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INTRODUCTION |
The coordinated expression of genes is required for the control of
cell proliferation and differentiation during early development and
homeostasis of the adult organism. Coactivator complexes containing histone acetyl transferases, such as p300/CBP and P/CAF, play a
pivotal role in the regulation of gene expression and facilitate transcriptional activation by acetylating conserved lysine residues of
the amino-terminal tails of core histones (1-3). Similarly, high
molecular weight complexes consisting of histone deacetylases and corepressors such as N-CoR/SMRT, mSin3 and
ETO1
(Eight-Twenty-One or MTG8) induce
transcriptional repression when recruited by transcription factors
(3-8). Unbalancing and perturbations of these processes are the causes
of many diseases and contribute to the development of cancer (9), as is
the case for the leukemia-associated fusion genes AML1/ETO,
PML/RAR
, and PLZF/RAR
(2,
10-12).
Apart from the association of ETO with transcriptional
repression, the physiological role of the nuclear protein ETO is still largely unknown. ETO was first identified in a frequent form of acute
myeloid leukemia (AML) with translocation t(8;21) (13), resulting in
the AML1/ETO fusion gene, which occurs in about 40% of
cases of acute leukemia with the M2 French-American-British subtype
(14, 15). In the AML1/ETO translocation product, the transactivation
domain of transcription factor AML1, which would normally bind to the
transcriptional coactivators p300/CBP (16), is replaced by
almost the entire ETO protein. Thus, the fusion protein recruits a
corepressor complex containing HDAC activity instead of the
coactivators p300/CBP. The translocation partner ETO, normally
expressed in brain, shows strong homology with the Drosophila nervy gene, especially in four regions
named nervy homology regions (NHR 1-4). The highly
conserved NHR4 region contains two zinc finger motifs and has been
reported to be essential for the interaction between ETO and N-CoR/SMRT
(10, 17-19). Furthermore, it has been shown that a corepressor complex
containing ETO also binds to mSin3 and HDAC2 (10), although it was not
clear whether these interactions are direct. Another conserved element
is the amphipathic helix structure, NHR2 (20), which induces
homodimerization and binding to homologous family members, such as
MTGR1 (21). Recent reports indicate that the oncogenic potential and
transcriptional repressor activity of the translocation product
AML1/ETO requires NHR2-induced dimerization and oligomerization (22).
Despite the insight into the function of the oncogene
AML1/ETO, the precise physiological role of ETO and its
family members is not yet clear, because they show no DNA binding
activity. However, they can potentiate transcriptional repression
induced by other transcription factors, such as the promyelocytic
leukemia zinc finger protein, by synergistically recruiting
corepressors and histone deacetylase (23). In this study we define a
"core repressor domain" in ETO, which contributes strongly to
repressor activity, homo- and heterodimerization, and high molecular
weight complex formation. Our data indicate that multiple regions of
ETO work synergistically to repress transcription but have little
repressor activity on their own. These data give new insights into the
formation and function of the corepressor complex and may help to
identify new strategies for the treatment of AML1/ETO-induced leukemias.
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MATERIALS AND METHODS |
Cell Culture and Plasmids--
293T cells were maintained in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum (Biochrom KG, Berlin, Germany),
50 units per ml penicillin, 50 µg per ml streptomycin, and 2 mM L-glutamine (all from Life Technologies,
Inc.). The expression plasmids for GAL4-ETO fusion proteins were
generated by subcloning the diverse ETO DNA fragments in frame with the
GAL4 DNA binding domain (residues 1-147) of pCMX-GAL4 (5). Additional
XhoI sites were introduced into pcDNA3-ETO (kindly
provided by Olaf Heidenreich, Department of Molecular and Cellular
Biology, University of Tübingen, Tübingen, Germany) by
site-directed mutagenesis (Stratagene, La Jolla, CA) at the following positions (amino acid residues indicated):
pcDNA3-ETO/XhoI-133 (upstream primer
5'-CCTTACTACCCTCGAGCAGTTTGGCA-3');
pcDNA3-ETO/XhoI-234 (upstream primer
5'-TTTCGTTCACCTCGAGAAGCAGCTCT-3');
pcDNA3-ETO/XhoI-323 (upstream primer
5'-CCACAGGGACCTCGAGGACAGAA ACA-3');
pcDNA3-ETO/XhoI-399 (upstream primer
5'-GTACAGTGACCTCGAGGACTTAAAAA-3'). The XhoI-XbaI(blunt) fragments of ETO were then
cloned between the SalI-EcoRV restriction sites
of pCMX-GAL4. To construct GAL4-ETO
1-501, a SalI site
was created in pcDNA3-ETO at residue 500. The
SalI-XbaI(blunt) fragment was then inserted
between the SalI and EcoRV sites of pCMX-GAL4.
