Distinct function of 2 chromatin remodeling
complexes that share a common subunit, Williams
syndrome transcription factor (WSTF)
Kimihiro Yoshimuraa,1, Hirochika Kitagawaa,1,Ryoji Fujikia,b, Masahiko Tanabea, Shinichiro Takezawaa,b, Ichiro Takadaa,
Ikuko Yamaokaa,b, Masayoshi Yonezawaa, Takeshi Kondoa, Yoshiyuki Furutanic, Hisato Yagid, Shin Yoshinagac,e,
Takeyoshi Masudac,e, Toru Fukudaa, Yoko Yamamotoa, Kanae Ebiharab, Dean Y. Lif, Rumiko Matsuokac,d,
Jun K. Takeuchig, Takahiro Matsumotoa,b, and Shigeaki Katoa,b,2
aInstitute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1–1-1, Bunkyo-ku, Tokyo 113-0032, Japan;dDivision of Genomic Medicine,
Institute of Advanced Biomedical Engineering and Science, Graduate School of Medicine, andcThe Heart Institute of Japan, Tokyo Women’s Medical
University, 8–1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan;eDivision of Electrical Engineering and Computer Science, Graduate School of
Engineering, Shibaura Institute of Technology, Shibaura 3–9-14, Minato-ku, Tokyo 108-0023, Japan;fProgram in Human Molecular Biology and Genetics,
Department of Medicine, University of Utah, Salt Lake City, UT 84112-5330;gCardiovascular Research, Global-Edge Institute, Tokyo Institute of Technology
Frontier Research Center, 4259 Nagatsuda, Midori-ku, Yokohama, Kanagawa 226-8503, Japan; andbExploratory Research for Advanced Technology, Honcho
4–1-8, Kawaguchi, Saitama 332-0012, Japan
Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved April 16, 2009 (received for review February 3, 2009)
A number of nuclear complexes modify chromatin structure and
operate as functional units. However, the in vivo role of each
component within the complexes is not known. ATP-dependent
chromatin remodeling complexes form several types of protein
complexes, which reorganize chromatin structure cooperatively
with histone modifiers. Williams syndrome transcription factor
(WSTF) was biochemically identified as a major subunit, along with
2 distinct complexes: WINAC, a SWI/SNF-type complex, and WICH,
an ISWI-type complex. Here, WSTF?/?mice were generated to
investigate its function in chromatin remodeling in vivo. Loss of
WSTF expression resulted in neonatal lethality, and all WSTF?/?
neonates and ?10% of WSTF?/?neonates suffered cardiovascular
abnormalities resembling those found in autosomal-dominant Wil-
liams syndrome patients. Developmental analysis of WSTF?/?
embryos revealed that Gja5 gene regulation is aberrant from E9.5,
conceivably because of inappropriate chromatin reorganization
around the promoter regions where essential cardiac transcription
fibroblast (MEF) cells also showed impaired transactivation func-
tions of cardiac transcription activators on the Gja5 promoter, but
the effects were reversed by overexpression of WINAC compo-
ATPase, to PCNA and cell survival after DNA damage were both
defective, but were ameliorated by overexpression of WICH com-
ponents. Thus, the present study provides evidence that WSTF is
shared and is a functionally indispensable subunit of the WICH
complex for DNA repair and the WINAC complex for transcriptional
WICH ? WINAC ? heart development ? SWI/SNF ? ISWI
nuclear rearrangement. Two major classes of chromatin-
modifying complexes that support nuclear events on chromo-
somes have been well characterized (1). One class is a histone-
modifying complex (2, 3), and the other class is an ATP-
dependent chromatin-remodeling complex (4). This complex
uses ATP hydrolysis to rearrange nucleosomal arrays in a
noncovalent manner to facilitate, or prevent, access of nuclear
factors to nucleosomal DNA. These ATP-dependent chromatin-
remodeling complexes have been classified into 4 subfamilies,
the SWI/SNF-type complex, the ISWI-type complex, INO80
complex, and the NuRD-type complex. Each complex contains
a major catalytic component that possesses DNA-dependent
ATPase activity, such as Brg-1/Brm (SWI/SNF-type complex) or
hromatin structure is reorganized through chromatin re-
modeling and epigenetic modifications in the process of
Snf2h (ISWI-type complex) (5, 6). Selection of catalytic ATPase
subunits, combined with other complex components, defines the
role of these complexes in various nuclear events including
transcription, DNA replication, or DNA repair (7). Genetic
analyses have shown that core components of the chromatin
remodeling complexes are indispensable for embryonic devel-
opment whereas coregulatory subunits appear to support the
spatiotemporal function of the complexes (8, 9). BAF60c was
recently identified as a heart-specific subunit of the SWI/SNF-
type complex (10).
