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J. Biol. Chem., Vol. 277, Issue 20, 17520-17530, May 17, 2002
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From the Eppley Institute for Research in Cancer and Allied
Diseases, University of Nebraska Medical Center,
Omaha, Nebraska 68198-6805
Received for publication, October 30, 2001, and in revised form, February 19, 2002
Previous studies demonstrated that
differentiation of mouse embryonal carcinoma cells leads to
transcriptional up-regulation of the mouse type II transforming growth
factor- Transforming growth factor- Both T In the case of the T Although most cells express TGF- Materials--
Dulbecco's modified Eagle's (DME) medium was
purchased from Invitrogen. Fetal bovine serum (FBS) was obtained from
HyClone (Logan, UT). Unless otherwise stated all chemicals were
purchased from Sigma.
Cloning the Promoter Region of the Murine Type II TGF- Cell Culture and Differentiation--
F9 EC cells were cultured
on gelatinized dishes in DME medium supplemented with 10% FBS and
antibiotics. Differentiation of F9 EC cells was induced by treatment
with 5 µM retinoic acid (RA) for the times indicated
(20). HeLa and 293T cells were cultured in DME medium supplemented with
10% FBS and antibiotics. All cells were cultured at 37 °C in a
humidified atmosphere of 5% CO2 in air.
Northern Blot Analysis and Primer Extension
Analysis--
Poly(A)+ RNA was isolated from F9 EC cells
and F9-differentiated cells using the Invitrogen FastTrack 2.0 kit
(Invitrogen) according to the manufacturer's instructions. Five µg
of each mRNA was fractionated on 1.2% agarose gels containing 0.22 M formaldehyde, transferred to nylon membrane by capillary
transfer, and immobilized by UV cross-linking (20). Probes were labeled
with [ Generation of Promoter/Reporter Gene Constructs--
Various
regions of the mT Construction of Expression Vectors--
Three FLAG-tagged mElf-3
expression vectors were cloned from mRNA using reverse
transcriptase-PCR. One contained a cDNA for full-length mElf-3
(amino acid residues 1-371); a second contained sequences coding for
the AT-hook domain plus the DNA binding domain of mElf-3 (amino acids
234-371) (ATH + DBD); and the third contained sequences for the DNA
binding domain of mElf-3 (amino acids 271-371) (DBD).
Poly(A)+ RNA was extracted from F9-differentiated cells
using the Invitrogen FastTrack 2.0 kit (Invitrogen, San Diego, CA).
Reverse transcription was performed at 42 °C for 1 h with
SuperScript IITM reverse transcriptase (Invitrogen) using
the antisense primer. PCR amplification reaction was performed by using
Pfu polymerase (Stratagene). The cycling conditions were
94 °C for 2 min followed by 35 cycles of 94 °C for 15 s,
55 °C for 30 s, and 70 °C for 1 min. The sequences of the
sense primer for full-length, ATH + DBD, and DBD
mElf-3 were
5'-CTCAAGCTTGCCACCATGGACTACAAGGACGACGACGATAAGATGGCTGCCACCTGTGAGATCAGC-3', 5'-ACGAGGATCCGACTATAAGAAGGGGGAACC-3', and
5'-AATTGGATCCAGAGGTACTCACCTGTGGGAGTTTATCC-3', respectively. The sense
primer for full-length mElf-3 contained a HindIII site, a
Kozac sequence, the FLAG sequence, and the 5' mElf-3 coding
sequences. The sense primers for ATH + DBD and
DBD contained a BamHI site and the coding
sequences. The sequence of the antisense primer was
5'-CGCTCTAGATTAATTCCGACTCTCTCCAACCTC-3'. It contained the
sequence for an XbaI site followed by coding sequences for
mElf-3. The PCR products were digested with restriction enzymes and
directionally cloned into the polylinker region of the mammalian
expression vector FNpcDNA3.0, which was kindly provided by Dr.
Craig Hauser (Birnham Institute, La Jolla, CA), and described in Galang
et al. (30). The resulting mElf-3 expression plasmids encoded a protein with an N-terminal FLAG tag. Site-directed
mutagenesis of the mElf-3 cDNA was performed using the
QuickChangeTM Site-directed Mutagenesis kit (Stratagene).
All sequences were verified by DNA sequencing.
Verification of mELF-3 cDNA Sequence--
Our sequence of
mElf-3 cDNA did not agree fully with its published
sequence (31, 32). To confirm the mElf-3 cDNA sequence, genomic DNA isolated from F9 EC cells using standard protocols (33) was
used as a template for PCR amplification. Oligonucleotide primers were
designed for the regions in question (exons 2, 4, and 7) to generate
PCR products containing the exon and the intron sequences. The
sequences of the primer sets were 5'-CCCTGAACAACCAACAGATGAC-3' (sense, exon 2) and 5'-TAGGCTCTCTTGGAAGGACATG-3' (antisense, exon 4)
with an expected amplification product of 852 bp, and
5'-CCTCCAACTCTTCTGATGAACTC-3' (sense, exon 4) and
5'-CAGTATTCCTTGCTCAGCTTTCTG-3' (antisense, exon 7) with an
expected amplification product of 870 bp. PCR amplification reaction
was performed using Pfu polymerase (Stratagene). PCR
conditions were 94 °C for 2 min followed by 35 cycles of 94 °C
for 15 s, 55 °C for 30 s, and 70 °C for 1 min. The PCR
products were gel-fractionated, and the visualized band was purified
using a QIAEX II gel extraction kit (Qiagen, Chatsworth, CA). The PCR products were sequenced in both directions and compared with the cDNA sequences.
Transient Transfection and CAT Reporter Gene
Analysis--
F9-differentiated cells were transfected by the calcium
phosphate precipitation method (34). The pCMV- Preparation of Nuclear Extracts and Performance of Gel Mobility
Shift Analysis--
Nuclear extracts were prepared from HeLa cells
transiently transfected with 10 µg of one of the three FLAG
epitope-tagged mElf-3 expression plasmids described above. Nuclear
extracts were prepared using the NE-PERTM nuclear and
cytoplasmic extraction kit (Pierce). Protein concentrations were
determined using the Micro BCA protein assay kit (Pierce). Annealed,
double-stranded oligonucleotides were labeled by a fill-in reaction
using the Klenow fragment of DNA polymerase I (New England Biolabs,
Beverly, MA). Nuclear extracts (12 µg) were incubated for 20 min at
room temperature in 20 µl of binding buffer containing 50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM
MgCl2, 1 mM dithiothreitol, 1 mM
EDTA, 5% glycerol, 1 µg of poly(dI-dC), 100 µg/ml bovine serum
albumin, and ~20,000 cpm of probe. In competitive binding or
supershift assays, unlabeled competitor DNAs or a monoclonal antibody
to the FLAG epitope (M2) was preincubated with the nuclear extract for
40 min at room temperature before addition of the labeled probe.
DNA-protein complexes were resolved on a 6% native polyacrylamide gel
in 0.25× Tris/boric/EDTA buffer as described previously (20). The gels
were dried and visualized by PhosphorImager (Molecular Dynamics,
Sunnyvale, CA). Oligonucleotide probes corresponding to nucleotides +3
to +34 of the mT Chromatin Immunoprecipitation (ChIP)--
293T cells were seeded
at a density of 5 × 106 on 150-mm plates. Cells were
co-transfected 24 h after seeding by the calcium phosphate
precipitation method (34) with the promoter/reporter vector
mT Western Blot Analysis--
Aliquots of nuclear extracts prepared
from cells (293T or HeLa) transfected with FLAG-tagged expression
vectors coding for full-length and truncated forms of mELF-3, as well
as aliquots of the cell extracts from transfected cells used in the CAT
assays, were immunoblotted and probed with an anti-FLAG antibody as
described previously (36). Visualization was accomplished using the
enhanced chemifluorescence kit (Amersham Biosciences). Quantitation was performed using the ImageQuant analysis software (Molecular Dynamics).
