Proteomic Study of Megakaryocyte Transcriptional Control

描述(申请人提供)：巨核细胞是大的多倍体骨髓细胞，通过产生血小板在止血中发挥重要作用。巨核细胞的增殖和发育障碍，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血小板减少症，与人类的高发病率和死亡率有关。虽然对血小板生物学和巨核细胞生成的细胞因子调控已知很多，但对巨核细胞发育和生长的转录调控还不完全了解。先前的工作表明，锌指转录因子GATA-1在这两个过程中都扮演着重要的角色。GATA-1基因缺失导致小鼠巨核细胞明显过度增殖和成熟障碍，并导致进行性骨髓纤维化。导致GATA-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。凝胶过滤层析的初步研究表明，GATA-1参与了L8057小鼠巨核细胞系中至少两个稳定的约360 kDa和230 kDa的多蛋白复合体(仅GATA-1的相对分子质量为48 kDa)。本建议利用蛋白质组学方法鉴定这些复合体的组成并检验下列假设：(1)含有GATA-1的稳定多蛋白复合体的形成在调控巨核细胞特异性基因表达和生长控制方面具有重要的功能；(2)包含GATA-1的复合体的组成在巨核细胞分化过程中发生变化，(3)包含GATA-1的不同的复合体存在于巨核细胞和红系细胞中，以及(4)与巨核白血病相关的GATA-1的氨基末端截短突变导致多蛋白复合体的形成和/或活性改变。采用代谢生物素标记和亲和素亲和层析的新方法分离含GATA-1的复合体。成分将通过LC/MS/MS和数据库搜索进行鉴定。结果将通过原代巨核细胞的免疫共沉淀和凝胶位移分析来验证。最后，通过在诱导的L8057细胞中稳定的RNAi基因沉默来评估所鉴定的成分的功能意义。这项研究的结果将为巨核细胞生成的转录调控提供新的见解，并可能为巨核细胞增殖和发育障碍的治疗提供新的靶点。

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.