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Single-cell multi-omics of human clonal hematopoiesis reveals that DNMT3A R882 mutations perturb early progenitor states through selective hypomethylation

Abstract

Somatic mutations in cancer genes have been detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, because mutated and wild-type cells are admixed, we have limited ability to link genotypes with phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing genotype, transcriptomes and methylomes in progenitors from individuals with DNMT3A R882 mutated clonal hematopoiesis. DNMT3A mutations result in myeloid over lymphoid bias, and an expansion of immature myeloid progenitors primed toward megakaryocytic–erythroid fate, with dysregulated expression of lineage and leukemia stem cell markers. Mutated DNMT3A leads to preferential hypomethylation of polycomb repressive complex 2 targets and a specific CpG flanking motif. Notably, the hypomethylation motif is enriched in binding motifs of key hematopoietic transcription factors, serving as a potential mechanistic link between DNMT3A mutations and aberrant transcriptional phenotypes. Thus, single-cell multi-omics paves the road to defining the downstream consequences of mutations that drive clonal mosaicism.

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Fig. 1: GoT demonstrates comingling of mutated and wild-type cells in DNMT3A R882-clonal hematopoietic differentiation.
Fig. 2: DNMT3A R882 mutated CH cells demonstrate distinct differentiation biases at key junctures.
Fig. 3: Differential gene expression analysis between mutated and wild-type cells reveals markers of lineage aberrancies and dysregulated MYC activity.
Fig. 4: DNMT3A R882 promotes selective hypomethylation of PRC2 targets in human hematopoiesis.
Fig. 5: DNMT3A R882 displays flanking sequence specificity associated with MYC binding motif.
Fig. 6: Bone marrow CH progenitor cells display megakaryocytic–erythroid differentiation bias, MYC target gene expression and enhanced accessibility for the R882 hypomethylated motif.

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Data availability

All of the processed counts matrix data and raw fastq files for murine experiments are available via Gene Expression Omnibus (GEO) under the accession number GSE158067. The raw fastq files for human samples are available via European Genome-Phenome Archive under the accession number EGAS00001006364.

Code availability

IronThrone v.2.2 pipeline is available at https://github.com/landau-lab/IronThrone-GoT

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Acknowledgements

The work was enabled by the Weill Cornell Epigenomics Core and Flow Cytometry Core. We thank A. Melnick (Weill Cornell Medicine) for a critical review of the manuscript. A.S.N. is supported by the Burroughs Wellcome Fund Career Award for Medical Scientists, the National Institutes of Health Director’s Early Independence Award (DP5 OD029619) and the Starr Cancer Consortium. N.D. is supported by a F30 Predoctoral Fellowship from the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health (F30HL156496). N.D. and R.M.M. are supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the National Institutes of Health under award number T32GM007739 to the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program. F.I. is supported by the American Society of Hematology Fellow-to-Faculty Scholar Award. R.C. is supported by Lymphoma Research Foundation and Marie Skłodowska-Curie fellowships. I.G. is supported by a grant from the Dr Miriam and Sheldon G. Adelson Medical Research Foundation, and a Stand Up To Cancer Dream Team Research Grant (SU2C-AACR-DT-28-18). D.A.L. is supported by the Burroughs Wellcome Fund Career Award for Medical Scientists, Valle Scholar Award, the National Institutes of Health Director’s New Innovator Award (DP2-CA239065), the Chan Zuckerberg Initiative Award, the Leukemia Lymphoma Society Career Development Program Award and the Mark Foundation Emerging Leader Award. This work was also supported by the NHLBI (R01HL145283) and the National Human Genome Research Institute, Center of Excellence in Genomic Science (RM1HG011014).

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Contributions

A.S.N., N.D., F.I., R.M., R.C., T.H.M., I.G. and D.A.L. conceived the project, designed the study and interpreted results. I.G. and L.A.G. provided clinical samples. R.C., R.M.M., J.S., S.B. and M.W. performed the experiments. A.S.N., N.D., F.I. and R.M. performed the analyses. A.S.N., N.D., F.I., R.M., R.M.M., R.C., I.G. and D.A.L. wrote the manuscript. A.S.N., N.D., F.I., R.M., T.H.M., J.S., S.B., F.G., S.T., L.A.G., R.C., R.L., O.A.-W., I.G. and D.A.L. helped interpret results. All authors reviewed and approved the final manuscript.

