Correction and Retractions CORRECTION

BIOCHEMISTRY Correction for “Mod5 binds to tRNA complexes and issue 33, August 13, 2013, of Proc Natl Acad Sci USA (110: affects local transcriptional silencing,” by Matthew Pratt-Hyatt, E3081–E3089; first published July 29, 2013;10.1073/pnas. Dave A. Pai, Rebecca A. Haeusler, Glenn G. Wozniak, Paul D. 1219946110). Good, Erin L. Miller, Ian X. McLeod, John R. Yates III, The authors note that Fig. 5 appeared incorrectly. The cor- Anita K. Hopper, and David R. Engelke, which appeared in rected figure and its legend appear below.

A ATG/GTP binding site S. cerevisiae H. sapiens A. thaliana

S. cerevisiae H. sapiens A. thaliana

S. cerevisiae H. sapiens A. thaliana RETRACTIONS CORRECTION AND

DMAPP binding site

S. cerevisiae H. sapiens A. thaliana

S. cerevisiae H. sapiens A. thaliana

Zn-Finger motif

S. cerevisiae H. sapiens A. thaliana

S. cerevisiae H. sapiens A. thaliana

B +His -His mod5Δ

S. cerevisiae

A. thaliana

H. sapiens

Fig. 5. Conservation of the Mod5 protein and silencing function in eukaryotes. (A) The alignment of three Mod5 from S. cerevisiae, A. thaliana,and H. sapiens is depicted, with areas of conservation indicated by shaded boxes. All variants contain motifs for ATP/GTP binding and DMAPP binding, including E. coli (14, 40). Eukaryotic homologs also have a motif in the C-terminal extensions. (B) tgm silencing functions of yeast Mod5 are conserved in plant and human proteins. It was previously shown that human and Arabidopsis Mod5 homologs are able to restore i6A modification of tRNAs in yeast in an mod5Δ strain (14, 24). Plasmids expressing these ORFs were tested for supporting tgm silencing in an mod5Δ strain containing a tgm silencing reporter plasmid, with loss of silencing shown by growth on media lacking histidine (−His). Although both homologs restore silencing, the H. sapiens version of Mod5 (TRIT1) is consistently more robust in preventing growth without histidine.

www.pnas.org/cgi/doi/10.1073/pnas.1400058111

www.pnas.org PNAS | February 11, 2014 | vol. 111 | no. 6 | 2397–2398 Downloaded by guest on September 26, 2021 RETRACTIONS

CELL BIOLOGY CELL BIOLOGY Retraction for “Distinct function of 2 chromatin remodeling Retraction for “Testis-specific protein on Y (TSPY) complexes that share a common subunit, Williams syndrome represses the activity of the androgen in androgen- (WSTF),” by Kimihiro Yoshimura, Hirochika dependent testicular germ-cell tumors,” by Chihiro Akimoto, Kitagawa, Ryoji Fujiki, Masahiko Tanabe, Shinichiro Takezawa, Takashi Ueda, Kazuki Inoue, Ikuko Yamaoka, Matomo Sakari, Ichiro Takada, Ikuko Yamaoka, Masayoshi Yonezawa, Takeshi Wataru Obara, Tomoaki Fujioka, Akira Nagahara, Norio Kondo, Yoshiyuki Furutani, Hisato Yagi, Shin Yoshinaga, Takeyoshi Nonomura, Syuichi Tsutsumi, Hiroyuki Aburatani, Tsuneharu Masuda, Toru Fukuda, Yoko Yamamoto, Kanae Ebihara, Dean Y. Miki, Takahiro Matsumoto, Hirochika Kitagawa, and Shigeaki Li, Rumiko Matsuoka, Jun K. Takeuchi, Takahiro Matsumoto, Kato, which appeared in issue 46, November 16, 2010, of Proc and Shigeaki Kato, which appeared in issue 23, June 9, 2009, of Natl Acad Sci USA (107:19891–19896; first published November Proc Natl Acad Sci USA (106:9280–9285; first published May 26, 1, 2010; 10.1073/pnas.1010307107). 2009; 10.1073/pnas.0901184106). The authors wish to note, “Recently it has come to our at- The authors note, “Recently it has come to our attention that tention that inappropriate data arrangements and manipulations Fig. 3D contained inappropriate data arrangements and manipu- were made in Figs. 1, 2C,S4A,S4B, and 5. To avoid further lations. To avoid further confusion to the related scientific com- confusion to the related scientific community, we hereby retract munity, we hereby retract this report. We sincerely apologize for this report. We sincerely apologize for any inconvenience from any inconvenience from this outcome. All of the authors agreed this outcome. All of the authors agree with the retraction.” with the retraction.” Chihiro Akimoto Kimihiro Yoshimura Takashi Ueda Hirochika Kitagawa Kazuki Inoue Ryoji Fujiki Ikuko Yamaoka Masahiko Tanabe Matomo Sakari Shinichiro Takezawa Wataru Obara Ichiro Takada Tomoaki Fujioka Ikuko Yamaoka Akira Nagahara Masayoshi Yonezawa Norio Nonomura Takeshi Kondo Syuichi Tsutsumi Yoshiyuki Furutani Hiroyuki Aburatani Hisato Yagi Tsuneharu Miki Shin Yoshinaga Takahiro Matsumoto Takeyoshi Masuda Hirochika Kitagawa Toru Fukuda Shigeaki Kato Yoko Yamamoto www.pnas.org/cgi/doi/10.1073/pnas.1323399111 Kanae Ebihara Dean Y. Li Rumiko Matsuoka Jun K. Takeuchi Takahiro Matsumoto Shigeaki Kato