We reported that Williams syndrome transcription factor
(WSTF) (11) is a subunit of WINAC, which is a subclass of the
SWI/SNF-type ATP-dependent chromatin remodeling com-
plexes (12). WSTF is crucial for gene regulation by the vitamin
D receptor (VDR) and is expressed during embryogenesis (12,
13). WSTF is also reported to assemble with Snf2h to form
WICH, an ISWI-type chromatin complex (14). Colocalizing with
(15). WSTF was initially found as 1 of several genes (including
Cyln2, Limk1, Elastin, Bcl7b, and Fzd) deleted in patients with
autosomal-dominant Williams syndrome, a disease that displays
a wide spectrum of developmental defects, including cardiovas-
cular abnormalities (11, 16). In the present study, to address the
physiological significance of WSTF as a component of chromatin
remodeling complexes, we ablated WSTF expression in mice. All
WSTF?/?mice and 10% of WSTF?/?mice exhibited overt
cardiovascular abnormalities similar to those observed in Wil-
liams syndrome patients. Detailed analysis of the mutant mice
and cells revealed that the function of both WINAC and WICH
complexes was impaired in the absence of WSTF. Our findings
suggest that WSTF is shared and is a functionally indispensable
subunit of 2 distinct chromatin remodeling complexes; WICH
for DNA repair and WINAC for transcriptional control.
Author contributions: K.Y., H.K., J.K.T., T. Matsumoto, and S.K. designed research; K.Y.,
H.K., R.F., M.T., S.T., I.T., I.Y., M.Y., T.K., Y.F., H.Y., S.Y., T. Masuda, T.F., Y.Y., K.E., D.Y.L.,
R.M., J.K.T., and T. Matsumoto performed research; K.Y., H.K., J.K.T., and T. Matsumoto
analyzed data; and K.Y., H.K., J.K.T., T. Matsumoto, and S.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1K.Y. and H.K. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
June 9, 2009 ?
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Results and Discussion
WSTF Is Essential for Life and Exhibits a Specific Expression Pattern in
Embryos. The physiological impacts of WSTF were addressed by
born with Mendelian frequency but died within a few days. They
had smaller body sizes but were of normal appearance (Fig. 1 A
and B). Expression of the WSTF gene was not detected in
WSTF?/?pups as expected, but transcript levels of several other
candidate Williams syndrome genes (Cyln2, Limk1, Elastin,
Bc17b, and Fzd9) were not affected (Fig. 1C). The reported role
of WSTF in gene regulation (9, 11) was then tested by expression
analysis of the key enzymes regulating vitamin D biosynthesis
and catabolism, which are the 25(OH)1?-hydroxylase
[1?(OH)ase] and 25(OH)24-hydroxylase [24(OH)ase] genes (18).
Activation of the VDR by binding of 1?, 25(OH)2D3 served as
a transcriptional repressor of 1?(OH)ase, but was a transcrip-
tional activator of 24(OH)ase (12, 13, 18). Although up-
regulated expression of 1?(OH)ase was observed, no clear
induction of 24(OH)ase was detected despite an excess of
1?,25(OH)2D3observed in WSTF?/?embryos (Fig. 1D). Sup-
porting these observations, serum calcium levels were elevated
in WSTF?/?pups (Fig. 1E). Infantile hypercalcemia found in
Williams syndrome patients thus appears attributed, at least in
part, to a malfunction of WSTF in a VDR-mediated gene
cascade (12, 19).
To address a possible unique role of WSTF in WINAC during
embryogenesis, gene expression of BAF60c and BAF180, specific
components of other SWI/SNF-type complex subclasses respon-
sible for heart development (20, 21), was examined by in situ
hybridization at E9.5 (12). No exclusive expression patterns for
WSTF, BAF60c, or BAF180 were found in the trabeculae of heart
ventricles. In the pharyngeal arch 1 (Pa1), expression of WSTF
and BAF180, but not BAF60c, was detected in E9.5 embryos.
Conversely, ubiquitous expression of Snf2h was observed (Fig. 1F).
