Phosphorylation of the DNA damage repair factor 53BP1 by ATM kinase controls neurodevelopmental programs in cortical brain organoids

Abstract

53BP1 is a well-established DNA damage repair factor that has recently emerged to critically regulate gene expression for tumor suppression and neural development. However, its precise function and regulatory mechanisms remain unclear. Here, we showed that phosphorylation of 53BP1 at serine 25 by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical brain organoids. Dynamic phosphorylation of 53BP1-serine 25 controls 53BP1 target genes governing neuronal differentiation and function, cellular response to stress, and apoptosis. Mechanistically, ATM and RNF168 govern 53BP1's binding to gene loci to directly affect gene regulation, especially at genes for neuronal differentiation and maturation. 53BP1 serine 25 phosphorylation effectively impedes its binding to bivalent or H3K27me3-occupied promoters, especially at genes regulating H3K4 methylation, neuronal functions, and cell proliferation. Beyond 53BP1, ATM-dependent phosphorylation displays wide-ranging effects, regulating factors in neuronal differentiation, cytoskeleton, p53 regulation, as well as key signaling pathways such as ATM, BDNF, and WNT during cortical organoid differentiation. Together, our data suggest that the interplay between 53BP1 and ATM orchestrates essential genetic programs for cell morphogenesis, tissue organization, and developmental pathways crucial for human cortical development.

Fig 1. ATM binds 53BP1, is required for pS25-53BP1, and promotes cortical organoid differentiation.

(A) WB of the nuclear extract of hESCs and hNPCs showed marked increase of 53BP1-pS25 in hNPCs. WB analysis of IgG, (B) 53BP1, and (C) ATM co-immunoprecipitation in the nuclear extract of hESCs. (D) Quantification of the relative ATM protein levels (normalized to β-ACTIN) in 5 replicate WB analyses of hESCs and hNPCs. (E) Schematic diagram of the cortical organoid differentiation. Aggregates were formed in the induction media for 17 days, embedded in Matrigel droplets and cultured in cortical differentiation medium for 16 days, and then cultured in cortical maturation media thereafter. (F) WB analysis of WT and ATM-KO cortical organoids at day 35 of differentiation. Immunofluorescence of (G) PAX6 and CTIP2 and (J) KI67 in cryosections of cortical organoids at day 35 of differentiation. Bar, 100 μm. At day 35 of differentiation, the (H) area and (I) thickness of VZ-like regions were compared between groups. Data points represent single organoids. The mean ± SEM values were compared by one-way ANOVA with Dunnett’s multiple comparisons test to yield **** indicating p < 0.0001. n = 13 organoids/group. Underlying numerical values for figures are found in S1 Data. ATM, ataxia telangiectasia mutated; DMEM, Dulbecco’s Modified Eagle Medium; GMEM, Glasgow Modified Essential Medium; hESC, human embryonic stem cell; hNPC, human neural progenitor cell; IgG, immunoglobulin G; KO, knockout; KSR, Knockout Serum Replacement; VZ, ventricular zone; WB, western blot; WT, wild type; 53BP1, p53 binding protein 1; 53BP1-pS25, 53BP1 phosphorylated at serine 25.

Fig 2. Analysis of neuronal differentiation and cell organization in cortical organoids.

Quantification of PAX6/CTIP2 ratios in (A) D28 and (B) D37 cortical organoids. (C) Quantification of NEUN/DAPI in D37 cortical organoids. (D) In D37 cortical organoids, 6 organoids were surveyed to count ZO-1-positive apical surfaces and proportions of PAX6-positive NPCs that are organized around the apical surfaces. (E) Proportions of PH3-positive cells that are adjacent to ZO-1-positive apical surfaces (“rings”). Data from 53BP1-S25A and S25D were included for comparison. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant by Welch’s t test in (A-C) and two-way ANOVA test in (D). From GSEA, functional terms that are highly enriched in (F) up-regulated and (G) down-regulated genes in ATM-KO vs. WT NPCs. % Match, % of genes in the enriched term that overlap the differentially expressed genes or proteins. Underlying numerical values for figures are found in S1 Data. ATM, ataxia telangiectasia mutated; GSEA, gene set enrichment analysis; KO, knockout; NES, normalized enrichment score; NPC, neural progenitor cell; WT, wild type; 53BP1, p53 binding protein 1.

