From the Department of Geriatric Research, National
Institute for Longevity Sciences, Aichi 474-8522, Japan and
§ Division of Morphogenesis, National Institute of Basic
Biology, Aichi 444-8585, Japan
Received for publication, September 20, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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Dlx5, a member of the Dlx family of homeodomain
proteins, plays a critical role in bone development and fracture
healing. To understand the molecular mechanism underlying the
transcriptional regulation by Dlx5, we performed yeast two-hybrid
screening and isolated a novel protein, Dlxin-1, that binds Dlx5 and
regulates its transcriptional function. Dlxin-1 cDNA encodes a
775-amino acid protein that has a partial homology with necdin at the C terminus and 25 repeats of hexapeptides
(WQXPXX) in the middle region. Dlxin-1
mRNA is expressed in various adult tissues, but not the
spleen, and also in osteoblastic and chondrogenic cell lines. During
embryogenesis, a strong signal for Dlxin-1 mRNA was found in cell
layers surrounding cartilaginous elements in bone rudiment during digit
formation. Dlxin-1 binds not only Dlx5 but also Dlx7 and Msx2 and forms
homomultimers in vivo. Transfection and reporter gene
assays indicate that Dlxin-1 activates the transcriptional function of
Dlx5. Therefore, Dlxin-1 may act as a regulator of the function of Dlx
family members in bone formation.
The Dlx gene family, which comprises at least six mammalian
homologues of the Drosophila homeodomain protein
Distal-less, is expressed predominantly in forebrain, limbs, and
branchial arches during fetal development (1, 2). Among this family, Dlx5 is expressed in most developing skeletal elements and is induced
during the process of bone fracture healing (2-6), suggesting that it
may play a crucial role in bone development and formation. Msx2 is
expressed in skeletal elements and many other organs (7-9). The
expression of Dlx5 mRNA, as well as Msx2, is induced by bone morphogenetic protein (5, 7, 10). It has been suggested that
Dlx5 is a transcriptional activator and regulates osteoblastic functions positively (4, 5, 11, 12). In contrast, Ryoo et
al. (13) have demonstrated, using a reporter gene assay, that Dlx5
is a potential negative regulator of the expression of the rat
osteocalcin gene, which is specifically expressed in bone. Recently,
Acampora et al. (14) and Depew et al. (15) have
reported the phenotype of mice homozygous for targeted deletion of the
dlx5 gene, in which the expression of the osteocalcin gene in osteoblasts was markedly increased, suggesting that Dlx5 represses the expression of the osteocalcin gene in vivo (14).
To understand the molecular mechanism(s) underlying the transcriptional
regulation involving Dlx5, we attempted to identify a molecule that
binds Dlx5 and regulates its function. Here we report the isolation and
characterization of Dlxin-1, a novel protein that binds Dlx5 and
regulates its transcriptional function. Dlxin-1 is a unique member of
the necdin/melanoma-associated antigen (MAGE)1 family (16-18).
Cells--
KUSA/A1 (19), a mouse osteoblastic cell line, and
HT1080, a human fibrosarcoma cell line, were kindly provided by Drs. A. Umezawa (Keio University, Japan) and A. Fukamizu (University of Tsukuba, Japan), respectively. A yeast strain, Y153, was a gift from
Dr. S. Kato (University of Tokyo).
Plasmid Construction--
The pBIND vector was purchased from
Promega (Madison, WI). Mouse Dlx5 and Msx2 cDNAs were isolated by
reverse transcriptase-polymerase chain reaction, and the N-terminal
domains (amino acids 2-132 for Dlx5 and 2-136 for Msx2) were inserted
into pBIND, generating pBIND-Dlx5 Yeast Two-hybrid Screening--
Yeast two-hybrid screening was
performed in accordance with the manufacturer's instructions
(Matchmaker, CLONTECH). Briefly, a yeast strain,
Y153, was cotransformed with pGBT-Dlx5 Trasfection--
Transfection was carried out using
LipofectAMINE Plus (Life Technologies, Inc.) or FuGENE6 (Roche
Diagnostics, Mannheim, Germany). pFDlx5 or pcDNA3 was introduced into
P19 embryonic carcinoma cells, and stable transfectants were selected
by the addition of Geneticin (400 µg/ml; Life Technologies). Colonies
were taken and expanded to examine the expression of FLAG-tagged Dlx5
protein. A Dlx5-expressing P19 cell line (P19Dlx5) and a
mock-transfected line (P19neo) were used in this study. There was no
difference in proliferative activity and morphology between P19Dlx5 and P19neo.
