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The Study of the Molecular Mechanism of EDAG Regulating Hematogenesis

Author: XuChengWang
Tutor: YangXiaoMing
School: PLA Military Academy of Medical Sciences
Course: Biochemistry and Molecular Biology
Keywords: EDAG interaction protein HSP70 GATA-1
Type: PhD thesis
Year: 2008
Downloads: 36
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EDAG is a hematopoietic tissue-specific gene which involved in the regulation of hematopoietic cell proliferation, differentiation and apoptosis. But the mechanism is still unknown. The study in this paper is focused on the interaction between EDAG and binding partner HSP70, and makes a scientific approach to the molecular mechanism of EDAG regulating hematogenesis. We propose the regulation model of the complex of EDAG, HSP70 and GATA-1.In this study, a lot of EDAG binding partners were screened and identified from HEK293 or K562 cells by combination co-immunoprecipitation with mass spectrum. After analysis of locations, functions, dieases associations, phenotypes of mouse models and repeatabilities, twenty high confident proteins were obtained. Six proteins were selected to perform co-immunoprecipitation testing. Five of them are positively interacted with EDAG. Based on previous results, HSP70, which involved in the regulation of GATA-1, was performed further investigation. The physical interaction of EDAG and HSP70 was tested by co-localization and co-immunoprecipitation, the ATPase domain of HSP70 was found to be necessary for EDAG binding. We find that HSP70 may enhance the transactivation of EDAG in dose-dependant manner, while, EDAG may increase the nucleus translocation of HSP70. It can enhance the transcription activities of GATA-1, down regulation of EDAG expression may inhibit the expressions of GATA-1’s target genes. Binding of EDAG to the promoter sequence of GATA-1 target genes was confirmed by ChIP. EDAG, HSP70 and GATA-1 were testified in the same immunocomplex by Western blot. Taken together, these results suggest that EDAG may regulate hematopoiesis by interacting with HSP70 and GATA-1.Based on our data, we have proposed a hypothesis: EDAG, HSP70 and GATA-1 form a complex in vivo, by interacting each other, EDAG may increase the nucleus translocation of HSP70, on the other hand, HSP70 may stabilize the complex, especially stabilize GATA-1, and assist EDAG to recruit some transcription cofactors then enhance transactivation activity and transcription activities of GATA-1. By this way, EDAG, HSP70 and GATA-1 may work together to regulate the expressions of GATA-1 target genes and regulate hematopoiesis.

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