Physical and Functional Interactions between
Daxx and DNA Methyltransferase
1-Associated Protein, DMAP1
This information is current as
of June 15, 2017.
Ryuta Muromoto, Kenji Sugiyama, Akie Takachi, Seiyu
Imoto, Noriko Sato, Tetsuya Yamamoto, Kenji Oritani,
Kazuya Shimoda and Tadashi Matsuda
J Immunol 2004; 172:2985-2993; ;
doi: 10.4049/jimmunol.172.5.2985
http://www.jimmunol.org/content/172/5/2985
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References
The Journal of Immunology
Physical and Functional Interactions between Daxx and DNA
Methyltransferase 1-Associated Protein, DMAP11
Ryuta Muromoto,* Kenji Sugiyama,† Akie Takachi,* Seiyu Imoto,* Noriko Sato,*
Tetsuya Yamamoto,* Kenji Oritani,‡ Kazuya Shimoda,§ and Tadashi Matsuda2*
D
axx was first identified as a Fas-binding protein by yeast
two-hybrid screening and was known as a proapoptotic
protein that can enhance Fas-mediated apoptosis
through c-Jun N-terminal kinase (JNK)3 activation (1). However,
disruption of Daxx in mice increased apoptosis during the embryonic development, suggesting that Daxx acts as an antiapoptotic
protein in the embryo (2). Because of the diverse effects of Daxx
between in vitro and in vivo experimental systems, the roles of
Daxx in apoptotic signals still remain unclear. Although the interaction between Daxx and Fas indicated the importance of Daxx in
cytoplasm, nuclear localization of Daxx was observed in various
cell lines, and the interactions of Daxx with several nuclear proteins such as the centromeric protein C, DNA methyltransferase 1
(DNMT1), Pax3, Pax5, E26 avian leukemia oncogene ETS1,
Ubc9, small ubiquitin-like modifier-1, and promyelocytic leukemia protein (PML) were reported (2–9). Thus, Daxx is likely to
play alternative roles in shuttling between nucleus and cytoplasm.
Our experiments using Tyk2-deficient mice revealed that Tyk2
is essential for the IFN-␣␤-induced inhibition of colony formation
*Department of Immunology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, †Nippon Boehringer Ingelheim, Kawanishi Pharmaceutical Research Institute, Hyogo, ‡Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Osaka, and §First Department
of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan
Received for publication October 6, 2003. Accepted for publication December
15, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants-in-aid for Scientific Research from the Ministry
of Education, Science, Sports and Culture in Japan, the Osaka Foundation for Promotion of Clinical Immunology, the Akiyama Foundation, the Suhara Memorial
Foundation, Mochida Memorial Foundation for Medical and Pharmceutical Reasearch, and the Uehara Memorial Foundation.
2
Address correspondence and reprint requests to Dr. Tadashi Matsuda, Department of
Immunology, Graduate School of Pharmaceutical Sciences, Hokkaido University,
Kita-Ku Kita 12 Nishi 6, Sapporo 060-0812, Japan. E-mail address: tmatsuda@
pharm.hokudai.ac.jp
3
Abbreviations used in this paper: JNK, c-Jun N-terminal kinase; DNMT, DNA
methyltransferase; DMAP, DNMT1 associated protein; PML, promyelocytic leukemia protein; ETS, E26 avian leukemia oncogene 1; FKBP, FK506 binding protein;
MMTV, murine mammary tumor virus; LUC, luciferase; HA, hemagglutinin; GR,
glucocorticoid receptor, HDAC, histone deacetylase.
Copyright © 2004 by The American Association of Immunologists, Inc.
of B lymphocyte progenitors in response to IL-7 as well as the
up-regulation and nuclear translocation of Daxx (10). Because
Daxx plays crucial roles in IFN-␣-induced growth suppression of
B lymphocyte progenitors (11), it is very informative to identify
potential regulators of Daxx involved in the growth arrest and/or
apoptosis in early B cell development.
Recent studies implied that Daxx might function as a transcriptional coregulator. Daxx has been shown to possess transcriptional
repression activity by inhibiting several transcription factors such
as Pax3, ETS1 and glucocorticoid receptor (GR) through direct
protein-protein interactions (4, 6, 12). Daxx is also shown to act as
a transcriptional coactivator or corepressor of Pax5 in different cell
types (5). Although the exact mechanism accounting for these observations is still unclear, the recruitment of nuclear factors possessing either histone acetyltransferase or histone deacetylase
(HDAC) activity by Daxx to modulate Pax5 transcriptional activity was proposed (5). In addition, the transcriptional repression
effect of Daxx could be modulated by subnuclear compartmentalization through protein-protein interactions. Furthermore, PML
has been shown to relieve the transrepression effect of Daxx on
Pax3 or GR transcriptional activity through sequestering Daxx into
the PML oncogenic domains (12, 13).
In an attempt to identify novel Daxx partners, we screened a
mouse embryo cDNA library with a yeast two-hybrid system using
the N-terminal (Daxx/N) domain or C-terminal (Daxx/C) domain
of Daxx as bait. In this study, we identify DNMT1-associated protein (DMAP1) as a protein that interacts specifically with Daxx/N.
