CHARACTERIZATION OF THE SATB1 PHOSPHORYLATIONS UPON IONIZATION RADIATION

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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.