Gal4-ETO
1-236
C was constructed by inserting two XbaI
sites in Gal4-ETO
1-236 at residues 382 (upstream primer
5'-CTAAAGCGGTGTCTAGAAGCAGACC-3') and 430 (upstream primer 5'-GACGCGCATCTAGAATTCCTTCAC-3') and by removing the sequence in between the two XbaI sites.
To generate GAL4-ETO
1-236
NHR2, two XbaI sites were
created in Gal4-ETO
1-236 at residues 337 (upstream primer
5'-GCATGGCACACGTCTAGAAGAAATGATTG-3') and 382 (upstream primer
5'-CTAAGGCGGTGTCTAGAAGCAGACC-3'). The sequence
between these two sites was then deleted. GAL4-NHR2+C was created by
removing the sequence in between the EcoRI sites (from
residue 432 to the carboxyl terminus) in GAL4-ETO
1-321. GAL4-N was constructed by deleting the sequence in between the MscI and EcoRI sites (from residue 306 to the
carboxyl terminus) in GAL4-ETO
1-236. To generate GAL4-NHR2
and GAL4-C, the corresponding inserts were cut with SalI and
HindIII (blunt) out of pGEX-AHK-NHR2 and pGEX-AHK-C (see
below), respectively, and subcloned into the SalI and
EcoRV sites of pCMX-GAL4. All constructs were verified by
automated DNA sequencing on an ALF DNA sequencer (Amersham Pharmacia Biotech).
The GST fusion constructs were generated by subcloning the ETO DNA
fragments, in frame, with the GST domain of pGEX-AHK (5). GST·AML1/ETO and GST·ETO were constructed by inserting a
SalI restriction site in pcDNA3-AML1/ETO and
pcDNA3-ETO (both provided from Olaf Heidenreich, Tübingen,
Germany), respectively, immediately in front of the coding region and
by subcloning the SalI-XbaI fragments into the
respective sites of pGEX-AHK. GST·NHR3+4 was generated by inserting
the EcoRI-XbaI fragment of pcDNA3-ETO between the respective sites of pGEX-AHK. GST·NHR4 was created by subcloning the SalI-XbaI fragment of
pcDNA3-ETO-SalI-512 into the respective sites of
pGEX-AHK. GST·N, GST·N+NHR2, GST·NHR2, GST·NHR2+C, and GST·C
were generated by ligation of the
XbaI-HindIII-digested polymerase chain reaction
products of pcDNA3-ETO into the respective sites of pGEX-AHK. For
the amplification of polymerase chain reaction fragments, the following
primers were used: 5'-CGCTCTAGACTCGATGTGAACGAAAACGGG-3' as a
common upstream primer for GST·N and GST·N+NHR2,
5'-CGCTCTAGAAGGGACCTCAGGGACAGAAAC-3' as a common upstream primer for
GST·NHR2 and GST·NHR2+C, 5'-CGCTCTAGAGCAGACCGGGAAGAATTG-3' as
upstream primer for GST·C, 5'-CGTCCCAAGCTTGTTTCTGTCCCTGAGGTCCCT-3' as
downstream primer for GST·N, 5'-CGTCCCAAGCTTCAATTCTTCCCGGTCTGC-3' as
a common downstream primer for GST·N+NHR2 and GST·NHR2, and 5'-CGTCCCAAGCTTGAATTCCCGATGCGCGTCTAG-3' as a common downstream primer for GST·NHR2+C and GST·C. All constructs were verified by
DNA sequencing.
