SUPPLEMENTARY INFORMATION

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1 SUPPLEMENTARY INFORMATION doi: /nature19357 Figure 1a Chd8 +/+ Chd8 +/ΔSL Chd8 +/+ Chd8 +/ΔL E10.5_Whole brain E10.5_Whole brain E10.5_Whole brain E14.5_Whole brain E14.5_Whole brain E14.5_Whole brain E18.5_Whole brain E18.5_Whole brain E18.5_Whole brain E18.5_Whole brain 1

2 RESEARCH SUPPLEMENTARY INFORMATION Figure 1a Chd8 +/+ Chd8 +/ΔSL Chd8 +/+ Chd8 +/ΔL OB CTX HIP STR TH HY MB CB P 2

3 SUPPLEMENTARY INFORMATION RESEARCH Katayama et al. Supplementary Figure 1 +/+ Chd8 Figure 1a OB CTX HIP STR +/ΔSL Chd8 OB CTX HIP +/+ Chd8 STR +/ΔL Chd8 TH OB HIP STR TH CTX TH HY MB CB P HY MB CB P OB HIP CTX CB HY MB TH STR P CB MB HY P W W W. N A T U R E. C O M / N A T U R E 3

4 RESEARCH SUPPLEMENTARY INFORMATION Extended Data Figure 2e, f Chd8 +/+ Chd8 +/ΔSL Chd8 +/+ Chd8 +/ΔL Heart Thymus Spleen Testis Heart Spleen Thymus Testis 4

5 SUPPLEMENTARY INFORMATION RESEARCH Figure 4e Extended Data Figure 1f IP IgG Anti- Input Chd8+/+ Chd8+/ΔL Chd8+/+ Chd8+/ΔL Chd8+/+ Chd8+/ΔL E10.5 Whole brain E14.5 Whole brain E18.5 Whole brain Adult Olfactory bulb Chd8+/+ Chd8+/ΔL REST Extended Data Figure 6a CHD8 antibodies : A A Chd8 genotype : Chd8+/+ Chd8L F/F A A Chd8+/+ Chd8L F/F Anti- mab Chd8+/+ Chd8L F/F Anti-CHD8 pab Chd8+/+ Chd8L F/F Extended Data Figure 1c 4-OHT (1 μm) : kb Chd8+/+ Chd8+/ΔL Wild-type Deletion 3 Extended Data Figure 10f Extended Data Figure 10h Input IP: FLAG Chd8 +/+ Chd8 +/ΔL Chd8 ΔL/ΔL Extended Data Figure 1d Chd8+/+ CAG-CreER T2 /Chd8+/+ Chd8L F/F CAG-CreER T2 /Chd8L F/F FLAG : - FOXP1 - FOXP1 REST CHX (h): REST FOXP

6 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Discussion Antibody specificity We used rabbit polyclonal antibodies to pan-chd8 (anti-chd8 pab) for immunoblot analysis and a rat monoclonal antibody to CHD8 L (anti-chd8 L mab) for ChIP and immunoprecipitation in the present study. We examined the specificity of these antibodies generated in-house by immunoblot and ChIP analyses with lysates prepared from WT or CHD8 L -null MEFs (the latter of which comprised CAG-CreER T2 /Chd8 F/F L MEFs treated with 4-hydroxytamoxifen [4-OHT]). The signal corresponding to CHD8 L was not detected in the CHD8 L -null MEFs by immunoblot analysis with either anti-chd8 pab or anti-chd8 L mab (Extended Data Fig. 6a). ChIP with anti-chd8 L mab also revealed that CHD8 L binding to target genes identified in previous studies, including Pten, Nras, and Rac1, was greatly diminished in CHD8 L -null MEFs (Extended Data Fig. 6b). These results together show that our anti-chd8 L mab is highly specific for CHD8 L, and they suggest that the data obtained with this antibody in the present study are reliable. Unexpectedly, ChIP signals with one of the antibody preparations (A A, Bethyl Laboratories) used in previous studies 20,46 were not diminished in CHD8 L -null cells in our experiments (Extended Data Fig. 6b), suggesting that the results of ChIP analysis with this antibody preparation may include nonspecific artifacts. CHD8 L and CHD8 S expression We examined the abundance of CHD8 L and CHD8 S isoforms at both mrna and protein levels in tissues of E10.5, E14.5, E18.5, and adult Chd8 +/+, Chd8 +/ SL, and Chd8 +/ L mice including various brain regions as well as tissues such as heart, thymus, spleen, and testis in which CHD8 is relatively abundant in adult mice 12 (Fig. 1a and Extended Data Fig. 2). In most cases, CHD8 L protein levels in the whole brain or brain regions of Chd8 +/ SL and Chd8 +/ L mice were ~70% of those in WT mice (Fig. 1a), whereas this value was ~% for the other tissues examined (Extended Data Fig. 2e). The amounts of Chd8 L and Chd8 S 6