www.pnas.org/cgi/doi/10.1073/pnas.1323397111

2398 | www.pnas.org Downloaded by guest on September 26, 2021 See Retraction Published January 21, 2014 Distinct function of 2 chromatin remodeling complexes that share a common subunit, Williams syndrome transcription factor (WSTF)

a,1 a,1 a,b a a,b a Kimihiro Yoshimura , Hirochika Kitagawa , Ryoji Fujiki , Masahiko Tanabe , Shinichiro Takezawa , Ichiro Takada , 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, and cThe 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; and bExploratory 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 Snf2h (ISWI-type complex) (5, 6). Selection of catalytic ATPase operate as functional units. However, the in vivo role of each subunits, combined with other complex components, defines the component within the complexes is not known. ATP-dependent role of these complexes in various nuclear events including chromatin remodeling complexes form several types of protein transcription, DNA replication, or DNA repair (7). Genetic complexes, which reorganize chromatin structure cooperatively analyses have shown that core components of the chromatin with histone modifiers. Williams syndrome transcription factor remodeling complexes are indispensable for embryonic devel- (WSTF) was biochemically identified as a major subunit, along with opment whereas coregulatory subunits appear to support the 2 distinct complexes: WINAC, a SWI/SNF-type complex, and WICH, spatiotemporal function of the complexes (8, 9). BAF60c was ؊ ؊ an ISWI-type complex. Here, WSTF / mice were generated to recently identified as a heart-specific subunit of the SWI/SNF- investigate its function in chromatin remodeling in vivo. Loss of type complex (10). ؊/؊ WSTF expression resulted in neonatal lethality, and all WSTF We reported that Williams syndrome transcription factor Ϸ ؉/؊ neonates and 10% of WSTF neonates suffered cardiovascular (WSTF) (11) is a subunit of WINAC, which is a subclass of the abnormalities resembling those found in autosomal-dominant Wil- SWI/SNF-type ATP-dependent chromatin remodeling com- ؊/؊ liams syndrome patients. Developmental analysis of WSTF plexes (12). WSTF is crucial for gene regulation by the vitamin embryos revealed that Gja5 gene regulation is aberrant from E9.5, D receptor (VDR) and is expressed during embryogenesis (12, conceivably because of inappropriate chromatin reorganization 13). WSTF is also reported to assemble with Snf2h to form around the promoter regions where essential cardiac transcription WICH, an ISWI-type chromatin complex (14). Colocalizing with factors are recruited. In vitro analysis in WSTF؊/؊ mouse embryonic replication foci, WICH is considered to support DNA replication fibroblast (MEF) cells also showed impaired transactivation func- (15). WSTF was initially found as 1 of several (including tions of cardiac transcription activators on the Gja5 promoter, but Cyln2, Limk1, Elastin, Bcl7b, and Fzd) deleted in patients with the effects were reversed by overexpression of WINAC compo- nents. Likewise in WSTF؊/؊ MEF cells, recruitment of Snf2h, an ISWI autosomal-dominant Williams syndrome, a disease that displays ATPase, to PCNA and cell survival after DNA damage were both a wide spectrum of developmental defects, including cardiovas- defective, but were ameliorated by overexpression of WICH com- cular abnormalities (11, 16). In the present study, to address the ponents. Thus, the present study provides evidence that WSTF is physiological significance of WSTF as a component of chromatin shared and is a functionally indispensable subunit of the WICH remodeling complexes, we ablated WSTF expression in mice. All Ϫ/Ϫ ϩ/Ϫ complex for DNA repair and the WINAC complex for transcriptional WSTF mice and 10% of WSTF mice exhibited overt control. cardiovascular abnormalities similar to those observed in Wil- liams syndrome patients. Detailed analysis of the mutant mice WICH ͉ WINAC ͉ heart development ͉ SWI/SNF ͉ ISWI and cells revealed that the function of both WINAC and WICH complexes was impaired in the absence of WSTF. Our findings hromatin structure is reorganized through chromatin re- suggest that WSTF is shared and is a functionally indispensable Cmodeling and epigenetic modifications in the process of subunit of 2 distinct chromatin remodeling complexes; WICH nuclear rearrangement. Two major classes of chromatin- for DNA repair and WINAC for transcriptional control. 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- Author contributions: K.Y., H.K., J.K.T., T. Matsumoto, and S.K. designed research; K.Y., dependent chromatin-remodeling complex (4). This complex 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., uses ATP hydrolysis to rearrange nucleosomal arrays in a R.M., J.K.T., and T. Matsumoto performed research; K.Y., H.K., J.K.T., and T. Matsumoto noncovalent manner to facilitate, or prevent, access of nuclear analyzed data; and K.Y., H.K., J.K.T., T. Matsumoto, and S.K. wrote the paper. factors to nucleosomal DNA. These ATP-dependent chromatin- The authors declare no conflict of interest. remodeling complexes have been classified into 4 subfamilies, This article is a PNAS Direct Submission. the SWI/SNF-type complex, the ISWI-type complex, INO80 1K.Y. and H.K. contributed equally to this work. complex, and the NuRD-type complex. Each complex contains 2To whom correspondence should be addressed. E-mail: [email protected]. a major catalytic component that possesses DNA-dependent This article contains supporting information online at www.pnas.org/cgi/content/full/ ATPase activity, such as Brg-1/Brm (SWI/SNF-type complex) or 0901184106/DCSupplemental.