Transcriptional Repression Assays--
293T cells were
transfected in triplicate with 0.75 µg of the indicated
pCMV-Gal4-ETO plasmids, 1.5 µg of 2xUAS-thymidine kinase-luciferase plasmid (5), and 1 µg of a promoterless renilla luciferase plasmid by calcium phosphate coprecipitation (5). 48 h
after transfection the cells were lysed, and luciferase activity was
measured using the Dual-Luciferase Reporter Assay system (Promega Corporation, Madison, WI) following the protocols provided by the
manufacturer. Repression is given relative to the luciferase activity
obtained by the DNA binding domain of Gal4 alone. Experiments were
repeated at least five times, and results are indicated as the means
with S.D.
In Vitro Protein Interaction Analysis: Glutathione S-Transferase
(GST) Pull-down Assays--
Assays were performed as described
elsewhere (5). In short, GST and the indicated GST fusion proteins were
expressed in Escherichia coli BL21 codon+ cells (Stratagene,
La Jolla, CA), and equal amounts of each were immobilized on
glutathione-Sepharose beads (Sigma-Aldrich). Full-length ETO as well as
the indicated ETO fragments were transcribed and translated in
vitro in the presence of [35S]methionine (Amersham
Pharmacia Biotech) by using the TNT T7 coupled reticulocyte lysate
system (Promega Corporation) according to the manufacturer's
instructions. For precipitation assays, equal amounts of GST fusion
proteins were incubated with adequate amounts of the ETO-TNT reaction
mixture in 100 µl of PPI buffer (50 mM HEPES, pH 7.8, 50 mM NaCl, 5 mM EDTA, 1 mM
dithiothreitol, 0.02% Nonidet P-40 containing a protease inhibitor
mixture (Roche Diagnostics GmbH), and 0.5 mM
phenylmethylsulfonyl flouride (Sigma-Aldrich) for 20 min at 37 °C.
The beads were washed four times, and the bound proteins were eluted by
boiling in Laemmli buffer (Roth, Karlsruhe, Germany) and
subjected to SDS-polyacrylamide gel electrophoresis (PAGE). The gel was
then fixed in gel drying solution (Bio-Rad) for 30 min, dried, and
subjected to autoradiography.
Coimmunoprecipitation Experiments--
In vitro
translated mSin3A was incubated with the indicated
35S-labeled translated ETO polypeptides in 50 µl of NETN
buffer (20 mM Tris, pH 8, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol,
0.5% Nonidet P-40) for 30 min at 37 °C. 1 µg of anti-mSin3A
rabbit polyclonal antibody (K20, Santa Cruz Biotechnology, Inc., Santa
Cruz, CA) or 1 µg of anti-GAL4 rabbit polyclonal antibody (Upstate
Biotechnology, Lake Placid, NY) was added to the mixture, and
immunoprecipitation was performed at 4 °C. 35 µl of a 50% slurry
of protein A/G-agarose (Santa Cruz Biotechnology, Inc.) was then added
for 1 h to precipitate the immune complexes. The immune complexes
were washed five times with NETN buffer, the precipitated proteins were
then separated by SDS-PAGE, and the dried gel was subjected to autoradiography.
For coprecipitation experiments using whole cell extracts, 293T cells
(5 × 106 cells seeded in 10-mm-diameter dishes
24 h prior to transfection) were transfected with 20 µg of the
diverse Gal4-ETO constructs. 48 h after transfection, cells were
lysed in NETN buffer supplemented with 0.5 mM
phenylmethylsulfonyl fluoride (Sigma-Aldrich) and a protease inhibitor
mixture (Roche Diagnostics Gmbh). After centrifugation for 5 min at
4 °C, the supernatants were collected and immunoprecipitated for
1 h with 1 µg of anti-mSin3A primary antibody. 40 µl of a 50%
slurry of protein A/G-agarose (Santa Cruz Biotechnology, Inc.) was
added for 1 h to collect the immune complexes, which were then
washed five times with NETN buffer. The precipitated proteins were
eluted from the immune complexes by boiling for 5 min in Laemmli buffer
(ROTH), separated by SDS-PAGE, and transferred to a polyvinylidene
difluoride membrane (Roth). Western blots were blocked for 2 h
with 5% milk and incubated with anti-Gal4 (DBD) primary antibody
(RK5C1; Santa Cruz Biotechnology, Inc.) at 4 °C overnight. After
extensive washing the blots were incubated with a peroxidase-conjugated
secondary antibody for 30 min. After further washing, the proteins were
visualized by enhanced chemiluminescence (Pierce).
