CRISPR/Cas9-mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in cerebral organoids derived from iPS cells

Ping Wang, Ryan Mokhtari, Erika Pedrosa, Michael Kirschenbaum, Can Bayrak, Deyou Zheng, Herbert M. Lachman

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

Background: CHD8 (chromodomain helicase DNA-binding protein 8), which codes for a member of the CHD family of ATP-dependent chromatin-remodeling factors, is one of the most commonly mutated genes in autism spectrum disorders (ASD) identified in exome-sequencing studies. Loss of function mutations in the gene have also been found in schizophrenia (SZ) and intellectual disabilities and influence cancer cell proliferation. We previously reported an RNA-seq analysis carried out on neural progenitor cells (NPCs) and monolayer neurons derived from induced pluripotent stem (iPS) cells that were heterozygous for CHD8 knockout (KO) alleles generated using CRISPR-Cas9 gene editing. A significant number of ASD and SZ candidate genes were among those that were differentially expressed in a comparison of heterozygous KO lines (CHD8 +/−) vs isogenic controls (CHD8 +/−), including the SZ and bipolar disorder (BD) candidate gene TCF4, which was markedly upregulated in CHD8 +/− neuronal cells. Methods: In the current study, RNA-seq was carried out on CHD8 +/− and isogenic control (CHD8 +/+) cerebral organoids, which are 3-dimensional structures derived from iPS cells that model the developing human telencephalon. Results: TCF4 expression was, again, significantly upregulated. Pathway analysis carried out on differentially expressed genes (DEGs) revealed an enrichment of genes involved in neurogenesis, neuronal differentiation, forebrain development, Wnt/β-catenin signaling, and axonal guidance, similar to our previous study on NPCs and monolayer neurons. There was also significant overlap in our CHD8 +/− DEGs with those found in a transcriptome analysis carried out by another group using cerebral organoids derived from a family with idiopathic ASD. Remarkably, the top DEG in our respective studies was the non-coding RNA DLX6-AS1, which was markedly upregulated in both studies; DLX6-AS1 regulates the expression of members of the DLX (distal-less homeobox) gene family. DLX1 was also upregulated in both studies. DLX genes code for transcription factors that play a key role in GABAergic interneuron differentiation. Significant overlap was also found in a transcriptome study carried out by another group using iPS cell-derived neurons from patients with BD, a condition characterized by dysregulated WNT/β-catenin signaling in a subgroup of affected individuals. Conclusions: Overall, the findings show that distinct ASD, SZ, and BD candidate genes converge on common molecular targets - an important consideration for developing novel therapeutics in genetically heterogeneous complex traits.

Original languageEnglish (US)
Article number11
JournalMolecular Autism
Volume8
Issue number1
DOIs
StatePublished - Mar 20 2017

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Clustered Regularly Interspaced Short Palindromic Repeats
Organoids
Induced Pluripotent Stem Cells
Gene Knockout Techniques
Gene Regulatory Networks
DNA-Binding Proteins
Autistic Disorder
Genes
Schizophrenia
Bipolar Disorder
Catenins
Homeobox Genes
Neurons
Stem Cells
RNA
Exome
Telencephalon
Untranslated RNA
Chromatin Assembly and Disassembly
Neurogenesis

Keywords

  • Autism
  • Beta-catenin
  • Bipolar disorder
  • Cancer
  • Distal-less homeobox
  • DLX6-AS1
  • Gabaergic
  • HMGA2
  • Schizophrenia
  • TCF4
  • Wnt
  • ZNF132

ASJC Scopus subject areas

  • Molecular Biology
  • Developmental Neuroscience
  • Developmental Biology
  • Psychiatry and Mental health

Cite this

CRISPR/Cas9-mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in cerebral organoids derived from iPS cells. / Wang, Ping; Mokhtari, Ryan; Pedrosa, Erika; Kirschenbaum, Michael; Bayrak, Can; Zheng, Deyou; Lachman, Herbert M.

In: Molecular Autism, Vol. 8, No. 1, 11, 20.03.2017.

