE luc (3
EBS-CRE) or 3
EBS-AP1 luc (3
EBS-AP1). Normalized relative luciferase
values are shown. C, effect of dominant negative forms of CRE
site-binding proteins on ERM-induced transcriptional activity. NIH 3T3
cells were transfected with 1.2 µg of the 3
EBS-CRE luc
reporter plasmid and 2 µg of either pEF-ERM or a control vector
pEF-BOS in combination with various amounts of one of the following
expression vectors: the dominant negative CREB expression vector (KCREB), CRE-BP1 leucine zipper expression vector (CRE-BP1LZ), or expression vector encoding
an amino-terminal deletion mutant of CRE-BP1 (NT253).
Normalized relative luciferase values are as in A. Data are
representative of three independent experiments with similar
results.
We used the EBS-CRE as a target element for ERM to further
study the factor(s) cooperating with ERM. We first tested the effects
of several dominant negative forms of CRE-binding proteins on the
ERM-induced transcriptional activity through the EBS-CRE site (Fig. 1C). Overexpression of either CRE-BP1 LZ without
the basal region of CRE-BP1 necessary for DNA binding, or NT253, an
amino-terminal deletion mutant of CRE-BP1, efficiently reduced the
ERM-activated 3 EBS-CRE-driven gene expression in a
dose-dependent manner. CRE-BP1 LZ acted as a dominant negative form by
dimerizing with endogenous CRE-BP1 or proteins capable of dimerizing
with CRE-BP1 LZ and alleviating their DNA-binding activities to the CRE
site. Overexpressed NT253, devoid of transcriptional activity, likely
replaces the endogenous CRE-binding proteins. On the other hand,
overexpression of KCREB, a dominant negative form of CREB, increased
the ERM-activated 3
EBS-CRE-driven gene expression in a
dose-dependent manner. These results suggested that cooperation between
ERM and a factor(s) acting on the adjacent CRE motif are required for
ERM to function efficiently, and the endogenous proteins are most
likely ones capable of dimerizing with the CRE-BP1 leucine zipper,
possibly CRE-BP1 itself or other proteins including c-Jun.
To
determine which transcription factor(s) can cooperate with ERM on the
EBS-CRE, NIH 3T3 cells were transfected with the ERM expression vector
and various amounts of an expression vector for either CREB, CRE-BP1,
c-Jun, JunB, JunD, or c-Fos together with the 3 EBS-CRE
luciferase reporter construct. Among the factors tested, only c-Jun
showed synergy with ERM (Fig. 2A). Since this synergy
between ERM and c-Jun was not observed when 3
EBS-pBSB luc
reporter was used (data not shown), it is unlikely that over-expression
of c-Jun enhances the expression level of ERM. The synergy was evident
when smaller amounts of c-Jun expression vector were used, suggesting
that overexpression of c-Jun may sequestrate another factor(s)
necessary for ERM to be transcriptionally active. Exogenous expression
of CRE-BP1 showed little effect on ERM-induced transcriptional activity (Fig. 2C). On the other hand, CREB at any concentration
tested inhibited ERM-induced transcription (Fig. 2B), consistent
with the positive effect of KCREB on ERM-induced transcriptional
activity (Fig. 1C). Neither c-Fos, JunB, nor JunD
showed cooperative activity with ERM (data not shown). These results
suggested that the endogenous protein cooperating with ERM on EBS-CRE
might be c-Jun itself. It is also possible that a heterodimer of
CRE-BP1 and c-Jun, known to recognize a typical CRE(51) , is
involved.
Figure 2:
ERM
and c-Jun synergistically activated transcription through a response
element containing an EBS and a CRE site. The 3 EBS-CRE luc
reporter construct was transfected into NIH 3T3 cells with 2 µg of
the ERM expression vector, pEF-ERM (
) or the control pEF-BOS
(
) in combination with various amounts of the expression
plasmids of c-Jun (A), CREB (B), and CRE-BP1 (C). The total amount of DNA used for transfections was
adjusted with the control expression plasmids without an insert.
Normalized luciferase values with standard deviations are expressed
relative to the basal activity of the control reporter plasmid (pBSB
luc).
Then we examined whether exogenous expression of some members of the CREB-activating transcription factor family or the Jun-Fos family enhance the transcription activity of ERM. For that purpose, we used a fusion protein (GAL4-ERM) of the Gal4 (1-147AA) DNA-binding domain and a full-length ERM (Fig. 3). Neither the control vectors, the Gal4 expression vector lacking the ERM cDNA, nor the expression vectors without an insert, showed any effect on the reporter gene expression. The GAL4-ERM fusion protein had little activity and its transcriptional activity dramatically increased by around 30-fold in the presence of exogenously expressed c-Jun (Fig. 3). This increase in transcription activity was not due to the increase in the amount or in the DNA-binding activity of the GAL4-ERM fusion protein, because co-expression of c-Jun did not change either of these factors (data not shown). Neither JunD, c-Fos, CREB, nor CRE-BP1 showed significant effects on the transcriptional activity of the GAL4-ERM fusion protein (Fig. 3). These results indicated that c-Jun specifically enhances the transcriptional activity of ERM.
