Functional Analysis of TBX5 Missense Mutations
Associated with Holt-Oram Syndrome*
Chun
Fan
,
Mugen
Liu
§, and
Qing
Wang
¶
From the
Center for Molecular Genetics, Department of
Molecular Cardiology, Lerner Research Institute, and the Center for
Cardiovascular Genetics, Department of Cardiovascular Medicine, The
Cleveland Clinic Foundation, Cleveland, Ohio, 44195, and the
§ Institute of Genetics, Fudan University,
Shanghai, P. R. China 200433
Received for publication, August 8, 2002, and in revised form, December 23, 2002
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ABSTRACT |
TBX5 is a T-box transcription factor that
plays a critical role in organogenesis. Seven missense mutations
in TBX5 have been identified in patients with Holt-Oram syndrome
characterized by congenital heart defects and upper limb abnormalities.
However, the functional significance and molecular pathogenic
mechanisms of these mutations are not clear. In this study we describe
functional defects in DNA binding, transcriptional activity,
protein-protein interaction, and cellular localization of mutant TBX5
with these missense mutations (Q49K, I54T, G80R, G169R, R237Q, R237W,
and S252I). Mutations G80R, R237Q, and R237W represent a group of mutations that dramatically reduce DNA-binding activity of TBX5, leading to reduced transcription activation by TBX5 and the loss of
synergy in transcriptional activation between TBX5 and NKX2.5. The
second group of mutations includes Q49K, I54T, G169R, and S252I, which
have no or moderate effect on DNA-binding activity and the function of
transcription activation of TBX5 but cause the complete loss of
synergistic transcription activity between TBX5 and NKX2.5. All seven
missense mutations greatly reduced the interaction of TBX5 with NKX2.5
in vivo and in vitro. Immunofluorescent staining showed that wild type TBX5 was localized completely into the
nucleus, but mutants were localized in both nucleus and cytoplasm. These results demonstrate that all seven missense mutations studied here are functional mutations with a spectrum of defects ranging from
decreases in DNA-binding activity and transcriptional activation to the
dramatic reduction of interaction between TBX5 and NKX2.5, and loss of
synergy in transcriptional activation between these two proteins, as
well as impairment in the nuclear localization of TBX5. These
defects are likely central to the pathogenesis of Holt-Oram syndrome.
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INTRODUCTION |
T-box transcription factors belong to a conserved family of
proteins that contribute to early cell fate determination,
differentiation, and organogenesis (1). Genetic mutations in human
T-box transcription factors cause a number of human congenital
disorders such as Holt-Oram syndrome (TBX5) (2, 3), ulnar-mammary
syndrome (TBX3) (4), X-linked cleft palate and ankyloglossia (TBX22)
(5), and DiGeorge syndrome (TBX1) (6, 7, 8). T-box genes have been
identified in animals from ctenophores (comb jellies) and nematodes to
mammals, and may account for ~0.1% of animal genomes.
The Tbx5 gene is located on chromosome 12q24 and encodes a
protein of 518 amino acids (2, 3). TBX5 contains a highly conserved
DNA-binding domain, T-box domain. Tbx5 is expressed in
embryonic heart and limb tissues (9-12). During embryogenesis, Tbx5 expression starts at the earliest stages of heart
formation and is co-localized with genes encoding other important
cardiac transcription factors, Nkx2.5 and Gata4
(10). Over-expression of Tbx5 under the control of the
-myosin heavy chain promoter in mice inhibits ventricular chamber
development and loss of ventricular-specific gene expression (10).
Homozygous knockout mice deficient in Tbx5 die early in
embryogenesis at D9.5, and heterozygous mice showed the forelimb and
congenital heart defects (13). These results indicate that
Tbx5 is critical to development of the heart and skeletal structures.
