CHD8 suppresses p53-mediated apoptosis through histone H1 ...

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CHD8 suppresses p53-mediated apoptosis through histone H1 recruitment during early embryogenesis Masaaki Nishiyama1,2, Kiyotaka Oshikawa1,2, Yu-ichi Tsukada1,2, Tadashi Nakagawa1,2, Shun-ichiro Iemura3, Tohru Natsume3, Yuhong Fan4,5, Akira Kikuchi6, Arthur I. Skoultchi4 and Keiichi I. Nakayama1,2,7 The chromodomain helicase DNA-binding (CHD) family of enzymes is thought to regulate gene expression, but their role in the regulation of specific genes has been unclear. Here we show that CHD8 is expressed at a high level during early embryogenesis and prevents apoptosis mediated by the tumour suppressor protein p53. CHD8 was found to bind to p53 and to suppress its transactivation activity. CHD8 promoted the association of p53 and histone H1, forming a trimeric complex on chromatin that was required for inhibition of p53-dependent transactivation and apoptosis. Depletion of CHD8 or histone H1 resulted in p53 activation and apoptosis. Furthermore, Chd8–/– mice died early during embryogenesis, manifesting widespread apoptosis, whereas deletion of p53 ameliorated this developmental arrest. These observations reveal a mode of p53 regulation mediated by CHD8, which may set a threshold for induction of apoptosis during early embryogenesis by counteracting p53 function through recruitment of histone H1. Although apoptosis has a key role in organization of the developing embryo, it is not fully understood how apoptosis is regulated during embryogenesis. The tumour suppressor protein p53 mediates the induction of apoptosis in response to DNA damage caused by genotoxic stress. It activates the transcription of numerous genes, and thereby triggers cell-cycle arrest, senescence or apoptosis to prevent tumorigenesis1–5. Activation of transcription by p53 is regulated, at least in part, by the amount of p53 as well as by post-translational modifications of p53 (refs 6–8). In addition, certain chromatin-associated proteins that change chromatin configuration interact with p53 and thereby modulate its transactivation activity5,9–12. Although these findings suggest that chromatin configuration may affect the transactivation activity of p53, the mechanism by which the structure of chromatin changes, as well as the biological outcome of such regulation, have remained largely unknown. Certain classes of molecules recognize modified histones and are thought to translate the modification code into specific functions. Such proteins include members of the chromodomain helicase DNA-binding (CHD) family of enzymes, which also belong to the SNF2 superfamily of ATP-dependent chromatin remodellers13–15. Chd1 of Saccharomyces cerevisiae is a component of the multi-subunit histone acetyltransferase complexes SAGA and SLIK16, and is required for methylation of histone

H3 at Lys 4 (H3K4; ref. 17). Human CHD1 catalyses the ATP-dependent transfer of histones from the NAP‑1 chaperone to DNA, resulting in the assembly of active chromatin18,19. Nine genes for CHD1-related proteins have been identified in mammalian species. Among these proteins, CHD8 (Duplin) was originally isolated as a negative regulator of the Wnt–β-catenin signalling pathway20. The carboxy-terminal region of CHD8 interacts with the insulator-binding protein CTCF, and this interaction is important for insulator activity21. We previously generated Chd8–/– mice and showed that these animals die in utero between embryonic day (E) 5.5 and E7.5, manifesting widespread apoptosis22. However, Wnt activation was not seen in the Chd8–/– embryos before their death. Although these observations suggest that CHD8 may possess antiapoptotic activity that is independent of Wnt signalling inhibition, it has been unclear how the loss of CHD8 induces apoptosis. We now show that CHD8 binds to both p53 and histone H1, and that these interactions facilitate recruitment of histone H1 to p53 target genes, resulting in suppression of their expression and of apoptosis induced by genotoxic insults. These results thus suggest that loss of CHD8 in mice induces apoptosis as a result of unrestrained p53 activity at the early stage of embryonic development. Consistent with this conclusion, deletion of p53 in Chd8–/– mice ameliorated the developmental arrest. The physiological role of CHD8 may thus be to prevent such unwanted apoptosis during early embryogenesis.

1 Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3‑1‑1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812‑8582, Japan. 2CREST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332‑0012, Japan. 3National Institute of Advanced Industrial Science and Technology (AIST), Biological Information Research Center (JBIRC), Kohtoh-ku, Tokyo 135‑0064, Japan. 4Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. 5School of Biology and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, IBB 2313, 315 Ferst Drive, Atlanta, GA 30332‑0363, USA. 6Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, 1‑2‑3 Kasumi, Minami-ku, Hiroshima 734‑8551, Japan. 7 Correspondence should be addressed to K.I.N. (e-mail: [email protected])

Received 29 July 2008; accepted 28 October 2008; published online 18 January 2009; DOI: 10.1038/ncb1831

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nature cell biology volume 11 | number 2 | FEBRUARY 2009 © 2009 Macmillan Publishers Limited. All rights reserved.

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Figure 1 Anti-apoptotic activity of CHD8. (a–c) NIH 3T3 cells overexpressing CHD8S or CHD8L were subjected to immunoblot (IB, a) analysis with antiCHD8 and exposed to genotoxic stress. Cells were examined by phasecontrast microscopy (b) and the percentage of dead cells was determined by trypan blue staining (c). Data in c are mean ± s.d., n = 3. (d) NIH 3T3 cells overexpressing CHD8S were exposed to etoposide (ETOP, 50 µM), cycloheximide (CHX, 100 µg ml–1), staurosporine (STR, 1 µM), UV radiation or c‑Myc overexpression. Data in d are mean ± s.d., n = 3 (**P 0.05; Student’s t-test). (e) U2OS cells were infected with retroviral vectors for CHD8S or p53 and were stained with trypan blue

(left panel). Data shown in the right panel are mean ± s.d., n = 3. (f) U2OS cells were infected with retroviral vectors encoding shRNAs specific for p53, both CHD8S and CHD8L (CHD8S+L‑1) or EGFP (control), subjected to immunoblotting (left panel), stained with trypan blue and the percentage of dead cells determined (right panel). Data shown in the right panel are mean ± s.d., n = 3. (g, h) p53+/+ or p53–/– MEFs infected with retroviral vectors for CHD8S+L‑1 or CHD8S+L‑2 shRNAs were examined by phasecontrast microscopy (g) and the percentage of dead cells determined by trypan blue staining (h). Data in h are mean ± s.d., n = 3. Scale bars are 100 μm (b, e, g).

RESULTS CHD8 suppresses p53-dependent apoptosis Mouse Chd8 consists of 37 exons spanning about 40 kb. Alternative splicing of exon 9 generates two transcripts that encode a protein (CHD8L) with a relative molecular mass of 280,000 (Mr 280K), containing two chromodomains

(a helicase/ATPase domain and a DNA binding domain) and a 110K protein (CHD8S, also known as Duplin), which contains only the amino-terminal chromodomain (Supplementary Information, Fig. S1a, b). These molecules are expressed in most cell lines and tissues (Supplementary Information, Fig. S1c, d), but the ratio of CHD8L to CHD8S varies.


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Figure 2 CHD8 interacts with and inhibits transactivation by p53. (a) HeLa cells expressing Myc–CHD8S were immunostained with anti-Myc or p53. Scale bar, 5 μm. (b) U2OS cells were incubated with etoposide and then subjected to immunoprecipitation (IP) with an anti-CHD8 antibody, and immunoblot analysis with an anti‑p53 antibody. (c) Immunoprecipitation of U2OS lysates with anti‑p53 and immunoblot analysis with an anti-CHD8 antibodies. (d) In vitro binding assay for recombinant CHD8S and p53.

(e) U2OS cells overexpressing CHD8S were incubated with doxorubicin (DXR) and then subjected to immunoblot analysis with the indicated antibodies. (f) U2OS cells overexpressing CHD8S were treated with the genotoxic agents DXR (0.5 µM) and etoposide (ETOP, 20 µM) and then subjected to qRT– PCR. (g) U2OS cells infected with a retroviral vector encoding CHD8S+L‑1 shRNA were subjected to qRT–PCR. (h) Luciferase assay using either wildtype or mutant CHD8SΔ53. Data are mean ± s.d., n = 3 (f–h).

