Histone H1 Recruitment by CHD8 Is Essential for Suppression of the Wnt–β-Catenin Signaling Pathway

Masaaki Nishiyama; Arthur I Skoultchi; Keiichi I Nakayama

doi:10.1128/MCB.06409-11

. 2012 Jan;32(2):501–512. doi: 10.1128/MCB.06409-11

Histone H1 Recruitment by CHD8 Is Essential for Suppression of the Wnt–β-Catenin Signaling Pathway

Masaaki Nishiyama ^a,^b, Arthur I Skoultchi ^c, Keiichi I Nakayama ^a,^b,^✉

PMCID: PMC3255766 PMID: 22083958

Abstract

Members of the chromodomain helicase DNA-binding (CHD) family of proteins are thought to regulate gene expression. Among mammalian CHD proteins, CHD8 was originally isolated as a negative regulator of the Wnt–β-catenin signaling pathway that binds directly to β-catenin and suppresses its transactivation activity. The mechanism by which CHD8 inhibits β-catenin-dependent transcription has been unclear, however. Here we show that CHD8 promotes the association of β-catenin and histone H1, with formation of the trimeric complex on chromatin being required for inhibition of β-catenin-dependent transactivation. A CHD8 mutant that lacks the histone H1 binding domain did not show such inhibitory activity, indicating that histone H1 recruitment is essential for the inhibitory effect of CHD8. Furthermore, either depletion of histone H1 or expression of a dominant negative mutant of this protein resulted in enhancement of the response to Wnt signaling. These observations reveal a new mode of regulation of the Wnt signaling pathway by CHD8, which counteracts β-catenin function through recruitment of histone H1 to Wnt target genes. Given that CHD8 is expressed predominantly during embryogenesis, it may thus contribute to setting a threshold for responsiveness to Wnt signaling that operates in a development-dependent manner.

INTRODUCTION

The Wnt–β-catenin signaling pathway plays key roles in development, specification of cell fate, and adult stem cell proliferation (2, 21, 27, 44). Abnormal activation of this pathway is associated with various human cancers, including colorectal cancer and head and neck squamous cell carcinoma. Many genes associated with tumor growth, including those for c-Myc, matrix metalloproteinases, and cyclin D1, have been identified as Wnt target genes, with β-catenin functioning as a transcriptional coactivator at such genes (11, 16, 22, 35). In the absence of β-catenin binding, the promoters of Wnt target genes are occupied by Tcf/Lef family proteins, the corepressor Groucho (also known as TLE1), and histone deacetylase 1 (3, 5, 9, 34). Wnt signaling results in the accumulation of β-catenin in the cytosol and nucleus, and nuclear β-catenin displaces Groucho from Tcf by binding Tcf at the promoters of Wnt target genes. β-Catenin then recruits chromatin-remodeling complexes or other transcriptional coactivators to stimulate gene transcription (2, 12, 21, 27, 43, 44). Several members of the Snf2 superfamily of ATP-dependent chromatin-remodeling enzymes, including BRG1 (1), Snf2H, and p400 (39), were recently found to be recruited by β-catenin in the induction of target gene transcription. Although these findings suggest that ATP-dependent chromatin remodeling plays a fundamental role in the regulation of β-catenin-dependent transcription, it remains unclear how such enzymes contribute to the regulation of chromatin at Wnt target genes.

Members of the chromodomain helicase DNA-binding (CHD) family of proteins also belong to the Snf2 superfamily of ATP-dependent chromatin remodelers. Among the nine mammalian members of this family, CHD1 is thought to play an important role in gene transcription. The tandem chromodomains of human CHD1 thus specifically recognize and bind to the trimethylated form of lysine 4 of histone H3 (H3K4me3), a hallmark of actively transcribed chromatin (7), and mediate the recruitment of transcriptional initiation and pre-mRNA splicing factors (40). In mammals, CHD3 and CHD4 are subunits of nucleosome-remodeling and histone deacetylase (NURD) complexes, which contain histone deacetylases and function as transcriptional repressors (47). Mutations in CHD7 result in CHARGE syndrome, a multiple-malformation syndrome in humans for which more than 40 alleles have been defined (42). Chd7^+/− mice recapitulate several aspects of this human disease, including inner-ear vestibular dysfunction (13). Molecular studies suggest that CHD7 contributes to transcriptional activation of tissue-specific genes during differentiation (38). However, most of the biological functions mediated by members of the CHD family remain to be elucidated.

CHD8 exists in two isoforms, short (CHD8_S) and long (CHD8_L), that are generated as a result of alternative mRNA splicing. CHD8_S (also known as duplin) was originally isolated as a negative regulator of the Wnt–β-catenin signaling pathway (36) and binds directly to the Armadillo repeats of β-catenin (36, 41). The COOH-terminal region of full-length CHD8 (CHD8_L) interacts with the insulator-binding protein CTCF, with this interaction being important for insulator activity (14). CHD8 has also been implicated as a positive or negative transcriptional regulator of various genes (19, 32, 33, 45, 46). We previously showed that Chd8^−/− mice die early during embryogenesis, manifesting widespread apoptosis (28), whereas additional deletion of the tumor suppressor gene p53 ameliorated this developmental arrest (29). Both isoforms of CHD8 bind to p53 and suppress its transactivation activity by recruiting histone H1, with formation of a p53-CHD8-histone H1 trimeric complex on chromatin being required for inhibition of p53-dependent transactivation and apoptosis. These observations led us to examine whether such histone H1 recruitment by CHD8 might also contribute to the regulation of other genes such as Wnt target genes. We now show that CHD8 mediates the recruitment of histone H1 to Wnt target genes, resulting in suppression of the expression of these genes induced by activation of Wnt–β-catenin signaling. Our results thus suggest that CHD8 may target multiple transcriptional activators, including β-catenin and p53, for suppression of transcriptional activity through recruitment of histone H1.

MATERIALS AND METHODS

Antibodies and reagents.

