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Phosphorylation of the DNA damage repair factor 53BP1 by ATM kinase controls neurodevelopmental programs in cortical brain organoids [1]

['Bitna Lim', 'Department Of Developmental Neurobiology', 'St Jude Children S Research Hospital', 'Memphis', 'Tennessee', 'United States Of America', 'Yurika Matsui', 'Seunghyun Jung', 'Mohamed Nadhir Djekidel', 'Center For Applied Bioinformatics']

Date: 2024-09

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.

Funding: This work was supported by the American Lebanese Syrian Associated Charities ( https://www.stjude.org/ to JCP), American Cancer Society ( https://www.cancer.org/ ; 132096-RSG-18-032-01-DDC to JCP), and NIH ( https://www.nih.gov/ ; 1R01GM134358-05 to JCP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Lim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The studies mentioned above have contributed to a model of 53BP1, where posttranslational modifications of its different residues and domains coordinate various activities. Most prominently, numerous residues of 53BP1 are phosphorylated by ATM (ataxia telangiectasia mutated) kinase [ 10 , 15 – 17 ]. ATM-mediated phosphorylation of 53BP1 or 53BP1-interacting proteins controls protein interactions, cellular localization, and DNA repair mechanisms [ 11 , 12 , 18 – 20 ]. Despite these discoveries, the impact of phosphorylation on the gene regulatory activity of 53BP1 remains unknown. Here, we report that phosphorylation of 53BP1-serine 25 by ATM is crucial for the proper expression of genetic programs during the growth and development of cortical brain organoids. ATM-dependent phosphorylation controls the chromatin binding of 53BP1 to genomic targets functioning in several key pathways, including neuronal differentiation, cytoskeleton, p53, and ATM, BNDF, and WNT signaling pathways. These results highlight the essential role of 53BP1 phosphorylation in regulating genetic programs for the differentiation of cortical brain organoids.

Studies of 53BP1 have primarily focused on its role in the DNA damage response. To localize to chromatin with double-stranded breaks, 53BP1 uses its BRCT domain to bind to γH2AX, the Tudor domain to bind to H4K20 dimethylation, and its UDR segment to bind to ubiquitinated H2AK15 [ 7 – 10 ]. Additionally, the phosphorylated SQ/TQ motif of 53BP1 coordinates the docking of RIF1 or SCAI, selectively promoting nonhomologous end-joining or reducing homologous recombination [ 11 , 12 ]. These interactions are likely relevant to the gene regulatory activities of 53BP1. For example, γH2AX recruits 53BP1 and is required for resolving R-loops, DNA demethylation, transcription activation, and transcription elongation [ 13 , 14 ]. These findings suggest that the activities of 53BP1 in DNA damage response are interconnected with its gene regulatory functions.

Transcription ensures the proper expression of genetic information for the development and function of the organism, whereas DNA repair maintains the integrity of the genetic code. These 2 processes share cross-functional factors, including CSB, TFII, and XPG, which repair DNA damage caused by torsional stress from transcription-initiating RNA polymerase II [ 1 – 3 ]. Conversely, some proteins initially believed to function exclusively in DNA repair have been found to regulate gene expression. For example, 53BP1 (p53 binding protein 1) is a key regulator of DNA repair mechanisms, promoting nonhomologous end-joining over homologous recombination [ 4 ]. During the DNA damage response, 53BP1 plays a pivotal role in p53-mediated activation of tumor suppressive genetic programs [ 5 ]. Recent research has also revealed that 53BP1 collaborates with the chromatin modifier UTX in neural progenitor cells (NPCs), promoting an open chromatin to facilitate the activation of neurogenic or corticogenic programs [ 6 ]. Intriguingly, the 53BP1–UTX interaction is observed in humans but not in mice [ 6 ]; the mechanism is not well conserved and regulates primate neurodevelopment. These discoveries highlight the importance of 53BP1 in gene regulation for tumor suppression and neural development. However, the precise mechanisms underlying 53BP1’s role in gene regulation and its upstream mechanism are yet to be fully understood.

Results

Phosphorylated 53BP1-S25 increases during differentiation of hESCs into NPCs Although 53BP1 is required for human embryonic stem cells (hESCs) to differentiate into NPCs [6], its levels did not change during differentiation (Fig 1A). Therefore, its regulation is likely posttranslational during neural differentiation. Human NPCs were analyzed by RNA-seq and immunofluorescence to validate successful NPC generation (S1A-S1E Fig). 53BP1 is targeted by various kinases, including ATM, and we hypothesized that 53BP1 phosphorylation regulates the differentiation of hESCs into NPCs. Intriguingly, we found that the levels of 53BP1 phosphorylated at serine 25 (53BP1-pS25) were markedly increased in NPCs compared to hESCs (Figs 1A and S1F-S1H). The levels of the DNA damage marker γH2AX were similar in NPCs and hESCs (S1I Fig), suggesting that the increase in 53BP1-pS25 levels during NPC differentiation is not due to increased DNA damage. PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3002760.g001

