From the Department of Orthopedics and Sports
Medicine, University of Washington School of Medicine, Seattle,
Washington 98195-6500 and the § Department of Orthopedic
Surgery, Osaka University Medical School, 2-2 Yamadaoka, Suita,
Osaka 565-0871, Japan
Received for publication, January 7, 2003, and in revised form, January 27, 2003
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ABSTRACT |
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A specific t(21;22) chromosomal translocation
creates the chimeric EWS/ERG gene in some cases of Ewing's
sarcoma. In the resultant EWS/ERG fusion protein, the N-terminal part
of the ETS family protein ERG is replaced by the N terminus of the
RNA-binding protein EWS. We found that both the EWS/ERG and
COL11A2 genes are expressed in the Ewing's sarcoma cell
line, CADO-ES1. To investigate a potential role for EWS/ERG in
COL11A2 gene expression, we characterized the
COL11A2 promoter and tested the ability of wild-type ERG
and EWS/ERG sarcoma fusion protein to transactivate COL11A2
promoter using a luciferase assay. We found that expression of EWS/ERG, but not wild-type ERG, transactivated the COL11A2 promoter
and that this transactivation required not only the N-terminal region of EWS but also an intact DNA-binding domain from ERG. Electrophoretic mobility shift assay using COL11A2 promoter sequence showed
involvement of EWS/ERG in the formation of DNA-protein complexes, and
chromatin immunoprecipitation assay revealed direct interaction between COL11A2 promoter and EWS/ERG fusion protein in
vivo. EWS/ERG, but not wild-type ERG, bound to RNA polymerase II.
Treatment of cells with the histone deacetylase inhibitor trichostatin
A enabled ERG to transactivate the COL11A2 promoter,
therefore abolishing the differential effects of EWS/ERG and ERG. Taken
together, these findings indicate that the COL11A2 gene is
regulated both by potential ERG association with a histone deacetylase
complex and by direct EWS/ERG recruitment of RNA polymerase II.
Ewing's sarcoma is a highly aggressive bone and soft tissue tumor
that occurs in adolescents and is of unknown tissue origin (1). Most
Ewing's sarcomas exhibit a specific t(11;22) chromosomal translocation
that results in the fusion of EWS to the ETS family member Fli-1. In a
subset of patients, a specific t(21;22) chromosomal translocation
involving the Ewing's sarcoma gene (EWS) and the ETS-related gene (ERG) generates instead an EWS/ERG fusion
protein (2). Due to the sequence homology between ERG and Fli-1, it is
believed that EWS/ERG and EWS/Fli-1 function similarly in causing malignant transformation (3). EWS is known to be an RNA-binding protein
(4), and the N-terminal domain of EWS was recently shown to interact
with RNA Pol1 II and the
transcriptional coactivator CREB-binding protein (5, 6). The C-terminal
domain of EWS recruits splicing factors such as TASR and YB1 (5, 7).
ERG protein, with structural motifs shared by other ETS family members
such as Fli-1, binds to a specific DNA sequence and is a well
characterized transcriptional activator (8). The fusion of N-terminal
EWS and C-terminal ERG therefore generates a hybrid molecule with the
potential to recruit RNA Pol II.
CADO-ES1 is an established human Ewing's sarcoma cell line that can
differentiate into neural and mesenchymal cell lineages (9, 10).
Because expression of mouse Col11a2 has been linked to
neural and mesenchymal cell phenotypes (11, 12), we analyzed this cell
line for expression of the COL11A2 collagen gene. We confirmed that CADO-ES1 cells express both COL11A2 mRNA
and EWS/ERG fusion protein. Since the ERG transcription factor is known
to participate in cartilage differentiation from mesenchyme
(chondrogenesis), a marker of which is the transactivation of
COL11A2 (13, 14), we further investigated whether ERG and/or
EWS/ERG participate in regulating COL11A2 expression. Using
mouse and human promoter constructs, we demonstrate that EWS/ERG
sarcoma fusion protein, but not wild-type ERG, transactivates
COL11A2. We also show that the ETS DNA-binding domain of the
EWS/ERG fusion protein is necessary for transactivation of
COL11A2 and present what we believe is the first evidence
that transactivation by wild-type ERG is repressed by histone
deacetylases. Unlike wild-type ERG, the EWS/ERG sarcoma fusion protein
appears to activate genes through direct interaction with RNA Pol II
and through avoiding repression by histone deacetylases.