Isolation of the 5'-Flanking Region of the mT Regulatory Regions of the mT
To test the role of the EBS in the mT Increased Expression of the mT mELF-3 Transactivates the mT
The mElf-3 mammalian expression vector described above was used to test
whether enhanced expression of mElf-3 would boost the activity of the
mT Truncated Forms of mElf-3 Inhibit mElf-3-mediated
Transactivation--
To better understand the action of mElf-3, we
examined the effects of deleting several domains of mElf-3. In addition
to the Ets domain, which is responsible for DNA binding, and the
transactivation domain, which maps to amino acids 229-259, mElf-3
contains two other domains, a pointed domain and an AT-hook domain
(Fig. 6A) (31). The pointed
domain exhibits sequence homology to a similar domain contained in
Ets-1 (42), and the AT-hook domain is similar to a domain first
identified in the high mobility group protein HMG-I(Y) (43). In this
study, we examined a truncated form of mElf-3 (amino acids 234-371)
that lacks the pointed domain and the transactivation domain and a
smaller form (amino acids 271-371) that also lacks the AT-hook domain.
We determined that mammalian expression vectors for both truncated
forms did not stimulate the activity of the mT
One possible explanation for the inhibitory effects of the truncated
forms of mElf-3 is that they interfere with the expression of
full-length mElf-3. We examined this possibility by performing Western
blot analysis of the expression of mElf-3 in the presence of the
truncated form that contained both the AT-hook and the DNA binding
domain. Due to low transfection efficiency of the F9-differentiated
cells, we performed this study in 293T cells, which can be transfected
at high efficiency. We determined that the truncated form reduces the
response of the reporter gene to mElf-3 (Fig.
7A) without reducing the
expression of mElf-3 in 293T cells (Fig. 7B). Taken
together, our results argue that the truncated forms of mElf-3 act as
dominant negatives and reduce the activity of the mT DNA Binding of mElf-3--
There are over 30 members in the Ets
family of transcription factors. Previous studies (45-51) have shown
that several members of the Ets family of transcription factors contain
auto-inhibitory domains that influence their ability to bind DNA
in vitro. To examine whether this was also true for mElf-3,
we performed gel mobility shift analysis using nuclear extracts
prepared from HeLa cells transfected with expression vectors for mElf-3
or one of the two truncated forms of mElf-3 described above. The
smaller truncated protein, which consists of only the DNA binding
domain, formed one major and one minor DNA-protein complex (Fig.
8A), and both complexes were
supershifted with the M2 monoclonal antibody that recognizes the FLAG
epitope placed at the N terminus of the protein. In other studies, we
determined that the intensity of both complexes is greatly diminished
when a 100-fold excess of the unlabeled probe was added to the reaction
mixture (data not shown). In contrast, these complexes were unaffected
by the addition of a 100-fold excess of an unlabeled competitor that
contained a scrambled EBS, and no DNA-protein complexes formed when
this sequence was used as our probe (data not shown). The reason why two complexes form with the DNA binding domain of mElf-3 is unclear. However, the slower migrating complex may consist of two molecules of
the DNA binding domain bound to the probe. In this regard, the probe
used in this analysis consists of the region +3 to +34 (Fig.
1A), which contains the two closely spaced Ets-binding
sites.
In direct contrast to our findings with the DNA binding domain of
mElf-3, we did not observe DNA binding of full-length mElf-3 in
vitro. Similarly, relatively little binding was observed in vitro by the truncated form of mElf-3 that contains both the
AT-hook domain and the DNA binding domain (Fig. 8A). To
ensure that mElf-3 and the longer truncated form of mElf-3 were
expressed adequately, we performed Western blot analysis on the nuclear
extracts that were used in the gel mobility shift analysis (Fig.
8B). Although mElf-3 was expressed at a level ~65% lower
than expression of the truncated form containing the DNA binding
domain, this is unlikely to explain the failure of full-length mElf-3
to bind DNA in vitro. This is evident in the case of the
larger truncated form of mElf-3 (ATH + DBD), which bound weakly to DNA
and which was expressed at a higher level than the smaller truncated
protein containing only the DBD.
Importantly, the DNA binding characteristics of mElf-3 and its
truncated counterparts do not appear to be cell type-specific. Similar
results were obtained with nuclear extracts prepared from 293T cells,
where ectopic expression of the three proteins was similar to each
other (data not shown). Furthermore, similar findings were observed
using F9-differentiated cells. For this purpose, F9-differentiated
cells were transfected with either the expression vector for mElf-3 or
the expression vector for the truncated form of Elf-3 containing only
the DBD. Although we did not observe the formation of a DNA-protein
complex using nuclear extract prepared from cells transfected with
full-length mElf-3, we observed a prominent DNA-protein complex using
nuclear extracts prepared from cells transfected with the DNA binding
domain of mElf-3 (Fig. 8C). Moreover, as in the case of HeLa
cells and 293T cells, a second DNA-protein complex, which migrated more
slowly, was observed when a higher concentration of the nuclear extract
was tested. Hence, it appears that mElf-3, like several other members
of the Ets family of transcription factors, contains one or more
domains that limit its binding to DNA in vitro.
Although full-length mElf-3 does not bind to DNA in vitro,
it should be able to bind in vivo, because it stimulates the
expression of the mT Transactivation Domain of mElf-3 and the Steady-state Levels of
mElf-3--
A critical domain required for the function of virtually
all transcription factors is their transactivation domain. Previous studies (52) employed Gal4-hELF-3 fusion proteins, containing different
domains of hELF-3, to localize the position of its transactivation domain. This work identified a 13-amino acid acidic core, which is able
to form an
It is possible that the weak transactivation by mElf-3(L142P) was due
to its expression being lower than that of unmodified mElf-3. We
examined this possibility in 293T cells, which were transiently
transfected with expression vectors for mElf-3, mElf-3(D133A), or
mElf-3(L142P) (Fig. 10C). As in F9-differentiated cells,
mElf-3(D133A) and mElf-3(L142P) exhibited lower levels of
transactivation than mElf-3. Moreover, they were expressed at higher
levels than that of mElf-3 (Fig. 10D). In the case of
mElf-3(L142P), its ability to transactivate was nearly 5-fold lower
than that of mElf-3, even though it was expressed at a level that was
3.7-fold higher than mElf-3. This finding not only supports the
argument that the acidic region of mElf-3 (residues 128-159) is
required for transactivation, it also suggests that there is an inverse
relationship between the transactivation by mElf-3 and its steady-state
level. The latter finding is particularly interesting given recent
findings that the turnover of several transcription factors correlates inversely with their ability to activate gene expression (53-55). This
intriguing possibility is discussed below.
Previous studies (20) demonstrated that the differentiation of EC
cells leads to up-regulation of the mT mElf-3 is one of over 30 members in the Ets family of transcription
factors. Sequence homology within this family is primarily limited to a
highly conserved 80-90-amino acid DNA binding domain (the Ets domain),
which interacts with a purine-rich GGA(A/T) or a GGA core
sequence flanked by different nucleotides that determine the affinity
and specificity of binding for a particular Ets family member (37).
This family of transcription factors plays critical roles in the
transcriptional regulation of genes involved in tissue development,
angiogenesis, immune response, cell cycle regulation, cell
proliferation, and apoptosis (56). ELF-3, also named ESE-1 (57), ESX
(58), Jen (59), or ERT (22), is the prototype of a new subclass of Ets
transcription factors, because it exhibits no striking homology to any
particular subclass of the Ets family of transcription factors. The
most obvious structural difference distinguishing mouse and human Elf-3
from other Ets family members is the presence of an AT-hook domain,
which was first described in the high mobility group, non-histone
chromosomal protein HMG-I(Y) (43). Until now, hELF-3 was believed to be
expressed exclusively in epithelial cells (39, 57, 59, 60). In this
study, we demonstrate low expression of mElf-3 in F9 EC
cells and higher levels of mElf-3 expression when EC cells
are induced to differentiate into cells that exhibit the properties of
parietal extra-embryonic endoderm, which is one of the first cell types
to form during mammalian embryogenesis. In addition to its activation
of the T The work described in our study provides three lines of evidence that
mElf-3 plays a key role in the regulation of the mT Previous studies (22, 31, 57, 58) demonstrated hELF-3 binding to DNA
in vitro. Each of these studies involved hELF-3 produced by
in vitro translation or recombinant hELF-3 purified from
bacteria. In this study, we provide the first direct evidence that
mElf-3 binds to the T Several mechanisms could account for the regulation of mElf-3 binding
to DNA. Expression of mElf-3 in mammalian cells could lead to
post-translational modifications, such as phosphorylation, which
modulate its binding to DNA. For example, phosphorylation of Ets-1
outside of its DNA binding domain reduces DNA binding 50-fold (62).
