From the Department of Animal Biology and Mari Lowe
Center for Comparative Oncology, School of Veterinary Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania 19104, ¶ Science Applications International Corporation, Frederick Cancer
Research and Development Center, Frederick, Maryland 21702, and the
Program in Cancer Genetics, Cancer Center, Medical College of
Pennsylvania and Hahnemann University,
Philadelphia, Pennsylvania 19102
Received for publication, January 24, 2001, and in revised form, February 20, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We demonstrate that dexamethasone-mediated
transcription activation of the cytochrome P-450c27 promoter involves a
physical interaction and functional synergy between glucocorticoid
receptor (GR) and Ets2 factor. Ets2 protein binding to a "weak"
Ets-like site of the promoter is dependent on GR bound to the adjacent cryptic glucocorticoid response element. Coimmunoprecipitation and
chemical cross-linking experiments show physical interaction between GR
and Ets2 proteins. Mutational analyses show synergistic effects of Ets2
and GR in dexamethasone-mediated activation of the cytochrome P-450c27
promoter. The DNA-binding domain of GR, lacking the transcription
activation and ligand-binding domains, was fully active in synergistic
activation of the promoter with intact Ets2. The DNA-binding domain of
Ets2 lacking the transcription activation domain showed a dominant
negative effect on the transcription activity. Finally, a fusion
protein consisting of the GR DNA-binding domain and the transcription
activation domain of Ets2 fully supported the transcription activity,
suggesting a novel synergy between the two proteins, which does not
require the transactivation domain of GR. Our results also provide new
insights on the role of putative weak consensus Ets sites in
transcription activation, possibly through synergistic interaction with
other gene-specific transcription activators.
Cytochrome P-450c27
(CYP27)1 is a multifunctional
enzyme with major activities for the c27 hydroxylation of cholesterol
and c25 hydroxylation of vitamin D3 (1). Cholesterol c27
hydroxylation in the liver mitochondria is a rate-limiting step in the
acidic bile acid pathway and also in the feedback regulation of
cholesterol biosynthesis (2-4). Vitamin D3 25 hydroxylation in liver mitochondria, on the other hand, represents an
important first step toward the conversion of inactive vitamin into the
active hormonal form. Thus, the enzyme plays important roles in
different pathways of cholesterol homeostasis, bile acid metabolism,
and Ca2+ homeostasis (1, 2, 4). In the rat, the CYP27 gene
is expressed as a major species of 2-kb mRNA and a minor species of
5'-extended 2.3-kb mRNA (5). The physiological function of the
latter species remains unclear. Previous studies from our and other
laboratories (5-7) showed that CYP27 gene expression, particularly the
transcription of the 2.0-kb mRNA, is under the control of pituitary
growth hormone, glucocorticoids, and also bile
acid-dependent diurnal regulation (8). We have
characterized the rat CYP27 gene structure and found that the
transcription initiation site and the basal promoter elements required
for transcription of the major 2-kb mRNA are localized within the
5' end of the second exon (6). In the present study, we have
investigated mechanisms of glucocorticoid-mediated transcription
activation of the 2-kb mRNA and found that the essential elements
needed for hormone-mediated activation are localized in the
5'-proximal, putative intron region, upstream of the 2-kb mRNA
start site. Throughout this study, this downstream promoter will be
referred to as the CYP27 promoter.
GR is a steroid receptor family zinc finger protein that activates
transcription of many liver-specific genes (9). Binding of Dx, a
synthetic glucocorticoid hormone, and related ligands to GR induces
structural changes in the receptor resulting in the transcription
activation of target genes (9). Variant forms of GREs with different
affinities for GR binding and hence various activation potentials have
been reported (10, 11). In most cases, the 15-nucleotide-long GRE
consists of two half-sites, with consensus sequence
(T/G)GTACAXXXTGTTCT (9). One molecule of dimeric GR
binds to each of the half-sites, TGTACA and TGTTCT, in a cooperative
manner (12, 13). A duplicated GRE motif is a more potent transcription
activator when compared with a single GRE motif (14, 15).
GR has been reported to regulate various signaling events by
interacting with other transcription factors, such as members of the
Ap-1 family, Jun and Fos (16-19), RelA protein of the NF Some studies suggest that transcription activation by GR is also
subject to regulation by Ets family transcription factors. Transient
transfection studies showed that PU.1 and GR reciprocally modulate the
activity of each factor, resulting in transcriptional repression (27,
28). The precise mechanisms of repression, however, remain unclear.
Another study showed that mutations targeted to the Ets1-binding site,
overlapping the GR binding motif of the tyrosine aminotransferase
promoter, resulted in a 2-fold reduction in transcription activity,
suggesting synergistic effects of the two factors (29). However, the
precise mode of physical interaction between the factors was not
investigated. In the present study we show that Dx-mediated activation
of CYP27 gene expression involves GR binding to a variant GRE site and
its synergistic interaction with factors binding to an adjacent
"weak" consensus Ets site, referred to in this study as an Ets-like
site. Interestingly, the activation appears to require a novel
functional and physical interaction between the DNA-binding domain of
GR and the transcription activation domain of the ubiquitously
expressed Ets2 transcription factor.
Plasmid Construction--
The CYP27 promoter constructs
were generated by PCR amplification of the
The rat GR expression cDNA (p6RGR) encoding the full-length
receptor protein and the truncated rat GR DNA-binding domain (GRDBD, amino acids 407-556 of the GR protein) expression plasmid (p6R-X556) were kindly provided by Dr. K. Yamamoto (31). Expression plasmid PSV2Ets2, encoding the full-length Ets2
protein, and a dominant negative mutant
PSV2tEts2, expressing only the DNA-binding
domain of Ets2 were described before
(32).2 CMVEts1
encoding an intact murine Ets1 was obtained from Dr. Michael Atchison.
A cDNA encoding the DNA-binding region of Ets1 (tEts1, amino acids
260-440 of murine Ets1) was generated by PCR amplification and cloned
in the pCMV vector. A chimeric construct of the GRDBD and the
transcription activation domain of Ets2 was generated by cloning the
GRDBD cDNA (NdeI and SalI fragment,
nucleotides 1206-1656 of 6GR cDNA) into similarly cut pGEM5z
plasmid DNA. cDNA corresponding to the transcription activation
domain of murine Ets2 (amino acid residues 40-280) was amplified
using the sense primer containing a SmaI site and antisense
primer containing a SalI site and cloned into similarly
digested 5z GRDBD plasmid DNA to produce a contiguous open reading
frame. The resulting cDNA expressed a 26-kDa fusion protein
containing a DNA-binding domain of GR with a C-terminally fused (in
frame) transcription activation domain of Ets2. Finally, the chimeric
cDNA construct was cloned into the mammalian expression vector
PCMV4 and was designated as pGRDBD-Ets2. This clone was
used to generate deletion constructs pGRDBD Cell Culture and Transfection--
Mouse Balb/c 3T3 fibroblast
cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 50 mM glutamine,
and 50 µg/ml gentamicin at 37 °C. Murine NIH 3T3 fibroblast
cells were maintained as described before (6). Cells were transfected
in replicate plates with CsCl2 gradient-purified CYP27
promoter plasmid DNA by the calcium phosphate coprecipitation method or
in some cases using the Fugene 6 reagent (Roche Molecular Biochemicals). The latter procedure routinely yielded transcription efficiency of >60%. Cells (100-mm plates) were transfected with 5 µg of test plasmid DNAs and 1.5 µg of CMV Preparation of Nuclear Extracts and Proteins--
Nuclear
extract from NIH 3T3 cells were prepared by the method of Dignam
et al. (34). Bacterially expressed purified GRDBD protein
(apparent molecular mass of 17.0 kDa) was a generous gift from Dr. K. Yamamoto (35). Ets2 protein was overexpressed in BL21 cells
by isopropyl-1-thio- Northern Hybridization--
RNA from transfected and
mock-transfected Balb/c 3T3 cells was prepared by the guanidinium
thiocyanate method (36). RNA (30 µg each) was resolved by
electrophoresis through a formaldehyde-containing gel, transblotted to
Nytran membranes, and hybridized with the 32P-labeled rat
CYP27 cDNA or cytochrome c oxidase subunit IV cDNA probes under moderate stringency conditions. The blots were quantified using a Bio-Rad GS 525 Molecular Imager system.