Biochemical Purification of Gal4-ETO High Molecular Weight
Complexes--
500 µl of cell extracts obtained from 293T cells
transfected with Gal4-ETO deletion mutants were loaded onto a Superose
6 HR 10/30 size exclusion column (Amersham Pharmacia Biotech) to determine the native molecular weight. The column was run in
Dulbecco's phosphate-buffered saline (PAA, Linz, Austria) supplemented
with 1 mM dithiothreitol at a flow rate of 0.5 ml/min.
Fractions of 1 ml were collected and analyzed for the presence of
complexes containing Gal4-ETO mutants by Western blotting with an
anti-ETO antibody (Calbiochem-Oncogene Research Products, Cambridge,
UK) or an anti-Gal4 antibody (Santa Cruz Biotechnology, Inc.), respectively.
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RESULTS |
Determinants of ETO-mediated Transcriptional Repression--
To
functionally dissect the repressor domains of ETO and to determine
their role in ETO-mediated repression, we constructed a series of
amino-terminal deletion mutants leaving NHR4, the presumed binding site
for the corepressor N-CoR, intact (Fig. 1A). Like other corepressors,
the ETO protein cannot bind to DNA by itself. We therefore used
Gal4-ETO fusion proteins to repress transcription from a reporter
construct containing two Gal4 binding sites upstream of a thymidine
kinase promoter driving firefly luciferase gene expression
(2xUAS-thymidine kinase-luciferase) in transient transfection assays in
293T cells. Transfection efficiencies were normalized to the activity
of a cotransfected promoterless renilla luciferase reporter plasmid
(p
prom-Renilla Luciferase). A fusion protein of GAL4 and wild type
ETO (Gal4-ETOwt) showed 80-fold reduction in luciferase activity
compared with a control plasmid, expressing only the Gal4 DNA binding
domain (Fig. 1B). The repressor activity of all other
constructs was given relative to that of Gal4-ETOwt, which was set as
100% repressor activity. The deletion of the first 236 amino acids of
ETO including NHR1 (Gal4
1-236) did not significantly affect
repressor activity, indicating that the amino-terminal region is not
essential for transcriptional repression (Fig. 1B). Deletion
of a further 85 amino acids strongly reduced repressor activity to only
22.5% of ETOwt levels. This construct (Gal4
1-321) lacks NHR1 and
the region between NHR1 and NHR2 (Fig. 1B), revealing a
previously unrecognized role of this region in transcriptional
repression. Gal4 constructs containing either NHR3 and NHR4 (Gal4
1-401) or NHR4 alone (Gal4
1-510) showed only 5.4 or 3.8%
transcriptional repression, respectively, as compared with Gal4-ETOwt
(Fig. 1B). All GAL4-ETO constructs were expressed at similar
levels, as estimated from Western blots of lysates obtained from
transfected 293T cells (data not shown).

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Fig. 1.
Transcriptional repression of ETO
mutants. ETO requires all but its amino-terminal region for
maximum repressor activity. A, schematic diagram of Gal4-ETO
mutants used. PST, proline-serine-threonine-rich region. The
numbers 1-4 denote nervy homology regions 1-4
(NHR1-NHR4). B, 293T cells were transfected with 1.5 µg
of the 2xUAS-thymidine kinase promoter-luciferase plasmid, 0.75 µg of
the promoterless renilla luciferase plasmid, and 1.0 µg of the
indicated Gal4-ETO plasmids. Transcriptional repression of the
ETO mutants is given as the mean of five experiments and compared with
that of ETOwt, which was set to 100% repression.
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ETO-N-CoR Interaction Requires the Presence of NHR3 and
NHR4--
The observation that extended amino-terminal deletions of
ETO (ETO
1-401 and ETO
1-510) display drastically reduced
repressor activity, prompted us to investigate whether they would be
impaired in respect to binding to the corepressor N-CoR. In pull-down
experiments with GST fusions of ETO mutants, a fusion protein
containing NHR3 and NHR4 (GST·NHR3+4) but not NHR4 alone (GST·NHR4)
interacted with N-CoR (Fig. 2). Together
with the repressor activity (Fig. 1), these data suggest that N-CoR
binding per se is not sufficient to mediate significant
transcriptional repression. We mapped the minimal ETO binding site of
N-CoR between aa 1147 and 1213, because a Gal4-N-CoR construct
containing aa 970-1258 (Fig. 2), but not a smaller fragment
(Gal4-N-CoR 970-1147) or a more carboxyl-terminal region (Gal4-N-CoR
1213-1502), can be precipitated by GST·NHR3+4 (data not shown).

