CRISPR/Cas9-mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in neurodevelopment.
ABSTRACT: Disruptive mutation in the CHD8 gene is one of the top genetic risk factors in autism spectrum disorders (ASDs). Previous analyses of genome-wide CHD8 occupancy and reduced expression of CHD8 by shRNA knockdown in committed neural cells showed that CHD8 regulates multiple cell processes critical for neural functions, and its targets are enriched with ASD-associated genes.To further understand the molecular links between CHD8 functions and ASD, we have applied the CRISPR/Cas9 technology to knockout one copy of CHD8 in induced pluripotent stem cells (iPSCs) to better mimic the loss-of-function status that would exist in the developing human embryo prior to neuronal differentiation. We then carried out transcriptomic and bioinformatic analyses of neural progenitors and neurons derived from the CHD8 mutant iPSCs.Transcriptome profiling revealed that CHD8 hemizygosity (CHD8 (+/-)) affected the expression of several thousands of genes in neural progenitors and early differentiating neurons. The differentially expressed genes were enriched for functions of neural development, ?-catenin/Wnt signaling, extracellular matrix, and skeletal system development. They also exhibited significant overlap with genes previously associated with autism and schizophrenia, as well as the downstream transcriptional targets of multiple genes implicated in autism. Providing important insight into how CHD8 mutations might give rise to macrocephaly, we found that seven of the twelve genes associated with human brain volume or head size by genome-wide association studies (e.g., HGMA2) were dysregulated in CHD8 (+/-) neural progenitors or neurons.We have established a renewable source of CHD8 (+/-) iPSC lines that would be valuable for investigating the molecular and cellular functions of CHD8. Transcriptomic profiling showed that CHD8 regulates multiple genes implicated in ASD pathogenesis and genes associated with brain volume.
Project description:Recent studies implicate chromatin modifiers in autism spectrum disorder (ASD) through the identification of recurrent de novo loss of function mutations in affected individuals. ASD risk genes are co-expressed in human midfetal cortex, suggesting that ASD risk genes converge in specific regulatory networks during neurodevelopment. To elucidate such networks, we identify genes targeted by CHD8, a chromodomain helicase strongly associated with ASD, in human midfetal brain, human neural stem cells (hNSCs) and embryonic mouse cortex. CHD8 targets are strongly enriched for other ASD risk genes in both human and mouse neurodevelopment, and converge in ASD-associated co-expression networks in human midfetal cortex. CHD8 knockdown in hNSCs results in dysregulation of ASD risk genes directly targeted by CHD8. Integration of CHD8-binding data into ASD risk models improves detection of risk genes. These results suggest loss of CHD8 contributes to ASD by perturbing an ancient gene regulatory network during human brain development.
Project description:Truncating mutations of chromodomain helicase DNA-binding protein 8 (CHD8), and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA sequencing) with genome-wide CHD8 binding (ChIP sequencing). Suppressing CHD8 to levels comparable with the loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8-binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci (P < 10(-8)) and CHD8-bound genes (P = 0.0043), which align with previously identified coexpression modules during fetal development. We also find an intriguing enrichment of cancer-related gene sets among CHD8-bound genes (P < 10(-10)). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene-expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis.
Project description:CHD8, encoding Chromodomain helicase DNA binding protein 8, is a top autism spectrum disorders (ASDs) risk gene. To better understanding the molecular links between CHD8 functions and ASD, we have applied the CRISPR/Cas9 technology to knockout one copy of CHD8 in induced pluripotent stem cells (iPSCs) to mimic the loss of function status that would exist in the developing human embryo prior to neuronal differentiation. Transcriptome profiling (RNA-seq) in neural progenitors and early differentiating neurons revealed that CHD8 hemizygosity (CHD8+/-) affected the expression of several thousands of genes, enriched for functions of neural development, β-catenin/Wnt signaling, extracellular matrix, and skeletal system development. Moreover, CHD8 regulates multiple genes implicated in ASD, schizophrenia and genes associated with brain volume. iPSCs derived from a healthy subject were transduced with CRISPR/Cas9 vectors with single guide RNA sequences to target the N-terminal of CHD8 protein to generate truncated mutation seach of the two target sequences. Two clones, one with a 2-bp (KO1) and the other with a 10-bp (KO2) heterozygous deletion were found.The CHD8+/- iPSC lines were used to generate NPCs and early differentiating neurons for RNA-seq analysis, together with samples prepared from the parental clones, for a total of 8 samples (two biological replicates of wild-type (WT) and CHD8+/- at two neurodevelopmental stages).