Reporter Gene Assay--
The plasmids, pBIND and pG5luc, and the
dual luciferase assay system were purchased from Promega. Transfected
cells were lysed in Passive Lysis Buffer (Promega) and assayed for
firefly and Renilla luciferase activities (Lumat LB 9507, EG & G Berthold, Bad Wildbad, Germany). Each assay was carried out at
least in triplicate.
Northern Blotting and in Situ Hybridization--
Northern
blotting was carried out using a 0.7-kilobase pair fragment of mouse
Dlxin-1 cDNA on mouse MTN and Embryo set blot (CLONTECH). Total RNA isolated from mouse long
bones, bone marrow cells in long bones, and calvaria was denatured by
glyoxal/Me2SO and electrophoresed in agarose gels. The
digoxigenin-labeled antisense and sense RNA probes were synthesized
using T7 or Sp6 RNA polymerase (Promega) with a nucleotide mix
containing digoxigenin-labeled CTP (Roche Diagnostics). Hybrid-ready
tissue slides were obtained from Novagen (Madison, WI), and in
situ hybridization was performed as reported previously (21).
GST Pull-down Assay--
GST and GST fusion proteins were
expressed in DH5 Immunoprecipitation and Immunoblotting--
The HA
epitope-tagged Dlxin-1 expression vector (pHA-Dlxin1) and the FLAG
epitope-tagged expression vectors (pFDlx3, pFDlx5, pFDlx7, pFMsx2,
pF-Dlxin1) were cotransfected transiently into COS7 cells using
LipofectAMINE reagent (Life Technologies). At 24 h after
transfection, cells were lysed with radioimmune precipitation buffer
(10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, and 1% sodium
deoxycholate) supplemented with a mixture of proteinase inhibitors
(CompleteTM; Roche Diagnostics). Precleared cell lysates
were subjected to immunoprecipitation with anti-FLAG M2 antibody
(Sigma) following adsorption to protein G-Sepharose beads (Amersham
Pharmacia Biotech). In some experiments, agarose-conjugated anti-FLAG
antibody was used, with FLAG peptide as a competitor (Sigma). Bound
proteins were eluted from beads by boiling in SDS sample buffer,
separated by SDS-PAGE, and transferred to polyvinylidene difluoride
membrane. Immunoblotting was performed using anti-FLAG M5 antibody
(Sigma) or anti-HA 3F10 antibody and visibilized by ECL Plus reagents (Amersham Pharmacia Biotech).
Transcriptional Activator Function of the N-terminal Domain of
Dlx5--
To evaluate the transcriptional activity of Dlx5, we
constructed a reporter gene assay system. The reporter construct,
DlxRE, which consists of Dlx5-responsive elements and basal promoter with the luciferase gene (Fig.
1A), was introduced into
Dlx5-expressing (P19Dlx5) or mock-transfected P19 cells (P19neo). As
shown in Fig. 1B, a high activity of DlxRE-driven reporter
was observed in P19Dlx5 cells, but not in P19neo cells. When a core
sequence of DlxRE was mutated (Dlxm), reporter activity was not
detected in either P19neo or P19Dlx5 cells. These results suggest that Dlx5 has a transcriptional activation function in P19 cells.
The N-terminal domain of Dlx5 has similarities to those of Dlx2, Msx1,
and Msx2 (4). To characterize the function of this shared domain, the
N-terminal domain of Dlx5 (Dlx5 Isolation of a Molecule That Binds to the N-terminal Domain of
Dlx5--
In an attempt to search for a protein(s) that associates
with the N-terminal domain of Dlx5 and regulates its function, yeast two-hybrid screening was performed. From screening of the mouse embryo
(embryonic day 11) cDNA library, seven independent and overlapping
clones were obtained on the basis of selection for histidine
requirement for growth and lacZ expression (Fig.