Mutations in Tbx5 have been found to cause Holt-Oram
syndrome, an autosomal-dominant disease characterized by upper limb
malformations and congenital heart defects including atrial and
ventricular septal defects or cardiac conduction disease (2, 3, 14, 15). To date, twenty-four different Tbx5 mutations (15-19)
have been described in patients with Holt-Oram syndrome. Many
Tbx5 mutations are nonsense, frameshift, and splice
site mutations, or large chromosomal rearrangements (translocations,
deletions) that are expected to produce truncated TBX5 proteins or no
TBX5 at all (e.g. nonsense-mediated mRNA decay,
translocation in intron 1). Based on these findings, Tbx5
haploinsufficiency has been proposed as a mechanism underlying
pathogenesis of Holt-Oram syndrome. This hypothesis is further
supported by the finding that heterozygous Tbx5del/+ mice modeled Holt-Oram syndrome (13).
It is interesting to note that increased Tbx5 dosage, such
as a chromosome 12q2 duplication, also results in Holt-Oram syndrome
(19), suggesting that Tbx5 dosage is critical to the
development of the heart and limbs. However, seven missense mutations
in the Tbx5 gene, which would not be expected to alter the
dosage of Tbx5, have been reported in patients with
Holt-Oram syndrome. It remains to be determined whether these
Tbx5 missense mutations are functional mutations, and if
they are, what are the molecular pathogenic mechanisms by which these
mutations act?
Biochemical studies have demonstrated that TBX5 protein interacts with
the homeobox transcription factor NKX2.5 to synergistically transactivate expression of target genes (13, 20). The homeodomain of
NKX2.5 was shown to be necessary and sufficient for its interaction with TBX5, although the NK domain (a17 amino acid domain
specific to NK-class proteins) located C-terminal to the homeodomain
can significantly enhance the interaction (13, 20). For TBX5, the
region including the N-terminal domain and the N-terminal portion of
the T-domain (amino acids 1-90) is sufficient for its association with
NKX2.5 (20).
The target genes identified for Tbx5 include the genes for
atrial natriuretic factor
(ANF)1 (20) and
connexin 40 (Cx40) (13, 20). Synergistic activation of both
ANF and Cx40 promoters by TBX5 and NKX2.5 were
demonstrated (13, 20). The promoters of ANF and
Cx40 harbor binding sites for both TBX5 and NKX2.5, and
these sites were shown to be capable of binding to both transcriptional
factors (13, 20). Using an in vitro selection procedure with
double-stranded random 26-mer oligonucleotides, a consensus sequence
for TBX5 binding ((A/G)GGTGT(C/T/G)(A/G)) was identified (21). This
consensus sequence is part of a half-site from the Brachyury T-box
target sequence (21). TBX5 can bind to either a single site or paired
non-palindromic sites in vitro (21).
In the present study, we have used a biochemical system comprising of
TBX5, NKX2.5, and the ANF promoter to characterize the TBX5
missense mutations identified in Holt-Oram syndrome patients. We
demonstrate that the TBX5 missense mutations are functional mutations
that cause a spectrum of defects in DNA binding, transcription activation, interaction with NKX2.5, synergistic transactivation with
NKX2.5, and nuclear localization. These results also provide insights
into structure-function relationship of TBX5.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs--
The region from
270 to +1 bp upstream
from the transcription start site of the ANF promoter was
PCR-amplified and cloned into the pGL3-Basic vector, resulting in the
ANF-Luc reporter gene. The full-length Tbx5
cDNA was cloned into the pcDNA3.1 vector, resulting in the
over-expression constructs (20). Three forms of Tbx5
expression constructs were used: one with and the other without the
FLAG-epitope tag as well as one construct for expressing His6-tagged TBX5. The expression constructs for
hemagglutinin (HA)-tagged-NKX2.5 and GST-NKX2.5 fusion protein were as
described previously (a gift from Dr. Issei Komuro) (20). The missense mutations in Tbx5 were introduced into the wild type
construct by PCR-based site-directed mutagenesis and verified by DNA
sequencing. The full-length Tbx5 cDNA was also cloned
into the pET-28b vector, resulting in the bacterial expression
construct for TBX5.