Antibodies to β-catenin and to Hsp90 were obtained from BD Biosciences (San Jose, CA); those to FLAG (M2 or M5) were from Sigma (St. Louis, MO); those to the hemagglutinin (HA) epitope (HA.11) were from Covance (Princeton, NJ); those to Myc (9E10) were from Roche (Indianapolis, IN); those to glutathione S-transferase (GST) were from MBL (Nagoya, Japan); those to histone H1 were from Abcam (Cambridge, United Kingdom); and those to CHD8 were generated in rabbits by injection with a recombinant fragment of mouse CHD8 (residues 484 to 670). Recombinant human β-catenin was obtained from Millipore (Billerica, MA), and recombinant mouse Wnt3a was obtained from R&D Systems (Minneapolis, MN). Alexa Fluor 488-conjugated goat antibodies to mouse immunoglobulin G (IgG) were obtained from Molecular Probes-Invitrogen (Carlsbad, CA).

Plasmids.

Complementary DNAs encoding wild-type or mutant forms of mouse CHD8_S tagged with the FLAG or Myc epitope at the NH₂ terminus were subcloned into pcDNA3 (Invitrogen) with the use of the Gateway vector conversion system (Invitrogen); those encoding wild-type or mutant forms of mouse histone H1c with three copies of the FLAG epitope at the NH₂ terminus were also subcloned into pcDNA3. A cDNA for mouse β-catenin tagged at its NH₂ terminus with the HA epitope was subcloned into pCGN. His₆-tagged proteins were expressed in Escherichia coli strain BL21(DE3)pLys(S) (Merck, Darmstadt, Germany).

Cell culture, immunoprecipitation, and immunoblot analysis.

HEK293T and HeLa cells were cultured under an atmosphere of 5% CO₂ at 37°C in Dulbecco's modified Eagle's medium (DMEM) (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (Invitrogen). NIH 3T3 cells were cultured under the same conditions in DMEM supplemented with 10% bovine serum (Invitrogen). Embryonic stem (ES) cells were cultured as described previously (25, 26). In some experiments, HeLa or NIH 3T3 cells were cultured in the presence of LiCl (20 mM) or Wnt3a (20 ng/ml) for 4 h and ES cells were incubated with LiCl (2.5 mM) or Wnt3a (2.5 ng/ml) for 4 h. Cell lysis, immunoprecipitation, and immunoblot analysis were performed as described previously (15).

Retrovirus expression system.

Complementary DNAs encoding wild-type or mutant forms of mouse CHD8_S, human CHD8_L, or mouse histone H1c tagged with the FLAG epitope at the NH₂ terminus were subcloned into pMX-puro (kindly provided by T. Kitamura, University of Tokyo, Japan) with the use of the Gateway vector conversion system. The resulting vectors were used to transfect Plat E packaging cells and thereby to generate recombinant retroviruses (23). NIH 3T3 cells as well as HeLa cells stably expressing the mouse ecotropic retrovirus receptor (mCAT-1) were infected with retroviruses produced by Plat E cells and were then cultured in the presence of puromycin (Sigma) at 10 μg/ml.

RNA interference.

The pMX-puro II vector was constructed by deletion of the U3 portion of the 3′ long terminal repeat of pMX-puro. The mouse U6 gene promoter, followed by DNA corresponding to a short hairpin RNA (shRNA) sequence, was subcloned into the NotI and XhoI sites of pMX-puro II, yielding pMX-puro II-U6/siRNA. The DNA for the shRNA encoded a 21-nucleotide hairpin sequence specific to the mRNA target, with a loop sequence (-TTCAAGAGA-) separating the two complementary domains, and contained a tract of five T nucleotides to terminate transcription. The hairpin sequences specific for human β-catenin (β-catenin-1 and β-catenin-2), human CHD8, and enhanced green fluorescent protein (EGFP) mRNAs (Clontech, Mountain View, CA) corresponded to nucleotides 1774 to 1794 (β-catenin-1), 501 to 521 (β-catenin-2), 138 to 158 (CHD8), and 126 to 146 (EGFP) of the respective coding regions. The resulting vectors were used to transfect Plat E cells and thereby to generate recombinant retroviruses.

RT-PCR.

Total RNA (1 μg) isolated from HeLa, NIH 3T3, or ES cells with the use of Isogen (Nippon Gene, Tokyo, Japan) was subjected to reverse transcription (RT) with ReverTra Ace α (Toyobo, Osaka, Japan). The resulting cDNA was subjected to PCR with Power SYBR green PCR master mix in an ABI-Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). The relative amounts of each target mRNA were calculated as described previously (31). The primer sequences for RT-PCR analysis (sense and antisense, respectively) are 5′-CTGGCTTTGGTGAACTGTTG-3′ and 5′-AGTTGCTCACAGCCAAGACA-3′ for hAxin-2, 5′-ATCTGTATGTCCTGTCTGCCAGCG-3′ and 5′-TGTCCTGATTCCCAAGTCAAGCAC-3′ for mAxin-2, 5′-CTACCCTCTCAACGACAGCA-3′ and 5′-AGAGCAGAGAATCCGAGGAC-3′ for hc-Myc, 5′-TCCTGACGACGAGACCTTCATCAAG-3′ and 5′-TGAGAAACCGCTCCACATACAGTCC-3′ for mc-Myc, 5′-GCAAATTCCATGGCACCGT-3′ and 5′-TCGCCCCACTTGATTTTGG-3′ for hGAPDH, and 5′-GGACCCGAGAAGACCTCCTT-3′ and 5′-GCACATCACTCAGAATTTCAATGG-3′ for mRplp0. Human and mouse genes are indicated by h and m, respectively.

ChIP.

Chromatin immunoprecipitation (ChIP) assays were performed with a ChIP assay kit (Millipore) and 1 × 10⁶ cells for each reaction. Precipitated DNA was quantitated by real-time PCR analysis as described previously (31). The primer sequences for ChIP analysis (sense and antisense, respectively) are 5′-CTGGAGCCGGCTGCGCTTTGATAA-3′ and 5′-CGGCCCCGAAATCCATCGCTCTGA-3′ for hAxin-2 (Wnt-responsive element [WRE]), 5′-CTGGAGCCGGCTGCGCTTTGATAA-3′ and 5′-TGGCCCCGAAATCCATCGCGAACGG-3′ for mAxin-2 (WRE), 5′-CTGGCTTTGGTGAACTGTTG-3′ and 5′-AGTTGCTCACAGCCAAGACA-3′ for hAxin-2 (control region [CR]), 5′-ATCTGTATGTCCTGTCTGCCAGCG-3′ and 5′-TGTCCTGATTCCCAAGTCAAGCAC-3′ for mAxin-2 (CR), 5′-GCGGGTTACATACAGTGCACTTCA-3′ and 5′-TGGAAATGCGGTCATGCACAAA-3′ for hc-Myc (WRE), 5′-CTACCCTCTCAACGACAGCA-3′ and 5′-AGAGCAGAGAATCCGAGGAC-3′ for hc-Myc (CR), and 5′-CCGCCGCCGCAACCAATGGAT-3′ and 5′-GGAGTCGCAGAGCCGTGAGCA-3′ for hp27.

