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The Autism-Associated Gene Chromodomain Helicase DNA-Binding Protein 8 (CHD8) Regulates Noncoding Rnas and Autism-Related Genes

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The Autism-Associated Gene Chromodomain Helicase DNA-Binding Protein 8 (CHD8) Regulates Noncoding Rnas and Autism-Related Genes OPEN Citation: Transl Psychiatry (2015) 5, e568; doi:10.1038/tp.2015.62 www.nature.com/tp ORIGINAL ARTICLE The autism-associated gene chromodomain helicase DNA-binding protein 8 (CHD8) regulates noncoding RNAs and autism-related genes B Wilkinson1, N Grepo2, BL Thompson3, J Kim2,4, K Wang2,4, OV Evgrafov2,4,WLu5,6, JA Knowles2,4 and DB Campbell2,4 Chromodomain helicase DNA-binding protein 8 (CHD8) was identified as a leading autism spectrum disorder (ASD) candidate gene by whole-exome sequencing and subsequent targeted-sequencing studies. De novo loss-of-function mutations were identified in 12 individuals with ASD and zero controls, accounting for a highly significant association. Small interfering RNA-mediated knockdown of CHD8 in human neural progenitor cells followed by RNA sequencing revealed that CHD8 insufficiency results in altered expression of 1715 genes, including both protein-coding and noncoding RNAs. Among the 10 most changed transcripts, 4 (40%) were noncoding RNAs. The transcriptional changes among protein-coding genes involved a highly interconnected network of genes that are enriched in neuronal development and in previously identified ASD candidate genes. These results suggest that CHD8 insufficiency may be a central hub in neuronal development and ASD risk. Translational Psychiatry (2015) 5, e568; doi:10.1038/tp.2015.62; published online 19 May 2015 INTRODUCTION abnormality leading to disruption of CHD8.10 CHD8 LoF mutations Autism spectrum disorder (ASD) is a complex neurodevelopmental have not been found in any of the 8792 controls included in these disorder characterized by impairments in social interaction, analyses, emphasizing the impact of CHD8 LoF mutations on ASD 9 communication and behavioral flexibility.1 Due to the vast clinical risk. Phenotypic characterization of individuals with CHD8 and genetic heterogeneity of ASD, the identification of causal disrupting mutations indicate a subset of ASD that includes genetic determinants has proven challenging.2–4 However, multi- macrocephaly, distinct facial features and gastrointestinal fi 8 ple independent studies have now provided substantial evidence dif culties. Although a critical role of CHD8 in development is revealed by for the contribution of de novo loss-of-function (LoF) mutations in 11 chromodomain helicase DNA-binding protein 8 (CHD8) to ASD.5–9 the embryonic lethality of Chd8 knockout mice, the function of Initially, exome sequencing on 209 ASD probands and their CHD8 in neural cell lineages has been largely unexplored. As CHD8 actively associates with core transcriptional machinery,12 tran- unaffected family members discovered two de novo LoF mutations scription factors13,14 and histone-modifying complexes,15 tran- in CHD8.6 Subsequent targeted sequencing of 44 candidate genes scriptional dysregulation conferred by CHD8 insufficiency may in 2446 ASD probands identified an additional seven de novo LoF provide evidence for the neurodevelopmental phenotypes mutations in CHD8, establishing a frequency of mutation that is fi 7 observed in ASD. To emulate the potential effects of the identi ed the highest of all genes screened. Furthermore, a second LoF mutations, we performed small interfering RNA (siRNA)- targeted-sequencing study of an additional 3370 children with mediated knockdown of CHD8 followed by genome-wide fi ASD or developmental delay identi ed three de novo LoF transcriptional profiling through RNA sequencing (RNA-seq). Here, 8 fi fi mutations in CHD8. Altogether, these highly signi cant ndings we show that knockdown of CHD8 in SK-N-SH human neural have placed CHD8 as a genuine ASD risk factor and account for progenitor cells results in altered expression of a highly 0.2% (12/6,176) of ASD cases. The CHD8 LoF mutations have been interconnected network of genes, which are enriched in several found throughout the coding region of the gene, with truncating processes essential for neuronal development. Remarkably, mutations as early as amino acid 62 of the 2581 amino acid CHD8 numerous previously identified ASD candidate genes are also protein. Truncating mutations were found in the chromodomain, differentially expressed in response to knockdown of CHD8, the dex domain and the helicase domain. A detailed map of all the suggesting a convergence of multiple genes contributing to ASD identified CHD8 LoF mutations was published recently.9 In onset. The data presented here point toward a role of CHD8 in addition to de novo mutations