Cell Culture, Transfections, Western Blot, and Luciferase
Assay--
COS-7, HeLa, or NIH-3T3 cells were grown to 90% confluence
in Dulbecco's minimum essential medium (DMEM) supplemented with 10%
fetal bovine serum and transfected with LipofectAMINE 2000 and 50 ng of
DNA for the expression construct, 1 µg of DNA for the reporter gene,
and 50 ng of internal control plasmid pSV-
-galactosidase. Cells were
harvested and lysed 48 h after transfection.
The efficiency of transfection was examined by Western blot analysis.
Forty µg of total cellular lysates were separated by 12% SDS-PAGE
and electro-transferred to a polyvinylidene fluoride membrane. The
membrane was probed with mouse monoclonal anti-FLAG M2 antiserum
(Sigma) as the primary antibody and the rabbit anti-mouse IgG
horseradish peroxidase-conjugated secondary antibody (Santa Cruz
Biotechnology, Santa Cruz, CA). ECL Western blotting detection reagents (Amersham Pharmacia Biotech) were used to visualize the protein signal.
Luciferase assay was performed using a Dual-Luciferase assay kit
according to the manufacturer's instructions (Promega).
-galactosidase activity expressed from pSV-
-galactosidase was
used to normalize the transfection efficiency. The experiments were
repeated three times in triplicate. Data are expressed as mean
±S.E.
In Vitro Translation of TBX5 Protein and Electrophoretic Mobility
Shift Assay (EMSA)--
The synthetic TBX5-binding site was generated
by annealing double-stranded oligonucleotides (5'-aataTCACACCTgtac-3';
5'-gtacAGGTGTGAtatt-3'), labeled with [
-32P]dCTP and
Klenow enzyme, and used in EMSA. The labeled TBX5-binding site was
incubated with 2 µg of poly(dI-dC) and 4 µl of TBX5 protein synthesized using the TNT Quick Coupled Transcription/Transcription system in 20 µl of binding buffer. The TBX5-DNA complex was then separated from free DNA using 6% native polyacrylamide gels. The gels
were run at 200 volts for 40 min, dried, and exposed to x-ray films.
TBX5-NKX2.5 Interactions in Vivo and in Vitro--
For in
vivo protein-protein interactions, HeLa cells were transiently
co-transfected with expression plasmids for His-tagged TBX5 and
HA-tagged NKX2.5 for 48 h. The transfected cells were harvested,
washed with PBS, incubated in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1%
Nonidet P-40, a mini-protease inhibitor mixture (Roche Molecular
Biochemicals)), and digested with DNase I. Total cell lysate was
dialyzed with dialysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 20% glycerol, 0.5 mM
phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol). The
cell lysate was then mixed with 20 µl of Ni-NTA magnetic agarose
beads (Qiagen) and incubated for 4-12 h at 4 °C. After extensive
washing with the lysis buffer, the bound proteins were eluted with
Laemmli sample buffer (Bio-Rad), separated by SDS-PAGE, and transferred
onto a nitrocellulose membrane. The membrane was probed with an anti-HA
antibody (Sigma), and protein signal was visualized by enhanced
chemiluminescence according to the manufacturer's instructions
(Amersham Biosciences).
For in vitro protein-protein interactions, GST or GST-NKX2.5
fusion proteins were over-expressed in Escherichia coli BL21 (DE3), purified using standard protocols, and immobilized on
glutathione-Sepharose beads. Aliquots (20 µl) of GST and GST-NKX2.5
beads were incubated with in vitro-translated TBX5
(described earlier) for 4 h at 4 °C. After washing with PBS
buffer, bound proteins were eluted from beads with PBS containing 0.1 M glutathione and analyzed by Western blot with a
monoclonal anti-His antibody (Sigma).
Immunocytochemistry--
Transfected cells (NIH-3T3, COS-7, and
HEK-293) were seeded on chamber slides at a density of 1 × 105 cells and incubated at 37 °C and 5% CO2
for 24-48 h. Cells were then fixed in 2% paraformaldehyde,
washed in PBS, and incubated with the primary antibody (1:1000
dilution) in PBS/3% nonfat milk at 4 °C overnight. The mouse
anti-FLAG M2 primary antibody recognizes the FLAG-tagged TBX5 protein.