7 SUPPLEMENTARY INFORMATION RESEARCH mrnas were reduced by ~% in all tissues and at all developmental stages in the corresponding mutant mice (Extended Data Fig. 2a d), suggesting that the dosage compensation for CHD8 L haploinsufficiency in the brain occurs at a posttranscriptional level. We did not detect shorter proteins that were present only in the Chd8 +/ L brain (Extended Data Fig. 1f) or in CHD8 L -null MEFs (Extended Data Fig. 6a), suggesting that any such truncated forms of CHD8 produced from the mutant allele are degraded immediately after translation or otherwise unstable. Unfortunately, our in-house rabbit polyclonal antibodies to pan-chd8 (anti-chd8 pab) detected a nonspecific band with an electrophoretic mobility similar to that of CHD8 S in mouse brain (unpublished results), making it technically difficult to quantify CHD8 S protein and therefore to determine the stoichiometry of CHD8 L and CHD8 S isoforms in the brain. Of note, the abundance of Chd8 S mrna was up-regulated in the brain at certain developmental stages and in most regions of the adult brain in Chd8 +/ L mice (Extended Data Fig. 2b), suggesting that CHD8 might suppress its own gene promoter in a negative feedback loop. This moderate increase in the abundance of Chd8 S mrna in the brain might result in a corresponding increase in the amount of CHD8 S protein in Chd8 +/ L mice. Furthermore, it is difficult to experimentally exclude the possibility that such a compensatory up-regulation of CHD8 S protein might affect the ASD-like phenotypes of Chd8 +/ L mice. However, given that the results of the behavioural tests were similar for Chd8 +/ SL and Chd8 +/ L mice (Fig. 1c g, Fig. 2, and Extended Data Fig. 4) and that many mutations in the human CHD8 gene are localized in the region specific to the CHD8 L isoform (Extended Data Fig. 1a), it is likely that the abundance of CHD8 L protein, rather than that of CHD8 S, is an important determinant of ASD phenotypes. The presence of the CHD8 S isoform thus does not appear to substantially affect ASD phenotypes in human or mouse. Presence of the CHD8 S isoform in humans In our previous study 12, immunoblot analysis with our in-house anti-chd8 pab revealed the presence of the CHD8 S isoform in HEK293T, U2OS, SaOS2, HeLa, and HCT116 cell lines, all of which are derived from human. Indeed, we recently isolated a cdna encoding CHD8 S 7

8 RESEARCH SUPPLEMENTARY INFORMATION from human-derived cells (unpublished results). We have no evidence for an interaction between the long and short protein products. Although we cannot exclude the possibility that CHD8 S might function in a dominant negative manner, this appears unlikely given the absence of substantial phenotypic differences between Chd8 +/ SL and Chd8 +/ L mice. Genes whose expression is altered in Chd8 mutant mice We found only a few genes whose expression level was changed by a factor of >2 or <0.5 and with a P value of <0.05 in the brain of Chd8 mutant mice, and even these genes only just fulfilled these criteria (Fig. 3c and Extended Data Fig. 9b f). Such genes include those for noncoding RNAs and a pseudogene and have no known connection to neural function (Extended Data Fig. 9g). It is therefore unlikely that the changes in the expression of such genes contribute to the ASD-like phenotypes of Chd8 mutant mice. CHD8 haploinsufficiency thus does not appear to result in prominent changes in the expression of a few specific genes but rather gives rise to small but global changes in gene expression in mouse brain, reminiscent of the brain of human ASD patients. CHD8 binding pattern in Chd8 mutant mice Comparison of the ChIP-seq results between WT and Chd8 +/ L mice revealed that the pattern of CHD8 binding to transcription start sites (TSSs) in adult brain did not differ significantly between the two genotypes (Extended Data Fig. 9h) regardless of changes in gene expression (Extended Data Fig. 9i). CHD8 binding thus appears to decrease uniformly in the brain of Chd8 +/ L mice with no change in the binding pattern. The specific changes in gene expression apparent in the brain of Chd8 +/ L mice are thus not likely attributable to changes in the pattern of CHD8 binding. Instead, the sensitivity to CHD8 haploinsufficiency may differ among genes. Our results suggest that many REST target genes are included in such genes whose expression is sensitive to CHD8 haploinsufficiency. Of course, REST target genes that do not associate with CHD8 are not influenced by CHD8 haploinsufficiency (Fig. 4f). 8

