Aberrant DNA methylation occurs in RUNX1 heterozygous mutations harboring hematopoietic progenitor cells [CUT&RUN]

Background: Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by a heterozygous germline mutation inRUNX1. The patients with FPDMM show not only thrombocytopenia with platelet dysfunction, but also a high level of developing hematological malignancies, strongly suggesting that FPDMM is in a precancerous state. However, the DNA methylation status of FPDMM has not yet been elucidated due to no animal models for FPDMM and the difficulty in obtaining the patient-derived samples. Results: We found that differentiation efficiencies into HPCs and megakaryocytes was reduced in the FPDMM-mimicking cells, which were established by genome editing for human iPS cells as a FPDMM-model, compared with those of wild-type cells. The FPDMM-mimicking HPCs were subjected to DNA methylation analysis, and the HPCs showed the distinct DNA methylation patterns compared to wild-type HPCs. Furthermore, we identified FLI1 as a putative causative transcription factor of the differential DNA hypermethylation involved in both promoting the binding site-directed DNA demethylation and regulating megakaryopoiesis. Overall design: To establish the human iPS cell lines with FPDMM-mimicking RUNX1 heterozygous mutations, we performed CRISP-Cas9-mediated genome editing of RUNX1, followed by single-cell cloning of the resulting genome-edited cells. We performed cleavage under targets and release using nuclease (CUT&RUN) sequencing for FLI1 to confirm its potential binding in the vicinity of hypermethylated differentially methylated cytosine-guanine dinucleotides (DMCs) in FPDMM-mimicking HPCs.