in CHD8, six potentially disrupting maintaining the active transcription of neural-specific genes and mutations with unknown or inherited modes of transmission have begins to elucidate the potential contributions of decreased also been identified8,9 along with a balanced chromosomal functional CHD8 to the pathogenesis of ASD. 1Development, Stem Cells, and Regeneration Ph.D. Program, University of Southern California, Los Angeles, CA, USA; 2Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA; 3Division of Occupational Science and Occupational Therapy, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, USA; 4Department of Psychiatry and the Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA; 5Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA and 6Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. Correspondence: Dr DB Campbell, Keck School of Medicine, University of Southern California, 213 Zilkha Neurogenetic Institute, 1501 San Pablo Street, Los Angeles, CA 90089, USA. E-mail: [email protected] Received 29 October 2014; revised 12 February 2015; accepted 31 March 2015 CHD8 insufficiency in neural progenitor cells B Wilkinson et al 2 MATERIALS AND METHODS with a supplied GTF file of the corresponding transcriptome to be Cell culture analyzed. Cuffdiff analysis was performed with default settings with the exception of the masking of all annotated ribosomal RNAs and the To measure gene expression in human neural progenitor cells, SK-N-SH no-effective-length-correction option, which was used to reduce over- cells (American Type Culture Collection; Manassas, VA, USA) were estimation of short transcript expression. Genes with a false discovery rate maintained in minimal essential medium supplemented with 10% heat- (Q-value) of o0.05 were considered significant. All gene ontology (GO) inactivated fetal bovine serum, 1% penicillin/streptomycin, non-essential − and protein–protein interaction analyses were carried out using the list of amino acids, and 1.5 g l 1 sodium bicarbonate in 183-cm flasks at 37 °C significantly differentially expressed genes identified using the UCSC Hg19 and 5% CO . 2 human genome annotation. To explore changes in genes transcribing ncRNAs, we also carried out the RNA-seq analysis using the ENSEMBL siRNA transfection GrCH37, release 75, gene annotation, which contains a much larger To determine the impact of CHD8 knockdown on gene expression in quantity of ncRNA transcripts compared with the UCSC Hg19 annotated human neural progenitor cells, SK-N-SH cells were seeded into six-well 10- transcriptome. cm plates and grown for 24 h (~70% confluency) before transfection. Transfections were carried out with either siRNA silencer select negative Quantitative PCR control No. 1 (catalog no. 4390843, Ambion/Life Technologies; Carlsbad, To measure the degree to which siRNA knockdown of CHD8 results in CA, USA) or siRNA targeting CHD8 (catalog no. 33582, Ambion/Life 16 decreased transcript levels and verify the quality of RNA-seq data analysis, Technologies) at a concentration of 20 nM using Lipofectamine RNAiMAX cell pellets were processed for RNA isolation using the Qiagen RNeasy kit Reagent (Invitrogen/Life Technologies; Carlsbad, CA, USA) according to the (Qiagen). Superscipt III (Life Technologies; Grand Island, NY, USA) was used manufacturer's protocol. Cells were then collected 72 h post siRNA for reverse transcription of cDNA libraries. Quantitative PCR (qPCR) was transfection and processed for downstream applications. Experiments performed using Life Technologies Taqman Gene Expression qPCR kits on were performed in quadruplicate. a Life Technologies OneStepPlus real-time PCR machine. Each sample was run in triplicate along with the housekeeping gene, GAPDH. Assays used Western blot analyses for validation (Life Technologies) include ABCA1 (Hs01059118_m1), CHD8 To determine the degree to which siRNA knockdown of CHD8 transcript (Hs00394229_m1), BDNF (Hs02718934_s1), CUL1 (Hs01117001_m1), GAPDH results in decreased CHD8 protein, total protein was isolated using the (Hs9999905_m1), GJA1 (Hs00748445_s1), HMGA2 (Hs00171569_m1), HSPB3 Illustra triplePrep kit (GE Healthcare; Waukesha, WI, USA) and protein (Hs00937412_s1), IGF2 (Hs04188276_m1), IGFBP3 (Hs00365742_g1), IL1R1 concentration was determined using the DC protein assay (Bio-Rad; (Hs00991002_m1), IL8 (Hs00174103_m1), LINC00473 (Hs00293257_m1), Hercules, CA, USA). Total protein (10 μg) was then separated on a 4–20% LINC01021 (Hs01388536_m1), MMP3 (Hs00968305_m1), PITX3 gradient criterion TGX gel (Bio-Rad) and transferred to a nitrocellulose (Hs01013935_g1), SCN2A (Hs01109877_m1), SERPINE1 (Hs01126606_m1), membrane by capillary transfer at 80 V for 3 h using a Bio-Rad Criterion SEMA3D (Hs00380877_m1), SYTL2 (Hs00909223_m1), TGM2 blotter system.
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