Cell Culture and Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)--
CADO-ES1 cells were cultured in RPMI 1640 medium with
10% fetal bovine serum, under 5% CO2 at 37 °C (9).
RT-PCR of EWS/ERG mRNA and the alternatively spliced N-terminal
portion of COL11A2 mRNA were performed as described (2, 15), and
the correct products were confirmed by sequencing. A 415-bp fragment of
the cDNA encoding the C terminus of COL11A2 (16, 17) was amplified using the forward primer (exons 63-64) 5'-agagcttcccgatggagagta-3' and
the reverse primer (exon 66) 5'-gtctgagaaggaggcatccag-3'.
Plasmid Constructs--
Various lengths of the DNA sequences for
mouse Col11a2 promoter regions (9, 10) were cloned into
MluI/XhoI sites of pGL3 basic luciferase reporter
vector (Promega, Madison, WI) (Fig. 1c). To characterize the
minimum human COL11A2 promoter sequence between
The cDNAs for ERG and EWS/ERG were cloned into the pSG5-FL
expression vector (18). The resultant proteins are FLAG epitope-tagged at the N termini. EWS/ERG Transfection and Luciferase Assay--
For promoter analysis,
two duplicate wells of 70% confluent CADO-ES1 or NIH3T3 cells
(American Type Culture Collection) in six-well plates were transfected
with 3.5 µg of pGL3 reporter plasmid, 1 µg of pSG5-FL effector
plasmid, and 0.5 µg of pRL-SV40 Renilla luciferase control
using 30 µl of DOTAP transfection reagent (Roche Molecular
Biochemicals). Forty-eight hours after transfection, cells were assayed
for luciferase activity using Dual Luciferase Reporter Assay System
(Promega) on a TD-20e luminometer (Turner Designs, Sunnyvale, CA). For
inhibition of histone deacetylase, transfected cells were treated with
100 ng/ml trichostatin A (TSA) (Sigma) 18 h before luciferase
assay and compared with transfected cells without TSA treatment.
Transfection was repeated at least three times, and the luciferase
activity was normalized to internal control. The results are shown as
average ± S.E.
Electrophoretic Mobility Shift Assay--
CADO-ES1 nuclear
extract was prepared as described previously (19). Double-stranded
oligonucleotide containing wild-type human COL11A2 promoter
sequence between Chromatin Immunoprecipitation Assay--
To cross-link DNA and
protein, 1 × 108 of HeLa (American Type Culture
Collection) or CADO-ES1 cells were treated with 1% formaldehyde for 5 min at room temperature. Chromatin solution was then prepared as
described previously (20). For immunoprecipitation, 10 µl of antibody
against acetylated histone H3 (anti-Ac-H3; Upstate Biotechnology, Inc.,
Lake Placid, NY) or antibody against the N-terminal domain of EWS
(N-18; Santa Cruz Biotechnology) was incubated with chromatin solutions
overnight at 4 °C on a rotating wheel. The immunocomplexes were
collected with salmon sperm DNA/Protein A-Sepharose beads and washed
sequentially with 200 µl of each of the following buffers containing
protease inhibitor mixture (Sigma): once with wash buffer I (20 mM Tris, pH 8.0, 2 mM EDTA, 0.1% SDS, 1%
Triton X-100, 150 mM NaCl), once with wash buffer II (20 mM Tris, pH 8.0, 2 mM EDTA, 0.1% SDS, 1%
Triton X-100, 500 mM NaCl), once with wash buffer III (10 mM Tris, pH 8.0, 1 mM EDTA, 1% Nonidet P-40,