DMAP1 was isolated as a DNMT1-interacting protein by yeast
two-hybrid screening (14). DMAP1 interacts directly with the first
120 amino acids of DNMT1. The N-terminal noncatalytic domain
of DNMT1 binds to HDAC2 and DMAP1 and can mediate transcriptional repression (14).
In this study, we have characterized both biochemical and functional interactions between Daxx and DMAP1. Daxx and DMAP1
with DNMT1 formed a complex and colocalized in the nucleus.
DMAP1 enhanced Daxx-mediated repression of GR transcriptional activity. Furthermore, Daxx protected protein degradation of
DMAP1 in vivo. These results provide an important linkage between Daxx and DNMT1, which establishes an efficient repressive
transcription complex in the nucleus.
0022-1767/04/$02.00
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Daxx has been shown to play an essential role in type I IFN-␣␤-mediated suppression of B cell development and apoptosis.
Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and translocation of Daxx, which may
result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify how Daxx regulates B cell development, we
examined Daxx interacting partners by yeast two-hybrid screening. DNA methyltransferase 1 (DNMT1)-associated protein
(DMAP1) was identified and demonstrated to interact with Daxx. The interaction regions in both proteins were mapped, and the
cellular localization of the interaction was examined. Both Daxx and DMAP1 formed a complex with DNMT1 and colocalized in
the nucleus. DMAP1 enhanced Daxx-mediated repression of glucocorticoid receptor transcriptional activity. Furthermore, Daxx
protected protein degradation of DMAP1 in vivo. These results provide the novel molecular link between Daxx and DNMT1, which
establishes a repressive transcription complex in the nucleus. The Journal of Immunology, 2004, 172: 2985–2993.
2986
Materials and Methods
Reagents and Abs
Construction of fusion proteins with the Daxx/N or Daxx/C and
the Gal4 DNA-binding domain
Full-length mouse Daxx cDNAs were obtained from Dr. X. Yang. To
generate a bait construct with Daxx/N or Daxx/C, PCR was used to amplify
the portion of the cDNA encoding amino acid residues 1–500 or residues
501–739, respectively (primer sequences are available upon request). The
PCR product was digested with BamHI and SalI and inserted into pGBKT7
digested with BamHI and SalI (downstream of the Gal4 activation domain).
All constructs were sequenced to verify integrity of the constructs.
Yeast two-hybrid screen
Gal4-Daxx/N was constructed by fusing Daxx/N coding sequence in-frame
to the Gal4 DNA-binding domain in the pGBKT7 vector as previously
described. Saccharomyces cerevisiae, strain AH109 cells transformed with
pGal4-Daxx/N, followed by mating with a pretransformed mouse 11-day
embryo Matchmaker cDNA library in Y187 cells (Clontech Laboratories,
Palo Alto, CA), were plated onto medium that lacked tryptophan, leucine,
and histidine and had been supplemented with 5 mM 3-amino-1, 2, 4-triazole (Sigma-Aldrich). Approximately 5 ⫻ 106 colonies were screened for
growth in the absence of histidine. Plasmid DNAs derived from positive
clones were extracted from yeasts, and sequenced Clones were reintroduced into yeast strain AH109 along with either empty pGBKT7, or
pGBKT7-Daxx/N or pGBKT7-Daxx/C to verify the Daxx-clone
interaction.
Cell culture, transfection, luciferase assays, and cell viability assays
An IL-3-dependent murine pro-B cell line, BAF3 was maintained in RPMI
1640 medium supplemented with 10% FCS and 10% conditioned medium
from WEHI 3B cells as a source of IL-3 (15). A stable transforming cell
line expressing FK506 binding protein (FKBP)-Fas, FLAG-tagged Daxx,
and Myc-tagged DMAP1, BAF/FD7/DM1, was established as previously
described (15) and maintained in the above medium in the presence of
G418 (1 mg/ml). AP20187 stimulation was also performed in the presence
of IL-3. Human embryonic kidney carcinoma cell line, 293T, was maintained in DMEM containing 10% FCS and transfected by the standard
calcium precipitation protocol (17). The cells were harvested 48 h after
transfection and lysed in 100 ␮l of PicaGene Reporter Lysis buffer (Toyo
Ink, Tokyo, Japan) and assayed for luciferase and ␤-galactosidase activities
according to the manufacturer’s instructions. Luciferase activities were
normalized to the ␤-galactosidase activities. Three or more independent
experiments were conducted for each assay. Cell viability was determined
by Cell Counting kit 8 (Wako Chemicals, Tokyo, Japan) according to the
manufacturer’s instructions.
Immunoprecipitation and immunoblotting
The immunoprecipitation and Western blot assays were performed as previously described (17). Cells were harvested and lysed in lysis buffer (50
mM Tris-HCl, pH 7.4, 0.15 M NaCl, containing 1% Nonidet P-40, 1 ␮M
sodium orthovanadate, 1 ␮M PMSF, and 10 ␮g/ml each of aprotinin, pepstatin and leupeptin). The immunoprecipitates from cell lysates were resolved on SDS-PAGE and transferred to Immobilon filter (Millipore, Bedford, MA). The filters were then immunoblotted with each Ab.
Immunoreactive proteins were visualized using an ECL detection system
(Amersham Pharmacia Biotech, Piscataway, NJ).