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Fig. 1. Neonatal lethality in mice lacking WSTF Generation of WSTF knockout mice was confirmed as described in Fig. S1.(A) External appearance of wild-type (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 Ϯ SD; n ϭ 3. (D) Aberrant expression of (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 WSTFϩ/Ϫ mice and wild type (WSTFϩ/ϩ) 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.)

Results and Discussion ventricles. In the pharyngeal arch 1 (Pa1), expression of WSTF WSTF Is Essential for Life and Exhibits a Specific Expression Pattern in and BAF180, but not BAF60c, was detected in E9.5 embryos. Embryos. The physiological impacts of WSTF were addressed by Conversely, ubiquitous expression of Snf2h was observed (Fig. 1F). gene disruption in mice (17) (Fig. S1). WSTF-deficient mice were ؊ ؊ born with Mendelian frequency but died within a few days. They WSTF / Mice Exhibit Cardiovascular Abnormalities. Histological Ϫ Ϫ had smaller body sizes but were of normal appearance (Fig. 1 A analysis of embryos revealed severe heart defects in all WSTF / ϩ/Ϫ and B). Expression of the WSTF gene was not detected in and Ϸ10% of WSTF E9.5 embryos and neonates (P0) (Fig. 2 CELL BIOLOGY WSTFϪ/Ϫ pups as expected, but transcript levels of several other A–C). Heart trabeculation was disorganized with dilation of both candidate Williams syndrome genes (Cyln2, Limk1, Elastin, ventricles, and multiple atrial and muscular ventricular septal Ϫ Ϫ Bc17b, and Fzd9) were not affected (Fig. 1C). The reported role defects (ASD and VSD) were visible in both WSTF / and ϩ Ϫ of WSTF in gene regulation (9, 11) was then tested by expression WSTF / neonates (Fig. 2A). Hypertrophy of both ventricles and analysis of the key enzymes regulating vitamin D biosynthesis double-outlet right ventricles (DORV) was observed in Ϫ Ϫ and catabolism, which are the 25(OH)1␣-hydroxylase WSTF / embryos at low frequencies, 17.9% and 2.6%, respec- [1␣(OH)ase] and 25(OH)24-hydroxylase [24(OH)ase] genes (18). tively. WSTF ablation also induced an infantile-type coarctation Activation of the VDR by binding of 1␣, 25(OH)2D3 served as of the aorta (CoA) at P0 (Fig. 2B). The fourth pharyngeal arch a transcriptional repressor of 1␣(OH)ase, but was a transcrip- artery was hypoplastic at E10.5 (Fig. 2D), reflecting a narrowed tional activator of 24(OH)ase (12, 13, 18). Although up- aorta between the left carotid and subclavian arteries at P0 (Fig. ϩ Ϫ regulated expression of 1␣(OH)ase was observed, no clear 2C, arrow). Approximately 10% of WSTF / embryos exhibited induction of 24(OH)ase was detected despite an excess of a wide spectrum of cardiac defects (Fig. 2E). The molecular basis Ϫ/Ϫ 1␣,25(OH)2D3 observed in WSTF embryos (Fig. 1D). Sup- of this genetic penetrance in mice remains unknown, but it is porting these observations, serum calcium levels were elevated reminiscent of the cardiac defects observed in Williams syn- in WSTFϩ/Ϫ pups (Fig. 1E). Infantile hypercalcemia found in drome patients (19). Williams syndrome patients thus appears attributed, at least in From the phenotypic observations of elastin-deficient mice, part, to a malfunction of WSTF in a VDR-mediated gene elastin was considered to be responsible for the vascular defor- cascade (12, 19). mities, for instance, supravalvular aortic stenosis (SVAS), in To address a possible unique role of WSTF in WINAC during Williams syndrome patients (11, 16). However, elastin gene embryogenesis, gene expression of BAF60c and BAF180, specific expression was not detected, even in wild-type WSTFϩ/ϩ em- components of other SWI/SNF-type complex subclasses respon- bryos at E9.5 (Fig. 2F). Considering also that no overt cardiac sible for heart development (20, 21), was examined by in situ defects were obvious in elastinϪ/Ϫ embryos at E9.5 (Fig. 2A), lack hybridization at E9.5 (12). No exclusive expression patterns for of WSTF expression appeared to be responsible for the cardiac WSTF, BAF60c,orBAF180 were found in the trabeculae of heart defects seen in this hereditary disease.

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Fig. 2. Cardiac defects and aortic arch disorganization in WSTFϩ/Ϫ and WSTFϪ/Ϫ embryos. (A) WSTF mutant (WSTFϩ/Ϫ and WSTFϪ/Ϫ) mice exhibited severe heart defects. Coronal sections of typical dilation of both ventricles of WSTF mutant mice and elastinϪ/Ϫ 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 coarctation of the aorta (arrow) are indicated. (D) The fourth aortic arch artery of WSTF mutant mice at E10.5 was hypoplastic. Aortic arch arteries were visualized 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 WSTFϩ/Ϫ and WSTFϪ/Ϫ hearts at E9.5. [Scale bars, 500 ␮minA, 100 ␮minB, 800 ␮minC, 300 ␮minD, and 1mminH.]