The secondary antibody, a fluorescein isothiocyanate-conjugated sheep
anti-mouse IgG (1:500 dilution), was then added and incubated at room
temperature for 1 h. Slides were mounted using anti-fading
vectashield with DAPI and cells were viewed under a Zeiss
Axioskop fluorescence microscope equipped with Photometrics
SmartCapture. The images were analyzed with the Melanie software
(Geneva Bioinformatics) for the relative distribution of the protein in
the nucleus versus cytoplasm.
 |
RESULTS |
Functional Defects of TBX5 Missense Mutations in DNA
Binding--
Direct binding of TBX5 transcriptional factor to DNA is
required for activation of its target genes including ANF
encoding the atrial natriuretic factor. To identify the mechanism by
which TBX5 missense mutations cause Holt-Oram syndrome, we assessed the
ability of mutant TBX5 proteins to bind to its target DNA-binding site
using EMSA. Seven missense mutations in TBX5 were analyzed, and they
include Q49K and I54T at the N terminus, G80R, G169R, R237Q, and Q237W
in the T-domain responsible for DNA binding, and S252I in the C
terminus (Fig. 1). The promoter region of
ANF (
252 upstream from the transcription start site)
contains a TBX5-binding site, and this site was chemically synthesized
as pairs of complementary single strand oligonucleotides
(5'-aataTCACACCTgtac-3'; 5'-gtacAGGTGTGAtatt-3'). The synthetic site
was labeled and used in EMSA with in vitro-translated TBX5
proteins. As shown in Fig. 2, the wild
type TBX5 protein can bind to DNA and forms a specific DNA-protein
complex. Competition experiments with unlabeled synthetic TBX5-binding
site dramatically reduced the TBX5-DNA complex signal, and a synthetic
DNA fragment having a sequence unrelated to the TBX5-binding site
failed to form TBX5-DNA complex (data not shown). These data suggest
that the observed binding of TBX5 to the synthetic binding site is specific.

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Fig. 1.
TBX5 missense mutations identified in
patients with Holt-Oram syndrome. Among more than 20 TBX5
mutations, Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I represent
missense mutations identified in patients with Holt-Oram syndrome. TBX5
consists of 9 exons with exons 3-7 encoding the T-domain responsible
for DNA binding. Note that two mutations (Q49K and I54T) are in the N
terminus, four mutations (G80R, G169R, R237Q, and R237W) are within the
T-box domain, and one mutation (S252I) is in the C terminus.
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Fig. 2.
The effect of missense mutations of TBX5 on
DNA-binding activity. The wild type and mutant TBX5 proteins were
synthesized in vitro using the TNT-coupled
transcription/translation system. The TBX5-binding site is a synthetic
double-stranded DNA fragment corresponding to the region from 257 to
242 bp upstream from the ANF transcriptional start site
(5'-aataTCACACCTgtac-3'). A, EMSA. Lane " ,"
no TBX5 protein; WT, wild type TBX5; Q49K,
I54T, G80R, G169R, R237Q,
R237W, and S252I, TBX5 with individual missense
mutations. B, a silver-stained protein gel showing the
approximately equal level of synthesis of wild type and various mutant
TBX5 proteins used in EMSA. Lane 1, no TBX5 expression
plasmid DNA; lane 2, wild type TBX5; lanes 3-9
represent Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I
mutant TBX5, respectively. C, EMSA with various amounts of
wild type TBX5 and mutants G80R and R237Q. D, the amount of
the TBX5-DNA complex in panel C was plotted against TBX5
concentrations. The fitted slope for specific binding of wild type TBX5
(WT) and mutants G80R and R237Q is 79.5, 19.5, and 11.7, respectively.
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Three mutations in the T-domain, including G80R, R237Q, and R237W,
dramatically reduced the binding ability of TBX5 to DNA (Fig.
2A). The relative DNA-binding abilities of wild type and mutant G80R and R237Q TBX5 were determined using various concentrations of proteins. As shown in Fig. 2, C and D, both
mutant proteins have much lower affinity to DNA than wild type TBX5.