ATM safeguards transcriptional and translational programs in differentiating cortical organoids To investigate the molecular basis of the cellular defects we observed in ATM-KO, we performed RNA-seq to compare ATM-KO to WT NPCs and D35 cortical organoids derived from WT and ATM-KO hESCs. The expression of forebrain markers was similar between WT and ATM-KO cortical organoids (and low expression of midbrain and hindbrain markers; S2 Table), suggesting that the ATM-KO cortical organoids specified to the forebrain lineage. A false discovery rate (FDR) <0.05 was used to identify differentially expressed genes. Gene set enrichment analysis (GSEA) showed that up-regulated genes in ATM-KO NPCs were enriched in forebrain development, axis specification, and metabolic pathways (Figs 2F and S6A), whereas down-regulated genes were enriched in neuronal differentiation, epithelial mesenchymal transition, and tube morphogenesis (Fig 2G). Comparison of transcriptomes profiles of D35 cortical organoids from 8 ATM-KO versus 6 WT datasets yielded similar GSEA terms (Fig 3A and 3B). These data suggest that ATM regulates genetic programs related to forebrain development, metabolism, and neuronal differentiation in NPCs and cortical organoids. Dysregulated genetic programs likely contributed to enhanced neuronal maturation and cellular disorganization in ATM-KO cortical organoids. PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3002760.g003 As ATM kinase is crucial for many cell and developmental processes, we aimed to analyze its effect on the proteome and phosphoproteome of differentiating cortical organoids. First, we used multiplexed tandem mass tag-based quantification and 2D liquid chromatography-tandem mass spectrometry (TMT LC-MS/MS) to profile the proteome of WT and ATM-KO D35 cortical organoids (S3B Fig, Methods). We quantified 10,895 proteins between 4 WT, 4 ATM-KO2, 3 ATM-KO3, and 3 ATM-KO14 D35 cortical organoid samples by using the criteria of fold change >1.5 and FDR <0.05 (Fig 3C). Consistency between replicate datasets is supported by principal component analysis (S3B Fig). GSEA showed that compared to WT, up-regulated proteins in ATM-KO were enriched in terms related to neurotransmission, neuron spine, dendrite, synapse, and axon (S3C Fig), whereas down-regulated proteins were enriched in BMP/TGFβ and WNT signaling, epithelial morphogenesis, and stem cell differentiation (S3D Fig). These data suggest that ATM controls posttranscriptional and translational gene regulation to suppress neuronal function and promote stem cell differentiation, epithelial morphogenesis, and TGFβ and WNT signaling pathways in D35 cortical organoids. We have observed distinct patterns in the transcriptomics and proteomics data in ATM-KO versus WT cortical organoids. Interestingly, while transcriptomic programs related to neuronal differentiation were down-regulated (Fig 3B), proteomic programs related to neuronal function were up-regulated (S3C Fig) in ATM-KO versus WT cortical organoids. These differential patterns in transcriptomics and proteomics are likely a consequence of the regulatory role of ATM in multiple cellular processes. It is possible that the higher protein expression related to neuronal functions in ATM-KO lead to down-regulation of transcriptional expression of neuronal differentiation programs. This would suggest that the dysregulated transcriptomic and proteomic programs in ATM-KO cortical organoids are interconnected and result from the complex interplay of ATM’s regulation of various cellular pathways. These findings shed light on the intricate role of ATM in coordinating gene expression and protein levels, influencing neuronal differentiation and function in cortical organoids.