0.25 M LiCl, 1% sodium deoxycholate), and twice with PBS.
Formaldehyde cross-linking was reversed by overnight incubation at
65 °C in 0.2 M NaCl plus 200 µg/ml proteinase K
(Sigma). The mixture was then extracted with phenol/chloroform, and the
DNA was precipitated with ethanol and resuspended in 20 µl of
H2O. 20 ng of the DNA template was then used for PCR
amplification of GAPDH and COL11A2 promoter
regions. Primers for GAPDH were 5'-tcctcctgtttcatccaagc-3'
( Immunoprecipitation and Western Blotting--
CADO-ES1 cells or
transfected NIH3T3 cells in a 10-cm dish were lysed with 1.2 ml of
buffer A (10 mM Tris, pH 7.4, 100 mM NaCl, 2.5 mM MgCl2, 0.5% Triton X-100, 10 mM
DTT) supplemented with protease inhibitor mixture and phosphatase
inhibitor mixture (Sigma). The 8WG16 mouse monoclonal anti-RNA Pol II
antibody (Research Diagnostics, Inc., Flanders, NJ) or a control mouse
IgG (Santa Cruz Biotechnology) was incubated with 40 µl of Protein
A/G PLUS-agarose (Santa Cruz Biotechnology) and 0.2 ml of buffer A for
50 min at 4 °C. The complex was then incubated with 0.2 ml of fresh
cell lysate for 60 min at 4 °C on a rotating wheel. After four
washes with radioimmune precipitation buffer (50 mM Tris,
pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS) at 4 °C, 40 µl of 1× SDS sample buffer
was added to the agarose beads. The protein samples were denatured for
5 min at 95 °C, separated by 8% SDS-PAGE, transferred onto a
polyvinylidene difluoride membrane, and subjected to Western blotting
with the C-20 rabbit polyclonal anti-ERG antibody (Santa Cruz
Biotechnology), followed by the anti-Pol II antibody. Protein bands
were visualized with the ECL Western blotting analysis system (Amersham
Biosciences).
CADO-ES1 Cells Express Both COL11A2 and
EWS/ERG--
Our previous RT-PCR analysis revealed that the
expression and splicing patterns of COL11A2 are linked to a
chondrocytic phenotype in osteochondrogenic tumors (15). The Ewing's
sarcoma cell line CADO-ES1 also expressed COL11A2
transcripts, but the splicing pattern of COL11A2 transcripts
in Ewing's sarcoma cells differed from that in chondrocytes (Fig.
1a, lanes
1 and 2). Specifically, most COL11A2
transcripts in CADO-ES1 cells retained combinations of alternative
exons 6-8, whereas those in chondrocytes did not. Further analysis by
RT-PCR and DNA sequencing showed that CADO-ES1 cells also express
EWS/ERG fusion transcript of type I (Fig. 1, a,
third panel from top, and
b).
Although there are no reported effects of EWS/ERG fusion protein on
collagen gene expression, a function for wild-type ERG protein in
chondrogenesis is well documented (14, 21). Chondrogenesis can be
monitored by the induction of chondrocyte-specific collagen genes
(e.g. type II, IX, and XI collagens) (13). We hypothesized that ERG and/or EWS/ERG are directly involved in the expression of
collagen genes and, in particular, the COL11A2 gene in
CADO-ES1 Ewing's sarcoma cells. It has been reported that the mouse
Col11a2 promoter sequence,
In order to identify the promoter sequence responsible for
COL11A2 expression in CADO-ES1 cells, a range of pGL3
luciferase reporter plasmids containing various lengths of mouse
Col11a2 promoter sequence were generated (Fig.
1c). The mouse Col11a2 promoter sequence from
The Human COL11A2 Promoter Contains Multiple Regulatory
Subregions--
Inspection of the DNA sequence between
Collagen gene regulation by Sp1 has been documented extensively (25),
whereas protein interactions with the TRT sequence have not been
thoroughly investigated. To detect potential interactions between
CADO-ES1 nuclear proteins and the TRT sequences within human
COL11A2 promoter, an electrophoretic mobility shift assay was performed using a 40-bp DNA oligonucleotide matching
We next performed a chromatin immunoprecipitation assay to determine
whether EWS/ERG fusion protein selectively binds to COL11A2 promoter in CADO-ES1 cells. Acetylated histone molecules are known to
be enriched in areas near actively transcribed genes such as GAPDH (20). HeLa cells do not express COL11A2;
therefore, the COL11A2 promoter region was not
co-precipitated with an anti-Ac-H3 antibody in the chromatin
immunoprecipitation assay (Fig. 3c, compare lanes
9 and 10). In CADO-ES1 cells, the
COL11A2 gene is active, and its promoter sequence becomes
cross-linked to acetylated histone H3 after treatment with formaldehyde
(Fig. 3c, compare lanes 11 and
12). Interestingly, COL11A2 promoter sequence was enriched in immunocomplexes using an antibody that recognizes the
N-terminal domain of EWS/ERG, and this enrichment appeared to be
specific as GAPDH DNA was absent from the same
immunocomplexes (Fig. 3c, lane 13).
These results suggest that EWS/ERG fusion protein interacts with
COL11A2 promoter in vivo.