Mutations I54T and S252I had moderate effect on DNA binding of TBX5. In contrast, there was no significant difference in DNA binding between the wild type TBX5 and mutants Q49K and G169R (Fig. 2A). A
silver-stained protein gel showed that both wild type and mutant TBX5
proteins were successfully synthesized at approximately equal amounts
(Fig. 2B). Our results suggest that a functional defect in
DNA binding is the molecular mechanism for some TBX5 mutations (G80R,
R237Q, and R237W), but not a universal mechanism for all mutations.
Functional Defects of TBX5 Missense Mutations in Transcriptional
Activation--
TBX5 alone can activate transcription of its target
genes, but in the presence of NKX2.5, synergy in transcriptional
activation by these two proteins was observed (13, 20). We assessed the functional effect of TBX5 mutations on transcriptional activation using
a reporter gene (ANF-Luc, Fig.
3A) in which the
ANF promoter (region from
270 to + 1 bp) was fused to the
luciferase gene. The ANF-Luc reporter gene was
co-transfected with wild type or various mutant TBX5 expression
constructs into COS-7 cells. Transcriptional activity was examined and
expressed as relative luciferase units. As shown in Fig. 3B,
expression of wild type TBX5 activated transcription of the
ANF promoter. Three mutations, including G80R, R237Q, and R237W, reduced transcription activation by TBX5, consistent with our
data that these three mutations reduced DNA binding of TBX5. In
contrast, TBX5 mutations Q49K, I54T, G169R, and S252I had little effect
on transcriptional activation by TBX5 (Fig. 3B). Western blot analysis showed that both wild type and all mutant TBX5 proteins were successfully expressed in transfected cells (Fig.
3C).

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Fig. 3.
The effect of missense mutations of TBX5 on
transcription activation activity in the presence or absence of
NKX2.5. A, the reporter gene used for transcriptional
activation assay. The promoter region, from 270 to +1 bp upstream
from the transcriptional start site, of ANF was fused to the
luciferase gene (LUC). B, transcriptional
activation assay for all seven missense mutations of TBX5 in the
absence (left block) and presence (right block)
of NKX2.5. Transcriptional activity is shown as relative luciferase
activity on the y-axis. The transcriptional activity for the
vector only was set arbitrarily to 1. WT, wild type. Note
that all missense mutations of TBX5 abolished synergistic transcription
activation of the ANF promoter between TBX5 and NKX2.5.
C, Western blot analysis to determine whether mutant TBX5
were successfully expressed in transfected COS-7 cells. BE,
purified wild type TBX5 control from a bacterial over-expression
system; WT, lane with lysate from COS-7 cells containing
expressed FLAG-tagged wild type TBX5; Q49K, I54T,
G80R, G169R, R237Q, R237W,
and S252I, lanes with lysates from COS-7 cells containing
expressed TBX5 proteins with corresponding mutations.
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We then investigated whether the missense mutations in TBX5 could
disrupt the synergistic transactivation between TBX5 and NKX2.5. As
shown in Fig. 3B, either wild type TBX5 or NKX2.5 alone activated expression of the ANF promoter, but
co-transfection of TBX5 and NKX2.5 into COS-7 cells showed synergistic
activation of the ANF promoter as reported previously (12,
16). However, all seven missense mutations of TBX5 did not produce such
synergistic effect with NKX2.5 (Fig. 3B).
Defects of TBX5 Missense Mutations in TBX5 and NKX2.5
Interaction--
Because all seven TBX5 missense mutations caused loss
of synergistic activation of transcription by TBX5 and NKX2.5, we
determined whether these mutations affect the physical interaction
between these two transcriptional factors. We used both in
vivo and in vitro protein-protein interaction assays.
For the in vivo assay, His-tagged TBX5 and NKX2.5 tagged
with HA epitope at the C terminus were co-expressed in HeLa cells.
Western blot analysis showed that both TBX5 and NKX2.5 were
successfully expressed in the same cells (Fig.