2A). We found, by BLAST search
(22), that several expressed sequence tag sequences were identical with
the isolated clones, and the 5'-nucleotide sequence was determined by
5'-rapid amplification of cDNA ends. As shown in Fig.
2B, the amino acid sequence predicted from the full-length
cDNA reveals a novel protein of 775 amino acids, and the predicted
molecular mass of the protein is 85.7 kDa. We propose that the novel
protein be called Dlxin-1, for Dlx interaction.
Dlxin-1 has a partial similarity to necdin (23) and MAGEs at the
C-terminal region, as shown in Fig.
3A. A putative human
counterpart of mouse Dlxin-1 has been reported as MAGE-D1 (18). In the
data base, SNERG-1 (GenBankTM accession number
AF274043) from rat testis showed a high homology (96%) to mouse
Dlxin-1, suggesting that SNERG-1 is a rat orthologue of mouse Dlxin-1
(Fig. 3B). There is no significant hydrophobic stretch
corresponding to a signal sequence or transmembrane domain, suggesting
that Dlxin-1 is an intracellular protein. Notably, 25 hexapeptide
repeats in tandem were found in the middle part of the protein, with a
consensus sequence of WQXPXX (Fig.
3C). The shortest clone (clone 16) isolated in the
two-hybrid screening encodes 18 out of the 25 repeats, and the sequence
was included in all of the overlapping clones, suggesting that the
WQXPXX repeat region represents the binding site
of the N-terminal domain of Dlx5 (Fig. 2A). No significant
homology to any protein sequences was found in the N-terminal part of
Dlxin-1 by searching data bases.
Expression of Dlxin-1 mRNA--
To determine the expression of
Dlxin-1 mRNA in various tissues and cell lines, Northern blot
analyses were performed. Most adult mouse tissues were found to express
a 3.0-kilobase pair transcript of Dlxin-1, except for the spleen (Fig.
4A). As Dlx5 has been reported
to be expressed predominantly in skeletal elements (1, 14, 15), Dlxin-1
mRNA was detected in bone, but not in bone marrow, suggesting that
Dlxin-1 is expressed in adherent cells rather than hematopoietic cells
in bone. The Dlxin-1 mRNA was expressed at embryonic stages
(embryonic days 7-17 (ED 7-17)) in mouse development (Fig.
4A). Dlxin-1 was also widely expressed in various human and
mouse cell lines, including osteoblastic (KUSA/A1 and MC3T3-E1) and
chondrogenic (ATDC5) cells (data not shown).
Next, we performed in situ hybridization to localize the
expression of the Dlxin-1 gene in embryonic and adult tissues. The expression detected by the antisense probe for Dlxin-1 mRNA was ubiquitous in tissues on mouse embryo days 13 and 15 (not shown). Notably, the strongest signal was detected in the cell layers surrounding cartilaginous elements in bone rudiment during embryonic digit formation (Fig. 4B).
Dlxin-1 Binds to Dlx5 in Vivo--
Although the clones for
Dlxin-1 were isolated by interaction trap with Dlx5, the protein
interaction remained to be confirmed in vitro and in
mammalian cells. In the GST pull-down assay, in vitro
translated Dlx5 coprecipitated with GST-Dlxin-1, indicating that
Dlxin-1 associates with Dlx5 in vitro, probably via direct binding (data not shown). Interestingly, Msx2, another member of the
Dlx/Msx homeodomain protein family, also bound to Dlxin-1 (data not shown).
Next, the association of Dlx5 with Dlxin-1 was determined in COS7
cells. As shown in Fig. 5, FLAG-tagged
Dlx5 protein coprecipitated with HA-tagged Dlxin-1. Dlx7, Msx2,
and FLAG-tagged Dlxin-1 also coprecipitated with HA-tagged Dlxin-1.
These results indicate that Dlxin-1 associates not only with Dlx5 but
also with Dlx7 and Msx2 and forms multimers in vivo.