Conversely, phosphorylation of the C-terminal transcriptional
activation domain of Elk-1 enhances both DNA binding and
transcriptional activation (65). Alternatively, certain cells, such as
the HeLa cells and 293T cells used in our DNA binding studies, may
express a cofactor that binds to mElf-3 and limits its binding to DNA
in vitro. We think this is unlikely given that mElf-3
stimulates T The work reported in this study also extends our understanding of the
transactivation domain of mElf-3. Previous studies (52) identified a
small acidic region of hELF-3 (amino acids 129-159) that could
strongly stimulate a viral promoter when fused to the DNA binding
domain of the yeast transcription factor Gal4. In our study, we have
extended this finding by focusing on mElf-3 itself and one of its
likely in vivo targets. Specifically, we demonstrate that a
single amino acid substitution at residue 142 (L142P) is sufficient to
cause a 5-fold reduction in the ability of mElf-3 to stimulate the
mT In the course of these studies, we determined that the steady-state
levels of mElf-3 were consistently lower than mutant forms of mElf-3,
in particular mElf-3(L142P), which exhibits significantly lower
transactivation activity. Similarly, the truncated forms of mElf-3,
which act as dominant negatives, also exhibited significantly higher
steady-state levels of expression than mElf-3 (Fig. 8B). Currently, the mechanisms responsible have not been determined. However, it may be the result of a slower rate of mElf-3 turnover. Recent studies (54, 55, 71) have shown that many transcription factors
turn over by a proteosome-mediated pathway and that the rate of
transcription factor degradation is inversely correlated with their
ability to activate transcription. Interestingly, the domains
responsible for turnover of transcription factors have been shown to
overlap with their transactivation domains (72), even though they are
ubiquitinated at other sites. Furthermore, recent studies (55)
suggest that ubiquitination of at least some transcription factors
serves a dual role of stimulating both transactivation and
proteosome-mediated turnover. Given these new developments,
further studies regarding the connection between mElf-3
transactivation and turnover are warranted.
In conclusion, the work reported in this study argues strongly that
mElf-3 plays a key role in the regulation of the mT We thank A. Harris for reading this
manuscript and Heather Rizzino for editorial suggestions. The Core
facilities of the University of Nebraska Medical Center, Eppley Cancer
Center, were supported in part by a Cancer Center Support Grant CA 36727.
*
This work was supported in part by NCI Grant CA 79491 from
the National Institutes of Health.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF118264.
§
To whom correspondence should be addressed. Tel.: 402-559-6338;
Fax: 402-559-4651; E-mail: arizzino@unmc.edu.
Published, JBC Papers in Press, March 13, 2002, DOI 10.1074/jbc.M110434200
1
The abbreviations used are: TGF-
Activation of the Murine Type II Transforming Growth Factor-
Receptor Gene
UP-REGULATION AND FUNCTION OF THE TRANSCRIPTION FACTOR
Elf-3/Ert/Esx/Ese-1*
, and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor (mTR-II) gene. To elucidate the
molecular mechanisms regulating transcription of this gene, we isolated
the 5'-flanking region of the mTR-II gene and
characterized its expression in F9-differentiated cells. Analysis of
mTR-II promoter/reporter gene constructs demonstrates that two conserved Ets-binding sites play an important role in the
activity of the mTR-II promoter. Importantly, we present evidence that mElf-3, a member of the Ets family, plays a key role in
the activation of the mTR-II promoter. Northern blot analysis reveals that the steady-state levels of mTR-II
mRNA increase in parallel with those of mElf-3 mRNA
during the differentiation of F9 embryonal carcinoma cells. We also
demonstrate that mElf-3 contains one or more domains that influence its
binding to DNA. Finally, we report that a single amino acid
substitution in the transactivation domain of mElf-3 reduces its
ability to transactivate and elevates its steady-state levels of
expression. In conclusion, our data argue that mElf-3 plays a key role
in the regulation of the mTR-II gene, and Elf-3 itself
is regulated at multiple levels.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-)1 is a family of
genetically distinct polypeptides that are secreted by virtually all
cells. These growth factors play important roles in cell proliferation, differentiation, and synthesis of extracellular matrix proteins (1, 2).
Three major TGF- receptors have been identified and designated as
type I (TR-I), type II (TR-II), and type III (TR-III)
receptors. TR-III is a glycoprotein (~280 kDa) that appears to
lack intrinsic signaling activity. However, TR-III can present
TGF- to its signaling receptors, TR-I (~55 kDa) and TR-II
(~75 kDa) (3). Appropriate cellular responses to extracellular
TGF- are initiated upon binding of TGF- to TR-II, which is a
constitutively active serine/threonine kinase. When bound to ligand,
TR-II forms a heteromeric signaling complex with TR-I and
phosphorylates TR-I at serine and threonine residues within its
kinase domain (4). This activates a series of downstream signaling
pathways (2).
R-I and TR-II are required for TGF--mediated growth
suppression. Hence, loss of either receptor leads to TGF-
resistance, which often contributes to malignant progression. TGF-
resistance caused by defects in TR-II expression has been
reported in numerous tumor cell lines (5-7). Moreover, several studies
(8, 9) have established a clear relationship between defective TR-II expression and malignant progression. Importantly, transfection of
several different tumor cell lines with an expression vector for
TR-II restored sensitivity of the cells to TGF- and reduced their
malignant behavior (5, 9, 10). Similarly, transfection of a
TR-I-defective colon carcinoma cell line with a TR-I expression vector reversed its malignant phenotype (11). Together, these findings
illustrate the importance of both TR-I and
TR-II as tumor suppressor genes.
R-II gene, gross structural mutations
of both TR-II alleles have been observed in 71-90% of
colorectal tumors and in 71% of gastric cancer cell lines with
microsatellite instability (6, 12, 13). However, in several different types of tumors, which exhibit either reduced or undetectable expression of the TR-II gene at the mRNA or protein
level, no structural mutations were apparent within the coding region
of the gene (6, 14-16). This suggests that defects in the mechanisms regulating the transcription of the TR-II gene may play
important roles in the aberrant expression of TR-II. In this regard,
it has been argued that the low levels of TR-II mRNA
expressed by A431 tumor cells are due to a point mutation located in
the 5'-flanking region of the TR-II promoter (17).
Additionally, it has been reported that this point mutation increases
CDP/Cut transcription factor binding affinity, and overexpression of
CDP/Cut reduces transcription from TR-II promoter (18).
During the last several years, the promoter region of the human
TR-II gene (hTR-II) was examined in several
cell culture model systems (19-22). Initial analysis identified three
positive cis-regulatory elements within 200 bp of the major
transcription start site, i.e. a CRE/ATF motif, a GC box,
and two Ets-binding sites (EBS) that are separated by 3 bp (19).
receptors, mouse embryonal
carcinoma (EC) cells lack detectable TGF- binding (23). After EC
cells undergo differentiation, TGF- binding is readily detected, and
the growth of the differentiated cells becomes responsive to TGF-
(23). Subsequent studies demonstrated that the differentiation of mouse
EC cells leads to transcriptional up-regulation of both the
TR-II gene (20) and the gene for one of its ligands,
TGF-2 (24). Because mouse EC cells provide an excellent model system for studying both mammalian embryogenesis (25) and the transcriptional regulation of genes that play key roles during development and cancer
(26), we set out to clone and characterize the regulatory regions of
the mouse TR-II gene (mTR-II). Cloning the mouse promoter would make it possible to study the transcriptional
up-regulation of the TR-II gene in a well characterized
mouse model system. It would also enable us to identify and then focus
on conserved cis-regulatory elements. In this study, we describe the
isolation and characterization of the 5'-flanking region of the
mTR-II gene. In the course of characterizing the
mTR-II promoter, we focused our attention on the
conserved EBS that are located just downstream of the major
transcription start site in the human and mouse TR-II
genes. We show that mElf-3 binds to the EBS and strongly stimulates
expression of the mTR-II promoter in differentiated cells
derived from mouse F9 EC cells. We also demonstrate that differentiation of EC cells leads to parallel increases in the expression of mRNA for mTR-II and mElf-3.
