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NEOPLASIA
From the Department of Hematology and Oncology,
Graduate School of Medicine, University of Tokyo, Japan.
Evi-1 is a zinc finger nuclear protein whose inappropriate
expression leads to leukemic transformation of hematopoietic cells in
mice and humans. This was previously shown to block the
antiproliferative effect of transforming growth factor The Evi-1 gene was first identified as a
common locus of retroviral integration in myeloid tumors in AKXD
mice.1,2 It encodes a transcriptional regulator with 2 zinc finger domains. Evi-1 is shown to be highly expressed
in human myeloid leukemias and myelodysplastic syndromes by chromosomal
rearrangements involving 3q26, to which Evi-1 is
mapped,3-6 although it is expressed at a very low level in
a limited stage of normal myeloid cell differentiation. The most
frequent rearrangements involving 3q26 are the t(3;3)(q21;q26) and the
inv(3)(q21;q26). Cases of myelodysplastic syndrome with these anomalies
are characterized by the increase in the platelet count and the
dysplastic features of megakaryocytes and are designated as 3q21q26
syndrome.7 Aberrant expression of Evi-1 as a
fusion transcript with AML1 (AML1/Evi-1) leads to
blastic transformation in patients with chronic myelogenous
leukemia.8 Even in the absence of cytogenetically evident
abnormalities at chromosome 3q26, overexpression of Evi-1
gene has been shown in a variety of myelogenous
leukemias.9 These facts strongly suggest a critical role
for Evi-1 in human leukemogenesis.
Our previous studies revealed that Evi-1 possesses diverse functions as
an oncoprotein.10-12 Among these studies is our
demonstration that Evi-1 and AML1/Evi-1 repress transforming growth
factor Intracellular mechanisms that transmit TGF- Evi-1 and AML1/Evi-1 antagonize the growth-inhibitory effect of TGF- C-terminal binding protein (CtBP) was originally identified as a
protein that interacts with the C-terminal region of adenoviral oncoprotein E1A, which results in the reduced ability of E1A to transform cells.26,27 To date, 2 highly related
homologues, CtBP1 and CtBP2, have been identified in vertebrates. It is
known that CtBP2 is expressed primarily during embryogenesis in mice, whereas CtBP1 is widely expressed throughout the developmental stages.
Differences in function between them, however, remain elusive.28 CtBP has been shown to have a transcriptional
repression activity when fused to the heterologous DNA-binding
motif.29 The Drosophila homologue of CtBP was
shown to mediate transcriptional repression by Knirps, Krüppel,
and Snail in the early embryo.29,30 In vertebrates, CtBP
was also shown to act as a cofactor of certain transcriptional
repressors, including basic Krüppel-like factor (BKLF), friend of
GATA (FOG), and T-cell factor (TCF).31-33 These facts suggest a critical role for CtBP in transcriptional repression of
genes in a wide variety of cells. CtBP was shown to interact with E1A
of human adenoviruses 12 and 2 through the 5-amino acid sequences
PLDLS and PVDLS (single-letter amino acid codes), respectively. These sequences are highly conserved among the E1A
proteins.26 Repressor proteins that are described as
interacting with CtBP so far also contain similar 5-amino acid
sequences that are called CtBP-binding motifs.31-33 We
have reported a repressor domain of Evi-1 in the region between amino
acids 608 and 732.14 In this study, we identified another
region of Evi-1, spanning amino acids 544 to 607, that is required for
full repressor activity. Recently, Turner and Crossley31
identified 2 CtBP-binding-motif-like sequences in this region of Evi-1
and showed physical interaction between mCtBP2 and a portion of Evi-1,
including this region by the yeast 2-hybrid system. However, it remains
to be determined whether Evi-1 interacts with CtBP in vivo and whether
Evi-1-mediated repression depends on the interaction with CtBP.
Furthermore, the most important point is to elucidate the biological
phenomenon and its signaling pathway, where the physical interaction
between Evi-1 and CtBP occurs.