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Fig. 2.
ETO requires NHR3 and NHR4 for binding to
N-CoR. GST alone and GST·ETO fusion constructs were expressed in
E. coli and purified on glutathione-agarose beads. These
purified proteins were then incubated with in vitro
translated 35S-labeled Gal4 fusions of N-CoR (aa
970-1258). After extensive washing, the material that remained bound
to the beads was subjected, together with 10% of the input,
to SDS-PAGE and visualized by autoradiography.
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A "Core Repressor Region" Confers Transcriptional Repression by
ETO--
Because NHR3 and NHR4 do not have substantial repressor
activity on their own, despite binding to N-CoR, we investigated which other regions in ETO contribute to repressor activity. To this end we
generated internal and carboxyl-terminal deletions of a Gal4-ETO
deletion mutant (Gal4-
1-236) that showed maximum repression in our
assay system. Removing the region between NHR2 and NHR3 (Gal4-
1-236
C;
aa 384-432) reduced repressor activity to
49.3% repression compared with 102.6% repression with Gal4-
1-236
(Fig. 3B). To evaluate the
role of NHR2, we deleted the NHR2 region to generate the construct
Gal4-
1-236
NHR2. This deletion reduced transcriptional repression
to 24.3% of Gal4-ETOwt levels (Fig. 3B). To our surprise, a
construct containing 196 amino acids, including the central NHR2 domain
and surrounding regions (Gal4-CRD; aa 236-432) but lacking the N-CoR
binding site, showed significant repressor activity (42.7% of
Gal4-ETOwt; Fig. 3B). This region of ETO was designated
"core repressor domain" (CRD). Gal4-ETO fusions containing only
subfragments of the core repressor domain were considerably less
effective in repressing transcription. A fusion of Gal4 with a region
between NHR1 and NHR2 (Gal4-N; aa 236-306) conferred only 1.7%
repression, whereas a region between NHR2 and NHR3 (Gal4-C; aa
384-432) or the NHR2 region alone (Gal4-NHR2; aa 321-388) induced 8.8 and 13.5% repression, respectively (Fig. 3B). From these
data we conclude that ETO requires all but its first 236 amino acids to
induce maximal repression, whereby a region containing NHR2 and
neighboring amino-terminal and carboxyl-terminal sequences (CRD)
represents the smallest deletion construct conferring significant
repressor activity on its own.

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Fig. 3.
A core repressor domain confers
transcriptional repression. A, schematic diagram of
Gal4-ETO mutants used in these studies. PST,
proline-serine-threonine-rich region. The numbers 1-4
denote nervy homology regions 1-4 (NHR1-4). B,
a domain comprising carboxyl- and amino-terminal regions surrounding
NHR2 is the smallest deletion construct of ETO that confers substantial
transcriptional repression. 293T cells were transfected with 2.0 µg
of the 2xUAS-thymidine kinase promoter-luciferase plasmid, 1.0 µg of
the promoterless renilla luciferase plasmid, and 100 ng of the
indicated Gal4-ETO plasmids. Transcriptional repression of the
ETO mutants is given as the mean of five experiments and compared with
that of ETOwt, which was set to 100% repression.
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mSin3A Interacts Strongly with the Core Repressor
Domain--
Based on these results we tested whether the core
repressor domain binds to another corepressor molecule, mSin3A. We
performed immunoprecipitation experiments using in vitro
translated mSin3A together with in vitro translated
35S-labeled mutants of ETO. An antibody against mSin3A
coprecipitated full-length ETO, amino-terminally deleted ETO
(Gal4-
1-236), and carboxyl-terminally deleted ETO (Gal4-CRD),
indicating that the interaction domain is placed within the core
repressor domain (Fig. 4). To further map
the ETO-mSin3A interaction, we tested internal deletion mutants of ETO.