Given that lack of both CHD8 S and CHD8 L results in widespread apoptosis in mouse embryos22, we hypothesized that CHD8 may possess anti-apoptotic activity. Indeed, overexpression of either CHD8S or CHD8L inhibited the induction of apoptosis in

mouse NIH 3T3 cells exposed to etoposide or ultraviolet (UV) radiation (Fig. 1a–c; Supplementary Information, Fig. S2a). The apoptosis-related cleavage of both caspase‑3 and poly(ADP-ribose) polymerase (PARP) was also inhibited by overexpression of CHD8

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Figure 3 CHD8 binds to the promoters of p53 target genes. (a) U2OS cells infected with a retroviral vector encoding p53 shRNA were incubated with the genotoxic agents etoposide (ETOP, 20 µM) and doxorubicin (DXR, 0.5 µM). The cells were then subjected to ChIP with an anti-CHD8 antibody and the precipitated DNA was quantified by real-time PCR with primers specific for p53-responsive elements (p53REs) 1 or 2 or a control

region (CR) of the p21 promoter or for the p27 promoter. (b) U2OS cells overexpressing CHD8S were treated with ETOP (20 µM) and DXR (0.5 µM) and subjected to ChIP with an anti‑p53 antibody. Data are mean ± s.d., n = 3 (a, b). (c) U2OS cells overexpressing CHD8S were incubated with ETOP and then subjected to ChIP with an anti-CHD8 antibody. The immunoprecipitates were subjected to ChIP with an anti‑p53 antibody.

(Supplementary Information, Fig. S2b). CHD8 markedly suppressed apoptosis induced by etoposide, UV radiation or c‑Myc, all of which are dependent on p53, but did not affect apoptosis induced by cycloheximide or staurosporine, which are independent of p53 (Fig. 1d). Furthermore, apoptosis induced by overexpression of p53 was inhibited by CHD8S (Fig. 1e) or CHD8L (Supplementary Information, Fig. S3a). Conversely, depletion of both CHD8S and CHD8L by RNA interference (RNAi) induced cell death in U2OS human osteosarcoma cells (Fig. 1f), which harbour wild-type p53 alleles, as well as in HeLa and HCT116 cells (Supplementary Information, Fig. S3b–d). Depletion of CHD8L alone had no such effect. We confirmed that cell death triggered by depletion of CHD8 is due to apoptosis (Supplementary Information, Fig. S2c, d). Together, these observations indicate that both CHD8S and CHD8L possess anti-apoptotic activity and that the presence of CHD8S alone in cells is sufficient to prevent apoptosis, suggesting that the anti-apoptotic activity of CHD8 is dependent on the common region of CHD8S and CHD8L. Apoptosis induced by depletion of CHD8 was blocked by the caspase inhibitor Z‑VAD–fmk and was associated with retardation of cell growth (Supplementary Information, Fig. S2e, f). To investigate whether the apoptosis induced by CHD8 depletion was dependent on p53, we depleted U2OS cells of both CHD8 and p53 and found that additional depletion of p53 in cells depleted of CHD8 restored cell survival (Fig. 1f). Depletion of CHD8 resulted in a marked increase in apoptosis in p53+/+ MEFs, but had virtually no effect in p53–/– MEFs (Fig. 1g, h), indicating that CHD8 depletion results in p53 activation. These data thus indicate that CHD8 is an anti-apoptotic molecule and a negative regulator of p53.

CHD8 interacts with and inhibits p53 The subcellular localization of CHD8S was found to be almost identical to that of p53, with both proteins being largely restricted to the nucleus (Fig. 2a). Reciprocal co-immunoprecipitation analysis revealed that endogenous CHD8 specifically interacted with endogenous p53 in U2OS cells (Fig. 2b, c; Supplementary Information, Fig. S3e). Similar experiments with a series of deletion mutants of CHD8S revealed that the N-terminal region of CHD8 is responsible for binding to p53 (Supplementary Information, Fig. S4a). Reciprocal analysis showed that CHD8 binds to the central core domain of p53 (Supplementary Information, Fig. S4b). Furthermore, pulldown assays revealed that recombinant CHD8S and recombinant p53 bound to each other in vitro, suggesting that the interaction is direct (Fig. 2d). We next examined whether CHD8 affects transcriptional activation by p53 in U2OS cells. Overexpression of CHD8S or CHD8L markedly inhibited the etoposide- or doxorubicin-induced upregulation of Mdm2, p21 and Noxa, all of which are encoded by p53 target genes, in U2OS cells (Fig. 2e, f; Supplementary Information, Figs S3f, S5a–c). In contrast, depletion of CHD8 resulted in an increase in the expression of p53 target genes, including those for p21 and Noxa (Fig. 2g), suggesting that CHD8 antagonizes p53 function. Furthermore, overexpression of CHD8S inhibited in a concentration-dependent manner the p53induced increase in luciferase activity in human osteosarcoma SaOS2 cells (p53-null) harbouring a luciferase gene fused to the promoter of the p53 target genes for p21 or PG13 (Fig. 2h). CHD8S overexpression did not affect the activity of a p21 promoter in which the two p53 binding sites are mutated (Supplementary Information, Fig. S5d). The inhibitory effect of CHD8S was not mimicked by the CHD8SΔ53 mutant (Fig. 2h; Supplementary Information, Fig. S5d), which lacks


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Figure 4 CHD8 recruits histone H1 to the promoters of p53 target genes. (a) HEK293T cells expressing 3 × Flag–histone H1c were subjected to immunoprecipitation with anti-Flag and immunoblot analysis with antiCHD8 antibodies. (b) Immunoprecipitation of HEK293T lysates with antiCHD8 and immunoblot analysis with anti-histone H1 antibodies. (c) In vitro binding assay for recombinant CHD8S and histone H1c. (d) U2OS cells overexpressing CHD8S were treated with the genotoxic

agents etoposide (ETOP, 20 µM) and doxorubicin (DXR, 0.5 µM) and subjected to ChIP with an anti-histone H1 antibody. (e) ChIP was performed for the BAX promoter (n.d., not detected). (f) U2OS cells infected with a retroviral vector for CHD8S+L‑1 shRNA were subjected to ChIP. (g) U2OS cells overexpressing CHD8S were infected with a retroviral vector for p53 shRNA, incubated with ETOP and then subjected to ChIP. Data are mean ± s.d., n = 3 (d–g).

the p53-binding domain (Supplementary Information, Fig. S5e), suggesting that binding of CHD8 to p53 is required for suppression of p53 function. CHD8S did not affect the activity of the transcription factor NF‑κB induced by TRAF2 (Supplementary Information, Fig. S5f), suggesting that it is not a general inhibitor of transcription but rather, a specific inhibitor of p53-dependent transactivation. Together, these data indicate that CHD8 interacts with p53 and thereby negatively regulates its function.

CHD8 recruits histone H1 to inhibit p53 We next investigated the possible association of CHD8 and p53 with the promoter regions of p53 target genes using chromatin immunoprecipitation (ChIP; Supplementary Information, Fig. S6). ChIP using anti-CHD8 antibodies revealed that CHD8 specifically associated with p53-responsive elements 1 and 2 of the p21 promoter in U2OS cells treated with etoposide or doxorubicin but not in untreated cells (Fig. 3a). No substantial association of CHD8 with a control region of the p21

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Figure 5 Interaction of CHD8 with p53 and histone H1 is necessary for recruitment of histone H1 to the promoters of p53 target genes. (a) Schematic representation of CHD8S derivatives. (b–d) U2OS cells overexpressing CHD8S

derivatives were exposed to genotoxic stress (etoposide, ETOP, 20 µM and doxorubicin, DXR, 0.5 µM) and then subjected to ChIP (b), qRT–PCR (c) and trypan blue staining (d). Data are mean ± s.d., n = 3.

