Integrative functional genomics following suppression of CHD8 identifies transcriptional signatures that are enriched for autism genes and macrocephaly. M. Biagioli1,4, A. Sugathan1,2,4, C. Golzio3, I. Blumenthal1,2, S. Erdin1,2, P. Manavalan1, A. Ragavendran1,2, D. Lucente1, J. Miles5, S. D. Sheridan1, A. Stortchevoi1,2, S. J. Haggarty1,2,4,6, J. F. Gusella1,4,6,7, N. Katsanis3, M. E. Talkowski1,2,4,6 1) Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114; 2) Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114; 3) Center for Human Disease Modeling and Department of Cell biology, Duke University, Durham, NC 27710; 4) Department of Neurology, Harvard Medical School, Boston, MA 02114; 5) Departments of Pediatrics, Medical Genetics and Pathology, The Thompson Center for Autism and Neurodevelopmental Disorders, University of Missouri Hospitals and Clinics, Columbia, MO 65201; 6) Broad Institute of M.I.T. and Harvard, Boston, MA 02142; 7) Department of Genetics, Harvard Medical School, Boston, MA 02115.

   Inactivating mutations of CHD8, and of a spectrum of genes with diverse functions, act as strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We perturbed the transcriptional networks that CHD8 regulates early in neurodevelopment by suppressing its expression in iPS-derived neural precursors using 5 independent shRNAs. We integrated transcriptome sequencing with genome-wide CHD8 binding from three independent antibodies. Suppressing CHD8 altered expression of 1,756 genes, most of which were up-regulated (~65%), consistent with its putative function as a transcriptional repressor. ChIP-seq revealed pervasive CHD8 binding, with 7,324 replicated sites from all three antibodies marking 5,658 genes, yet just 9% of these genes were differentially expressed. These data suggest that a limited array of direct regulatory effects of CHD8 produces a larger network of expression changes through secondary indirect regulatory mechanisms. Interestingly, the networks associated with direction of CHD8 regulation are functionally distinct. Genes indirectly down-regulated (i.e., without CHD8 binding sites) are strongly enriched for genes associated with ASD (p = 1.0x10-9) and reflect pathways involved in brain development. In contrast, genes with CHD8 binding sites are associated with cell maintenance and transcriptional regulation and are enriched for cancer related genes from three independent cancer datasets (enrichment p < 1.0x10-10 in all datasets). Among the most significant genes differentially expressed were known ASD genes (e.g., SCN2A, SHANK3) as well as a series of cell adhesion molecules. The most significant pathway and phenotypic enrichment observed was related to abnormality of skull size, prompting us to study in vivo effects. We observed macrocephaly in zebrafish models of chd8 suppression initially using morpholino knockdown that was replicated by CRISPR, comparable to the phenotype reported in humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates gene expression changes that include indirect down-regulation of many other ASD risk genes. Perturbation of other genes within this ASD network are ongoing, and our results collectively suggest that some genes associated with ASD and neurodevelopmental disorders may converge on shared mechanism of pathogenesis.

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