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Chd8 mediates cortical neurogenesis via transcriptional regulation of cell cycle and Wnt signaling

Abstract

De novo mutations in CHD8 are strongly associated with autism spectrum disorder, but the basic biology of CHD8 remains poorly understood. Here we report that Chd8 knockdown during cortical development results in defective neural progenitor proliferation and differentiation that ultimately manifests in abnormal neuronal morphology and behaviors in adult mice. Transcriptome analysis revealed that while Chd8 stimulates the transcription of cell cycle genes, it also precludes the induction of neural-specific genes by regulating the expression of PRC2 complex components. Furthermore, knockdown of Chd8 disrupts the expression of key transducers of Wnt signaling, and enhancing Wnt signaling rescues the transcriptional and behavioral deficits caused by Chd8 knockdown. We propose that these roles of Chd8 and the dynamics of Chd8 expression during development help negotiate the fine balance between neural progenitor proliferation and differentiation. Together, these observations provide new insights into the neurodevelopmental role of Chd8.

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Figure 1: Chd8 regulates neural progenitor proliferation in developing cortex.
Figure 2: Chd8 is important for the expression of cell cycle genes in developing brain.
Figure 3: Direct and indirect regulation of genes necessary for early cortical development by Chd8.
Figure 4: Chd8 is a positive regulator of canonical Wnt signaling in neuronal cells.
Figure 5: Increased β-catenin rescues phenotypes associated with Chd8 knockdown.
Figure 6: Chd8 Knockdown in upper cortical layer neurons results in behavioral deficits, which can be rescued via induction of Wnt signaling.

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Acknowledgements

We thank R. Madabhushi, J. Penney and A. Mungenast for critical reading of the manuscript. We are thankful to R. Madabhushi, J.D. Cheng, J. Penney and Y.T. Lin for technical help and suggestions with the project. The Super8xTOPFLASH luciferase reporter construct was a kind gift from Dr. R. Moon (University of Washington, WA). O.D. is a Henry Singleton (1940) Fellow (Brain and Cognitive Sciences, Massachusetts Institute of Technology). Y.J.K.-W. and L.A.W. were supported by a postdoctoral fellowship from the Simons Foundation (Simons Center for the Social Brain, Massachusetts Institute of Technology). L.A.W. is supported by postdoctoral fellowship from Natural Science and Engineering Council of Canada. We would like to thank the JPB Foundation for supporting our study. This work was partially supported by a NIH U01 grant (MH106018-03) to L.-H.T.

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Authors and Affiliations

Authors

Contributions

O.D. and L.-H.T. designed the study, and L.-H.T. directed and coordinated the study. O.D. initiated, planned and performed the experiments. F.G. conducted the bioinformatics analysis. Y.J.K.-W. cloned the human CHD8 construct and contributed to sample preparation for FAC-sorting. R.R. prepared cultured various cell lines and helped with sample preparation for FAC-sorting. A.J.M. conducted some of the luciferase assays. A.J.M. and A.N. contributed to behavioral experiments. C.Y.L. conducted the neuronal morphology experiments. L.A.W. conducted the in situ hybridization assay. O.D. and L.-H.T. wrote the manuscript with critical input from all of the authors.

Corresponding author

Correspondence to Li-Huei Tsai.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Chd8 is highly expressed in developing mouse and human embryonic brain

(a) Chd8 is highly expressed in early embryonic mouse cortex (n=2 for E12 and n=3 for the rest). (b-c) CHD8 expression in the developing human dorsolateral prefrontal cortex (DFC) (Early fetal (8 post-conceptional weeks (PCW) ≤ Age < 13 PCW); n=5, Early mid-fetal (13 PCW ≤ Age < 19 PCW); n=7, Late mid-fetal (19 PCW ≤ Age < 24 PCW); n=2, Late fetal (24 PCW ≤ Age < 38 PCW); n=3, Infancy (0 months (M) (Birth) ≤ Age < 12 M); n=3, Childhood (1 postnatal year (Y) ≤ Age < 12 Y); n=7) and medial prefrontal cortex (MFC) (Early fetal; n=4, Early mid-fetal; n=7, Late mid-fetal; n=2, Late fetal; n=2, Infancy; n=3, Childhood; n=6). (d) In situ hybridization for Chd8 in E12 and E16 mouse brain. “n” refers to number of different brain tissue samples.