promoter that does not bind p53 or with the p27 promoter was detected. Moreover, depletion of p53 by RNAi reduced the extent of CHD8 association with the p53-responsive elements of the p21 promoter, suggesting that p53 mediates the association of CHD8 with chromatin. ChIP using anti‑p53 antibodies revealed that CHD8S overexpression did not affect the association of p53 with chromatin in response to genotoxic stress (Fig. 3b), suggesting that p53 binds to the p21 promoter in a CHD8independent manner. A sequential ChIP experiment revealed that p53 and CHD8 occupy the p21 promoter region simultaneously (Fig. 3c). To elucidate the mechanism by which CHD8 inhibits p53 function, we attempted to identify molecules that associate with Flag–CHD8S in HEK293T cells using a ‘shotgun’ proteomics approach. The results of several independent experiments revealed that histone H1 was consistently the most prominent of the proteins associated with immunoprecipitated Flag–CHD8S (Supplementary Information, Table S1). Reciprocal co-immunoprecipitation analysis showed that Flag-tagged histone H1c interacted with endogenous CHD8 (Fig. 4a). We also confirmed that endogenous CHD8 associated with endogenous histone H1 (Fig. 4b). Furthermore, pulldown assays revealed that recombinant CHD8S and recombinant histone H1c bound to each other in vitro, suggesting that the interaction is direct (Fig. 4c). We examined which histone H1 subtypes interact with CHD8 in a co-immunoprecipitation assay. This analysis revealed that all five major H1 subtypes (H1a, H1b, H1c, H1d, H1e) interacted with CHD8S (Supplementary Information, Fig. S4c). In vitro binding assays with a series of deletion mutants showed that residues 500–600 is the region of CHD8S required for binding to histone H1 (Supplementary Information, Fig. S4d). The histone H1-binding domain thus includes the nuclear localization signal of CHD8S and is distinct from the chromodomain at the C terminus. Given that CHD8 was found to interact with histone H1, we also examined whether histone H1 is recruited to the p53-responsive elements of the p21 promoter. ChIP using an anti–histone H1 antibody showed that histone

H1 was indeed recruited to the p21 promoter on treatment of U2OS cells with etoposide or doxorubicin, and this effect was markedly enhanced by overexpression of CHD8S (Fig. 4d). Similar results were obtained with the promoter of the p53 target gene BAX (Fig. 4e). Conversely, depletion of either CHD8 or p53 markedly reduced the amount of histone H1 associated with the p21 promoter (Fig. 4f, g). This series of ChIP experiments thus suggests that a p53–CHD8–histone H1 complex forms at the promoters of p53-inducible genes in response to genotoxic stress. p53–CHD8–histone H1 complex is necessary for suppression of p53 function We next examined the ability of CHD8 mutants that lack either the p53 binding domain (CHD8SΔ53) or the histone H1 binding domain (CHD8SΔH1) to recruit histone H1 to the promoters of p53 target genes (Fig. 5a). Given that the CHD8SΔH1 mutant lacks residues 500–600, which include the nuclear localization signal, we added the nuclear localization signal of the large T antigen of simian virus 40 to the C terminus of the mutant protein. Deletion of either of the two binding domains markedly impaired the ability of CHD8S to recruit histone H1 to the p53-responsive elements of the p21 promoter (Fig. 5b). Furthermore, neither CHD8SΔ53 nor CHD8SΔH1 had an inhibitory effect on p53-dependent transactivation (Fig. 5c) or apoptosis (Fig. 5d). Together, these data suggest that CHD8 recruits histone H1 to p53-responsive elements, and that such recruitment of histone H1 results in suppression of transcriptional activation by p53. Histone H1 is essential for repression of p53-dependent transcription Our observation that the CHD8SΔH1 mutant is unable to repress p53 function led us to investigate the requirement for histone H1 in such repression. To this end, we adopted three independent approaches.


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Figure 6 Requirement for histone H1 in repression of p53-mediated transcription. (a, b) Wild-type (WT) or HH1c–/–HH1d–/–HH1e–/– triple-knockout (TKO) ES cells were incubated with etoposide (ETOP, µ2 M) and doxorubicin (DXR, 0.05 µM) and then subjected to qRT–PCR (a) and trypan blue staining

(b). (c) U2OS cells were infected with a retroviral vector for histone H1 (HH1) shRNA and then subjected to immunoblot analysis. (d) The cells were incubated with ETOP (2 µM) and DXR (0.05 µM) and then subjected to qRT–PCR. Data are mean ± s.d., n = 3 (a, b, d).

First, CHD8-dependent suppression of p53 function was examined in HH1c–/–HH1d–/–HH1e–/– triple-knockout (TKO) embryonic stem (ES) cells23. Expression of p21 and Noxa genes (Fig. 6a), as well as the level of apoptosis (Fig. 6b) induced by genotoxic stress, were markedly increased in TKO cells, compared with those in control cells. Second, U2OS cells were subjected to RNAi with a short hairpin RNA (shRNA) designed to deplete all five major subtypes of histone H1 (Fig. 6c). Such depletion of histone H1 resulted in a substantial increase in etoposide- or doxorubicin-induced upregulation of p21 or Noxa mRNA levels (Fig. 6d). Third, expression of a dominant-negative mutant of histone H1 (N-fusion) reduced the amount of endogenous histone H1, as described previously24, and also increased that of p21, resulting in suppression of cell proliferation (Supplementary Information, Fig. S7). Expression of a histone H1 mutant that does not have dominant-negative activity (C-fusion) did not affect the abundance of p21 or cell proliferation. Together, these three lines of evidence support our conclusion that CHD8 negatively regulates p53 function by recruiting histone H1 to the promoters of p53 target genes, resulting in suppression of apoptosis triggered by p53.

of Chd8–/– mice may be attributable to unscheduled activation of p53. To test this hypothesis, we examined whether deletion of p53 ameliorated the abnormalities of Chd8–/– embryos. Most of the Chd8–/– mice died in utero between E5.5 and E7.5 and none survived beyond E8.5, whereas Chd8–/–p53–/– embryos survived until E10.5 (Supplementary Information, Table S2). Histopathological examination revealed that the growth retardation seen in Chd8–/– embryos was markedly less pronounced in Chd8–/–p53–/– and Chd8–/–p53+/– embryos at E7.5 and E8.5 (Fig. 7c). Cells with condensed nuclei were not seen in Chd8–/–p53–/– embryos at E8.5. The extent of recovery was greater in Chd8–/–p53–/– embryos than in Chd8–/–p53+/– embryos, suggesting that it was related to the reduction in the amount of p53. Mesoderm formation, a crucial event in early embryonic development that does not occur in Chd8–/– mice, was observed in Chd8–/–p53–/– mice (Fig. 7d). Chd8–/–p53–/– embryos died in utero at E10.5 and had severe haemorrhage, indicative of a defect in the cardiovascular system (M.N and K.I.N., unpublished data), suggesting that CHD8 also has another function (or functions) that is required for mid-stage embryonic development. We also observed growth of E3.5 embryos (blastocysts) in culture. Chd8–/– blastocysts degenerated between E5.5 and E7.5, whereas Chd8–/–p53–/– blastocysts, like wild-type controls, were alive at E9.5 (Fig. 7e). It is thus likely that the embryonic death of Chd8–/– mice is attributable to the unscheduled activation of p53-dependent apoptosis. To investigate the physiological role of CHD8 during early embryogenesis, we examined embryos at various stages. In ES cells (equivalent to E3.5 embryos), endogenous CHD8 was shown to interact with both endogenous p53 (Fig. 8a) and endogenous histone H1 (Fig. 8b). Furthermore,

Survival of Chd8–/–p53–/– embryos The abundance of CHD8 mRNA or protein in mouse embryos was greater during early embryogenesis than at later embryonic stages or in newborns (Fig. 7a, b). In addition to mouse embryos, all human and mouse cancer cell lines tested in this study were found to express CHD8 at relatively high levels (Supplementary Information, Fig. S1c, e, f). Given that CHD8 is a negative regulator of p53, we reasoned that the early embryonic death 178


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Figure 7 Deletion of p53 rescues the phenotype of CHD8-deficient mice. (a) qRT–PCR for CHD8S and CHD8L mRNAs in mouse embryos at the indicated stages. Data are mean ± s.d., n = 3. (b) Immunoblot analysis of CHD8S in mouse embryos as well as in U2OS cells and those overexpressing (OE) CHD8S. (c) Histopathological examination of

Chd8+/+p53+/+, Chd8–/–p53+/+, Chd8–/–p53+/– and Chd8–/–p53–/– embryos stained with haematoxylin and eosin. (d) Higher-magnification views of the boxed regions in c. Arrowheads indicate a layer of mesoderm. (e) In vitro culture of blastocysts of the indicated genotypes. Scale bars are 100 μm (c–e).