EWS/ERG, but Not ERG, Activates the COL11A2 Promoter in
NIH3T3 Cells--
To investigate whether ERG and EWS/ERG can
transactivate the COL11A2 promoter, ERG and EWS/ERG
expression constructs were generated for co-transfection with the
COL11A2 promoter reporter construct. Our initial experiments
showed minimal effects of ERG or EWS/ERG on COL11A2 promoter
activity when transfected into CADO-ES1 cells (data not shown). We
suspected that this might be caused by abundant endogenous EWS/ERG in
CADO-ES1 cells; therefore, the experiments were repeated with NIH3T3
cells that do not express EWS/ERG or
Col11a2 (26). Whereas wild-type ERG protein had no effect on
COL11A2 promoter activity, the EWS/ERG sarcoma fusion protein potently transactivated human COL11A2 promoter
construct H149 (Fig. 4). To investigate
whether the transactivation was dependent on the ability of EWS/ERG to
bind DNA, we constructed EWS/ERG mutants that lacked the EDB domain or
had either of two EWS/ERG point mutants (W351R or R367L) within the EDB
domain (Fig. 4a). The W351R mutation of EWS/ERG is
equivalent to the W321R mutation of Fli-1, an ETS protein with high
sequence homology to ERG. Both point mutations are known to eliminate
the DNA binding activity of Fli-1 and ERG (27, 28). When co-transfected
into NIH3T3 cells with the COL11A2 promoter construct H149,
neither the EWS/ERG EWS/ERG, but Not ERG, Is Physically Associated with RNA
Pol II, and EWS/ERG Is Less Susceptible than ERG to
Repression by Histone Deacetylases--
Although both ERG and EWS/ERG
have identical DNA binding domains, our findings show that only EWS/ERG
is able to transactivate the COL11A2 promoter construct. It
is important to understand the mechanism underlying this intriguing
finding, since EWS is known to fuse with the DNA-binding domains of a
variety of transcription factors in different sarcomas. Part of the
explanation for the differential action may lie in the properties of
the EWS component of the EWS/ERG fusion protein. Both wild-type EWS and
EWS/Fli-1 sarcoma fusion protein have been reported to associate with
RNA Pol II (5). If the N-terminal region of EWS is indeed responsible for binding to RNA Pol II, then EWS/ERG should likewise interact with
RNA Pol II. To test for this, CADO-ES1 cell lysate was treated with an
anti-RNA Pol II antibody. Endogenous EWS/ERG but not ERG was
co-precipitated with the RNA-Pol II, whereas a control mouse IgG did
not immunoprecipitate either protein (Fig.
5a). With lysate from
transfected NIH3T3 cells, the same anti-RNA Pol II antibody pulled down
the FLAG-tagged EWS/ERG but not ERG (Fig. 5b). Together, these results show that EWS/ERG, but not ERG, binds to RNA Pol II.
Another property contributing to this dichotomous behavior could be a
loss of function from the absence of the N-terminal ERG domain in the
EWS/ERG sarcoma fusion protein. Our recent cloning and analysis of an
ERG-associated protein with a SET domain (ESET) revealed that ESET
protein acts as a histone H3-specific methyltransferase (29). Histone
methyltransferases usually function in complexes with histone
deacetylases to silence gene expression (30-32), and ESET has been
shown to recruit histone deacetylases as well as mSin3 transcriptional
corepressors (33). Since EWS/ERG lacks the N-terminal domain of ERG
responsible for binding to ESET, we suspected that ESET-associated
histone deacetylases could suppress ERG-mediated transactivation,
whereas these same histone deacetylases would have no such effect on
EWS/ERG.
Our approach in testing this hypothesis is based upon prior studies of
histone deacetylases using reporter constructs (34-36). Transiently
transfected plasmid DNA does not usually assume a native chromatin
structure, but published observations indicate that histone molecules
do associate with plasmid DNA to form nucleosome-like particles (37,
38). A shift toward increased acetylation of these nucleosome-like
particles can result in a more open structure that is accessible to the
transcriptional machinery. In fact, inhibitors of histone deacetylases
have been reported to similarly up-regulate promoter constructs in both
transiently and stably transfected cells (39, 40).
We co-transfected NIH3T3 cells in duplicate plates with the
COL11A2 promoter construct H149 plus either ERG or EWS/ERG
expression plasmid and then treated cells in one plate with TSA,
a well known inhibitor of histone deacetylases (41). Transfected cells
in the other plate were not treated with TSA; therefore, they
were used as a control to calculate -fold derepression by TSA. Whereas the ability of wild-type ERG protein to transactivate the
COL11A2 promoter was significantly increased in the presence
of TSA, transactivation by EWS/ERG fusion protein was not as profoundly
influenced by TSA-mediated inhibition of histone deacetylases (Fig.