ATM-dependent phosphorylation controls signaling pathways for neurogenesis, stem cell differentiation, and morphogenesis in cortical organoids To investigate how ATM exerts its modulatory control during cortical organoid formation, we performed phosphoproteomics analysis of WT and ATM-KO cortical organoids. Using TMT LC-MS/MS, we quantified 22,646 phosphopeptides and normalized their abundance based on the protein abundance measured in the total proteomics analysis. A comparison between WT and ATM-KO lines revealed that 198 proteins had consistently lower levels of phosphorylation in at least 2 of the 3 ATM-KO lines (log2(fold change >1.5) and FDR <0.05; Fig 3D and S3 Table). Among these proteins, 53BP1 and EIF4EBP1 were known substrates of ATM (Fig 3E) [15,16,22,23], validating the approach to identify putative ATM substrates in cortical organoids. However, it is essential to note that this approach does not distinguish between direct and indirect effects, and, therefore, some of the identified proteins could be phosphorylated by protein kinases that require ATM for their activity. Notably, many ATM-dependent phosphorylated proteins were found to be key neurodevelopmental regulators (Fig 3E) and enriched in functions related to neurodevelopment, neurogenesis, cell morphogenesis, and cytoskeleton (Fig 3F). These findings suggest that ATM plays a critical role in regulating the phosphorylation of proteins involved in essential processes for neurodevelopment and neuronal function in cortical organoids. We further explored the effects of ATM by identifying protein kinases that had ATM-dependent phosphorylation. We used the IKAP machine learning algorithm [24] to analyze substrates (inferred from literature curation) and deduce the activities of those kinases. For example, in ATM-KO compared to WT, we found reduced phosphorylation of proteins related to MAPK9 activities, such as DCX, MAPT, and NFATC4 (S7A Fig and S4 Table) [24]. On the other hand, we found higher phosphorylation of proteins related to CDK5 activities, including ADD2, ADD3, DCX, DNM1L, DPYSL3, MAPT, and SRC (S7B Fig and S4 Table) [24]. In ATM-KO, we inferred lower activities in MAPK9, CDK2, CHEK1, ATR, CSNK1A1, MTOR, CAMK2A, and PRKACA (Figs 3G and S7C), with enriched functions in ATM signaling, BNDF signaling, and axon guidance (Figs 3G, 3H, and S7C). On the other hand, we inferred higher activities in GSK3B, MAPK3, PAK1, CSNK2A1, CDK5, CDK1, and PRKDC (Figs 3G, 3I, and S7C), with enriched function in ATM signaling, WNT signaling, G2/M checkpoint, and p53 regulation in ATM-KO (Fig 3H and 3I). ATM KO leads to both lower and higher activities of kinases in ATM signaling. Additionally, some of the altered kinase activities could be secondary to ATM-KO, as CHEK1, ATR, and PRKDC were known substrates of ATM [23,25]. Overall, these data suggest that the activities of kinases related to ATM signaling, BNDF signaling, WNT signaling, G2/M checkpoint, and p53 regulation became dysregulated in ATM-KO D35 cortical organoids. We thus conclude that ATM plays a crucial role in controlling key neurodevelopmental regulators. The dysregulated phosphorylation and activities of these regulators disrupt the normal transcriptomic program responsible for neuronal differentiation, leading to higher proteomic programs associated with neuronal function. As a consequence, the dysregulated programs in ATM-KO cortical organoids are likely responsible for the observed defects in neurogenesis and morphogenesis (formation of ventricular zone–like regions). These findings provide valuable insights into the role of ATM in neurodevelopment and shed light on potential molecular mechanisms underlying neurological disorders associated with ATM dysfunction.

53BP1-S25A and S25D predominantly alter the expression of 53BP1 target genes To obtain further mechanistic insights into the role of 53BP1 in controlling gene expression, we reanalyzed 53BP1 ChIP-seq data (using 2 separate anti-53BP1 antibodies) in WT NPCs [6]. Using SICER [27] and MACS2 [28] with a criterion of FDR <0.05, we identified 37,519 targets bound by 53BP1. About 41% of these 53BP1 targets localize to promoter regions, suggesting a transcriptional regulatory role of 53BP1 (S14D Fig). Remarkably, more than 82% of the differentially expressed genes in 53BP1-S25A and 53BP1-S25D D35 cortical organoids were found to be targets bound by 53BP1 (Figs 7A, S13E, and S13F). 53BP1 target genes with increased transcript levels in the mutant organoids were highly enriched in neuronal development, axonogenesis, neuron projection, synapse organization, and neurotransmitter transport, transmission, and signaling (Fig 7B). On the other hand, 53BP1 targets with reduced transcript levels in the mutant organoids were enriched in IRE1-mediated unfolded protein response, cellular response to stress, iron import, and apoptosis regulation (S13G Fig). Of note, genes involved in IRE1-mediated unfolded protein response and apoptosis regulation showed reduced expression upon loss of ATM or mutation of 53BP1-S25 and were identified as direct targets of 53BP1 in NPCs. This suggests that ATM-mediated phosphorylation of 53BP1-S25 directly promotes the expression of these genes to maintain NPCs during formation of cortical organoids. PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3002760.g007 We wanted to test whether ATM alters 53BP1 binding, considering ATM is required for 53BP1-pS25 in D35 cortical organoids (Fig 1F) and NPCs (S2B Fig). A comparison of 53BP1 ChIP-seq in WT and ATM-KO NPCs showed that ATM-KO altered 53BP1 binding to chromatin (S14A-S14C Fig). ATM-KO reduced 53BP1 binding at specific sites, with 96.3% of these sites being promoters (S14C Fig). To explore the impact of 53BP1-pS25, we performed CUT&RUN in 2 separate WT NPC lines. Our analysis revealed that 67.1% of 53BP1-pS25 targets localize to promoter regions, suggesting a transcriptional regulatory role (S14D Fig). Under the criteria of fold-change >2 and p < 0.05, 58.6% (3,390/5,789) of 53BP1-pS25 targets overlapped with 53BP1 targets (Fig 7C); the nonoverlapped sites may be attributed to differences in ChIP versus CUT&RUN procedures and the accessibility of 53BP1 versus 53BP1-pS25 antibodies. 53BP1-pS25 targets were significantly enriched in 414 up-regulated genes in NPCs versus ESCs (Fig 7D), suggesting a role of 53BP1-pS25 in promoting their expression in NPCs. Genes having overlapped 53BP1 ChIP-seq and 53BP1-pS25 CUT&RUN signals were enriched in chromatin remodeling, DNA metabolism, RNA splicing, translation, transcription, cell cycle, and neuron development (S14E Fig). These data suggest that ATM can alter 53BP1 binding and that 53BP1-pS25 is enriched in the promoters of genes regulating cellular processes and neurodevelopment.