RESULTS

Both forms of CHD8 interact with β-catenin on chromatin in a Wnt signaling-dependent manner.

Forced expression of CHD8_S or CHD8_L in mammalian cells revealed that both isoforms associated with β-catenin (36, 41). We examined whether endogenous CHD8_S and CHD8_L also interact with endogenous β-catenin in HEK293T cells. Immunoblot analysis revealed that immunoprecipitates prepared with antibodies to β-catenin contained both CHD8_S and CHD8_L (Fig. 1A). In addition, reciprocal analysis also showed that endogenous β-catenin was present in immunoprecipitates prepared with antibodies to CHD8 (Fig. 1B and C). Both endogenous CHD8_S and CHD8_L thus specifically interacted with endogenous β-catenin in HEK293T cells, and such association was markedly increased in cells that were exposed to LiCl (Fig. 1B) or to Wnt3a (Fig. 1C) as activators of the Wnt–β-catenin signaling pathway. Given that CHD8 is localized in the nucleus (29), the binding between CHD8 and β-catenin may increase with the accumulation and nuclear translocation of β-catenin induced by activation of Wnt signaling.

Fig 1 — CHD8 interacts with and inhibits transactivation by β-catenin. (A) Whole-cell lysates of HEK293T cells were subjected to immunoprecipitation (IP) with mouse antibodies to β-catenin or with control mouse IgG. The resulting precipitates, as well as 1% of the original cell lysates (input), were subjected to immunoblot analysis (IB) with antibodies to CHD8 or to β-catenin. (B and C) HEK293T cells were incubated in the absence (−) or presence (+) of LiCl (20 mM) (B) or Wnt3a (20 ng/ml) (C) for 4 h and were then subjected to immunoprecipitation with rabbit antibodies to CHD8 or with control rabbit IgG. The resulting precipitates, as well as 1% of the original cell lysates (input), were subjected to immunoblot analysis with antibodies to β-catenin, to CHD8, or to Hsp90 (loading and negative control). (D and E) HeLa cells infected with retroviruses encoding FLAG-tagged forms of CHD8_S or CHD8_L (or with the empty retrovirus) were incubated in the absence (−) or presence of LiCl or Wnt3a for 4 h. The cells were then subjected to ChIP with antibodies to FLAG, and the precipitated DNA was quantitated by real-time PCR analysis with primers specific for Wnt-responsive elements (WRE) or a control region (CR) of the axin2 (D) or c-Myc (E) gene promoter or for the p27 gene promoter. The cells were also subjected to immunoblot analysis with antibodies to CHD8 or to Hsp90 (D). (F and G) HeLa cells infected with a retrovirus for CHD8_S or the empty vector were incubated with LiCl or Wnt3a for 4 h and were then subjected to RT and real-time PCR analysis of axin2 (F) and c-Myc (G) mRNAs. All quantitative data are means ± standard deviations (SD) from three independent experiments.

We next examined whether CHD8 might be recruited to the promoter regions of Wnt target genes with the use of ChIP. ChIP with antibodies to the FLAG epitope revealed that FLAG-tagged forms of both CHD8_S and CHD8_L were specifically associated with Wnt-responsive elements of the axin2 gene promoter in HeLa cells only when the cells were exposed to LiCl or Wnt3a (Fig. 1D). Similar results were obtained with the promoter of the c-Myc gene (Fig. 1E). These data thus suggested that both CHD8_S and CHD8_L are recruited to the promoters of Wnt target genes in a Wnt signaling-dependent manner and that CHD8 and β-catenin may interact with each other but are not located at such gene promoters in unstimulated cells.

We next examined whether CHD8 might affect transcriptional activation by β-catenin in cancer cells. Forced expression of CHD8_S in HeLa cells markedly inhibited the LiCl- or Wnt3a-induced upregulation of mRNAs derived from the Wnt target genes for axin2 (Fig. 1F) and c-Myc (Fig. 1G). Collectively, these results thus suggested that both CHD8_S and CHD8_L interact with β-catenin and inhibit its transactivation activity on chromatin.

We investigated whether the association of CHD8 with chromatin at Wnt target genes is dependent on β-catenin. ChIP with antibodies to β-catenin revealed that forced expression of CHD8_S did not substantially affect the association of β-catenin with chromatin in response to Wnt signaling (Fig. 2A). We also depleted HeLa cells of β-catenin with the use of two independent shRNAs targeting nucleotides 1774 to 1794 (shRNA-1) or nucleotides 501 to 521 (shRNA-2) of the coding region (Fig. 2B), with the decrease in the abundance of endogenous β-catenin being greater with shRNA-1 than with shRNA-2. ChIP with antibodies to CHD8 revealed that the amount of CHD8 associated with Wnt-responsive elements of the axin2 gene promoter was decreased in β-catenin-depleted cells stimulated with LiCl or Wnt3a and that the extent of this decrease was correlated with that in the total abundance of β-catenin (Fig. 2C). These results thus indicated that β-catenin mediates the association of CHD8 with Wnt-responsive elements of the axin2 gene promoter. Collectively, our observations thus suggested that β-catenin binds to the axin2 gene promoter in a CHD8-independent manner, whereas CHD8 association with this promoter is dependent on β-catenin. They are also consistent with the results of ChIP analysis showing that CHD8 was not localized to a substantial extent at Wnt target genes in unstimulated cells (Fig. 1D and E).