Equally important, we demonstrate that mElf-3 itself is subject to
other levels of regulation. Hence, characterization of the mechanisms
that regulate mElf-3 may help identify ways to enhance the expression
of the TR-II gene in diseased tissues that exhibit
aberrantly low levels of TR-II.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Receptor (mTR-II) Gene--
To assist in cloning the 5' end of the
mTR-II gene, we designed a set of PCR primers based on
the 5' end of the cDNA sequence from Suzuki et
al. (see Ref. 27; GenBankTM D32072):
5'-CCTCCGGGCCTCCGAGCTCC-3' (sense) and 5'-CCGTCGCTCGTCATAGACCG-3' (antisense). Using these primers, Genome Systems Inc. (St. Louis, Missouri) screened a murine strain 129/O1a genomic library and identified a P1 clone that contained a 75-100-kb genomic fragment. To
obtain a smaller genomic fragment, the P1 clone was digested with
PstI, and the resulting fragments were ligated into
Bluescript KS+ (Stratagene) and transformed into DH5
F' bacteria.
Using the same set of primers used to screen the P1 genomic library, we identified a clone (~4 kb) that contained the 5'-flanking region of
the mTR-II gene. Sequence analysis of both strands over a region of 1021 bp indicated that it extended more than 500 bp upstream
of the two major transcription start sites (see below). Sequencing was
performed with an Applied Biosystems 373 DNA Sequencer (PerkinElmer
Life Sciences). Alignment of the sequencing results was accomplished
using version 1.1 of the Sequence Navigator program (PerkinElmer Life
Sciences/Applied Biosystems). Comparisons (alignments) with other
sequences were performed using programs of the Wisconsin Package
version 9.1, Genetics Computer Group (GCG, Madison, WI).
-32P]dCTP by using the Prime-It II kit
(Stratagene, La Jolla, CA). mElf-3 cDNA (see below) was
used to generate a probe that was 864 bp in length and encoded amino
acid residues 1-287. mTR-II cDNA was used to
generate a probe that was 699 bp in length and encoded the amino acid
residues 90-322. A mTR-I (ALK-5) cDNA was used to
generate a probe that was 657 bp in length and encoded the amino acid
residues 152-370. Hybridization was performed at 68 °C in the
ExpressHybTM hybridization solution
(CLONTECH, Palo Alto, CA) for 1.5 h, and the
blots were washed according to the manufacturer's instructions. Hybridization of GAPDH was used as a control for equal
loading. Quantitation was accomplished using the PhosphorImager and
ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Sizes of the
transcripts were estimated by comparison to Millennium RNA markers
(Ambion, Austin, TX). The transcription start site of the
mTR-II gene was examined by primer extension as described
previously (28). For this purpose, we used the antisense primer
mTR-II-PE (5'-GCGCGCGCGGGGGGTGTCGTCGGTCGGTGC-3' (+96 to +67))
and mRNA isolated from F9 EC cells induced to differentiate by
exposure to RA for 4 days.
R-II promoter were amplified by PCR from
the 4-kb genomic mTR-II DNA fragment, which we had
subcloned into Bluescript KS+. Restriction sites (HindIII or
BglII) were incorporated at the ends of all primers to
permit directional cloning of the amplified fragments into the
HindIII and BglII sites in the polycloning region
of the promoterless chloramphenicol acetyltransferase (CAT) expression
plasmid pBLCAT7 (29). Site-directed mutagenesis of the
mTR-II promoter CAT construct was performed using the
QuickChangeTM Site-directed Mutagenesis kit (Stratagene).
All promoter sequences were verified by DNA sequencing.
-gal plasmid
containing the -galactosidase reporter gene under the control of the
CMV immediate early promoter was cotransfected to normalize
for differences in transfection efficiency. F9 EC cells were plated at
a density of 1 × 105 cells per 100-mm dish in DME
medium containing 10% FBS and 5 µM RA. After 72 h,
the cells were transfected and incubated with the DNA-calcium phosphate
precipitate for 22 h and then were refed with DME medium
containing 10% FBS and 5 µM RA. CAT activities were
determined 72 h after transfection using the method of Seed and
Sheen (35) and were normalized to -galactosidase activity. 293T
cells were seeded at 2 × 106 cells per 100-mm dish in
DME medium containing 10% FBS. After 24 h, the cells were
transfected using the calcium phosphate precipitation method (34). The
cells were incubated with the precipitate for 22 h and then were
refed with DME medium containing 10% FBS. CAT activities were
determined 24 h after transfection and were normalized as
described above. When different amounts of the expression plasmid for
mElf-3 were transfected into F9-differentiated and 293T cells, the
total amounts of transfected DNA were kept constant by addition of null
plasmid DNA, FNpcDNA3.0, as indicated in the figure legends. HeLa
cells were seeded at 7.5 × 105 cells per 100-mm dish
in DME medium containing 10% FBS. Transfections were performed as
described for 293T cells. HeLa cell nuclear extracts were prepared
24 h after transfection and were used for Western blot analysis
and gel mobility shift analysis as described below. All transfection
experiments were repeated at least twice with different plasmid preparations.
R-II gene were as follows: wild
type, 5'-TGGCGAGGAGTTTCCTGTTTCCCTCTCGGCGC-3'; mutant,
5'-TGGCGAGGAGTTTGGATCCACCCTCTCGGCGC-3' (underline
indicates mutated sequences).
R-II(
108/+56) and a FLAG-tagged mElf-3 expression
vector containing either full-length Elf-3 or the DBD of Elf-3. After incubation with the precipitate for 20 h, the medium was changed, and ChIP analysis was initiated 4 h later. Cross-linking was
performed by addition of formaldehyde to the media at a final
concentration of 1% for 10 min at room temperature. The cross-linking
reaction was quenched with glycine (0.125 M) for 5 min at
room temperature. Cells were then washed 3 times with PBS and scraped
into 1 ml of phosphate-buffered saline. Cell pellets were resuspended
in Cell Lysis Buffer (5 mM PIPES, 85 mM KCl,
0.5% IGEPAL CA-630, 0.5 mM phenylmethylsulfonyl fluoride),
incubated for 15 min on ice, and the nuclei pelleted. The nuclei were
lysed in Nuclei Lysis Buffer (50 mM Tris-Cl, 10 mM EDTA, 1% SDS, 0.5 mM phenylmethylsulfonyl fluoride) on ice for 10 min. Shearing of the DNA was then performed using a W-225 cup horn sonifier (Misonix, Inc.), and cell debris was
pelleted. The supernatant was precleared with protein G PLUS-agarose beads (pre-blocked with bovine serum albumin, Santa Cruz Biotechnology) for 1 h at 4 °C. Following preclearing, the supernatant was
diluted 2-fold with Dilution Buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl, pH 8, 167 mM NaCl), and 5% was removed and save as an "Input"
sample. The remaining sample was divided into 2 aliquots. One aliquot
was incubated with FLAG (M2) antibody conjugated to agarose beads
(Sigma), and the other aliquot was incubated with Gal4 (DBD) antibody
conjugated to agarose beads (Santa Cruz Biotechnology). Both aliquot
mixtures were incubated overnight at 4 °C. The beads were then
washed 4 times with TBS (20 mM Tris, 140 mM
NaCl, pH 7.6), and complexes were eluted by two incubations with 100 µl of Elution Buffer (1% SDS, 50 mM NaHCO3) at 65 °C for 10 min (a total of 200 µl for each sample). The
eluted immunoprecipitated samples and Input sample were decross-linked by addition of NaCl to a final concentration of 0.2 M and
incubation at 65 °C overnight. Following decross-linking, the
samples were treated with 20 µg of proteinase K in Proteinase K
Buffer (10 mM Tris-Cl, pH 7.5, 5 mM EDTA,
0.25% SDS) for 2 h at 42 °C. DNA fragments were purified using
the Geneclean Turbo kit (Qbiogene). The M13-21 FOR primer
(5'-TGTAAAACGACGGCCAG-3'), specific for a sequence located in the
plasmid backbone, and the primer ELF3R (5'-ACGATGCCATTGGGATA-3'),
specific for a sequence within the 108/+56 region of
mTR-II, were used to amplify specifically the
mTR-II promoter region located in the
transfected promoter/reporter gene construct. PCR was performed on
equivalent volumes of each sample and analyzed at multiple cycles.