RNAi-mediated depletion of CHD8 promoted both apoptosis (Fig. 8c, d) and expression of the p53 target genes p21 and Noxa (Fig. 8e). Cultured Chd8–/– blastocysts (equivalent to E5.5 embryos) also showed higher levels of p21 and Noxa mRNAs than did wild-type blastocysts (Fig. 8f). In E7.5 embryos, TUNEL assay showed substantial apoptosis in Chd8–/– embryos, whereas no apoptosis was observed in wild-type or Chd8–/–p53–/– embryos (Fig. 8g). Furthermore, Mdm2 expression was upregulated in Chd8–/– embryos in a p53-dependent manner (Fig. 8h). This genetic evidence supports the notion that CHD8 is a physiological antagonist of p53 in vivo, and that loss of CHD8 allows unrestrained p53 activity, which induces apoptosis at the early stage of embryonic development. We therefore propose that the biological role of CHD8 is to suppress unwanted apoptosis during early embryogenesis (Fig. 8i).

influence chromatin structure, nucleosome mobility and gene regulation25–32. Histone H1 is not essential for growth or cell division in several unicellular eukaryotes33–36, but ablation of three of eight genes that encode isoforms of histone H1 in mouse ES cells, resulting in a 50% reduction in histone H1 content, led to embryonic death of the mutant mice23,37. Such downregulation of histone H1 elicited marked changes in chromatin structure, including a global reduction in nucleosome spacing, local reduction in chromatin compaction and changes in the modification of core histones. Despite these changes, microarray analysis of the mutant ES cells revealed that expression of only a few genes was altered23. These observations suggest that histone H1 participates in the regulation of specific genes that are essential for survival. In response to genotoxic stress, mammalian cells activate a complex network of proteins, a key element of which is p53. Post-translational modification of p53 results in transcriptional activation of its target genes1,6,7. Regulation of p53 by checkpoint signalling was originally thought to be mediated almost exclusively at the post-translational level1,6,8. More recently, p53 has also been found to be regulated at the transcriptional38 and translational39 levels. We have now uncovered another mode of p53 regulation mediated by CHD8-dependent recruitment of histone H1 to the promoters of its target genes. Cells proliferate extensively with short G1 and G2 phases during early embryogenesis. The DNA replication checkpoint may therefore be readily activated at this time, with the consequent risk of inducing p53-dependent apoptosis. We propose that the biological role of CHD8 is to suppress unwanted apoptosis during early embryogenesis (Fig. 8i). Consistent with this notion, we found that the amounts of CHD8 mRNA and protein are higher during the early and mid phases of mouse embryonic development

DISCUSSION We have shown that CHD8 negatively regulates p53 function by recruiting histone H1 to the promoters of p53 target genes. Formation of the p53–CHD8–histone H1 complex requires expression of CHD8 and stabilization of p53 by genotoxic stress. CHD8 is preferentially expressed in embryonic tissues and in cancer cell lines that are thought to reflect the embryonic state. Loss of CHD8 induced hyperactivation of p53, resulting in apoptosis, which was prevented by depletion of p53. The biochemical and genetic evidence provided by our study may explain how p53 function is regulated by histone H1. Histone modification is a fundamental mechanism for epigenetic control of gene expression. The role of core histones in gene regulation has been studied extensively, but that of the linker histone H1 has remained unclear. In vitro studies suggest that linker histone molecules nature cell biology volume 11 | number 2 | FEBRUARY 2009

© 2009 Macmillan Publishers Limited. All rights reserved.

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Figure 8 CHD8 sets a threshold for induction of apoptosis during early embryogenesis by counteracting p53 function. (a, b) Immunoprecipitation of ES cell lysates with an anti-CHD8 antibody, and immunoblot analysis with either anti‑p53 (a) or anti-histone H1 (b) antibodies. (c–e) ES cells infected with retroviral vectors for CHD8S+L‑1 or CHD8S+L‑2 shRNAs were subjected to trypan blue staining (c, quantification shown in d) and qRT–PCR (e). (f) Chd8+/+ or Chd8–/– blastocysts were subjected to qRT–PCR for p21 and Noxa. Data are mean ± s.d., n = 3 (d–f). (g, h) Sections of

Chd8+/+p53+/+, Chd8–/–p53+/+ and Chd8–/–p53–/– embryos were subjected to TUNEL assay (g) or to immunohistochemistry (h) with anti-Mdm2 antibody. Scale bars are 100 μm (c, g, h). (i) Model for the biological role of CHD8. When cells proliferate extensively during early embryogenesis, CHD8 is expressed at high levels and suppresses p53 function through histone H1 recruitment to prevent unwanted apoptosis. Once the level of CHD8 expression decreases, during mid to late embryogenesis, some cells undergo apoptosis for organogenesis.

than in newborns. Genetic ablation of CHD8 results in extensive p53dependent apoptosis in mouse embryos at a stage when Chd8 is expressed at high levels in wild-type mice. On the other hand, apoptosis is necessary for organogenesis, which occurs mainly during mid to late embryogenesis, when the level of Chd8 expression decreases. CHD8 may therefore regulate a threshold for apoptosis induction in a developmental-stage-specific manner: The threshold for apoptosis triggered by p53 is high during early embryogenesis, whereas it is lower after the mid-stage. In cancer cell lines, the level of CHD8 is relatively high, suggesting that cancer cells generally show an undifferentiated phenotype and reflect embryonic stages of development, or that cells expressing CHD8 may have a selective advantage in terms of cell growth or acquisition of immortality as a result of the suppressing p53 function. Thus we suggest that p53 function is, at least in

part, suppressed by CHD8 in such cancer cell lines. Furthermore, haploinsufficiency at the CHD8 locus was recently implicated in the pathogenesis of a human developmental anomaly40, providing further genetic evidence in support of a crucial role for CHD8 in development.

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METHODS Antibodies. Anti-p53 (FL393, Pab240), anti-Mdm2 (SMP14) and anti-His6 (H15) antibodies were obtained from Santa Cruz Biotechnology; anti-caspase‑3 cleaved at Asp‑175 and anti-PARP antibodies were from Cell Signalling; anti-histone H1 antibodies were from Abcam; anti-p21 antibodies from BD Pharmingen; antiHsp70 and anti-Hsp90 antibodies from Transduction Laboratories; anti-HA (HA11) antibodies from Babco; anti-Myc (9E10) antibodies from Roche; anti-Flag (M2) from Sigma; anti-α‑tubulin (TU01) antibodies from Zymed; anti- glyceraldehyde‑3-phosphate dehydrogenase (GAPDH; 1D4) from Stressgen; and anti-CHD8 antibodies were generated by A. Kikuchi (Hiroshima University). Alexa 488- or nature cell biology volume 11 | number 2 | FEBRUARY 2009