Gel Mobility Shift Assays--
DNA-protein binding was
assayed by gel mobility shift EMSA as described (37). Binding was
carried out with a 32P end-labeled 135-bp DNA fragment from
the glucocorticoid hormone-responsive region of the promoter (amino
acids Immunoprecipitation and Immunoblotting--
Varied amounts of
bacterially expressed GRDBD protein (35) and Ets2 protein were
incubated in phosphate-buffered saline (20 mM
KH2PO4, pH 7.0, 150 mM NaCl) for 30 min at room temperature to facilitate protein-protein interaction and
immunoprecipitated with Ets2 antibody (Santa Cruz Biotechnology, Santa
Cruz, CA) by the protein A-agarose (Sigma) binding method (39). In some experiments nuclear extracts from cells transfected with various cDNAs were used for coimmunoprecipitation by the same procedure. The antibody-antigen complexes were dissociated at 56 °C for 1 h in nonreducing Laemmli's sample buffer (42), and proteins were
resolved on 10% SDS-polyacrylamide gel and transblotted to Nytran
membrane. The blot was successively probed with monoclonal antibody to
GR (BUGR2) and polyclonal antibody to Ets2 by using the Pierce
Super-glo chemiluminescence kit (Pierce) and visualized with a Bio-Rad
FluorS imager. The Ets2 antibody used in this study also cross-reacted
with Ets1 protein.
In Vitro Translation and Chemical Cross-linking--
Various
cDNAs cloned in pGEM 7zf vectors (Promega) were used as templates
for generating 35S-labeled in vitro translation
products in the TNT reticulocyte translation system
(Promega) according to the manufacturer's protocol. Adx cDNA was
cloned in pCRTM II vector as described elsewhere (40).
35S-Labeled translation products, 20,000 cpm in the case of
GRDBD and Adx proteins or 50,000 cpm in the case of Ets1 and Ets2
proteins, were used for protein cross-linking. Cross-linking was
carried out in 50 µl of reaction mix containing 10 mM
HEPES, pH 8.0, 1 mM EDTA, 50 mM NaCl, 0.5%
Nonidet P-40, 10% glycerol, and 35S-labeled proteins. The
mixture was preincubated for 2 h at 4 °C to facilitate
protein-protein interaction, followed by the addition of cross-linking
agents dithiobis[succinimidylpropionate] (DSP) or
m-maleimidobenzyl-N-hydroxysulfosuccinimide ester
(sulfo-MBS), both from Pierce, to a final concentration of 1 mM. Cross-linking was carried out at room temperature for
30 min, and the reaction was quenched by adding Tris-HCl, pH 7.5, to a
final of 20 mM (in the case of DSP) or 100 mM
Gly containing 5 mM Localization of Dx Response Region of the Rat CYP27
Promoter--
It is known that Dx induces the rat hepatic CYP27
mRNA, specifically the shorter 2-kb species, by 15-20-fold of
control animals (7). In the present study, the putative hormone
response region of the CYP27 promoter was mapped by progressive 5'
deletion of the Protein Binding Property of the Dx Response Region of the
Promoter--
The nature of proteins binding to the Dx response region
was studied by EMSA using the 135-bp (sequence
To further characterize the protein in complex A that was competed by
excess Ets DNA (Fig. 2A), we tested the binding of
bacterially expressed, purified Ets2 protein to the 135-bp DNA probe
both in the presence and absence of added GRDBD protein. EMSA in Fig. 2C shows that Ets2 protein by itself did not bind to the DNA
probe, whereas a combination of GRDBD and Ets2 proteins resulted in the formation of a slow migrating complex (complex a), in addition to the
GRDBD-specific complex b. Interestingly, addition of 20-fold molar
excess of Ets DNA effectively abolished complex a formation without affecting complex b. These results suggest that Ets2 protein binding to the Ets-like site of the DNA probe is conditional, depending
on the presence of GRDBD. This possibility was further investigated by
testing the effects of variable concentrations of GRDBD on the extent
of Ets2 protein binding using a more restricted DNA probe containing
only the cryptic GRE and the downstream Ets-like sites. The gel shift
in Fig. 2D shows that addition of GRDBD protein alone
results in the formation of complex b, whose band intensity increases
with increasing levels of the protein from 0.2 to 0.8 mg. Formation of
complex a was observed only in the presence of added Ets2 and GRDBD and
the extent of its formation was directly proportional to the amount of
added GRDBD. Furthermore, 20-fold molar excess of cryptic CYP27
GRE DNA inhibited both complexes, whereas the cryptic CYP27 Ets2 DNA
selectively inhibited complex a. Notably, mutations targeted to the
core sequences of both GRE-like and Ets-like motifs drastically reduced
their ability to compete with either of the complexes. These results
further support the possibility that nucleation of the GRE site by
GRDBD (complex b) is essential for Ets2 protein binding and that the
slow migrating complex a represents a higher order complex containing
the Ets2 protein. Results also show that both GRE-like and
Ets-like motifs of the promoter are needed for the formation of the
higher order complex a.
Functional Analysis of GR and Ets Sites in Dx-mediated
Transcription Stimulation--
The functional importance of the
cryptic GR, Ets, and other potential protein binding motifs was tested
by mutational analysis. Mutations targeted to different protein binding
motifs are shown at the bottom of Fig.
3. The promoter activity of wild type and mutant constructs in the presence or absence of added Dx and
coexpression with full-length GR construct (p6RGR) were tested in 3T3
cells. As shown in Fig. 3, the transcription activity of the wild type
In companion experiments, we also investigated the requirements for
specific domains of the GR and Ets2 proteins and possible synergistic
activation by tissue-specific Ets1 protein. The nature of the
full-length GR construct expressing 6RGR and the GRDBD cDNA
construct are shown at the top of Fig.
4. Results of CAT activity in transfected
3T3 cells (Fig. 4A) show that using the
Results in Fig. 4B show that coexpression with the GRDBD
cDNA yielded a 3.5-fold higher transactivation of the Dominant Negative Effects of tEts2 on the Expression of Endogenous
CYP27 mRNA--
We investigated the effects of Dx and
overexpression of GR on the CYP27 mRNA levels in Balb/c 3T3 cells.
These cells are known to respond to glucocorticoid hormone and also
express low, albeit measurable levels of GR protein (41). As shown in
the Northern blot in Fig. 5A, addition of 100 nM
Dx alone resulted in a 5-7-fold induction of 2-kb CYP27 mRNA under
in vitro culture conditions. Similarly, transfection with 1 and 2 µg of full-length GR cDNA (p6RGR) without added Dx resulted
in 5-6- and 15-fold induction, respectively. Addition of 100 nM Dx to cells transfected with 2 µg of GR cDNA
caused a further increase in mRNA level to about 23-fold of
control. The 2.3-kb mRNA is expressed at very low levels in these
cultured cells, and the bands were visible only when the blots are
overexposed (results not shown). These results demonstrate that the
endogenous mouse CYP27 gene is under the regulation of GR and
glucocorticoid hormones. The Northern blot in Fig. 5B shows that cotransfection with tEts2 cDNA reduced the endogenous mRNA levels by about 60-70% of the control cell level. Furthermore, coexpression with tEts2 cDNA also nearly completely abolished the
GR2 + Dx-mediated induction of CYP27 mRNA levels from near 25-fold
to a mere 1.8-fold of control (Fig. 5B, last
lane). The level of ubiquitously expressed cytochrome oxidase
subunit IV mRNA (Fig. 5B, COX IV mRNA)
level was affected only marginally (less than 20%) by transfection
with various cDNAs. These results suggest a physiological role for
the transcription activation domain of Ets2 factor in the
glucocorticoid hormone responsiveness of the promoter.
Physical Interaction of GR and Ets2 Proteins--
Physical
interaction between Ets family proteins and GRDBD under in
vitro and in vivo conditions was studied by
coimmunoprecipitation and chemical cross-linking. As shown in Fig.
6, indicated amounts of bacterially
expressed and purified GRDBD and Ets2 proteins were preincubated and
immunoprecipitated either with preimmune IgG or Ets2 antibody. The
immunoprecipitates were successively probed with polyclonal antibody
against Ets2 (Fig. 6A, upper panel) and
monoclonal antibody to GR proteins (Fig. 6A, lower
panel) by immunoblot analysis. Results show that preimmune sera
failed to immunoprecipitate either of the proteins from the incubation mixture. Antibody to Ets2, on the other hand, coprecipitated the 17-kDa
GRDBD (Fig. 6A, lower panel). A 2-fold increase
of GRDBD in the incubation mixture resulted in a correspondingly
increased amount of GRDBD coimmunoprecipitation by Ets2 antibody.
