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Regulation of expression and funcional analysis of Down syndrome gene

唐氏综合症基因表达调控及功能分析

基本信息

批准号：
14370323
负责人：
SHIMIZU Yoshiko
金额：
$ 8.96万
依托单位：
Kyorin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370323/
关键词：
Down Syndrome SIM2 DSCR4 MNB DYRK1A Regulation of transcription nuclear localization signal ubiquitination target gene ARNT2 デイファレンシャルデイスプレイ法 CpGアイランドのメチル化恒常発現細胞定量RT-PCR CpGアイランドメチル化

项目摘要

1.Promoter analysis and identification of transcription factors for Down syndrome genes such as SIM2, DSCR4 and MNB/DYRK1ABy transient transfection assay and gel shift assay in T98G glioblastoma cells, we determined cis-elements for transcription factors such as c-myb, C/EBP are critical for the transcription of SIM2 gene. SIM2 promoter region was found in the immunoprecipitates of anti-c-Myb, anti-C/EBP β or anti-C/EBP ε using the chromatin immunoprecipitation assay. Furthermore, we made transgenic mouse with 2.2kb of SIM2 promoter region and analyzed the expression of the reporter gene in mouse embryo. DSCR4 gene was expressed only in human placenta and human choriocarcinoma cell lines, JEG3 and BeWo. We performed the promoter analysis and showed OLF-1 and E47 binding sites were important for DSCR4 transcription. Similarly, human MNB/DYRK1A transcription was influenced by SP1, Oct1 and c-Myb binding sites.2.Novel nuclear localization signal in the human SIM1 and SIM2We found that SIM … More 1 and SIM2 proteins are localized in nuclei without any stimuli. Using the transient expression analysis of EGFP-fusion protein, a novel nuclear localization signal (NLS) exists in aa368-388 of SIM1 and aa367-389 of SIM2. Further analysis with amino acid substitution of this small region of SIM2 revealed the critical role of five amino acid residues (Arg367, Lys373, Pro385, Tyr386 and Gln389) for the NLS activity.3.Heterodimerization of SIM1, SIM2/ARNT, ARNT2NLS-deficient SIM1 and SIM2 move into nuclei in the cells by cotransfection with ARNT or ARNT2. PAS1 and PAS2 domains of SIM2 and PAS2 domain of ARNT were necessary for the association with ARNT. On the other hand, only PAS2 domain of SIM1 or SIM2 and PAS2 domain of ARNT2 were needed for the association with ARNT2.4.Identification of target genes of SIM2 transcription factorWe have established the stable transfectant with SIM2 (HeLa-SIM2) and isolated the candidate genes for the targets of SIM2 by the differential display analysis between HeLa-mock and HeLa-SIM2. After real time RT-PCR analysis we focussed on two genes (HAS2, HLCS) for further analysis of their promoters.5.Polyubiquitination of SIM2Transient expression of SIM2 in HEK293 cells revealed polyubiquitination of SIM2. Polyubiquitinated SIM2 was immunoprecipitated with the RING-IBR-RING-type E3 ligases such as Parkin and HHARI. Less

1.唐氏综合征基因启动子分析及转录因子SIM 2、DSCR 4和MNB/DYRK 1A的鉴定通过瞬时转染和凝胶迁移实验，我们确定了转录因子c-myb、C/EBP的顺式元件对SIM 2基因的转录起关键作用。染色质免疫沉淀法在抗c-Myb、抗C/EBP β和抗C/EBP ε的免疫沉淀物中发现了SIM 2启动子区。此外，我们还制备了带有2.2kb SIM 2启动子区的转基因小鼠，并分析了报告基因在小鼠胚胎中的表达。DSCR 4基因仅在人胎盘和人绒毛膜癌细胞系JEG 3和BeWo中表达。我们进行了启动子分析，并显示OLF-1和E47结合位点是重要的DSCR 4转录。同样，人MNB/DYRK 1A的转录也受到SP1、Oct 1和c-Myb结合位点的影响。2.人SIM 1和SIM 2中新的核定位信号 ...更多信息 1和SIM 2蛋白在没有任何刺激的情况下定位于细胞核中。利用EGFP融合蛋白的瞬时表达分析，一个新的核定位信号（NLS）存在于SIM 1的aa 368 -388和SIM 2的aa 367 -389。通过对SIM 2的这一小区域的氨基酸替换进一步分析，发现5个氨基酸残基（Arg 367，Lys 373，Pro385，Tyr 386和Gln 389）对NLS活性起关键作用。3.通过与ARNT或ARNT 2共转染，SIM 1、SIM 2/ARNT、ARNT 2 NLS缺陷型SIM 1和SIM 2的异源二聚体进入细胞核。SIM 2的PAS 1和PAS 2结构域以及ARNT的PAS 2结构域是与ARNT结合所必需的。另一方面，只需要SIM 1或SIM 2的PAS 2结构域和ARNT 2的PAS 2结构域就可以与ARNT 2结合。4. SIM 2转录因子靶基因的鉴定我们建立了SIM 2的稳定转染子（HeLa-SIM 2），并通过HeLa-mock和HeLa-SIM 2的差异显示分析，分离了SIM 2靶基因的候选基因。真实的RT-PCR分析后，我们将重点放在两个基因（HAS 2，HLCS）上，以进一步分析它们的启动子。5. SIM 2的多泛素化SIM 2在HEK 293细胞中的瞬时表达显示SIM 2的多泛素化。用RING-IBR-RING型E3连接酶如Parkin和HHARI免疫沉淀多泛素化的SIM 2。少