9 SUPPLEMENTARY INFORMATION RESEARCH Comparison of our results with those of previous studies We compared our ChIP-seq results with those of previous studies 20,21,46 with regard to the degree of overlap in CHD8 binding peaks (Extended Data Fig. 8a) and in CHD8 target genes (Extended Data Fig. 8b e). Our data were found to overlap with those of the previous studies to some extent. Given that the samples and antibodies used differ among these various studies, some level of inconsistency in results is to be expected. As mentioned above with regard to antibody specificity, we found that ChIP signals obtained with one of the anti-chd8 preparations used in two of the previous studies 20,46 were not diminished in CHD8-null cells, suggesting that the results of ChIP experiments with these antibodies may include nonspecific artifacts. We also compared our RNA-seq results with those of the previous studies 20,21. We found that the gene set for REST was markedly affected in the brain of Chd8 mutant mice and of human ASD patients (Fig. 4a, b, d), whereas such a decrease in REST target gene expression was not detected by GSEA with the results of these two previous studies 20,21 (Extended Data Fig. 10b, c). This difference in the detection of these genes between our study and previous studies might be attributable to the difference in the samples analyzed: We examined Chd8 mutant mouse brain (30% to % reduction in CHD8 protein abundance), whereas the other groups used neural stem cells differentiated from human induced pluripotent stem cells (ipscs) and subjected to RNA interference mediated knockdown of CHD8 (70% to 90% reduction in CHD8 protein abundance). The relation between CHD8 and REST is thus revealed only in our study. REST expression in hetmt mice GSEA with Hallmark gene sets revealed that the Wnt/ -catenin pathway appeared to be slightly activated in the brain of Chd8 +/ L mice only at E14.5 (P = 0.043) (Extended Data Fig. 9k). REST was previously shown to be activated by the Wnt signaling pathway 47. Indeed, we found that the amount of Rest mrna tended to be increased in the brain of hetmt mice at E14.5 (P = for Chd8 +/ SL mice and P = for Chd8 +/ L mice) (Extended Data Fig. 9

10 RESEARCH SUPPLEMENTARY INFORMATION 10d), consistent with the Wnt activation apparent at this stage. Given that REST was shown to increase its own expression through the action of the microrna mir-9 in a positive feedback loop 48,49, this small increase in the amount of Rest mrna might reflect a secondary effect of REST activation by CHD8 haploinsufficiency. Mechanistic insight It will be important to explore the mechanistic connection between changes in gene expression profiles and the behavioural abnormalities in our Chd8 mutant mice. We found that CHD8 physically binds to REST and suppresses its function. The importance of the CHD8-REST axis in ASD etiology is supported by two additional observations. First, GSEA revealed that expression of REST target genes was down-regulated in the brain of human ASD patients (Fig. 4d), as seen in our mutant mice (Fig. 4b). Second, expression of REST target genes that bind CHD8 was down-regulated in the brain of Chd8 +/ L mice compared with WT mice, whereas that of such genes that do not bind CHD8 did not show such enrichment (Fig. 4f). These results thus suggest that the CHD8-REST axis plays a key role in ASD pathogenesis. There are four possible mechanisms by which CHD8 might interfere with REST: (1) It inhibits REST activity; (2) it induces dissociation of REST from target genes; (3) it attenuates the expression of Rest; and (4) it promotes REST degradation. With regard to possibility (2), genome-wide ChIP-seq analysis for REST with the brain of WT and CHD8-haploinsufficient mouse embryos at E14.5 revealed no significant difference in REST binding to the genes with or without bound CHD8 between the two genotypes (Extended Data Fig. 10g). These results thus indicate that CHD8 does not prevent or reverse the binding of REST to its target genes. With regard to possibility (3), the amount of Rest mrna was slightly increased in the brain of the heterozygous mutant mice at E14.5 as mentioned above. Regardless of this finding, however, ChIP-seq data showing that the amount of REST bound to its target genes was unchanged in Chd8 heterozygous mutant mice (Extended Data Fig. 10g) indicate that the slight increase in Rest mrna abundance is not likely responsible for 10

