Regulation of expression and funcional analysis of Down syndrome gene

1.唐氏综合征基因SIM2、DSCR4和MNB/DYRK1转录因子的启动子分析及鉴定通过对T98G胶质母细胞瘤细胞的瞬时转染和凝胶漂移实验，确定了转录因子c-myb、C/EBP的顺式元件对SIM2基因的转录起关键作用。染色质免疫沉淀法在抗c-Myb、抗C/EBpβ和抗C/EBpε免疫沉淀物中发现SIM2启动子区域。此外，我们制备了2.2kb SIM2启动子区域的转基因小鼠，并对报告基因在小鼠胚胎中的表达进行了分析。DSCR4基因仅在人胎盘和人绒毛癌细胞株JEG3和BeWo中表达。我们进行了启动子分析，发现OLF-1和E47结合位点对DSCR4转录具有重要作用。类似地，人类MnB/DYRK1a转录受Sp1、Oct1和c-Myb结合位点的影响。2.人类SIM1和SIM2中新的核定位信号我们发现SIM…在没有任何刺激的情况下，更多的1和SIM2蛋白定位在细胞核中。通过对融合蛋白的瞬时表达分析，发现SIM1的aa368-388和SIM2的aa367-389存在一个新的核定位信号(NLS)。进一步分析SIM2这个小区域的氨基酸替换，发现5个氨基酸残基(Arg367、Lys373、Pro385、Tyr386和Gln389)对NLS活性起关键作用。3.SIM1、SIM2/Arnt、ARNT2NLS缺失的SIM1和SIM2通过与Arnt或ARNT2共转染进入细胞核。ARNT的SIM2和PAS2结构域的PAS1和PAS2结构域是与ARNT结合所必需的。另一方面，只需要SIM1或SIM2的PAS2结构域和ARNT2的PAS2结构域就可以与ARNT2.4结合。4.SIM2转录因子靶基因的鉴定我们建立了稳定的SIM2转染体(HeLa-SIM2)，并通过HeLa-mock和HeLa-SIM2的差异显示分析，分离了SIM2靶基因的候选基因。实时RT-PCR分析后，我们聚焦于两个基因(HAS2，HLCS)以进一步分析它们的启动子。5.SIM2的多泛素化SIM2在HEK293细胞中的瞬时表达表明SIM2的多泛素化。多泛素化的SIM2与Parkin和HHARI等环-IBR-环型E3连接酶免疫共沉淀。较少

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