Results are presented for one cycle within the linear range of amplification.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R-II Gene--
The
5'-flanking region of the mTR-II gene was isolated from a
P1 genomic library that was screened by PCR using primers based on the
5' end of the published mTR-II cDNA sequence (27).
The P1 genomic clone identified in this screen was digested with
PstI, subcloned into pBlueScript II KS+, and rescreened with
the same set of primers used to identify the P1 genomic clone. This led to the isolation of a 4-kb PstI genomic fragment that
contained the 5' end of the mTR-II cDNA. Sequencing
of this 4-kb genomic subclone demonstrated that it contained the
5'-flanking region of the mTR-II gene and extended 3'
beyond the exon I/intron I border. The sequence of this region has been
assigned the GenBankTM accession number AF118264.
Comparison of the immediate 5'-flanking region of the murine and human
TR-II genes revealed considerable homology (Fig.
1A). Previous work (19-22)
with hTR-II promoter/reporter gene constructs identified
several cis-regulatory elements, including a CRE/ATF motif, a CCAAT box
motif, a GC box, and two closely spaced EBS. The sequences and
positions of these putative cis-regulatory elements are fully conserved
in the mTR-II gene. In addition, the 5'-flanking region
of the mTR-II gene, like its human counterpart, does not
contain a canonical TATA box. Examination of the human TR-II promoter by 5'-RACE indicated that transcription is
initiated at multiple sites over a 70-bp region (19). By using primer extension, we identified three sites of initiation for the
mTR-II promoter (Fig. 1B). Two sites are
closely spaced, and a third is ~37 bp downstream. One of the sites,
indicated in the Fig. 1B, corresponds to the major
transcription initiation site reported for the hTR-II
gene. This site in the hTR-II gene was designated as the
+1 position (19). For the purposes of alignment, we designated the
corresponding G residue in the mTR-II promoter region as +1 in this report (Fig. 1A).
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Fig. 1.
Sequence of the mT R-II
gene. A, comparison of the immediate 5'-flanking
regions of the mTR-II gene and the hTR-II
gene. Putative CRE/ATF-binding motif, CCAAT box, GC box, and Ets family
transcription factor-binding motifs are boxed. The
nucleotide designated +1 is number 557 in the mouse genomic
sequence deposited with the GenBankTM accession number
AF118264. The human sequence was assembled from the published cDNA
(GenBankTM M85079; number 1 is +1) and promoter
(GenBankTM U37070, number 1883 is 1)
sequences. B, primer extension analysis to identify the
transcription start sites of the mTR-II gene in
F9-differentiated cells. Three micrograms of poly(A)+ RNA
isolated from F9-differentiated cells was hybridized to the
radiolabeled antisense primer mTR-II-PE. Primer extension was
performed using Superscript II reverse transcriptase. The resulting
cDNA product was fractionated by electrophoresis and analyzed as
described in the "Experimental Procedures."
R-II Gene--
To understand better
how the TR-II gene is regulated in mouse
F9-differentiated cells, we prepared a series of mTR-II
promoter/reporter gene constructs (Fig.
2A). These constructs were
generated with the plasmid pBLCAT7, which contains a CAT reporter gene
cassette and a multiple cloning site into which different lengths of
the 5'-flanking region of the mTR-II gene were inserted.
The largest construct, mTR-II(300/+56), contains all of
the regulatory regions identified in the hTR-II gene as
well as the major transcription start sites identified for the mouse
gene. The mTR-II promoter/reporter gene constructs were
transiently transfected into F9-differentiated cells (Fig.
2B). The constructs mTR-II(300/+56),
-(232/+56), and -(108/+56) exhibited the highest levels of promoter
activity. Deletion of sequences from 300 to 233 resulted in a minor
increase, and further deletion of sequence from 232 to 109, which
contains a putative CRE/ATF-binding site (20), resulted in a minor
decrease in promoter activity. However, deletion of the sequence
between +2 and +56, which contains the potential EBS, markedly
decreased mTR-II promoter activity. These results
suggested that the EBS are likely to play an important role in the
expression of mTR-II gene, although other cis-regulatory
elements in the mTR-II promoter are also likely to
influence the expression of this gene.
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Fig. 2.
Functional analysis of the
mT R-II promoter. A, schematic
representation of mTR-II promoter/reporter gene
constructs. Overlapping segments of the 5'-flanking region of the
mTR-II promoter were generated by PCR and inserted into
the multiple cloning site upstream of the CAT reporter gene in a
promoterless vector, pBLCAT7. The dotted line represents
deleted regions (+3 to +56). B, mTR-II
promoter activity in F9-differentiated cells. Cells were transiently
transfected with 15 µg of each construct described in A,
along with 1 µg of an internal control plasmid pCMV--gal.
Activities of the CAT and -galactosidase reporter genes were assayed
and normalized as described under "Experimental Procedures." The
promoter activity of each construct is calculated relative to the
expression of the CAT reporter gene observed with pBLCAT7, which was
set to 1. Data shown are means and S.D. for duplicate measurements from
one representative transfection. This experiment was performed three
times, and similar results were obtained in each case.
R-II promoter, the
Ets-binding sites located between +14 and +24 were disrupted by
site-directed mutagenesis. Because both Ets-binding sites in the
hTR-II promoter were found to influence its expression
(19), both sites in the construct mTR-II(108/+56) were
modified by site-directed mutagenesis. Specifically, two different
mutant constructs were generated, mTR-II(108/+56EBSmut1) and
mTR-II(108/+56EBSmut2) (Fig.
3A). mTR-II(108/+56EBSmut1) inactivated both GGAA core
element EBS but contained three GGA sequences. Because GGA sequences
have been reported to provide a core-binding element for some Ets
family members (37), each of the three GGA sequences in
mTR-II(108/+56EBSmut1) were modified to create
mTR-II(108/+56EBSmut2). When these modified constructs
and their wild type counterpart were transiently transfected into
F9-differentiated cells, we determined that disruption of the EBS in
both mutant constructs reduced promoter activity by ~60% (Fig.
3B). Interestingly, a larger reduction (~80%) is observed when the region between +3 and +56 is deleted (Fig. 2B).
However, removal of this region also removes one of the two potential
transcription start sites in our promoter/reporter gene constructs. In
the case of hTR-II promoter/reporter gene constructs,
removal of a comparable region reduced promoter activity by 60% (19).
Hence, our studies and those conducted with hTR-II
promoter/reporter gene constructs argue that the EBS play an important
role in the function of both the human and the mouse
TR-II promoter.
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Fig. 3.
Functional analysis of the EBS located
between nucleotide +2 and +56 of the mT R-II
promoter region. A, the sequences of the wild
type and the mutant EBS. The sequences shown begin at +7 and end with
+30. The EBS in the wild type construct
mTR-II(108/+56) are underlined. The
nucleotides modified in the mutant promoter/reporter gene constructs
are underlined. In addition, the nucleotides that differ
between the two mutant constructs are indicated by
asterisks. B, F9-differentiated cells were
transiently transfected with wild type
(mTR-II(108/+56)) and two mutant
promoter/reporter gene constructs,
mTR-II(108/+56EBSmut1) and
mTR-II(108/+56EBSmut2) along with 1 µg of plasmid
CMV--gal as an internal control. Promoter activity of the mutant
construct is calculated relative to the wild type construct, which was
assigned a relative activity of 1. Data shown are mean and S.D. for
duplicate measurements from one representative transfection.