To address these issues, we further investigated the mechanism for
Smad-inhibition by Evi-1 and demonstrated that CtBP acts as an
essential cofactor for Evi-1-mediated repression of TGF- Cell culture and establishment of stable clones
Plasmids
Luciferase assay For analysis of luciferase activities, HepG2 cells were seeded in 12-well culture plates at a density of 4 × 104 per well. At 12 hours after seeding, the cells were transfected with 1 µg p3TP-Lux or p800neoLUC reporter plasmid along with the effector plasmids (400 ng for pME-Evi-1 or the equivalent molar for their derivatives, and 400 ng for Smads in pcDNA3) with SuperFect (Qiagen, Valencia, CA) according to the manufacturer's instructions. For analysis of the luciferase activity derived from cotransfection with several expression plasmids, the total amount of DNA in terms of weight was adjusted to be equal by adding the plasmid pUC13. As an internal control of transfection efficiency, a plasmid expressing -galactosidase was cotransfected. The cells were harvested 48 hours after transfection and assayed for the luciferase activity by
means of the luciferase assay system (Promega, Madison, WI) and a
luminometer (Lumat, Berthold, Badwildbod, Germany). The data were
normalized to the -galactosidase activity. Cells were treated with
200 pM TGF- (Roche Diagnostics, Mannheim, Germany) for 24 hours
before harvesting. For the luciferase assay using the histone
deacetylase (HDAc) inhibitor, 50 ng/mL trichostatin A (Waco, Osaka,
Japan), dissolved in ethanol, was added to culture medium for 8 hours
before harvesting. The same amounts of ethanol were added to the
control cells.
Immunoprecipitation and Western blotting COS7 cells were transfected by the diethylaminoethyl-dextran method as described previously.34 The cells were cultured for 48 to 72 hours after transfection and were lysed in the TNE buffer.13 For immunoprecipitation, cell lysates were incubated with the anti-T7 monoclonal antibody (Novagen, Madison, WI) for 3 hours at 4°C. Then the samples were incubated with protein A-Sepharose (Sigma, St Louis, MO) for 1 hour at 4°C. Immunoprecipitates were washed 5 times with the TNE buffer and were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analysis by Western blotting. Immunoblotting was performed with anti-Evi-1 serum10 and anti-T7 and was detected with the enhanced chemiluminescence system (Amersham Pharmacia Biotech).GST pull-down assay [35S]methionine-labeled CtBP1 was synthesized in vitro by means of TNT-coupled reticulocyte lysate system (Promega) according to the manufacturer's instructions. We collected 5 µg bacterially produced GST fusion proteins on Glutathione Sepharose 4B beads (Amersham Pharmacia Biotech) and incubated them with [35S]methionine-labeled CtBP1 for 3 hours at 4°C in the TNE buffer. Then, the beads were washed 5 times with the TNE buffer and subjected to SDS-PAGE followed by detection with autoradiography.Growth-inhibition assay The stable clones derived from Mv1Lu cells were seeded in duplicate at a density of 1 × 104 per well in 24-well culture plates. At 12 hours later, different doses of TGF- were
added, and the cells were incubated for 24 hours. During the last 4 hours, the cells were labeled with 1 µCi/mL
[3H]thymidine (Amersham Pharmacia Biotech). The cells
were washed with phosphate-buffered saline, fixed with 10%
trichroloacetate, and solubilized with 0.5 M NaOH. The cell extracts
were neutralized with HCl, and [3H] radioactivity was
measured in a liquid scintillation -counter (Aloka, Mitaka,
Tokyo, Japan).
The region between amino acids 544 and 607 of Evi-1 is required
to block TGF- signaling.14 One is the first zinc finger domain, through which Evi-1 binds to Smad3 directly (Figure
1A). The Evi-1 mutant that lacks this
domain has lost the ability to suppress TGF- signaling. Using
C-terminal-truncated mutants of Evi-1, we found that the other domain,
the region between amino acids 608 and 732 of Evi-1, is also required
for efficient repression of TGF- signaling, although it does not
contribute to binding to Smad3. We have therefore termed this region
the repression domain.