Supplementary Figure 2 Chd8 shRNA efficiency

(a) Both Chd8 sh1 and sh2 significantly reduces Chd8 expression assessed by qRT-PCR (Control; n=5, Chd8 sh1; n=3, Chd8 sh2; n=6). (b) Expression of previously identified target of Chd8 is significantly reduced by shRNAs (n=3 for all). (c) CHD8 shA significantly reduces the expression of CHD8 in human embryonic kidney (HEK) 293T cell line. Chd8 sh1 and sh2 does not target human CHD8 (n=3 for all). All analyses, one-way analysis of variance (one-way ANOVA) followed by Dunnett’s Multiple Comparison Test; *, p-value<0.05; ***, p-value<0.001. “n” refers to number of transfected cell culture samples.

Supplementary Figure 3 Chd8 is necessary to maintain Sox2+ neural progenitor pool but does not affect apoptosis in developing cortex

(a) Images of E16 mouse cortices electroporated at E13 with non-targeting (Control) or Chd8-directed small hairpin (Chd8 shRNA), and GFP expression plasmid. Images were stained for GFP (green), cleaved caspase 3 (CC3) (red), and Sox2 (cyan). (b) Chd8 knockdown did not change the percentage of CC3-positive cell in GP+ population (Control, n=4; Chd8 sh1, n=3; Chd8 sh2, n=4). (c) Chd8 knockdown resulted in reduced percentage of Sox2+ neural progenitors (Control, n=3; Chd8 sh1, n=3; Chd8 sh2, n=3). All analyses, one-way analysis of variance (one-way ANOVA) followed by Dunnett’s Multiple Comparison Test; *, p-value<0.05. “n” refers to number of different brain samples analyzed. Scale bars: 100µm

Supplementary Figure 4 Scatter plots for RNA-seq datasets

(a) Volcano plots showing the correlation between Log2(Fold Change) and Log10(P-value) for each dataset (Left, sh1; right, sh2). (b) Scatter plots for gene expression (Log10(Fold Change + 1)) comparing each replicate within hairpins. The Pearson correlation is given at the top of each plot. (c) Scatter plots for gene expression (Log10(Fold Change + 1)) comparing each replicate across hairpins. The Pearson correlation is given at the top of each plot. (d) Bar graph showing overlap between DEGs between Chd8 sh1 and sh2 at different significance levels.

Supplementary Figure 5 Gene ontology of pathways

(a) Analysis of Gene Ontology of WikiPathways for down-regulated genes from Chd8 knockdown RNA-seq datasets.

Supplementary Figure 6 Chd8 knockdown causes dysregulation of ASD risk genes

(a-b) Overlap between differentially expressed genes following Chd8 knockdown and SFARI ASD risk genes. SFARI ASD; 550 for sh1 and 537 for sh2 genes, Chd8 sh1 DEG; 3281 genes, Chd8 sh2 DEG; 4098 genes. P-values are calculated using hypergeometric distribution test.

Supplementary Figure 7 Chromatin states of E12 mouse cortex

Chromatin state profiling of E12 mouse cortex based on combinatorial patterns of seven histone marks. These chromatin marks include histone3-Lys36-trimethylation (H3K36me3, associated primarily with transcribed regions), H3K4me1 (enhancers), H3K27 acetylation (H2K27ac; enhancer/promoter activation), H3K9ac (active promoters), H3K4me2 (enhancer/promoter activation), H3K4me3 (active promoters) and H3K27me3 (Polycomb repression). TssA: active promoter, TssD: downstream promoter, TssU: upstream promoter, Tx: transcribed, Enh: enhancer, Bival: bivalent, ReprPC: repressed Polycomb, Low: low signal. Color intensity indicates the enrichment of the measured histone mark in the particular state.

Supplementary Figure 8 Chd8 binds to the promoters of Wnt signaling genes both in human and mouse neuronal cells

(a) Chd8/CHD8 binds to the promoter region of multiple genes involved in Wnt signaling in hNPCs, hNSCs and E17.5 mouse brain identified by ChIP-seq12,16. (b) Confirmation of Chd8 binding to the promoters of Fzd1, Dvl3 and Ctnnb1 in E12 mouse brain cortex. Values are percentage input normalized to IgG (Fzd1; n=3; Dvl3; n=3, Ctnnb1; n=4). “n” refers to number of different brain samples used.

Supplementary Figure 9 Chd8 does not regulate expression of Wnt signaling genes in HEK293T cells

(a) No significant change in expression of Wnt signaling genes (FZD1, DVL3, CTNNB1) in Chd8 knockdown samples from HEK293T cells assessed using qRT-PCR (n=3 for all samples; two-tailed student’s t-test; ***, p-value<0.001). “n” refers to number of transfected cell culture samples.