Conversely, a 2-fold increase of Ets2 protein in the incubation mixture
resulted in the increased level of Ets2 protein accompanied by a
marginal increase in the level of GRDBD in the immunoprecipitate. Thus, the coimmunoprecipitation is specific and varies depending on the input
levels of GRDBD as well as Ets2 proteins. Although not shown, Ets1
protein also physically interacted with GRDBD as seen by antibody
pull-down experiments.
The in vivo interaction between these proteins was studied
by coimmunoprecipitation using nuclear extracts from cells transfected with various combinations of cDNA constructs. Fig. 6B
shows that GRDBD failed to interact with Ets2 transcription activation
domain (amino acids 40-280 of murine Ets2, TA-Ets2) because antibody to Ets2 immunoprecipitated only the TA-Ets2 and GR antibody
immunoprecipitated only the 17-kDa GRDBD. The latter protein, however,
efficiently interacted with wild type Ets2 or the DNA-binding domain of
Ets2 because both pairs of proteins were immunoprecipitated by GR
antibody. These results show that GRDBD interacts with Ets2 protein
under in vivo conditions and that the interaction requires
the DNA-binding domain of Ets2 protein. The results also indicate that
transiently expressed truncated GRDBD, tEts2, and TA-Ets2 proteins are
translocated into the nuclear compartment.
In the second approach, we used chemical cross-linking to test the
physical interaction between GRDBD and Ets2 or Ets1 proteins, using DSP
or sulfo-MBS, which show a high preference for positively charged and
sulfhydryl-containing side chains. Furthermore, DSP is sensitive
to treatment with reducing agents, such as BME, whereas sulfo-MBS is
not. 35S-Labeled Ets2 or Ets1 proteins were incubated with
35S-labeled GRDBD in the presence or absence of DSP, and
the products were immunoprecipitated with antibodies to either Ets2 or
GR and resolved on polyacrylamide gels. In some experiments human
adrenodoxin (Adx, see Ref. 40 for details) was used along with either
Ets1, Ets2, or GRDBD proteins as negative controls. In this case
immunoprecipitation was carried out with antibodies to either Adx,
Ets2, or GR. Results in Fig.
7A, using DSP as the
cross-linking agent show that rabbit or mouse preimmune sera did not
immunoprecipitate either Ets1 or GRDBD proteins. Similarly, Adx and
Ets1 pairs failed to produce any cross-linked products as tested by
immunoprecipitation with Adx antibody as well as Ets2 antibody.
Immunoprecipitation of the reaction mixture containing GRDBD and Ets2
proteins without added cross-linker, with GR antibody, yielded only the
17-kDa GRDBD protein, and that with Ets2 antibody yielded only the
50-kDa Ets1 protein. Immunoprecipitation of reaction mixtures with DSP, in the absence of added BME, on the other hand yielded an ~67-kDa cross-linked product with both of the antibodies. It is noted that both
GRDBD and Ets1 translation products yielded double bands, possibly
because of translation initiation from alternate ATG codons. The 67-kDa
species detected in the immunoprecipitate (Fig. 7A,
last lane) indeed represents the cross-linked product
because use of Laemmli's sample buffer (42) containing 5 mM BME dissociated the product into 50-kDa Ets1 and 17-kDa
GRDBD proteins. Fig. 7B shows that reactions with
35S-labeled Ets2 protein also yielded a 69-kDa cross-linked
product, excepting that in the case of GR antibody, an additional
species of about 48 kDa was also immunoprecipitated. This latter
species might represent an unknown protein from the reticulocyte lysate cross-linked with GRDBD. These results suggest that both Ets1 and Ets2
can physically interact with GRDBD even when they are not bound to DNA.
Although not shown, sulfo-MBS also yielded similar cross-linked
products, except that they were resistant to treatment with BME.
Functional Interaction between Ets2 and GR and Possible Sharing of
Domains--
To test the roles of the DNA-binding domain of GR and
transcription activation domain of Ets2 in the synergistic activation, we constructed fusion proteins consisting of the wild type or mutated
GRDBD with C-terminally fused transcription activation domains from
Ets2 or Ets1. These proteins were tested for transcription activation
of wild type and various mutant promoter constructs. As shown in Fig.
8, the GRDBD-Ets2 fusion construct was
able to stimulate the activity of the wild type Expression and Nuclear Localization of Proteins in Transfected
Cells--
Fig. 9 shows immunoblot
analyses of whole cell extracts and nuclear extracts from 3T3 cells
transfected with various intact (wild type), deletion, and fusion
cDNA constructs. Full-length Ets1, Ets2, GR, truncated GRDBD, and
also GRDBD-Ets1/Ets2 fusion proteins are not only expressed efficiently
under the present transfection conditions but also are localized to the
nuclear compartment. It is also seen that GRDBD-Ets1 and also
GRDBD-Ets2 fusion proteins migrated with an apparent molecular mass of
26 kDa instead of the expected 39-41 kDa. This anomalous migration may
be due to the high Pro contents (44, 45) of the transcription activation domains of both Ets1 and Ets2 proteins. Although not shown,
GRDBD Transcription regulation of mRNA coding genes in mammalian
cells requires a complex cooperative interaction of gene-specific transcription activators and coactivators with the basal transcription machinery, which includes the TFIID complex and polymerase II holoenzyme complex (46, 47). In this paper we demonstrate that
Dx-mediated transcription activation of the rat CYP27 gene involves a
cooperative interaction between GR and Ets2 factors. Functional
synergies between gene/tissue-specific and ubiquitous housekeeping
varieties of sequence-specific DNA-binding transcription factors can
be classified into the following categories. The first type involves
ligand or physiological pathway-specific transcription factors and
ubiquitous constitutive factors binding to the same sequence motif as
heteromeric complexes. Examples of heteromeric factors include myogenic
factors MyoD and E2A (38), proteins belonging to the Ap1/Ap2/CAAT
family (Jun/Fos, NFATc and CEBP) (48-50), and ligand-specific factors
belonging to the steroid superfamily nuclear receptors (51). The second
type involves cooperativity between the same class or homologous
factors bound to tandemly duplicated DNA-binding sites leading to
functional synergy. Transcription activation of promoter sites with
duplicated binding sites by GR (51, 52) and Ets family GABP
factors (53, 54) belong to this class. The third type involves synergy
between transcription factors bound to spatially separated DNA sites
possibly through interaction with common coactivator proteins.
Synergistic activation of the low density lipoprotein receptor promoter
by sterol response element-binding protein (SREBP) and the ubiquitous
factor Sp1 through interaction with coactivator cAMP-response
element-binding protein-binding protein is a classical example
of this type (52, 55). The fourth type of functional synergy is seen in
the recruitment of factor Pu.1 interacting protein by DNA-bound
PU.1 or E2A in the activation of immunoglobulin Physical and functional analyses of the Dx-responsive region of the
promoter by site-directed mutations (Figs. 1 and 3) suggest that the
cryptic GRE motif and a downstream Ets-like motif are important for
Dx-mediated transcription activation of the promoter. Cotransfection
with GR and Ets2 cDNAs resulting in synergistic activation of CAT
activity further substantiated the role of these cis-DNA
elements (Figs. 1, 3, and 4). In support of these functional data, the
cryptic GRE motif (TGCTGT) bound to proteins from nuclear extracts,
which cross-reacted with GR-specific antibody as seen by a supershift
in EMSA (Fig. 2A). Additionally, bacterially expressed purified GRDBD also bound to this motif (Fig. 2B). However,
the Ets-like motif by itself failed to form a detectable complex with both purified Ets2 protein (Fig. 2C) and 3T3 nuclear extract
(not shown). These results are in full support of general consensus in
the field that the core sequences (G/T)GGAA(T/A) are weak motifs for
binding to ubiquitously expressed Ets2 and GABP (variably called NRF2)
as well as tissue-specific Ets1 and other members of Ets family
proteins (58-60). EMSA with the nuclear extract and 135-bp hormone
response region DNA probe also yielded a slow migrating complex, which
was competed by both cryptic GRE and Ets consensus DNA (Fig. 2,
A and C), suggesting that GR binding to the
cryptic GRE site helps recruiting an Ets family protein. Use of
bacterially expressed purified proteins and competition with
site-specific DNA indeed supported this possibility (Fig.