Interestingly, constructs where NHR2 (Gal4-
1-236
NHR2),
the carboxyl-terminal region between NHR2 and NHR3
(Gal4-
1-236
C), or the amino-terminal 85 amino acids
(Gal4-
1-321) are deleted showed reduced or no binding to mSin3A
(Fig. 4). As expected, no mSin3A interaction was seen with a construct
containing only the carboxyl-terminal NHR3 and NHR4 regions
(Gal4-
1-401). Furthermore, we could not detect coprecipitation of
mSin3A with ETO deletions that contained only the amino-terminal (Gal4-N) or the carboxyl-terminal sequences (Gal4-C; data not shown)
surrounding NHR2 or NHR2 (Gal4-NHR2) alone. However, ETO mutants
containing NHR2 and the carboxyl-terminal region (Gal4-NHR2+C; aa
321-432) or NHR2 and the amino-terminal region (Gal4-N+NHR2; aa
236-389) were still able to interact weakly with mSin3A (Fig. 4).

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Fig. 4.
mSin3A interacts with the core repressor
domain in vitro. In vitro translated
mSin3A was incubated with various in vitro translated
35S-labeled Gal4-ETO mutants and then immunoprecipitated
with anti-mSin3A IgG. After extensive washing, the bound material
(lanes B), together with 10% of each input (lanes
I), was subjected to SDS-PAGE.IP,
immunoprecipitation.
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These results were confirmed using cellular extracts from 293T cells
transfected with Gal4-ETO deletion constructs. All ETO mutants
containing the core repressor domain could be coimmunoprecipitated with
an antibody against Sin3A (Fig. 5). We
found strong interaction of Sin3A with Gal4-ETOwt, Gal4-
1-236, and
the construct containing only the core repressor domain (Gal4-CRD) by
coprecipitating 10 to 20% of ETO protein in cellular lysates with an
antibody to Sin3A. In contrast, 10-fold less ETO protein (1-2% of
input) could be coprecipitated with Sin3A from cellular lysates that
expressed ETO mutants lacking the amino-terminal region of the core
repressor domain (Gal4-
1-321) or the NHR2 region (Gal4-
1-401).
No interaction could be found with shorter mutants consisting only of
NHR2 and the neighboring carboxyl-terminal region (Gal4-NHR2+C) or a
Gal4 fusion with NHR3 and NHR4 (Gal4-
1-401) (Fig. 5). The ETO
binding site within mSin3A was mapped to the paired amphipathic helix 2 domain in mSin3A (data not shown), which has also been described to
interact with the repression domain of Mad I (24). Interaction of N-CoR
and HDAC2 with ETO was only seen with the ETOwt construct but not with
the construct containing the core repressor domain (data not
shown).

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Fig. 5.
mSin3A interacts with the core repressor
domain in vivo. Endogenous Sin3A from 293T cell
lysates containing different Gal4-ETO mutant proteins was
immunoprecipitated with anti-mSin3A IgG or carrier alone (protein
A/G-Sepharose), subjected to SDS-PAGE, and transferred onto a
polyvinylidene difluoride membrane. Blotting with an antibody directed
against the DNA binding domain of Gal4 allowed detection of
coimmunoprecipitated Gal4-ETO fusion constructs. IP,
immunoprecipitation; WB, Western blot.
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ETO Mutants Lacking the Carboxyl- or Amino-terminal Part of the CRD
Can Still Homodimerize--
Because deletion of amino- or
carboxyl-terminal sequences within the CRD led to impaired repressor
activity, we investigated whether these mutants are defective in
NHR2-mediated homodimerization. NHR2 has been shown to induce homo- and
heterodimerization between ETO and related family members such as MTGR1
(21). All constructs containing the NHR2 domain, but not those lacking
NHR2 (e.g. Gal4-
1-236
NHR2), were able to bind to
GST·ETO in pull-down experiments, confirming the structural integrity
of NHR2 (Fig. 6). Homodimerization was also seen in a construct, Gal4
1-321, that showed only little repressor activity (22.5%, Fig. 1B), indicating that ETO
repressor activity depends only in part on the NHR2 amphipathic helix
structure, whereas a proline-rich region amino-terminal to NHR2 appears
to be critically required for maximum repressor activity.

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Fig. 6.
Gal4-ETO mutants containing NHR2 dimerize
with ETO. GST or GST·ETO constructs were expressed in
E. coli and purified on glutathione-agarose beads. The
purified proteins were then incubated with the indicated in
vitro translated 35S-labeled Gal4-ETO mutants. After
extensive washing, the material that remained bound to the beads
(lanes B for GST·ETO; lanes - for GST),
together with 10% of the input of each labeled Gal4-ETO (lanes
I), was subjected to SDS-PAGE and visualized by autoradiography.