Dysregulation of Cardiac Transcription Activators in Developing heart and that WINAC is required for normal function of cardiac Hearts. As severe defects were observed in WSTFϪ/Ϫ E9.5 transcriptional regulators. embryonic hearts, we presumed that the function of cardiac transcription factors in E9.5 embryos was impaired due to WINAC Components Support the Function of Cardiac Transcription dysregulation of WSTF-containing chromatin remodeling com- Factors. WISH analysis of developing hearts suggested that the plexes. To test this idea, a cardiac conduction system-related major transcriptional activators responsible for cardiac develop- gene Gja5, which encodes connexin40 (Cx40) (22, 23), their ment require WSTF, presumably as a WINAC component, for upstream transcriptional regulators, Nkx2.5, Tbx5, and Gata4 their transcriptional regulation. The association of endogenous (22, 24, 25), and the cardiac trabeculation marker, Irx3 (26), were WSTF with endogenous Brg1 or Snf2h in MEF cells was verified analyzed by RT-PCR assay in E9.5 embryonic hearts (Fig. 2G). by coimmunoprecipitation (Fig. 3A). Although Brg1 was coim- Although WSTF ablation appeared unlikely to affect the expres- munoprecipitated with all of the tested BAF components, BAF sion of cardiac transcription factors, a significant reduction in the 180 was not seen in WSTF immunoprecipitates in MEF cells or expression of Gja5(Cx40) and Irx3 was found (Fig. 2 G and H). in cardiogenic-P19.CL6 cells (RIKEN) (Fig. 3A and Fig. S3C). Such reduced expression of Gja5(Cx40) and Irx3 genes was seen To test transcriptional regulation by WINAC, a reporter assay even in WSTFϩ/Ϫ hearts (Fig. 2G). WISH analysis of E9.5 was performed by using a luciferase reporter construct contain- embryos confirmed the altered gene expression patterns (Fig. ing the Gja5(Cx40) promoter region, which includes direct 2H). However, cardiac muscle markers, Mlc2a and Mlc2v (27), binding sites for cardiac transcription factors (22, 23). Impair- were expressed normally in WSTFϪ/Ϫ hearts and thus appeared ment in transactivation functions of all of the tested cardiac to be independent of WSTF-mediated gene regulation. Such activators was observed in WSTFϪ/Ϫ MEF cells and impairment dysregulated expression of cardiac development markers was was restored by coexpression of WSTF/Brg1, but not by coex- further studied by in situ hybridization of E9.5 hearts (Fig. S2). pression of WSTF/Snf2h (Fig. 3B). Physical association of WSTF These findings imply that WSTF, presumably as a WINAC with transcriptional activators was consistently observed subunit, is crucial for the normal gene cascades in the developing (Fig. S3).

9282 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901184106 Yoshimura et al. See Retraction Published January 21, 2014