Fig 2 — β-Catenin mediates the association of CHD8 with chromatin. (A) HeLa cells stably overexpressing CHD8_S or infected with the empty vector were incubated in the absence or presence of LiCl (20 mM) or Wnt3a (20 ng/ml) for 4 h. The cells were then subjected to ChIP with antibodies to β-catenin, and the precipitated DNA was quantitated by real-time PCR analysis with primers specific for Wnt-responsive elements or a control region of the axin2 gene promoter or for the p27 gene promoter. (B) HeLa cells infected with retroviruses encoding shRNAs specific for β-catenin mRNA or for EGFP mRNA (control) were incubated in the absence or presence of LiCl or Wnt3a for 4 h, after which the cells were lysed and subjected to immunoblot analysis with antibodies to β-catenin, to CHD8, or to Hsp90 (loading control). (C) HeLa cells infected with retroviruses for β-catenin or EGFP (control) shRNAs were incubated in the absence or presence of LiCl or Wnt3a for 4 h and were then subjected to ChIP analysis with antibodies to CHD8 and with PCR primers as in panel A. All quantitative data are means ± SD from three independent experiments.

CHD8 recruits histone H1 to Wnt target genes.

We previously identified histone H1 as a molecule that associates with CHD8 with the use of a “shotgun” proteomics approach (29). Given that histone H1 was shown to be recruited to p53-responsive elements by CHD8 to suppress the transactivation activity of p53, we examined whether CHD8 also recruits histone H1 to Wnt-responsive elements and forms a β-catenin–CHD8–histone H1 trimeric complex. Pulldown assays in vitro with recombinant glutathione S-transferase (GST)-tagged β-catenin, HA-tagged CHD8_S, and FLAG-tagged histone H1 proteins that had been produced in bacteria revealed that histone H1 directly interacted with CHD8_S but not with β-catenin; histone H1 thus associated with β-catenin only in the presence of CHD8_S (Fig. 3A). We also examined the association of 3×FLAG-tagged histone H1 with HA-tagged β-catenin and with Myc epitope-tagged CHD8_S or CHD8_SΔH1, a CHD8_S mutant that lacks the histone H1 binding domain, in HEK293T cells. Coimmunoprecipitation analysis revealed that the interaction between histone H1 and β-catenin was markedly increased by forced expression of CHD8_S but not by that of CHD8_SΔH1 (Fig. 3B). Collectively, these results indicated that β-catenin directly interacts with CHD8 and that CHD8 directly interacts with histone H1, whereas β-catenin indirectly interacts with histone H1 through CHD8.

ChIP with antibodies to histone H1 showed that histone H1 was indeed recruited specifically to the axin2 gene promoter in HeLa cells in a manner dependent both on exposure of the cells to LiCl or Wnt3a and on forced expression of CHD8_S (Fig. 3C). Similar results were obtained with the Wnt-responsive elements of the c-Myc gene promoter (Fig. 3D). These results thus suggested that upregulation of CHD8 might antagonize transactivation of Wnt target genes by β-catenin through recruitment of histone H1.

We also depleted HeLa cells of both CHD8_S and CHD8_L with a specific shRNA (Fig. 3E) and then performed ChIP analysis with antibodies to histone H1 (Fig. 3F). The amount of histone H1 associated with the Wnt-responsive elements of the c-Myc gene promoter was specifically and markedly reduced in the cells depleted of CHD8. Together with the results obtained by CHD8 overexpression, these data indicated that CHD8 recruits histone H1 specifically to Wnt-responsive elements.

Histone H1 recruitment by CHD8 is necessary for suppression of β-catenin function.

Our previous study with a series of deletion mutants delineated the region of CHD8_S required for binding to histone H1 as that comprising residues 500 to 600 (29). Consistent with the previous finding that β-catenin interacts with rat CHD8 through residues 667 to 749 of the latter (36), which correspond to residues 669 to 751 of mouse CHD8, coimmunoprecipitation analysis in HEK293T cells revealed that β-catenin interacted with the mouse mutant protein CHD8_SΔH1 (which lacks residues 500 to 600) to a similar extent as it did with wild-type CHD8_S (Fig. 4A). We also performed ChIP analysis with antibodies to FLAG in HeLa cells expressing FLAG-tagged CHD8_S or CHD8_SΔH1. Both CHD8_S and CHD8_SΔH1 were recruited to similar extents to the Wnt-responsive elements of the axin2 (Fig. 4B) or c-Myc (Fig. 4C) gene promoters in a Wnt signaling-dependent manner.

Fig 4 — The CHD8_SΔH1 mutant binds to the promoters of Wnt target genes. (A) HEK293T cells expressing FLAG-tagged CHD8_S or CHD8_SΔH1 were subjected to immunoprecipitation with antibodies to FLAG. The resulting precipitates, as well as 3% of the original cell lysates (input), were subjected to immunoblot analysis with antibodies to β-catenin or to FLAG. (B and C) HeLa cells infected with retroviruses encoding FLAG-tagged forms of CHD8_S or CHD8_SΔH1 (or with the empty retrovirus) were incubated in the absence or presence of Wnt3a (20 ng/ml) for 4 h. The cells were then subjected to ChIP with antibodies to FLAG, and the precipitated DNA was quantitated by real-time PCR analysis with primers specific for Wnt-responsive elements or a control region of the axin2 (B) or c-Myc (C) gene promoter. All quantitative data are means ± SD from three independent experiments.

Whereas wild-type CHD8_S promoted the recruitment of histone H1 to the Wnt-responsive elements of the axin2 gene promoter, CHD8_SΔH1 failed to do so (Fig. 5A). Similar results were obtained for the c-Myc gene promoter (Fig. 5B). Furthermore, in contrast to wild-type CHD8_S, CHD8_SΔH1 did not substantially inhibit the upregulation of axin2 (Fig. 5C) or c-Myc (Fig. 5D) mRNAs induced by exposure of cells to LiCl or Wnt3a. These results suggested that the inability of CHD8_SΔH1 to suppress transcriptional activation by β-catenin is indeed attributable to the lack of histone H1 binding, rather than to an altered localization of the mutant CHD8 on chromatin. We therefore conclude that CHD8 recruits histone H1 to Wnt-responsive elements and that such recruitment of histone H1 results in suppression of transcriptional activation by β-catenin.

Fig 5 — Interaction of CHD8 with histone H1 is necessary for recruitment of histone H1 to the promoters of Wnt target genes. (A and B) HeLa cells stably expressing CHD8_S or CHD8_SΔH1 were incubated in the absence or presence of LiCl (20 mM) or Wnt3a (20 ng/ml) for 4 h and then subjected to ChIP assays as in Fig. 3C and D, respectively. (C and D) HeLa cells stably expressing CHD8_S or CHD8_SΔH1 were incubated in the absence or presence of LiCl or Wnt3a for 4 h, after which the abundance of axin2 (C) and c-Myc (D) mRNAs was determined by quantitative RT-PCR analysis. All quantitative data are means ± SD from three independent experiments.

The COOH-terminal domain of histone H1 plays a major role in determination of linker DNA conformation and chromatin condensation (10). We generated deletion mutants of histone H1 that lack the NH₂-terminal, central globular, or COOH-terminal domain (Fig. 6A) and tested these mutants for their ability to bind to CHD8 with the use of coimmunoprecipitation analysis (Fig. 6B). We found that CHD8 interacted with the COOH-terminal domain of histone H1, suggesting that the biological relevance of this domain relies, at least in part, on the interaction with CHD8. To examine the effect of deletion of the COOH-terminal domain of histone H1 on the suppression of Wnt-mediated transcriptional activation, we forcibly expressed wild-type histone H1 or the mutants lacking the NH₂-terminal or COOH-terminal domain in NIH 3T3 cells (Fig. 6C). The Wnt3a-induced increase in the abundance of axin2 mRNA was inhibited in cells expressing wild-type histone H1 and the mutant lacking the NH₂-terminal domain compared with that in control cells, whereas it was unaffected in cells expressing the mutant lacking the COOH-terminal domain. These results also suggested that the association of histone H1 with CHD8 is essential for the suppression of Wnt target genes.

Fig 6 — The COOH-terminal domain of histone H1 is necessary for repression of β-catenin-dependent transcription. (A) Schematic representation of full-length (FL) histone H1 (HH1) and its derivatives. The NH₂-terminal domain, central globular domain, and COOH-terminal domain are shown. (B) HEK293T cells expressing 3×FLAG-tagged histone H1 derivatives were subjected to immunoprecipitation with antibodies to FLAG. The resulting precipitates, as well as 3% of the original cell lysates (input), were subjected to immunoblot analysis with antibodies to CHD8 or to FLAG. (C) NIH 3T3 cells stably expressing HH1 derivatives were incubated in the absence or presence of Wnt3a (20 ng/ml) for 4 h, after which the abundance of axin2 mRNA was determined by quantitative RT-PCR analysis. Data are means ± SD from three independent experiments.

Histone H1 is essential for repression of β-catenin-dependent transcription.

Our observation that the CHD8_SΔH1 mutant is impaired in the ability to repress β-catenin function led us to investigate the requirement for histone H1 in such repression. To this end, we adopted two independent approaches. First, we examined the expression of Wnt target genes in HH1c^−/− HH1d^−/− HH1e^−/− triple-knockout (TKO) mouse ES cells. In these mutant ES cells, the abundance of histone H1 is reduced to ∼50% of that in wild-type ES cells, resulting in marked changes in chromatin structure; microarray analysis revealed that the expression of only a few genes is altered in the mutant cells, however (6). Nevertheless, expression of axin2 (Fig. 7A) and c-Myc (Fig. 7B) genes was markedly increased in the TKO cells by low concentrations of LiCl or Wnt3a that do not activate Wnt signaling in wild-type control cells. The poor responsiveness of the wild-type cells to stimulation was likely attributable to the inhibition of Wnt signaling by endogenous CHD8, the expression of which is much greater in ES cells than in any adult tissues examined (29). These results thus suggested that CHD8 regulates cellular sensitivity to Wnt–β-catenin signaling in early development. Second, we examined the effects both of a dominant negative mutant of histone H1 (EGFP-HH1) that reduces the amount of endogenous histone H1 and of a reciprocal mutant (HH1-EGFP) that does not exhibit dominant negative activity (Fig. 7C and D) (8). ChIP analysis revealed that both EGFP-HH1 and HH1-EGFP were equally recruited to the Wnt-responsive elements of the axin2 gene promoter (Fig. 7E). Expression of EGFP-HH1 potentiated the stimulatory effects of LiCl and Wnt3a on expression of the axin2 gene, whereas that of HH1-EGFP had no such effect (Fig. 7F). Together, these two approaches thus supported our conclusion that CHD8 negatively regulates the transactivation function of β-catenin by recruiting histone H1 to the promoters of Wnt target genes (Fig. 8). CHD8 is highly expressed in the early phase of embryonic development and in cancer cell lines, whereas its expression ceases during late embryogenesis and its abundance is generally low in normal adult tissues. CHD8 may therefore serve to set a threshold for responsiveness to the Wnt signaling pathway in a development-dependent manner.

Fig 7 — Requirement for histone H1 in repression of β-catenin-dependent transcription. (A and B) Wild-type (WT) or *HH1c*^−/− *HH1d*^−/− *HH1e*^−/− (HH1 TKO) ES cells were incubated with LiCl (2.5 mM) or Wnt3a (2.5 ng/ml) for 4 h and then subjected to quantitative RT-PCR analysis of axin2 (A) and c-Myc (B) mRNAs. (C) Schematic representation of histone H1 (HH1) tagged at its NH₂ terminus (EGFP-HH1) or COOH terminus (HH1-EGFP) with EGFP. Both fusion proteins were also tagged at their NH₂ termini with HA. (D) NIH 3T3 cells infected with retroviruses encoding EGFP-HH1 or HH1-EGFP (or with the empty vector) were then subjected to immunoblot analysis with antibodies to HA, to histone H1, or to Hsp90. (E) NIH 3T3 cells infected as in panel D were incubated in the absence or presence of Wnt3a (20 ng/ml) for 4 h. The cells were then subjected to ChIP with antibodies to HA, and the precipitated DNA was quantitated by real-time PCR analysis with primers specific for Wnt-responsive elements or a control region of the axin2 gene promoter. (F) NIH 3T3 cells infected as in panel D were incubated with LiCl (20 mM) or Wnt3a (20 ng/ml) for 4 h and then subjected to quantitative RT-PCR analysis of axin2 mRNA. All quantitative data are means ± SD from three independent experiments. ∗∗, P < 0.01 versus the corresponding value for cells infected with the empty virus (unpaired Student's t test).

Fig 8 — Model for CHD8 function. CHD8 interacts with β-catenin and subsequently facilitates recruitment of histone H1, resulting in chromatin compaction and transcriptional silencing.

DISCUSSION

With the use of biochemical and genetic approaches, we have shown that CHD8 negatively regulates β-catenin function by recruiting histone H1 to the promoters of Wnt target genes. Formation of the β-catenin–CHD8–histone H1 complex requires two conditions: expression of CHD8 and stabilization of β-catenin by activation of Wnt signaling. CHD8 is preferentially expressed in embryonic tissues and in cancer cell lines, which are thought to reflect the embryonic state.

CHD8_S, also known as duplin, was originally isolated as a negative regulator of the Wnt–β-catenin signaling pathway (36). Recent studies have indicated that CHD8_L interacts directly with β-catenin and negatively regulates β-catenin-dependent gene expression through the ATP-dependent modulation of chromatin structure (41). On the other hand, the COOH-terminal region of CHD8_L interacts with the insulator-binding protein CTCF, with this interaction being important for insulator activity (14). However, our present study suggests that not only CHD8_L but also CHD8_S is able to inhibit Wnt- and β-catenin-dependent transactivation. Given that CHD8_S contains the chromodomain but lacks the Snf2 helicase domain and the CTCF binding domain of CHD8_L, neither the ATP-dependent remodeling activity mediated by the Snf2 helicase domain nor CTCF binding through the COOH-terminal region of CHD8_L is necessary for this suppressive effect of CHD8 on the Wnt–β-catenin signaling pathway. In contrast, this suppressive activity requires the region present in both CHD8_S and CHD8_L that mediates binding to histone H1. A CHD8_S mutant that lacks the histone H1 binding domain thus did not exhibit this inhibitory activity, and either depletion of histone H1 or expression of a dominant negative mutant thereof increased the cellular sensitivity to Wnt signaling. Our results thus indicate that histone H1 recruitment mediated by CHD8 is fundamental to negative regulation of the Wnt–β-catenin signaling pathway.

Histone H1 molecules are highly mobile, and the interaction of a specific H1 molecule with a specific nucleosome is transient (18, 20), suggesting that the molecules are continuously exchanged among chromatin binding sites according to a “stop-and-go” process in which a histone H1 molecule remains at a binding site for a limited time before dissociating and moving rapidly to another such site. Most chromatin fibers thus likely always contain histone H1, but there is a continuous turnover of histone H1 molecules at the level of the individual nucleosome. Given that histone H1 is implicated in regulation of the expression of specific genes, we postulated the existence of a factor such as CHD8 that extends the residence time of histone H1 at the corresponding specific chromatin sites. It is also possible that CHD8 alters the interaction of histone H1 with linker DNA and represses transcription by enhancing the intrinsic stability of nucleosomes. The detailed mechanisms of transcriptional suppression for specific genes remain to be elucidated.

The identification of various proteins that associate with β-catenin has provided insight into its function as a transcriptional regulator. Many of these proteins, including components of histone acetylation and methylation complexes as well as chromatin-binding proteins, contribute to regulation of chromatin structure or of RNA polymerase II. Several transcriptional complexes or coactivators, including Bcl-9 (also known as Lgs), Pygopus (Pygo), polymerase-associated factor 1 (Paf1), and SET1 (trithorax), have been found to be recruited by β-catenin for target gene regulation (17, 24, 39). At active target genes, Pygo binds through its PHD finger to methylated H3K4 and, by using Bcl-9 as an adaptor, retains β-catenin near Wnt-responsive elements independently of Tcf (37). Pygo-dependent binding of β-catenin to methylated histones frees Tcf to recruit its corepressors, such as Groucho, that counteract the activating β-catenin-induced chromatin-remodeling processes. We have now uncovered another mode of β-catenin regulation mediated by the CHD8-dependent recruitment of histone H1 to the promoters of Wnt target genes.

We previously showed that apoptosis mediated by p53-dependent transactivation is also suppressed by histone H1 recruited by CHD8 (29). Loss of CHD8 induced hyperactivation of p53, resulting in apoptosis, which was prevented by the additional depletion of p53. Mice deficient in CHD8 die during early embryogenesis (around embryonic day 7.5) as a result of aberrant apoptosis induced by the unscheduled activation of p53 (28, 29). It is likely that such apoptosis dominates and masks possible phenotypes induced by hyperactivation of Wnt signaling. Additional deletion of p53 in CHD8-deficient mice ameliorated the developmental arrest, resulting in extension of survival until embryonic day 10.5. Death of the Chd8^−/− p53^−/− mice is associated with severe hemorrhage, probably as a result of a defect in cardiovascular development (M. Nishiyama and K. I. Nakayama, unpublished observations). Given that mice homozygous for a mutant Apc allele encoding a product truncated at position 716 (Apc^Δ⁷¹⁶) die in utero as a result of the unscheduled activation of Wnt signaling (30) and that Wnt signaling plays an essential role in cardiovascular development during embryogenesis (4), the embryonic death of Chd8^−/− p53^−/− mice may be attributable to a defect in the cardiovascular system resulting from unscheduled activation of Wnt signaling.

The mode of action of CHD8 in antagonism of Wnt–β-catenin signaling appears almost identical to that for p53 inhibition. The histone H1 binding domain that is present in both CHD8_S and CHD8_L is thus indispensable for CHD8-mediated inhibition of transactivation by p53 or β-catenin. Furthermore, genetic evidence from cells depleted of histone H1 also supports this similarity. Given that CHD8 is preferentially expressed in embryonic tissues and in cancer cell lines, which are thought to reflect the embryonic state, CHD8 may counteract p53 and Wnt–β-catenin function through recruitment of histone H1 during early embryogenesis to set a threshold for induction of apoptosis, cell proliferation, and axis formation. It is also possible that CHD8 may bind to transcriptional factors other than p53 and β-catenin and act as a more general inhibitor of transactivation activity through recruitment of histone H1.

ACKNOWLEDGMENTS

We thank T. Kitamura for pMX-puro; J. M. Cunningham and K. Hanada for the mCAT-1 plasmid; F. Ishikawa and R. Funayama for plasmids encoding the EGFP-HH1 and HH1-EGFP mutants of histone H1; A. Kikuchi for Wnt3a; M. Sato, H. Takeda, C. Mitai, N. Nishimura, and K. Oyamada for technical assistance; A. Ohta for help in preparation of the manuscript; and members of our laboratories for discussion.

A.I.S. was supported by NIH grant CA79057.

Footnotes

Published ahead of print 14 November 2011

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34. Roose J, et al. 1998. The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors. Nature 395: 608–612 [DOI] [PubMed] [Google Scholar]
35. Rubinfeld B, et al. 1997. Stabilization of β-catenin by genetic defects in melanoma cell lines. Science 275: 1790–1792 [DOI] [PubMed] [Google Scholar]
36. Sakamoto I, et al. 2000. A novel β-catenin-binding protein inhibits beta-catenin-dependent Tcf activation and axis formation. J. Biol. Chem. 275: 32871–32878 [DOI] [PubMed] [Google Scholar]
37. Sampietro J, et al. 2006. Crystal structure of a β-catenin/BCL9/Tcf4 complex. Mol. Cell 24: 293–300 [DOI] [PubMed] [Google Scholar]
38. Schnetz MP, et al. 2009. Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns. Genome Res. 19: 590–601 [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Sierra J, Yoshida T, Joazeiro CA, Jones KA. 2006. The APC tumor suppressor counteracts β-catenin activation and H3K4 methylation at Wnt target genes. Genes Dev. 20: 586–600 [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Sims RJ, 3rd, et al. 2007. Recognition of trimethylated histone H3 lysine 4 facilitates the recruitment of transcription postinitiation factors and pre-mRNA splicing. Mol. Cell 28: 665–676 [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Thompson BA, Tremblay V, Lin G, Bochar DA. 2008. CHD8 is an ATP-dependent chromatin remodeling factor that regulates β-catenin target genes. Mol. Cell. Biol. 28: 3894–3904 [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Vissers LE, et al. 2004. Mutations in a new member of the chromodomain gene family cause CHARGE syndrome. Nat. Genet. 36: 955–957 [DOI] [PubMed] [Google Scholar]
43. Willert K, et al. 2003. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423: 448–452 [DOI] [PubMed] [Google Scholar]
44. Willert K, Jones KA. 2006. Wnt signaling: is the party in the nucleus? Genes Dev. 20: 1394–1404 [DOI] [PubMed] [Google Scholar]
45. Yates JA, Menon T, Thompson BA, Bochar DA. 2010. Regulation of HOXA2 gene expression by the ATP-dependent chromatin remodeling enzyme CHD8. FEBS Lett. 584: 689–693 [DOI] [PubMed] [Google Scholar]
46. Yuan CC, et al. 2007. CHD8 associates with human Staf and contributes to efficient U6 RNA polymerase III transcription. Mol. Cell. Biol. 27: 8729–8738 [DOI] [PMC free article] [PubMed] [Google Scholar]
47. Zhang Y, LeRoy G, Seelig HP, Lane WS, Reinberg D. 1998. The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities. Cell 95: 279–289 [DOI] [PubMed] [Google Scholar]

[B1] 1. Barker N, et al. 2001. The chromatin remodelling factor Brg-1 interacts with beta-catenin to promote target gene activation. EMBO J. 20: 4935–4943 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2. Bienz M, Clevers H. 2000. Linking colorectal cancer to Wnt signaling. Cell 103: 311–320 [DOI] [PubMed] [Google Scholar]

[B3] 3. Billin AN, Thirlwell H, Ayer DE. 2000. β-Catenin-histone deacetylase interactions regulate the transition of LEF1 from a transcriptional repressor to an activator. Mol. Cell. Biol. 20: 6882–6890 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4. Cohen ED, Tian Y, Morrisey EE. 2008. Wnt signaling: an essential regulator of cardiovascular differentiation, morphogenesis and progenitor self-renewal. Development 135: 789–798 [DOI] [PubMed] [Google Scholar]

[B5] 5. Daniels DL, Weis WI. 2005. Beta-catenin directly displaces Groucho/TLE repressors from Tcf/Lef in Wnt-mediated transcription activation. Nat. Struct. Mol. Biol. 12: 364–371 [DOI] [PubMed] [Google Scholar]

[B6] 6. Fan Y, et al. 2005. Histone H1 depletion in mammals alters global chromatin structure but causes specific changes in gene regulation. Cell 123: 1199–1212 [DOI] [PubMed] [Google Scholar]

[B7] 7. Flanagan JF, et al. 2005. Double chromodomains cooperate to recognize the methylated histone H3 tail. Nature 438: 1181–1185 [DOI] [PubMed] [Google Scholar]

[B8] 8. Funayama R, Saito M, Tanobe H, Ishikawa F. 2006. Loss of linker histone H1 in cellular senescence. J. Cell Biol. 175: 869–880 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9. Graham TA, Weaver C, Mao F, Kimelman D, Xu W. 2000. Crystal structure of a β-catenin/Tcf complex. Cell 103: 885–896 [DOI] [PubMed] [Google Scholar]

[B10] 10. Happel N, Doenecke D. 2009. Histone H1 and its isoforms: contribution to chromatin structure and function. Gene 431: 1–12 [DOI] [PubMed] [Google Scholar]

[B11] 11. He TC, et al. 1998. Identification of c-MYC as a target of the APC pathway. Science 281: 1509–1512 [DOI] [PubMed] [Google Scholar]

[B12] 12. He X, Semenov M, Tamai K, Zeng X. 2004. LDL receptor-related proteins 5 and 6 in Wnt/β-catenin signaling: arrows point the way. Development 131: 1663–1677 [DOI] [PubMed] [Google Scholar]

[B13] 13. Hurd EA, et al. 2007. Loss of Chd7 function in gene-trapped reporter mice is embryonic lethal and associated with severe defects in multiple developing tissues. Mamm. Genome 18: 94–104 [DOI] [PubMed] [Google Scholar]

[B14] 14. Ishihara K, Oshimura M, Nakao M. 2006. CTCF-dependent chromatin insulator is linked to epigenetic remodeling. Mol. Cell 23: 733–742 [DOI] [PubMed] [Google Scholar]

[B15] 15. Kamura T, et al. 2004. VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases. Genes Dev. 18: 3055–3065 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16. Korinek V, et al. 1997. Constitutive transcriptional activation by a β-catenin-Tcf complex in APC^−/− colon carcinoma. Science 275: 1784–1787 [DOI] [PubMed] [Google Scholar]

[B17] 17. Kramps T, et al. 2002. Wnt/wingless signaling requires BCL9/legless-mediated recruitment of pygopus to the nuclear β-catenin-TCF complex. Cell 109: 47–60 [DOI] [PubMed] [Google Scholar]

