CHD8 exhibits nucleosome-stimulated ATPase activity. (A) SDS-PAGE of purified recombinant CHD8 (rCHD8). (B) rCHD8 or rSnf2H was incubated with [γ-³²P]ATP in the presence or absence of DNA or purified HeLa cell nucleosomes. The release of ³²P_i was measured by thin-layer chromatography, followed by exposure to a phosphorimager for analysis. (C) Mutation of lysine 842 of CHD8 to arginine (K842R) results in a loss of ATPase activity. rCHD8 or rCHD8(K842R) was assayed for nucleosome-stimulated ATPase activity as for panel B.

CHD8 is an ATP-dependent remodeling factor. (A) Recombinant CHD8 or CHD8(K842R) was assayed for increased restriction enzyme accessibility of mononucleosomes. (Inset) Representative data. The top band is the uncut template, and the bottom band is the resulting cut fragment. (B) Time course of restriction enzyme cleavage in the presence and absence of CHD8. (C) Recombinant CHD8 was assayed for nucleosome sliding activity with nucleosomes reconstituted on a DNA template without a nucleosome positioning sequence (lanes 3 to 6). Also included were mobility standards reconstituted on DNA templates with a centrally (Mid) or terminally (End) located nucleosome positioning sequence (lanes 1 and 2, respectively).

CHD8 is a component of a large complex in HeLa nuclear extracts. (A) HeLa nuclear extract was fractionated by phosphocellulose (P11) chromatography utilizing stepwise elution with the indicated KCl concentrations (0.1 M, 0.3 M, 0.5 M, 1.0 M). Western blot analysis was performed using affinity-purified anti-CHD8 antibodies. The 0.5 M P11 fraction was further fractionated by DEAE-Sephacel chromatography and eluted stepwise with 0.35 M KCl. Samples were further resolved by chromatography on a Superose 6 HR 10/30 column. Western blotting was performed using affinity-purified anti-CHD8 antibodies. Arrows (bottom) indicate the elution position of thyroglobulin (670 kDa) and the void volume of the column (∼2 MDa). (B) Immunoprecipitation of CHD8. HeLa nuclear extract was incubated with anti-CHD8 antibodies (α-CHD8) or normal rabbit IgG (α-Control) cross-linked to protein A agarose. After extensive washes to remove nonspecific interacting proteins, immunoprecipitates were subjected to SDS-PAGE, and proteins were visualized by Colloidal Blue staining. (C) 293 cells were transfected with Flag-WDR5 or the control vector. Immunoprecipitations (IP) were performed with anti-Flag M2 antibodies. After washing, purified samples were subjected to SDS-PAGE followed by Western blot analysis (WB) using the antibodies indicated. (D) Recombinant CHD8 was incubated with recombinant GST or GST-WDR5 as indicated at the top. After washing, glutathione-agarose-purified samples were subjected to SDS-PAGE. The top of the gel was subjected to Western blot analysis using anti-CHD8 antibodies. The bottom of the gel was analyzed by Coomassie staining.

CHD8 interacts with β-catenin in vivo and in vitro. (A) Recombinant GST-β-catenin fusion proteins (given above gels) were incubated with recombinant CHD8. After washing, purified samples were subjected to SDS-PAGE (left), followed by Western blot analysis (right). α-CHD8, anti-CHD8 antibody. (B) Diagram of GST-β-catenin fusion proteins used in panel A. (C) A nuclear extract was prepared from HeLa cells stably transfected with Flag-β-catenin^active or from the parental cell line. Immunoprecipitations (IP) were performed with anti-Flag-M2 antibodies. After washing, purified samples were subjected to SDS-PAGE followed by Western blot analysis using the antibodies given on the right.

CHD8 is bound to β-catenin-responsive promoters. (A) Chromatin from the HCT116 colorectal cell line was cross-linked in vivo with formaldehyde. Cells were lysed, and ChIPs were performed with the indicated antibodies. Immunoprecipitates were extensively washed, and the cross-linking was reversed. Bound DNA was detected by quantitative PCR with primer pairs to the 5′ or 3′ end of the gene (given below the graphs). MeH3K4, trimethyl-histone H3 Lys4; AcH4, acetyl-histone H4. (B) Mapping of CHD8 along the Axin2 gene. Bound DNA identical to that in panel A was detected by quantitative PCR with primer pairs to the indicated locations in the Axin2 gene (given below the graphs). Control IgG-precipitated samples in all experiments were less than 0.001% of input and therefore are not shown.

CHD8 regulates β-catenin target genes. (A) CHD8 shRNA in HCT116 cells. HCT116 cells were transfected with a control shRNA or a CHD8 shRNA. Following selection of the transfected cells, total RNA was harvested, and expression of the indicated genes was analyzed by real-time RT-PCR. Shown is a typical result from multiple experiments. (B) Western blot analysis following CHD8 shRNA transfection of HCT116 cells. Shown is a Western blot of cells transfected with a CHD8 shRNA or a control shRNA from panel A. α-CHD8, antibody against CHD8. Actin is blotted as a loading control. (C) kismet RNAi in Drosophila S2 cells. S2 cells were treated with control RNAi, axin RNAi, kismet RNAi, or both axin and kismet RNAi. Total RNA was harvested, and expression of nkd was analyzed by real-time RT-PCR. Shown is a typical result from multiple experiments. *, P < 0.05 by Student's t test; **, P < 0.001 by Student's t test.