4A). The protein mixture was
then incubated with Ni-NTA beads (which bind His-tagged TBX5 or
His-TBX5-NKX2.5-HA complex). The protein or protein-complex bound to
Ni-NTA beads was eluted and analyzed by Western blot with an anti-HA
antibody to detect the presence of NKX2.5. As expected, interaction
between wild type TBX5 and NKX2.5 was observed (Fig. 4A,
lane 2). Faint signal (very weak interaction) was observed
for TBX5 proteins with mutation Q49K or G169R. No interactions were
found for mutants I54T, G80R, R237Q, R237W, or S252I. These results
suggest that seven missense mutations studies here dramatically reduce
the interaction between TBX5 and NKX2.5.

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Fig. 4.
The effect of missense mutations of TBX5 on
the interaction between TBX5 and NKX2.5 in vivo
(A) and in vitro
(B). A, HeLa cells were
transiently transfected with expression plasmids for His-TBX5 and
NKX2.5-HA. Total cell lysates containing His-tagged TBX5 and/or
HA-tagged NKX2.5 were incubated with Ni-NTA beads, separated by 12%
SDS-PAGE, and analyzed by Western blot with an anti-His antibody
(upper panel) or with an anti-HA monoclonal antibody
(middle panel). Proteins bound to Ni-NTA beads were washed
with washing buffer, eluted, and fractionated by 12% SDS-PAGE and
analyzed by Western blot with anti-mouse HA for NKX2.5. Lane
1, wild type (WT) TBX5 without co-transfection of
NKX2.5; lane 2, wild type TBX5 with co-transfection of
NKX2.5; lanes 2-9, TBX5 with Q49K, I54T, G80R, G169R,
R237Q, R237W, and S252I co-transfected with NKX2.5-HA, respectively.
B, GST-NKX2.5 fusion protein and in
vitro-translated His-tagged TBX5 were incubated with
glutathione-Sepharose 4B beads, and proteins bound to the beads were
washed with PBS, eluted, fractionated by 12% SDS-PAGE, and analyzed by
Western blot with an anti-His antibody for detecting TBX5.
Approximately equal amounts of wild type and various mutant TBX5
proteins were used in the GST pull-down assay as shown in the legend to
Fig. 2B.
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We also used GST pull-down assay to determine the interaction of
various TBX5 mutants with NKX2.5. GST-NKX2.5 fusion protein was
purified, immobilized to glutathione-Sepharose 4B beads, and mixed with
in vitro-translated TBX5. We separated TBX5 bound to GST-NKX2.5 and analyzed it using Western blot. Wild type TBX5, but none
of the mutant TBX5, showed direct physical interaction with NKX2.5
(Fig. 4B). These results further confirm the in
vivo data that missense mutations severely disrupt the direct
interaction between TBX5 and NKX2.5.
Defects in Nuclear Localization by TBX5 Missense
Mutations--
TBX5 is a transcription factor, and it is expected to
be localized in the nucleus. We hypothesized that missense mutations in
TBX5 may cause conformational changes of the protein and result in
protein trafficking defects. Such defects will prevent TBX5 from
exerting its function as a transcription factor. To test this
hypothesis, we expressed wild type and mutant TBX5 proteins tagged with
a FLAG-epitope into NIH-3T3 cells and studied cellular localization of
TBX5 by immunofluorescence staining. The relative amount of the nuclear
and cytoplasmic protein was quantified and shown in Fig.
5B. As expected, wild type
TBX5 was localized completely into the nucleus (Fig. 5, WT,
green signal). Mutant Q49K TBX5 exhibited a minor defect in
trafficking with most of the protein molecules in the nucleus and few
molecules in the cytoplasm (the protruded bud) (Fig. 5A).
Mutation R237Q caused the most severe defect in TBX5 trafficking with
most of the signals in the cytoplasm and few in the nucleus (Fig.
5A). The rest of mutations (I54T, G80R, G169R, R237W, and
S252I) had moderate effect on nuclear localization of TBX5 with
immunofluorescence signals found in both the nucleus and cytoplasm
(Fig. 5A). Similar results were obtained with HEK-293 or
COS-7 cells (data not show).