Phosphorylation of 53BP1-S25 controls the localization of 53BP1 to chromatin for gene regulation To investigate the impact of 53BP1-S25 on the genomic distribution of 53BP1, we performed ChIP-seq in 53BP1-WT, S25A, and S25D NPCs. Two independent NPC lines were used for each group, and the ChIP-seq data were subjected to principal component analysis, which showed high consistency between the replicate dataset (S8A Fig). We used SICER [27] and MACS2 [28] with the criteria of fold-change >2 and p < 0.05 to perform pairwise comparisons of the merged datasets from 53BP1-WT, S25A, and S25D ChIP-seq experiments. The pairwise comparisons identified thousands of 53BP1-bound regions that were significantly different between 53BP1-WT, S25A, and S25D. Notably, the regions that significantly gained binding in 53BP1-S25A or S25D versus WT were highly enriched at promoters (within 2 kb of transcription start sites), constituting 82% and 71.1%, respectively (S8B and S8C Fig). In contrast, the regions that significantly lost binding in 53BP1-S25A or S25D versus WT were not as enriched at promoters, constituting 32.6% and 33%, respectively (S8B and S8C Fig). We generated heatmaps to visualize the genomic regions with significantly different 53BP1 binding intensity (compared against control regions). The heatmaps confirmed consistent changes in 53BP1 binding patterns between 53BP1-S25A and S25D versus WT, and between 53BP1-S25A versus S25D (Figs 5C, 5D, and S8D). These data support that 53BP1-S25 and its phosphorylation control the genomic distribution of 53BP1 on chromatin. We next set out to examine the correlation between changes in 53BP1 distribution on chromatin and changes in gene expression in 53BP1-WT, S25A, and S25D cortical organoids. We performed GSEA and made some notable observations. Firstly, regions that gained 53BP1 binding in 53BP1-S25A or S25D cortical organoids, as compared to WT, were enriched with up-regulated genes (Fig 5E and 5F). Similarly, regions that had lower 53BP1 binding were enriched with down-regulated genes in 53BP1-S25A or S25D versus WT (S8E and S8F Fig). These results suggest that the 53BP1-S25A or S25D mutation directly influences 53BP1 binding and gene expression and subsequently regulates gene expression, particularly at promoters where higher 53BP1 binding leads to higher gene expression. Interestingly, genes that lost 53BP1-S25A or S25D protein binding had minimal overlap in GSEA terms, except promoters occupied with H3K4me3 and regulation of epithelial-mesenchymal transition (S8E and S8F Fig). In contrast, the genes that gained 53BP1-S25A or S25D protein binding were enriched with promoters marked by bivalent histone marks (H3K4me3 and H3K27me3) or occupied by H3K27me3 alone [29] (Fig 5E and 5F), suggesting that 53BP1-S25D or S25A proteins preferentially bind to these promoters and subsequently up-regulate gene expression. Moreover, genes that gained 53BP1-S25A or S25D binding shared common functions related to sodium ion transmembrane transporter, DNA replication, positive regulation of cell division, and regulation of histone H3K4 methylation (Fig 5E and 5F). This suggests that despite the 1,187 regions showing different 53BP1 bindings between 53BP1-S25A and S25D (S8D Fig), both mutations impact genes involved in neuronal functions and cell proliferation. Altogether, these findings show that 53BP1-S25A and S25D mutations have a direct impact on 53BP1 binding to chromatin and subsequently affecting gene regulation. We propose that 53BP1-pS25 likely inhibits 53BP1 binding to promoters associated with bivalent and H3K27me3-occupied promoters. This inhibition may lead to the reduced expression of genes involved in the regulation of H3K4me3, neuronal functions, and cell proliferation.

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