CHARACTERIZATION OF THE SATB1 PHOSPHORYLATIONS UPON IONIZATION RADIATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我的实验室的一个主要目标是了解涉及核结构蛋白SATB1的分子信号机制。SATB1被认为是一个全球性的基因调节因子，在受限的细胞类型中组织更高阶的染色质结构。SATB1对于T细胞的正常发育是必不可少的。SATB1在胸腺细胞核中呈笼状分布，基因组序列锚定在其上，并与染色质重塑复合体和转录因子组装在一起。SATB1是一种磷酸化的蛋白质，在胸腺细胞中有不同的SATB1群体，每个群体与其靶DNA序列具有不同的亲和力。我们希望确定在电离辐射诱导的胸腺细胞中SATB1的翻译后修饰。我们将验证这一假设，即电离辐射诱导的SATB1磷酸化(和其他形式的修饰)会导致SATB1s基因调控的变化。我们以前开发的用于从胸腺细胞中纯化SATB1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的蛋白质组学研究，以表征SATB1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SATB1的修饰位点。然后将通过定点突变对关键的磷酸化位点(和其他修饰位点)进行功能测试，然后将其导入野生型和SATB1基因敲除的胸腺细胞。我们的研究结果将对SATB1在细胞发育和DNA损伤后的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。

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.