Differential DNA methylation occurs in RUNX1 heterozygous mutations harboring hematopoietic progenitor cells [FLI1_EM]

Background: Familial platelet disorder (FPD) is an autosomal dominant disease caused by a heterozygous germline mutation inRUNX1. FPD patients show not only thrombocytopenia with platelet dysfunction, but also a high level of developing hematological malignancies, strongly suggesting that FPD is in a precancerous state. However, the DNA methylation status of FPD has not yet been elucidated due to no animal models for FPD and the difficulty in obtaining FPD patient-derived samples. Results: We found that differentiation efficiencies into HPCs and megakaryocytes was reduced in the FPD-mimicking cells, which were established by genome editing for human iPS cells as a FPD-model, compared with those of wild-type cells. The FPD-mimicking HPCs were subjected to DNA methylation analysis, and the HPCs showed the distinct DNA methylation patterns compared to wild-type HPCs. Furthermore, we demonstrated a putative causative transcription factor of the differential DNA hypermethylation, which are involved in both promoting the binding site-directed DNA demethylation and regulating megakaryopoiesis. We performed the methylome analysis of iPS cells overexpressed FLI1, a putative causative transcription factor for differential DNA hypermethylation in FPD-mimicking HPCs, by doxycycline (DOX)-inducible manner to validate whether FLI1 induces the DNA demethylation at its binding sites in vitro.

背景：家族性血小板疾病（Familial platelet disorder, FPD）是一类由RUNX1基因杂合生殖系突变引发的常染色体显性遗传病。FPD患者不仅表现为血小板减少症伴血小板功能异常，还存在较高的血液系统恶性肿瘤发病风险，这强烈提示FPD处于癌前状态。然而，由于缺乏FPD相关动物模型且难以获取患者来源样本，目前学界尚未阐明FPD的DNA甲基化状态。
结果：本研究通过对人类诱导多能干细胞（induced pluripotent stem cells, iPSCs）进行基因组编辑，构建了FPD模型细胞——FPD模拟细胞。相较于野生型细胞，该细胞向造血祖细胞（hematopoietic progenitor cells, HPCs）与巨核细胞（megakaryocytes）的分化效率显著降低。我们对FPD模拟造血祖细胞开展DNA甲基化分析，发现其DNA甲基化模式与野生型造血祖细胞存在显著差异。此外，本研究还鉴定出一种可能介导差异DNA高甲基化的转录因子，该因子既可参与结合位点导向的DNA去甲基化过程，又可调控巨核细胞生成。为验证转录因子FLI1是否可在体外诱导其结合位点发生DNA去甲基化，我们采用强力霉素（doxycycline, DOX）诱导系统，对过表达FLI1（FPD模拟造血祖细胞中差异DNA高甲基化的潜在致病转录因子）的诱导多能干细胞开展甲基化组分析。