Anti-apoptotic activity of CHD8. (a–c) NIH 3T3 cells overexpressing CHD8_S or CHD8_L were subjected to immunoblot (IB, a) analysis with anti-CHD8 and exposed to genotoxic stress. Cells were examined by phase-contrast 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 CHD8_S 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 < 0.01; n.s., not significant; P > 0.05; Student’s t-test). (e) U2OS cells were infected with retroviral vectors for CHD8_S 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 CHD8_S and CHD8_L (CHD8_S+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 CHD8_S+L-1 or CHD8_S+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).

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 CHD8_S 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 CHD8_S 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.

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 CHD8_S derivatives. (b–d) U2OS cells overexpressing CHD8_S 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.

Deletion of p53 rescues the phenotype of CHD8-deficient mice. (a) qRT–PCR for CHD8_S and CHD8_L mRNAs in mouse embryos at the indicated stages. Data are mean ± s.d., n = 3. (b) Immunoblot analysis of CHD8_S in mouse embryos as well as in U2OS cells and those overexpressing (OE) CHD8_S. (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).

CHD8 interacts with and inhibits transactivation by p53. (a) HeLa cells expressing Myc–CHD8_S 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 CHD8_S and p53. (e) U2OS cells overexpressing CHD8_S were incubated with doxorubicin (DXR) and then subjected to immunoblot analysis with the indicated antibodies. (f) U2OS cells overexpressing CHD8_S 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 CHD8_S+L-1 shRNA were subjected to qRT–PCR. (h) Luciferase assay using either wild-type or mutant CHD8_S^Δ53. Data are mean ± s.d., n = 3 (f–h).

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 anti-CHD8 antibodies. (b) Immunoprecipitation of HEK293T lysates with anti-CHD8 and immunoblot analysis with anti-histone H1 antibodies. (c) In vitro binding assay for recombinant CHD8_S and histone H1c. (d) U2OS cells overexpressing CHD8_S 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 CHD8_S+L-1 shRNA were subjected to ChIP. (g) U2OS cells overexpressing CHD8_S 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).

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).

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 CHD8_S+L-1 or CHD8_S+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.