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Mechanism of cytarabine-induced neurotoxicity

Nature volume 643, pages 1400–1409 (2025)Cite this article

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Abstract

Postmitotic neurons have high levels of methylated cytosine and its oxidized intermediates such as 5-hydroxymethylcytosine1. However, the functional relevance of these epigenetic modifications of DNA are poorly understood. Here we show that some cytidine analogues, such as cytarabine, cause DNA double-strand breaks during TET-mediated active 5-methylcytosine demethylation by interrupting TDG-dependent base excision repair. These double-strand breaks are frequently converted into deletions and translocations by DNA ligase 4. In vivo, Purkinje and Golgi cells in the cerebellum are the only neuronal populations that exhibit high levels of DNA damage due to cytarabine. In Purkinje cells, TET targets highly expressed gene bodies marked by enhancer-associated histone modifications. Many of these genes control movement coordination, which explains the long-recognized cerebellar neurotoxicity of cytarabine2. We show that other cytidine analogues, such as gemcitabine, cause only single-strand breaks in neurons, which are repaired by DNA ligase 3 with minimal toxicity. Our findings uncover a mechanistic link between TET-mediated DNA demethylation, base excision repair and gene expression in neurons. The results also provide a rational explanation for the different neurotoxicity profiles of an important class of antineoplastic agents.

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Fig. 1: Active DNA demethylation mediated by TET and TDG contributes to cytarabine-induced neurotoxicity.
Fig. 2: Cytarabine induces DSBs and chromosome translocations at neuronal enhancers.
Fig. 3: Neuronal DSBs are repaired by canonical NHEJ.
Fig. 4: Cytarabine induces transcriptional changes in Purkinje cells in vivo.
Fig. 5: Highly expressed Purkinje-specific genes are directly targeted by cytarabine.

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Data availability

The CRISPRi screen, END-seq, S1-END-seq, RNA-seq, LAM-HTGTS, ATAC–seq, SAR-seq, targeted deep sequencing (amplicon-seq), PS-seq and CUT&RUN data have been deposited into the GEO database under accession number GSE293256. The following data were obtained from publicly available sources: single-nucleus RNA-seq in SCA1 cerebellar tissues (GSE246183); BS-seq and oxBS-seq in Purkinje cells (GSE166423); ATAC–seq in Purkinje and granule cells (GSE174842); ChIP–seq of H3K4me1 in primary cortical neurons (GSE164265); ChIP–seq of H3K4me1 (GSE167259); and dideoxycytosine S1-END-seq and dideoxycytosine END-seq in i3Neurons (GSE210317). The hg19 (human) and mm10 (mouse) genome assemblies were used as references for alignment. Source data are provided with this paper.

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Acknowledgements

We thank L. Boxer and R. Sakr for help with the isolation of primary mouse cortical neurons; B. Adamson for the AX227 sgRNA library; D. Zong for comments on the manuscript; A. Vogt for purifying the XRCC4–LIG4 complex; M. Greenberg for discussions; staff at the National Cancer Institute/Center for Cancer Research Genomics Core for help with sequencing; staff at the Laboratory Animal Science Program for mouse work; and staff at the Center for Cancer Research/Laboratory of Genome Integrity Flow Cytometry Core for cell sorting. The computational resources of the NIH High-Performance Computing Biowulf cluster were used for data analyses. This work was supported by the Intramural Research Program of the NIH (National Cancer Institute contract HHSN2612015000031 to A.N., F.L. and P.D.A.), an Ellison Medical Foundation Senior Scholar in Aging Award (AG-SS-2633-11 to A.N.), Department of Defense Awards (W81XWH-16-1-599 and W81XWH-19-10652 to A.N.), an International Partnership Program of Chinese Academy of Sciences grant (318GJHZ2023004MI to W.W.), a National Natural Science Foundation of China grant (32370575 to W.W.), the Shanghai Rising-Star Program (24QA2710100 to W.W.) and NIH grants (NS-37956 and NS-123081 to P.J.M., and R01GM144559 and P01CA092584 to Y.H.).