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Correspondence to Irene Ghobrial or Dan A. Landau.

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Competing interests

O.A.-W. has served as a consultant for H3B Biomedicine, Foundation Medicine Inc., Merck Pfizer, and Janssen, and is on the scientific advisory board of Envisagenics Inc. and AIChemy; O.A.-W. has received prior research funding from H3B Biomedicine and LOXO Oncology unrelated to the current manuscript. I.G. serves on the advisory board of Bristol Myers Squibb, Takeda, Janssen, Sanofi and GlaxoSmithKline. D.A.L. has served as a consultant for Abbvie, AstraZeneca and Illumina, and is on the scientific advisory board of Mission Bio, Alethiomics, Pangea and C2i Genomics; D.A.L. has received prior research funding from BMS, 10x Genomics, Ultima Genomics, Abbvie and Illumina unrelated to the current manuscript.

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Nature Genetics thanks Michael Rehli, Vijay Sankaran and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 GoT captures genotyping information of thousands of CD34+ cells in scRNA-seq.

a, Summary of Genotyping of Transcriptomes (GoT) data from clonal hematopoiesis (CH) patient samples with DNMT3A R882 mutations. b, Number of genes per cell (left) and number of unique molecular identifiers (UMIs) per cell (right) from CD34+ sorted hematopoietic progenitors by patient sample after quality control (QC) filters (CH01 n = 6133, CH02 n = 5372, CH03 n = 7379, CH04 n = 8440). c, DNMT3A R882 mutant fraction of single cells determined by GoT versus DNMT3A R882 mutation variant allele frequencies (VAF) in bulk sequencing of matched unsorted stem cell product31. d, Fraction of cells by number of DNMT3A UMIs in standard 10x Genomics data without genotyping information (left), DNMT3A UMIs with R882 locus coverage in standard 10x data (middle), and DNMT3A UMIs with R882 locus coverage in GoT amplicon library (right). e, Species-mixing experiment data in which mouse cells (Ba/F3) with a human mutant CALR transgene were mixed with human cells (UT-7) with a human wildtype CALR transgene29. Mouse and human genome alignment of 10x data with genotyping data from GoT pre (top) and post (bottom) implementation of UMI consensus assembly based on Levenshtein distance (methods). f, Number of duplicate reads supporting cell barcode-UMI pair in the GoT library that is identified in the 10x gene expression (GEX) library as a DNMT3A gene (left), no gene (middle), or a non−DNMT3A gene (right). P-values from two-sided Wilcoxon rank sum test. g, Heatmap of relative expression of genes ordered by chromosome/chromosomal position following copy number variation analysis using the InferCNV package33. Cells (y-axis) are stratified by patient and DNMT3A R882 genotype status. h, Heatmap of relative expression of Y-chromosome genes following copy number variation analysis and cell stratification as in g. WT, wildtype, MUT, mutant.

Extended Data Fig. 2 Integration of DNMT3A R882 mutation and assignment of progenitor subsets in clonal hematopoiesis patient samples.

a, Uniform manifold approximation and projection (UMAP) of CD34+ progenitor cells from samples CH01-CH04 after integration using the Seurat package (methods). b, Heatmap of top 10 differentially expressed genes for progenitor subsets. c, Lineage-specific genes (left) and modules from Velten et al.41 (right, Supplementary Table 2) are scored and projected onto the UMAP representation of CD34+ cells. d, UMAP of CD34+ cells overlaid with cluster assignments, split by patient sample. HSPC, hematopoietic stem progenitor cells; IMP, immature myeloid progenitors; IMP-ME, megakaryocytic-erythroid biased IMP; IMP-GM, granulo-monocytic biased IMP; LMPP, lympho-myeloid primed progenitors; CLP, common lymphoid progenitor; MEP, megakaryocytic-erythroid progenitors; E/B/M, eosinophil, basophil, and mast cell progenitors; EP, erythroid progenitor; MkP, megakaryocytic progenitor; NP, neutrophil progenitor.