For a schematic representation of Gal4-ETO constructs, see Figs. 1 and
3.
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Integrity of ETO Protein Is Required for Stable High Molecular
Weight Complex Formation--
In a recent paper by Minucci et
al. (22), high molecular weight (HMW) complex formation was shown
to be required for efficient repressor activity of AML-ETO,
PML-RAR, and promyelocytic leukemia zinc finger-RAR. To
investigate the ability of our constructs to form HMW complexes, we
expressed various GAL4-ETO mutants in 293T cells and determined the
molecular weight of the complexes by size-exclusion chromatography. HMW
complexes obtained with Gal4 fusions of full-length ETO had an apparent
molecular mass of 1,600 kDa. Similarly, ETO deletions
1-236,
Gal4-
1-236
NHR2, and
1-321 formed complexes with the same
molecular weight, but the formation of smaller complexes, which were
eluted in all fractions, from 500 to 1,600 kDa could be noted, probably
because of destabilization of the 1,600-kDa complex. Furthermore,
deletions within the CRD, such as deletion of the 85 amino acids
amino-terminal to NHR2 (Gal4-
1-321) or an internal deletion of NHR2
(Gal4-
1-236
NHR2), shifted the peak elution volume to a lower
molecular mass of about 990 kDa. A complete deletion of the CRD
in the construct Gal4
1-401 had its elution maximum at 500 kDa,
similar to a Gal4 fusion containing the CRD alone (Gal4-CRD), whereas
the construct Gal4
1-510 was eluted in its monomeric and dimeric
form only (Fig. 7). These data highlight
the importance of the integrity of the CRD in HMW complex formation and
in ETO-induced repression, suggesting that both phenomena
correlate.

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Fig. 7.
A core repressor domain contributes to high
molecular weight complex formation. Cellular extracts from 293T
cells transfected with the indicated Gal4-ETO fusion constructs were
fractionated by size-exclusion chromatography and analyzed by Western
blotting using an anti-ETO antibody. The fraction number and the
molecular mass of standard protein markers and their peak
elution fraction are indicated on the top. For a schematic
representation of Gal4-ETO constructs, see Figs. 1 and 3.
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|
In summary, our data clearly indicate that various regions of ETO
cooperate to mediate repressor activity. The amphipathic helix
structure of NHR2 and adjacent carboxyl- and amino-terminal sequences
provide the structural basis for strong mSin3A binding, which
contributes to transcriptional repression and HMW complex formation.
N-CoR binding through NHR3 and NHR4, however, does not mediate
repression by itself but is required in cooperation with additional
factors for maximum transcriptional repression.
 |
DISCUSSION |
Our data indicate that transcriptional repression of the
leukemia-associated protein ETO is based on a modular structure that mediates high molecular weight complex formation and maximum
transcriptional repression. Furthermore, we have defined a core
repressor domain containing NHR2 and the neighboring carboxyl- and
amino-terminal sequences that mediates strong interaction with Sin3A
and represents the smallest deletion with significant, albeit reduced
repressor activity. NHR1 (25), however, which shows sequence similarity to a central 80-amino acid region of the transcriptional coactivators hTAF 130 (TBP-associated factor 130), hTAF 105, and
Drosophila TAF 110 (21, 26-28), is not required for
repressor function in the context of our experimental system. This is
in agreement with published data showing that deletion of NHR1 does not
affect ETO-mediated repression of the MDR-1 promoter. In this context
it is worth noting that although a lack of NHR1 did not effect
repressor activity, it destabilized the formation of high molecular
weight complexes, as indicated by the appearance of smaller-sized
complexes compared with ETOwt.
