Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Blood. 2010 Apr 15;115(15):3128-35. doi: 10.1182/blood-2009-04-214601. Epub 2010 Feb 24.
Haploinsufficiency of RUNX1 (also known as CBFA2/AML1) is associated with familial thrombocytopenia, platelet dysfunction, and predisposition to acute leukemia. We have reported on a patient with thrombocytopenia and impaired agonist-induced aggregation, secretion, and protein phosphorylation associated with a RUNX1 mutation. Expression profiling of platelets revealed approximately 5-fold decreased expression of 12-lipoxygenase (12-LO, gene ALOX12), which catalyzes 12-hydroxyeicosatetraenoic acid production from arachidonic acid. We hypothesized that ALOX12 is a direct transcriptional target gene of RUNX1. In present studies, agonist-induced platelet 12-HETE production was decreased in the patient. Four RUNX1 consensus sites were identified in the 2-kb promoter region of ALOX12 (at -1498, -1491, -708, -526 from ATG). In luciferase reporter studies in human erythroleukemia cells, mutation of each site decreased activity; overexpression of RUNX1 up-regulated promoter activity, which was abolished by mutation of RUNX1 sites. Gel shift studies, including with recombinant protein, revealed RUNX1 binding to each site. Chromatin immunoprecipitation revealed in vivo RUNX1 binding in the region of interest. siRNA knockdown of RUNX1 decreased RUNX1 and 12-LO proteins. ALOX12 is a direct transcriptional target of RUNX1. Our studies provide further proof of principle that platelet expression profiling can elucidate novel alterations in platelets with inherited dysfunction.
RUNX1（也称为 CBFA2/AML1）的单倍体不足与家族性血小板减少症、血小板功能障碍和急性白血病易感性有关。我们曾报道过一名血小板减少症患者，其血小板激动剂诱导的聚集、分泌和蛋白磷酸化受损，与 RUNX1 突变有关。对血小板进行表达谱分析发现，12-脂氧合酶（12-LO，基因 ALOX12）的表达降低了约 5 倍，该酶可催化花生四烯酸生成 12-羟基二十碳四烯酸。我们假设 ALOX12 是 RUNX1 的直接转录靶基因。在本研究中，患者的血小板 12-HETE 产生减少。在 ALOX12（从 ATG 开始的 -1498、-1491、-708、-526）的 2kb 启动子区域中鉴定出 4 个 RUNX1 共有序列。在人红白血病细胞中的荧光素酶报告基因研究中，每个位点的突变均降低了活性；过表达 RUNX1 可上调启动子活性，而 RUNX1 位点的突变则使其丧失活性。凝胶迁移实验，包括重组蛋白，揭示了 RUNX1 与每个位点的结合。染色质免疫沉淀实验显示在感兴趣区域有 RUNX1 的体内结合。RUNX1 的 siRNA 敲低降低了 RUNX1 和 12-LO 蛋白。ALOX12 是 RUNX1 的直接转录靶基因。我们的研究进一步证明了血小板表达谱分析可以阐明遗传性功能障碍血小板的新变化。
