Transcription-associated DNA DSBs activate p53 during hiPSC-based neurogenesis

Abstract

Neurons are overproduced during cerebral cortical development. Neural progenitor cells (NPCs) divide rapidly and incur frequent DNA double-strand breaks (DSBs) throughout cortical neurogenesis. Although half of the neurons born during neurodevelopment die, many neurons with inaccurate DNA repair survive leading to brain somatic mosaicism. Recurrent DNA DSBs during neurodevelopment are associated with both gene expression level and gene length. We used imaging flow cytometry and a genome-wide DNA DSB capture approach to quantify and map DNA DSBs during human induced pluripotent stem cell (hiPSC)-based neurogenesis. Reduced p53 signaling was brought about by knockdown (p53^KD); p53^KD led to elevated DNA DSB burden in neurons that was associated with gene expression level but not gene length in neural progenitor cells (NPCs). Furthermore, DNA DSBs incurred from transcriptional, but not replicative, stress lead to p53 activation in neurotypical NPCs. In p53^KD NPCs, DNA DSBs accumulate at transcription start sites of genes that are associated with neurological and psychiatric disorders. These findings add to a growing understanding of how neuronal genome dynamics are engaged by high transcriptional or replicative burden during neurodevelopment.

Figure 1

Neural p53^KD increases cell number, DNA DSBs, and mitotic activity. (A,B) Marked reduction in p53 protein in p53^KD neurogenesis (Unpaired t-test, p < .01). Blot is cropped, original blot presented in Supplementary Figure S7A. (C) After 6 weeks of neuronal differentiation, p53^KD cultures have > threefold more cells than control (Error Bars = Mean +/− SEM, unpaired t-test, p < .001). (D,E) Representative images of Control and p53^KD neuronal cultures stained with DAPI and Fluoropan (Neuron Specific Cocktail antibody). (F) Pooled stacked histogram of γH2AX puncta per nucleus from two neurotypic cell lines (BJ, 9429). p53^KD neurons (Tuj1 +) have significantly more DNA DSBs (N = 47,406, Chi-squared test, p < .0001). (G) Representative IFC images of neurons with γH2AX foci. (H,I) p53^KD (Tuj1 +) vs Control have fewer cells in G1/G0 (Two-way ANOVA, Tukey’s Test p < .01), and more cells in G2/M (Two-way ANOVA, Tukey’s Test p < .01).

Figure 2

Nutlin treatment rescues p53^KD phenotypes in early neurogenesis. Nutlin treatment rescues delayed neurogenesis phenotypes in p53^KD at 1 week of neuronal differentiation. (A) One week treatment with 15 μM Nutlin-3 partially restores total p53 levels in p53^KD neural cells. Blot is cropped, original blot presented in Supplementary Figure S7B. (B,C) p53^KD neuronal cultures have more Ki67 + cells vs control and nutlin treated p53^KD (One-way ANOVA, Dunnett’s Test p < .01). (D) p53^KD have less cell death vs controls (One-way ANOVA, Dunnett’s Test, p < .05). (E) Control cells had the highest percentage of Tuj1 + cells vs p53^KD cells (One-way ANOVA, Dunnett’s test, p < .05). No significant difference between controls vs Nutlin p53^KD (One-way ANOVA, Dunnett’s Test p = .10). (F) Nestin +/Tuj1 + cells were strikingly enriched in p53^KD cultures (One-way ANOVA, Dunnett’s Test p < .01). These account for the Tuj1 + cells in G2/M from Fig. 1. p53^KD cells treated with DMSO had significantly more double positive cells, nutlin treatment significantly reduced this population (One-way ANOVA, Dunnett’s Test, p = .51). (G,H,I) Representative imaging flow cytometry plots of samples of control, p53^KD, and p53^KD treated with Nutlin. (J) Nutlin treatment did not reduce DNA DSB level in p53^KD vs control (p < .0001, KS test) (p < .0001, KS test). (K) Representative images from imaging flow cytometry.

Figure 3

p53^KD leads to an acute increase in TSS-associated DSBs. In highly transcribed genes (n = 2542), DNA DSBs are enriched at the TSS of NPCs treated for 1 day (A) and 4 days (B). (C) Elevated DNA DSB levels in p53^KD NPCs are associated with gene expression levels in 1 day cultures. Boxplot of DSB coverage (TSS ± 500 bp) in 10 equal bins based on the gene expression. p53^KD NPCs have more DNA DSBs in highly transcribed genes compared to DMSO Control cells (Wilcoxon signed-rank test, two-sided, paired test, p < .00001). (D) After 4 days APH treatment, control NPCs treated with APH now have more DNA DSBs compared to untreated, p53^KD, and p53^KD treated with APH in highly transcribed genes (Wilcoxon signed-rank test, two-sided, paired test, p < .00001). The box plot shows the 25th and 75th percentile; the middle line is the median; the whiskers span 5% to 95%, and outliers are not shown.

Figure 4

TOP1 inhibition elicits a robust p53 response in human NPCs. (A) NPCs treated with either 400 nM APH or 100 nM Camptothecin (CPT) had significantly more DNA DSBs per nucleus (Chi-squared, p < .0001, N = 63,436 cells). CPT cells had more DSB/cell (KS test, p < .0001, N = 63,436 cells). (B,C) NPCs exposed to either APH or CPT show significant S-phase arrest (Two-way ANOVA, Dunnett’s Test p < .0001). (D,E) NPCs have more pATM foci per nucleus after CPT treatment (One-way ANOVA, Dunnett’s Test, p < .0001) and less pATM foci per nucleus in replication stress (APH) (One-way ANOVA, Dunnett’s Test, p < .01). (F) Western blots identify elevated pChk2 (unpaired t-test, p < .05), phosphorylated p53 (unpaired t-test, p < .001), and γH2AX (unpaired t-test, p < .001). (G) Caspase-3 positive cells were elevated only in CPT treated NPCS (One-way ANOVA, Dunnett’s Test p < .0001) (One-way ANOVA, Dunnett’s Test, p = .98). (H) Single-cell RNA seq reveals that CPT-treated NPCs have differential expression of p53 target genes involved in DNA damage response, cell cycle arrest, and cell death (N = 4598, n is number of cells). TSNE plots shown for each condition. TSNE 1 and TSNE 2 are the same for each condition. (I) Functional pathway analysis of the top 100 highest variant genes from the 592 cells in the red cluster (enriched in CPT treatment) are involved cellular processes related to DNA damage checkpoint (p < 1.37E−22), cell death (p < 1.16E−22), and p53 mediators (p < 1.16E−22). (J) The percentage of cells in each cluster per condition is shown in a pie chart.

Figure 5

Recurrent sites of p53-susceptible DNA DSBs are associated with neurodevelopment and psychiatric disease. (A) 61 genes had > 1.5 fold DNA DSB increase in p53^KD NPCs vs control and were analyzed for known associations with disease. 47% of these genes are involved in the nervous system. Among them, more than 40% play role in intellectual disability and 33% in psychiatric disorders (right). (B) An IGV plot of three genes associated with disorders of the nervous system showing an increase in DNA DSBs in p53^KD vs controls are shown (from top to bottom: STAT3, MAN1B1, STARD9). Red boxes highlight the TSS where the differences in DNA DSBs were quantified.