Fig. 3. WSTF coactivates the transcriptional properties of cardiac transcription factors. (A) Coimmunoprecipitation of WSTF with WINAC and WISH complex 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 Ϯ 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 (WSTFϩ/ϩ) and WSTF homozygous mice (WSTFϪ/Ϫ). 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. CELL BIOLOGY To test the physiological impact of the WINAC complex on the repair after damage (28). Thus, we determined whether WICH function of cardiac transcription factors, regulation of was involved in DNA repair by assessing cell survival after Gja5(Cx40) expression by WINAC components in cardiogenic treatment with methyl methanesulfonate (MMS), an inducer of P19 cells was tested. Gja5(Cx40) expression was attenuated by DNA damage (29). Recovery from DNA double-strand breaks siRNA of the tested WINAC components, but not by the appeared lower in WSTFϪ/Ϫ MEF cells compared with wild-type non-WINAC component BAF180 (Fig. 3C). Consistently, re- cells. Furthermore, reduced survival could be normalized when cruitment of WINAC components and activators was observed both WSTF and Snf2h were over-expressed (Fig. 4B). After by using a ChIP assay (12) with the Gja5(Cx40) gene promoter MMS-induced DNA damage in WSTFϪ/Ϫ MEF cells, recruit- in embryos at E9.5, but WSTF was dispensable for the recruit- ment of Snf2h to Pcna was not detected by coimmunoprecipi- ment (Fig. 3D). In E18.5 hearts, expected recruitment of Nkx2.5, tation (Fig. 4D). Consistently, chromatin-bound Snf2h was not Gata4, Tbx5, and WINAC components were clearly impaired by seen in MMS-treated WSTFϪ/Ϫ MEF cells by immunofluores- WSTF ablation, and histone modification markers of activated cence analysis (Fig. 4E). Together, these findings imply that chromatin were reduced (Fig. 3E). However, recruitment of certain phenotypic abnormalities in WSTFϪ/Ϫ mice, and pre- PBAF components onto known PBAF-specific target gene pro- sumably shared with Williams syndrome patients, may be caused moters (21) appeared unaffected in WSTFϪ/Ϫ hearts (Fig. S4). by an impaired DNA repair process owing to the dysfunction of WICH (Fig. 4F). WICH Components Are Essential for the DNA Repair Process but Not The present findings suggest that WSTF is a chromatin for DNA Replication. In previous reports, both WICH and WINAC remodeler that is essential for physiological functions of certain were shown to support DNA replication in vitro (12, 15). sequence-specific transcriptional regulators other than VDR Therefore, using WSTFϪ/Ϫ MEF cells, we asked whether WSTF (12, 13) (Fig. 4F). Although WSTF was indispensable for proper mediated DNA replication. From flow cytometric analyses of the in vitro function of transcriptional activators specific for cardio- distribution of DNA content (12) (Fig. 4A), cell cycle regulation vascular development like other BAF components (12, 30), appeared intact in the absence of WSTF. WSTF and Snf2h are WSTFϪ/Ϫ embryos did not exhibit the same abnormalities seen anchored to Pcna (15), a major protein for controlling DNA in the embryos deficient in either BAF components (10, 21) or