Analysis of protein stability
To analyze the stability of DMAP1 in 293T cells, cells were transfected
with Myc-tagged DMAP1 and full-length or Daxx mutant by the standard
calcium precipitation protocol. After 36 h of incubation, the transfected
cells were treated with or without 10 ␮M MG132 for 1 h and then with
cycloheximide at a final concentration of 25 ␮g/ml for the indicated periods. The cell lysates were prepared and followed by Western blotting with
an anti-Myc Ab or an anti-actin Ab.
Indirect immunofluorescence
Monkey COS7 or human HeLa cells were maintained in DMEM containing 10% FCS transfected with DMAP1 and/or Daxx and/or DNMT1 1–126
by the calcium phosphate precipitation protocol. After 48 h of transfection,
cells were fixed with a solution containing 4% paraformaldehyde and reacted with an anti-FLAG Ab, an anti-Myc Ab, or an anti-Daxx Ab. The
cells were then reacted with an FITC-conjugated anti-rabbit IgG or rhodamine-conjugated anti-mouse IgG (Chemicon International) and observed
under a confocal laser fluorescent microscope. Images were obtained by
using a Zeiss LSM 510 laser scanning microscope with an Apochromat
⫻63/1.4 oil immersion objective and ⫻4 zoom.
Results
Identification of DMAP1 as an interaction partner of Daxx
To identify proteins that could be involved in the IFN/Daxx-mediated signaling, we screened a mouse 11-day embryo cDNA library using mouse Daxx/N Daxx/C as bait. Several Daxx-interacting proteins were identified from a screening of ⬃5 ⫻ 106
transforming yeast. DNA sequencing analysis revealed that one of
the positive clones that interacted specifically with Gal4 DNAbinding domain-fused Daxx/N was identical with a DMAP1 that
contains a whole amino acid insertion (residue 1– 467). To demonstrate specificity of binding, the plasmid was isolated from the
positive two-hybrid clone and introduced back into S. cerevisiae
along with either the Daxx/N or the Daxx/C fused to the DNAbinding domain of Gal4 or empty vector (Gal4 DNA-binding domain alone). Neither the Daxx/N nor the Daxx/C resulted in activation of the reporter genes (data not shown). After mating the
indicated yeast, growth occurs only in the presence of the Daxx/N
(Fig. 1A), demonstrating that DMAP1 interacted with the Daxx/N
in this assay. As previously described, Daxx had been reported to
interact with a number of cellular proteins. Most of these factors
have in common is that they require the Daxx/C for binding (see
detailed listing in Fig. 1B) (1–9, 18 –22).
To investigate the association of DMAP1 with Daxx in mammalian cells, 293T cells were transfected with FLAG-tagged Daxx
together with or without Myc-tagged DMAP1. As shown in Fig.
1C, Western blot analysis of the immunoprecipitates with an antiFLAG Ab revealed that DMAP1 interacts with Daxx in 293T cells.
To delineate the regions of Daxx that are involved in Daxx/
DMAP1 interaction, various deletion constructs of Daxx were engineered (Fig. 1B) and subjected to analyze in coimmunoprecipitation assays in 293T cells. As shown in Fig. 1D, the N-terminal
deletion mutant of Daxx (Daxx 493–740) failed to interact with
DMAP1. In contrast, the C-terminal deletion mutant of Daxx
(Daxx 1–240 or Daxx 241– 492) was capable of interacting with
DMAP1. This is consistent with results observed in the yeast twohybrid assay. Together, these results implicated Daxx/N as necessary and sufficient for interaction with DMAP1. We then examined
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Dexamethasone and cycloheximide were purchased from Wako Pure
Chemical (Osaka, Japan). MG132 was purchased from Peptide Institute
(Osaka, Japan). A synthetic dimerizer, AP20187, was kindly provided by
ARIAD Gene Therapeutics (Cambridge, MA) (15). Expression vectors,
FLAG-tagged Daxx (15), murine mammary tumor virus-luciferase
(MMTV-LUC) (16), and hemagglutinin (HA)-tagged Daxx (1) were kindly
provided by Dr. H. Ariga (Hokkaido University, Sapporo, Japan), Dr. T.
Taira (Hokkaido University), Dr. X. Yang (Massachusetts Institute of
Technology, Cambridge, MA), and Dr. G. L. Hager (National Cancer Institute, National Institutes of Health, Bethesda, MD), respectively. Myctagged Daxx and DMAP1 mutants were generated by PCR methods and
sequenced (primer sequences are available upon request). Myc-tagged
mouse DNMT1 encoding amino acid residues 1–126 was also generated by
PCR methods and sequenced (primer sequences are available upon request). Anti-HA and anti-Myc, anti-DNMT1 and anti-Daxx Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-FLAG M2
Ab was purchased from Sigma-Aldrich (St. Louis, MO). Anti-actin Ab was
purchased from Chemicon International (Temecula, CA). Anti-DMAP1 Ab
for Western blotting was purchased from Abcam (Cambridge, U.K.). We
also generated anti-DMAP1 Ab for immunoprecipitation by immunizing
GST-DMAP1 into a rabbit. JNK activation was determined by PhosphoPlus JNK Ab kit (Cell Signaling Technology, Beverly, MA) according to
the manufacturer’s instructions.