2C). Direct evidence for the functional role of Ets2 factor
and the physiological significance of its in vitro
functional synergy with GR is presented by experiments showing the
dominant negative effects of tEts2 (only the DNA-binding domain of
Ets2) on the activity of the promoter construct and also the endogenous
CYP27 gene expression in 3T3 fibroblasts.
Ets2 factor binding to the hormone response region of the promoter is
dependent on GR or GRDBD bound to the cryptic GRE motif as well as the
downstream Ets-like sequence motif, the latter thought to be a weak
consensus sequence for binding to Ets family proteins (58, 59). This
conclusion is based on EMSA results showing that slow migrating complex
a obtained with purified GRDBD and Ets2 proteins (Fig. 2, C
and D) was effectively competed by Ets-like DNA but not by a
mutant form. A DNA probe carrying mutations at the Ets-like sequence of
the GHR region probe formed only complex b but not the higher order
complex a even in the presence of added GRDBD and Ets2 proteins
(results not shown). Similarly, the intact Ets-like sequence motif is
required for the synergistic activation of the promoter by
cotransfection with GR (or GRDBD) and Ets2 cDNAs (Fig. 3). Results
of cotransfection also show that only the Ets2 protein with an intact
DNA-binding domain, but not the deletion mutant lacking this domain, is
able to induce transcription activation (results not presented). These
results suggest the need for DNA binding by Ets2 protein as an
essential part of the functional synergy with the adjacently bound GR.
Thus, a novel finding of this work relates to the role of the putative
weak Ets consensus site in the transcription activation. The putative weak Ets consensus sites are widely distributed on many promoters. In
view of our results, the roles of the ubiquitously expressed Ets2
protein and the weak consensus Ets-like sites, particularly those
located in the close proximity of other sequence-specific factor
binding sites, need to be further evaluated.
It is known that Ets1 and Ets2 factors can interact with members of
Jun/Fos and other AP-1 family proteins (16-19, 48) and also other
leucine zipper proteins such as Myb (15, 32), modulating their
transcription activity. Results of coimmunoprecipitation (Fig. 6) and
chemical cross-linking experiments (Fig. 7) reported in this study for
the first time demonstrate a direct physical interaction between GRDBD
and Ets2 as well as Ets1 proteins. Furthermore, the interaction
requires the DNA-binding domains of both proteins because the
transcription activation domain of Ets2, lacking the DNA-binding
domain, showed no detectable interaction with the GRDBD protein.
Thus, it is likely that the two factors interact physically in their
DNA-bound form on the hormone response region of the CYP27 promoter. It
is known that GRDBD, which lacks the ligand-binding domain, has a
constitutively activated DNA binding activity (61). It was, however,
surprising that GRDBD lacking both the N- and C-terminal activation
domains (62, 63) and the ligand-binding domain was able to
synergistically activate the promoter with Ets2 (Fig. 4B).
The possible requirement for physical interaction between GRDBD and
Ets2 for functional synergy was further supported in experiments
showing that a fusion protein containing GRDBD and the Ets2
transcription activation domain could efficiently activate the
promoter. Interestingly, mutation of the weak Ets-like site did not
affect the fusion protein-induced activity as long as the cryptic GRE
site was intact. Notably, the GRDBD-Ets2 fusion construct with mutated
zinc finger domains of GRDBDB showed vastly reduced transcription
activation of the promoter. Based on these results, we hypothesize that
the role of the GR or GRDBD bound to the cryptic GRE of the promoter is to help recruit the Ets2 protein to the complex. Interaction with GR
may induce conformational change(s) in the Ets protein (64) such that
it can bind to the weak Ets consensus site. Apparently, binding of Ets2
factor to the weak Ets site alone in the absence of adjacently bound GR
is not strong enough for resolution of the complex through the gel
matrix in EMSA. Thus, the requirement for physical interaction between
GR and Ets2 for functional synergy is uniquely different from the
synergistic activation by SREBP and Sp1, which does not involve
intermolecular protein-protein interaction (55). The requirement for
the transcription activation domain of only Ets2 but not that of GR for
synergistic interaction is reminiscent of the synergy between Pu.1 and
Pu.1 interacting protein, Fos, and Jun for the activation of
immunoglobulin A current model on the mechanism of synergistic activation by SREBP and
Sp1 suggests that the transactivation domains of both of the proteins
are essential for transactivation (50). It is proposed that the
transcription activation domain of Sp1 may be involved in interaction
with the basal transcription machinery, whereas that of SREBP might be
involved in recruiting factors with HAT activity (50). The synergy
observed with GR and Ets2 factors, on the other hand, appears to be
highly dependent on the transcription activation domain of the latter,
without requiring the presence of the former. It should be noted that
tissue-specific Ets1 factor also binds to the promoter DNA in a
GRDBD-dependent manner (results not shown) and physically
interacts with GRDBD as tested by coimmunoprecipitation and chemical
cross-linking (Figs. 7 and 8). However, cotransfection with Ets1 and GR
or GRDBD cDNAs failed to show any synergistic activation of the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B pathway
(20-22), liver-specific factor HNF3 (23, 24), and Stat-5 (25). GR also
modulates transcription activity through receptor-receptor interaction between proteins bound to tandem GRE sites (15, 26).
Recently Zhang et al. (22) demonstrated a complex
pattern of interaction between interleukin-6 response element-bound
protein and GR bound to an adjacent GRE site. This interaction was
suggested to help synergize transcriptional effects of the
interleukin-6 response element and GRE sites through Stat-3, which acts
as a potent coactivator of GRE-mediated transcription.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
841/+23mCAT DNA from the
rat CYP27 gene described before (6) and cloned into the promoterless
and enhancerless basic CAT plasmid pCATbasic (Promega Corp., Madison,
WI) in the HindIII and SalI sites. The
329/+23mCAT was generated by digesting the
841/+23mCAT DNA with
PstI (at position
329) and SalI (at position
+23), and the fragment was cloned into the same sites of pCATbasic DNA.
The 5' progressive deletions
194/+23mCAT,
75/+23mCAT, and
45/+23mCAT were derived by digesting the SalI-digested
841/+23mCAT DNA with SacI (
194), XhoI (
76),
or SmaI (
45) enzymes, respectively. All the fragments were
gel purified, blunt ended with T4 DNA polymerase, and recloned into
pCATbasic plasmid at blunt ended SphI and SalI sites. Mutations at the cryptic GRE, Ets-like, Ap-1-like sites within
the
329 to
194 promoter sequence were introduced by overlapping PCR
using primers containing appropriate substitutions and further amplified using sense primer containing a HindIII site and
antisense primer containing a SacI site for cloning. The
mutant DNA fragments were cloned in the HindIII and
SacI sites of pCATbasic vector. The nucleotide sequence of
all the constructs was verified by the dideoxy chain termination
sequencing (30).
ZF-Ets2 (lacking amino
acids 455-485 of GRDBD) and pGRDBD-Ets2
200 (lacking the N-terminal
200 residues of Ets2) by standard or overlap PCR. A cDNA encoding
GRDBD and the N-terminal 40-280 amino acid residues of murine Ets1
fusion protein was generated using a similar approach.
-gal expression
cDNA, pCH110 (33). For cotransfection experiments, 5 µg of the
reporter plasmid was cotransfected with either 2 µg of rat GR
cDNA, 0.8 µg of CMVEts1/Ets2 cDNAs,
1.5-3.0 µg of GRDBD-Ets2, GRDBD
ZF-Ets2, GRDBD-Ets2
200, and
GRDBD-Ets1 chimeric cDNA constructs, or 1 µg of GRDBD cDNA
construct. The total DNA in each transfection was normalized to 15 µg
with CMV DNA as the filler. In some experiments the medium was changed
with that containing 2% fetal bovine serum after 40 h of
transfection, and cells were exposed to 100 nM Dx (Sigma)
for 8-10 h before harvesting. Control cells not subjected to Dx
induction were harvested after 48-50 h of transfection. Cell extracts
were prepared by standard procedure and used for assaying CAT activity
(33) or by the colorimetric method using a CAT enzyme-linked
immunosorbent assay kit from Roche Molecular Biochemicals. The
-galactosidase activity of each of the extracts was used as an
internal control for normalizing the transfection efficiency (33).