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Fig. 4. Impaired DNA repair in WSTFϪ/Ϫ MEF cells. (A) DNA replication was intact in WSTFϪ/Ϫ MEF cells. DNA content from wild-type (WT) and WSTFϪ/Ϫ (KO) mice was measured in MEFs by flow cytometry, as described in ref. 34. (B) Cell survival after DNA damage was impaired in WSTFϪ/Ϫ mice. Results are expressed as the mean Ϯ SD of 6 independent experiments (*, P Ͻ 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 WICH complexes.

cardiac activators (22, 24, 25). This clearly suggests a unique role a chromatin remodeler and is a component of 2 functionally for WINAC among the SWI/SNF-type chromatin remodeling distinct complexes. complex subclasses in cardiovascular development. Considering that WINAC shares most of its components with other complex Materials and Methods subclasses of the SWI/SNF-type (12), it is likely that a particular Whole-Mount in Situ Hybridization. Whole-mount section in situ hybridization combination of common components with a subclass-specific by using digoxigenin-labeled probes was performed as described in ref. 12. subunit enables each complex to carry out its specific function in Embryos were fixed with 4% paraformaldehyde, stored in 10% methanol, and ␮ gene regulation. The detailed phenotypic analysis of E9.5 hearts 10- m paraffin sections were cut. In situ hybridization was performed by using digoxigenin-labeled probes generated by in vitro transcription (Roche) and showed similar but distinct abnormalities found in Baf60c standard procedures. The following mouse cDNAs were used as templates for knocked-down mice (see Fig. S2) (10). Thus, we presume that riboprobe synthesis: Gja5(Cx40), Irx3, Gata4, WSTF, BAF60c, BAF180, Snf2h, Baf60c cooperatively works with WSTF on common target gene Nkx2.5, Tbx5, Mlc2a, and Mlc2v. Light microscopy was performed at room tem- promoters presumably by forming a single protein complex (see perature by using a microscope (AX10; Zeiss) fitted with an Axio Cam CCD camera Fig. 3 A and C) at a specific developmental stage in cardiac (Zeiss) through a EC Plan-Neofluar 20 0.5 objective with acquisition software Axio development. Vision 4.6 (Zeiss). Images of embryos were taken with a stereomicroscope (Leica WSTF is also a WICH component (14, 15) so certain pheno- MZ 16FA) under a Planapo 1.0ϫ objective equipped with an Axio Cam CCD types of WSTFϪ/Ϫ mice may be attributed to dysfunction of camera (Zeiss). Images were processed by using Adobe Photoshop. WICH. Although both WICH and WINAC were reported to affect DNA replication (15), no overt defects in the cell cycle or Ink Injection. At E10.5, India ink was injected into the left ventricle of the growth were found in WSTFϪ/Ϫ MEF cells, as was seen in 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 Xenopus eggs (31). Thus, WSTF appears dispensable for DNA several hours in 2 volumes of benzyl benzoate and 1 volume of benzyl alcohol. replication in developing mice. Conversely, it is likely that WICH is indispensable for repair of DNA damage because restored MEF Preparation. Primary murine embryonic fibroblasts (MEFs) were isolated expression of WSTF, together with the WICH component Snf2h, and cultured as described in ref. 13. For the luciferase assay, MEFs were Ϫ/Ϫ ameliorated impaired survival after DNA damage in WSTF transfected with the indicated plasmids by using Lipofectamine plus reagents MEF cells. In conclusion, the WSTF subunit appears to serve as (Invitrogen).