INTERACTIONS BETWEEN Daxx AND DMAP1
The Journal of Immunology
2987
the interacting domains on DMAP1 with Daxx. To delineate the
domains in the DAMP1 that mediate the protein-protein interaction with Daxx, coimmunoprecipitation experiments were performed with a series of mutant DMAP1 proteins (Fig. 2A). Expression vectors encoding FLAG-tagged Daxx and a series of
Myc-tagged DMAP1 mutants were transiently transfected into
293T cells. Cells were lysed and subjected to immunoprecipitation
with an anti-FLAG Ab. Immunoprecipitates were then used in
Western blot analysis with an anti-Myc Ab. As shown in Fig. 2B,
the C-terminal domain of DMAP1 (DMAP1/C; amino acid residues 235– 467) strongly interacted with Daxx. Furthermore, the
middle region of the C-terminal domain of DMAP1 (DMAP1/C2;
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FIGURE 1. Physical interactions between Daxx and DMAP1. A, Interactions
between Daxx and DMAP1 in a yeast
two-hybrid assay. Growth of transformed
S. cerevisiae demonstrating an interaction
between either Daxx/N or Daxx/C and
DMAP1. pGBKT7-Daxx/N, pGBKT7Daxx/C, pGBKT7-p53, or empty pGBKT7
in AH109 were mated with pACT2DMAP1, pACT2-SV40 T antigen (T), or
empty pACT2 in Y187 as indicated. Colonies were then restreaked onto highstringency plates. B, Schematic overview
depicting interaction domains within
Daxx required for association with previously published cellular proteins (Refs.
1–9, 18 –22). The amino acids of human
Daxx required for the respective interactions are indicated. Daxx mutant fragments used in experiments are also schematically shown. C, 293T cells (1 ⫻ 107)
were transfected with FLAG-tagged
Daxx (10 ␮ g) and/or Myc-tagged
DMAP1 (10 ␮g). Cell lysates were then
immunoprecipitated with anti-FLAG Ab
and immunoblotted with anti-Myc Ab
(upper panel) or anti-FLAG Ab (middle
panel). Total cell lysates (1%) were blotted with anti-Myc Ab as indicated (lower
panel). D, 293T cells (1 ⫻ 107) were
transfected with a series of Myc-tagged
Daxx mutants (10 ␮g) and FLAG-tagged
DMAP1 (10 ␮g). After 48-h transfection,
cells were lysed and immunoprecipitated
with an anti-FLAG Ab and immunoblotted with anti-Myc Ab (upper panel) or
anti-FLAG Ab (middle panel). Total cell
lysates (1%) were blotted with anti-Myc
Ab (lower panel). The asterisks indicate
the migration position of Daxx deletion
mutants.
amino acid residues 293– 411) strongly mediated the protein-protein interaction between DMAP1 and Daxx. These results indicate
that Daxx/N interacts with DMAP1/C. To examine the interactions
between Daxx and DMAP1 under more physiological conditions
through endogenous proteins, we first transfected FLAG-tagged
DMAP1 alone into 293T cells. Cells were lysed and subjected to
immunoprecipitation with an anti-FLAG Ab. Immunoprecipitates
were then used in Western blot analysis with an anti-Daxx Ab. As
shown in Fig. 2C, FLAG-tagged DMAP1 interacted with endogenous
Daxx. Furthermore, we performed coimmunoprecipitation experiments using untransfected 293T cells. As shown in Fig. 2C, we found
that the immunoprecipitate with anti-DMAP1 Ab contained Daxx
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INTERACTIONS BETWEEN Daxx AND DMAP1
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FIGURE 2. Mapping of Daxx interacting domain on DMAP1 and endogenous interactions between Daxx and DMAP1. A, Domain structure of DMAP1
and mutant fragments are schematically shown. B, 293T cells (1 ⫻ 107) were transfected with a series of Myc-tagged DMAP1 mutants (10 ␮g) and
FLAG-tagged Daxx (10 ␮g). After 48-h transfection, cells were lysed and immunoprecipitated with an anti-FLAG Ab and immunoblotted with anti-Myc
Ab (upper panel) or anti-FLAG Ab (middle panel). Total cell lysates (1%) were blotted with anti-Myc Ab (lower panel). The asterisks indicate the migration
position of the full-length DMAP1 or deletion mutants. C, 293T cells (3 ⫻ 107 cells) were transiently transfected with expression vectors containing
FLAG-tagged DMAP1 (10 ␮g) alone. After transfection (48 h), cells were lysed and immunoprecipitated with control IgG (lane 1) or an anti-FLAG Ab
(lane 2), and immunoblotted with anti-Daxx Ab (top panel), anti-FLAG Ab (middle panel). Total cell lysates (1%) were blotted with an anti-Daxx Ab
(bottom panel). 293T cells (4 ⫻ 107 cells) were lysed and immunoprecipitated with control IgG (lane 3) or an anti-DMAP1 Ab (lane 4), and immunoblotted
with anti-Daxx Ab (top panel), anti-DMAP1 Ab (middle panel). Total cell lysates (1%) were blotted with respective Ab (bottom panel).
proteins. These results strongly suggest that DMAP1 forms a complex
with Daxx in 293T cells.
Association of Daxx with DNMT1 via DMAP1
DMAP1 was first identified as an interaction partner with DNMT1
through its N-terminal 120 amino acids without the C-terminal
catalytic domain (14). The noncatalytic, N-terminal portion of
DNMT1 is shown to function as a transcriptional repressor that
directly interacts with, and is partially dependent on, the activity of
HDAC2 (14). In a previous report, a direct interaction between
Daxx and DNMT1 in yeast was described, although no data were
presented (2).