Dlxin-1 Activates Dlx5 Dlxin-1 Plays a Role in Dlx5-dependent
Transcription--
Finally, to test the hypothesis that Dlxin-1
stimulates Dlx5-dependent transcription, a reporter gene
assay was carried out using a Dlx5/Dlx5-responsive element reporter
system. The Dlx5-binding domain of Dlxin-1 (Dlxin-DlxBD, clone 16, in
Fig. 7A) was transfected into
P19Dlx5 cells, which express a relatively high level of Dlxin-1 mRNA, and reporter activity was then measured. The reporter
activity was reduced to the basal level by the addition of Dlxin-DlxBD, whereas cotransfection of full-length Dlxin-1 (Dlxin-FL)
showed little effect (Fig. 7B). It is suggested that Dlxin-1
is required for maximal Dlx5-dependent transcriptional
activation in P19 cells.
Dlxin-1, a Novel Dlx5-binding Protein--
In this paper, we
describe the identification of a novel Dlx5-binding protein, named
Dlxin-1. When compared with MAGE-D1, a human orthologue of Dlxin-1, the
N-terminal section, flanking the WQXPXX repeats,
is longer in mouse Dlxin-1, raising the possibility that the initiation
codon of mouse Dlxin-1 is located downstream of the predicted most 5'
ATG codon in the reading frame. However, a purified polyclonal antibody
raised against GST-Dlxin1-C reacted with endogenous 90-kDa proteins
(data not shown) corresponding to the molecular mass of the
transfected, epitope-tagged Dlxin-1. Although there remains the
possibility that Dlxin-1 is subjected to some post-translational
modification, we predict the open reading frame to code for 775 amino acids.
Dlxin-1 shows similarities to MAGE/necdin family proteins. Necdin, one
of the most characterized proteins in the family, is a postmitotic
neuron-specific nuclear protein (23). Necdin associates with E2F1 and
mimics the function of Rb protein, thereby regulating cell cycle
progression (24, 25). We could not detect any Rb-like activity or
binding to E2F1 of Dlxin-1 (data not shown). We isolated a RING finger
protein that binds to the necdin homology domain of
Dlxin-1.2 It is therefore
suggested that this domain, structurally similar to necdin, is involved
in protein-protein interaction. Translated regions of MAGE/necdin
family proteins are encoded by a single exon (17), whereas the coding
sequence of the MAGE/necdin homologous domain is split into at least
three exons in the mouse Dlxin-1 gene,2 which are unique to
the Dlxin-1 gene among known MAGE/necdin family members. Thus, Dlxin-1
gene may be evolutionarily distant from other MAGE/necdin family proteins.
The WQXPXX repeats in the middle of Dlxin-1
molecule are unique to this molecule and show no homology with any
sequence in the data base, other than MAGE-D1 and SNERG-1, human and
rat orthologues of Dlxin-1, respectively. Although some variations were
observed, the repeats are highly conserved between species.
Interestingly, the domain for binding to the Dlx/Msx proteins is mapped
in the tandem repeats. It has been shown that the Dlx and Msx
homeodomain proteins form homo- or heterodimers through their
homeodomain, thereby regulating each other's functions. The N-terminal
domain of Dlx5, which shares a weak homology with other Dlx/Msx family proteins, is a primary binding site for Dlxin-1, because the domain, but not the homeodomain, was used for the bait in isolating Dlxin-1 by
the two-hybrid screening. Tryptophan is included in many protein motifs
for molecular recognition, like the WW domain and WD40 repeat proteins
(26-28). It is tempting to speculate that Dlxin-1 binds to multiple
proteins via the WQXPXX repeats, due to the presence of multiple tryptophan residues in tandem. In light of the
recent identification of Miz1 (29) and MINT (30) that bind to Msx2 via
proline-rich domains, it is also possible that proline residues are
involved in the interaction between Dlxin-1 and Msx2 and/or Dlx
homeodomain proteins.