To define the full picture of the Evi-1 domains that contribute to
inhibition of TGF- Evi-1 interacts with CtBP1 through its consensus motif Recently, it was reported that Evi-1 contains 2 amino acid sequences, PFDLT (single-letter amino acid codes) and PLDLS, that fit to the CtBP-binding motif (Figure 2A).31 These sequences are included in the region between amino acids 544 and 607 of Evi-1, which we showed as participating in efficient inhibition of TGF-
signaling, and they are completely conserved between mouse and human
Evi-1 proteins.42 These findings allowed us to perform a
coprecipitation assay to confirm whether Evi-1 interacts with CtBP
in mammalian cells. We introduced T7-tagged CtBP1 with wild-type or the
deletion mutants of Evi-1 into COS7 cells. Cell lysates were subjected
to immunoprecipitation with anti-T7, followed by immunoblotting with
anti-Evi-1. We observed that wild-type Evi-1 was coprecipitated
with T7-CtBP1. However,  544-607 or 544-732, which lacks putative
CtBP-binding motifs, was not. In contrast, 608-732, which retains
the domain including the motifs, was coprecipitated with T7-CtBP1
(Figure 2B). From these results, we concluded that Evi-1 interacts with
CtBP1 in vivo and that the region between amino acids 544 and 607 is
responsible for binding to CtBP1. Next we examined the relative
contribution of the 2 putative CtBP-binding motifs to the interaction
with CtBP. For this purpose, we constructed Evi-1 mutants that harbor
specific amino acid substitutions. The previous report demonstrated
that substituting the AS residues for the DL residues in the
CtBP-binding motif of E1A completely eliminates binding to
CtBP1.26 Therefore, we introduced the same substitution
into Evi-1 at the PFDLT (AS/DL), the PLDLS (DL/AS), or both (AS/AS)
(Figure 2C). We transfected these mutants with T7-CtBP1 into COS7 cells
and performed immunoprecipitation experiments. Each mutant is expressed
comparably at the anticipated size. Both wild-type Evi-1 and the mutant
for PFDLT (AS/DL) interacted with T7-CtBP1. However, neither of the 2 mutants for PLDLS (DL/AS and AS/AS) retained the ability to interact
with CtBP (Figure 2D). Thus, of the 2 CtBP-binding-motif-like
sequences, PLDLS at amino acid 584 is responsible for the interaction
between Evi-1 and CtBP1, and PFDLT at amino acid 553 is not.
To determine the region of CtBP1 that interacts with Evi-1, we
overexpressed wild-type Evi-1 with full-length or truncated forms of
CtBP1 fused to GST in COS7 cells (Figure
3A). Whereas a full-length CtBP1 strongly
binds to Evi-1, none of the truncated forms of CtBP1 interacted with
Evi-1 (Figure 3B). These results suggest that the integrity of the CtBP
protein is required for interacting with Evi-1.
To determine whether Evi-1 binds to CtBP in vitro, we performed a
pull-down assay using GST fusion proteins. Evi-1 [544-607] and its
mutants for the CtBP-binding motifs were fused to GST (Figure
4A) and expressed in bacteria. These
proteins are immobilized onto Glutathione Sepharose 4B beads and
incubated with [35S]methionine-labeled, in
vitro-translated CtBP1. GST-DL/AS and GST-AS/AS failed to bind to
CtBP, whereas GST-DL/DL and GST-AS/DL interacted with in
vitro-translated CtBP1 (Figure 4B). These data indicate that Evi-1
interacts with CtBP1 in vitro.
Evi-1 requires CtBP as a corepressor in inhibition of Smad3 CtBP is shown to act as a transcriptional corepressor in Drosophila and in vertebrates.28,29,43 To investigate a role for CtBP in Evi-1-mediated repression, we tested whether the AS mutation, which abrogates the binding of CtBP1 to Evi-1, would impair the ability of Evi-1 to repress TGF- signaling. We
transfected p3TP-Lux into HepG2 cells together with the effector
plasmids as indicated in Figure 5. The
AS/DL mutant, which retains the ability to interact with CtBP1,
repressed TGF--mediated transactivation as efficiently as wild-type
Evi-1 (Figure 5A). In contrast, DL/AS or AS/AS mutant, in which the
interaction with CtBP1 is specifically abrogated, showed a severely
reduced ability of repression. We also performed these experiments
using overexpression of Smad3 and Smad4 instead of exposure to TGF-,
and similar results were obtained (Figure 5B). The experiments using
p800neoLUC as a reporter plasmid demonstrated similar results (data not
shown). Taken together, these data indicate that recruitment of CtBP
plays an important role in full repression of TGF- signaling
by Evi-1.