Supplementary Figure 10 Induced Wnt–β-catenin signaling rescues transcriptional dysregulations associated with Chd8 knockdown.

(a) Overlap between differentially expressed genes following Chd8 knockdown with sh2 and differentially expressed genes in β-catenin rescue condition. (b) Rescue of gene expression is validated in subset of genes in independent set of samples using qRT-PCR (Control; n=4, Chd8 sh2+β-catenin: n=3, two-tailed paired-student’s t-test; **, p-value<0.01). “n” refers to number of different FAC sorted samples analyzed.

Supplementary Figure 11 Chd8 knockdown in upper cortical layer neurons does not alter basic locomotor activity or learning behavior

(a-c) Images of adult mouse cortices bilaterally electroporated at E15 with control or Chd8 sh2 or Chd8 sh2+S/A-β-catenin, and membrane-bound GFP expression construct. Scale bars: 1 mm. (d-h) Chd8 knockdown mice exhibit normal basic locomotory activity. All three groups of mice were subjected to open-field test for 10 minutes (Control; n=17, Chd8 sh2; n=12, Chd8 sh2+S/A-β-cat; n=10; one-way ANOVA followed by Dunnett’s Multiple Comparison Test; ns, p-value>0.05). (d) Total distance traveled (m), (e) total time in motion (s), (f) velocity (cm/s), (g) total time spent in the center (s), (h) total time spent in the periphery (s). (i) Chd8 knockdown did not affect learning and memory based on context-dependent freezing in fear conditioning paradigm. “n” refers to number of animals.

Supplementary Figure 12 Chd8 knockdown mice exhibit deficits in social interaction

(a) All three groups of mice did not show any preference for side chambers during the 10 minute habituation period (Control; n=13, Chd8 sh2; n=12, Chd8 sh2+S/A-β-cat; n=10; one-way ANOVA followed by Bonferroni’s Multiple Comparison Test; ns, p-value>0.05). (b) Total time spent with either wired cages (Stranger + Empty) did not show any significant difference between groups (Control; n=6, Chd8 sh2; n=8; Chd8 sh2+S/A-β-cat; n=10; one-way ANOVA followed by Bonferroni’s Multiple Comparison Test; ns, p-value>0.05). “n” refers to number of animals used.

Supplementary Figure 13 Abnormal neuronal positioning and reduced dendritic spine density in Chd8 knockdown animals

(a) Images of 3-month adult mouse cortices electroporated at E15 with control or Chd8 sh2, and cytoplasmic-GFP expression construct. Scale bars: 100 µm. (b) Chd8 knockdown in embryonic brain caused reduced number of neurons produced in adult brain. Same size images are taken from each brain and the total number of GFP+ in each image is counted. Total number of cells are divided by the number of tissue slices used for each brain (Control; n=4, Chd8 sh2; n=5; two-tailed student’s t-test; **, p-value<0.01). “n” refers to number of different brain samples analyzed. (c) Distribution of GFP+ cells in adult mouse brain is shifted away from the brain surface in Chd8 knockdown animals (left panel, in 0.05 mm bins; right panel, 0.5 mm bins). The bins represent the distance from the surface of the brain (Control; n=4, Chd8 sh2; n=5; Two-way ANOVA followed by Bonferroni’s Multiple Comparison Test; *, p-value<0.05). “n” refers to number of different brain samples analyzed. (d) Representative images of dendritic spines of upper cortical layer neurons from control and Chd8 knockdown animals. (e) Chd8 knockdown resulted in reduced spine density in upper cortical layer neurons (Control; n=15 cells from 4 animals, Chd8 sh2; n=16 from 4 animals, Chd8 sh2+ S/A-β-cat; n=18 from 4 animals; one-way ANOVA followed by Bonferroni’s Multiple Comparison Test; *, p-value<0.05; **, p-value<0.01; ***, p-value<0.001). “n” refers to number of neurons analyzed.

Supplementary Figure 14 Full-length western blots.

(a) Full-length western blots of Chd8, Ezh2 and α-tubulin for cropped images in Figure 3f.

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Durak, O., Gao, F., Kaeser-Woo, Y. et al. Chd8 mediates cortical neurogenesis via transcriptional regulation of cell cycle and Wnt signaling. Nat Neurosci 19, 1477–1488 (2016). https://doi.org/10.1038/nn.4400

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