This experiment was repeated, and similar results were obtained. The
reduced activity of mTR-II(108/+56EBSmut1)
relative to mTR-II(108/+56) was observed in
multiple experiments.
R-II Gene during the
Differentiation of F9 EC Cells Correlates with Increased Expression of
the mElf-3 Gene--
Previous studies (22) have shown that the
transcription factor hELF-3 can bind to the EBS present in the
hTR-II promoter. Moreover, recent studies (38) have shown
that ectopic expression of hELF-3 increases expression of hTR-II in
human breast cancer cells and reduces their malignant phenotype.
Because we had shown previously (20) that the differentiation of EC
cells leads to increases in the steady-state levels of
mTR-II mRNA, we examined the expression of the
mElf-3 gene in EC cells and their RA-induced differentiated
cells. For this purpose, we initially compared mElf-3
mRNA expressed by F9 EC cells and by F9 cells that had been treated
with RA for 5 days. Northern blot analysis demonstrated that the
steady-state levels of mElf-3 were 7-8-fold higher in F9-differentiated cells than in EC cells (data not shown). The finding
that differentiation leads to significant increases in expression of
mElf-3 prompted us to examine the temporal relationship between increases in mElf-3 mRNA and
mTR-II mRNA. If, in fact, increases in mElf-3 are
responsible, at least in part, for the up-regulation of the
mTR-II gene, increases in mElf-3 mRNA
should precede or parallel the increases in mTR-II. To
address this issue, mRNA was prepared from EC cells and from EC
cells that had been treated with RA for different periods up to 5 days.
A Northern blot was prepared and sequentially probed for the expression of mElf-3, TR-II, GAPDH, and
mTR-I transcripts. As reported by other investigators
(31, 39) studying mElf-3 in other cell types, multiple
mElf-3 transcripts were observed, and the most abundant
transcript exhibited a size of 2.2 kb (Fig.
4). Our analysis revealed that
mElf-3 mRNA increased ~3-fold after 24 h of exposure to RA and more than 6-fold after 3 days (Fig. 4). Moreover, analysis of
the PhosphorImager data by ImageQuant demonstrated that each of the
three transcripts exhibited similar increases during the 5-day period
(data not shown). Interestingly, mTR-II mRNA
increased over 6-fold during the same 5-day period, and the increases
in the expression of mElf-3 and mTR-II
correlated closely with one another, in particular during the initial
stages of differentiation (the first 48 h). Hence, these findings
are consistent with the possibility that the increase in mElf-3
expression during the differentiation of EC cells plays a key role in
the elevated expression of the mTR-II gene. Surprisingly,
the steady-state levels of TR-I mRNA did not change
significantly when F9 EC cells differentiate. Both EC cells and their
differentiated counterparts express similar levels of a 5.5-kb
TR-I transcript (Fig. 4), which is the transcript size
observed in various mouse tissues (40).
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Fig. 4.
Northern blot analysis of
mElf-3, mT R-II, and
TR-I mRNA prepared from F9 EC cells and
F9-differentiated cells. A, poly(A)+ RNA
was isolated from F9 EC and F9-differentiated EC cells at 24-h
intervals (1-5 days). Each lane was loaded with 5 µg of
poly(A)+-rich RNA. The blot was sequentially hybridized
with radiolabeled mElf-3-, mTR-II-,
GAPDH-, and mTR-I-specific probes under
stringent conditions as described under "Experimental Procedures."
The sizes of the transcripts are shown on the left.
B, expression of the three genes (mElf-3,
mTR-II, and mTR-I) are shown relative to
GAPDH. Relative expression levels were determined as
described under "Experimental Procedures."
R-II Promoter Specifically through
Binding to the EBS Located between +14 and +24 in the mTR-II
Promoter--
To determine whether mElf-3 influences expression of the
mTR-II gene in F9-differentiated cells, we generated an
mElf-3 expression vector. For this purpose, we employed reverse
transcriptase-PCR and mRNA prepared from F9-differentiated cells.
The cDNA product was inserted into the multiple cloning site of the
mammalian expression vector FNpcDNA 3.0 and sequenced. Previous
reports (31, 32) of the coding sequence of mElf-3 differed at six amino
acid residues. Sequence analysis of our cDNA agrees with one of the
published sequences (32) at four of the amino acid residues and
agrees with the other report for the two remaining differences
(31) (Table I). Each of the differences
identified in our study was confirmed at the nucleotide level in four
separate sequencing reactions, including the sequencing of both strands
of PCR products generated from genomic DNA isolated from F9 EC cells.
Currently, it is unclear whether any of the differences are due to
single nucleotide polymorphisms. Our study and one of the published
reports (32) utilized mouse strain 129. The mouse strain used in the other study (31) could not be determined.
Differences observed in the coding sequences of mElf-3
R-II promoter in F9-differentiated cells. For this
purpose, the cells were transiently transfected with the
mTR-II(108/+56) promoter/reporter gene construct and
increasing amounts of the mElf-3 expression vector. This resulted in a
dose-dependent increase in the activity of the
mTR-II promoter, which exceeded 20-fold (Fig.
5). In contrast, little increase was
observed when either mTR-II(108/+2) or
mTR-II(108/+56EBSmut1), which lack the EBS, was
co-transfected with the mElf-3 expression vector. These findings
indicate that overexpression of mElf-3 can increase the activity of the
mTR-II promoter and further argues that the ability of
mElf-3 to stimulate the mTR-II promoter occurs
predominantly through the EBS located between +14 and +24. Furthermore,
we tested the ability of Ets-2 to stimulate the promoter/reporter gene
construct mTR-II(108/+56), because Ets-2 is expressed
by EC-differentiated cells (41). However, unlike mElf-3, ectopic
expression of Ets-2 in F9-differentiated cells did not stimulate the
expression of the reporter gene (data not shown).
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Fig. 5.
Effect of mElf-3 on the expression of
mT R-II promoter/reporter gene constructs.
F9-differentiated cells were transiently transfected with 15 µg of
either mTR-II(108/+56),
mTR-II(108/+56EBSmut1), or
mTR-II(108/+2) and increasing amounts of the plasmid
DNA coding for mElf-3. The cells were also transfected with 1 µg of
pCMV--gal plasmid to normalize for differences in transfection
efficiencies. Total amount of DNA transfected was kept constant using
the null plasmid DNA, FNpcDNA3.0. Activities of CAT and
-galactosidase were assayed and normalized as described under
"Experimental Procedures." Data are presented as fold activation
relative to cells transfected with either
mTR-II(108/+56),
mTR-II(108/+56EBSmut1), or
mTR-II(108/+2) alone. Data shown are means and S.D. for
duplicate measurements from one representative transfection. This
experiment was repeated twice, and similar results were obtained in
each case.
R-II
(108/+56) promoter/reporter construct. In fact, they reduced basal
expression of the reporter gene (Fig. 6B). These findings
suggested that the truncated forms of mElf-3, like the truncated forms
of other Ets family members (30, 44), were behaving as dominant
negatives and blocking the function of an endogenous factor. This
possibility was examined by transfecting F9-differentiated cells with
an expression vector for full-length mElf-3 and increasing amounts of
expression vectors for the truncated forms of mElf-3 (Fig.
6C). At 1 µg of the full-length mElf-3 expression vector,
the level of the reporter gene was elevated more than 12-fold. As the
concentrations of the expression vectors for the truncated forms were
increased, the expression of the reporter gene decreased. Moreover, the
truncated form that contained both the AT-hook and the DNA binding
domain reduced the expression of the reporter gene to the level
observed in the absence of added full-length mElf-3.
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Fig. 6.
Effects of truncated forms of mElf-3 on the
expression of mT R-II promoter/reporter gene
constructs in F9-differentiated cells. A, schematic
representation of full-length and two truncated forms of mElf-3.
PNT, TAD, ATH, and Ets indicate
Pointed domain, transactivation domain, AT-hook domain, and Ets DNA
binding domain, respectively. B, F9-differentiated cells
were transfected with 15 µg of the mTR-II(108/+56)
promoter/reporter construct plus an expression plasmid encoding
full-length mElf-3 or one of the two truncated forms of mElf-3 (ATH + DBD, DBD). The cells were also transfected with 1 µg of pCMV--gal
plasmid to correct for any differences in transfection efficiency.
