Proteomic Study of Megakaryocyte Transcriptional Control

描述（由申请人提供）：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起重要作用。巨核细胞增殖和发育障碍，包括巨核母细胞白血病、骨髓纤维化、原发性血小板增多症和巨核母细胞血小板减少症，在人类中与高发病率和死亡率相关。虽然我们对血小板生物学和巨核细胞生成的细胞因子调控有很多了解，但对巨核细胞发育和生长的转录调控还不完全了解。先前的研究表明锌指转录因子GATA-1在这两个过程中都起着至关重要的作用。GATA-1的缺乏导致巨核细胞明显过度增殖和成熟受损，并导致小鼠进行性骨髓纤维化。导致GATA-1氨基末端截断的突变与唐氏综合征患者的巨核母细胞白血病高度相关。凝胶过滤色谱的初步研究表明，GATA-1在L8057小鼠巨核细胞系中至少参与了两个稳定的多蛋白复合物，约为360和230 kDa （GATA-1单独的分子质量为48 kDa）。本研究利用蛋白质组学方法鉴定了这些复合物的组成部分，并验证了以下假设：(1)含有GATA-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的功能；(2)巨核细胞分化过程中含GATA-1复合物的组成发生变化；(3)巨核细胞与红系细胞中存在不同的含GATA-1复合物；(4)巨核母细胞白血病相关的GATA-1氨基末端截断突变导致多蛋白复合物形成和/或活性改变。本文将采用代谢生物素标记和亲和层析相结合的新方法分离GATA-1复合物。成分将通过LC/MS/MS质谱和数据库检索进行鉴定。结果将通过原代巨核细胞的共免疫沉淀和凝胶转移试验进行验证。最后，在诱导的L8057细胞中，通过稳定的RNAi基因沉默来评估鉴定组分的功能意义。本研究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提供新的治疗靶点。

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.