WSTF?/?Mice Exhibit Cardiovascular Abnormalities. Histological
analysis of embryos revealed severe heart defects in all WSTF?/?
and ?10% of WSTF?/?E9.5 embryos and neonates (P0) (Fig. 2
A–C). Heart trabeculation was disorganized with dilation of both
ventricles, and multiple atrial and muscular ventricular septal
defects (ASD and VSD) were visible in both WSTF?/?and
double-outlet right ventricles (DORV) was observed in
WSTF?/?embryos at low frequencies, 17.9% and 2.6%, respec-
tively. WSTF ablation also induced an infantile-type coarctation
of the aorta (CoA) at P0 (Fig. 2B). The fourth pharyngeal arch
artery was hypoplastic at E10.5 (Fig. 2D), reflecting a narrowed
aorta between the left carotid and subclavian arteries at P0 (Fig.
2C, arrow). Approximately 10% of WSTF?/?embryos exhibited
of this genetic penetrance in mice remains unknown, but it is
reminiscent of the cardiac defects observed in Williams syn-
drome patients (19).
From the phenotypic observations of elastin-deficient mice,
elastin was considered to be responsible for the vascular defor-
mities, for instance, supravalvular aortic stenosis (SVAS), in
Williams syndrome patients (11, 16). However, elastin gene
expression was not detected, even in wild-type WSTF?/?em-
bryos at E9.5 (Fig. 2F). Considering also that no overt cardiac
of WSTF expression appeared to be responsible for the cardiac
defects seen in this hereditary disease.
(Left), heterozygous (Center), and homozygous (Right) mice at postnatal day 0 (P0). (B) All homozygous WSTF mice died by P2. (C) Expression of the putative
genes responsible for Williams syndrome (WS) was unaffected by WSTF ablation. Total RNA from P0 heart of WSTF wild-type/mutant mice were subjected to
qRT-PCR analysis. Values are mean ? SD; n ? 3. (D) Aberrant expression of vitamin D receptor (VDR) target genes. Northern blot analyses of P0 embryos were
performed: 1a(OH)ase (Top), 24(OH)ase (Middle), and ?-actin (Bottom). (E) Serum calcium concentrations in WSTF?/?mice and wild type (WSTF?/?) mice. Serum
samples from 4 mice of each type were collected at the indicated days after birth. (F) WSTF expression patterns in the heart overlapped with those of BAF60c,
BAF180, and Snf2h. Expression patterns in E9.5 embryos were tested by in situ hybridization. Pa1, pharyngeal arch 1. (Scale bar, 100 ?m.)
Neonatal lethality in mice lacking WSTF Generation of WSTF knockout mice was confirmed as described in Fig. S1. (A) External appearance of wild-type
Yoshimura et al.PNAS ?
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Dysregulation of Cardiac Transcription Activators in Developing
Hearts. As severe defects were observed in WSTF?/?E9.5
embryonic hearts, we presumed that the function of cardiac
transcription factors in E9.5 embryos was impaired due to
dysregulation of WSTF-containing chromatin remodeling com-
plexes. To test this idea, a cardiac conduction system-related
gene Gja5, which encodes connexin40 (Cx40) (22, 23), their
upstream transcriptional regulators, Nkx2.5, Tbx5, and Gata4
(22, 24, 25), and the cardiac trabeculation marker, Irx3 (26), were
analyzed by RT-PCR assay in E9.5 embryonic hearts (Fig. 2G).
Although WSTF ablation appeared unlikely to affect the expres-
expression of Gja5(Cx40) and Irx3 was found (Fig. 2 G and H).
Such reduced expression of Gja5(Cx40) and Irx3 genes was seen
even in WSTF?/?hearts (Fig. 2G). WISH analysis of E9.5
embryos confirmed the altered gene expression patterns (Fig.
2H). However, cardiac muscle markers, Mlc2a and Mlc2v (27),
were expressed normally in WSTF?/?hearts and thus appeared
to be independent of WSTF-mediated gene regulation. Such
dysregulated expression of cardiac development markers was
further studied by in situ hybridization of E9.5 hearts (Fig. S2).
These findings imply that WSTF, presumably as a WINAC
subunit, is crucial for the normal gene cascades in the developing
WINAC Components Support the Function of Cardiac Transcription
Factors. WISH analysis of developing hearts suggested that the
major transcriptional activators responsible for cardiac develop-
ment require WSTF, presumably as a WINAC component, for
their transcriptional regulation. The association of endogenous
WSTF with endogenous Brg1 or Snf2h in MEF cells was verified
by coimmunoprecipitation (Fig. 3A). Although Brg1 was coim-
munoprecipitated with all of the tested BAF components, BAF
180 was not seen in WSTF immunoprecipitates in MEF cells or
in cardiogenic-P19.CL6 cells (RIKEN) (Fig. 3A and Fig. S3C).
