| Project/Area Number |
13680771
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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 |
Molecular biology
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| Research Institution | Aichi Cancer Center |
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Principal Investigator |
HIROSE Fumiko Div. of Biochemistry, Senior researcher, 発がん制御研究部, 主任研究員 (60208882)
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| Co-Investigator(Kenkyū-buntansha) |
YAMAGUCHI Masamitsu Kyoto inst. of technology, Faculty of textile sciences, Professor, 繊維学部, 教授 (00182460)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Chromatin remodeling / teanscription factor / Mi-2 / DREF / regulation of gene expression / クロマチン / 転写調節因子 / ショウジョウバエ |
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| Research Abstract |
(1) Interaction between DREF and Mi-2 : We have carried out a yeast two-hybrid screening with DREF polypeptide as bait and identified Mi-2 as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA binding domain of dDREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits DNA binding activity of dDREF. Ectopic expression of dDREF and dMi-2 in eye imaginal discs resulted in severe and mild rough eye phenotypes, respectively, while flies simultaneously expressing both proteins exhibited almost normal eyes. Half dose reduction of the dMi-2 gene enhanced the DREF-induced rough eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that dDREF and dMi-2 bind in mutually exclusive ways. The line of evidence define a novel function of dMi-2 in negative regulation of dDREF by its DNA-binding activity. Finally, we postulate the hypothesis that dDREF and dMi-2 may
… More
demonstrate reciprocal regulation of their functions.
(2) Characterization of human DREF : A human homologue of Drosophila DREF (hDREF) was identified by BLAST search. A consensus binding sequence (5'-TGTCGC/TGAC/TA) for hDREF, determined by the CASTing method, overlapped with that for the Drosophila DREF (5'-TGTCGATA). We found hDREF binding sequences in the promoter regions of human genes related to cell proliferation. Analyses using a specific antibody revealed that an hDREF binds to the promoter region of the histone H1 gene. Co-transfection experiments with an hDREF-expressing plasmid and a histone H1 promoter directed-luciferase reporter plasmid in HeLa cells revealed possible activation of the histone H1 promoter. Immunohistochemical analysis demonstrated that hDREF is localized in the nuclei. Although the expression level of the factor was found to be low in serum-deprived human normal fibroblasts, the amount was increased by adding serum to cultures and reached a maximum during S phase. RNA interference experiments targeting hDREF resulted in inhibition of S-phase entry and reduction of histone H1 mRNA in HeLa cells. These results suggest that expression of hDREFmay have a role in regulation of human genes related to cell proliferation. Less
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