Project description: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.
Project description:Truncating mutations of CHD8, encoding a chromodomain helicase, and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA-seq) with genome-wide CHD8 binding (ChIP-seq). Suppressing CHD8 to levels comparable with loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8 binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci (p = 1.01x10-9) and CHD8-bound genes (p = 4.34x10-3), which align with previously identified co-expression modules during fetal development. We also find an intriguing enrichment of cancer related gene-sets among CHD8-bound genes (p < 1.9x10-11). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis. RNA-seq in NPCs treated with shRNAs targeting CHD8. For controls, NPCs were treated with shRNAs targeting GFP and LacZ. Infection and sequencing was carried out in two separate batches, with one GFP and one LacZ sample in each batch. All samples were sequenced in two technical replicates.
Project description:The packaging of DNA into chromatin determines the transcriptional potential of cells and is central to eukaryotic gene regulation. Case sequencing studies have revealed mutations to proteins that regulate chromatin state, known as chromatin remodeling factors, with causal roles in neurodevelopmental disorders. Chromodomain helicase DNA binding protein 8 (CHD8) encodes a chromatin remodeling factor with among the highest de novo loss-of-function mutation rates in patients with autism spectrum disorder (ASD). However, mechanisms associated with CHD8 pathology have yet to be elucidated. We analyzed published transcriptomic data across CHD8 in vitro and in vivo knockdown and knockout models and CHD8 binding across published ChIP-seq datasets to identify convergent mechanisms of gene regulation by CHD8. Differentially expressed genes (DEGs) across models varied, but overlap was observed between downregulated genes involved in neuronal development and function, cell cycle, chromatin dynamics, and RNA processing, and between upregulated genes involved in metabolism and immune response. Considering the variability in transcriptional changes and the cells and tissues represented across ChIP-seq analysis, we found a surprisingly consistent set of high-affinity CHD8 genomic interactions. CHD8 was enriched near promoters of genes involved in basic cell functions and gene regulation. Overlap between high-affinity CHD8 targets and DEGs shows that reduced dosage of CHD8 directly relates to decreased expression of cell cycle, chromatin organization, and RNA processing genes, but only in a subset of studies. This meta-analysis verifies CHD8 as a master regulator of gene expression and reveals a consistent set of high-affinity CHD8 targets across human, mouse, and rat in vivo and in vitro studies. These conserved regulatory targets include many genes that are also implicated in ASD. Our findings suggest a model where perturbation to dosage-sensitive CHD8 genomic interactions with a highly-conserved set of regulatory targets leads to model-specific downstream transcriptional impacts.
Project description:BACKGROUND:Autism spectrum disorder (ASD) is a common and etiologically heterogeneous neurodevelopmental disorder. Although many genetic causes have been identified (> 200 ASD-risk genes), no single gene variant accounts for > 1% of all ASD cases. A role for epigenetic mechanisms in ASD etiology is supported by the fact that many ASD-risk genes function as epigenetic regulators and evidence that epigenetic dysregulation can interrupt normal brain development. Gene-specific DNAm profiles have been shown to assist in the interpretation of variants of unknown significance. Therefore, we investigated the epigenome in patients with ASD or two of the most common genomic variants conferring increased risk for ASD. Genome-wide DNA methylation (DNAm) was assessed using the Illumina Infinium HumanMethylation450 and MethylationEPIC arrays in blood from individuals with ASD of heterogeneous, undefined etiology (n = 52), and individuals with 16p11.2 deletions (16p11.2del, n = 9) or pathogenic variants in the chromatin modifier CHD8 (CHD8+/-, n = 7). RESULTS:DNAm patterns did not clearly distinguish heterogeneous ASD cases from controls. However, the homogeneous genetically-defined 16p11.2del and CHD8+/- subgroups each exhibited unique DNAm signatures that distinguished 16p11.2del or CHD8+/- individuals from each other and from heterogeneous ASD and control groups with high sensitivity and specificity. These signatures also classified additional 16p11.2del (n = 9) and CHD8 (n = 13) variants as pathogenic or benign. Our findings that DNAm alterations in each signature target unique genes in relevant biological pathways including neural development support their functional relevance. Furthermore, genes identified in our CHD8+/- DNAm signature in blood overlapped differentially expressed genes in CHD8+/- human-induced pluripotent cell-derived neurons and cerebral organoids from independent studies. CONCLUSIONS:DNAm signatures can provide clinical utility complementary to next-generation sequencing in the interpretation of variants of unknown significance. Our study constitutes a novel approach for ASD risk-associated molecular classification that elucidates the vital cross-talk between genetics and epigenetics in the etiology of ASD.
