Department of Medicine, Cardeza Foundation for Hematologic Research, Sidney Kimmel Medical School at Thomas Jefferson University, Philadelphia, Pennsylvania, United States.
Department of Human & Molecular Genetics, Baylor College of Medicine, Houston, Texas, United States.
Thromb Haemost. 2019 May;119(5):716-725. doi: 10.1055/s-0039-1678694. Epub 2019 Feb 7.
Megakaryopoiesis produces specialized haematopoietic stem cells in the bone marrow that give rise to megakaryocytes which ultimately produce platelets. Defects in megakaryopoiesis can result in altered platelet counts and physiology, leading to dysfunctional haemostasis and thrombosis. Additionally, dysregulated megakaryopoiesis is also associated with myeloid pathologies. Transcription factors play critical roles in cell differentiation by regulating the temporal and spatial patterns of gene expression which ultimately decide cell fate. Several transcription factors have been described as regulating megakaryopoiesis including myocyte enhancer factor 2C (MEF2C); however, the genes regulated by MEF2C that influence megakaryopoiesis have not been reported. Using chromatin immunoprecipitation-sequencing and Gene Ontology data we identified five candidate genes that are bound by MEF2C and regulate megakaryopoiesis: , , , and . To study expression of these genes, we silenced gene expression in the Meg01 megakaryocytic cell line and in induced pluripotent stem cells by CRISPR/Cas9 editing. We also knocked down expression in cord blood-derived haematopoietic stem cells by siRNA. We found that absent or reduced expression resulted in defects in megakaryocytic differentiation and reduced levels of the candidate target genes. Luciferase assays confirmed that genomic sequences within the target genes are regulated by MEF2C levels. Finally, we demonstrate that small deletions linked to a platelet count-associated single nucleotide polymorphism alter transcriptional activity, suggesting a mechanism by which genetic variation in alters platelet production. These data help elucidate the mechanism behind MEF2C regulation of megakaryopoiesis and genetic variation driving platelet production.
巨核细胞生成在骨髓中产生专门的造血干细胞，这些干细胞最终产生巨核细胞，而巨核细胞又产生血小板。巨核细胞生成缺陷可导致血小板计数和生理学改变，导致止血功能障碍和血栓形成。此外，调节异常的巨核细胞生成也与骨髓病理有关。转录因子通过调节基因表达的时空模式在细胞分化中发挥关键作用，最终决定细胞命运。已经描述了几种转录因子来调节巨核细胞生成，包括肌细胞增强因子 2C（MEF2C）；然而，受 MEF2C 调节并影响巨核细胞生成的基因尚未报道。使用染色质免疫沉淀测序和基因本体数据，我们确定了五个受 MEF2C 结合并调节巨核细胞生成的候选基因：、、、和。为了研究这些基因的表达，我们通过 CRISPR/Cas9 编辑沉默 Meg01 巨核细胞系和诱导多能干细胞中的基因表达，并通过 siRNA 敲低脐带血来源的造血干细胞中的表达。我们发现，缺失或减少表达导致巨核细胞分化缺陷和候选靶基因水平降低。荧光素酶测定证实靶基因内的基因组序列受 MEF2C 水平调节。最后，我们证明与血小板计数相关的单核苷酸多态性相关的小缺失改变了转录活性，这表明了在改变血小板生成中遗传变异的机制。这些数据有助于阐明 MEF2C 调节巨核细胞生成和遗传变异驱动血小板生成的机制。