An HDAc inhibitor alleviates Evi-1-mediated Smad repression An HDAc is known to mediate transcriptional repression by rendering the nearby chromatin inaccessible to transcriptional activators through deacetylation of histone proteins.44,45 HDAc forms a large multiprotein complex with corepressor proteins, including the nuclear hormone corepressor (N-CoR), the silencing mediator for retinoid and thyroid-hormone receptors, and the mammalian homologue of yeast transcriptional repressor SIN3 (mSin3).46,47 The recent study demonstrates that CtBP interacts with HDAc1, a member of HDAc family, both in vitro and in vivo.48 We also performed the immunoprecipitation study by coexpressing CtBP1 and HDAc1 in COS7 cells and confirmed the interaction between them (data not shown). These observations suggest that CtBP might play a role as a corepressor by recruiting HDAcs. To investigate whether Evi-1-mediated repression involves histone deacetylation, we performed a luciferase assay using a specific HDAc inhibitor, trichostatin A.49 Trichostatin A has been shown to relieve transcriptional repression by Mad-Max complex or by other repressor proteins, which are mediated by HDAc.50,51 We cultured HepG2 cells with 50 ng/mL trichostatin A for 8 hours before harvesting, and the treatment had little effect on the basal or TGF--induced activity of p3TP-Lux
(Figure 6). In contrast, repression of
TGF- signaling elicited by Evi-1 was significantly attenuated by the
treatment with trichostatin A. To determine whether the effect of
trichostatin A is dependent on recruitment of CtBP, we tested Evi-1
mutants with altered CtBP-binding properties for trichostatin
A-induced derepression in this assay. DL/AS mutant and 544-607,
both of which do not interact with CtBP, were little affected by
trichostatin A treatment. However, 608- 732 mutant, which lacks the
previously identified repression domain but preserves binding to CtBP,
was sensitive to trichostatin A treatment (Figure 6). These results suggest that an HDAc activity is involved in repression of TGF- signaling by Evi-1 and that this is mediated chiefly through the interaction with CtBP.
Association with CtBP is required for Evi-1 to inhibit
TGF- -mediated growth inhibitory signals.
Overexpression of Evi-1 in Mv1Lu cells abrogates the antiproliferative
effect of TGF-.14 To investigate a role for CtBP in the
Evi-1-mediated block of antiproliferative effect of TGF-, we
established Mv1Lu clones that stably express either wild-type Evi-1 or
the DL/AS mutant. We introduced the expression vector for wild-type
Evi-1 or the DL/AS mutant that enables concomitant expression of the neomycin-resistant gene into Mv1Lu cells and selected them for neomycin
resistance in the presence of G418. Of the resultant G418-resistant
clones, Western blot analyses using anti-Evi-1 revealed 4 representative clones that express high levels of wild-type Evi-1 (W-55
and W-60) or the DL/AS mutant (A-72 and A-82) (Figure 7A). Two independent Mv1Lu clones, M-2
and M-4, which were transfected with the empty vector, were used as
controls. In these experiments, intrinsic Evi-1 was below the
detectable level in the naive Mv1Lu cells. When cultured in complete
medium without TGF-, all of these clones showed comparable
viabilities and proliferative abilities (data not shown). We then
evaluated growth-inhibitory effects of TGF- on these Mv1Lu clones
using [3H]thymidine-incorporation assays. Shown in Figure
7B are the effects on [3H]thymidine uptake when these
clones were exposed to increasing amounts of TGF-. The Mv1Lu clones,
which overexpress Evi-1 (W-55 and W-60), showed reduced responsiveness
to TGF-, in comparison with mock-transfected clones (M-2 and M-4).
In contrast, the growth of A-72 and A-82 clones, which overexpress the
DL/AS mutant, was inhibited in response to TGF- as efficiently as
that of mock-transfected clones. These results suggest that recruitment
of CtBP is required for Evi-1 to release cells from the
growth-inhibitory effect of TGF- in vivo.
In this study, we investigated the mechanism through which Evi-1
blocks TGF- Involvement of corepressor proteins in human leukemogenesis has been
proposed with the demonstration that PML/RAR In the previous study, we showed that Evi-1 directly binds to the MH2
domain of Smad3 in the nucleus through its first zinc finger domain and
affects the interaction of the complex of Smad3 and Smad4 with its
DNA-binding sequence. A C-terminal portion of Evi-1 spanning amino
acids 608 to 732 (Evi-1 [608-732]), which is not involved in the
binding to Smad3, also contributes to the repression.14 In
the present study, we identified another region of Evi-1, spanning
amino acids 544 to 607, that is also required for repressing TGF- It remains to be determined how CtBP works as a transcriptional
corepressor. One possibility is that CtBP recruits an HDAc complex.
