RESUMO
In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they 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 wild-type cells. Here, 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 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 patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.
Assuntos
Cromatina , Epigênese Genética , Genótipo , Mutação , Análise de Célula Única , Animais , Feminino , Humanos , Masculino , Camundongos , Antígenos CD34/metabolismo , Diferenciação Celular/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética/genética , Epigenoma/genética , Genoma Mitocondrial/genética , Técnicas de Genotipagem , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Inflamação/genética , Inflamação/patologia , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Megacariócitos/metabolismo , Megacariócitos/patologia , Proteínas de Membrana/genética , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , RNA/genética , Células Clonais/metabolismoRESUMO
DNA double-strand breaks (DSBs) are nucleolytically processed to generate single-stranded DNA tails for homologous recombination. In Saccharomyces cerevisiae meiosis, this 5'-to-3' resection involves initial nicking by the Mre11-Rad50-Xrs2 complex (MRX) plus Sae2, then exonucleolytic digestion by Exo1. Chromatin remodeling adjacent to meiotic DSBs is thought to be necessary for resection, but the relevant remodeling activity was unknown. Here we show that the SWI/SNF-like ATPase Fun30 plays a major, non-redundant role in resecting meiotic DSBs. A fun30 null mutation shortened resection tract lengths almost as severely as an exo1-nd (nuclease-dead) mutation, and resection was further shortened in the fun30 exo1-nd double mutant. Fun30 associates with chromatin in response to meiotic DSBs, and the constitutive positioning of nucleosomes governs resection endpoint locations in the absence of Fun30. We infer that Fun30 directly promotes both the MRX- and Exo1-dependent steps in resection, possibly by removing nucleosomes from broken chromatids. Moreover, we found that the extremely short resection in the fun30 exo1-nd double mutant is accompanied by compromised interhomolog recombination bias, leading to defects in recombination and chromosome segregation. Thus, this study also provides insight about the minimal resection lengths needed for robust recombination.
RESUMO
SARS-CoV-2 infection and vaccination elicit potent immune responses. Our study presents a comprehensive multimodal single-cell analysis of blood from COVID-19 patients and healthy volunteers receiving the SARS-CoV-2 vaccine and booster. We profiled immune responses via transcriptional analysis and lymphocyte repertoire reconstruction. COVID-19 patients displayed an enhanced interferon signature and cytotoxic gene upregulation, absent in vaccine recipients. B and T cell repertoire analysis revealed clonal expansion among effector cells in COVID-19 patients and memory cells in vaccine recipients. Furthermore, while clonal αß T cell responses were observed in both COVID-19 patients and vaccine recipients, expansion of clonal γδ T cells was found only in infected individuals. Our dataset enables side-by-side comparison of immune responses to infection versus vaccination, including clonal B and T cell responses. Our comparative analysis shows that vaccination induces a robust, durable clonal B and T cell responses, without the severe inflammation associated with infection.