RT Journal Article SR Electronic T1 Integrated Single-Cell Genotyping and Chromatin Accessibility Charts JAK2V617F Human Hematopoietic Differentiation JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.05.11.491515 DO 10.1101/2022.05.11.491515 A1 Robert M. Myers A1 Franco Izzo A1 Sanjay Kottapalli A1 Tamara Prieto A1 Andrew Dunbar A1 Robert L. Bowman A1 Eleni P. Mimitou A1 Maximilian Stahl A1 Sebastian El Ghaity-Beckley A1 JoAnn Arandela A1 Ramya Raviram A1 Saravanan Ganesan A1 Levan Mekerishvili A1 Ronald Hoffman A1 Ronan Chaligné A1 Omar Abdel-Wahab A1 Peter Smibert A1 Bridget Marcellino A1 Ross L. Levine A1 Dan A. Landau YR 2022 UL http://biorxiv.org/content/early/2022/05/11/2022.05.11.491515.abstract AB 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.Competing Interest StatementM.S. has served as a consultant for Curis Oncology, Haymarket Media, and Boston Consulting, and is on the Scientific Advisory Board of Novartis and Kymera. R.H. has served as a consultant for Protagonist Therapeutics, Inc., received research funding from Kartos Therapeutics, Inc., Novartis, and AbbVie Inc, and is on the Data Safety Monitoring Board of Novartis and AbbVie Inc. 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, LOXO Oncology, and Nurix Therapeutics unrelated to the current manuscript. P.S. and E.P.M. are current employees of 10x Genomics and Immunai, respectively. R.L.L. is on the supervisory board of Qiagen and is a scientific advisor to Imago, Mission Bio, Bakx, Zentalis, Ajax, Auron, Prelude, C4 Therapeutics and Isoplexis. R.L.L. has received research support from Abbvie, Constellation, Ajax, Zentalis and Prelude. R.L.L. has received research support from and consulted for Celgene and Roche and has consulted for Syndax, Incyte, Janssen, Astellas, Morphosys, and Novartis. R.L.L. has received honoraria from Astra Zeneca and Novartis for invited lectures and from Gilead and Novartis for grant reviews. D.A.L. has served as a consultant for Abbvie and Illumina and is on the Scientific Advisory Board of Mission Bio and C2i Genomics. D.A.L. has received prior research funding from BMS, 10x Genomics and Illumina unrelated to the current manuscript. R.M.M., F.I., E.P.M., R.C., P.S., and D.A.L. have filed a patent for GoT-ChA (#63/288,874). No other authors report competing interests.