6). The differential effects of EWS/ERG
and ERG on COL11A2 promoter thus appear to result in part
from their differing abilities to recruit RNA Pol II and to associate
with histone deacetylases.
Our findings indicate that ERG and the sarcoma fusion protein
EWS/ERG differentially regulate expression of the COL11A2
gene. Although both wild-type ERG and the EWS/ERG fusion protein share the same DNA-binding domain and are expected to bind to the same promoter sequence, the human COL11A2 promoter construct is
transactivated by EWS/ERG but not by ERG. Recent reports have shown
that EWS fusion proteins can inhibit expression of several genes (3, 42). In contrast, our results together with a recent report on
induction of the Id2 gene by EWS-ETS (43) reveal that an EWS
fusion protein is able to activate an authentic promoter that is not
activated by its wild-type partner. We also show that mutations within
the tandem repeat of tcc trinucleotides of the COL11A2 promoter sequence or within the ETS-DNA binding domain of EWS/ERG protein interfere with COL11A2 activation. Furthermore,
electrophoretic mobility shift and chromatin immunoprecipitation assays
confirm the direct interaction of EWS/ERG and/or ERG with the
COL11A2 promoter. Thus, transactivation is at least
partially mediated by interaction between the COL11A2
promoter and the DNA binding domain of EWS/ERG.
In this study, we also investigated why EWS/ERG, but not ERG,
transactivates COL11A2. Our study points to at least two
potential mechanisms. First, since inhibition of histone deacetylase
significantly enhanced the effects of ERG on COL11A2
promoter activity, transactivation by ERG appears to be epigenetically
suppressed. This is very similar to the proposed mechanism for Fli-1
inhibition of COL1A2 transcription (25). Interestingly,
recent studies have shown that a murine Kruppel-associated box-zinc
finger protein represses Col11a2 promoter activity (44), and
human ESET (also called SETDB1) is known to bind to the universal,
obligatory corepressor of Kruppel-associated box-zinc finger proteins
(45). On the other hand, a recent study showed that the N-terminal
region of wild-type EWS interacts with CREB-binding protein that has
histone acetyltransferase activity (6). It may be that EWS/ERG also
associates with CREB-binding protein and promotes derepression of
transcription via acetylation of histones bound to its target
promoters. Second, as evidenced by immunoprecipitation experiments,
EWS/ERG fusion protein recruits RNA Pol II in vivo, whereas
wild-type ERG does not. By fusing the N terminus of EWS to the
DNA-binding domain of ERG, the EWS/ERG oncogenic protein is able to
recruit RNA Pol II directly to the site of transcription, and this
could serve to bypass suppression by histone deacetylase complexes.
These findings therefore provide new insights into the mechanisms
underlying collagen gene expression in general and COL11A2
expression in particular.
In addition to transcriptional deregulation, EWS sarcoma fusion
proteins are reported to interfere with basic cellular processes such
as RNA splicing. Unlike wild-type EWS protein, the C-terminal domains
of EWS fusion products are unable to recruit serine-arginine splicing
factors (5), which may partly explain the abnormal splicing patterns in
Ewing's sarcoma cells. The finding that EWS/ERG binds RNA Pol II also
suggests a coupled mechanism for stimulated gene expression and altered
RNA splicing. Since gene transcription and RNA splicing are physically
linked via the RNA Pol II holoenzyme, EWS/ERG has the potential to
deregulate gene activation, epigenetic gene repression, and RNA
splicing. All of these processes are integral to cell growth and differentiation.