Figure 3: Effect of CRE-binding proteins on the transcriptional activity of the GAL4-ERM fusion protein. The 5GBS luc reporter plasmid was transfected into NIH 3T3 cells with 2 µg of the GAL4-ERM fusion protein expression vector (openbar) or the control Gal4 expression vector (blackbar), pSG424, expressing only the 147-amino acid DNA-binding domain in combination with 2 µg of either the expression vectors indicated or the vector control lacking an insert DNA. Normalized luciferase values are expressed relative to the basal activity of the control Gal4 expression vector transfected alone. Values are representative of three independent experiments.
To investigate which
part of ERM has transcription activation domain and which part is
required for c-Jun enhancement of the transcriptional activity of ERM,
we tested a series of fusion proteins containing the Gal4 DNA-binding
domain and different segments of ERM for the ability to activate
5 Gal4-site-driven gene expression without or with exogenous
c-Jun expression (Fig. 4). Deletion of the carboxyl-terminal
region including the ETS domain of ERM (pGAL326) increased
transcription activity by 4-fold as compared with the basal activity of
Gal4 protein alone. Further deletion of the carboxyl-terminal portion,
pGAL276, resulted in a 10-fold increase in transcription activity. The
amino-terminal portion containing an acidic region (pGAL165) showed a
69-fold increase in transcription activity. However, regions other than
the amino-terminal portion of ERM (pGAL166-510,
pGAL327-510) seemed not to have transcription activity. The part
from amino acid 166 to 326 of the ERM protein may contain a negative
regulatory domain(s). Among these fusion proteins, the enhancing effect
of c-Jun was evident only when the fusion proteins contained both the
region of amino acids 166-276 and the amino-terminal portion of
amino acids 1-165, such as in pGAL510, pGAL326, and pGAL276.
Consistent with this, c-Jun did not activate the transcription activity
of pGAL166-510 nor that of pGAL327-510. In contrast to the
situation with GAL4-ERM, c-Jun had no effect on the transcription
activity of GAL4-ER81, a fusion protein of the GAL4 DNA-binding domain,
and a full-length ER81 that is closely related to ERM (Fig. 4),
suggesting the specificity of ERM-c-Jun cooperation.
Figure 4: Effect of the exogenous c-Jun expression on the transactivation potential of a series of fusion proteins containing the Gal4 (1-147 amino acids) DNA-binding domain and different segments of ERM or a full-length ER81 protein. The structures of the constructs examined are depicted schematically at the left. The reporter 5GBS luc plasmid was transfected into NIH 3T3 cells with 2 µg of the indicated GAL4-ERM construct in combination with either 2 µg of the c-Jun expression plasmid or control plasmid. The basal activity of the 5GBS luc was defined as 1. Normalized luciferase values are expressed relative to the basal activity of the 5GBS luc without expression vectors. Data are representative of three independent experiments.
Figure 5: Chromosomal localization of the human ERM gene by fluorescence in situ hybridization. A, twin-spot signals specific for ERM (arrows) were detected on both sister chromatids of the long arm of chromosome 3. B, G-band patterns of the same chromosomes were delineated through a UV filter, indicating that the human ERM gene is located on 3q27.
ERM together with PEA3(13) , ER81(14) , and
EIA-F, the human homolog of PEA3(41) , comprises a subgroup in
the ets gene family based on their sequence similarity
throughout the entire molecules. In contrast to PEA3, which is
expressed in a tissue-restricted manner, most abundant in the mouse
epididymis and brain, and in cell lines of fibroblast and epithelial
cell origin(13) , both ERM and ER81 mRNA are expressed in a
wide variety of tissues and cell lines(14, 39) .
The members of the PEA3 subfamily have an acidic region in
their amino termini, which has been suggested as a transcription
activation domain (13, 24) . We showed that using a
Gal4 system, the amino-terminal portion of ERM did indeed have
transcription activity (Fig. 4). Interestingly, in the same
assay system, we showed that the region from amino acids 166-326
of ERM might contain a negative regulatory domain. This notion was
supported by our result showing that the Gal4 fusion protein containing
the ERM region (amino acids 166-326) exerted a repressor activity
on the herpes simplex virus thymidine kinase promoter in the assay
systems using a reporter plasmid that contained five copies of
Gal4-binding sites upstream of the thymidine kinase promoter. Since the GAL4-ER81 was not activated by c-Jun and the
proline-rich region (amino acids 146-195), not present in ER81,
was located in the region of ERM essential for the activation of ERM by
c-Jun (Fig. 4), it was possible that the proline-rich region was
one of the targets for the action of c-Jun.