The Journal of Immunology
protein only in the presence of DMAP1. These results suggest that
Daxx forms a complex with DMAP1 and DNMT1 in the nucleus.
Colocalization of Daxx with DMAP1 and DNMT1
To determine whether DMAP1 colocalizes with Daxx in the nucleus, expression vectors for Myc-tagged DMAP1 and FLAGtagged Daxx were transfected into COS7 cells. After 48-h transfection, the cells were stained with anti-Myc and anti-FLAG Abs
and were visualized with rhodamine and FITC-conjugated secondary Abs, respectively, under a confocal laser microscope (Fig. 4A).
In the previous study, DMAP1 was shown to colocalize with
DNMT1 to small punctate structures characteristic of S phase replication in the nucleus (14). Our results showed that both DMAP1
and Daxx were located in the nucleus, and they were found to be
colocalized after demonstration of the merged figure in which the
red and green colors turned yellow (Fig. 4A), suggesting that both
DMAP1 and Daxx colocalize in the nucleus. We also examined
whether both DMAP1 and Daxx colocalize with DNMT1, expression vectors for Myc-tagged DNMT1 (1–126), and FLAG-tagged
DMAP1 and HA-tagged Daxx were transfected into COS7 cells.
After transfection (48 h), the cells were stained with anti-Daxx and
anti-Myc, anti-FLAG Abs, and were visualized with rhodamine
and FITC-conjugated secondary Abs, respectively, under a confocal laser microscope (Fig. 4B). As shown Fig. 4B, they were found
to be colocalized in the nucleus.
We next examined whether Daxx and DMAP1 colocalized in
the nucleus under more physiological conditions. Expression vectors for FLAG-tagged DMAP1 alone were transfected into HeLa
cells. After 48-h transfection, the cells were stained with anti-Daxx
and anti-FLAG Abs, and they were visualized with rhodamine and
FITC-conjugated secondary Abs, respectively, under a confocal
laser microscope (Fig. 4C). As shown in Fig. 4B, endogenous
Daxx and FLAG-tagged DMAP1 were found to be colocalized in
the nucleus. These results indicate that Daxx and DMAP1 with
DNMT1 colocalize in the nucleus.
Effect of DMAP1 on Daxx-mediated apoptosis in murine
pro-B cells
FIGURE 3. Daxx forms a complex with DMAP1 and DNMT1. A, 293T
cells (1 ⫻ 107 cells) were transiently transfected with expression vectors
containing HA-tagged Daxx (10 ␮g), FLAG-tagged DMAP1 (10 ␮g) and
Myc-tagged DNMT1(1–126) (15 ␮g) as indicated. After transfection (48
h), cells were lysed and immunoprecipitated with an anti-HA Ab and immunoblotted with anti-Myc Ab (upper panel) or anti-HA Ab (top middle
panel). Total cell lysates (1%) were blotted with anti-FLAG (bottom middle panel) or anti-Myc Ab (lower panel). B, 293T cells (1 ⫻ 107 cells) were
transiently transfected with expression vectors containing FLAG-tagged
Daxx (10 ␮g) and Myc-tagged DMAP1 (10 ␮g) as indicated. After transfection (48 h), cells were lysed and immunoprecipitated with control IgG
or an anti-DNMT1 Ab, and immunoblotted with anti-FLAG Ab (upper
panel), anti-Myc Ab (middle panel) or anti-DNMT1 Ab (lower panel).
Total cell lysates (1%) were blotted with respective Ab.
In our previous study, we established a murine pro-B cell line,
BAF/FD7, which expresses the fusion protein composed of FKBP
and membrane-anchored intracellular domain of Fas (FKBP-Fas)
and FLAG-tagged Daxx after transfecting these expression constructs into original BAF3 cells (15). Aggregation of FKBP-Fas
proteins by the addition of a bivalent FKBP ligand, AP20187,
strongly induced cell death. However, original BAF3 as well as
BAF3 cells expressing FKBP-Fas alone did not die after the treatment with AP20187 (15). Furthermore, AP28187 treatment of
BAF/FD7 cells induced the marked JNK activation, when we monitored by immunoblotting using an Ab directed to phospho-JNK
Thr183/Tyr185 (15).
To examine whether DMAP1 has an effect on Fas/Daxx-mediated cell death, we transfected Myc-tagged DMAP1 into BAF/FD7
cells and established a stable transformation with BAF/FD7DM1,
which expresses both Daxx and DMAP1. We first examined the
interaction between Daxx and DMAP1 in BAF/FD7/DM1 cells.
Coimmunoprecipitation experiments were performed using cell lysates obtained from original BAF3, BAF/FD7, or BAF/FD7DM1
cells that were untreated with AP20187. Cell lysates were subjected to immunoprecipitation with an anti-FLAG Ab. Immunoprecipitates were then used in Western blot analysis with an antiMyc Ab. As shown in Fig. 5A, we found that FLAG-tagged Daxx
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We then examined whether Daxx forms the complex with
DMAP1 and DNMT1 in mammalian cells. Expression vectors encoding HA-tagged Daxx and/or FLAG-tagged DMAP1, Myctagged N-terminal region of DNMT1 (amino acid residues 1–126)
were transiently transfected into 293T cells. Cells were lysed and
subjected to immunoprecipitation with an anti-HA Ab. Immunoprecipitates were then used in Western blot analysis with an antiMyc Ab. As shown in Fig. 3A, the immunoprecipitates with antiHA Ab contained the N-terminal domain of DNMT1 only in the
presence of DMAP1, suggesting that Daxx forms a complex with
DNMT1 in the presence DMAP1.