The expression of COL11A2 in CADO-ES1 cells supports a
neural/mesenchymal origin of Ewing's sarcoma (10, 46). Interestingly, the regulatory elements of the COL11A2 active in CADO-ES1
cells differ from those active in cells of cartilage and neural
lineages (12, 47). The COL11A2 promoter shares many features
with certain housekeeping promoters (23), such as the presence of
G/C-rich sequences, the absence of a TATA box, and multiple
transcription start sites. Similar to the findings here with the
COL11A2 promoter, the TATA-less, GC-rich promoters of the
human heparanase-1 and the mouse thymidylate synthase genes are
synergistically regulated by ETS family transcription factors and the
Sp1 protein (48, 49). It is likely that other genes with promoter
properties in common with COL11A2 are also differentially
regulated by EWS/ERG and ERG, and the implications, we believe, are
therefore fundamental to an understanding of Ewing's tumor pathobiology.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
149 and
+27 bp (H149 construct in Figs. 1c and 2a),
deletion constructs were created as H84 (
84 to +27 bp), H68 (
68 to
+27 bp), and H40 (
40 to +27 bp). In addition, mutations were
introduced into human COL11A2 promoter constructs as
follows. mTRT (mutated tandem repeat of the tcc trinucleotides)
contains a 4-base mutation from tccctcc to
tAActAA located between
130 and
124 bp of the H149 construct; dH84
(deletion mutant of H84 construct) contains sequences of
84 to
59
bp and
40 to +27 bp; mSp1-u (mutated upstream Sp1 site) contains an
8-base mutation from gggcgggcgg to TTTcTTTcTT located
between
82 and
73 bp of the H149 construct; mSp1-d (mutated
downstream Sp1 site) contains a 5-base change mutation from
gggcgg to TTTcTT located between
50 and
45 bp of the
H149 construct; mSp1-u/d contains mutations at both upstream and
downstream Sp1 sites of the H149 construct.
ETS DNA-binding (EDB) domain was created by digestion with restriction enzymes BamHI/NcoI
and subsequent self-ligation, resulting in the deletion of 39 residues
from amino acids 343-381 of ERG within the EDB domain. Point mutations
were introduced into the EDB domain of EWS/ERG using the Gene
EditorTM site-directed mutagenesis kit (Promega).
149 and
110 bp was used in the electrophoretic
mobility shift assay. An otherwise identical probe mutating
tccctcc to tAActAA (Mut) was also used in the
assay. 32P-Labeled DNA probe was incubated with CADO-ES1
nuclear extract (1 µg of protein) for 20 min at room temperature in
the presence of poly(dI-dC) (50 µg/ml). The DNA-protein complexes
were analyzed on a 5% nondenaturing polyacrylamide gel. For
competition assay, unlabeled wild type or Mut was added to the reaction
10 min before the addition of the 32P-labeled wild type.
Supershift experiments were done by the addition of C-20X rabbit
polyclonal anti-ERG antibody (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA) to the reaction mixture 1 h after the formation of the
DNA-protein complexes. The reaction was continued for 3 h at
4 °C. Equal amount of normal rabbit IgG was added to a control sample to exclude nonspecific interactions.
358 to
377 bp from the transcriptional start site) and
5'-tagtagccgggccctacttt-3' (
141 to
160 bp from the transcriptional
start site). Primers for COL11A2 were
5'-caggagagagcgagcgatag-3' (
167 to
147 bp from the transcriptional
start site) and 5'-cccggcccggcccccgcctccagccgcccgcccacagcca-3' (
89 to
50 bp from the transcriptional start site). The resulting products, 218 bp for GAPDH and 118 bp for
COL11A2, were separated by agarose gel electrophoresis.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of COL11A2 and EWS/ERG transcripts
in CADO-ES1 Ewing's sarcoma cells. a, RT-PCR analysis
of COL11A2 and EWS/ERG transcripts from
CADO-ES1 cells (lane 1), human chondrocytes
(lane 2), and human skin fibroblasts
(lane 3). Splice variants containing different
combinations of exons 6-8 covering the N-terminal COL11A2-coding
region are identified. Exon 0 lacks any of the three exons.
b, sequence analysis of the EWS/ERG transcript
indicates that CADO-ES1 cells express the type I EWS/ERG
fusion transcript. c, the head-to-tail arrangement of the
mouse Col11a2 and Rxrb genes (top) and
the human COL11A2 and RXRB genes
(bottom). The transcription start site of mouse
Col11a2 corresponds to 327 bp from the human
COL11A2 transcription start site. Cartilage-specific
(filled circle) and neural tissue-specific
(filled square) cis-elements are shown in the
mouse Col11a2 promoter sequence. Horizontal
lines mark the mouse Col11a2 and human
COL11A2 promoter constructs to scale. d, firefly
luciferase reporter constructs were transfected into CADO-ES1 cells,
and the promoter activity was normalized to the Renilla
luciferase control.
742 to +380 bp from the
transcriptional start site, is sufficient to drive reporter gene
expression (11), and multiple subregions between
742 and
453 bp are
known to be involved in cartilage- and neural tissue-specific promoter activities (12). The mouse Col11a2 gene is located next to
the retinoic acid receptor
(Rxrb) gene in a head-to-tail
arrangement (Fig. 1c, top line) (11).