A RT I C L E S Alexa 546-conjugated goat antibodies to mouse or rabbit IgG were obtained from Molecular Probes. Antibodies were used at a dilution of 1:2,000. Plasmids. Complementary DNAs encoding wild-type or mutant forms of mouse CHD8S or human CHD8L, each tagged at its N terminus with Flag, Myc or HA epitopes, were subcloned into pcDNA3 (Invitrogen). Complementary DNAs encoding wild-type or mutant forms of human p53, each tagged at its N terminus with the HA epitope, were subcloned into pCGN. Complementary DNAs encoding mouse histone H1 variants, each tagged at its N terminus with three copies of the Flag epitope, were subcloned into pcDNA3. His6-tagged proteins were expressed in Escherichia coli strain BL21(DE3)pLys(S) (Novagen). Cell culture, transfection and infection. HEK293T, HeLa, HCT116, U2OS and SaOS2 cells were cultured in an atmosphere of 5% CO2 at 37°C in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). NIH 3T3 cells were cultured under the same conditions in DMEM supplemented with 10% bovine serum (Invitrogen). MEFs and ES cells were cultured as described previously41,42. HEK293T, HeLa and SaOS2 cells were transfected with vectors using FuGENE6 (Roche). For retroviral infection, cDNAs were subcloned into pMX-puro. Immunoprecipitation and immunoblot analysis. Cell lysis, immunoprecipitation and immunoblot analysis were performed as described previously43. For solubilization of chromatin-bound histone H1, cells were suspended in a solution containing 50 mM Tris-HCl (pH 7.5), 0.5% Triton X‑100, 300 mM NaCl, 60 mM MgCl2, 10 mM CaCl2 and DNase I (167 U ml–1, Roche) and then incubated at 30 °C for 50 min. Induction of apoptosis. Cells were incubated with etoposide (50 μM; Sigma) for 24 h, with cycloheximide (100 μg ml–1; Wako) for 24 h, with staurosporine (1 μM; Sigma) for 5 h or with doxorubicin (0.5 μM or the indicated concentrations; Sigma) for 24 h. Cells were exposed to UV radiation (50 J/m2) with an ultraviolet crosslinker UVC500 (Hoefer) and examined after 12 h. RNAi. The retroviral vector for expression of shRNAs was described previously43. The hairpin sequences specific for human or mouse CHD8 (CHD8S+L‑1, CHD8S+L‑2, CHD8L‑1, CHD8L‑2), for human p53, for human histone H1A (also effective for H1B, H1C, H1D and H1E, given the high similarity of the target region) and for enhanced green fluorescent protein (EGFP, Clontech) mRNAs corresponded to nucleotides 138–158 (CHD8S+L‑1), 814–834 (CHD8S+L‑2), 3808– 3828 (CHD8L‑1), 4413–4433 (CHD8L‑2), 775–793 (p53), 262–282 (histone H1A) and 126 –146 (EGFP) of the respective coding regions. The resulting vectors were used to transfect Plat E cells and thereby to generate recombinant retroviruses. Protein identification by LC–MS/MS analysis. CHD8S-associated proteins were digested with Achromobacter protease I and the resulting peptides were analysed with a nanoscale liquid chromatography–tandem mass spectrometry (LC–MS/ MS) system as described previously44. Reverse transcription–polymerase chain reaction (RT–PCR) and luciferase assays. RT–PCR analysis and luciferase assays were performed as described previously45. Purification of mRNA from cultured blastocysts was performed with a TurboCapture mRNA kit (Qiagen). The primer sequences for RT–PCR are listed in Supplementary Information, Table S3. For luciferase assays, SaOS2 cells were transfected using FuGENE6 with expression vectors encoding human p53 or Flag-tagged wild-type or mutant versions of mouse CHD8S together with pRLTk (Promega) as an internal control and luciferase reporter plasmids containing promoter sequences of PG13 or human p21 (wild-type or mutant) genes. The cells were collected 24 h after transfection, lysed and assayed for luciferase activity with a dual-luciferase reporter assay system (Promega). ChIP. ChIP assays were performed with a ChIP assay kit (Upstate Biotechnology) and 106 cells for each reaction. Precipitated DNA was quantified by real-time PCR as described previously45. The primer sequences are listed in Supplementary Information, Table S3. Generation of Chd8–/–p53–/– mice, histopathology and culture of pre-implantation embryos. Chd8+/– mice generated in our laboratory22 were crossed with

p53–/– mice (Taconic Biotechnology). All mice used in this study were backcrossed to the C57BL/6 background for more than six generations. Histopathological analysis, TUNEL assay, immunohistochemistry and culture of pre-implantation embryos were performed as described previously22. The primer sequences for genotyping of embryos by nested PCR are listed in Supplementary Information, Table S3. All animal experiments were performed in accordance with institutional guidelines. Note: Supplementary Information is available on the Nature Cell Biology website. Acknowledgements We thank T. Kitamura for pMX-puro; J. M. Cunningham and K. Hanada for the mCAT‑1 plasmid; S. Miyake for the PG13‑, p21‑, and p21 mutant–Luc plasmids; T. Takemori for the NF‑κB Luc plasmid; F. Ishikawa and R. Funayama for the N‑fusion and C‑fusion plasmids; M. Kitagawa for HCT116 and SaOS2 cells; M. Sato, Y. Yamada, T. Moroishi, Y. Katayama, N. Nishimura and K. Oyamada for technical assistance; M. Kimura and A. Ohta for help with preparation of the manuscript; and T. Ushijima, K. Hayashi and members of the authors’ laboratories for discussion. K.I.N. was supported by Takeda Science Foundation. Y.F. and A.I.S. were supported by NIH grant CA79057. Y.F. is a GCC Cancer Scholar supported by Georgia Cancer Coalition. Author contributions M.N. performed and planned all experiments, except some of ChIP and coimmunoprecipitation experiments, which were performed by K.O., Y.T. and T. Nakagawa; K.I.N. coordinated the study, oversaw the results and wrote the manuscript; S.I. and T. Natsume contributed to proteomic analysis; Y.F. and A.I.S. provided histone H1 triple-knockout cells and many suggestions; A.K. provided antibodies to CHD8. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Published online at http://www.nature.com/naturecellbiology/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. Vousden, K. H. & Lu, X. Live or let die: the cell’s response to p53. Nature Rev. Cancer 2, 594–604 (2002). 2. Harris, S. L. & Levine, A. J. The p53 pathway: positive and negative feedback loops. Oncogene 24, 2899–2908 (2005). 3. Vogelstein, B., Lane, D. & Levine, A. J. Surfing the p53 network. Nature 408, 307–310 (2000). 4. Laptenko, O. & Prives, C. Transcriptional regulation by p53: one protein, many pos‑ sibilities. Cell Death Differ. 13, 951–961 (2006). 5. Aylon, Y. & Oren, M. Living with p53, dying of p53. Cell 130, 597–600 (2007). 6. Toledo, F. & Wahl, G. M. Regulating the p53 pathway: in vitro hypotheses, in vivo veritas. Nature Rev. Cancer 6, 909–923 (2006). 7. Lavin, M. F. & Gueven, N. The complexity of p53 stabilization and activation. Cell Death Differ. 13, 941–950 (2006). 8. Zhang, Y. & Xiong, Y. A p53 amino-terminal nuclear export signal inhibited by DNA damage-induced phosphorylation. Science 292, 1910–1915 (2001). 9. Tanaka, T., Ohkubo, S., Tatsuno, I. & Prives, C. hCAS/CSE1L associates with chromatin and regulates expression of select p53 target genes. Cell 130, 638–650 (2007). 10. Das, S. et al. Hzf Determines cell survival upon genotoxic stress by modulating p53 transactivation. Cell 130, 624–637 (2007). 11. Lee, D. et al. SWI/SNF complex interacts with tumor suppressor p53 and is necessary for the activation of p53-mediated transcription. J. Biol. Chem. 277, 22330–22337 (2002). 12. Kim, K. et al. Isolation and characterization of a novel H1.2 complex that acts as a repressor of p53-mediated transcription. J. Biol. Chem. 283, 9113–9126 (2008). 13. Becker, P. B. Nucleosome remodelers on track. Nature Struct. Mol. Biol. 12, 732–733 (2005). 14. Marfella, C. G. & Imbalzano, A. N. The Chd family of chromatin remodelers. Mutat. Res. 618, 30–40 (2007). 15. Hall, J. A. & Georgel, P. T. CHD proteins: a diverse family with strong ties. Biochem. Cell Biol. 85, 463–476 (2007). 16. Pray-Grant, M. G., Daniel, J. A., Schieltz, D., Yates, J. R., 3rd & Grant, P. A. Chd1 chromodomain links histone H3 methylation with SAGA- and SLIK-dependent acetyla‑ tion. Nature 433, 434–438 (2005). 17. Zhang, L., Schroeder, S., Fong, N. & Bentley, D. L. Altered nucleosome occupancy and histone H3K4 methylation in response to ‘transcriptional stress’. EMBO J. 24, 2379–2390 (2005). 18. Flanagan, J. F. et al. Double chromodomains cooperate to recognize the methylated histone H3 tail. Nature 438, 1181–1185 (2005). 19. Lusser, A., Urwin, D. L. & Kadonaga, J. T. Distinct activities of CHD1 and ACF in ATPdependent chromatin assembly. Nature Struct. Mol. Biol. 12, 160–166 (2005).