Research output: Contribution to journalArticle

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abstract = "Background: CHD8 (chromodomain helicase DNA-binding protein 8), which codes for a member of the CHD family of ATP-dependent chromatin-remodeling factors, is one of the most commonly mutated genes in autism spectrum disorders (ASD) identified in exome-sequencing studies. Loss of function mutations in the gene have also been found in schizophrenia (SZ) and intellectual disabilities and influence cancer cell proliferation. We previously reported an RNA-seq analysis carried out on neural progenitor cells (NPCs) and monolayer neurons derived from induced pluripotent stem (iPS) cells that were heterozygous for CHD8 knockout (KO) alleles generated using CRISPR-Cas9 gene editing. A significant number of ASD and SZ candidate genes were among those that were differentially expressed in a comparison of heterozygous KO lines (CHD8 +/−) vs isogenic controls (CHD8 +/−), including the SZ and bipolar disorder (BD) candidate gene TCF4, which was markedly upregulated in CHD8 +/− neuronal cells. Methods: In the current study, RNA-seq was carried out on CHD8 +/− and isogenic control (CHD8 +/+) cerebral organoids, which are 3-dimensional structures derived from iPS cells that model the developing human telencephalon. Results: TCF4 expression was, again, significantly upregulated. Pathway analysis carried out on differentially expressed genes (DEGs) revealed an enrichment of genes involved in neurogenesis, neuronal differentiation, forebrain development, Wnt/β-catenin signaling, and axonal guidance, similar to our previous study on NPCs and monolayer neurons. There was also significant overlap in our CHD8 +/− DEGs with those found in a transcriptome analysis carried out by another group using cerebral organoids derived from a family with idiopathic ASD. Remarkably, the top DEG in our respective studies was the non-coding RNA DLX6-AS1, which was markedly upregulated in both studies; DLX6-AS1 regulates the expression of members of the DLX (distal-less homeobox) gene family. DLX1 was also upregulated in both studies. DLX genes code for transcription factors that play a key role in GABAergic interneuron differentiation. Significant overlap was also found in a transcriptome study carried out by another group using iPS cell-derived neurons from patients with BD, a condition characterized by dysregulated WNT/β-catenin signaling in a subgroup of affected individuals. Conclusions: Overall, the findings show that distinct ASD, SZ, and BD candidate genes converge on common molecular targets - an important consideration for developing novel therapeutics in genetically heterogeneous complex traits.",
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AU - Wang, Ping

AU - Mokhtari, Ryan

AU - Pedrosa, Erika

AU - Kirschenbaum, Michael

AU - Bayrak, Can

AU - Zheng, Deyou

AU - Lachman, Herbert M.

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AB - Background: CHD8 (chromodomain helicase DNA-binding protein 8), which codes for a member of the CHD family of ATP-dependent chromatin-remodeling factors, is one of the most commonly mutated genes in autism spectrum disorders (ASD) identified in exome-sequencing studies. Loss of function mutations in the gene have also been found in schizophrenia (SZ) and intellectual disabilities and influence cancer cell proliferation. We previously reported an RNA-seq analysis carried out on neural progenitor cells (NPCs) and monolayer neurons derived from induced pluripotent stem (iPS) cells that were heterozygous for CHD8 knockout (KO) alleles generated using CRISPR-Cas9 gene editing. A significant number of ASD and SZ candidate genes were among those that were differentially expressed in a comparison of heterozygous KO lines (CHD8 +/−) vs isogenic controls (CHD8 +/−), including the SZ and bipolar disorder (BD) candidate gene TCF4, which was markedly upregulated in CHD8 +/− neuronal cells. Methods: In the current study, RNA-seq was carried out on CHD8 +/− and isogenic control (CHD8 +/+) cerebral organoids, which are 3-dimensional structures derived from iPS cells that model the developing human telencephalon. Results: TCF4 expression was, again, significantly upregulated. Pathway analysis carried out on differentially expressed genes (DEGs) revealed an enrichment of genes involved in neurogenesis, neuronal differentiation, forebrain development, Wnt/β-catenin signaling, and axonal guidance, similar to our previous study on NPCs and monolayer neurons. There was also significant overlap in our CHD8 +/− DEGs with those found in a transcriptome analysis carried out by another group using cerebral organoids derived from a family with idiopathic ASD. Remarkably, the top DEG in our respective studies was the non-coding RNA DLX6-AS1, which was markedly upregulated in both studies; DLX6-AS1 regulates the expression of members of the DLX (distal-less homeobox) gene family. DLX1 was also upregulated in both studies. DLX genes code for transcription factors that play a key role in GABAergic interneuron differentiation. Significant overlap was also found in a transcriptome study carried out by another group using iPS cell-derived neurons from patients with BD, a condition characterized by dysregulated WNT/β-catenin signaling in a subgroup of affected individuals. Conclusions: Overall, the findings show that distinct ASD, SZ, and BD candidate genes converge on common molecular targets - an important consideration for developing novel therapeutics in genetically heterogeneous complex traits.

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KW - HMGA2

KW - Schizophrenia

KW - TCF4

KW - Wnt

KW - ZNF132

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