Extended Data Fig. 3 Classification of IMPs showing lineage biases and pseudotime analysis between mutated and wildtype cells.

a, UMAP of CD34+ cells, overlaid with cluster assignment of the IMP subsets. b, Neutrophil and Megakaryocytic-Erythroid lineage specific gene module scores from Velten et al.41 compared across the three IMP clusters. P-value was calculated from two-sided Wilcoxon rank sum test. c, UMAP of CD34+ cells overlaid with mutation status for wildtype (WT), DNMT3A R882 mutant (MUT), or unassigned (NA), split by genotype for all samples (top) and by patient sample (bottom). d, UMAP with projected pseudotime values (top left). Pseudotime comparison between WT and MUT cells for all samples (top right) and for individual samples (bottom) as estimated by Monocle39. P-value was calculated from likelihood ratio test of linear mixed model (LMM) with/without mutation status for aggregate analysis (methods, top) and two-sided Wilcoxon rank sum test for individual samples (bottom).

Extended Data Fig. 4 Comparison between mutated and wildtype progenitor cells of cell cycle module expression and RNA velocity derived transition probabilities.

a, Cell cycle module score represents the union of S-phase and G2M-phase gene-module expression (Supplementary Table 2)42. P-value was calculated from likelihood ratio test of linear mixed model with/without mutation status (methods). Analysis was performed for clusters with at least 200 genotyped cells across all patient samples. b, Single cell mean IMP → IMP-ME and c, IMP → IMP-GM transition probabilities, as measured via RNA velocity34, between wildtype or DNMT3A R882 mutant IMPs for each sample. P-values from two-sided Wilcoxon rank-sum test.

Extended Data Fig. 5 Comparison of differential expression analysis between permutation test and linear mixed model and MYC gene expression.

a, P-values from permutation test and linear mixed model (methods) are plotted per gene. Correlation coefficient R calculated using Pearson’s Correlation. P-values derived from two-sided Student’s t-distribution. Shading denotes 95% confidence interval. b, Normalized MYC gene expression between mutated and wildtype cells in MEP and EP. P-value was calculated from likelihood ratio test of linear mixed model with/without mutation status (methods).

Extended Data Fig. 6 Multi-omics single cell methylome, transcriptomic, and somatic genotyping reveals hypomethylation of PRC2 targets in DNMT3A R882 CH.

a, UMAP dimensionality reduction (n = 528 cells) based on scRNA-seq data (Smart-seq2) after integration and batch correction of six plates (methods). b, UMAP dimensionality reduction showing cluster gene markers for the transcriptome data. c, Number of CpG sites captured per cell after quality filtering (methods). The metrics for each sample according to enzymatic digestion with Msp1 (Single) or Msp1 plus HaeIII (Double) are shown. d, Average single cell methylation at all regions (global, double digest), promoters, introns or exons. P-values from likelihood ratio test of LMM with/without mutation status (methods). e, Average single cell methylation at CpH (that is CpA, CpC or CpT) sites. P-values from likelihood ratio test of LMM with/without mutation status (methods). f, Average single cell methylation at 269 hypomethylated promoters identified with differentially methylated region (DMR) analysis (shown in Fig. 4e, promoters with P-value < 0.05 and at least −5% methylation change) in CH02 and CH04. P-values from two-sided Wilcoxon rank sum test. g, Average single cell methylation at SUZ12 (top panel) and EZH2 (bottom panel) ENCODE chromatin immunoprecipitation sequencing (ChIP-seq) peaks intersected with bivalently H3K27me3, H3K4me3-marked regions in CD34+ cells for CH02 and CH04. P-values from likelihood ratio test of LMM with/without mutation status. h, Normalized expression of PRC2 target genes with preferentially hypomethylated transcription start site (TSS) (from Fig. 4e) in GoT data of WT versus MUT cells by progenitor subtype. P-values from likelihood ratio test of LMM with/without mutation status. i, Comparison of average methylation values for TSS ± 1 kb regions in normal HSPCs79 and DNMT3A WT (n = 6) versus DNMT3A R882, NPM1 mutated acute myeloid leukemia (AML; n = 7) samples in regions without (left) or with (right) PRC2 ChIP-seq peaks, controlling for CpG content. P-values from two-sided Wilcoxon rank sum test. j, Comparison of average methylation values for promoter regions in WT (n = 122) versus DNMT3A R882 mutated AML (n = 9) samples from TCGA in regions without (left) or with (right) PRC2 ChIP-seq peaks, controlling for CpG content. P-values from two-sided Wilcoxon rank sum test.