Author information

Author notes

  1. Dongpeng Wang

    Present address: State Key Laboratory of Epigenetic Regulation and Intervention, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

  2. These authors contributed equally: Jia-Cheng Liu, Dongpeng Wang

Authors and Affiliations

  1. Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA

    Jia-Cheng Liu, Dongpeng Wang, Elsa Callen, Chuanyuan Chen, Santiago Noriega, Nancy Wong, Yongge Zhao, Ferenc Livak & André Nussenzweig

  2. Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

    Yafang Shang

  3. Department of Biomedicine, University of Basel, Basel, Switzerland

    David Schürmann & Primo Schär

  4. Key Laboratory of Multi-Cell Systems, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China

    Yawei Song & Wei Wu

  5. Gladstone Institutes, San Francisco, CA, USA

    Gokul N. Ramadoss

  6. Genome Modification Core, Frederick National Laboratory for Cancer Research, Frederick, MD, USA

    Raj Chari

  7. Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA

    Yuan He

  8. Department of Biology, Johns Hopkins University, Baltimore, MD, USA

    Yuan He

  9. Genetics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA

    Peter D. Aplan

  10. National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA

    Michael E. Ward

  11. Laboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA

    Nathaniel Heintz

  12. La Jolla Institute for Immunology, La Jolla, CA, USA

    Anjana Rao

  13. Center for Pediatric Neurological Disease Research, St Jude Pediatric Translational Neuroscience Initiative, Department of Cell and Molecular Biology, St Jude Children’s Research Hospital, Memphis, TN, USA

    Peter J. McKinnon

  14. Genome Damage and Stability Centre, University of Sussex, Brighton, UK

    Keith W. Caldecott

  15. Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Academy of Natural Sciences (SANS), Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China

    Fei-Long Meng

Authors
  1. Jia-Cheng Liu
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Contributions

J.-C.L., D.W., E.C., F.L. and A.N. conceived, designed and analysed the experiments. C.C., W.W. and Y. Song designed the bioinformatic pipelines and performed data analyses. J.-C.L., C.C. and W.W. designed the figures. J.-C.L. and E.C. prepared the i3Neurons, performed CRISPRi screens, gene knockdown and survival experiments. M.E.W. helped with the CRISPRi screen. J.-C.L., E.C. and S.N. performed immunofluorescence imaging. J.-C.L. performed CRISPR knockout, END-seq, S1-END-seq, ATAC–seq, PS-seq, targeted deep sequencing, CUT&RUN and RT–qPCR. J.-C.L. and D.W. prepared cortical neurons and performed RNA-seq and SAR-seq experiments. J.-C.L., D.W., F.L. and E.C. performed FACS. Y. Shang performed LAM-HTGTS and analysed the data, supervised by F.-L.M. G.N.R. generated the virus-like particles used for LAM-HTGTS. R.C. performed cloning experiments for TDGdegron. D.S. performed in vitro reconstitution of BER and analyses, supervised by P.S. Y.H. provided purified the XRCC4–LIG4 complex. N.W. and E.C. performed genotyping of mutant mice. N.W. performed western blotting. Y.Z. assisted in intraperitoneal injections. N.H. and A.R. provided gene knockout mice. P.D.A., N.H., P.J.M. and K.W.C. provided critical insights. J.-C.L., F.L. and A.N. wrote the paper with input from all co-authors. A.N. supervised the study.

Corresponding author

Correspondence to André Nussenzweig.

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Extended data figures and tables

Extended Data Fig. 1 CRISPR interference screen identifies genes involved in AraC-induced neurotoxicity.

a, Pairwise sample correlations of triplicates collected from different time points (day 0, day 3, and day 9) in iN either treated or not with AraC. The correlation is displayed by a pie graph on the upper right and values are indicated on the bottom left. b, Scatter plot showing the beta score of individual 476 genes in the CRISPRi screen. Top 10 “resistant” genes (red) and top 10 “susceptible” genes (blue) are highlighted. c,d, Interaction network of positive (c) and negative (d) hits with the enriched pathways indicated and color coded. e, Knockdown efficiency of individual sgRNA. Y-axis represents the expression of DNMT1/TDG/UHRF1 in knockdown iN (sgDNMT1/sgTDG/sgUHRF1) relative to that in wild-type iN transfected with a control vector (WT). Data are presented as mean ± SD, n = 3 biologically independent experiments. Statistical significance was determined using two-sided paired t test. f, Western blot analysis of DNMT1/TDG protein levels in wild-type (WT) and DNMT1/TDG knockdown (sgDNMT1/sgTDG) iN. For the gel source data, see Supplementary Fig. 1. g, Images of γ-H2AX foci in wild-type, and TDG/DNMT1/UHRF1 knockdown iN either treated or not with AraC (40 μM) on day 6. 4′,6-diamidino-2-phenylindole (DAPI) (blue); γ-H2AX (red). Scale bar, 20 μm. h, Images of NeuN and γ-H2AX in wild-type, Tdg−/− primary cortical neurons treated with AraC (40 μM) on day 6. 4′,6-diamidino-2-phenylindole (DAPI) (blue); NeuN (green); γ-H2AX (red). 4-Hydroxytamoxifen (4-OHT) was added in vitro to knockout TDG in primary cortical neurons isolated from CreERT2 Tdgfl/fl embryos. Scale bar, 20 μm. i,j, Expression of Tet1/2/3 in wild-type cortical neuron (i) and Purkinje neuron (j). Y-axis represents the expression level measured by RNA-seq. Data are presented as mean ± SD. n = 3 biologically independent samples (cortical neuron), n = 6 mice (Purkinje neuron). Statistical significance was determined using the two-sided paired t test, p value is indicated. k, Images of NeuN and γ-H2AX in wild-type and Tet−/− primary cortical neurons treated with AraC (40 μM) on day 6. 4′,6-diamidino-2-phenylindole (DAPI) (blue); NeuN (green); γ-H2AX (red). 4-Hydroxytamoxifen (4-OHT) was added in vitro to knockout TET1/2/3 in primary cortical neurons isolated from CreERT2 Tet1/2/3fl/fl embryos. Scale bar, 20 μm. l, Survival of wild-type and Tet−/− primary cortical neurons one day after AraC treatment. Y-axis represents viability relative to non-treated cells. Data are presented as mean ± SD, n = 4 biologically independent samples. Statistical significance was determined using the two-sided paired t test, p value is indicated. m, Survival of iPSC with indicated treatments. Y-axis represents viability relative to non-treated cells, X-axis represents days after treatment. Data are presented as mean ± SD, n = 3 biologically independent samples. n, Heatmap of expression of p53-pathway genes in wild-type iN with indicated treatments.

Source Data

Extended Data Fig. 2 AraC induces double-strand breaks that are TET- and TDG-dependent.

a, Structure of Deoxycytidine (dC), Dideoxycytidine (ddC), Cytarabine (AraC), Gemcitabine (dFdC) and Adenine arabinoside (AraA). b, Representative images of iN with immunofluorescence staining for DAPI (blue) and PARylation (green) in iN treated or not with AraC. Scale bar, 10 μm. c, Heatmaps of AraC-S1-END-seq in TDGdegron iN treated or not with dTAG. Similar results were observed in at least 2 independent experiments. d, Schematic diagram of pyridine borane sequencing (PS-seq). 5-Formylcytosine (5fC) and 5-carboxycytosine (5caC) are converted to dihydrouracil (DHU) by pyridine borane and read as T after PyroMark PCR amplification. e, Bar graph depicting the percentage of (5fC + 5caC)/total C in 25 CpGs at four targeted AraC-induced DSB sites in TDGdegron iN treated overnight or not with dTAG as measured by PS-seq. f, Heatmap of AraC-END-seq in wild-type iN treated or not with DMOG (1 mM). g, Composite DNA sequence motif analysis 4 bp on either side of AraC-END-seq peak summits in iN. Peak fraction and p value are indicated. Statistical significance was determined using the two-sided binomal test.

Extended Data Fig. 3 AraC induces expression of immediate early genes and late response genes in cortical neurons.