-D-galactopyranoside induction using
the pET vector (Novagen, Inc., Madison, WI) and purified to near
homogeneity by affinity binding to the nickel-agarose matrix (Qiagen
Inc., Velenica, CA).
329 to
194), which was generated by digesting pCAT-329/+23
DNA with PstI and SacI restriction enzymes. In
some experiments, a shorter 65-bp DNA (amino acids
233 to
298)
generated by PCR was used as a probe. Typically, binding reactions were
run in 20-µl volumes and contained ~0.1-0.2 ng of labeled DNA
(10,000-15,000 cpm), 5-8 µg of nuclear extract, and 1 µg of
poly(dI-dC) under conditions described previously (37). Binding
reactions with the bacterially expressed purified GR derivative X556
(GRDBD protein) were performed in a similar way except that 100-800 ng
of protein, diluted in a zinc-containing buffer (10 mM
Tris, pH 7.6, 40 mM NaCl, 15 µM
ZnCl2), was used (38). DNA-protein complexes were resolved
on 5% nondenaturing polyacrylamide gels in 0.25× TBE (1× TBE: 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA, pH 8.0). For competition studies, stated amounts
of unlabeled competitor DNAs were preincubated with the assay mixture
for 10 min, at 25 °C, prior to the addition of labeled probes. For
antibody supershift assays, 1 µl of BuGR2 monoclonal antibody
(Affinity Bioreagents, Neshanic Station, NJ) or preimmune IgG was added
to the reaction and incubated with the protein for 10 min at room
temperature before the addition of DNA probe.
-mercaptoethanol (in the case of
sulfo-MBS). Immunoprecipitation of cross-linked products was carried
out essentially as described before (39), except that the buffer system
contained 1 mM Met. The immunoprecipitates were dissolved
in 1× Laemmli's buffer (42), without added
-mercaptoethanol (BME)
in the case of DSP cross-linked products, by heating at 95 °C for 5 min. The cross-linked products were resolved by electrophoresis on
SDS-polyacrylamide gels, and the gels were scanned through a Bio-Rad
Molecular Imager.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
841/+23CAT promoter construct, which contains
essential elements for the expression of 2-kb mRNA (6). For this
purpose we used NIH 3T3 cells, which efficiently supported the activity
of the CYP27 promoter in our previous study (6). Fig.
1A shows the CAT activity of
cells transfected with the full-length and deletion constructs under
different conditions. A physical map of the promoter indicating the
location of sequence motifs with full or partial consensus for binding
to various transcription factors has been shown in the bottom
panel of Fig. 1A. The activity of the full-length
841/+23CAT promoter construct was induced by about 3-fold either by
cotransfection with GR cDNA or addition of 100 nM Dx. A
combination of these two treatments, however, induced the transcription
activity by about 6.5-fold. The
329/+23 CAT construct yielded a
similar pattern of induction by cotransfection with GR cDNA and
addition of Dx. Deletion to amino acid
194, however, drastically
reduced the promoter activity to a level lower than the activities of the
75/+23 and
45/+23 constructs. These results suggest the occurrence of a negative enhancer element within the
194 to
75 sequence region. The
75/+23 construct yielded less than half of the
activity of the full-length promoter, whereas the
45/+23 construct
yielded further reduced activity, which was previously shown to be the
basal promoter activity (6). Results also show that deletion to
194
and beyond completely abolished responsiveness to GR and Dx, suggesting
that the GHR region is located between sequence
329 and
194.
As shown before (6), the activity of the full-length
841/+23 CAT
construct was >70-fold of the pCAT basic plasmid DNA. Fig.
1B shows the nucleotide sequence of the putative GHR region
(sequence
329 to
194), which includes a cryptic GRE (TGCTGT), two
weak Ets sites (TGGAAG), an AP-1-like site, and an upstream Sp1-like
site.
View larger version (33K):
[in a new window]
Fig. 1.
Localization of the glucocorticoid-responsive
region of the promoter by progressive 5' deletion. A,
the 841/+23 region of the promoter cloned in the pCAT basic plasmid
vector (
841/+23CAT) was used to generate deletion constructs
329/+23CAT,
194/+23CAT,
75/+23CAT, and
45/+23CAT as described
under "Materials and Methods." NIH 3T3 cells were transfected with
5 µg of the CAT constructs and 1.5 µg of the
-gal expression
plasmid (CMV-gal). In some cases, 6RGR cDNA (1 µg/plate)
expressing full-length GR protein was also used for cotransfection. Dx
(100 nM) was added at 40 h after transfection, and
cells from both with and without added Dx plates were harvested at
48 h after transfection. Cell extracts were assayed for CAT
activity, which was normalized to the
-gal activity. Values
represent the averages ± S.E. (n = 4-6). The
cartoon underneath shows the locations of various cryptic
protein binding motifs on the rat CYP27 promoter. B, the
nucleotide sequence of the
329 to
194 region of the CYP27 promoter
responsive to Dx is shown. Various consensus sites based on computer
analysis are indicated. Transfection was carried out using the calcium
phosphate precipitation method.
194 to
329) or 65-bp (
233 to
298) DNA probes. As shown in Fig.
2A, the 135-bp DNA probe
formed a major complex (complex B) and a minor slow migrating complex
(complex A), both of which were competed by 20-fold molar excess
of unlabeled cryptic GRE DNA (CYP27 GRE). Further, a 20-fold molar
excess of Ets-like DNA motif from the 135-bp region of the promoter (TTGGAAGCC) and also a high affinity Ets DNA motif (TCGGAAGC) competed with complex A, with marginal effects on complex B. The AP-1-like motif, on the other hand, failed to compete with both complexes. Although not shown, the Sp1-like sequence from upstream of
the Ets-like sequence also did not compete with either of the complexes. Furthermore, antibody to GR but not preimmune IgG caused a
supershifted complex, suggesting that a GR-related protein indeed binds
to the 135-bp DNA probe. For yet unknown reasons the preimmune IgG
marginally affected the intensity of complex A. The possible GR protein
binding to the cryptic GRE motif (CYP27 GRE) was further investigated
by EMSA using bacterially expressed, purified Zn2+-loaded
GRDBD protein. As shown in Fig. 2B, the CYP27 GRE motif formed a complex with GRDBD protein, which was supershifted by GR-specific antibody but not by preimmune IgG. Furthermore, the complex
with GRDBD was competed by an unlabeled 20-fold molar excess
CYP27 GRE DNA (ACTGCTGTGC). These results show that the cryptic GRE
sequence indeed binds to GR protein.
View larger version (47K):
[in a new window]
Fig. 2.
Protein binding property of the Dx response
region DNA by gel mobility shift analysis. A, EMSA
using the 135-bp DNA probe (0.1 ng, 20,000 cpm) and 6 µg of nuclear
extract from NIH 3T3 cells. The cryptic GRE, Ets-like, and AP-1-like
DNA sequences were as shown in Fig. 1B except that each
motif contained EcoRI and BamHI sequence
overhangs used for cloning. A 20-fold molar excess of unlabeled DNA in
each case was used for competition. The Ets motif consisted of a CGGAAG
core sequence flanked by EcoRI and BamHI sites. 1 µg each of preimmune IgG or anti-GR-IgG (1 µl of antibody) was
added in some reactions as indicated. B, EMSA using the
cryptic GRE DNA probe (0.1 ng, 25,000 cpm) described for A
and 50 ng of bacterially expressed purified GRDBD protein. 1 µg each
of preimmune IgG or anti-GR IgG was used. SS represents
supershifted complex. C, EMSA using the 135-bp DNA as in
A and 50 ng each of bacterially expressed GRDBD and Ets2
proteins. A 20-fold molar excess of Ets DNA (CGGAAG core
sequence) described in A was used for competition.
D, EMSA using the 65-bp DNA containing only the cryptic GRE
and the downstream Ets-like sequence carried out under excess probe
conditions. EMSA was carried out in the presence of variable amounts of
bacterially expressed and purified GRDBD (0-0.8 µg) and Ets2 (0-0.5
µg) proteins as indicated. A 20-fold molar excess of cryptic GRE and
Ets-like sequence of the CYP27 promoter shown in Fig. 1B and
the respective mutant DNAs as shown in Fig. 3B were used for
competition. Details of probe preparation and conditions of protein
binding and gel electrophoresis were as described under "Materials
and Methods."