Fig 3. Transcriptomic and proteomic profiles of WT versus ATM-KO cortical organoids.

From GSEA, functional terms that are highly enriched in (A) up-regulated and (B) down-regulated genes in ATM-KO D35 cortical organoids. % Match, % of genes in the enriched term that overlap the differentially expressed genes or proteins. (C) Schematic diagram outlining TMT LC-MS/MS profiling of total proteomics and phosphoproteomics of D35 WT and ATM-KO cortical organoids. TMT signals from total proteomics were used to normalize those of phosphopeptides. (D) Using FC>1.5 and FDR<0.05, 198 phosphoproteins were found to be lower in 2 ATM-KO versus WT. (E) Normalized levels of phosphoproteins that have ATM-dependent phosphorylation in D35 cortical organoids. 53BP1 and EIF4EBP1 were known substrates of ATM. The error bars depict the mean and standard error of the mean values, which were calculated based on the normalized levels of each phosphopeptide in the protein. (F) Enrichment of proteins with ATM-dependent phosphorylation in specific functional categories. (G) Heatmap showing altered activities of kinases between D35 ATM-KO2 and WT cortical organoids. Relative changes in kinase activity are shown as row Z‐scores. Kinase activity was inferred by IKAP [25] based on normalized substrate phosphorylation levels from phosphor-proteome. The normalization was performed by dividing phosphor-peptide abundance of each protein by corresponding protein abundance [57]. Circos plots showing kinases with inferred (H) higher and (I) lower activities in D35 ATM-KO versus WT cortical organoids and their corresponding enriched pathways. Underlying numerical values for figures are found in S1_Data.xlsx. ATM, ataxia telangiectasia mutated; FC, fold-change; FDR, false discovery rate; GSEA, gene set enrichment analysis; KO, knockout; NES, normalized enrichment score; TMT LC-MS/MS, liquid chromatography-tandem mass spectrometry; WT, wild type; 53BP1, p53 binding protein 1.

Fig 4. 53BP1-pS25 is required for the differentiation of cortical organoids.

(A) In the endogenous 53BP1 locus, the codon TCT encoding serine-25 in was mutated to GCT and GAT encoding alanine and glutamate, respectively. (B) Bright-field images of cortical organoids formed by 4 53BP1-S25A lines, 4 53BP1-S25D lines, and 2 WT control at day 35 of differentiation. Bar, 1.5 mm. At day 35 of differentiation, the (C) organoid size and (F) area of ventricular zone–like region were compared between groups. Data points represent single organoids. The mean ± SEM values were compared by one-way ANOVA with Dunnett’s multiple comparisons test to yield ****, ***, **, *, and ns indicating p < 0.0001, 0.001, 0.01, 0.05, and not significant, respectively. n = 39–47 organoids/group for (C) and 15–33 organoids/group for (F). (D) Immunofluorescence of PAX6 and CTIP2 in cryosections of cortical organoids at day 35 of differentiation. Bar, 100 μm. (E) Illustration of ventricular zone–like areas in cortical organoids. Underlying numerical values for figures are found in S1 Data. WT, wild type; 53BP1, p53 binding protein 1; 53BP1-pS25, 53BP1 phosphorylated at serine 25.

Fig 5. 53BP1-S25A and S25D lower cell proliferation in cortical organoids.

