Single-cell multiomic profiling of gene mutation, chromatin accessibility, and gene expression in TET2-mutant clonal hematopoiesis Free

Age-related clonal hematopoiesis (CH) and clonal cytopenia of undetermined significance (CCUS) represent pre-leukemic conditions associated with predisposition to hematological malignancies, particularly myeloid neoplasms. Frequently associated with mutations in epigenetic regulators, such as DNMT3A, TET2, and ASXL1, deregulated epigenomes are implicated in the aberrant gene expression involved in the pathogenesis of CH and CCUS, whose molecular mechanism, however, has not been investigated. The major difficulty lies in the technical limitation to simultaneously analyze gene mutation, transcriptome, and epigenetics in a small fraction of mutant cells in comparison with wild-type cells within the same patients, particularly in those cases where mutant cell fractions are very small.

Aim/Methods In this study, we developed a novel platform that enabled simultaneous measurements of gene mutations and chromatin accessibility at the single-cell level based on the modified Mission Bio Tapestri system, which was applied to the analysis of 11 samples with TET2-mutated (TET2-MUT) CH and CCUS to investigate the impact of TET2 mutations on chromatin accessibility. In a subset of the samples (n = 3), we performed 10x Chromium single-cell ATAC+RNA-seq (Multiome) experiments and then predicted single-cell TET2 genotypes by employing ATAC peak accessibility-based logistic regression models trained on the paired Tapestri multimodal data, enabling the analysis of effects of TET2 mutation on gene regulation and transcriptional outputs at single-cell resolution.

Results First, our platform was validated using a mixed cell line sample (HL-60 and SKM-1). ATAC data alone clearly separated the two cell lines, and genotyping accuracy for cell line-discriminative SNPs reached 94-99%, confirming robust integration of ATAC-seq and genotyping at the single-cell level.

Next, to investigate the consequences of TET2 mutation on hematopoiesis and chromatin accessibility, we applied the platform to a total of 37,190 cells from TET2-MUT CH/CCUS patients, achieving successful genotyping in 83% (74–92%) of the cells. TET2-MUT cells exhibited a differentiation bias toward myeloid and plasmacytoid dendritic cell (pDC) lineages, with reduced mature lymphoid output. In line with this skewed differentiation, TET2-MUT hematopoietic stem/progenitor cells (HSPCs) showed an increased accessibility to the motifs of transcription factors (TFs) associated with myeloid/pDC differentiation (e.g., CEBPE, RUNX1, and IKZF1), compared to wild-type (TET2-WT) HSPCs, whereas TET2-MUT B cells had a reduced accessibility to the motifs of B cell development-associated TFs (e.g., BACH2, IRF4, and IRF8), compared with TET2-WT counterparts. In erythroblasts, TET2-MUT cells exhibited a decreased accessibility to erythroid maturation-related genes and an increased accessibility to genes involved in cell proliferation, including E2F and MYC targets, compared with TET2-WT cells, suggesting dysregulated maturation of TET2-MUT erythroblasts. Collectively, these results revealed TET2 mutation-driven alterations in chromatin accessibility affecting lineage maturation.

Next, to investigate combined effects of TET2 mutation on TF-mediated gene regulation and transcriptional programs, we analyzed 10x Multiome data with predicted single-cell TET2 genotypes. We first analyzed TET2-MUT cell fractions within monocyte subsets and observed a depletion of mutants in the CD16(+) subset, suggesting impaired maturation of TET2-MUT cells. To uncover the underlying mechanisms, we then compared TF activity between genotypes, using SCENIC+, which integrates paired single-cell ATAC-seq and RNA-seq data to infer TF activity based on both accessibility at TF-binding enhancers and expression of their target genes. For both accessibility- and expression-based TF activities, TET2-MUT monocytes exhibited up-regulation of cell cycle-regulating TFs (e.g., TFDP1, TFDP2, and MYBL2) and down-regulation of key monocyte maturation-related TFs (e.g., CEBPB, CEBPD, and SPI1). These results suggest that TET2 mutation may impair monocyte development by epigenetically and transcriptionally suppressing maturation-related programs and sustaining proliferative states.

ConclusionOur innovative single-cell multi-omics approaches revealed that TET2 mutations rewire chromatin accessibility and TF regulatory networks, leading to dysregulated lineage maturation and aberrant cell cycle control in CH/CCUS.