a, Genome browser screenshot showing a double-strand break (END-seq) induced by AraC (40 μM) that colocalizes with the enhancer mark H3K4me1 (non-treated) in primary cortical neurons. b, Heatmaps of END-seq (DSB) in non-treated and AraC-treated primary cortical neurons, and ChIP-seq of H3K4me1 in non-treated primary cortical neurons. The peaks are ordered by END-seq intensity and plotted 1 kb on either side of DSB summits. c, Composite DNA sequence motif analysis 4 bp on either side of AraC-END-seq peak summits in primary cortical neurons. Peak fraction and p value are indicated. Statistical significance was determined using the two-sided binomal test. d, Motif analysis by HOMER. Top, the Fos binding motif downloaded from JASPAR motif database. Bottom, the most enriched motif identified from sequences within 200 bp of the AraC-END-seq peak summits in primary cortical neurons. Peak fraction and p value are indicated. Statistical significance was determined using the two-sided binomal test. e, Aggregate plot showing the Fos motif distribution at AraC-END-seq peak summits in primary cortical neurons 1 kb on either side of AraC-END-seq peak summits. f, Volcano plot showing differentially expressed genes upon AraC treatment in primary cortical neurons. Genes whose expression were upregulated and downregulated significantly (log2fold change>1 and p value < 0.01) are indicated in red and blue respectively. The 10 most significantly upregulated genes are indicated. Statistical significance was determined using the two-sided Wald test and adjusted by the Benjamini & Hochberg method. g, Heatmap of immediate early genes (IEGs) and late response genes (LRGs) expression in non-treated and AraC-treated primary cortical neurons. h, Top 15 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways identified from upregulated genes upon AraC treatment in primary cortical neurons. Statistical significance was determined using the one-sided hypergeometric test. i, qRT-PCR measurement of Fos, Fosb, Npas4, Junb, Bdnf and Vgf gene expression in non-treated or AraC/dFdC (40 μM for 16 hr) treated primary cortical neurons. Statistical significance was determined using the two-sided paired t test, p value is indicated. Data are presented as mean ± SD, n = 3 biologically independent samples. j, qRT-PCR measurement of Fos and Npas4 gene expression in non-treated or etoposide (ETO, 50 μM for 16 hr) treated primary cortical neurons. Statistical significance was determined using two-sided paired t test, p value is indicated. Data are presented as mean ± SD, n = 3 biologically independent samples. k, qRT-PCR measurement of Fos, Fosb, Npas4, Junb, Bdnf and Vgf gene expression in wild-type or Tdg−/− primary cortical neurons treated with AraC (40 μM for 16 hr). 4-Hydroxytamoxifen (4-OHT) was added in vitro to deplete TDG in primary cortical neurons isolated from CreERT2 Tdgfl/fl embryos. Statistical significance was determined using two-sided paired t test, p value is indicated. Data are presented as mean ± SD, n = 3 biologically independent samples.

Source Data

Extended Data Fig. 4 DSBs induced by AraC leads to chromosome translocations and indels.

a, Schematic overview of LAM-HTGTS. b, Aggregate plot of B2M-captured HTGTS signals in wild-type iN treated with (B2M+AraC) or without (B2M) AraC relative to AraC-END-seq summits. Two of the three replicates are plotted, with the remaining shown in Fig. 2g. c, Enhancers (H3K4me1) colocalizing with DNA repair synthesis hotspots (SAR-seq), AraC-induced DSBs (AraC-END-seq) and translocations (AraC-HTGTS) at a prey gene MEX3A and the bait gene NHLH1 on chromosome 1. The translocation hotspot and HTGTS bait are highlighted by orange and red dashed box respectively. d, Schematic overview of targeted deep sequencing. e, Length distribution of deletions induced by AraC. The minimal (1 bp), maximum (89 bp), median (26 bp) and mean (28 bp) deletion lengths are indicated. f, Expression of POLQ in iPSC, day7-iN and day17-iN. Y-axis represent the expression level measured by RNA-seq. Data are presented as the mean, n = 2 biologically independent samples. g, Western blot analysis of LIG4 protein levels in wild-type and lig4 knockout iN. Two independent lig4 knockout clones c13 and c40 were tested. For the gel source data, see Supplementary Fig. 1. h,i, Top, Genome browser screenshot of DSBs induced by AraC on chromosome 1 (h) and 6 (i). Bottom, the deletion profile of the above DSB in wild-type, lig4 knockouts (c13 and c40) and DNA-PKcs inhibitor (NU7441, 5 μM) treated wild-type iN. Y-axis represents the deletion frequency; X-axis represents the chromosome location.

