Autism-associated CHD8 keeps proliferation of human neural progenitors in check by lengthening the G1 phase of the cell cycle

Abstract

De novo mutations (DNMs) in chromodomain helicase DNA binding protein 8 (CHD8) are associated with a specific subtype of autism characterized by enlarged heads and distinct cranial features. The vast majority of these DNMs are heterozygous loss-of-function mutations with high penetrance for autism. CHD8 is a chromatin remodeler that preferentially regulates expression of genes implicated in early development of the cerebral cortex. How CHD8 haploinsufficiency alters the normal developmental trajectory of the brain is poorly understood and debated. Using long-term single-cell imaging, we show that disruption of a single copy of CHD8 in human neural precursor cells (NPCs) markedly shortens the G1 phase of the cell cycle. Consistent with faster progression of CHD8+/- NPCs through G1 and the G1/S checkpoint, we observed increased expression of E cyclins and elevated phosphorylation of Erk in these mutant cells - two central signaling pathways involved in S phase entry. Thus, CHD8 keeps proliferation of NPCs in check by lengthening G1, and mono-allelic disruption of this gene alters cell-cycle timing in a way that favors self-renewing over neurogenic cell divisions. Our findings further predict enlargement of the neural progenitor pool in CHD8+/- developing brains, providing a mechanistic basis for macrocephaly in this autism subtype.

Keywords: ASD; Autism; CHD8; CRISPR/Cas gene editing; Cell cycle; Chromatin; Cortical development; Stem cell.

Fig. 1.

Disruption of a single allele of CHD8 in hESCs by CRISPR/Cas9 gene editing. (A) Genomic organization of the CHD8 locus. Boxed in red are the first two conserved exons of the two CHD8 variants against which sgRNAs were designed. The regions across exon–exon boundaries amplified by proximal and distal TaqMan primers are indicated in green. (B) Single-allele sequencing reveals the insertion of a single nucleotide C (G in the opposite strand sequenced here) at a frequency consistent with a heterozygous mutation (C). (D) DNA and protein sequence alignments of the wt (top) and mutated (bottom) allele of the E11 clone. The one base pair insertion introduces a stop codon shortly after the edited site. (E) TaqMan RT-qPCR showing fold-change in the CHD8 mRNA transcript in E11 relative to wt hESCs (n=3). Error bars indicate s.t.d. (F,G) Immunofluorescence staining of CHD8 in WT and E11 hPSCs. (G) Quantification of CHD8 fluorescence intensity in individual nuclei of wt (n=114) and E11 (n=101) hPSCs. P-value was obtained by an unpaired t-test.

Fig. 2.

Flow cytometry reveals subtle differences in cell cycle profile of CHD8^+/− hESCs and hNPCs. (A-D) Immunostaining of Oct4 (green) and Pax6 (red) in CHD8^+/+ (A,C) and CHD8^+/− (B,D) hESCs (A,B) and hNPCs (B,D). (E-I) Cell cycle profile by DNA content analysis. (E) PI Histogram of wt hESCs (cell number versus PI intensity) showing the distribution of cells in G0-G1, S and G2-M. (F,H) fraction of wt and mutant cells in G0-G1, S and G2/M for hESCs (F) and hNPCs (H). (G,I) Mean G2-M/G1-G0 ratio for hESCs (G) and hNPCs (I). n=3, error bars indicate 95% CI. P-values obtained by Mann–Whitney U-test.

Fig. 3.

Minimal perturbation of cell cycle timing in CHD8^+/− hESCs. (A,B) Color-based detection of G1 and S/G2/M by the FUCCI fluorescent reporter. The G1 and S/G2/M probes are expressed as a single open reading frame using the T2A self-cleaving peptide sequence ensuring stochiometric expression of both probes. (C) Measurement of G1 and S/G2/M duration based on FUCCI intensity traces (see Materials and Methods). (D-H) High-content FUCCI imaging in wt (d,f,h) and mutant (e,g,h) hESCs. (D,E) Snapshots of CHD8^+/+ (D) and CHD8^+/− (E) hESCs during cell cycle progression (∼17 h). White arrows show a cell initially in S/G2 undergoing mitosis and cytokinesis and its two daughter cells transitioning from G1 to S/G2. Time is in h and min. Scale bar: 20 µm. (F,G) FUCCI intensity traces of mother and daughter cells marked by an arrow in D and E. The green y axis (left) indicates mAG-Geminin_1-110 intensity (S/G2/M), the red y axis (right) indicates mKO2-Cdt1_30-120 intensity (G1). Grey bars indicate cytokinesis (M/G2). Orange bars indicate G1/S transitions. (H) Box plots showing duration of G1 and S/G2/M in CHD8^+/+ (n=48) and CHD8^+/− (n=49) cells. P-values were measured using a Mann–Whitney U-test.

Fig. 4.

Marked reduction of G1 length in CHD8^+/− hNPCs. (A,B) Time-lapse imaging of clonal lineages in wt (A) and mutant (B) NPCs going through several rounds of cell division over the course of ∼70 h. Cells corresponding to a single lineage are marked by white arrows. Time is indicated in h and min. Scale bar: 20 µm. (C,D) Lineage reconstruction and FUCCI intensity traces of individual cells marked by an asterisk in A and B for wt (C) and mutant (D) NPCs. The green y axis (left) indicates mAG-Geminin_1-110 intensity (S/G2/M), the red y axis (right) indicates mKO2-Cdt1_30-120 intensity (G1). Orange bars show G1/S transitions. (E) Box plots show duration of G1 and S/G2/M in CHD8^+/+ (n=23) and CHD8^+/− (n=34) cells. P-values were measured using a Mann–Whitney U-test.

Fig. 5.

CHD8 represses cyclins E and MAPK signaling. (A) RT-qPCR analysis of CHD8^+/+ and CHD8^+/− hNPCs. Fold-change (mut/wt) in mRNA expression is shown for the indicated genes (n=4, error bars indicate s.t.d.). (B,C) pErk (B) and Pan-Erk (C) immunostaining in CHD8^+/+ and CHD8^+/− hNPCs. Arrows point to pERK in the nucleus. Scale bar: 20 µm. (D,E) Quantification of pERK total intensity (D) and nucleus to cytoplasm intensity ratio (E) in CHD8 wt (n=109) and mutant cells (n=116). (F) Quantification of total Erk intensity in CHD8 wt (n=97) and mutant (n=80) cells. Intensity data are visualized in swarmplots and bar graphs (mean Intensity±95% CI). P-values were measured using a Mann–Whitney U-test.

Fig. 6.

A model for CHD8 regulation of G1 length and S phase entry. Loss-of-function of a single CHD8 allele shortens the G1 phase of the cell cycle in neural stem cells by relieving transcriptional repression of the MAPK pathway and cyclins E. Truncated G1 causes overproliferation of cortical progenitors by accelerating the cell cycle and by promoting self-renewing divisions at the expense of neurogenic ones.