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Fig. 5.
Immunostaining of NIH-3T3 cells expressing
human TBX5. A, WT, cell transfected with the
FLAG-tagged wild type TBX5 construct; Q49K, I54T,
G80R, R237Q, R237W, G169R,
and S252I represent cells over-expressing various mutant
TBX5 proteins. Transfected cells were immunostained with anti-FLAG
(green) for TBX5 and the nucleus was stained with DAPI
(blue). No detectable immunofluorescence staining was
observed in non-transfected cells. Note that wild type TBX5 is
completely localized into the nucleus, whereas TBX5 proteins with
various missense mutations are distributed in both the nucleus and
cytoplasm. B, percentage of nuclear
versus cytoplasm distribution of TBX5 for wild type
(WT) and mutant (Q49K, I54T,
G80R, G169R, R237Q, R237W,
and S252I). The data were based on the images in panel
A.
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DISCUSSION |
This study provides evidence that the seven TBX5 missense
mutations identified in Holt-Oram syndrome patients are functional mutations with a spectrum of defects. We evaluated the functional effect of these missense mutations on DNA binding and transcriptional activation by TBX5 as well as synergistic activation by the interaction between TBX5 and NKX2.5. Based on our results, TBX5 missense mutations can be divided into two different functional groups. The first group
consists of mutations G80R, R237W, and R237Q, which exert their effect
mainly by disrupting the DNA-binding function of TBX5. Reduced DNA
binding could lead to observed reduction in transcription activation
and loss of synergy between TBX5 and NKX2.5 in transactivation of
target genes. The second group consists of mutations Q49K, I54T, G169R,
and S252I, which had no or moderate effect on DNA binding. This group
of mutations did not affect the transcriptional activation by TBX5, but
they caused dramatic reduction in the interaction between TBX5 and
NKX2.5 and the complete loss of synergy between TBX5 and NKX2.5 in
transcriptional activation.
Our study represents the most comprehensive analysis of all missense
mutations identified in TBX5 to date. Two previous studies reported
partial characterization of a few missense mutations in TBX5. Ghosh
et al. (21) reported analysis of the effect of four point
mutations, G80R, G169R, R237Q and S252I, on DNA binding to an in
vitro selected fragment with two half TBX5-binding sites oriented
tail-tail using a truncated form of TBX5 (amino acids 1-279). These
authors, however, did not assay the effect of these mutations on
transcription activation. Mutations G80R and R237Q were found to
eliminate DNA binding of TBX5, whereas mutations G169R and S252I
retained DNA-binding activity (21). Our DNA binding analysis with the
full-length TBX5 and a native TBX5-binding site in the ANF
promoter (Fig. 2) yielded similar results on mutations G80R, G169R,
R237Q, and S252I. Hiroi et al. (20) analyzed the effect of
mutations G80R and R237Q on transcription activity of the
ANF promoter but did not examine their effect on DNA
binding. Mutation G80R caused significant reduction of transcriptional activation activity of TBX5 and loss of synergistic activation with
NKX2.5. Surprisingly, mutation R237Q, which eliminated DNA binding, had
minor effects on transcriptional activation by TBX5 or synergistic
activation by TBX5 and NKX2.5 (20) In our study, similar results were
obtained for mutation G80R; however, we found much more dramatic effect
by mutation R237Q, which is consistent the DNA binding data on this
mutation. Moreover, we analyzed the effects of seven missense mutations
on the direct interaction and synergy in transactivation between TBX5
and NKX2.5. Our study provides further definition of molecular
mechanisms of TBX5 mutant protein dysfunctions.