C, F9-differentiated cells were transfected with 15 µg of
the mTR-II(108/+56) promoter/reporter gene construct
along with an expression plasmid encoding full-length mElf-3 on its own
or together with increasing amounts of an expression plasmid encoding
the truncated forms of mELF-3. The cells were also transfected with 1 µg of pCMV--gal. The total amount of DNA transfected was kept
constant by the addition of FNpcDNA3.0. Data are presented as fold
activation relative to cells transfected with
mTR-II(108/+56) alone. Data shown in B and
C are means and S.D. for duplicate measurements from one
representative transfection. Each experiment was repeated twice, and
similar results were obtained in each case.
R-II
promoter.
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Fig. 7.
Effects of a truncated form of mElf-3 on the
expression of mT R-II promoter/reporter gene
constructs in 293T cells. A, 293T cells were
co-transfected with 15 µg of the mTR-II(108/+56)
promoter/reporter construct and 3 µg of the expression plasmid DNA
encoding full-length mElf-3 on its own or together with 5 µg of the
expression plasmid encoding truncated mELF-3(ATH + DBD). The
cells were also transfected with 1 µg of pCMV--gal plasmid to
correct for any differences in transfection efficiency. The total DNA
transfected was kept constant by addition of FNpcDNA3.0. Data are
presented as fold activation relative to the activity observed in cells
transfected with mTR-II(108/+56) alone. Data shown are
means and S.D. for duplicate measurements from one representative
transfection. This experiment was repeated twice, and similar results
were obtained. B, Western blot analysis of cells transfected
with expression vectors encoding full-length mElf-3 protein or the
truncated form of mElf-3(ATH-DBD). Western blot analysis was
performed with an anti-FLAG monoclonal antibody as described under
"Experimental Procedures." The sizes (kDa) and positions of
molecular weight standards are indicated on the right. The
predicted molecular mass of full-length and ATH + DBD is 42.2 and 16.7 kDa, respectively, plus the mass of the FLAG epitope and nuclear
localization sequence added to the N terminus. Western blot analysis
was repeated in a separate experiment, and similar results were
obtained.
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Fig. 8.
Gel mobility shift analysis with the EBS of
mT R-II. A, gel mobility shift
analysis was performed with nuclear extracts prepared from HeLa cells
transfected with expression vectors for mElf-3 or truncated forms of
mElf-3 (ATH + DBD or DBD) as described under "Experimental
Procedures." The arrow indicates the supershifted DBD
complex. B, immunoblots were performed with the M2 anti-FLAG
monoclonal antibody (mAb) and nuclear extracts used in
A. The sizes (kDa) and positions of molecular weight
standards are indicated on the left. The predicted molecular
mass of full-length, ATH + DBD, and DBD is 42.2, 16.7, and 12.3 kDa,
respectively, plus the mass of the FLAG epitope tag and the nuclear
localization signal inserted at the N terminus of the protein. The
relative levels of mElf-3, mElf-3(DBD), and mElf-3(ATH + DBD), which
were determined as described under "Experimental Procedures," are
shown below in parentheses. C, gel mobility shift
analysis was performed with nuclear extracts prepared from
mock-transfected F9-differentiated cells or F9-differentiated cells
transfected with expression vectors for mElf-3 or truncated form of
mElf-3 (DBD) as described under "Experimental Procedures." Nuclear
extracts were added at 5 µl (21.5 µg) or 12 µl (51.6 µg) as
indicated. The asterisk indicates the less intense
DNA-protein complex observed when a higher concentration of nuclear
extract from the cells transfected with the mElf-3 DBD was used.
Competitor DNAs (wild type and mutant) described under "Experimental
Procedures" were added at 100-fold molar excess.
R-II promoter/reporter constructs
(Figs. 5-7). To test this directly, we examined whether mElf-3 could
bind to the EBS in the TR-II promoter in the
promoter/reporter gene construct TR-II(108/+56)
using ChIP analysis. This study was performed in 293T cells, because
they can be transfected at much higher efficiency than
F9-differentiated cells and because mElf-3 stimulates the
TR-II promoter in these cells. In addition, because an
antibody to Elf-3 was not available, 293T cells were transfected with
expression vectors that code for FLAG epitope-tagged mElf-3
(full-length) or for FLAG epitope-tagged mElf-3 DNA binding domain. For
the immunoprecipitation step, two antibodies were employed, the M2 antibody, which recognizes the FLAG epitope, and a control antibody, which recognizes the DNA binding domain of the yeast transcription factor Gal4. Both antibodies were conjugated to agarose beads. ChIP
analysis indicates that both mElf-3 and its DNA binding domain can bind
to the TR-II promoter in vivo (Fig.
9). Relative to the Gal4 antibody
control, we observed nearly a 15-fold enrichment of TR-II
promoter DNA with the M2 antibody. This was true for cells transfected
with either the mElf-3 expression vector or the expression vector for
the mElf-3 DNA binding domain. To confirm these results, this
experiment was also performed with M2 and Gal4 antibodies that were not
conjugated to beads. In this case, the antibody-protein-DNA complexes
were collected using protein G-agarose beads. By using this protocol,
we observed a 10-fold enrichment of TR-II promoter DNA
when the cells were transfected with the expression vector for mElf-3
and an 8-fold enrichment when the cells expressed its DNA binding
domain (data not shown). Hence, although full-length mElf-3 does not
bind to DNA in vitro, it can do so in vivo.
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Fig. 9.
Binding of mElf-3 to the T R-II
promoter in vivo. 293T cells were
transfected with the promoter/reporter construct
TR-II(108/+56) and a FLAG-tagged expression vector for
full-length mElf-3 (FL) or its DNA binding domain
(DBD). ChIP analysis was performed as described under
"Experimental Procedures." Quantification of the PCR products was
performed using Kodak Electrophoresis Documentation and Analysis System
and Kodak ID Image Analysis Software.
-helical conformation in polar solvents (52). Moreover,
it was determined that changes in two specific amino acids within the
acidic core (D134A,L143P) greatly reduced transactivation by the
Gal4-hELF-3 fusion protein, which contained hELF-3 amino acids
129-159. Alignment of the amino acid sequences for hELF-3 and mElf-3
demonstrated that they are highly homologous in this region and that
there is perfect conservation of the 13-amino acid acidic region (Fig.
10A). This prompted us to
examine whether comparable changes in mElf-3 would influence its
ability to transactivate. For this purpose, we employed mElf-3 itself
rather than Gal4/fusion proteins. Specifically, we used site-directed
mutagenesis to prepare two modified forms of mElf-3. In one of the
modified forms, aspartate at position 133 was converted to alanine
(mElf-3(D133A)). In the second, leucine 142 was converted to proline
(mElf-3(L142P)). Initially, we compared the transactivation activity of
each mutant to the activity of mElf-3 in F9-differentiated cells (Fig.
10B). Although mElf-3(D133A) was only slightly less active
in stimulating the TR-II(108/+56) promoter/reporter
gene construct, mElf-3(L142P) was nearly 5-fold less active than
unmodified mElf-3. Hence, this study provides further evidence that the
transactivation domain of intact mElf-3, like its Gal4/fusion protein
counterpart, appears to be localized to an acidic region of the
protein.
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Fig. 10.
Effects of point mutations in the putative
transactivation domain of mElf-3. A, comparison of the
human and mouse Elf-3 transactivation domain. The underlined
residues represent a 13-residue acidic transactivation core in
human ELF-3. Amino acids 133 and 142 in the mouse sequence are
underlined. B, F9-differentiated cells were
transfected with 15 µg of the mT R-II promoter/reporter
gene construct mTR-II(108/+56) and 1 µg of an
expression vector for mElf-3, mElf-3(D133A), or mElf-3(L142P). The
cells were also transfected with 1 µg of pCMV--gal plasmid to
correct for any differences in transfection efficiency. The total DNA
transfected was kept constant by addition of FNpcDNA3.0.
Transactivation is represented as relative activity, compared with the
cells transfected with mTR-II(108/+56) construct alone.