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A RT I C L E S 20. Sakamoto, I. et al. A novel β‑catenin‑binding protein inhibits β‑catenin‑dependent Tcf activation and axis formation. J. Biol. Chem. 275, 32871–32878 (2000). 21. Ishihara, K., Oshimura, M. & Nakao, M. CTCF-dependent chromatin insulator is linked to epigenetic remodeling. Mol. Cell 23, 733–742 (2006). 22. Nishiyama, M. et al. Early embryonic death in mice lacking the β‑catenin‑binding protein Duplin. Mol. Cell. Biol. 24, 8386–8394 (2004). 23. Fan, Y. et al. Histone H1 depletion in mammals alters global chromatin structure but causes specific changes in gene regulation. Cell 123, 1199–1212 (2005). 24. Funayama, R., Saito, M., Tanobe, H. & Ishikawa, F. Loss of linker histone H1 in cellular senescence. J. Cell Biol. 175, 869–880 (2006). 25. Vignali, M. & Workman, J. L. Location and function of linker histones. Nature Struct. Biol. 5, 1025–1028 (1998). 26. Thomas, J. O. Histone H1: location and role. Curr. Opin. Cell Biol. 11, 312–317 (1999). 27. Lusser, A. & Kadonaga, J. T. Strategies for the reconstitution of chromatin. Nature Methods 1, 19–26 (2004). 28. Thoma, F., Koller, T. & Klug, A. Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin. J. Cell Biol. 83, 403–427 (1979). 29. Bednar, J. et al. Nucleosomes, linker DNA, and linker histone form a unique structural motif that directs the higher-order folding and compaction of chromatin. Proc. Natl Acad. Sci. USA 95, 14173–14178 (1998). 30. Pennings, S., Meersseman, G. & Bradbury, E. M. Linker histones H1 and H5 prevent the mobility of positioned nucleosomes. Proc. Natl Acad. Sci. USA 91, 10275–10279 (1994). 31. Shimamura, A., Sapp, M., Rodriguez-Campos, A. & Worcel, A. Histone H1 represses transcription from minichromosomes assembled in vitro. Mol. Cell. Biol. 9, 5573–5584 (1989). 32. Laybourn, P. J. & Kadonaga, J. T. Role of nucleosomal cores and histone H1 in regula‑ tion of transcription by RNA polymerase II. Science 254, 238–245 (1991).

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s u p p l e m e n ta r y i n f o r m at i o n

DOI: 10.1038/ncb1831

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tissues by quantitative RT-PCR analysis. Data are expressed relative to Figure S1 CHD8 has two splicing isoforms. (a) Organization of the mouse Chd8 gene. The region encompassing exons 8 to 10 (redNishiyama box) and et the al. corresponding Figure S1 amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and are means ± SD from three independent experiments. containing the alternative splicing site in exon 9 is expanded in the lower (e, f) Expression of Chd8 in mouse cancer cell lines and corresponding scheme. (b) Domain structure of proteins encoded by the two alternative normal cells. The amounts of CHD8S and CHD8L mRNAs in Neuro2a transcripts of Chd8. (c) Immunoblot analysis of lysates (80 µg of protein) neuroblastoma cells and normal neurons (e) as well as in Hepa-1 hepatoma of the indicated cell lines with anti-CHD8 and anti-Hsp70 (loading cells and normal hepatocytes (f) were determined by quantitative RT-PCR control). The positions of CHD8L (L) and CHD8S (S) are indicated. (d) analysis. Data are means ± SD from three independent experiments. Determination of the amounts of CHD8S and CHD8L mRNAs in mouse

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IB: PARP

82

IB: α-Tubulin

c

49

d

CHD8S+L-1 shRNA

Phase contrast

Control shRNA

kDa 19

IB: Cleaved caspase-3

104

TUNEL

PI fluorescence

10

Control shRNA 0.8

104

3.1

3

10

87.1

9.1 102

101

101

101

102

103

104

5.6

51.8

41.8

3

102

100 100

CHD8S+L-1 shRNA 0.8

100 100

101

102

103

104

Hoechst

FITC-Annexin V fluorescence

e 60


Cell number (105)

Cell death (%)

Control shRNA CHD8S+L-1 shRNA CHD8L-2 shRNA 20

50 40 30 20

15 10 5 0

10 0

f

Z-VAD (-)

Z-VAD (+)

0

24

Time (h)

48

72

Nishiyama et al. Figure S2 Figure S2 Inhibition of caspase-dependent apoptosis by CHD8. (a) NIH 3T3 cells stably infected with a retroviral vector for CHD8S or with the empty vector were exposed (or not) to etoposide and then stained with Hoechst 33258 for examination of nuclear morphology by fluorescence microscopy. Arrows indicate apoptotic cells with condensed or fragmented nuclei. Scale bar, 20 µm. (b) NIH 3T3 cells treated as in (a) were lysed and subjected to immunoblot analysis with antibodies to cleaved caspase-3, to PARP, or to α-tubulin (loading control). (c) HeLa cells were infected with retroviral vectors for CHD8S+L-1 or EGFP (control) shRNAs for 96 h and then either examined by phase-contrast microscopy, subjected to the terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end-labeling (TUNEL) assay, or stained with Hoechst 33258. Arrows indicate apoptotic cells with condensed

2

or fragmented nuclei. Scale bars, 100 µm (top panels) or 20 µm (middle and bottom panels). (d) HeLa cells infected as in (c) were stained with fluorescein isothiocyanate (FITC)–labeled Annexin V and propidium iodide (PI) and then analyzed by flow cytometry. (e) HeLa cells were infected with retroviral vectors for CHD8S+L-1 or EGFP (control) shRNAs for 48 h and then incubated in the absence or presence of 50 µM Z-VAD-fmk (Peptide Institute) for 20 h. The percentage of dead cells was determined by staining with trypan blue. Data are means ± SD from three independent experiments. (f) HeLa cells (1.5 × 105) infected with retroviral vectors for CHD8S+L-1, CHD8L-2, or EGFP (control) shRNAs were collected after 24 h and plated in 60-mm culture dishes. Cell number was determined with a hemocytometer after the indicated times. Data are means ± SD of triplicate cultures from a representative experiment.

www.nature.com/naturecellbiology © 2009 Macmillan Publishers Limited. All rights reserved.


b

30

10

8L -2

CHD

CHD

8S+

115 82

Hsp70 Vector

p53

d

50 40

U2OS

20

8L -2

8L -1

CHD

CHD

L -1

L -2

8S+

CHD

8S+

CHD

Con

trol

10

f -

-

+

IB: p53

64

Vector CHD8S CHD8L p53

12

p21


4

Figure S3 Both CHD8S and CHD8L inhibit p53 function. (a) U2OS cells were infected with retroviral vectors for CHD8L or p53 (or with the empty Nishiyama et vector) for 72 h. The percentage of dead cells was then determined by trypan blue staining. Data are means ± SD from three independent experiments. (b) HeLa cells were infected with retroviral vectors encoding CHD8S+L-1, CHD8S+L-2, CHD8L-1, CHD8L-2, or EGFP (control) shRNAs. After 96 h, the cells were subjected to immunoblot analysis with anti-CHD8 or antiHsp70. (c) HeLa cells infected with retroviral vectors encoding CHD8 or EGFP (control) shRNAs were collected after 72 h and stained with trypan blue for determination of the percentage of dead cells. Data are means ± SD from three independent experiments. (d) HeLa, U2OS, and HCT116

5 4 3 2 1 0

No tr

No tr

eatm

0

DXR

2

P

S

6

ETO

115

8

ent

IB: FLAG 180


10

L

Noxa

6

DXR

+

-

P

-

FLAG-CHD8L: kDa

ETO

FLAG-CHD8S:

ent

e

IP: FLAG

HCT116

CHD8S+L-1 shRNA

30

0

HeLa

Control shRNA

60

Cell death (%)

CHD

CHD8S

20

c

kDa

CHD8L

40

0

8S+

L -1

50

eatm

Cell death (%)

60

CHD

Co n

trol

70

8L -1

shRNA

Vector CHD8L

L -2

a

cells were infected with retroviral vectors for CHD8S+L-1 or EGFP (control) shRNAs for 72 h and then examined by phase-contrast microscopy. Scale al. Figure S3 bar, 100 µm. (e) HEK293T cells expressing FLAG-tagged CHD8S or CHD8L were incubated with 10 µM MG132 for 8 h and then subjected to immunoprecipitation with anti-FLAG. The resulting precipitates were subjected to immunoblot analysis with anti-p53 or anti-FLAG. (f) U2OS cells infected with retroviruses encoding CHD8S or CHD8L or with the empty vector were incubated in the absence or presence of etoposide (ETOP) or doxorubicin (DXR) for 24 h and then subjected to quantitative RT-PCR analysis of p21 and Noxa mRNAs. Data are means ± SD from three independent experiments.