Extended Data Fig. 7 Motif enrichment at hypomethylated CpGs and hypomethylated motif enrichment in regions around differentially expressed genes.

a, Base frequency odds ratio of hypo- versus hyper-methylated CpG flanking sequences at positions N-2, N-1, N + 1, and N + 2. The odds ratios were derived from base frequencies of flanking positions of the CpG sites hypo- or hyper-methylated in mutant versus wildtype cells above the thresholds shown in the x axis for minimum absolute CpG methylation difference (Pearson correlation, P-values derived from two-sided F-test, shading denotes 95% confidence interval). b, Reported motif logos derived from Emperle et al.87 for either hypomethylated (disfavored) or hypermethylated (favored) sites for DNMT3A R882 compared with its wildtype counterpart (left). c, Similarity scores between the reported and our de novo DNMT3A R882 hypo- and hypermethylated motifs as measured by correlation coefficients of the position weight matrices for the respective motifs excluding the CpG dinucleotide. d, Heatmap of expression of transcription factors with binding motif similarity >0.5 compared with hypomethylated motif of DNMT3A R882 (that do not meet the overall expression threshold, Fig. 5a,b). e, Frequencies of DNMT3A R882 hypomethylated motif within 30 kb of TSS of the differentially expressed genes between MUT and WT cells in progenitor subsets. P-values were calculated by two-sided Wilcoxon rank sum test. f, Frequencies of DNMT3A R882 hypomethylated motif within 10 kb, 30 kb or 50 kb of TSS of the differentially expressed genes between MUT and WT cells in HSPCs and EPs. P-values were calculated by two-sided Wilcoxon rank sum test. g, Ratio of frequencies of DNMT3A R882 hypomethylated motif to those of the control shuffled motif with CpG (Fig. 5e) within 10 kb of TSS of the differentially expressed genes between MUT and WT cells in HSPCs and EPs. P-values were calculated by two-sided Wilcoxon rank sum test. h, Average per-gene incidence of DNMT3A R882 hypomethylated motif within 50 kb of TSS by distance from TSS for differentially expressed genes between MUT and WT cells in HSPCs (top) and EPs (bottom).

Extended Data Fig. 8 Single nucleus ATAC-seq of Dnmt3a R878H Lin-, c-Kit + progenitors reveals enhanced accessibility of R882 hypomethylated motif and TF motifs with high similarity scores to the hypomethylated motif.

a, Distribution of fragment size in single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) data of Dnmt3a R878H and wildtype Lin-, c-Kit+ progenitors (n = 3 in each cohort). b, TSS enrichment of accessible fragments as a function of unique fragments per cell. c, UMAP of integrated datasets Dnmt3a R878H and wildtype Lin-, c-Kit+ progenitors, displayed per sample (n = 3 in each cohort). d, Heatmap of gene accessibility scores for differentially accessible progenitor identity marker genes across progenitor subsets. e, Scatterplot of similarity scores of mouse transcription factor (TF) motifs versus human TF motifs to the R882-hypomethylated motif (Pearson’s correlation, P-value derived from two-sided F-test). f, Binding motifs of mouse and human TFs with high similarity score to the R882-hypomethylated motif and expression in HSPCs (Fig. 5b, HOCOMOCO v11). g, Family-wise error rate (FWER) adjusted P-values calculated by two-sided Wilcoxon rank sum test for accessibility changes between wildtype and Dnmt3a R878H cells by progenitor identities for hypo-methylated motif and shuffled motifs controls (with and without CpG), as well as motif accessibility deviation of the TFs identified Fig. 5b (related to Fig. 5f). h, Accessibility of PRC2 targets between wildtype and Dnmt3a R878H and wildtype Lin-, c-Kit+ progenitor subsets. P-values from likelihood ratio test of LMM with/without mutation status.