329/+23CAT construct was induced by about 5.5-fold in cells
cotransfected with GR cDNA and added Dx. Interestingly,
cotransfection with Ets2 and GR cDNAs in the presence of added Dx
yielded a further increase in activity of about 9-fold as compared with
the control. Cotransfection with Ets2 cDNA alone yielded a modest,
albeit significant increase of activity (1.7-fold). The reporter
construct with mutated GR site (
329/+23CAT-GRmut) did not respond to
cotransfection with GR and Ets2 cDNAs or added Dx. Additionally,
the overall promoter activity was reduced by about 80% of the control.
Interestingly, mutations targeted to the cryptic Ets site, downstream
of the GR site, also abolished the response of the promoter to GR and added Dx in addition to Ets2. As expected, a construct containing both
mutated GR and Ets sites showed similar effects. The Ap1mut construct,
however, showed the overall activity as well as responses to GR, Ets2,
and also added Dx similar to the wild type construct. Although not
shown, mutations targeted to the upstream Ets site did not have any
effect on the promoter activity or inducibility with GR, Ets2, and Dx.
In support of the EMSA results in Fig. 2, these results show that both
the cryptic GR and the downstream Ets sites are important for
Dx-mediated stimulation of promoter activity and that protein factors
binding to these two motifs functionally synergize the transcription
activation.
View larger version (33K):
[in a new window]
Fig. 3.
Functional role of the cryptic GRE and
Ets-like sequence motifs in the Dx-mediated activation of the CYP27
promoter. NIH 3T3 cells were transfected with the wild type or
indicated mutant DNA constructs (5 µg each) and 1.5 µg of CMV-gal
DNA. Cells were cotransfected with the full-length GR expression
cDNA (1 µg) alone or in combination with Ets2 (full-length)
cDNA (0.8 µg each). Treatment with Dx (100 nM) was as
described in the legend to Fig. 1A. CAT activity was
normalized to the -gal activity, and the values represent the
averages of two assays. Mutations targeted to different motifs of the
promoter are indicated at the bottom of the figure. Details
of mutagenesis, transfection, and assays were as described under
"Materials and Methods" and in the legend to Fig. 1.
329/+23 CAT
reporter and full-length GR, maximal transcriptional activity is
obtained by adding the ligand, Dx, and cotransfection with Ets2
cDNA. Cotransfection with Ets1 cDNA by itself had no significant stimulatory effect on the activity of the
329/+23CAT promoter. Furthermore, Ets1 also failed to show any synergistic activation with GR and Dx in combination.
View larger version (22K):
[in a new window]
Fig. 4.
Requirements for the transcription activation
domain of Ets2 and DNA-binding domain of GR for synergistic activation
of the CYP27 promoter. The cartoon at the
top shows the different domains of GR and the GRDBD
construct used in cotransfection. A, effects of
cotransfection with full-length GR (1 µg) and Ets2 and Ets 1 (0.8 µg each) on the transcription activation of the CYP27 promoter, in
the presence or absence of added Dx as indicated. B, effects
of cotransfection with GRDBD (1 µg of DNA) and either intact Ets2,
Ets1, or tEts2 and tEts2 (lacking the transcription activation domains
but intact DNA-binding domains of Ets1 and Ets2) expression cDNA
plasmids (0.8 µg each) on transcription activation. In both cases
wild type 329/+23CAT construct (5 µg of DNA) was used. Details of
transfection, treatment with Dx, and assay of CAT activity were as
described under "Materials and Methods" and in the legend to Fig.
1. Results represent the averages and ± S.E. (n = 3).
329/+23 CAT promoter construct, which was further increased to about 8-fold of
control by cotransfection with Ets2 cDNA. As expected, addition of
Dx did not have any additive effect on transcription activity induced
by a combination of GRDBD and Ets2 cDNAs. Notably, tEts2 and tEts1
cDNA constructs lacking the transcription activation domain but
containing the DNA-binding domain (32) alone or in combination with GR
and Dx reduced the activity of the promoter by about 80%, showing a
dominant negative effect. These results demonstrate that constitutively
active GRDBD in the presence of Ets2 yields transcription stimulation
nearly similar to that obtained with intact GR and Ets2 in the presence
of added Dx. These results also show that although the transcription
activation domain of Ets2 is highly essential for the synergistic
activation of the promoter (Fig. 5), the
transcription activation domain of GR may not be critical for the
activity. Furthermore, as with the intact GR, the tissue-specific
factor Ets1 was unable to show any synergistic activation with
truncated GRDBD (Fig. 4B). Although not shown, the
ubiquitously expressed GA-binding protein
and
factors were ineffective in synergistic activation with GRDBD, suggesting specificity.
View larger version (24K):
[in a new window]
Fig. 5.
Induction by GR and Dx and dominant
negative effects of tEts2 on the CYP27 mRNA levels in Balb/c 3T3
cells. Balb/c 3T3 cells were transfected either with 1 µg
(GR1) or 2 µg (GR2) of full-length GR
expression cDNA, 6RGR, or 1.5 µg of tEts2 cDNAs. Control
cells were transfected with 2 µg of CMV vector DNA alone using the
Fugene-6 reagent. Some plates were treated with 100 nM Dx
for 8-10 h before harvesting. 30 µg of RNA from treated and
untreated cells was subjected to Northern blot hybridization with
32P-labeled rat P-450c27 cDNA or cytochrome oxidase
subunit IV cDNA probes (1.5 × 106 cpm/ml) as
indicated. The blots were imaged and quantified using a Bio-Rad GS 525 Molecular Imager. The stripped blots were rehybridized with
32P-labeled 18 S rRNA probe to assess the RNA loading
level. The levels of CYP27 and COX IV mRNAs in control cells were
considered to be 1 for determining the extent of induction in cells
transfected with various cDNAs.
View larger version (19K):
[in a new window]
Fig. 6.
Physical interaction between GRDBD and
Ets2 proteins by coimmunoprecipitation. A, indicated
amounts of purified recombinant GRDBD and Ets2 proteins were incubated
for 30 min in phosphate-buffered saline and subjected to
immunoprecipitation with Ets2 antibody or preimmune IgG as described
under "Materials and Methods." The immunoprecipitates were
subjected to electrophoresis on a 10% polyacrylamide-SDS gel, the
proteins were transblotted to Nytran membrane, and the blot was probed
sequentially with polyclonal antibody against Ets2 (upper
panel) and monoclonal antibody against GR (lower
panel). In the first two lanes Ets2 protein (100 ng)
and GRDBD protein (50 ng) were loaded as internal markers.
B, nuclear extracts (300 µg of protein each) from mock
transfected cells and cells transfected with GRDBD in combination with
either intact Ets2, TA-Ets2 (transcription activation domain only), or
tEts2 (DNA-binding domain only). cDNAs were immunoprecipitated
either with GR antibody or Ets antibody as indicated. Transfection was
carried out using the Fugene-6 reagent. The immunoprecipitates were
resolved on a 10% polyacrylamide-SDS gel and probed with a mixture of
GR and Ets antibodies.
View larger version (27K):
[in a new window]
Fig. 7.
Chemical cross-linking of GRDBD with Ets1 and
Ets2 proteins. 35S-Labeled GRDBD and Ets1
(A) or GRDBD and Ets2 (B) were subjected to
cross-linking with DSP and immunoprecipitated with indicated
antibodies. The immunoprecipitates were dissolved in sample buffer
lacking BME and subjected to electrophoresis on polyacrylamide-SDS gels
as described under "Materials and Methods." As a positive control,
immunoprecipitates from one cross-linking reaction each in A
and B (shown as + BME) were dissolved in sample
buffer containing 5 mM BME to dissociate the cross-linked
products. Reactions with bovine Adx and Ets2 proteins were also used as
an added control. The positions of GRDBD, Ets1, or Ets2 and the
cross-linked products (CL Prod.) are indicated with
arrows.