FACS quantified ratios of (A) KI67, (B) PAX6, and (C) CTIP2 to total cells in D28 cortical organoids. (D) Immunofluorescence of PAX6 and CTIP2 in D28 cortical organoids. Bar, 100 μm. Quantification of immunofluorescence signals of (E) PAX6/DAPI, (F) CTIP2/DAPI, and (G) PAX6/CTIP2 in D28 cortical organoids. Each data point represents quantification of cells in 1 cortical organoid. Quantification of (H) KI67/PAX6 and (I) PAX6/CTIP2 ratios in immunofluorescence of D35 cortical organoids. Each data point represents quantification of cells in 1 cortical organoid. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant by two-way ANOVA test. Underlying numerical values for figures are found in S1 Data.

Fig 6. 53BP1-S25 phosphorylation enforce the appropriate expression of genetic programs for cortical organoid differentiation.

(A) Number of differentially expressed genes identified by pairwise comparisons at FDR <0.05. At day 35 of differentiation, 53BP1-S25A and S25D cortical organoids are molecularly similar. (B) Differentially expressed genes in 53BP1-S25D versus WT overlap 87% (764/875) and 91% (361/396) of those in 53BP1-S25A versus WT. (C) Extensive overlap of up-regulated GSEA terms between 53BP1-S25A versus WT and 53BP1-S25D versus WT. Most terms relate to axon, synapse, and neurotransmitter. (D) Of 53BP1 target genes up-regulated by S25A and S25D, 212 genes require WT 53BP1 for expression in cortical organoids. (E) The 212 genes are enriched in functions related to transcriptional regulation, neuron projection, axonogenesis, synapse, neurotransmitter synthesis and transport, and membrane depolarization. (F) Venn diagrams depict high overlaps between down-regulated genes in all 3 groups of mutant versus WT pairwise comparisons. (G) GSEA graphs showed that down-regulated genes in 53BP1-S25A or S25D vs. WT had significant enrichment in down-regulated genes of ATM-KO vs. WT cortical organoids. P values were calculated by the hypergeometric test, assuming normal data distribution. (H) GSEA terms of the 115 genes that were down-regulated in all 3 groups (versus WT) revealed the genetic programs copromoted by ATM and 53BP1-pS25. ATM, ataxia telangiectasia mutated; FDR, false discovery rate; GSEA, gene set enrichment analysis; KO, knockout; WT, wild type; 53BP1, p53 binding protein 1.

Fig 7. 53BP1-pS25 positively and negatively regulate 53BP1 target genes.

(A) More than 82% of differentially expressed genes in 53BP1-S25A or S25D versus WT are chromatin targets bound by 53BP1 in WT NPCs. (B) 53BP1-S25A and S25D up-regulate 53BP1 targets that are involved in neuron development and projection, axonogenesis, synapse, and neurotransmitter synthesis and transport. (C) Heatmaps aligning peaks with 53BP1-pS25 CUT&RUN and 53BP1 ChIP-seq signals in WT NPCs. Input track was included as a negative control. n = numbers of peaks with differential and overlapped bindings. Criteria of FC>2 and p < 0.05 were used for comparison. (D) GSEA graph of 53BP1-pS25 CUT&RUN signals in genes that were lower in ESCs vs. NPCs, which were up-regulated in NPCs. P values were calculated by the hypergeometric test, assuming normal data distribution. Heatmaps aligning peaks with significantly different 53BP1 ChIP-seq signals in (E) 53BP1-S25A vs. WT and (F) 53BP1-S25D vs. WT, using the criterion of FC>2 and p < 0.05. Control peaks are those, after voom normalization, showed the least changes and served as semi-independent validation of differential ChIP-seq analysis. Bubble graphs present top enriched categories of genes that had significantly higher 53BP1 ChIP-seq in (G) 53BP1-S25A vs. WT and (H) 53BP1-S25D vs. WT. ESC, embryonic stem cell; FC, fold-change; GSEA, gene set enrichment analysis; NPC, neural progenitor cell; WT, wild type; 53BP1, p53 binding protein 1; 53BP1-pS25, 53BP1 phosphorylated at serine 25.