The fourth homology region (NHR4) consists of two zinc finger domains
that are necessary for ETO-N-CoR/SMRT interaction. Deletion, or even
point mutations, of this region completely abolish binding of ETO to
N-CoR/SMRT but reduce transcriptional repression only partially (10,
18, 19). This is in agreement with our data showing that the core
repressor domain alone mediates 50% of ETOwt repressor activity,
although it recruits only Sin3A but not N-CoR. Furthermore, we present
evidence that the NHR4 zinc finger motif alone is not capable of
interacting with the ETO binding region in N-CoR, which we mapped
within N-CoR repressor domain III. ETO-N-CoR interaction requires both
the presence of the zinc finger motif and the adjacent helical
structure of NHR3. In experiments similar to ours, Zhang et
al. (29) could also demonstrate binding of the highly homologous
corepressor SMRT to an ETO mutant containing NHR3 and NHR4 fused to the
Gal4 DNA binding domain. We conclude that NHR4 is necessary but
not sufficient to recruit N-CoR. Interestingly, the binding site for
N-CoR (NHR3 and NHR4) did not induce transcriptional repression,
suggesting that the interaction may be unable to efficiently recruit
histone deacetylases. However, N-CoR binding may serve to enhance
repression by stabilizing the corepressor complex with other
corepressors that are needed to induce repressor activity.
Recent evidence indicates that the oncogene AML1/ETO
requires homodimerization, mediated by the amphipathic helix structure NHR2 (17, 21), to form HMW complexes and induce transcriptional repression (22). We were able to confirm that the stability and
size of these complexes correlate with repressor function, entrusting
an important role to the core repressor domain for the correct
formation of HMW complexes. This was demonstrated with ETO
constructs lacking structural elements of the CRD, such as NHR2, the
amino-terminal 85 amino acids, or the carboxyl-terminal region, which
not only had reduced repressor activity but also led to the formation
of complexes with a lower molecular weight. Interestingly, both
Gal4-ETO mutants lacking either the amino-terminal or the
carboxyl-terminal region of the core repressor domain (Gal4-
1-321 and Gal4-
1-236
C) were still perfectly able to dimerize with full-length ETO, indicating that the amphipathic helix structure of
NHR2 was functional. These ETO deletions were, however, severely impaired in binding to Sin3A. We conclude that repressor activity and
HMW complex formation are not solely determined by the process of
NHR2-induced dimerization but also through the affinity of the ETO
molecule to Sin3A. In this context it is worth noting that only ETOwt
or the deletion construct Gal4-
1-236 could be eluted in the same
fraction as complexes containing Sin3A (data not shown). The ETO-Sin3A
interaction, however, appears to be more complex than anticipated. We
were still able to detect weak binding of Sin3A to ETO mutants lacking
NHR2 and sequences amino-terminal of NHR2 in cellular lysates. Similar
results have also been obtained in Cos-7 cells overexpressing mSin3A
and ETO deletion mutants (10), showing that binding of mSin3A to ETO
under these conditions occurs even in the absence of NHR2 (10). This
may indicate that Sin3A interacts with ETO not only directly, but also
indirectly through other corepressor molecules (5, 30, 31). To reduce the possibility of indirect interactions via secondary molecules, we
tested ETO-Sin3A interaction with in vitro translated
proteins. These experiments, however, support our in vivo
data showing weak interaction of mSin3A with ETO constructs lacking
sequences of the core repressor domain. Our data favor a model in which
complex structural requirements are needed for stable direct
interaction of Sin3A with ETO, whereas the indirect interaction of
Sin3A to ETO through binding to N-CoR and HDAC2 is not sufficient to
induce repressor activity.
Because Gal4-ETO mutants with deletions in the mSin3A binding site/core
repressor domain were impaired in their ability to repress
transcription, and neither the Sin3A binding site (CRD) nor the N-CoR
binding site (NHR3 and NHR4) were able to induce maximum
transcriptional repression or HMW complex formation by themselves, we
conclude that these two major domains in ETO cooperate for optimal
function. Although these results have been obtained in transient
transfection assays and may not properly reflect the physiological
situation at endogenous chromosomal loci (32), it raises the question
why binding to one corepressor molecule is not sufficient for ETO to
induce optimal repression. It appears that the corepressor complex
requires a certain stability, possibly mediated by the interaction of
corepressor molecules with each other. The concept that complexes with
multiple interacting subunits are required in the process of
transcriptional regulation has also been demonstrated for coactivator
complexes containing histone acetylase activity (3). The modular
structure of ETO opens the possibility to interfere with one or more
structural elements in the ETO molecule, thereby destabilizing complex
formation and reducing repressor activity. This in turn may ultimately
enable a therapeutic approach for the treatment of leukemias with the t(8;21) translocation.