Two major cis-acting motifs, 12-O-tetradecanoylphorbol-13-acetate-responsive element (AP1 site) (53, 54) and cAMP-responsive element (55, 56) have been shown to play key roles in the signal transduction processes that lead to cell growth or differentiation. In some cases, transcription factors acting on the AP1 site or CRE site cooperate with other transcription factors to achieve efficient gene activation(27, 57, 58) . Conversely, for optimal activity, Ets proteins seem to require another transcription factor binding to an adjacent site, including AP1(3, 4, 59) . Recent studies have indicated that cooperative functions between specific Ets and AP1 binding proteins are of significant importance in regulating inducible gene expression. For instance, Wasylyk et al.(1) showed that Ets-1 and Ets-2 functionally cooperated with AP1 composed of c-Fos and c-Jun in transcriptional activation from the PEA3-AP1 motif of the polyoma virus enhancer. More recently, Wang et al.(60) showed that in activated T cells, Elf-1, c-Fos, and JunB formed a complex binding to the PB1 motif (purine box 1 in the granulocyte macrophage colony-stimulating factor gene promoter), which is composed of an EBS and an adjacent AP1-binding site, both of which are required for inducible expression of the granulocyte macrophage colony-stimulating factor gene. Our data showed another case of synergy between an Ets family protein and c-Jun.
To elucidate the mechanisms
for the synergy between ERM and c-Jun, we examined two possibilities:
(i) cooperative binding of ERM and c-Jun to the response element and
(ii) enhancement of one's transcriptional activity by the other.
The first possibility was unlikely, since we did not detect cooperative
binding of ERM and c-Jun to the EBS-CRE even in the presence of CRE-BP1
(data not shown). For the second possibility, we have presented
evidence showing that c-Jun enhances the transcription activity of ERM
in an assay system using Gal4 fusion proteins. This enhancement
required both the amino-terminal transactivation domain and the portion
containing a negative regulatory domain of ERM. In the in vitro protein-protein interaction assays using S-labeled
c-Jun and various glutathione S-transferase-ERM fusion
proteins, we detected substantial direct interaction between c-Jun and
ERM through the carboxyl-terminal portion of ERM containing the Ets
domain, although we failed to detect a direct interaction of the
amino-terminal portion of ERM, including a negative regulatory domain,
and c-Jun.
Since the amino-terminal but not the
carboxyl-terminal portion of ERM was required for the activation of ERM
by c-Jun, a direct interaction between c-Jun and ERM may not occur. It
is conceivable that c-Jun may neutralize the squelching effect of the
negative regulatory domain of ERM on the transactivation domain. We
currently do not know the significance of the direct interaction of
c-Jun and the ERM carboxyl-terminal portion.
Recent studies have
indicated that phosphorylation of transcriptional activators is
important in either translocation of factors to the nuclei, binding to
the relevant sites in the control regions, or interaction with other
transcription factors or
coactivators(57, 61, 62, 63) . In
the ets gene family, Elk-1, acting on the c-fos serum
response element in concert with serum response factor as a ternary
complex, has been shown to be transcriptionally activated through
phosphorylation by a mitogen-activated protein (MAP)
kinase(31, 64) . PU.1 recruits the binding of a second
B cell-restricted nuclear factor, NF-EM5, to a DNA site in the
immunoglobulin 3` enhancer(32) . Recently,
phosphorylation of PU.1 at Ser
was shown to be necessary
for interaction with NF-EM5(65) . Since ERM cooperates with
c-Jun, a well-known signal-transducing transcription factor, such a
signal-transducing property might be expected for ERM. In relation to
this matter, ERM has three putative mitogen-activated protein kinase
phosphorylation sites (PX(S/T)P), at Thr
,
Thr
, and Ser
. The effects of
phosphorylation of ERM and c-Jun on their synergistic action should be
analyzed to establish the precise role of ERM in signal transduction.
It is of interest that combinations of adjacent Ets and CRE or
related motifs are found in several gene promoters, including junB(37) , Rb(66) , CD8(67) , and TCR
(28) .
The significance of the synergy between ERM and c-Jun in
transcriptional activation of such cellular genes, actual target genes,
remains to be established. Other functional aspects of ERM, such as
oncogenicity exerted in concert with c-Jun and the roles of
ubiquitously expressed ERM in development will be the focus of our
future work. Information on the location of the ERM gene at 3q27 will
be useful in searching for naturally occurring tumors caused by the
abnormal expression or activity of ERM.