Regulation of cell proliferation by a chromatin-remodeling factor, Mi-2

染色质重塑因子 Mi-2 对细胞增殖的调节

• 批准号: 13680771
• 负责人: HIROSE Fumiko
• 金额: $ 2.18万
• 依托单位: Aichi Cancer Center
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680771/
• 关键词: 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

(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

(1)DREF与Mi-2的相互作用：以DREF多肽为诱饵进行酵母双杂交筛选，确定Mi-2为DREF相互作用蛋白。生化分析表明，果蝇Mi-2(Dmi-2)的C末端区域与dDREF的DNA结合域特异结合。凝胶迁移率改变分析表明，DMI-2可抑制dDREF的DNA结合活性。DDREF和DMI-2在眼睛想象盘中的异位表达分别导致了重度和轻度粗糙的眼睛表型，而同时表达这两种蛋白的果蝇表现出几乎正常的眼睛。减半剂量的DMI-2基因增强了DREF诱导的粗糙眼表型。唾液腺多线染色体免疫组织化学染色显示，dDREF与Dmi-2以互斥方式结合。这一系列证据定义了DMI-2通过其DNA结合活性对dDREF进行负调控的新功能。最后，我们假设dDREF和dmi-2可能…人类DREF的特征：通过BLAST搜索鉴定出一个果蝇DREF的人类同源物(HDREF)。铸造法测定的hDREF的共同结合序列(5‘-TGTCGC/TGAC/TA)与果蝇DREF的序列(5’-TGTCGATA)重叠。我们在人类细胞增殖相关基因的启动子区域发现了hDREF结合序列。使用特定抗体的分析显示，hDREF与组蛋白H1基因的启动子区域结合。HDREF表达载体和组蛋白H1启动子导向的荧光素酶报告基因在HeLa细胞中的共转染实验显示，组蛋白H1启动子可能被激活。免疫组织化学分析表明，hDREF定位于细胞核。虽然在无血清培养的人正常成纤维细胞中，该因子的表达水平很低，但加入血清后，该因子的表达水平增加，并在S时达到最大值。针对hDREF的RNA干扰实验显示，HeLa细胞的组蛋白H1m RNA水平降低，进入S时相受到抑制。这些结果表明，hDREF的表达可能在调节与细胞增殖相关的人类基因中发挥作用。较少

项目成果

Kwon, E-J , Seto, H., Hirose, F., Ohshima, N., Takahashi, Y., Nishida, Y. and Yamaguchi, M.: "Transcription control of a gene for Drosophila transcription factor, DREF by DRE and cis-elements conserved between Drosophila melanogaster and virilis"Gene. in

Kwon, E-J、Seto, H.、Hirose, F.、Ohshima, N.、Takahashi, Y.、Nishida, Y. 和 Yamaguchi, M.：“果蝇转录因子基因的转录控制，DREF 通过 DRE 和顺式

Kwon, E-J.: "Transcription control of a gene for Drosophila transcription factor, DREF by DRE and cis-elements conserved between Drosophila ・・・・"Gene. (in press).

Kwon，E-J.：“果蝇转录因子基因的转录控制，DRE 的 DREF 和果蝇之间保守的顺式元件......”基因。

Hirose, F. et al.: "Ectopic expression of DREF induces DNA synthesis, apoptosis and unusual morphogenesis in the Drosophila eye imaginal disc"Mol.Cell.Biol.. 21. 7231-7242 (2001)

Hirose, F. 等人：“DREF 的异位表达诱导果蝇眼成象盘中的 DNA 合成、细胞凋亡和异常形态发生”Mol.Cell.Biol.. 21. 7231-7242 (2001)

Hirose F.: "Drosophila Mi-2 negatively regulates DREF by inhibiting its DNA-binding activity"Mol.Cell.Biol. 22. 5182-5193 (2002)

Hirose F.：“果蝇 Mi-2 通过抑制 DREF 的 DNA 结合活性来负调节 DREF”Mol.Cell.Biol。

Hirose, F., Ohshima, N., Shiraki, M., Inoue, Y- H.,Taguchi, O., Nishi, Y., Matsukage, A. and Yamaguchi, M.: "Ectopic expression of DREF induces DNA synthesis, apoptosis and unusual morphogenesis in the Drosophila eye imaginal disc : possible interaction w

Hirose, F.、Ohshima, N.、Shiraki, M.、Inoue, Y-H.、Taguchi, O.、Nishi, Y.、Matsukage, A. 和 Yamaguchi, M.：“DREF 的异位表达诱导 DNA 合成