Project description:Autism spectrum disorder (ASD) is highly heritable but genetically heterogeneous. The affected neural circuits and cell types remain unclear and may vary at different developmental stages. By analyzing multiple sets of human single cell transcriptome profiles, we found that ASD candidates showed relatively enriched gene expression in neurons, especially in inhibitory neurons. ASD candidates were also more likely to be the hubs of the co-expression gene module that is highly expressed in inhibitory neurons, a feature not detected for excitatory neurons. In addition, we found that upregulated genes in multiple ASD cortex samples were enriched with genes highly expressed in inhibitory neurons, suggesting a potential increase of inhibitory neurons and an imbalance in the ratio between excitatory and inhibitory neurons in ASD brains. Furthermore, the downstream targets of several ASD candidates, such as CHD8, EHMT1 and SATB2, also displayed enriched expression in inhibitory neurons. Taken together, our analyses of single cell transcriptomic data suggest that inhibitory neurons may be a major neuron subtype affected by the disruption of ASD gene networks, providing single cell functional evidence to support the excitatory/inhibitory (E/I) imbalance hypothesis.
Project description:Whole-exome sequencing studies have implicated chromatin modifiers and transcriptional regulators in autism spectrum disorder (ASD) through the identification of de novo loss of function mutations in affected individuals. Many of these genes are co-expressed in mid-fetal human cortex, suggesting ASD risk genes converge in regulatory networks that are perturbed in ASD during neurodevelopment. To elucidate such networks we mapped promoters and enhancers bound by the chromodomain helicase CHD8, which is strongly enriched in ASD-associated de novo loss of function mutations, using ChIP-seq in mid-fetal human brain, human neural stem cells (hNSCs), and embryonic mouse cortex. We find that CHD8 targets are strongly enriched for ASD risk genes that converge in ASD-associated co-expression networks in human midfetal cortex. CHD8 knockdown in hNSCs results in significant dysregulation of ASD risk genes targeted by CHD8, as well as additional genes important for neurodevelopment, including members of the Wnt/β-catenin signaling pathway. Integration of CHD8 binding data with genetic and gene co-expression data in ASD risk models provides support for additional ASD risk genes. Together, our results suggest that loss of CHD8 function contributes to ASD through regulatory perturbation of other ASD risk genes during human cortical development. Two biological replicates for each ChIP with appropriate Input control Four biological replicates for each condition in knockdown experiments (Ctrl construct, Chd8 target C, and Chd8 target G)
Project description:Oligodendrocyte precursor cells (OPCs) constitute the main proliferative cells in the adult brain, and deregulation of OPC proliferation-differentiation balance results in either glioma formation or defective adaptive (re)myelination. OPC differentiation requires significant genetic reprogramming, implicating chromatin remodeling. Mounting evidence indicates that chromatin remodelers play important roles during normal development and their mutations are associated with neurodevelopmental defects, with CHD7 haploinsuficiency being the cause of CHARGE syndrome and CHD8 being one of the strongest autism spectrum disorder (ASD) high-risk-associated genes. Herein, we report on uncharacterized functions of the chromatin remodelers Chd7 and Chd8 in OPCs. Their OPC-chromatin binding profile, combined with transcriptome and chromatin accessibility analyses of Chd7-deleted OPCs, demonstrates that Chd7 protects nonproliferative OPCs from apoptosis by chromatin closing and transcriptional repression of p53 Furthermore, Chd7 controls OPC differentiation through chromatin opening and transcriptional activation of key regulators, including Sox10, Nkx2.2, and Gpr17 However, Chd7 is dispensable for oligodendrocyte stage progression, consistent with Chd8 compensatory function, as suggested by their common chromatin-binding profiles and genetic interaction. Finally, CHD7 and CHD8 bind in OPCs to a majority of ASD risk-associated genes, suggesting an implication of oligodendrocyte lineage cells in ASD neurological defects. Our results thus offer new avenues to understand and modulate the CHD7 and CHD8 functions in normal development and disease.