Human COL11A2 and RXRB are arranged similarly.
The intergenic DNA sequences of mice and humans are highly homologous
(Fig. 1c, bottom line) (22). The
transcriptional start site in mouse corresponds to
327 bp from the
human transcriptional start site (Fig. 1c) (11, 23).
742 to +380 bp was activated when transfected into CADO-ES1 cells
(Fig. 1, c and d, construct
742).
Interestingly, in CADO-ES1 cells, the mouse promoter constructs
530
and
500 containing either the cartilage- or the neural
tissue-specific cis-elements (Fig. 1c, top
line) showed similar activity to the
453 construct
containing only the constitutive promoter sequence between
453 and
+380 bp (Fig. 1, c and d) (12). Further deletion analysis identified a necessary minimum sequence between +133 and +380
bp of the mouse promoter, which corresponds to
178 to +37 bp of the
human COL11A2 promoter (Fig. 1, c and
d, compare construct +133 and construct +264). The
evolutionally conserved human COL11A2 promoter sequence
between
149 and +27 bp was therefore cloned into the luciferase
reporter vector and was equivalent in activity to the intact mouse
Col11a2 promoter when transfected into CADO-ES1 cells (Fig.
1, c and d, construct H149).
149 and +27
bp of the human COL11A2 promoter revealed
tccctcc, a tandem repeat of the
tcc trinucleotides (TRT) (24), as well as at least two
gggcgg Sp1 binding sites (Fig.
2a, construct H149). To test
them for functional activity, a series of deletion and mutation
constructs were generated for transfection (Fig. 2a).
Deletion between
149 and
85 bp, containing the TRT sequence decreased promoter activity by 50% (Fig. 2, construct H84). Mutation in the TRT sequence, from tccctcc to tAActAA
(uppercase letters indicate mutated nucleotides), also decreased
promoter activity (Fig. 2, construct mTRT), suggesting that the TRT
sequence is involved in the expression of COL11A2 in
CADO-ES1 cells. Deletion between
149 and
69 bp, containing the TRT
sequence and the upstream Sp1 site (Sp1-u) decreased promoter activity
by 70% (Fig. 2, construct H68). Deletion of the TRT sequence and the
downstream Sp1 site (Sp1-d) also decreased promoter activity by 70%
(Fig. 2, construct dH84). Deleting the DNA sequence between
149 and
41 bp nearly abolished the promoter activity (Fig. 2, construct H40),
thus confirming the importance of the TRT sequence and Sp1
sites. Involvement of both Sp1 sites was further supported by mutations
from gggcgggcgg to TTTcTTTcTT for the upstream Sp1 site and
from gggcgg to TTTcTT for the downstream Sp1 site. Whereas
mutating just one of the two Sp1 sites resulted in a 50% decrease
(Fig. 2, constructs mSp1-u and mSp1-d), mutations at both Sp1 sites led
to a 75% decrease in promoter activity (Fig. 2, construct mSp1-u/d).
Together, these results indicate that in CADO-ES1 cells, the TRT site,
and the Sp1 sites coordinately regulate COL11A2 promoter
activity.
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Fig. 2.
Mapping of the minimum promoter sequence for
COL11A2 in CADO-ES1 cells. a, the human
COL11A2 promoter region between 149 and +27 bp is shown
with potential transcription factor-binding sites
underlined. Deletions and mutations that target the tandem
repeat of the tcc trinucleotides (TRT) sequence and/or the two Sp1
sites are illustrated. Mutated nucleotides are indicated with
uppercase letters. b, mutant reporter
constructs were transfected into CADO-ES1 cells, and luciferase
activity was normalized to the Renilla luciferase
control.
149 to
110
bp from the COL11A2 transcriptional start site (Fig.
2a). CADO-ES1 nuclear extract and the TRT sequence formed
DNA-protein complexes designated as C1 and C2 (Fig.
3a, compare lanes
1 and 2). The addition of wild-type TRT
competitor prevented formation of the C1 and C2 complexes (Fig.
3a, lane 3), but an excess of the
mutated TRT DNA had little effect on the complex formation (Fig.
3a, lane 4). Furthermore, both C1 and
C2 complex formation was eliminated by a specific anti-ERG antibody
that recognizes the C-terminal portion of EWS/ERG fusion protein but
not by a control IgG (Fig. 3b, lanes 7 and 8). Thus, EWS/ERG and/or ERG in CADO-ES1 nuclear extract
appear to specifically interact with the TRT sequence within the human
COL11A2 promoter.
