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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Molecular Autism 1/2017

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

Zeitschrift:
Molecular Autism > Ausgabe 1/2017
Autoren:
Ping Wang, Ryan Mokhtari, Erika Pedrosa, Michael Kirschenbaum, Can Bayrak, Deyou Zheng, Herbert M. Lachman
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s13229-017-0124-1) contains supplementary material, which is available to authorized users.

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.
Zusatzmaterial
Additional file 1: Supplemental methods (DOCX 153 kb)
13229_2017_124_MOESM1_ESM.docx
Additional file 2: Figure S1. (1) Immunohistochemistry (IHS) of control organoid section showing GABA immunoreactivity (GABAergic neurons) in a field of MAP2+ neurons. (2) IHS showing GABA and vGLUT2+ (glutamatergic) neurons. (3). quantitative IHS. CHD8 protein was quantified as described in “ Methods” section comparing a control organoid ( CHD8 +/+) with a heterozygous CHD8 KO organoid ( CHD8 +/−). The images were captured using the same parameters, such as exposure times, for each fluorescence channel was the same for the CHD8 +/+ and CHD8 +/− samples. Images edited in power point using the Picture Tools option were adjusted to the same brightness and contrast levels in CHD8 +/+and CHD8 +/− sections for CHD8 immunoreactivity and for Tuj1 reactivity. (PDF 1507 kb)
Additional file 3: Table S1. Summary of RNA-seq quality and number of differentially expressed genes. (DOCX 14 kb)
13229_2017_124_MOESM3_ESM.docx
Additional file 5: Table S3. Enriched gene ontology (GO) terms for DEGs: Sheet 1, upregulated DEGs; Sheet 2, downregulated DEGs; Sheet 3, IPA canonical pathways. (XLSX 62 kb)
13229_2017_124_MOESM5_ESM.xlsx
Additional file 6: Table S4. Lists of overlapping DEGs between CHD8 KO and idiopathic ASD organoids (sheet 1). Gene ontologies for overlapping genes between CHD8 KO and idiopathic ASD organoids days 11 and 31 (sheets 2 and 3, respectively). (XLSX 58 kb)
13229_2017_124_MOESM6_ESM.xlsx
Additional file 7: Table S5. List of overlapping DEGs between CHD8 KO organoids, NPCs and neurons, and non-responder and lithium (Li) responder patients with BD (sheet 1). Gene ontologies for overlapping genes between CHD8 KO and Li non-responders and Li responders (sheets 2 and 3, respectively). (XLSX 22 kb)
13229_2017_124_MOESM7_ESM.xlsx
Additional file 8: Table S6. Lists DEG from CHD8 KO organoids, NPCs, and neurons that overlap with ASD and SZ candidate genes (sheet 1). Gene ontologies for overlapping genes between CHD8 KO and the SZGWAS, AutismKB, and SFARI databases (sheets 2-4, respectively). (XLSX 46 kb)
13229_2017_124_MOESM8_ESM.xlsx
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