9284 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901184106 Yoshimura et al. See Retraction Published January 21, 2014

ChIP Assay. Preparation of soluble chromatin for PCR amplification was taining 10 mM Tris⅐HCl pH7.4, 2.5 mM MgCl2, 1 mM PMSF, and 0.5% Nonidet performed according to the protocol provided by Upstate Biotechnology. To P-40 for 8 min on ice, as described in refs. 15 and 32. The following immuno- test in vivo binding of each transcription factor to the mouse Cx40 promoter, fluorescence procedure was performed by using standard methods as de- heart and liver tissues from E9.5 animals were used (12). One gram of tissue scribed in ref. 33. Primary antibodies used were Pcna [PC10 (sc-56), Santa Cruz], was homogenized in 10 mL PBS and treated with 1% formaldehyde for 15 min WSTF (ab51256, Abcam), Snf2h (ab3749, Abcam), Brg-1(H-88 (sc-10768), Santa at 37°C. Primer pairs were as follows: Gja5 (Cx40) promoter: 5Ј-TTTC- Cruz), ␥H2AX (ab11174, Abcam), and secondary antibodies were Alexa Fluor CTCGGGGTGCTTCAGGAAGG-3Ј and 5Ј-TCTTGAGCCTGTTAGTTGCTCCCG-3Ј, 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG. All fluo- and for quantitative RT-PCR (qPCR) 5Ј-CTTTCTCGACTGGTGAGGAA-3Ј and rescence images were captured at room temperature by using a 63ϫ/1.4 5Ј-GAGCCTGTTAGTTGCTCCCG-3Ј; S100A13 promoter: 5Ј-GGAGTAGCAGTC- oil-immersion objective on a confocal microscope (LSM510; Zeiss) with LSM CCTCTAACACAGA-3Ј and 5Ј-GCAGTCAGGAAAAGTAACTCACCG-3Ј (21). 510 software (Zeiss). Images were processed by using Adobe Photoshop.

Cell Survival Assay. All experimental procedures were conducted as described ACKNOWLEDGMENTS. We thank A. Moorman (Academic Medical Center, Ϫ Ϫ in our previous report, with some modifications (29). MEF cells from WSTF / Amsterdam) for kindly providing the Gja5(Cx40) plasmid, T. Ogura (Tohoku and wild-type mice were plated on 60-mm dishes at 40% confluency. The University, Sendai, Japan) for Gata4 and Irx3 plasmids, W. Wang (National expression vectors, or Smartpool RNAi (Dharmacon), were transfected with Institute on Aging, Baltimore) and L. Chen (National Institute on Aging, Lipofectamine 2000 (Invitrogen). After 24 h, transfected cells were treated Baltimore) for the BAF 180 antibody, and B. Bruneau (Gladstone Institute of with medium containing 0.02% MMS for 1 h, washed with PBS, and main- Cardiovascular Disease, San Francisco) for Irx3, Cx40, Bmp10, Nppa, Tbx20, and tained for 4 days in fresh medium. Surviving cells were then counted. All values Bmp4 probes. We also thank Z. Wang for helpful discussions, Barbara Levene are means Ϯ SD from 6 independent experiments. P values (lanes 9–11) were and Alexander Kouzmenko for editing the manuscript, Haruko Higuchi, Hi- roko Yamazaki, and Mai Yamaki for manuscript preparation, and Yuko Shi- calculated by Student t test (n ϭ 6). rode and Keiko Komatsu for technical support. This work was supported by a grant from Human Frontier Science Program, Mitsubishi Foundation (to J.K.T), Ϫ Ϫ Immunofluorescence. MEF cells from WSTF / and wild-type cells were treated and the Encouraging Development of Strategic Research Centers, Special with 0.02% MMS for 30 min, washed with PBS, and maintained for 30 min in Coordination Funds for Promoting Science and Technology, Ministry of Edu- fresh medium. They were then treated with a hypotonic lysis solution con- cation, Culture, Sports, Science and Technology, Japan (H.K. and S.K.).

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Yoshimura et al. PNAS ͉ June 9, 2009 ͉ vol. 106 ͉ no. 23 ͉ 9285