Expression of Dlxin-1--
Dlxin-1 mRNA was ubiquitously
expressed in many tissues and also during embryonic development, with
some variation in the expression level depending on cell or tissue
types. In hematopoietic tissues, such as spleen and bone marrow, little
expression was detected, suggesting that Dlxin-1 expression is more
pronounced in adherent cells. Dlx5 is predominantly expressed in
skeletal elements and osteoblasts (1, 14, 15), in which Dlxin-1 is also
expressed. It is suggested that Dlxin-1 may play a role in
Dlx5-mediated osteoblastic function and that Dlxin-1 associates not
only with Dlx5 but also other proteins, to regulate the function in
tissues and cells other than bones. Notably, it has been reported that
a deletion in the dlx5/dlx6 locus causes the split
hand/split foot malformation in humans (31). It has also been described that digit abnormality is observed in mice that are deficient in both
msx1 and msx2 genes. Considering the strong
expression of Dlxin-1 in the digit (Fig. 4B), tissues
surrounding chondrocytes and the chondrocytes, it is suggested that
Dlxin-1 may be involved in digit formation activity in collaboration
with Dlx/Msx homeodomain proteins.
It has been reported that the expression of Dlx5 mRNA, as well as
Msx2, is up-regulated by bone morphogenetic protein. However, the
expression level of the Dlxin-1 gene did not significantly change
following treatment with bone morphogenetic protein 4 or transforming
growth factor Dlxin-1 Is a Transcriptional Regulator--
It has been reported
that Dlx5 is a positive regulator of mouse osteocalcin gene and
osteoblastic functions (4, 5, 20). In this study, we demonstrated that
the N-terminal region of Dlx5 bears an intrinsic transcription
activation domain. It has recently been reported, however, that
expression of the osteocalcin gene was greatly increased in the
osteoblasts of dlx5 knockout mice, suggesting that Dlx5 is a
negative regulator of osteocalcin gene expression in vivo.
There are some possibilities that may explain this apparent
inconsistency. First, the dose of Dlx5 may be critical for
transcriptional regulation. The homeodomain proteins form dimers to act
on DNA (4). Liu et al. (32) demonstrated that the dosage of
Msx2 affects the number of proliferative osteoblasts. In this study, we
showed that Dlxin-1 activates Dlx5-dependent transcription
in a dose-related manner. Thus, not only the ratio between Msx and Dlx
family proteins, but also the ratio between the homeodomain proteins
and Dlxin-1 may be important in transcriptional regulation. Second,
Dlx5 has two modes of transcriptional function, one through direct
binding to DNA and the other by protein-protein interaction without
binding to DNA. Newberry et al. (33) suggested that Dlx5
antagonizes the transcriptional repression by Msx2 to maintain the
basal promoter activity (33). Thus, Dlx5 may be involved in the basal
activity of the osteocalcin promoter and function as a potential
inhibitor for a transcription factor(s) that stimulates the expression
of the osteocalcin gene in a later phase of osteoblastic
differentiation. Third, the function may be regulated by alternative
form(s). It has been reported that multiple Dlx5 transcripts generated
by alternative splicing exist in mouse brain (34). We have found a
splice variant form of Dlx5,
Our results show that Dlxin-1 binds to Msx2 more strongly than to Dlx5
and other Dlx family proteins. It could be partially explained by the
lower expression of Dlx5, in comparison with that of Msx2. Although a
weak similarity has been suggested to exist between the N-terminal
regions of Msx and Dlx family proteins (4), it is surprising that most
Dlx/Msx family proteins show binding to Dlxin-1. Thus, Dlxin-1 may be a
common regulator for transcriptional function, mediated by the Dlx/Msx
homeodomain proteins.
We failed to detect any transactivation of Dlxin-1 itself using
GAL4-mediated transcriptional assays (data not shown). The overexpression of Dlxin-1 reduced the maximal transcriptional activation without affecting the basal level of activation in Dlx5
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
C and pBIND-Msx2
C, respectively.