To further examine whether the full-length DNMT1 forms the
complex with Daxx, coimmunoprecipitation experiments were
performed using endogenous DNMT1 proteins in 293T cells. Expression vectors encoding FLAG-tagged Daxx and/or Myc-tagged
DMAP1 were transiently transfected into 293T cells. Cells were
lysed and subjected to immunoprecipitation with control IgG or an
anti-DNMT1 Ab. Immunoprecipitates were then used in Western
blot analysis with an anti-FLAG or Myc Ab. As shown in Fig. 3B,
the immunoprecipitates with anti-DNMT1 Ab contained Daxx
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2990
INTERACTIONS BETWEEN Daxx AND DMAP1
immunoprecipitated from BAF/FD7/DM1 cells resulted in a complex with Myc-tagged DMAP1 and this interaction was independent on the presence of AP20187, suggesting that Daxx constitutively associates with DMAP1 in murine pro-B cells.
We next examined whether DMAP1 has an effect on Fas/Daxxmediated cell death using BAF/FD7DM1 cells. As previously described, BAF/FD7 cells underwent cell death with the addition of
AP20187 as shown in Fig. 5B. Similarly, BAF/FD7DM1 cells underwent cell death by AP20187 in a dose-dependent manner (Fig.
5B). Upon AP20187 stimulation, the strong-sustained phosphorylation of JNK was induced in both BAF/FD7 and BAF/FD7DM1
cells as shown in Fig. 5C. These results suggest that DMAP1 expression and association with Daxx has no effect on Fas/Daxxmediated cell death and JNK activation when we overexpressed
both of them in murine pro-B cells.
DMAP1 and Daxx cooperatively repress GR-mediated
transcriptional activity
Daxx has been reported to function as a transcriptional modulator
in the nucleus (4, 6, 9, 23, 24). It has been also demonstrated that
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FIGURE 4. Colocalization of Daxx with
DMAP1 and DNMT1 in the nucleus. A, COS7
cells were cotransfected with FLAG-tagged
Daxx and Myc-tagged DMAP1 by the calcium
phosphate precipitation protocol. After transfection (48 h), cells were fixed, reacted with an antiDaxx polyclonal Ab, and an anti-Myc mAb, and
visualized with an FITC-conjugated anti-rabbit
Ab (a) and a rhodamine-conjugated anti-mouse Ab
(b). These images were merged (c). B, COS7 cells
were cotransfected with HA-tagged Daxx, FLAGtagged DMAP1 and Myc-tagged DNMT1(1–126)
by the calcium phosphate precipitation protocol.
After transfection (48 h), cells were fixed, reacted
with an anti-Myc polyclonal Ab (a), an anti-FLAG
mAb (b), an anti-Daxx polyclonal Ab (c), and an
anti-Myc mAb (d), and visualized with an FITCconjugated anti-rabbit Ab (a and d) and a rhodamine-conjugated anti-mouse Ab (b and e). These images were merged (c and f). C, HeLa cells were
transfected with FLAG-tagged DMAP1 alone. After transfection (48 h), cells were fixed, reacted with
an anti-Daxx Ab (a), an anti-FLAG mAb (b), and
visualized with an FITC-conjugated anti-rabbit Ab
(a) and a rhodamine-conjugated anti-mouse Ab (b).
These images were merged (c).
overexpression of Daxx suppresses GR-mediated activation of the
MMTV promoter in a human embryonic kidney carcinoma cell
line, 293T cells (12). To examine the functional relevance of the
Daxx-DMAP1 interaction in the context of GR signaling pathway,
we performed the transient transfection assay using 293T. The
GR-mediated transcriptional responses were measured by MMTVLUC, which is one of the standard reporters for assessing GR
activity (16). When 293T cells were transfected with MMTV-LUC
together with an expression vector for GR and treated with dexamethasone, luciferase expression was increased by 70-fold (Fig.
6A). As shown in Fig. 6A, overexpression of Daxx suppressed
GR-mediated transactivation in a dose-dependent manner.
DMAP1 was shown to act as a corepressor with DNMT1 independent of HDACs (14). We then examined the effect of overexpression of DMAP1 on GR-mediated transactivation in 293T cells.
When 293T cells were transfected with an expression vector for
DMAP1, GR, and MMTV-LUC, overexpression of DMAP1
showed a moderate suppression in GR-induced MMTV-LUC activity (Fig. 6B). Interestingly, in the presence of Daxx, overexpression of DMAP1 effectively suppressed GR-induced MMTV-LUC
The Journal of Immunology
activity, although the effect was additive. The previous study demonstrated that the suppression of GR activity was mediated through
the C-terminal region of Daxx (16). We then tested whether
DMAP1 has any effect on the suppression of GR activity by Daxx
493–740, which did not interact with DMAP1. As shown in Fig.