Among seven missense mutations studied, only the three mutations in the
T-domain, G80R, R237W, and R237Q, dramatically reduced binding of TBX5
to the DNA target, further confirming that the T-domain is the
DNA-binding domain. It is surprising that TBX5 with mutation G169R,
also at a highly conserved position in the T-domain, retained nearly
full binding activity, suggesting that not all amino acid residues in
the T-domain are responsible for DNA binding. Based on the x-ray
crystallographic data from the T-domain of Xenopus laevis T
protein (22), both Gly-80 and Arg-237 are located immediately
next to the critical amino acid residues for DNA binding, whereas
Gly-169 is five residues to the left and four residues to the right of
the critical DNA-binding residues. The different spatial positioning in
relation to critical DNA-binding residues may explain the difference of
binding activity to DNA for mutations at Gly-80 and Arg-237 from that
for mutation G169R.
Deletion studies implicated the N terminus of TBX5 and the N-terminal
part of the T-domain (amino acids 1-90) as the binding site for NKX2.5
(21). Our results that mutations Q49K, I54T, and G80R disrupt the
physical interaction and synergistic transcriptional activity between
TBX5 and NKX2.5 further support that the N terminus of TBX5 is involved
in its interaction with NKX2.5. Our data also implicate other regions,
including residues Gly-169, Arg-237, and Ser-252, as critical residues
involved in the interaction and synergistic transcription activation
between TBX5 and NKX2.5. Future structure-function relationship studies
and x-ray crystallographic studies with the full TBX5 and NKX2.5 will
define all critical residues involved in their interaction.
Immunostaining of cells transfected with FLAG-tagged TBX5 showed that
TBX5 proteins with the missense mutations were localized in both the
nucleus and cytoplasm. The molecular mechanism for such trafficking
defects is unknown. We speculate that TBX5 missense mutations cause
improper folding of TBX5, which will prolong the time required for TBX5
to be transported into the nucleus. This slow trafficking of mutant
TBX5 proteins leads to the observation of mutant TBX5 in both the
nucleus and cytoplasm. Delayed entry of TBX5 molecules into the nuclei
will prevent these molecules from executing their transcriptional
activation function. These results demonstrate impaired nuclear
localization of mutant TBX5 as a molecular mechanism for TBX5
missense mutations. Similar protein trafficking defects have been
reported for other cardiovascular diseases including long
QT syndrome (23) and Brugada syndrome (24).
In summary, we have shown that the missense mutations in TBX5
investigated in this study are functional mutations that cause a
spectrum of biochemical and cellular defects. Our results indicate three distinct molecular mechanisms underlying the pathogenesis of
Holt-Oram syndrome. First, some TBX5 mutations cause a dramatic decrease in its DNA-binding activity, which leads to reduced
transcription activation by TBX5 and the loss of synergy between TBX5
and NKX2.5. Second, some TBX5 mutations do not affect the DNA binding
and transcriptional activation functions of TBX5 but cause the complete loss of synergy between TBX5 and NKX2.5 in transcription activation due
to dramatically reduced interaction between TBX5 and NKX2.5. Third,
impaired nuclear localization is a common defect shared by all missense
mutations studied here. All these mechanisms are likely central to the
pathogenesis of Holt-Oram syndrome. Our data are consistent with the
hypothesis that the missense mutations in TBX5 act by a
loss-of-function mechanism.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Rajkumar Kadaba for help and
Dr. Issei Komuro for the generous gift of expression plasmids.
 |
FOOTNOTES |
*
This study was supported by a Fourjay Foundation
Cardiovascular Research grant (to Q. W.) and National Institutes of
Health Grants R01 HL65630 and R01 HL66251 (to Q. 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.
¶
To whom correspondence should be addressed: Center for
Molecular Genetics, The Cleveland Clinic Foundation, ND4-38, Cleveland, OH 44195. Tel.: 216-445-0570; Fax: 216-444-2682; E-mail:
wangq2@ccf.org.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M208120200
 |
ABBREVIATIONS |
The abbreviations used are:
ANF, the
atrial natriuretic factor gene;
WT, wild type;
Luc, luciferase;
EMSA, electrophoretic mobility shift assay;
HA, hemagglutinin;
GST, glutathione S-transferase;
PBS, phosphate-buffered saline;
Ni-NTA, nickel-nitrilotriacetic acid;
DAPI, 4',6-diamidino-2-phenylindole.
 |
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