For construction of the bait plasmid (pGBT- Dlx5
C), the Dlx5
C
cDNA fragment was inserted into pGBT9
(CLONTECH, Palo Alto, CA). pcDNA-FLAG was constructed in pcDNA3 (HindIII/XhoI),
with a DNA fragment encoding the single FLAG epitope (5'-AAG CTT GCC
ACC ATG GAC TAC AAG GAT GAT GAC GAC AAA CTC GAG-3') after the
initiation codon (ATG) with the Kozak leader sequence, giving rise to
the N-terminal FLAG tag attachment vector. Mouse Dlx3, Dlx7, Dlx5, and
Msx2 cDNAs were inserted into pcDNA-FLAG, generating pFDlx3,
pFDlx7, pFDlx5, and pFMsx2, respectively. Nucleotide sequences of
all polymerase chain reaction products reported here were verified by
sequencing. pGL3-basic, pGL3-control, and pGL3-promoter were purchased
from Promega. The potential Dlx-responsive element of the
col1a1 promoter (20) was connected in tandem to the
pGL3-promoter vector. Briefly, oligonucleotides corresponding to the
Dlx-responsive element (DlxRE) and a sequence mutated in the core
binding region (TAAT to TACG; Dlxm) were designed, and the
complementary oligonucleotides were annealed and ligated. The
concatemerized DNA of five repeats was purified and introduced into the
BglII site on the pGL3-promoter vector, generating
pDlxRE-luc and pDlxm-luc. The nucleotide sequences for the
construction were as follows: Dlx5N, 5'-GGGATCCTGACAGGAGTGTTTGACAGA-3'; Dlx5
C, 5'-GGTCTAGATACCATTCACCATCCTCAC-3'; Msx2N,
5'-GGGATCCTGGCTTCTCCGACTAAAGGG-3'; Msx2
C,
5'-GGTCTAGAGGGTGCAGGTGGTGGGGCT-3'; mDlx5-F,
5'-CCTCGAGACAGGAGTGTTTGACAGAAG- 3'; mDlx5-R,
5'-GGTCTAGACTAATAAAGCGTCCCGGAGGC-3'; mMsx2-F,
5'-CCTCGAGGCTTCTCCGACTAAAGGCGG-3'; mMsx2-R,
5'-GGTCTAGATTAGGATAGATGGTACATGC-3'; mDlx3-F,
5'-GGCTCGAGAGCGGCTCCTTCGATCGCAAG-3'; mDlx3-R,
5'-GGTCTAGAGGTGGGTACTCAGTACACAGC-3'; mDlx7-F,
5'-GGCTCGAGACCTCTTTACCCTGTCCCTTC-3'; mDlx7-R,
5'-GGTCTAGACTCACATCATCTGAGGCAG-3'; DlxRE-U,
5'-GATCCTGCATTCCCTTTAATTATAGCCTCA-3'; DlxRE-R,
5'-GATCTGAGGCTATAATTAAAGGGAATGCAG-3'; Dlxm-U,
5'-GATCCTGCATTCCCTTACGTATAGCCTCA-3'; Dlxm-R,
5'-GATCTGAGGCTATACGTAAAGGGAATGCAG-3'.
C and the mouse embryo
(embryonic day 11) cDNA library constructed in pGAD10 (CLONTECH). The transformants were first selected
for HIS3 gene transactivation on the basis of growth in the
absence of Trp, Leu, and His and in the presence of 60 mM
3-amino-1,2,4-triazole. Colonies grown on the selection media
were then selected for lacZ gene transactivation on
the basis of
-galactosidase activity in the filter assay. pLAM5'
encoding GAL4 DNA-binding domain/human lamin C fusion protein was used
as a negative control. Prey plasmids were recovered from
-galactosidase-positive colonies, and sequence analysis was
performed with ABI PRISMTM 310 Genetic Analyzer
(PerkinElmer Life Sciences).