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Fig. 3.
Interaction of Col11A2 promoter sequence with
CADO-ES1 nuclear proteins. a, the DNA-protein complexes
(lane 2) were eliminated by the addition of an
excess amount of wild-type TRT sequence (lane 3)
but not by mutant TRT sequence (Mut) (lane
4). b, the DNA-protein complexes (lane
6) were eliminated by the addition of an anti-ERG antibody
that recognizes the C-terminal region of EWS/ERG (lane
7) but not by a control IgG (lane 8).
c, chromatin immunoprecipitation assay of a 218-bp
GAPDH genomic fragment ( 358 to
141 bp to the
transcriptional start site, top panel) and a
118-bp COL11A2 genomic fragment (
167 to
50 bp to the
transcriptional start site, bottom panel) was
performed with HeLa cells (lanes 9 and
10) and CADO-ES1 cells (lanes 11-13).
Antibodies used in the assay are indicated. Input DNA represents a
portion of the sonicated chromatin prior to immunoprecipitation.
EDB nor the two point mutations were able to
transactivate the promoter (Fig. 4b), suggesting that a
functional EDB domain within EWS/ERG is critical for activation of the
COL11A2 promoter.
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Fig. 4.
Differential effects of ERG and EWS/ERG on
the COL11A2 promoter. a, schematic of EWS, ERG,
EWS/ERG, and mutant EWS/ERG constructs. Deletion of the EDB domain is
shown by a dotted line, and point mutations are
indicated by asterisks. QSY, glutamate-, serine-,
and tyrosine-rich domain; RGG, regions with multiple
Arg-Gly-Gly repeats; RNP-CS, ribonucleoprotein consensus
sequence. b, effect of ERG and EWS/ERG expression constructs
on the activity of wild-type COL11A2 promoter construct H149
in NIH3T3 cells.
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Fig. 5.
Association of EWS/ERG with RNA Pol II.
a, CADO-ES1 cell lysate (lane 1) was
immunoprecipitated (IP) with 8WG16 anti-RNA Pol II antibody
(lane 2) or a control mouse IgG (lane
3). Lysate and IP samples were electrophoresed and blotted
with an antibody that recognizes both ERG and EWS/ERG. b,
NIH3T3 cells were transfected with ERG or EWS/ERG expression plasmid
(lanes 4 and 5). Lysates were
immunoprecipitated with the anti-Pol II antibody (lanes
6 and 7) or a control mouse IgG (lanes
8 and 9), blotted with the antibody recognizing
both ERG and EWS/ERG.
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Fig. 6.
Derepression of COL11A2 promoter activity by
TSA. NIH3T3 cells in duplicate plates were co-transfected with the
wild-type COL11A2 promoter construct H149 plus ERG or
EWS/ERG expression plasmid. Cells in one plate were assayed for
luciferase activity 18 h after treatment with TSA and compared
with cells from the other plate that were not treated with TSA.
Luciferase activities from TSA-treated cells relative to the
TSA-untreated cells are shown as -fold derepression.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Ken Kodama for the CADO-ES1 cells, Mirka M. Vuoristo for cosmid clone 505-1, Ituro Inoue for a PAC clone containing the human COL11A2 region, and Kristin Rosler for technical assistance in the chromatin immunoprecipitation assay.
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FOOTNOTES |
---|
* This work was supported by a Veterans Affairs Merit Review Award (to H. A. C.) and by National Institutes of Health Grants 1R01CA90941 (to L. Y.), 5R01AR37318, and 5R37AR36794 (to D. R. E.).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.
¶ To whom correspondence may be addressed: Dept. of Orthopedics and Sports Medicine, University of Washington School of Medicine, Box 358280, Seattle, WA 98108. E-mail: lyang@u.washington.edu.
To whom correspondence may be addressed: Dept. of Orthopedics
and Sports Medicine, University of Washington School of
Medicine, Box 356500, Seattle, WA 98195-6500. Tel.: 206-543-4700; Fax:
206-685-4700; E-mail: deyre@u.washington.edu.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M300164200
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ABBREVIATIONS |
---|
The abbreviations used are: Pol, polymerase; CREB, cAMP-response element-binding protein; RT-PCR, reverse transcription-PCR; EDB domain, ETS DNA-binding domain; ESET, ERG-associated protein with a SET domain; TSA, trichostatin A; TRT, tandem repeat of the tcc trinucleotides.
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