329/+23CAT promoter
by about 8-fold, which is nearly similar to the synergistic activation obtained by cotransfection with GRDBD and Ets2 cDNAs. The
GRDBD-Ets1 fusion construct, by contrast, yielded only about 2-fold
activation. The fusion construct with a deleted zinc finger region of
GRDBD (GRDBD
ZF-Ets2), which is known to affect its binding to GRE
DNA (35, 43), also drastically reduced transcription activation. Furthermore, deletion of most parts of the transcription
activation domain of Ets2 (GRDBD-Ets2
200) also reduced the
transcription activation to near basal level. Furthermore,
transactivation by the GRDBD-Ets2 fusion construct was completely
abolished by mutations targeted to the cryptic GRE site alone or a
combination of cryptic GRE and Ets-like sites of the promoter. In
contrast, mutations targeted to the Ets-like site alone did not affect
the extent of activation by GRDBD-Ets2 fusion construct. However,
fusion constructs GRDBD
ZF-Ets2 and GRDBD-Ets2
200 with the mutated
DNA-binding domain or transcription activation domains failed to induce
transcription of the promoter above the basal activity. These results
strongly suggest a novel physical association and domain sharing
between DNA-bound GR and Ets2 factors. These results also suggest that transcription activation is highly dependent on the transcription activation domain of the ubiquitously expressed Ets2 factor.
View larger version (45K):
[in a new window]
Fig. 8.
Sharing of DNA-binding domain of GR and
transcription activation domain of Ets2 proteins in the transcription
activation of the CYP27 promoter. Fusion cDNA constructs (1 µg each) expressing the DNA-binding domain of GR and transcription
activation domain of Ets2 or Ets1 (GRDBD-Ets2 fusion and GRDBD-Ets1
fusion) or mutations targeted to the zinc finger domain of GRDBD
(GRDBD ZF-Ets2) and deleted transcription activation domain of Ets2
(GRDBD-Ets2
200) were cotransfected with the wild type or mutant
329/+23/CAT promoter constructs, in 3T3 cells, as described under
"Materials and Methods." Cotransfection with GRDBD and full-length
Ets2 cDNAs was carried out using the calcium phosphate
precipitation method as described in Fig. 4. The CAT activities were
normalized to the
-gal activity of the respective cell extracts and
represent the averages ± S.E. (n = 4).
ZF-Ets2, GRDBD-Ets2
200, TA-Ets2, and tEts2 are not only
expressed under transient transfection conditions but also are
translocated to the nuclear compartment.
View larger version (22K):
[in a new window]
Fig. 9.
Immunoblot detection and nuclear
localization of proteins expressed in transfected cells. Total
cell extracts and nuclear extracts from cells transfected with various
cDNA constructs were isolated as described (2). Proteins were
subjected to electrophoresis on 12 or 14% polyacrylamide-SDS gels as
indicated, and the blots were probed with indicated antibodies (1:500
dilution each) as described under "Materials and Methods."
Cex, total cell extract; Nex, nuclear
extract.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3' enhancer
(56, 57). The association of Pu.1 interacting protein with the promoter
sites appears to involve both protein-protein interaction with
DNA-bound PU.1 or E2A (56, 57) and also direct interaction with DNA. Thus, the functional synergy between the GR and Ets2 proteins observed
in this study shares similarities with the last two mechanisms described above.
3' enhancer (65).
329/+23CAT promoter. Similarly, cotransfection with GRDBD-Ets1 fusion
protein failed to show significant transcription activation of the
promoter. These results suggest that the transcription activation
domain of Ets2 protein is critical for the synergistic activation.
Although the DNA-binding domains of Ets1 and Ets2 proteins share over
90% sequence homology, the N-terminal 300-amino acid region comprising the transcription activation domains of the two proteins shares only
about 38% sequence identity. Notably, the transcription activation domain of Ets2 protein contains higher hydrophobic helical content and
higher glutamine contents. It is likely that the structural features of
Ets2 protein may be essential for interaction with components of basal
transcription machinery or with specific coactivator proteins. In
summary, we describe a novel functional synergy between GR and Ets2
proteins in the glucocorticoid hormone-dependent activation of the P-450c27 promoter.
![]() |
ACKNOWLEDGEMENTS |
---|
We are thankful to Dr. Keath Yamamoto for generously providing the GR and GRDBD cDNA constructs and purified GRDBD protein used in this study. Thanks are also due to Drs. Michael Atchison and Haider Raza for a critical review of the manuscript and to Dr. Marie-Anne Robin for helping with the preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants GM34883 and CA22762.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Experimental Oncology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105.
** To whom correspondence should be addressed: Dept. of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-8819; Fax: 215-573-6651; E-mail: narayan@vet.upenn.edu.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M100671200
2 V. Rao, and E. S. Reddy, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CYP27, cytochrome P-450c27;
GR, glucocorticoid receptor;
Dx, dexamethasone;
GRE, glucocorticoid response element;
tEts2, DNA-binding domain of Ets2
protein;
GRDBD, DNA-binding domain of glucocorticoid receptor protein;
BME, -mercaptoethanol;
EMSA, electrophoretic mobility shift analysis
for DNA-protein binding;
kb, kilobase(s);
PCR, polymerase chain
reaction;
CMV, cytomegalovirus;
-gal,
-galactosidase;
CAT, chloramphenicol acetyltransferase;
bp, base pair(s);
DSP, dithiobis[succinimidylpropionate];
sulfo-MBS, m-maleimidobenzyl-N-hydroxysulfosuccinimide
ester;
Adx, adrenodoxin;
SREBP, sterol response element-binding
protein;
GBP, GA-binding protein..
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Su, P., Rennert, H., Shayiq, R. M., Yamamoto, R., Zhwng, Y. M., Addya, S., Strauss, J. F., and Avadhani, N. G. (1990) DNA Cell Biol. 9, 657-665[Medline] [Order article via Infotrieve] |
2. | Bjorkhem, I. (1992) J. Lipid Res 33, 455-471[Medline] [Order article via Infotrieve] |
3. | Russell, D. W., and Setchell, K. D. (1992) Biochemistry 31, 4737-4749[Medline] [Order article via Infotrieve] |
4. | Stravitz, R. T., Vlahcevic, Z. R., Russell, T. L., Heizer, M. L., Avadhani, N. G., and Hylemon, P. B. (1996) J. Steroid Biochem. Mol. Biol 57, 337-347[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Shayiq, R. M.,
and Avadhani, N. G.
(1992)
J. Biol. Chem.
267,
2421-2428 |
6. | Mullick, J., Addya, S., Sucharov, C., and Avadhani, N. G. (1995) Biochemistry 34, 13729-13742[Medline] [Order article via Infotrieve] |
7. | Vlahcevic, Z. R., Jairath, S. K., Heuman, D. M., Stravitz, R. T., Hylemon, P. B., Avadhani, N. G., and Pandak, W. M. (1996) Am. J. Physiol. 33, G646-G652 |
8. | Rao, Y. P., Vlahcevic, Z. R., Stravitz, R. T., Mallonee, D. H., Mullick, J., Avadhani, N. G., and Hylemon, P. B. (1999) J. Steroid Biochem. Mol. Biol. 70, 1-14[CrossRef][Medline] [Order article via Infotrieve] |
9. | Beato, M. (1989) Cell 56, 335-344[Medline] [Order article via Infotrieve] |
10. |
Pearce, D.,
Matsui, W.,
Miner, J. N.,
and Yamamoto, K. R.