To test transcriptional regulation by WINAC, a reporter assay
was performed by using a luciferase reporter construct contain-
ing the Gja5(Cx40) promoter region, which includes direct
binding sites for cardiac transcription factors (22, 23). Impair-
ment in transactivation functions of all of the tested cardiac
activators was observed in WSTF?/?MEF cells and impairment
was restored by coexpression of WSTF/Brg1, but not by coex-
pression of WSTF/Snf2h (Fig. 3B). Physical association of WSTF
with transcriptional activators was consistently observed
defects. Coronal sections of typical dilation of both ventricles of WSTF mutant mice and elastin?/?mice at P0 are shown. Left atrium, left ventricle, right atrium,
and right ventricle are indicated by la, lv, ra, and rv, respectively. Arrow indicates atrial septal defect (ASD); arrow head shows ventricular septal defect (VSD).
(B) Heart defects in WSTF mutant mice were evident at E9.5. Sagittal sections of the embryo of each genotype are shown. Atrial chamber (a), left ventricle (lv),
and right ventricle (rv) are shown. (C) Aortic arch defects of WSTF mutant mice. Stereomicroscopic images are shown. Aorta (ao), pulmonary artery (pa), and
by ink injection. 3, 4, and 6 indicate the number of aortic arch arteries. (E) The frequency of cardiovascular abnormalities of WSTF mutant hearts is shown.
Coarctation of the aorta (CoA) and double-outlet right ventricle (DORV) are displayed. (F and G) Cardiac gene expression patterns at E9.5 analyzed by RT-PCR.
Expression of elastin was not detected at E9.5 (F). Down-regulated expression of Gja5(Cx40), with normal expression of its activators, in WSTF mutant mice (G).
(H) Altered gene expression in WSTF mutant embryos at E9.5 by WISH analysis. Left ventricle (lv) and atrium (a) are shown. Expression of most cardiac genes,
Nkx2-5, Tbx5, Gata4, Mlc2a, and Mlc2v, is normal in WSTF?/?and WSTF?/?hearts at E9.5. [Scale bars, 500 ?m in A, 100 ?m in B, 800 ?m in C, 300 ?m in D, and
1 mm in H.]
www.pnas.org?cgi?doi?10.1073?pnas.0901184106Yoshimura et al.
function of cardiac transcription factors, regulation of
Gja5(Cx40) expression by WINAC components in cardiogenic
P19 cells was tested. Gja5(Cx40) expression was attenuated by
siRNA of the tested WINAC components, but not by the
non-WINAC component BAF180 (Fig. 3C). Consistently, re-
cruitment of WINAC components and activators was observed
by using a ChIP assay (12) with the Gja5(Cx40) gene promoter
in embryos at E9.5, but WSTF was dispensable for the recruit-
ment (Fig. 3D). In E18.5 hearts, expected recruitment of Nkx2.5,
Gata4, Tbx5, and WINAC components were clearly impaired by
WSTF ablation, and histone modification markers of activated
chromatin were reduced (Fig. 3E). However, recruitment of
PBAF components onto known PBAF-specific target gene pro-
moters (21) appeared unaffected in WSTF?/?hearts (Fig. S4).
WICH Components Are Essential for the DNA Repair Process but Not
for DNA Replication.Inpreviousreports,bothWICHandWINAC
were shown to support DNA replication in vitro (12, 15).
Therefore, using WSTF?/?MEF cells, we asked whether WSTF
distribution of DNA content (12) (Fig. 4A), cell cycle regulation
appeared intact in the absence of WSTF. WSTF and Snf2h are
anchored to Pcna (15), a major protein for controlling DNA
repair after damage (28). Thus, we determined whether WICH
was involved in DNA repair by assessing cell survival after
treatment with methyl methanesulfonate (MMS), an inducer of
DNA damage (29). Recovery from DNA double-strand breaks
appeared lower in WSTF?/?MEF cells compared with wild-type
cells. Furthermore, reduced survival could be normalized when
both WSTF and Snf2h were over-expressed (Fig. 4B). After
MMS-induced DNA damage in WSTF?/?MEF cells, recruit-
ment of Snf2h to Pcna was not detected by coimmunoprecipi-
tation (Fig. 4D). Consistently, chromatin-bound Snf2h was not
seen in MMS-treated WSTF?/?MEF cells by immunofluores-
cence analysis (Fig. 4E). Together, these findings imply that
certain phenotypic abnormalities in WSTF?/?mice, and pre-
sumably shared with Williams syndrome patients, may be caused
by an impaired DNA repair process owing to the dysfunction of
WICH (Fig. 4F).