Data shown are means and S.D. for duplicate measurements from one
representative transfection. This experiment was repeated twice, and
similar results were obtained in each case. C, 293T cells
were transfected with 15 µg of the mTR-II
promoter/reporter gene construct mTR-II(108/+56)
and 3 µg of expression vector for mElf-3, mElf-3(D133A), or
mElf-3(L142P). The cells were also transfected with 1 µg of
pCMV--gal plasmid to correct for any differences in transfection
efficiency. The total DNA transfected was kept constant by addition of
FNpcDNA3.0. This experiment was repeated, and similar results were
obtained. D, Western blot analysis was performed with the M2
anti-FLAG monoclonal antibody and the nuclear extracts used in
C. The relative levels of mElf-3, mElf-3(D133A), and
mElf-3(L142), which were determined as described under "Experimental
Procedures," are shown in parentheses. This experiment was
repeated, and similar results were obtained.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R-II gene. In this study, we cloned and functionally characterized the 5'-flanking region
of the mTR-II gene in differentiated cells derived from F9 EC cells. Analysis of the mTR-II promoter by transient
transfection of promoter/reporter gene constructs demonstrated the
importance of a cis-regulatory element located between +2 and +56,
which maps to the EBS. In addition, we demonstrate that the
transcription factor mElf-3 binds to the EBS and strongly activates the
mTR-II gene. Importantly, we demonstrate that
differentiation of EC cells leads to the up-regulation of both the
mTR-II gene and the mElf-3 gene. We also
provide evidence that mElf-3 is subject to two other levels of
regulation, one that affects DNA binding and one that influences its
steady-state level of expression.
R-II gene, hELF-3 has been reported to activate
the expression of several markers of epidermal cell differentiation,
including SPRR2A, transglutaminase 3, and profilaggrin (57, 59).
R-II gene in F9-differentiated cells. First, our co-transfection studies demonstrate that mElf-3 strongly stimulates the activity of
mTR-II promoter/reporter constructs containing functional
EBS. Deletion of the region between nucleotides +2 and +56 or
disruption of the EBS by site-directed mutagenesis eliminates the
response of mTR-II promoter/reporter gene constructs to
mElf-3. Second, the dominant negative forms of mElf-3 not only
drastically reduce the stimulation in response to mElf-3, but also
reduce the basal level of the mTR-II promoter/reporter
gene constructs in F9-differentiated cells. In this regard, it should
be noted that this is the first report of a dominant negative form of
ELF-3. Third, and very importantly, northern blot analysis indicates
that increases in the steady-state levels of mElf-3 mRNA
closely parallel increases in mTR-II mRNA, especially
during the first 48 h of differentiation. In fact, this finding
suggests that increases in mElf-3 play a key role in the mechanisms
responsible for the up-regulation of the mTR-II gene. In
the future, we plan to monitor the levels of mElf-3 in nuclear extracts
by western blot analysis. These studies will be performed once suitable
antibodies for mElf-3 become available. Interestingly, northern blot
analysis also demonstrated that the steady-state levels of the
mTR-I gene do not increase during the differentiation of
F9 EC cells. If the sole role of mTR-I is to form a heterodimer with
mTR-II for the purpose of mediating the effects of TGF-, one
would expect to see parallel increases in transcripts for both
receptors. Hence, mTR-I may play a separate role in EC cells.
R-II promoter in vivo
(Fig. 9). In addition, we demonstrate that mElf-3 contains one or more
domains that negatively influence its binding in vitro.
Specifically, we did not observe in vitro DNA binding of
full-length mElf-3 produced in HeLa cells (Fig. 8A),
F9-differentiated cells (Fig. 8C), or 293T cells (data not
shown). In contrast, a truncated mElf-3 containing the DNA binding
domain, but not the other domains, binds readily to DNA in
vitro. Thus, mElf-3 joins a growing list of Ets family members
that contain domains that influence their binding to DNA. To date, at
least eight other members of the Ets family of transcription factors
(SAP-1, Ets-1, Ets-2, Net, Elk-1, ERM, ESE-2, and PEA3) have been shown
to possess binding regulatory domains (45-51). In the case of Ets-1,
the autoinhibitory domains have been mapped and studied extensively
(61-64).
R-II promoter/reporter gene constructs
in F9-differentiated cells (Fig. 5) and 293T cells (Figs. 7 and 10). In
contrast to an inhibitory cofactor, mElf-3 binding to DNA may require
cooperative binding with a positive cofactor that does not interact
with mElf-3 in vitro under the conditions employed in our
gel mobility shift assays. In fact, cooperative binding of
transcription factors is believed to play a central role in promoting
gene activation and, at the same time, preventing promiscuous activation of inactive promoters. A good case in point is Ets-1 and
CBF2/AML1. Interaction of these two proteins counteracts the
autoinhibitory domains of Ets-1 and, as a result, stimulates Ets-1
binding to DNA and enhances Ets-1 transactivation (64, 66). Similarly,
binding of USF-1 to PEA3 acts as a switch that modifies the
autoinhibitory motifs in PEA3 to promote DNA binding activity (67).
Furthermore, other studies (68-70) have shown that the addition of
monoclonal antibodies that recognize specific Ets transcription factors
significantly increases Ets protein binding to their cognate DNA
target. Clearly, one or more of these mechanisms could regulate mElf-3
binding to DNA, and further study of the mechanism(s) involved is
warranted. Understanding the mechanisms involved is likely to shed
important light on the ability of mElf-3 to regulate the
TR-II promoter.
R-II promoter. This substitution is particularly
interesting, because it has been proposed to alter the ability of this
region in hELF-3 to form an -helical conformation (52).
R-II promoter. Our work also suggests that the up-regulation of mElf-3 is
responsible, at least in part, for the up-regulation of the mTR-II gene during the differentiation of EC cells.
Furthermore, our studies suggest that mElf-3 is subject to at least two
other levels of regulation. Given these findings, we suggest that
determining the mechanisms used to regulate the expression, DNA
binding, and turnover of ELF-3 could help identify methods to elevate
the levels of TR-II in diseased tissues where its expression is
aberrantly low.
ACKNOWLEDGEMENTS
FOOTNOTES
Supported in part by NCI Training Grant CA 00476 from the National
Institutes of Health.
,
transforming growth factor-; TR-I, type I TGF- receptor;
TR-II, type II TGF- receptor; TR-III, type III TGF-
receptor; hTR-II, human TR-II; EBS, Ets-binding sites; EC,
embryonal carcinoma; mTR-II, mouse TR-II; DME, Dulbecco's
modified Eagle's; FBS, fetal bovine serum; RA, retinoic acid; CAT,
chloramphenicol acetyltransferase; DBD, DNA binding domain; ATH,
AT-hook domain; ChIP chromatin immunoprecipitation; PIPES,
1,4-piperazinediethanesulfonic acid.
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 H.-J. Kim and R. Lotan Identification of Retinoid-Modulated Proteins in Squamous Carcinoma Cells Using High-Throughput Immunoblotting Cancer Res., April 1, 2004; 64(7): 2439 - 2448. [Abstract] [Full Text] [PDF]
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 K. E. Bethin, Y. Nagai, R. Sladek, M. Asada, Y. Sadovsky, T. J. Hudson, and L. J. Muglia Microarray Analysis of Uterine Gene Expression in Mouse and Human Pregnancy Mol. Endocrinol., August 1, 2003; 17(8): 1454 - 1469. [Abstract] [Full Text] [PDF]
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T. Nowling, C. Bernadt, L. Johnson, M. Desler, and A. Rizzino The Co-activator p300 Associates Physically with and Can Mediate the Action of the Distal Enhancer of the FGF-4 Gene J. Biol. Chem., April 11, 2003; 278(16): 13696 - 13705. [Abstract] [Full Text] [PDF]
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 I. Dozmorov, M. R. Saban, N. P. Gerard, B. Lu, N.-B. Nguyen, M. Centola, and R. Saban Neurokinin 1 receptors and neprilysin modulation of mouse bladder gene regulation Physiol Genomics, February 6, 2003; 12(3): 239 - 250. [Abstract] [Full Text] [PDF]
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