(1998)
J. Biol. Chem.
273,
30081-30085 |
11. | Chen, J. D., and Evans, R. M. (1995) Nature 377, 454-457[CrossRef][Medline] [Order article via Infotrieve] |
12. | Tsai, S. Y., Carlstedt-Duke, J., Weigel, N. L., Dahlman, K., Gustafsson, J. A., Tsai, M. J., and O'Malley, B. W. (1988) Cell 55, 361-369[Medline] [Order article via Infotrieve] |
13. | Espinas, M. L., Roux, J., Pictet, R., and Grange, T. (1995) Mol. Cell. Biol. 15, 5346-5354[Abstract] |
14. | Schule, R., Muller, M., Otsuka-Murakami, H., and Renkawitz, R. (1988) Nature 332, 87-90[CrossRef][Medline] [Order article via Infotrieve] |
15. | Strahle, U., Schmid, W., and Schutz, G. (1988) EMBO J. 7, 3389-3395[Abstract] |
16. | Jonat, C., Rahmsdorf, H. J., Park, K. K., Cato, A. C., Gebel, S., Ponta, H., and Herrlich, P. (1990) Cell 62, 1189-1204[Medline] [Order article via Infotrieve] |
17. | Schule, R., Rangarajan, P., Kliewer, S., Ransone, L. J., Bolado, J., Yang, N., Verma, I. M., and Evans, R. M. (1990) Cell 62, 1217-1226[Medline] [Order article via Infotrieve] |
18. | Grange, T., Roux, J., Rigaud, G., and Pictet, R. (1991) Nucleic Acids Res. 19, 131-139[Abstract] |
19. | Yang-Yen, H. F., Chambard, J. C., Sun, Y. L., Smeal, T., Schmidt, T. J., Drouin, J., and Karin, M. (1990) Cell 62, 1205-1215[Medline] [Order article via Infotrieve] |
20. | Caldenhoven, E., Liden, J., Wissink, S., Van de Stolpe, A., Raaijmakers, J., Koenderman, L., Okret, S., Gustafsson, J. A., and Van der Saag, P. T. (1995) Mol. Endocrinol. 9, 401-412[Abstract] |
21. | Ray, A., and Prefontaine, K. E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 752-756[Abstract] |
22. |
Zhang, Z.,
Jones, S.,
Hagood, J. S.,
Fuentes, N. L.,
and Fuller, G. M.
(1997)
J. Biol. Chem.
272,
30607-30610 |
23. | Rigaud, G., Roux, J., Pictet, R., and Grange, T. (1991) Cell 67, 977-986[Medline] [Order article via Infotrieve] |
24. | Roux, J., Pictet, R., and Grange, T. (1995) DNA Cell Biol. 14, 385-396[Medline] [Order article via Infotrieve] |
25. | Stocklin, E., Wissler, M., Gouilleux, F., and Groner, B. (1996) Nature 383, 726-728[CrossRef][Medline] [Order article via Infotrieve] |
26. | Grange, T., Roux, J., Rigaud, G., and Pictet, R. (1989) Nucleic Acids Res. 17, 8695-8709[Abstract] |
27. | Crepieux, P., Coll, J., and Stehelin, D. (1994) Crit. Rev. Oncol. 5, 615-638 |
28. | Gauthier, J. M., Bourachot, B., Doucas, V., Yaniv, M., and Moreau-Gachelin, F. (1993) EMBO J. 12, 5089-5096[Abstract] |
29. | Espinas, M. L., Roux, J., Ghysdale, J., Pictet, R., and Grange, T. (1994) Mol. Cell. Biol. 14, 4116-4125[Abstract] |
30. | Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467[Abstract] |
31. | Pearce, D., and Yamamoto, K. R. (1993) Science 259, 1161-1165[Medline] [Order article via Infotrieve] |
32. | Dudek, H., Tantravathi, R. V., Rao, V. N., Reddy, E. S., and Reddy, E. P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1291-1295[Abstract] |
33. |
Lenka, N.,
Basu, A.,
Mullick, J.,
and Avadhani, N. G.
(1996)
J. Biol. Chem.
271,
30281-30289 |
34. | Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489[Abstract] |
35. | Freedman, L. P., Luisi, B. F., Korszun, Z. R., Basavappa, R., Sigler, P. B., and Yamamoto, K. R. (1988) Nature 334, 543-546[CrossRef][Medline] [Order article via Infotrieve] |
36. | Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve] |
37. | Singh, H., Sen, R., Baltimore, D., and Sharp, P. A. (1986) Nature 319, 154-158[Medline] [Order article via Infotrieve] |
38. | Lassar, A. B., Davis, R. L., Wright, W. E., Kadesch, T., Murre, C., Voronova, A., Baltimore, D., and Weintraub, H. (1991) Cell 66, 305-315[Medline] [Order article via Infotrieve] |
39. |
Anandatheerthavarada, H. K.,
Biswas, G.,
Mullick, J.,
Sepuri, N. B. V.,
Otvos, L.,
Pain, D.,
and Avadhani, N. G.
(1999)
EMBO J.
18,
5494-5504 |
40. | Anandatheerthavarada, H. K., Addya, S., Mullick, J., and Avadhani, N. G. (1998) Biochemistry 37, 1150-1160[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Mayo, K. E.,
and Palmiter, R. D.
(1981)
J. Biol. Chem.
256,
2621-2624 |
42. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
43. | Jones, K., Yamamoto, K. R., and Tjian, R. (1985) Cell 42, 559-572[Medline] [Order article via Infotrieve] |
44. | Duter-Coquillaud, M., Leprince, D., Flourens, A., Henry, C., Ghysdale, J., Debuire, B., and Stehelin, D. (1988) Oncogene Res. 2, 335-344[Medline] [Order article via Infotrieve] |
45. | Reddy, E. S. P., and Rao, V. N. (1988) Oncogene Res. 3, 239-246[Medline] [Order article via Infotrieve] |
46. | Carey, M. (1998) Cell 92, 5-8[Medline] [Order article via Infotrieve] |
47. | Roeder, R. G. (1996) Trends Biochem. Sci. 21, 327-335[CrossRef][Medline] [Order article via Infotrieve] |
48. |
Basuyaux, J. P.,
Ferreira, E.,
Stehelin, D.,
and Buttice, G.
(1997)
J. Biol. Chem.
272,
26188-26195 |
49. | MacDougald, O. A., and Lane, M. D. (1995) Annu. Rev. Biochem. 64, 345-373[CrossRef][Medline] [Order article via Infotrieve] |
50. |
McCaffrey, P. G.,
Jain, J.,
Jamieson, C.,
Sen, R.,
and Rao, R.
(1992)
J. Biol. Chem.
267,
1864-1871 |
51. | Tsai, M. J., and O'Malley, B. W. (1994) Annu. Rev. Biochem. 63, 451-486[CrossRef][Medline] [Order article via Infotrieve] |
52. | Schmid, W., Strahle, U., Schutz, G., Schmitt, J., and Stunnenberg, H. (1989) EMBO J. 8, 2257-2263[Abstract] |
53. |
Carter, R. S.,
and Avadhani, N. G.
(1994)
J. Biol. Chem.
269,
4381-4387 |
54. | LaMarco, K., Thompson, C. C., Byers, B. P., Walton, E. M., and McKnight, S. L. Science 253, 789-792 |
55. |
Naar, A. M.,
Beaurang, P. A.,
Robinson, K. M.,
Oliner, J. D.,
Avizonis, D.,
Scheek, S.,
Zwicker, J.,
Kadonaga, J. T.,
and Tjian, R.
(1998)
Genes Dev.
12,
3020-3031 |
56. |
Nagulapalli, S.,
and Atchison, M. L.
(1998)
Mol. Cell. Biol.
18,
4639-4650 |
57. | Pongubala, J. M., Nagulapalli, S., Klemsz, M. J., McKercher, S. R., Maki, R. A., and Atchison, M. L. (1992) Mol. Cell. Biol. 12, 368-378[Abstract] |
58. | Seth, A., Ascione, R., Fisher, R. J., Mavrothalassitis, G. J., Bhat, N. K., and Papas, T. S. (1992) Cell Growth Differ. 3, 327-334[Medline] [Order article via Infotrieve] |
59. | Sharrocks, A. D., Brown, A. L., Ling, Y., and Yates, P. R. (1997) Int. J. Biochem. Cell Biol. 29, 1371-1387[CrossRef][Medline] [Order article via Infotrieve] |
60. | Sucharov, C., Basu, A., Carter, R. S., and Avadhani, N. G. (1995) Gene Expr. 5, 93-111[Medline] [Order article via Infotrieve] |
61. | Lefstin, J. A., Thomas, J. R., and Yamamoto, K. R. (1994) Genes Dev. 8, 2842-2856[Abstract] |
62. | Hollenberg, S. M., and Evans, S. M. (1988) Cell 55, 899-906[Medline] [Order article via Infotrieve] |
63. | Lees, J. A., Fawell, S. E., and Parker, M. G. (1989) Nucleic Acids Res. 17, 5477-5489[Abstract] |
64. | Petersen, J. M., Skalivky, J. J., Donaldson, L. E., McIntosh, L. P., Arber, T., and Graves, B. J. (1995) Science 269, 1866-1869[Medline] [Order article via Infotrieve] |
65. |
Pongubala, J. M.,
and Atchison, M. L.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
127-132 |