Source Data

Extended Data Fig. 5 AraC generates DSBs in Golgi and Purkinje cells in the cerebellum.

a, Flow cytometric analysis of nuclei isolated from 1 month old mouse cerebellum after PBS- or AraC treatment. Y- and X-axes indicate the expression of the Purkinje specific marker ITPR1 and the DNA damage marker γ-H2AX respectively. Purkinje cells and an unknown population are marked by the red and green box respectively. For the flow cytometry gating strategy, see Supplementary Fig. 2. b, Cell type composition of sorted Purkinje cells and the unknown population shown in (a) determined by RNA-seq. The estimated fraction is determined using single-cell sequencing data from mouse brain. Nuclei were isolated 3 h after AraC treatment. c,d, Top 10 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways identified from AraC-induced upregulated (c) and downregulated (d) genes in Purkinje cells. Statistical significance was determined using the one-sided hypergeometric test. e, Fos expression in Purkinje cells isolated from wild type mice receiving PBS or AraC. Statistical significance was determined using the two-sided Wald test and adjusted by the Benjamini & Hochberg method, p value = 0.03. Data are presented as mean ± SEM, six and five mice were used for analysis in the PBS and AraC groups, respectively. f, Flow cytometric analysis of nuclei isolated from the whole brain without cerebellum from PBS- or AraC-treated mice. Y- and X-axes represent the expression of the DNA damage marker γ-H2AX and the neuronal marker NeuN respectively. The NeuN and γ-H2AX positive population is highlighted in the red square. For the flow cytometry gating strategy, see Supplementary Fig. 2. g, Cell type composition of sorted γ-H2AX positive population upon AraC treatment shown in (f) determined by RNA-seq. The estimated fraction is determined using single-cell sequencing data from mouse brain. h, Bar graph depicting the percentage of (5fC + 5caC)/total C in 55 CpGs at seven sites in genes Grid2 (six sites) and Itpr1 (one site) in Purkinje nuclei isolated from wild type or Pcp2-Cre TDGfl/fl mice (Tdg−/−) as measured by PS-seq. i, Integrative Genomics Viewer (IGV) Screenshots of H3K27ac and RNA-seq for highly expressed genes in Purkinje cells (Itpr1 and GABBR2), Golgi cells (SGCD and TCF4), and highly expressed genes in both Purkinje and Golgi cells (KCNMA1 and FRMD4A). j, Aggregate END-seq signal at DNA methylation valleys (DMV) within 118 AraC-induced downregulated genes.

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Extended Data Fig. 6 The AraC-induced increase in γ-H2AX in Purkinje cells is TET- and TDG-dependent.

a, Representative examples of imaging flow cytometric analysis of Purkinje cell nuclei. BF (bright field), NeuN (green), γ-H2AX (yellow), ITPR-1 (red), DAPI (blue) and Merge (γ-H2AX and ITPR-1). TDG knockout (Tdg−/−) was induced by intraperitoneal injection of tamoxifen for CreERT2 TDGfl/fl mice. Pcp2-Cre Tet1/2/3fl/fl mice were used for Purkinje-specific knockout of Tet1/2/3 (Tet−/−). b, Quantification of γ-H2AX foci from imaging flow cytometric analysis. The genotype and treatment are indicated on the bottom. Data are presented as median with interquartile range, n = 110 and 579 (WT), 256 and 898 (WT with AraC), 101 and 129 (Tdg−/− with AraC), and 352 and 314 (Tet−/− with AraC) nuclei examined in 2 independent experiments. Statistical significance was determined using two-sided Mann-Whitney test, p value is indicated (p < 1e-15). c, Flow cytometric analysis of nuclei isolated from 6-month old mouse cerebellum after PBS or AraC treatment. Y- and X-axes indicate the expression of the Purkinje specific marker ITPR-1 and the DNA damage marker γ-H2AX respectively. γ-H2AX positive Purkinje cells are marked by red. For the flow cytometry gating strategy, see Supplementary Fig. 2. d,e, Quantification of γ-H2AX level by flow cytometry. The genotypes and treatments are indicated. Pcp2-Cre Tet1/2/3fl/fl mice were used for Purkinje-specific knockout of Tet1/2/3 (Tet−/−) in (e). For the flow cytometry gating strategy, see Supplementary Fig. 2.