+

200-751

-

+

Δ(267-483)

+

+

Δ(200-483)

+

+

1-266

-

-

646 706

267-751

267

1

97

Δ(98-292)

HA-p53:

FL Δ(267-483) 200-751

82

Δ(200-483) 267-751

IB: FLAG 64

+

+

64

FL

49

1-292 Δ(98-292) 98-292 CHD8S IP: FLAG

d CHD8S

p53BD

ChromoHH1 binding domain

HH1BD

500

200

600

00-4

00-6

0

Δ(4

1-60

-751

Δ(2

180

200

kDa

FL

His6-CHD8S:

His6-FLAG-HH1c:

00)

-

00)

+

Δ(500-600)

0

3FLAGHH1

Δ(400-500)

1-60

IB: FLAG 37

+

00-6

CHD8S

1-600

Δ(4

IB: CHD8 115

-

-751

3FLAGHH1

Δ(400-600)

00-4

IB: FLAG 37

+

Δ(2

CHD8S

+

+

+

+

+

+

+

+

+

+

115

IB: His6

200-751 HH1c

IB: His6

+

+

00) +

Δ(4

+

Δ(4 00-6

+

FL

00-6 Δ(4

180

FL

kDa

00-5 00) Δ(5 00-6 00)

IP: FLAG

00) Δ(4 00-5 00) Δ(5 00-6 00)

Input

His6-CHD8S:

FL Δ(200-400) 1-600 Δ(400-600)

49 37

His6-FLAG-HH1c:

+

400

200

IB: CHD8 115

+

751

646 706

Δ(200-400)

00)

e HH1

AG-

c

d HH1

AG-

3FL

1

200-751

3FL

b HH1

HH1

AG-

AG-

3FL

3FL

AG-

Vec to

FL

IP: FLAG

FL

IP: FLAG

3FL

r

HH1

a

c

Input

+

NLS Input

kDa

+

+

Input

484-751

49

+

IB: FLAG 115

p53

115

+

37

200 -7 5 1 Δ(2 67-4 83) Δ(2 00-4 83)

-7 5 1 484

267

FL

64

+

FLAG-CHD8S: kDa

-7 5 1

200 -7 5 1 Δ(2 67-4 83) Δ(2 00-4 83)

-7 5 1

484

267

FL

-7 5 1

IB: p53

393

98-292

IB: HA

FLAG-CHD8S: kDa

292

1-292

92 Δ(9 8-29 2)

-

484-751

751

2

+

600

C-terminal

9 8 -2

-

500

200

Central core

1 -2 9

+

1

FL

FL

+

FL

N-terminal

p53

92 Δ(9 8-29 2)

NLS

b

Nuclear localization

2

p53 binding

9 8 -2

Chromodomain

HH1BD

1 -2 9

p53BD

00)

CHD8S

FL

a

+

+

115

+

FL Δ(400-500) Δ(500-600) Δ(400-600)

49

HH1c

37

Input

IP: FLAG

cell lysates) were subjected to immunoblot analysis with anti-HA or antiFigure S4 Molecular dissection of p53-CHD8–histone H1 interaction. et al.FLAG. Figure S4five major variants of histone H1 interact with CHD8 in vivo. (c) All to p53. (a) Identification of the region of CHD8S responsible for bindingNishiyama HEK293T cells expressing mouse histones H1a, H1b, H1c, H1d, or H1e HEK293T cells transiently expressing the indicated FLAG-tagged CHD8S tagged with three copies of the FLAG epitope, or those transfected with the derivatives were subjected to immunoprecipitation with anti-FLAG, and the empty vector, were lysed and subjected to immunoprecipitation with antiresulting precipitates (as well as 3% of the input cell lysates) were subjected FLAG. The resulting precipitates, as well as 3% of the original cell lysates to immunoblot analysis with anti-p53 or anti-FLAG (lower panel). The results (input), were subjected to immunoblot analysis with anti-CHD8 or anti-FLAG. for p53 binding and nuclear localization [determined by immunofluorescence (d) Identification of the region of CHD8S responsible for binding to histone analysis (data not shown)] are summarized together with schematic H1. Recombinant His6-tagged CHD8S derivatives and His6- and FLAG-tagged representations of the CHD8S derivatives tested (upper panel). p53BD, p53 histone H1c (HH1c) were mixed and subjected to immunoprecipitation binding domain; NLS, nuclear localization signal; HH1BD, histone H1 binding with anti-FLAG, and the resulting precipitates as well as 10% of the original domain; FL, full-length. (b) Identification of the region of p53 responsible for binding mixtures (input) were subjected to immunoblot analysis with anti-His6 binding to CHD8S. HEK293T cells expressing the indicated HA-tagged p53 (right panels). The results of the binding assay are summarized together with derivatives and FLAG-tagged CHD8S were subjected to immunoprecipitation schematic representations of the CHD8S derivatives (left panel). with anti-FLAG, and the resulting precipitates (as well as 3% of the input

4


IB: CHD8

kDa 64

IB: p53

IB: CHD8

115

IB: Hsp90

P ETO

DX R

-

DX R

ETO

115

IB: Hsp90

82

82

Wild-type p21 Mutant p21 Relative luciferase activity

P ETO

DXR

-

DXR

P ETO

-

CHD8S

kDa 64

d Vector

CHD8S

IB: CHD8

82

U2OS cells

Vector

IB: p53

115

IB: Hsp90

c

kDa 64

IB: p53

HeLa cells

-

P ETO

-

DX R

CHD8S

DX R

Vector P

b ETO

NIH 3T3 cells

-

a

P


50 40 30 20 10 0

p53 (μg):

-

CHD8SΔ53 (μg):

-

-

-

-

0.5

-

0.5

-

0.5

0.5

-

-

-

0.5

HD8

G-C

FLA

G-C

f

FLAG-CHD8S CHD8S

FLAG-CHD8SΔ53 IB: FLAG 115

FLAG-CHD8S FLAG-CHD8SΔ53

IB: CHD8 115

FLAG-CHD8S

Input

6

CHD8S

NF-κB

5 4 3 2 1 0

TRAF2 (μg):

-

p53 (μg):

-

CHD8S (μg):

FLAG-CHD8SΔ53 IB: FLAG 115


IP: CHD8

IB: CHD8 115

FLA

kDa

Vec to

r

HD8

S

e

S Δ5

3

CHD8S (μg):

CYLD (μg):

-

-

-

-

0.5

-

-

0.5

-

-

-

0.5

-

-

0.5

0.5

-

-

-

-

-

-

-

-

0.5

0.5

-

FLAG-CHD8S FLAG-CHD8SΔ53

Figure S5 CHD8 is a specific inhibitor of p53-dependent transactivation. (a–c) Abundance of p53 and CHD8 in cells overexpressing CHD8 and et subjected to genotoxic stress. NIH 3T3 (a), HeLa (b), or U2OSNishiyama (c) cells infected with a retrovirus for CHD8S or with the empty vector were incubated in the absence (–) or presence of etoposide (ETOP) or doxorubicin (DXR) for 24 h. The cells were then subjected to immunoblot analysis with anti-p53, anti-CHD8, or anti-Hsp90. (d) SaOS2 cells were transfected with the indicated amounts of expression vectors for p53 and either wildtype or mutant (∆53) CHD8S together with a luciferase reporter plasmid containing wild-type or mutant forms of the p21 promoter, the latter of which harbored mutations in p53RE1 and p53RE2. The cells were then incubated for 24 h before assay of relative luciferase activity. Data are means ± SD of triplicates from a representative experiment. (e) HEK293T

cells transiently expressing FLAG-tagged wild-type or mutant (∆53) CHD8S were subjected to immunoprecipitation with anti-CHD8. The resulting al. Figure as S5well as 3% of the original cell lysates (input), were subjected precipitates, to immunoblot analysis with anti-CHD8 or anti-FLAG. (f) HEK293T cells were transfected with the indicated amounts of expression vectors for TRAF2, CHD8S, p53, and CYLD together with a luciferase reporter plasmid containing three binding sites for NF-κB. The cells were then incubated for 24 h before assay of relative luciferase activity. TRAF2 expression increased the luciferase activity derived from this construct, but this effect of TRAF2 was not inhibited by overexpression of CHD8S or p53. In contrast, CYLD, a deubiquitinating enzyme that is a negative regulator of the NF-κB signaling pathway, markedly inhibited the TRAF2 effect. Data are means ± SD of triplicates from a representative experiment.