The present findings suggest that WSTF is a chromatin
remodeler that is essential for physiological functions of certain
sequence-specific transcriptional regulators other than VDR
(12, 13) (Fig. 4F). Although WSTF was indispensable for proper
in vitro function of transcriptional activators specific for cardio-
vascular development like other BAF components (12, 30),
WSTF?/?embryos did not exhibit the same abnormalities seen
in the embryos deficient in either BAF components (10, 21) or
components in MEF cells. (B) WSTF and Brg1 cooperatively coactivate the transactivation function of cardiovascular activators on the Gja5(Cx40) promoter (23).
Values are mean ? SD for triplicate luciferase assays in MEFs. (C) Knock-down of WINAC components by siRNA lowered endogenous Gja5(Cx40) gene expression
estimated by qRT-PCR in cardiogenic-P19 cells. (D) Recruitment of WINAC components on the target gene promoter at E9.5. In vivo ChIP assays were performed
as described in Material and Methods. For the Re-IP assay, the immunoprecipitates (IP) and their supernatants (sup.) were sequentially applied for the following
ChIP analysis (13). (E) WINAC components were recruited to the Gja5(Cx40) promoter exclusively in E18.5 hearts, but not in livers. ChIP assays were performed
with the indicated tissues in both wild-type mice (WSTF?/?) and WSTF homozygous mice (WSTF?/?). The Right image shows a qPCR analysis using the same
chromatin samples from E18.5 hearts shown in the Left image using specific primers for qPCR.
WSTF coactivates the transcriptional properties of cardiac transcription factors. (A) Coimmunoprecipitation of WSTF with WINAC and WISH complex
Yoshimura et al. PNAS ?
June 9, 2009 ?
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cardiac activators (22, 24, 25). This clearly suggests a unique role
for WINAC among the SWI/SNF-type chromatin remodeling
complex subclasses in cardiovascular development. Considering
that WINAC shares most of its components with other complex
subclasses of the SWI/SNF-type (12), it is likely that a particular
combination of common components with a subclass-specific
subunit enables each complex to carry out its specific function in
gene regulation. The detailed phenotypic analysis of E9.5 hearts
showed similar but distinct abnormalities found in Baf60c
knocked-down mice (see Fig. S2) (10). Thus, we presume that
Baf60c cooperatively works with WSTF on common target gene
promoters presumably by forming a single protein complex (see
Fig. 3 A and C) at a specific developmental stage in cardiac
WSTF is also a WICH component (14, 15) so certain pheno-
types of WSTF?/?mice may be attributed to dysfunction of
WICH. Although both WICH and WINAC were reported to
affect DNA replication (15), no overt defects in the cell cycle or
growth were found in WSTF?/?MEF cells, as was seen in
Xenopus eggs (31). Thus, WSTF appears dispensable for DNA
replication in developing mice. Conversely, it is likely that WICH
is indispensable for repair of DNA damage because restored
ameliorated impaired survival after DNA damage in WSTF?/?
MEF cells. In conclusion, the WSTF subunit appears to serve as
a chromatin remodeler and is a component of 2 functionally
Materials and Methods
Whole-Mount in Situ Hybridization. Whole-mount section in situ hybridization
by using digoxigenin-labeled probes was performed as described in ref. 12.
digoxigenin-labeled probes generated by in vitro transcription (Roche) and
standard procedures. The following mouse cDNAs were used as templates for
riboprobe synthesis: Gja5(Cx40), Irx3, Gata4, WSTF, BAF60c, BAF180, Snf2h,
Nkx2.5, Tbx5, Mlc2a, and Mlc2v. Light microscopy was performed at room tem-
Vision 4.6 (Zeiss). Images of embryos were taken with a stereomicroscope (Leica
MZ 16FA) under a Planapo 1.0? objective equipped with an Axio Cam CCD
camera (Zeiss). Images were processed by using Adobe Photoshop.