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Extended Data Fig. 7 Adenine arabinoside and Gemcitabine do not cause neurotoxicity.

a, Images of γ-H2AX foci in wild-type iN treated with AraC (40 μM) or AraA (40 μM) on day 6. 4′,6-diamidino-2-phenylindole (DAPI) (blue); γ-H2AX (red). Scale bar, 10 μm. Similar results were observed in at least 2 independent experiments. b-d, Survival of iPSC-derived postmitotic neurons (b) and iPSCs (c,d) with indicated treatment. Y-axis represents viability relative to non-treated cells, X-axis represents days after treatment. Data are presented as mean ± SD, n = 3 biologically independent samples. e, Composite DNA sequence motif analysis 4 bp on either side of dFdC-S1-END-seq peak summits in iN. Peak fraction and p value are indicated. Statistical significance was determined using the two-sided binomal test. f, Schematic diagram represents the distance between the summits of dFdC-S1-END-seq and AraC-END-seq (with ‘GG’ motif). Median length: 21 bp. g, Enrichment of GG/CC and G/C motifs at AraC-END-seq peak summits within 25 bp of dFdC-S1-END-seq peak summits. Data are presented as mean ± SEM, n = 27234 (G/C) and 8510 (GG/CC) peaks. Statistical significance was determined using the two-sided Wilcoxon Rank Sum and Signed Rank Tests, p < 2e-16. h, Heatmaps of dFdC-S1-END-seq (SSB) and dFdC-END-seq (DSB) peaks. The peaks are ordered by SAR-seq intensity and plotted 1 kb on either side of SAR-seq peak summits in iN. i, Images of NeuN and γ-H2AX in wild-type primary cortical neurons treated with AraC or dFdC (40 μM) on day 6. 4′,6-diamidino-2-phenylindole (DAPI) (blue); NeuN (green); γ-H2AX (red). Scale bar, 20 μm. Similar results were observed in at least 2 independent experiments. j, In vitro reconstitution of TDG-initiated base excision repair. Top, the structure of DNA substrate (50 nM) containing 5-carboxycytosine (caC) on both strands, the DNA length and fluorescein label (*, red) are indicated. Bottom, native PAGE gel (15%) separating the base excision repair product, the nucleoside triphosphate (50 μM of each dCTP, dFdCTP and AraCTP), enzymes (50 nM of each TDG, APE1, PolB and XRCC1/LIG3), incubation time and product length are indicated. Two of the three replicates are shown. For gel source data, see Supplementary Fig. 1. k,l, In vitro reconstitution of TDG-initiated base excision repair using the same setting as in (j) but either replaced XRCC1/LIG3 by XRCC1/LIG4 (k) or omitting the final ligation step (l). For the gel source data, see Supplementary Fig. 1. m, Knockdown efficiency tested in neurons. X-axis represents the expression of XRCC1 in knockdown iN relative to that in wild-type iN transfected with a control vector (WT). Statistical significance was determined using two-sided paired t test, p = 2.1e-6. n, Western blot analysis of XRCC1 protein levels in wild-type (WT) and XRCC1 knockdown (sgXRCC1) iPSCs. o, Genome browser screenshot showing double-strand breaks (END-seq) detected in dFdC (40 μM) treated wild-type and XRCC1 knockdown iN on day 7. p, Proposed model for active DNA demethylation interrupted by AraC and dFdC at neuronal enhancers. Neurons employ sequential DNA demethylation of symmetrically methylated CpGs to prevent the formation of DSB at enhancers. This mechanism involves the ligation of dFdC by XRCC1/LIG3 during base excision repair, resulting in SSB but minimal DSB formation. However, the unligatable nature of AraC leads to generation of massive SSBs and DSBs at neuronal enhancers.

Source Data

Extended Data Fig. 8 Proposed Model for AraC-induced cerebellar toxicity.

In Purkinje cells (PCs) of the cerebellum, a subset of highly expressed genes essential for motor coordination (Itpr1, Grid2 and etc.) undergoes active DNA demethylation. These genes are extensively covered with broad H3K27ac domains associated with enhancer marks. Active DNA demethylation involves TET-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC), followed by TDG-mediated excision of 5fC/5caC. AraC can interrupt base excision repair leading to double-strand breaks (DSBs) within gene bodies. AraC-induced DSBs can directly decrease gene expression or be processed into insertion/deletions (indels) and translocations, which can contribute to the development of cerebellar ataxia.

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Supplementary Figs. 1 and 2, which contain the uncropped gels and the flow cytometry gating strategy.

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Liu, JC., Wang, D., Callen, E. et al. Mechanism of cytarabine-induced neurotoxicity. Nature 643, 1400–1409 (2025). https://doi.org/10.1038/s41586-025-09210-9

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