Proteomic Study of Megakaryocyte Transcriptional Control

描述（由申请方提供）：巨核细胞是大型多倍体骨髓细胞，通过生成血小板在止血中发挥重要作用。巨核细胞增殖和发育障碍，包括巨核细胞白血病、骨髓纤维化、原发性血小板增多症和巨核细胞性血小板减少症，与人类的高发病率和死亡率相关。虽然对血小板生物学和巨核细胞生成的细胞因子调节了解很多，但对巨核细胞发育和生长的转录调节还不完全了解。先前的工作表明，锌指转录因子加塔-1在这两个过程中起着至关重要的作用。加塔-1的缺乏导致巨核细胞的显著过度增殖和受损的成熟，并引起小鼠中的进行性骨髓纤维化。导致加塔-1氨基末端截短的突变与唐氏综合征患者的巨核细胞白血病高度相关。使用凝胶过滤色谱法的初步研究表明，加塔-1在L 8057鼠巨核细胞系中参与至少两种约360和230 kDa的稳定多蛋白复合物（单独的加塔-1的分子量为48 kDa）。本研究利用蛋白质组学方法鉴定了这些复合物的组分，并验证了以下假设：（1）含有加塔-1的稳定多蛋白复合物的形成在控制巨核细胞特异性基因表达和生长控制中具有重要功能;（2）含加塔-1复合物的组成在巨核细胞分化过程中发生变化，（3）在巨核细胞与红系细胞中存在不同的含有加塔-1的复合物，和（4）与巨核细胞白血病相关的加塔-1的氨基末端截短突变导致改变的多蛋白复合物形成和/或活性。一种新的方法，采用代谢生物素标记，然后亲和素亲和层析将用于分离加塔-1含有复合物。将通过LC/MS/MS质谱法和数据库检索鉴别组分。将通过原代巨核细胞中的免疫共沉淀和凝胶迁移试验验证结果。最后，通过在诱导的L 8057细胞中稳定的RNAi基因沉默来评估鉴定的组分的功能意义。这项研究的结果应该提供新的见解巨核细胞的转录调控，并可能为巨核细胞增殖和发育障碍的新的治疗靶点。

DESCRIPTION (provided by applicant): Megakaryocytes are large polyploid bone marrow cells that play essential roles in hemostasis through the generation of platelets. Disorders of megakaryocyte proliferation and development, including megakaryoblastic leukemia, myelofibrosis, essential thrombocythemia, and megakaryoblastic thrombocytopenia are associated with high morbidity and mortality in humans. While much is known about platelet biology and the cytokine regulation of megakaryopoiesis, the transcriptional regulation of megakaryocyte development and growth is incompletely understood. Prior work has shown that the zinc finger transcription factor GATA-1 plays an essential role in both of these processes. Deficiency of GATA-1 leads to marked hyperproliferation and impaired maturation of megakaryocytes, and causes progressive myelofibrosis in mice. Mutations that result in an amino terminal truncation of GATA-1 are highly associated with megakaryoblastic leukemia in patients with Down syndrome. Preliminary studies using gel filtration chromatography indicate that GATA-1 participates in at least two stable multiprotein complexes of about 360 and 230 kDa in the L8057 murine megakaryocytic cell line (molecular mass of GATA-1 alone is 48 kDa). This proposal utilizes a proteomic approach to identify the components of these complexes and test the following hypotheses: (1) the formation of stable multiprotein complexes containing GATA-1 is functionally important in controlling megakaryocyte-specific gene expression and growth control; (2) the composition of GATA-1 containing complexes changes during megakaryocyte differentiation, (3) distinct GATA-1 containing complexes exist in megakaryocyte versus erythroid cells, and (4) amino terminal truncation mutations of GATA-1 associated with megakaryoblastic leukemia result in altered multiprotein complex formation and/or activity. A novel method employing metabolic biotin tagging followed by avidin affinity chromatography will be used to isolate GATA-1 containing complexes. Components will be identified by LC/MS/MS mass spectrometry and database search. Results will be validated by co-immunoprecipitation and gel shift assays in primary megakaryocytes. Finally, the functional significance of identified components will be assessed by stable RNAi gene silencing in induced L8057 cells. The results of this study should provide new insights into the transcriptional regulation of megakaryopoiesis, and possibly new therapeutic targets for disorders of megakaryocyte proliferation and development.