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The autism-associated chromatin modifier CHD8 regulates other autism risk genes during human neurodevelopment.

Cotney J, Muhle RA, Sanders SJ, Liu L, Willsey AJ, Niu W, Liu W, Klei L, Lei J, Yin J, Reilly SK, Tebbenkamp AT, Bichsel C, Pletikos M, Sestan N, Roeder K, State MW, Devlin B, Noonan JP - Nat Commun (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Genetics, Yale School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, USA [2] Kavli Institute for Neuroscience, Yale School of Medicine, PO Box 208001, New Haven, Connecticut 06520, USA.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Depletion of CHD8 in hNSCs significantly affects CHD8 target genes.(a) Top, Schematic depicting functional domains within CHD8. Sites in CHD8 that are targeted by knockdown shRNA constructs C and G are indicated by vertical grey bars. Bottom, Representative western blot of hNSC protein lysates demonstrating depletion of CHD8 protein levels due to transfection of each shRNA construct compared with a non-targeting transfection control (shCTL). QPCR and western blots were performed for each knockdown experiment. (b) Conserved CHD8 targets are disproportionately affected by CHD8 depletion. For each subset of CHD8 target genes shown, the P value from a Wilcoxon rank test comparing the distribution of differential expression P values in that subset versus active genes not bound by CHD8 in hNSC is plotted on the y axis, and the number of genes in the subset is plotted on the x axis (Supplementary Information). The red curve shows the smoothed (quadratic) spline fit to the data. (c) Residual values for the indicated subsets of CHD8 targets calculated from the fit lines in b. The set of CHD8 targets conserved in mouse holds the greatest fraction of genes showing differential expression by each CHD8 knockdown.
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f4: Depletion of CHD8 in hNSCs significantly affects CHD8 target genes.(a) Top, Schematic depicting functional domains within CHD8. Sites in CHD8 that are targeted by knockdown shRNA constructs C and G are indicated by vertical grey bars. Bottom, Representative western blot of hNSC protein lysates demonstrating depletion of CHD8 protein levels due to transfection of each shRNA construct compared with a non-targeting transfection control (shCTL). QPCR and western blots were performed for each knockdown experiment. (b) Conserved CHD8 targets are disproportionately affected by CHD8 depletion. For each subset of CHD8 target genes shown, the P value from a Wilcoxon rank test comparing the distribution of differential expression P values in that subset versus active genes not bound by CHD8 in hNSC is plotted on the y axis, and the number of genes in the subset is plotted on the x axis (Supplementary Information). The red curve shows the smoothed (quadratic) spline fit to the data. (c) Residual values for the indicated subsets of CHD8 targets calculated from the fit lines in b. The set of CHD8 targets conserved in mouse holds the greatest fraction of genes showing differential expression by each CHD8 knockdown.

Mentions: ASD-associated de novo truncating mutations in CHD8 are likely to result in reduced levels of functional CHD8 proteins in vivo. To model this putative haploinsufficiency, we carried out knockdowns of CHD8 transcript levels in hNSCs using two independent short hairpin RNA (shRNA) constructs (Fig. 4a). Both western and quantitative PCR (qPCR) analysis confirmed knockdown of CHD8 transcript from each construct 48 h after transfection (Fig. 4a and Supplementary Fig. 7). Genome-wide analysis indicated these CHD8 shRNAs did not show specificity for any other expressed gene in hNSCs. However, they target different regions of the CHD8 gene and may target distinct CHD8 isoforms (Supplementary Data 5). The shRNAs may thus have different biological effects so we analysed each knockdown independently.


The autism-associated chromatin modifier CHD8 regulates other autism risk genes during human neurodevelopment.

Cotney J, Muhle RA, Sanders SJ, Liu L, Willsey AJ, Niu W, Liu W, Klei L, Lei J, Yin J, Reilly SK, Tebbenkamp AT, Bichsel C, Pletikos M, Sestan N, Roeder K, State MW, Devlin B, Noonan JP - Nat Commun (2015)

Depletion of CHD8 in hNSCs significantly affects CHD8 target genes.(a) Top, Schematic depicting functional domains within CHD8. Sites in CHD8 that are targeted by knockdown shRNA constructs C and G are indicated by vertical grey bars. Bottom, Representative western blot of hNSC protein lysates demonstrating depletion of CHD8 protein levels due to transfection of each shRNA construct compared with a non-targeting transfection control (shCTL). QPCR and western blots were performed for each knockdown experiment. (b) Conserved CHD8 targets are disproportionately affected by CHD8 depletion. For each subset of CHD8 target genes shown, the P value from a Wilcoxon rank test comparing the distribution of differential expression P values in that subset versus active genes not bound by CHD8 in hNSC is plotted on the y axis, and the number of genes in the subset is plotted on the x axis (Supplementary Information). The red curve shows the smoothed (quadratic) spline fit to the data. (c) Residual values for the indicated subsets of CHD8 targets calculated from the fit lines in b. The set of CHD8 targets conserved in mouse holds the greatest fraction of genes showing differential expression by each CHD8 knockdown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4355952&req=5

f4: Depletion of CHD8 in hNSCs significantly affects CHD8 target genes.(a) Top, Schematic depicting functional domains within CHD8. Sites in CHD8 that are targeted by knockdown shRNA constructs C and G are indicated by vertical grey bars. Bottom, Representative western blot of hNSC protein lysates demonstrating depletion of CHD8 protein levels due to transfection of each shRNA construct compared with a non-targeting transfection control (shCTL). QPCR and western blots were performed for each knockdown experiment. (b) Conserved CHD8 targets are disproportionately affected by CHD8 depletion. For each subset of CHD8 target genes shown, the P value from a Wilcoxon rank test comparing the distribution of differential expression P values in that subset versus active genes not bound by CHD8 in hNSC is plotted on the y axis, and the number of genes in the subset is plotted on the x axis (Supplementary Information). The red curve shows the smoothed (quadratic) spline fit to the data. (c) Residual values for the indicated subsets of CHD8 targets calculated from the fit lines in b. The set of CHD8 targets conserved in mouse holds the greatest fraction of genes showing differential expression by each CHD8 knockdown.
Mentions: ASD-associated de novo truncating mutations in CHD8 are likely to result in reduced levels of functional CHD8 proteins in vivo. To model this putative haploinsufficiency, we carried out knockdowns of CHD8 transcript levels in hNSCs using two independent short hairpin RNA (shRNA) constructs (Fig. 4a). Both western and quantitative PCR (qPCR) analysis confirmed knockdown of CHD8 transcript from each construct 48 h after transfection (Fig. 4a and Supplementary Fig. 7). Genome-wide analysis indicated these CHD8 shRNAs did not show specificity for any other expressed gene in hNSCs. However, they target different regions of the CHD8 gene and may target distinct CHD8 isoforms (Supplementary Data 5). The shRNAs may thus have different biological effects so we analysed each knockdown independently.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Genetics, Yale School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, USA [2] Kavli Institute for Neuroscience, Yale School of Medicine, PO Box 208001, New Haven, Connecticut 06520, USA.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus