| Project/Area Number |
17591019
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Hematology
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| Research Institution | Health Sciences University of Hokkaido (2006)
Sasaki Institute (2005) |
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Principal Investigator |
OIKAWA Tsuneyuki Medical Genetics, Dept. of Communication Disorders, Professor, 心理科学部, 教授 (80150241)
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| Co-Investigator(Kenkyū-buntansha) |
KIHARA Fumiko (NEGISHI Fumiko) Department of Pharmacology, Teikyo University, Lecturer, 薬学部・臨床薬学, 講師 (40177902)
OHTA Toru Inst. of Personalized Medicine, Associate Professor, 個体差健康科学研究所, 准教授 (10223835)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2006: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2005: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | transcription factors / ETS / Ets-1 / PU.1 / Gfi-1 / GfilB / cross-talk / apoptosis / hematopoiesis / Gfi-1 / bax / アポトーシス / 血球分化 |
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| Research Abstract |
The protooncogene Ets-1 and growth factor independent-1 (Gfi-1) are implicated in cell growth and differentiation in various types of cells, and their deregulated expression is involved in malignant formation. We found that Ets-1 and Gfi-1 interacted and affected gene expression through their cross-talk. Co-immunoprecipitation analysis and glutathione-S-transferase (GST) pull-down assay revealed that Ets-1 bound directly to Gfi-1 via its Ets domain, and Gfi-1 bound to Ets-1 via its zinc-finger domain. Luciferase (Luc) assays using artificial reports showed that Gfi-1 repressed Ets-l-mediated transcription activation and Ets-1 repressed Gfi-l-mediated transcriptional activation. However, in the bax promoter where the Ets-and Gfrbinding sites (EBS and GBS) are adjacent, Ets-1 and Gfi-1 cooperatively reduced activation. Site-directed mutagenesis on the EBS and GBS of the bax promoter showed that both binding sites were necessary for full repression. Chromatin immunoprecipitation analyses
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confirmed that an Ets-1-Gfi-1 complex formed on the bax promoter even when either EBS or GBS was mutated. Introduction of small interfering RNA against Ets-1 and/or GE-1 enhanced endogenous bax gene expression. Our results suggest that the interaction between ETs-1 and Gfi-1 facilitates their binding to specific sites on the bax promoter and repress bax expression in vivo. PU.1 is a mater regulator for myeloid development and Gfi-1B is a hematopoietic transcription factor essential for growth and differentiation of the erythroid/megakaryocytic lineages. We found that PU.1 interacted with Gfi-1B in vivo by immunoprecipitation assay. GST pull-down assay showed that the binding sites were in the Ets domain of PU.1 and the zinc finger domain of Gfi-1B. Luciferase reporter assays revealed that PU.1 and Gfi-1B antagonized each other's transcriptional activity in a dose dependent manner. Transduction of Gfi-1B in mouse early myeloid progenitor cells showed that expression of Mac-1 was reduced, although this reduction was not too strong to inhibit PU.1-induced myeloid differentiation completely. Furthermore, transduction of PU.1 with Gfi1B in human cord blood hematopoietic progenitors significantly inhibited growth and colony formation of erythroid cells which were strongly enhanced by transduction of Gfi-lb alone. Co-expression of Gfi-1B with a mutant PU.1 deleted at the N-terminus and the 133/134 region of the Ets domain (PU. lANA133/34), which did not bind to GATA-1 but still bound to Gfi-1B, also inhibited GFi-1B-induced erythroid colony formation. Our results suggest that PU.1 inhibit growth and differentiation of erythroid cells by blocking GFi-1B function as well as by blocking GATA-1 function. Less
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