6C, the DAMP1-mediated suppression of GR activity was enhanced by Daxx 493–740. These results suggest that Daxx and
DMAP1 can independently act as a corepressor for GR-mediated
transcription.
Daxx protects DMAP1 from the proteasomal degradation
During our experiments on the Daxx-DMAP1 interaction, we
found that DMAP1 was a short-lived protein in 293T cells in the
absence of Daxx. As shown in Fig. 7A, coexpression of Daxx
protein in 293T cells resulted in the accumulation of DMAP-1
protein in a dose-dependent manner. We also examined whether
the proteasome-specific inhibitor MG132 can block DMAP1 degradation in 293T cells. Treatment of 293T cells with MG132 also
resulted in the accumulation of DMAP1 in a dose-dependent manner (Fig. 7B). To further determine whether Daxx or the proteasome inhibitor, MG132, directly affects degradation of DMAP-1
FIGURE 6. DMAP1 and Daxx cooperatively repress GR-mediated transcriptional activity. A, 293T cells (12-well plate) were transfected with
MMTV-LUC (0.3 ␮g) and/or Daxx as indicated. After transfection (48 h),
cells were stimulated for an additional 12 h for dexamethasone (Dex) (10⫺7
M) as indicated. Luciferase activities were determined. B, 293T cells (12well plate) were transfected with MMTV-LUC (0.3 ␮g) and/or DMAP1
and/or Daxx as indicated. After 48-h transfection, cells were stimulated for
an additional 12 h for dexamethasone (Dex) (10⫺7 M) as indicated. Luciferase activities were determined. C, 293T cells (12-well plate) were
transfected with MMTV-LUC (0.3 ␮g) and/or DMAP1 and/or Daxx(493–
740) as indicated. After 48-h transfection, cells were stimulated for an
additional 12 h for dexamethasone (10⫺7 M) as indicated. Luciferase activities were determined. The results are presented as fold induction of
luciferase activity from triplicate experiments, and the error bars represent
the SD.
protein in 293T cells, we examined the stability of DMAP1 protein
under conditions in which protein synthesis had been blocked by
cycloheximide. Western blot analysis revealed that the DMAP-1
protein was stabilized when cells were transfected with Daxx but
not Daxx/C-Daxx 493–740, or in the presence of MG132 (Fig.
7C). These results suggest that Daxx but not Daxx 493–740 protects DMAP1 protein from proteasomal degradation in vivo.
Discussion
In this study, two-hybrid screening has led to our identification of
DNMT1 associated protein, DMAP1, as a Daxx-interacting protein. The interaction involves Daxx/N and DMAP1/C2. Daxx
formed a complex with DNMT1 via DMAP1 in the nucleus. Furthermore, Daxx and DMAP1 additively repressed GR-mediated
transcription, although DMAP1 had no effect on Fas/Daxx-mediated apoptosis in a murine pro-B cell line. Interestingly, Daxx
protected DMAP1 protein from proteasomal degradation in vivo.
Daxx was reported to be involved in the Fas- and TGF-␤-mediated apoptotic signaling pathway (1, 25). Daxx was originally
cloned as a Fas-associated protein and binds specifically to the
death domain of Fas, although Daxx by itself lacks a death domain
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FIGURE 5. Effect of DMAP1 on Daxx-mediated apoptosis in a murine
pro-B cells. A, BAF3, BAF/FD7, or BAF/FD7DM1 cells (1 ⫻ 107) were
lysed and then immunoprecipitated with anti-FLAG, and immunoblotted
with anti-Myc Ab (upper panel), anti-FLAG Ab (middle panel). Total cell
lysates (1%) were blotted with an anti-Myc Ab (lower panel). B, Each cells
(2 ⫻ 104) were treated with the indicated concentrations of AP20187. After
transfection (24 h), cell viability was determined by Cell Counting kit 8. B,
JNK activation by AP20187 in BAF/FD7 or BAF/FD7DM1 cells. Each
cells (2 ⫻ 106) was treated with AP20187 (1 nM) for the indicated time,
lysed, and immunoblotted (C) with an anti-phospho JNK Ab or an antiJNK Ab.
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(1). There are two independent signaling pathways downstream of
Fas, involving the adapter protein Fas-associated death domain and
Daxx (26). The activation of Fas-associated death domain induces
a protease cascade (27), whereas that of Daxx enhances JNK activation, leading to apoptosis (1, 27). Overexpression of Daxx enhances Fas-induced apoptosis (1, 15, 24), and the targeted disruption of the Daxx gene in mice results in embryonic lethality (2).
Daxx is also involved in coupling of type II TGF-␤ receptor signaling with components of the apoptotic machinery (25). Daxx
associates with the cytoplasmic domain of the type II TGF-␤ receptor and transduces apoptotic signals by TGF-␤ (25). Recently,
Daxx was also reported to be essential for IFN-induced suppression of B cell development (11). IFN-␣ enhances Daxx expression,
with concomitant increases in Daxx protein levels and nuclear
body translocation. Moreover, Daxx antisense oligonucleotides
rescue IFN-␣-treated pro-B cells from growth arrest and apoptosis.
We also demonstrated that Tyk2 is essential for the transduction of
IFN-␣-induced suppression of B cell development through the activation of some signaling molecules other than STAT1, followed
by the up-regulation and nuclear translocation of Daxx (10). The
apoptotic signaling pathway downstream of Daxx still remains unknown. One candidate of Daxx targets is a JNK signaling pathway.
