High-throughput single-cell methylation resolves epigenetic states in hematopoietic differentiation and age-related clonal expansions Free

DNA methylation at CpG dinucleotides is a critical regulator of cell identity and epigenetic memory, and it becomes universally disrupted during hematopoietic aging, preleukemic clonal expansions and their progression towards malignancy. Therefore, understanding how hematopoietic identity is specified, inherited and disrupted at the single-cell level is essential to develop therapeutic strategies for age-related hematologic disorders. However, current single-cell DNA methylation profiling technologies are limited by either low throughput (hundreds of cells) or low capture rates (10³-10⁵ CpGs per cell; <0.3% of CpGs the human genome) and are thus insufficient to characterize heterogeneous populations in the hematopoietic system.

To tackle this limitation, we developed DREAM-seq (Droplet-based Restriction Enzyme And Methylation sequencing), a high-throughput single-cell DNA methylation technology based on droplet microfluidics and MspI-based reduced representation chemistry. DREAM-seq profiles >5,000 single-cell methylomes per experiment, capturing 0.44M CpGs per cell (3.75-fold increase vs. existing methods) and 3.16 CpGs per read (4-fold increase), with each CpG detected in 1 out of 20 cells. This allows us to generate comprehensive datasets covering >50% of CpGs in the human genome, with marked improvements in clustering resolution and cell type identification.

We leveraged DREAM-seq to sequence 51,208 single-cell methylomes from human peripheral blood, CD34-enriched and whole bone marrow (BM) cells, generating the largest tissue-specific atlas of hematopoietic differentiation at single-cell methylome resolution to date. Unbiased selection of 71,115 variably methylated CpG sites identified all major hematopoietic cell subtypes, recapitulating cell type specific methylation patterns (monocyte-specific CEBPA and MPO hypomethylation) and cell states (TCF7 gene body methylation in effector CD8+ T cells compared to naïve subsets). Cell-type defining CpGs were depleted from CpG islands and TSS but strongly enriched in distal enhancers and CTCF sites (72.2 %), highlighting their role in specifying hematopoietic cell fates. Interestingly, 27.8% of cell-identity CpGs were located in heterochromatin, where DNA methylation provides an additional regulatory layer not captured by chromatin accessibility methods. These data suggest a model in which DNA methylation regulates both enhancer activity and higher-order chromatin structure at heterochromatin regions, which together control hematopoietic cell identity.

To gain insights into methylation-driven lineage specification, we computed enhancer methylation levels across hematopoietic populations. We discovered that >40% of CD34+ cells display hypomethylation of lineage-specific enhancers prior to commitment, suggesting methylation-based lineage priming at the HSPC level. We further traced the methylation patterns from HSPCs towards differentiated cells in myeloid, lymphoid and erythroid lineages, identifying early and late changes in the epigenome that occur upon lineage commitment. This highlights previously unrecognized roles of DNA methylation in lineage priming and hematopoietic differentiation dynamics.

To understand how these methylation states are disrupted during hematopoietic aging and age-related clonal expansions, we analyzed 18,268 single-cell methylomes from aged BM donors, including 3 TET2-mutant clonal hematopoiesis samples. We first identified clonal populations by leveraging genetic variants as clonal markers, finding significant oligoclonality in aged BM, with 3-fold less clonal clusters than young BM. By characterizing the methylation state of these populations, we are defining “high-fitness” methylation states, which could be implicated in driving clonal expansions and might be reversed to constrain age-related clonal hematopoiesis.In summary, we present DREAM-seq, a new single-cell DNA methylation technology which we leverage to generate the largest single-cell methylome atlas of human hematopoiesis to date. By defining methylation-based cell states predictive of lineage output and clonal fitness, we identify epigenetic drivers of hematopoietic differentiation and their disruption during age-related clonal expansions. Our framework provides a foundational resource for understanding epigenetic drivers of hematopoietic malignancies, improving leukemia classification and predicting therapeutic responses.