CtBP has been shown to interact with HDAc1 both in vitro and in
vivo.48 We demonstrated that trichostatin A, a specific inhibitor of HDAc, significantly alleviates the Evi-1-mediated repression. Moreover, Evi-1 mutants that cannot bind to CtBP are not
vulnerable to trichostatin A, which suggests that derepression by
trichostatin A is dependent on CtBP. Ski, Sno, and TGIF are transcriptional corepressor proteins that associate with Smad proteins
and inhibit their transcriptional activation with dependence on an HDAc
activity.22-25 Although these corepressors for Smad proteins are known to be endogenously expressed in a variety of cells,
the present study is hardly affected by them, if at all, because
trichostatin A treatment does not change the basal transcriptional activity or the activity induced by TGF- Our study provides a novel function of Evi-1 as a transcriptional corepressor that interacts with DNA-binding transcription factors. Thus far, Evi-1 is proposed as a DNA-binding protein because of the observation that it directly interacts with specific sequences of DNA in vitro.40,41 Although these sequences overlap with the binding site for the GATA-family transcription factors, target genes that Evi-1 directly regulates have not been identified so far. Thus, Evi-1 may, in addition to binding to DNA directly, regulate transcription by interacting with DNA-binding proteins, as in the case of Smad3, and by recruiting corepressor complexes, including CtBP. Identification of those target DNA-binding proteins may greatly contribute to understanding Evi-1-induced leukemogenesis.
We thank G. Chinnadurai (St Louis University, MO) for providing T7-hCtBP1, J. Massagué (Memorial Sloan-Kettering Cancer Center) for Smad4, R. Derynck (University of California) for Smad3, K. Miyazono (The Cancer Institute of the Japanese Foundation for Cancer Research) for p3TP-Lux, M. Abe (Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan) for p800neoLUC, B. J. Mayer (Howard Hughes Medical Institute, Harvard Medical School) for pEBG, S. L. Schreiber (Howard Hughes Medical Institute) for HDAc1, and K. Arai (The Institute of Medical Science, University of Tokyo, Japan) for pME18Sneo.
Submitted August 7, 2000; accepted December 26, 2000.
Supported in part by The Mitsubishi Foundation and grants-in-aid for cancer research from the Ministry of Health and Welfare and the Ministry of Education, Science and Culture of Japan.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hisamaru Hirai, Dept of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; e-mail: hhirai-tky{at}umin.ac.jp.
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S. G. Katz, A. B. Cantor, and S. H. Orkin Interaction between FOG-1 and the Corepressor C-Terminal Binding Protein Is Dispensable for Normal Erythropoiesis In Vivo Mol. Cell. Biol., May 1, 2002; 22(9): 3121 - 3128. [Abstract] [Full Text] [PDF]
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D. Liu, B. L. Black, and R. Derynck TGF-beta inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3 Genes & Dev., November 15, 2001; 15(22): 2950 - 2966. [Abstract] [Full Text] [PDF]
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S. Chakraborty, V. Senyuk, S. Sitailo, Y. Chi, and G. Nucifora Interaction of EVI1 with cAMP-responsive Element-binding Protein-binding Protein (CBP) and p300/CBP-associated Factor (P/CAF) Results in Reversible Acetylation of EVI1 and in Co-localization in Nuclear Speckles J. Biol. Chem., November 21, 2001; 276(48): 44936 - 44943. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
signaling
Top
Abstract
Introduction
799 to +81 of the 5' end of
the human PAI-1 gene
) or without (
) 50 ng/mL
trichostatin A. Luciferase activities relative to the basal activity of
the reporter are presented. Values and error bars depict the mean and
the SD, respectively.
, and W-60, 
), DL/AS (A-72, 
, and M-4, 
) were subjected
to a [3H]thymidine-incorporation assay in the presence of
the indicated concentrations of TGF-
, the fusion protein of promyelocytic leukemia (PML) and retinoic acid 
EF-1,