Integrated Single-Cell Genotyping and Chromatin Accessibility ChartsJAK2^V617FHuman Hematopoietic Differentiation

Abstract

In normal somatic tissue differentiation, changes in chromatin accessibility govern priming and commitment of precursors towards cellular fates. In turn, somatic mutations can disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wildtype cells. To chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed Genotyping of Targeted loci with single-cell Chromatin Accessibility (GoT-ChA). This high-throughput, broadly accessible platform links genotypes to chromatin accessibility at single-cell resolution, across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from myeloproliferative neoplasm (MPN) patients with JAK2V617F-mutated hematopoiesis, where the JAK2 mutation is known to perturb hematopoietic differentiation. Differential accessibility analysis between wildtype and JAK2V617F mutant progenitors revealed both cell-intrinsic and cell state-specific shifts within mutant hematopoietic precursors. An early subset of mutant hematopoietic stem and progenitor cells (HSPCs) exhibited a cell-intrinsic pro-inflammatory signature characterized by increased NF-κB and JUN/FOS transcription factor motif accessibility. In addition, mutant HSPCs showed increased myeloid/erythroid epigenetic priming, preceding increased erythroid and megakaryocytic cellular output. Erythroid progenitors displayed aberrant regulation of the γ-globin locus, providing an intrinsic epigenetic basis for the dysregulated fetal hemoglobin expression observed in MPNs. In contrast, megakaryocytic progenitors exhibited a more specialized inflammatory chromatin landscape relative to early HSPCs, with increased accessibility of pro-fibrotic JUN/FOS transcription factors. Notably, analysis of myelofibrosis patients treated with JAK inhibitors revealed an overall loss of mutant-specific phenotypes without modifying clonal burden, consistent with clinical responses. Finally, expansion of the multi-modality capability of GoT-ChA to integrate mitochondrial genome profiling and cell surface protein expression measurement enabled genotyping imputation and discovery of aberrant cellular phenotypes. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner. We envision that GoT-ChA will thus serve as a foundation for broad future explorations to uncover the critical link between mutated somatic genotypes and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Multiomic Profiling of Human Clonal Hematopoiesis Reveals Genotype and Cell-Specific Inflammatory Pathway Activation

“…Cell-intrinsic consequences may arise directly from the somatic mutation while extrinsic, indirect consequences may arise from altered cell-cell interactions or secreted immune effectors. Several technologies have sought to close this gap by selectively amplifying the mRNA transcriptome and using this to genotype cells [11][12][13][14] . This approach is effective in HSPCs that express DNMT3A and TET2, with a genotyping efficiency rate of ~1-9%; however, genotyping is less efficient in cells that do not express these genes, such as fully differentiated cells in the peripheral blood 12,13,15 .…”

“…Several technologies have sought to close this gap by selectively amplifying the mRNA transcriptome and using this to genotype cells [11][12][13][14] . This approach is effective in HSPCs that express DNMT3A and TET2, with a genotyping efficiency rate of ~1-9%; however, genotyping is less efficient in cells that do not express these genes, such as fully differentiated cells in the peripheral blood 12,13,15 . Another method, TARGET-seq, has sought to amplify specific, previously identified variants in mutated cells with high-efficiency genotyping along with gene expression, albeit lower throughput 16,17 .…”

Clonal architecture evolution in Myeloproliferative Neoplasms: from a driver mutation to a complex heterogeneous mutational and phenotypic landscape

“…More recent studies combining at the single cell level the genotyping of specific genes with the analysis of chromatin accessibility showed that JAK2 V617F mutated HSPCs exhibited specific proinflammatory signatures with accessibility to DNA motifs binding NF-kB or JUN/FOS factors. This technique also allowed to highlight epigenetic modifications specific to mutated erythroid or megakaryocytic progenitors, demonstrating that the consequences of the mutations are different according to the differentiation state and cellular context [55]. A similar approach showed that DNMT3A mutations resulted in myeloid over lymphoid bias and in expansion of immature myeloid progenitors primed toward megakaryocytic-erythroid fate, and also demonstrated the dysregulated expression of lineage and leukemia stem cell markers with the preferential hypomethylation of polycomb repressive complex 2 targets [56].…”

Unraveling Heterogeneity in the Aging Hematopoietic Stem Cell Compartment: An Insight From Single-cell Approaches

“…An alternative approach uses a combination of genotyping of targeted loci with single-cell ChA (GoT-ChA) and was able to show both cell-intrinsic and cell state-specific shifts within mutant human hematopoietic HSPCs in the context of JAK2V617F-mutated MPN. 113 Contributing further to MPN biology by using scRNA-Seq and genotyping of JAK2V617F mutation status showed increases in the expression of interferon-response genes. 99,114 In human cells, genotyping of the mitochondrial genome at the single-cell level has been recently combined with scATAC-Seq to study cell population dynamics and clonal properties in cancer of the hematopoietic system, showing that it is possible to infer subclonal population structures using natural genetic mitochondrial DNA barcodes.…”

“…An alternative approach uses a combination of genotyping of targeted loci with single-cell ChA (GoT-ChA) and was able to show both cell-intrinsic and cell state-specific shifts within mutant human hematopoietic HSPCs in the context of JAK2V617F -mutated MPN. 113 Contributing further to MPN biology by using scRNA-Seq and genotyping of JAK2V617F mutation status showed increases in the expression of interferon-response genes. 99,114…”