| Project/Area Number |
16590309
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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 |
Experimental pathology
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| Research Institution | Nagoya University |
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Principal Investigator |
ICHIHARA Masatoshi Nagoya University, School of Medicine, Associate Professor, 医学部, 助教授 (00314013)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Masahide Nagoya University, Graduate School of Medicine, Professor, 大学院・医学系研究科, 教授 (40183446)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | RET / GDNF / GZF1 / ureteric bud / BTB / POZ / HOXA10 |
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| Research Abstract |
By differential display analysis, we have recently identified a novel GDNF-inducible gene named GZF1 (GDNF-inducible zinc finger protein 1). GZF1 encodes a protein with a BTB/POZ domain at the N-terminus and 10 tandemly repeated C2H2 zinc finger motifs. The fact that antisense phosphorothioated oligodeoxynucleotides of the GZF1 gene markedly impair the ureteric bud branching in the metanephric organ culture suggests that GZF1 expression mediated by GDNF may play a role in renal branching morphogenesis. In this study, we first demonstrated the DNA binding property of GZF1 and its potential target gene. Using the cyclic amplification and selection of targets technique (CAST method), the consensus DNA sequence to which GZF1 binds was determined. Deletion mutants of GZF1 revealed that zinc fingers 1-6 of GZF1 are necessary for full DNA binding ability. Electrophoretic mobility shift assay revealed that GZF1 specifically binds to the determined consensus sequence and suppresses transcription of the luciferase gene from the HOXA10 gene regulatory element. Moreover, GZF1 target sequence was found in the 5' regulatory region of the HOXA10 gene that is a homeobox protein expressed during kidney development. Chromatin fragments containing GZF1 target sequence in HOXA10 promoter were specifically precipitated with anti-GZF1 antibody. These results demonstrated that GZF1 binds to the recognition sequence in the HOXA10 regulatory region in vivo (Morinaga T, Nucleic Acids Res. 2005). Next, we explored a GZF1 binding protein to further elucidate the molecular mechanism of GZF1. Using LC/MS/MS, we identified two known proteins immunoprecipitated with GZF1. We focused on one of these proteins. The endogenous protein was actually immunoprecipitated with GZF1 as well as the overexpressed one. Furthermore, this protein was co-localized with GZF1 in the cell nucleus. We have been now determining the GZF1-binding domain in this protein and a functional role in the kidney development.
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