, induced by 0.25 mM
isopropyl-1-thio-
-D-galactopyranoside, and purified by
affinity chromatography using glutathione-Sepharose beads (Amersham Pharmacia Biotech). [35S]Methionine-labeled Dlx5 and Msx2
proteins were generated using an in vitro
transcription/translation system (TNT; Promega). Aliquots were
incubated with GST- or GST-Dlxin1-conjugated glutathione-Sepharose beads in binding buffer (50 mM Tris-HCl, pH 7.5, 138 mM KCl, 1 mM EDTA, 0.2% Nonidet P-40, 5%
glycerol, and 5% bovine serum albumin). 35S-Labeled Dlx5
or Msx2 was synthesized as above and incubated with GST or GST-Dlxin1
deletion mutant proteins in the same binding buffer, followed by
precipitation with glutathione-Sepharose beads. Bound proteins were
eluted from the beads by boiling in SDS sample buffer, separated by
SDS-PAGE, and visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Dlx5 has transactivation function. The
reporter constructs shown in A were transfected into
Dlx5-expressing (P19Dlx5) or mock-transfected (P19neo) P19 cells
(B). The N-terminal region of Dlx5 acts as a transcriptional
activation domain (C). The GAL4-Dlx5 C was generated, with
the N-terminal region of Dlx5 (amino acids 2-132) fused to the GAL4
DNA binding domain (GAL4DBD), as indicated in the
inset. GAL4-Dlx5
C or GAL4 alone was transfected into 293 cells at the indicated doses (µg), with the reporter shown in the
inset (pG5luc). pGL3-control, used as a nonspecific reporter
instead of pG5luc, showed no GAL4-Dlx5
C dependent activation,
indicating that the activation by GAL4-Dlx5
C was GAL4
element-mediated. No activation was detected when pGL3-basic, a
promoterless reporter, was used.
C) was fused to the GAL4 DNA-binding
domain, and the transcriptional function was evaluated by reporter gene
assay in 293 cells. Although GAL4 DNA-binding domain itself did not
activate the transcription, the fusion construct (pBIND-Dlx5
C)
stimulated the transcriptional activity in a dose-dependent
manner (Fig. 1C). In contrast, the Msx2 fusion construct
(pBIND-Msx2
C) did not activate, but rather suppressed, transcription
(data not shown). These results suggest that the N-terminal domain of
Dlx5 contains a transcriptional activation domain.
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Fig. 2.
Primary structure of Dlxin-1. Schematic
presentation of the primary structure of Dlxin-1 and the clones
isolated by yeast two-hybrid screening (A). Tandem repeats
of WQXPXX (in black) are located in
the middle portion of Dlxin-1. The necdin homology region is indicated
by a gray box. The scale bar in the
right top of the figure corresponds to
100 amino acids. The clones isolated are indicated below the
full-length Dlxin-1. The domain encoded by the shortest clone, clone
16, corresponds to a putative Dlx5-binding domain. B, the
nucleotide and deduced amino acid sequences of Dlxin-1. The
boxed sequence is encoded by clone 16 (see
above). The underlined sequence indicates a
potential polyadenylation signal (AATAAA). The nucleotide sequence of
mouse Dlxin-1 has been submitted to GenBankTM/EMBL/DDBJ
with accession number AB029448.
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Fig. 3.
Alignment of Dlxin-1 related proteins and the
WQXPXX repeat. A,
necdin homology domain. The amino acid sequences of the necdin domain
were aligned. The boxes at the top of each
row indicated the identity: all (black) or three
(gray) of the four sequences are matched. The sequence data
were obtained from GenBankTM as accession numbers D76440,
AAC23618, and U10686, for mouse necdin, human MAGE-B3, and human
MAGE-11, respectively. The numbers correspond to the amino acid number
in each protein. B, Dlxin-1 homologues. The deduced amino
acid sequence of Dlxin-1 is compared with those of human MAGE-D1 and a
rat sequence showing high similarity submitted to GenBankTM
with the accession number AF274043, with identities of 91 and 96%,
respectively. C, the WQXPXX repeats of
Dlxin-1. The tandem hexapeptide repeats are aligned. 22, 16, and 21 residues out of 25 repeats are tryptophan, glutamine, and proline in
the position of the each repeat, respectively, showing a
consensus.
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Fig. 4.
Expression of Dlxin-1 mRNA.
A, mouse embryo MTN blot (left) and mouse MTN
blot (middle panel) were used for Northern blot
analyses. Total RNA (5 µg) from mouse long bones, bone marrow, and
calvaria were blotted and hybridized. ED, embryonic day. A
single band of Dlxin-1 transcript was estimated as 3.0 kilobase pairs
in length. B, in situ hybridization. DIG-labeled
Dlxin-1 RNA probe in antisense (left) or sense
(right) orientation was hybridized to adjacent parasagittal
sections of an embryonic day 15 mouse embryo. Bar, 0.1 mm.