Extended Data Fig. 9 Integration of CH05 and control bone marrow CD34+ scRNA-seq data, assignment of progenitor subsets, and differentiation skews of CH cells.

a, UMAP of CD34+ progenitor cells from samples CH05 and control bone marrow samples BM01-05 after integration using the Seurat package (methods). b, Number of genes per cell (top) and number of UMIs per cell (bottom) from CD34+ hematopoietic progenitors by patient sample after QC filters and down-sampling to equivalent geometric means of UMIs per patient (BM1 n = 6,683, BM2 n = 7,254, BM3 n = 16,759, BM4 n = 2,200, BM5 n = 5,690, CH05 n = 5,952). c, Heatmap of top 10 differentially expressed genes for progenitor subsets. d, UMAP representation of CD34+ cells showing cell marker gene expressions. e, Modules from Velten et al.41 (Supplementary Table 2) are scored and projected onto the UMAP representation of CD34+ cells. f, Per-patient comparison of megakaryocytic-erythroid module scores in control bone marrow versus CH05 IMPs (Supplementary Table 2). Cell number downsampled to the same number (n = 132 cells per sample). P-values were calculated from likelihood ratio test of LMM with/without CH status. g, Per-patient comparison of granulocytic-monocytic module scores in control versus CH IMPs (Supplementary Table 2). Cell number downsampled to the same number (n = 132 cells per sample). P-values were calculated from likelihood ratio test of LMM with/without CH status. h, Fraction of IMP-ME cells out of all biased IMP (IMP-ME + IMP-GM) cells in control versus CH populations. P-value was calculated from one-sample t-test. Bar represents mean values ± SEM.

Extended Data Fig. 10 Bone marrow clonal hematopoiesis patient sample confirms results from CH01-CH04 and Dnmt3a R878H mouse model.

a, Per-patient comparison of module scores for differentially down- or up-regulated genes in mutant DNMT3A HSPCs (identified in GoT data, Fig. 3a,c) in control versus CH HSPCs. P-values were calculated from likelihood ratio test of LMM with/without CH status. b, Per-patient comparison of module scores for differentially down- or up-regulated genes in mutant DNMT3A EPs (identified in GoT data, Fig. 3a,c) in control versus CH EPs. P-values were calculated from likelihood ratio test of LMM with/without CH status. c, Module scores for genes upregulated in at least 2 cell types (identified in GoT data, Fig. 3b) in control versus CH cells of major cell types. P-values from likelihood ratio test of LMM with/without CH status. d, Fraction of control BM or CH05 cells in EP1 versus EP2 cell clusters. e, UMAP of CH05 cells (clustered independently of the control BM samples) with progenitor cell assignments. f, UMAP of CH05 cells with genotyping data for WT (n = 397 cells) and DNMT3A R882 mutant (MUT; n = 290 cells). g, Normalized expression of differentially upregulated genes in at least 2 cell types, highlighted in Fig. 3b in wildtype versus mutated cells in CH05. P-values from two-sided Wilcoxon rank sum test. h, UMAP of CH05 cells with protein expression (CITE-seq) and gene expression for CD38 and CD9. i, UMAP of CH05 cells highlighting HSPCs, IMP-ME, and MkPs (left) included in the comparison of CD9 expression in wildtype versus mutated cells (right). P-values from likelihood ratio test of LMM with/without mutation status, and cell type as random effect. j, Distribution of fragment size in snATAC-seq data of patient CH05 with DNMT3A R882 CH. k, TSS enrichment of accessible fragments as a function of unique fragments per cell. l, Heatmap of the gene accessibility scores for cluster marker genes (false discovery rate (FDR) < 0.01 and Log2(Fold-Change) (Log2FC) > 1) by cell cluster. m, Pseudotime trajectories for either erythroid (left, n = 1,843 cells) or lymphoid (right, n = 1,740 cells) differentiation. n, Difference between hypomethylated and shuffled motif accessibility z-scores across either erythroid (n = 1,843 cells) or lymphoid (n = 1,740 cells) pseudotime trajectory quartiles. P-values were calculated by two-sided Wilcoxon rank sum test.

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Nam, A.S., Dusaj, N., Izzo, F. et al. Single-cell multi-omics of human clonal hematopoiesis reveals that DNMT3A R882 mutations perturb early progenitor states through selective hypomethylation. Nat Genet 54, 1514–1526 (2022). https://doi.org/10.1038/s41588-022-01179-9

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