11 SUPPLEMENTARY INFORMATION RESEARCH the ASD-like phenotypes. With regard to possibility (4), cycloheximide chase analysis revealed that the stability of REST protein did not differ among WT, Chd8 +/ L, and Chd8 L/ L MEFs (Extended Data Fig. 10h), excluding the possibility that CHD8 inhibits REST function by promoting REST degradation. We therefore conclude that CHD8 inhibits REST activity while REST remains bound to its target genes. CHD8 binding might interfere with the formation of a protein complex that usually associates with REST and mediates its suppressor function such as CoREST, Sin3, LSD1, or histone deacetylase 27,28, or CHD8 binding might antagonize the suppressor function of REST either by itself or by recruiting other transcriptional activators such as the MLL histone methyltransferase complex 46,. In general, however, it is technically difficult at present to explore the molecular mechanisms by which a global modulator of gene expression affects a particular phenotype. For example, mutation of MeCP2, one of the most well-studied molecules in the field of ASD-related disease, results in Rett syndrome with an autistic phenotype 51. It remains to be fully understood how MeCP2 mutation gives rise to ASD-like phenotypes, however, with studies to shed light on the mechanistic connection still being in progress 16, Another example is provided by Brg1, one of the most well-characterized chromatin-remodeling factors. Brg1 mutation results in a variety of phenotypes, and altered Brg1 function has been suggested to underlie many human diseases The precise causal relation between Brg1 mutation and associated phenotypes also remains unclear. These examples suggest that chromatin remodelers or epigenetic modifiers with broad specificity might target thousands of genes, and abnormalities resulting from their mutation might be attributable to combined effects on the expression of many of these genes. Such appears to be the case for CHD8. Although we believe that REST activation may be a key causal factor, we do not conclude that REST alone is responsible for ASD etiology. Rather, it is likely that the combination of altered expression of many genes including REST target genes induced by CHD8 haploinsufficiency shapes ASD phenotypes. The discovery of the physical and functional interaction between CHD8 and REST may prove to be important in itself, however, given that REST is a master regulator of neurogenesis. Indeed, GSEA revealed that 11

12 RESEARCH SUPPLEMENTARY INFORMATION expression of REST target genes is down-regulated in the brain of human ASD patients (Fig. 4d), as it is in our mutant mice (Fig. 4b). Deregulated activation of REST resulting from CHD8 haploinsufficiency might thus give rise to neurodevelopmental delay and the consequent development of ASD phenotypes. Identification of the CHD8-REST axis may therefore provide important insight into the pathogenesis of ASD as well as serve as the basis for development of new treatments for this substantial medical and social problem. 12

13 SUPPLEMENTARY INFORMATION RESEARCH Supplementary references 47 Lu, T. et al. REST and stress resistance in ageing and Alzheimer's disease. Nature 7, (2014). 48 Packer, A. N., Xing, Y., Harper, S. Q., Jones, L. & Davidson, B. L. The bifunctional microrna mir-9/mir-9* regulates REST and CoREST and is downregulated in Huntington's disease. J. Neurosci. 28, (2008). 49 Laneve, P. et al. A minicircuitry involving REST and CREB controls mir-9-2 expression during human neuronal differentiation. Nucleic Acids Res. 38, (2010). Dou, Y. et al. Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF. Cell 121, (2005). 51 Chahrour, M. & Zoghbi, H. Y. The story of Rett syndrome: from clinic to neurobiology. Neuron 56, (2007). 52 Shahbazian, M. et al. Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3. Neuron 35, (2002). 53 Guy, J., Gan, J., Selfridge, J., Cobb, S. & Bird, A. Reversal of neurological defects in a mouse model of Rett syndrome. Science 315, (2007). 54 Gabel, H. W. et al. Disruption of DNA-methylation-dependent long gene repression in Rett syndrome. Nature 522, (2015). 55 Sztainberg, Y. et al. Reversal of phenotypes in MECP2 duplication mice using genetic rescue or antisense oligonucleotides. Nature 528, (2015). 56 Wilson, B. G. & Roberts, C. W. SWI/SNF nucleosome remodellers and cancer. Nat. Rev. Cancer 11, (2011). 57 Hang, C. T. et al. Chromatin regulation by Brg1 underlies heart muscle development and disease. Nature 466, (2010). 58 Zhang, Z. et al. Autism-associated chromatin regulator Brg1/SmarcA4 is required for synapse development and myocyte rnhancer factor 2-mediated dynapse remodeling. Mol. Cell. Biol. 36, (2015). 59 Ho, L. et al. An embryonic stem cell chromatin remodeling complex, esbaf, is an essential component of the core pluripotency transcriptional network. Proc. Natl. Acad. Sci. U. S. A. 106, (2009). 13