Alternatively, the localization of Daxx is also important because
the localization of Daxx in either the cytoplasmic or nuclear compartment was reported to be dependent upon the cell type and/or its
functional status (28 –30). In this study, we have tested whether a
novel Daxx-interacting partner, DMAP1 involves in Daxx-mediated apoptotic signaling by using our conditional Fas/Daxx suicide
within a murine pro-B cell line. However, DMAP1 had no effect
on its signaling including JNK activation, although DMAP1
showed a constitutive association with Daxx in cells.
Recent studies demonstrated that Daxx might function as a transcriptional coregulator for several transcription factors such as
Pax3, ETS1, and GR through direct protein-protein interactions (4,
6, 12). Daxx recruits nuclear factors possessing either histone
acetyltransferase or HDAC to modulate transcriptional activity (5,
20). In fact, Daxx is shown to bind to HDAC2 (20). In addition, the
transcriptional repression effect of Daxx could be modulated by
subnuclear compartmentalization through protein-protein interactions. For example, PML has been shown to relieve the transrepression effect of Daxx through sequestering Daxx into the PML
oncogenic domain (9). A nucleolar 58-kDa microspherule protein
is also shown to relieve the transcriptional repression by Daxx
through protein-protein interactions (21). DMAP1 is shown to
have an intrinsic transcriptionally repressive activity (14). We report that Daxx and DMAP1 cooperatively repressed GR-mediated
transcription in 293T cells. To our interest, Daxx protected
DMAP1 protein degradation through proteasomal signaling pathway. These results strongly suggested that Daxx and DMAP1 form
an effective repression complex in the nucleus.
Furthermore, our experiments revealed that another DMAP1interacting partner also joined and formed a complex with DMAP1
and Daxx in the nucleus. DNMT1 is a major enzyme that maintains mammalian DNA methylation. DNA methylation is known to
contribute to transcriptional silencing through several transcriptionally repressive complexes, which include methyl-CpG binding
domain proteins and HDACs (31–33). The N-terminal noncatalytic
domain of DNMT1 binds to both HDAC2 and DMAP1 (14), and
can mediate transcriptional repression. DMAP1 is also shown to
directly interact with tumor susceptibility gene 101 (TSG101) (14),
a protein recently demonstrated to be a transcriptional corepressor
(34), although we do not have any evidence yet whether TSG101
also interacts with Daxx. It has been also demonstrated that
DMAP1 is targeted to replication foci through interaction with the
N-terminal domain of DNMT1 throughout S phase, whereas
HDAC2 associates with DNMT1 and DMAP1 only during late S
phase following DNA replication (14). Thus, DMAP1 may mediate to form more effective repression complex in the nucleus by
linking between Daxx and DNMT1, although DMAP1 by itself has
a transcriptionally repressive activity. At the present time, we do
not know whether the Daxx-DMAP1 interaction has any effect on
DNA methylation through methyltransferase activity by DNMT1.
This interaction may modify DNMT1 enzymatic activity to carry
out an effective repression in the nucleus. Further work will be
required to assess this possibility.
The present report describes both physical and functional interactions between Daxx and DMAP1. Daxx and DMAP1 with
DNMT1 formed a complex and colocalized in the nucleus.
DMAP1 enhanced Daxx-mediated repression of a GR transcriptional activity. Furthermore, Daxx protected protein degradation of
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FIGURE 7. Daxx protects DMAP1 from the proteasomal degradation.
A, 293T cells (1 ⫻ 107 cells) were transiently transfected with expression
vectors containing Myc-tagged DMAP1 (10 ␮g) and an increasing
amounts of FLAG-tagged Daxx as indicated. After (48 h) transfection,
cells were lysed, and total lysates (1%) were immunoblotted with an antiMyc Ab (upper panel), an anti-FLAG Ab (middle panel) or anti-actin Ab
(lower panel). B, 293T cells (1 ⫻ 107 cells) were transiently transfected
with expression vectors containing Myc-tagged DMAP1 (10 ␮g). After
transfection (48 h), cells were treated with DMSO or increasing amounts of
MG132 as indicated for 1 h and lysed. Total lysate (1%) were immunoblotted with an anti-Myc Ab (top panel) and an anti-Daxx Ab (bottom
panel). C, 293T cells (1 ⫻ 107 cells) were transiently transfected with
expression vectors containing Myc-tagged DMAP1 (10 ␮g) and empty
vector, Daxx or Daxx(493–740). After transfection (36 h), cells were
treated with or without MG132 for 1 h, followed by treatment of cycloheximide at indicated periods. Cell were lysed and total lysate (1%) were
immunoblotted with an anti-Myc Ab (upper panels), an anti-Daxx Ab
(middle panels) or anti-actin Ab (lower panels).
INTERACTIONS BETWEEN Daxx AND DMAP1
The Journal of Immunology
DMAP1 in vivo. These results provide an important linkage between Daxx and DNMT1, which forms an efficient transcription
repression complex in the nucleus.
Acknowledgments
We thank Dr. H. Ariga, Dr. T. Taira, Dr. X. Yang, and Dr. G. L. Hager, for
their kind gifts of reagents. We also thank Dr. S. Matsuzawa for critical
comments and Dr. J. Akiyama for encouraging our work.
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