Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
J Thromb Haemost. 2013 Sep;11(9):1742-50. doi: 10.1111/jth.12355.
Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt-related transcription factor 1 (RUNX1) of FPD patients.
To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1-mediated induction of the FPD phenotype.
We assessed whether RUNX1 and its mutants, in combination with E26 transformation-specific-1 (ETS-1), Core-binding factor subunit beta (CBFβ), and Friend leukemia virus integration 1 (FLI-1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real-time RT-PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos-7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos-7 cells.
We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS-1 and CBFβ, or FLI-1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA-binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS-1, CBFβ, and FLI-1.
RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.
家族性血小板减少症（FPD）是一种罕见的常染色体显性遗传疾病，其特征为血小板减少和血小板功能异常。FPD 患者中 runt 相关转录因子 1（RUNX1）基因的致病突变已被鉴定。
阐明 RUNX1 在调节血小板因子 4（PF4）表达中的作用，并提出 RUNX1 介导 FPD 表型的可能机制。
我们评估了 RUNX1 及其突变体与 E26 转化特异性 1（ETS-1）、核心结合因子亚基 β（CBFβ）和 Friend 白血病病毒整合 1（FLI-1）一起在巨核细胞分化过程中是否协同调节 PF4 表达。在胚胎干细胞分化系统中，通过实时 RT-PCR 测定内源性和外源性 RUNX1 和 PF4 的表达水平。通过 HepG2 细胞的报告基因测定评估转录因子的启动子激活。通过 Cos-7 细胞的电泳迁移率变动分析和免疫沉淀分析分别分析 DNA 结合活性和蛋白相互作用。通过 Cos-7 细胞的免疫细胞化学和 Western blot 分析蛋白质定位。
我们证明 RUNX1 在巨核细胞分化中激活内源性 PF4 表达。只有 RUNX1，而不是其突变体，与 ETS-1 和 CBFβ，或 FLI-1 一起，协同激活 PF4 启动子。每个 RUNX1 突变体都具有各种功能异常，包括 DNA 结合活性丧失、异常的亚细胞定位和/或对 ETS-1、CBFβ 和 FLI-1 的结合亲和力改变。
只有 RUNX1，而不是其突变体，与 ETS 家族蛋白一起强烈且协同地激活 PF4 表达。此外，RUNX1 的转录激活功能丧失是由各种功能异常引起的。
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