Wijgaerts Anouck, Wittevrongel Christine, Thys Chantal, Devos Timothy, Peerlinck Kathelijne, Tijssen Marloes R, Van Geet Chris, Freson Kathleen
Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KULeuven, Belgium.
Department of Haematology, University Hospitals Leuven, Belgium.
Haematologica. 2017 Apr;102(4):695-706. doi: 10.3324/haematol.2016.152777. Epub 2017 Jan 12.
Gray platelet syndrome is named after the gray appearance of platelets due to the absence of α-granules. It is caused by recessive mutations in , resulting in macrothrombocytopenia and myelofibrosis. Though using the term gray platelets for GATA1 deficiency has been debated, a reduced number of α-granules has been described for macrothrombocytopenia due to mutations. We compared platelet size and number of α-granules for two NBEAL2 and two GATA1-deficient patients and found reduced numbers of α-granules for all, with the defect being more pronounced for NBEAL2 deficiency. We further hypothesized that the granule defect for GATA1 is due to a defective control of NBEAL2 expression. Remarkably, platelets from two patients, and Gata1-deficient mice, expressed almost no NBEAL2. The differentiation of GATA1 patient-derived CD34 stem cells to megakaryocytes showed defective proplatelet and α-granule formation with strongly reduced NBEAL2 protein and ribonucleic acid expression. Chromatin immunoprecipitation sequencing revealed 5 GATA binding sites in a regulatory region 31 kb upstream of covered by a H3K4Me1 mark indicative of an enhancer locus. Luciferase reporter constructs containing this region confirmed its enhancer activity in K562 cells, and mutagenesis of the GATA1 binding sites resulted in significantly reduced enhancer activity. Moreover, DNA binding studies showed that GATA1 and GATA2 physically interact with this enhancer region. GATA1 depletion using small interfering ribonucleic acid in K562 cells also resulted in reduced NBEAL2 expression. In conclusion, we herein show a long-distance regulatory region with GATA1 binding sites as being a strong enhancer for NBEAL2 expression.
灰色血小板综合征因血小板缺乏α颗粒而呈现灰色外观得名。它由[基因名称缺失]的隐性突变引起，导致大血小板减少症和骨髓纤维化。尽管对于GATA1缺乏症使用灰色血小板这一术语存在争议，但已描述了因[基因名称缺失]突变导致的大血小板减少症中α颗粒数量减少的情况。我们比较了两名NBEAL2缺陷患者和两名GATA1缺陷患者的血小板大小和α颗粒数量，发现所有人的α颗粒数量均减少，且NBEAL2缺陷时这种缺陷更为明显。我们进一步推测，GATA1的颗粒缺陷是由于对NBEAL2表达的控制缺陷所致。值得注意的是，两名患者以及Gata1缺陷小鼠的血小板几乎不表达NBEAL2。来自GATA1患者的CD34干细胞向巨核细胞的分化显示前血小板和α颗粒形成存在缺陷，NBEAL2蛋白和核糖核酸表达大幅降低。染色质免疫沉淀测序揭示在[基因名称缺失]上游31 kb的一个调控区域中有5个GATA结合位点，该区域被一个指示增强子位点的H3K4Me1标记覆盖。包含该区域的荧光素酶报告构建体证实了其在K562细胞中的增强子活性，GATA1结合位点的诱变导致增强子活性显著降低。此外，DNA结合研究表明GATA1和GATA2与该增强子区域存在物理相互作用。在K562细胞中使用小干扰核糖核酸耗尽GATA1也导致NBEAL2表达降低。总之，我们在此表明具有GATA1结合位点的一个长距离调控区域是NBEAL2表达的一个强增强子。