[B18] 18. Lever MA, Th'ng JP, Sun X, Hendzel MJ. 2000. Rapid exchange of histone H1.1 on chromatin in living human cells. Nature 408: 873–876 [DOI] [PubMed] [Google Scholar]

[B19] 19. Menon T, Yates JA, Bochar DA. 2010. Regulation of androgen-responsive transcription by the chromatin remodeling factor CHD8. Mol. Endocrinol. 24: 1165–1174 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20. Misteli T, Gunjan A, Hock R, Bustin M, Brown DT. 2000. Dynamic binding of histone H1 to chromatin in living cells. Nature 408: 877–881 [DOI] [PubMed] [Google Scholar]

[B21] 21. Moon RT, Kohn AD, De Ferrari GV, Kaykas A. 2004. WNT and β-catenin signalling: diseases and therapies. Nat. Rev. Genet. 5: 691–701 [DOI] [PubMed] [Google Scholar]

[B22] 22. Morin PJ, et al. 1997. Activation of β-catenin-Tcf signaling in colon cancer by mutations in β-catenin or APC. Science 275: 1787–1790 [DOI] [PubMed] [Google Scholar]

[B23] 23. Morita S, Kojima T, Kitamura T. 2000. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7: 1063–1066 [DOI] [PubMed] [Google Scholar]

[B24] 24. Mosimann C, Hausmann G, Basler K. 2006. Parafibromin/Hyrax activates Wnt/Wg target gene transcription by direct association with β-catenin/Armadillo. Cell 125: 327–341 [DOI] [PubMed] [Google Scholar]

[B25] 25. Nakayama K, et al. 1996. Mice lacking p27^Kip1 display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 85: 707–720 [DOI] [PubMed] [Google Scholar]

[B26] 26. Nakayama K, et al. 2000. Targeted disruption of Skp2 results in accumulation of cyclin E and p27^Kip1, polyploidy and centrosome overduplication. EMBO J. 19: 2069–2081 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27. Nelson WJ, Nusse R. 2004. Convergence of Wnt, β-catenin, and cadherin pathways. Science 303: 1483–1487 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28. Nishiyama M, et al. 2004. Early embryonic death in mice lacking the β-catenin-binding protein Duplin. Mol. Cell. Biol. 24: 8386–8394 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29. Nishiyama M, et al. 2009. CHD8 suppresses p53-mediated apoptosis through histone H1 recruitment during early embryogenesis. Nat. Cell Biol. 11: 172–182 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30. Oshima M, et al. 1995. Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene. Proc. Natl. Acad. Sci. U. S. A. 92: 4482–4486 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31. Pfaffl MW. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29: e45. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32. Rodenberg JM, et al. 2010. Regulation of serum response factor activity and smooth muscle cell apoptosis by chromodomain helicase DNA-binding protein 8. Am. J. Physiol. Cell Physiol. 299: C1058–1067 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33. Rodriguez-Paredes M, Ceballos-Chavez M, Esteller M, Garcia-Dominguez M, Reyes JC. 2009. The chromatin remodeling factor CHD8 interacts with elongating RNA polymerase II and controls expression of the cyclin E2 gene. Nucleic Acids Res. 37: 2449–2460 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B34] 34. Roose J, et al. 1998. The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors. Nature 395: 608–612 [DOI] [PubMed] [Google Scholar]

[B35] 35. Rubinfeld B, et al. 1997. Stabilization of β-catenin by genetic defects in melanoma cell lines. Science 275: 1790–1792 [DOI] [PubMed] [Google Scholar]

[B36] 36. Sakamoto I, et al. 2000. A novel β-catenin-binding protein inhibits beta-catenin-dependent Tcf activation and axis formation. J. Biol. Chem. 275: 32871–32878 [DOI] [PubMed] [Google Scholar]

[B37] 37. Sampietro J, et al. 2006. Crystal structure of a β-catenin/BCL9/Tcf4 complex. Mol. Cell 24: 293–300 [DOI] [PubMed] [Google Scholar]

[B38] 38. Schnetz MP, et al. 2009. Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns. Genome Res. 19: 590–601 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B39] 39. Sierra J, Yoshida T, Joazeiro CA, Jones KA. 2006. The APC tumor suppressor counteracts β-catenin activation and H3K4 methylation at Wnt target genes. Genes Dev. 20: 586–600 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B40] 40. Sims RJ, 3rd, et al. 2007. Recognition of trimethylated histone H3 lysine 4 facilitates the recruitment of transcription postinitiation factors and pre-mRNA splicing. Mol. Cell 28: 665–676 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B41] 41. Thompson BA, Tremblay V, Lin G, Bochar DA. 2008. CHD8 is an ATP-dependent chromatin remodeling factor that regulates β-catenin target genes. Mol. Cell. Biol. 28: 3894–3904 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42. Vissers LE, et al. 2004. Mutations in a new member of the chromodomain gene family cause CHARGE syndrome. Nat. Genet. 36: 955–957 [DOI] [PubMed] [Google Scholar]

[B43] 43. Willert K, et al. 2003. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423: 448–452 [DOI] [PubMed] [Google Scholar]

[B44] 44. Willert K, Jones KA. 2006. Wnt signaling: is the party in the nucleus? Genes Dev. 20: 1394–1404 [DOI] [PubMed] [Google Scholar]

[B45] 45. Yates JA, Menon T, Thompson BA, Bochar DA. 2010. Regulation of HOXA2 gene expression by the ATP-dependent chromatin remodeling enzyme CHD8. FEBS Lett. 584: 689–693 [DOI] [PubMed] [Google Scholar]

[B46] 46. Yuan CC, et al. 2007. CHD8 associates with human Staf and contributes to efficient U6 RNA polymerase III transcription. Mol. Cell. Biol. 27: 8729–8738 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B47] 47. Zhang Y, LeRoy G, Seelig HP, Lane WS, Reinberg D. 1998. The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities. Cell 95: 279–289 [DOI] [PubMed] [Google Scholar]

PERMALINK

Histone H1 Recruitment by CHD8 Is Essential for Suppression of the Wnt–β-Catenin Signaling Pathway

Masaaki Nishiyama

Arthur I Skoultchi

Keiichi I Nakayama

Abstract

INTRODUCTION