Regulation of expression and funcional analysis of Down syndrome gene
| Project/Area Number |
14370323
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Embryonic/Neonatal medicine
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| Research Institution | Kyorin University |
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Principal Investigator |
SHIMIZU Yoshiko Kyorin University, School of Health Sciences, Professor, 保健学部, 教授 (50255410)
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| Co-Investigator(Kenkyū-buntansha) |
KUDOH Jun Keio University, School of Medicine, Associate Professor, 医学部, 助教授 (80178003)
MATSUDA Takao Kyusyu University, Medical Institute of Bioregulation, research associate, 生体防御医学研究所, 助手 (10304825)
TAMURA Takashi Kyorin University, School of Sciences, lecturer, 保健学部, 講師 (70146554)
YAMAKI Akiko Kyorin University, School of Sciences, Research Associate, 保健学部, 助手 (40296546)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥14,000,000 (Direct Cost: ¥14,000,000)
Fiscal Year 2004: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥6,800,000 (Direct Cost: ¥6,800,000)
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| Keywords | 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 |
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| Research Abstract |
1.Promoter analysis and identification of transcription factors for Down syndrome genes such as SIM2, DSCR4 and MNB/DYRK1A
By 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 SIM2
We 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, ARNT2
NLS-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 factor
We 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 SIM2
Transient 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
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Report
(4 results)
Research Products
(16 results)
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[Journal Article] Transcription Factor Single-Minded 2 (SIM2) is ubiquitinated by the RING-IBR-RING-type E3 ubiquitin ligases2005
Author(s)
Okui, M., Yamaki, A., Takayanagi, A., Kudoh, J., Shimizu, N., Shimizu, Y.
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Journal Title
Description
「研究成果報告書概要(欧文)」より
Related Report
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[Journal Article] Molecular mechanisms of human single-minded 2 (SIM2) gene expression : Identification of a promoter site in the SIM22001
Author(s)
Yamaki, A., Tochigi, J., Kudoh, J., Minoshima, S., Shimizu, N., Shimizu, Y.
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Journal Title
Genomic Sequence, Gene 270
Pages: 265-275
Description
「研究成果報告書概要(欧文)」より
Related Report
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