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Fig. 5.
Dlxin-1 binds to Dlx/Msx homeodomain proteins
in vivo. HA-tagged Dlxin-1 (HA-Dlxin1) was
coimmunoprecipitated with FLAG-tagged Dlx5 (F-Dlx5), Dlx7 (F-Dlx7),
Msx2 (F-Msx2), and Dlxin-1 (F-Dlxin1), but not when the FLAG peptide
was included in the precipitation (+).
C-mediated Transcriptional
Activity--
To determine whether and how the binding of Dlxin-1 to
Dlx5 modulates the transcriptional activity of Dlx5, we employed a GAL4-dependent transcriptional activation assay in HT1080
cells, which express a low level of Dlxin-1 mRNA. As shown in Fig.
6, Dlxin-1 stimulated the reporter
activity through GAL4-Dlx5
C, but not GAL4,
dose-dependently, suggesting that Dlxin-1 augments the
transcription function of the N-terminal domain of Dlx5. At higher a
dose of Dlxin-1, the stimulation was attenuated, but the basal activity
by Dlx5
C remained, raising the possibility that the ratio between
the amounts of the Dlxin-1 and Dlx5 complexes is important in
transcriptional regulation.
View larger version (18K):
[in a new window]
Fig. 6.
Effect of Dlxin-1 on
Dlx5 C-mediated transcriptional activity.
pFDlxin-1 at the indicated doses (µg) was cotransfected into HT1080
cells, with GAL4-Dlx5
C or GAL4 only and the reporter construct,
pG5-luc.
View larger version (25K):
[in a new window]
Fig. 7.
Dlxin-1 is required for
Dlx5-dependent transcription. The schematic diagram
(A) shows expression vectors for the full-length of Dlxin-1
(Dlxin-FL) and the Dlx-binding domain of the protein
(Dlxin-DlxBD), which represents to the region encoded by
clone 16 (see Fig. 2). Either construct (1 µg each) was cotransfected
with pDlxRE-luc into Dlx5-expressing P19 (P19Dlx5) or mock-transfected
P19 (P19neo) cells, and the luciferase activity was measured.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in MC3T3-E1 and ST2 cells, while treatment of P19
cells with forskolin slightly decreased the expression of Dlxin-1
mRNA (data not shown). It is speculated that the expression of
Dlxin-1 may be regulated during cell differentiation rather than being constitutive.
Dlx5, in mouse osteoblast cell
lines.3 This variant form
codes for only the N-terminal domain of Dlx5 and lacks both the
homeodomain and the C-terminal region. Although the physiological
function of
Dlx5 is not known at the present time, there is the
possibility that the splice variant interferes with binding between
Dlx5 and Dlxin-1.
C-
or Dlx5-dependent transcription (Fig. 6). These findings suggest that Dlxin-1 may bridge or stabilize the complex of Dlx5 and
coactivators rather than being directly involved in transcriptional activation of Dlx5. However, it remains to be determined whether Dlxin-1 has any histone acetylase or chromatin-remodeling activities.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. A. Umezawa (Keio University), A. Fukamizu (University of Tsukuba), and S. Kato (University of Tokyo) for providing cell lines and yeast strain. We also appreciate useful information on necdin from Dr. K. Yoshikawa (Osaka University). We thank Drs. A. Matsuura and N. Motoyama for critical comments and Dr. R. Thornhill for proofreading.
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FOOTNOTES |
---|
* This work was supported by the Research Grant for Longevity Sciences from the Ministry of Health and Welfare of Japan (to K. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB029448.
¶ To whom correspondence should be addressed: Dept. of Geriatric Research, National Institute for Longevity Sciences, 36-3 Gengo, Morioka-cho, Obu, Aichi 474-8522, Japan. E-mail: kwatanab@nils.go.jp.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M008590200
2 A. Sasaki and K. Watanabe, unpublished data.
3 Y. Masuda, A. Sasaki, and K. Watanabe, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: MAGE, melanoma-associated antigen; DlxRE, Dlx-responsive element; GST, glutathione S-transferase; HA, hemagglutinin.
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