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.