3 p5

HH

IP

2.5

1

No

5

ti-C

10

An

20

rm al

HD

8

Input (%)

IgG

b

1

D8

CH

Inp ut No rm a

l Ig

G

a

IB: CHD8

IP

0.5

0.25

ti-H

1

An

2.5

No

5

rm

al

IgG

Input (%)

H1

c

IB: HH1

Input (%)

7.5

3

IgG al

rm

15

No

30

ti-p 53

60

IP

An

d

IB: p53

Figure S6 Antibody specificity and immunoprecipitation efficiency for ChIP. (a) U2OS cells infected with a retroviral vector encoding CHD8S were incubated with etoposide for 24 h, lysed, and subjected to ChIP with antiCHD8, anti–histone H1, or anti-p53 or with control IgG. The precipitated DNA (as well as 1% of the input cell lysates) was subjected to PCR analysis with primers specific for p53RE1 of the p21 promoter. (b–d) U2OS cells

treated as in (a) were subjected to ChIP with anti-CHD8 (b), anti–histone H1 (c), or anti-p53 (d). The resulting precipitates as well as the indicated percentages of the original cell lysates (input) were subjected to immunoblot analysis with the corresponding antibodies. By comparing the amounts of input and immunoprecipitated proteins, we determined the ChIP efficiency to be ~5% for CHD8 (b), 0.1% for histone H1 (c), and 15% for p53 (d).

Nishiyama et al. Figure S6

6



kDa Vector

C-fu

r

N-fu

Vec to

sion

b sion

a 64

IB: HA

37

IB: HH1

IB: Hsp90

N-fusion

26

IB: p21

C-fusion

82

c

Vector N-fusion C-fusion

Cell number (106)

6 5 4 3 2 1 0

0

1

2

3

Time (day)

4

5

Figure S7 Effects of expression of histone H1 mutants. (a) NIH 3T3 cells were infected with retroviral vectors encoding histone H1 tagged at its NH2-terminus (N-fusion) or COOH-terminus (C-fusion) with EGFP (or with the empty vector) for 96 h and were then subjected to immunoblot analysis with anti-HA (both fusion proteins were also tagged at their NH 2-termini with HA), anti–histone H1, anti-p21, or anti-Hsp90 (loading control).

6

(b) NIH 3T3 cells infected as in (a) were examined by phase-contrast microscopy. Scale bar, 100 µm. (c) NIH 3T3 cells (1.5 × 105) infected as in (a) were collected after 24 h and plated in 60-mm culture dishes. Cell number was determined with a hemocytometer after the indicated times. Data are means ± SD of triplicate cultures from a representative experiment.




s u p p l e m e n ta r y i n f o r m at i o n 1f

2b

2e

2d

4b

6c

7b

8a

8b 4c

Figure S8 Full scans of key immunoblots in the indicated figures.

8




Table S1. Identification of CHD8-associated proteins by proteomics analysis. Sequence Gene symbol

Protein name coverage (%)

HIST1H1E/HIST1H1C/HIST1H1D

Histone 1; H1e or H1c or H1d

23.5

Karyopherin alpha 2 (RAG cohort 1, KPNA2

22.3 importin alpha 1)

KPNA1

Karyopherin alpha 1 (importin alpha 5)

18.2

KPNA3


16.6

KPNB1

Karyopherin (importin) beta 1

14.4

KPNA4


11.7

HEK293T

cells

transiently

expressing

FLAG-tagged

CHD8S

were

subjected

to

immunoprecipitation with anti-FLAG, and the resulting precipitates were subjected to LC–MS/MS analysis. Proteins reproducibly detected in four independent experiments are listed. The percentage sequence coverage for each protein is also shown.

1


Table S2. Genotype of embryos from Chd8+/–p53+/– mouse intercrosses. E7.5 Chd8+/+ Chd8+/–

p53+/+ 4 7

p53+/– 9 14

p53–/– 3 5

Total 16 26

Chd8–/–

2

5

3

10

Total

13

28

11

52

E8.5

p53+/+

p53+/–

p53–/–

Total

+/+

Chd8 Chd8+/–

2 9

7 26

3 11

12 46

Chd8–/–

1

5

3

9

Total

12

38

17

67

E9.5 Chd8+/+ Chd8+/–

p53+/+ 5 10

p53+/– 16 36

p53–/– 8 10

Total 29 56

Chd8–/–

0

4

8

12

Total

15

56

26

97

Embryos from Chd8+/–p53+/– mouse intercrosses were dissected from uteri between E7.5 and E9.5 and genotyped by PCR.

2


Table S3. Primer sequences (5′→3′) for nested PCR, RT-PCR, and ChIP analyses. Gene (primer)

Forward primer

Reverse primer

Nested PCR mp53 (Mutant-1st)

GTGTTCCGGCTGTCAGCGCA

AGCGTCTCACGACCTCCGTC

mp53 (Wild type-1st)

ACACACCTGTAGCTCCAGCAC

AGCGTCTCACGACCTCCGTC

mp53 (Mutant-2nd)

CCCGGTTCTTTTTGTCAAGAC

ATGTGCTGTGACTTCTTGTAG

mp53 (Wild type-2nd)

TGGGGAGGCCAAAGTGGGAGG

ATGTGCTGTGACTTCTTGTAG

mChd8 (Mutant-1st)

TGCTAAAGCGCATGCTCCAGACTG

AACTCCGTAACCATTTGTCTATTC

mChd8 (Wild type-1st)

TATAGATTTCCTGTTTGATTTTCC

AACTCCGTAACCATTTGTCTATTC

mChd8 (Mutant-2nd)

ATGCTCCAGACTGCCTTGGGAAAA

GAAACAATGTAAAACAGGCAAATG

mChd8 (Wild type-2nd)

AAAGAATCACACTAGATCTAATCC

GAAACAATGTAAAACAGGCAAATG

RT-PCR mChd8S

CAGATGAGACACTTCTTTCATGAA

TTCTCCGCGCCCAACTCAC

mChd8L

CAGATGAGACACTTCTTTCATGAA

TTTTACCAGGTAGTAAATTACAGG

mp21

TGTCTTGCACTCTGGTGTCTGAGC

TCTTGCAGAAGACCAATCTGCG

hp21

CTGAGACTCTCAGGGTCGAA

CGGCGTTTGGAGTGGTAGAA

mNoxa

ACTCAGGAAGATCGGAGACAAAGTG

ACACTCGTCCTTCAAGTCTGCTGG

hNOXA

AGAGCTGGAAGTCGAGTGT

GCACCTTCACATTCCTCTC

mGapdh

GCCTGGAGAAACCTGCCAAGTATG

GAGTGGGAGTTGCTGTTGAAGTCG

hGAPDH

GCAAATTCCATGGCACCGT

TCGCCCCACTTGATTTTGG

ChIP hp21 (p53RE1)

CAGGCTGTGGCTCTGATTGG

TTCAGAGTAACAGGCTAAGG

hp21 (p53RE2)

GGTCTGCTACTGTGTCCTCC

CATCTGAACAGAAATCCCAC

hp21 (CR)

GGTGCTTCTGGGAGAGGTGAC

TGACCCACTCTGGCAGGCAAG

hBAX (p53RE)

TTGGAAGGCTGAGACGGGGTTATC

AGAAGTTTCGGGCAGGGTTTGAG

hBAX (CR)

CCTGCTGATCTATCAGCACAG

GCTGGTCTCTGAACTCCCAGA

hp27

CCGCCGCCGCAACCAATGGAT

GGAGTCGCAGAGCCGTGAGCA

Mouse and human genes are indicated by m and h, respectively.

3