Ink Injection. At E10.5, India ink was injected into the left ventricle of the
embryo’s heart. Embryos were fixed in Carnoy’s fixative, dehydrated through
a series of graded ethanol/PBS solutions to 100% ethanol, and cleared for
MEF Preparation. Primary murine embryonic fibroblasts (MEFs) were isolated
and cultured as described in ref. 13. For the luciferase assay, MEFs were
transfected with the indicated plasmids by using Lipofectamine plus reagents
mice was measured in MEFs by flow cytometry, as described in ref. 34. (B) Cell survival after DNA damage was impaired in WSTF?/?mice. Results are expressed
as the mean ? SD of 6 independent experiments (*, P ? 0.05; NS, not significant). (C) Western blot analysis of the indicated proteins after knock-down by siRNA.
(D) WSTF ablation reduced Snf2h recruitment to Pcna after DNA damage in MEF cells. Immunoprecipitation assays were performed 1 h after MMS treatment
to induce DNA damage. Western blot analyses (WB) are shown. (E and F) Aberrant recruitment of Snf2h to Pcna after DNA damage. Immunofluorescence using
the indicated antibodies was performed 1 h after MMS treatment. (Scale bar, 10 ?m.) (G) Schematic illustration of WSTF as a shared component of WINAC and
Impaired DNA repair in WSTF?/?MEF cells. (A) DNA replication was intact in WSTF?/?MEF cells. DNA content from wild-type (WT) and WSTF?/?(KO)
www.pnas.org?cgi?doi?10.1073?pnas.0901184106Yoshimura et al.
ChIP Assay. Preparation of soluble chromatin for PCR amplification was Download full-text
performed according to the protocol provided by Upstate Biotechnology. To
test in vivo binding of each transcription factor to the mouse Cx40 promoter,
heart and liver tissues from E9.5 animals were used (12). One gram of tissue
at 37°C. Primer pairs were as follows: Gja5 (Cx40) promoter: 5?-TTTC-
CTCGGGGTGCTTCAGGAAGG-3? and 5?-TCTTGAGCCTGTTAGTTGCTCCCG-3?,
and for quantitative RT-PCR (qPCR) 5?-CTTTCTCGACTGGTGAGGAA-3? and
5?-GAGCCTGTTAGTTGCTCCCG-3?; S100A13 promoter: 5?-GGAGTAGCAGTC-
CCTCTAACACAGA-3? and 5?-GCAGTCAGGAAAAGTAACTCACCG-3? (21).
Cell Survival Assay. All experimental procedures were conducted as described
in our previous report, with some modifications (29). MEF cells from WSTF?/?
and wild-type mice were plated on 60-mm dishes at 40% confluency. The
expression vectors, or Smartpool RNAi (Dharmacon), were transfected with
Lipofectamine 2000 (Invitrogen). After 24 h, transfected cells were treated
with medium containing 0.02% MMS for 1 h, washed with PBS, and main-
are means ? SD from 6 independent experiments. P values (lanes 9–11) were
calculated by Student t test (n ? 6).
with 0.02% MMS for 30 min, washed with PBS, and maintained for 30 min in
fresh medium. They were then treated with a hypotonic lysis solution con-
taining 10 mM Tris?HCl pH7.4, 2.5 mM MgCl2, 1 mM PMSF, and 0.5% Nonidet
P-40 for 8 min on ice, as described in refs. 15 and 32. The following immuno-
fluorescence procedure was performed by using standard methods as de-
Cruz), ?H2AX (ab11174, Abcam), and secondary antibodies were Alexa Fluor
594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG. All fluo-
rescence images were captured at room temperature by using a 63?/1.4
oil-immersion objective on a confocal microscope (LSM510; Zeiss) with LSM
510 software (Zeiss). Images were processed by using Adobe Photoshop.
ACKNOWLEDGMENTS. We thank A. Moorman (Academic Medical Center,
Amsterdam) for kindly providing the Gja5(Cx40) plasmid, T. Ogura (Tohoku
University, Sendai, Japan) for Gata4 and Irx3 plasmids, W. Wang (National
Institute on Aging, Baltimore) and L. Chen (National Institute on Aging,
Baltimore) for the BAF 180 antibody, and B. Bruneau (Gladstone Institute of
Bmp4 probes. We also thank Z. Wang for helpful discussions, Barbara Levene
and Alexander Kouzmenko for editing the manuscript, Haruko Higuchi, Hi-
roko Yamazaki, and Mai Yamaki for manuscript preparation, and Yuko Shi-
rode and Keiko Komatsu for technical support. This work was supported by a
and the Encouraging Development of Strategic Research Centers, Special
Coordination Funds for Promoting Science and Technology, Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan (H.K. and S.K.).
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