CHARACTERIZATION OF THE SATB1 PHOSPHORYLATIONS UPON IONIZATION RADIATION

电离辐射下 SATB1 磷酸化的表征

• 批准号: 7602873
• 负责人: Terumi Kohwi-Shigematsu
• 金额: $ 4.2万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602873
• 关键词: Affinity; Biological; Cell Nucleus; Cells; Cellular biology; Chromatin Remodeling Factor; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Damage; DNA Sequence; Development; Exhibits; Funding; Gene Expression Regulation; Genomics; Goals; Grant; Higher Order Chromatin Structure; Institution; Ionizing radiation; Knock-out; Laboratories; Methods; Modification; Molecular; National Center for Research Resources; Nuclear; Phosphorylation; Phosphorylation Site; Population; Post-Translational Protein Processing; Proteins; Proteomics; Radiation; Regulator Genes; Research; Research Personnel; Resolution; Resources; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Source; T-Cell Development; Testing; Transfection; United States National Institutes of Health; base; cell type; insight; ionization; irradiation; thymocyte; transcription factor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A major goal of my laboratory is to understand the molecular signaling mechanisms involving the nuclear architectural protein, SATB1. SATB1 is known to be a global gene regulator, organizing higher-order chromatin structure in restricted cell types. SATB1 is essential for proper T cell development. SATB1 exhibits a cage-like nuclear distribution in thymocyte nuclei, onto which genomic sequences are anchored and assembled with chromatin remodeling complexes and transcription factors. SATB1 is a phosphorylated protein, and there are distinct populations of SATB1 within thymocytes, each exhibiting a different affinity to its target DNA sequences. We wish to determine post-translational modifications of SATB1 in thymocytes induced by ionizing radiation. We will test the hypothesis that SATB1 phosphorylation (and other forms of modification) induced by ionizing radiation results in changes in SATB1s gene regulation. A DNA affinity column, which we previously developed to purify SATB1 from thymocytes (Kohwi-Shigematsu et al., Methods in Cell Biology 53: 324-352, 1998), has enabled us to perform large-scale proteomics to characterize post-translational modification of SATB1. The NCRR high-sensitivity, high-resolution LC-MS/MS will be used to identify modification sites of SATB1 before and after ionization irradiation. Functional testing of key phosphorylation sites (and other modification sites) will then be performed by site-directed mutagenesis, followed by transfection into wild-type and SATB1 knockout thymocytes. Results from our study will provide valuable insights into the molecular basis of the important role of SATB1 for cell development and gene regulation following DNA damage, and help in evaluating the biological effects of ionization radiation.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我实验室的一个主要目标是了解涉及核结构蛋白SATB 1的分子信号机制。 已知SATB 1是一个全局基因调节器，在有限的细胞类型中组织高级染色质结构。 SATB 1对正常的T细胞发育至关重要。 SATB 1在胸腺细胞核中表现出笼状核分布，基因组序列锚定并与染色质重塑复合物和转录因子组装。 SATB 1是一种磷酸化蛋白，胸腺细胞内存在不同的SATB 1群体，每种群体对其靶DNA序列表现出不同的亲和力。我们希望确定电离辐射诱导的胸腺细胞中SATB 1的翻译后修饰。我们将检验电离辐射诱导的SATB 1磷酸化（和其他形式的修饰）导致SATB 1的基因调控。 我们先前开发的用于从胸腺细胞纯化SATB 1的DNA亲和柱（Kohwi-Shigematsu等人，Methods in Cell Biology 53：324-352，1998），使我们能够进行大规模蛋白质组学以表征SATB 1的翻译后修饰。NCRR高灵敏度、高分辨率LC-MS/MS将用于鉴定电离辐照前后SATB 1的修饰位点。 然后通过定点诱变进行关键磷酸化位点（和其他修饰位点）的功能检测，然后转染至野生型和SATB 1敲除胸腺细胞中。 我们的研究结果将为SATB 1在DNA损伤后细胞发育和基因调控中的重要作用的分子基础提供有价值的见解，并有助于评估电离辐射的生物学效应。