Eukaryotic chromatin is organized into loop domains, which might have both structural and functional roles such as differential gene expression and replication. SATB1 is a cell-type specific MAR-binding protein which is predominantly expressed in thymocytes. SATB1 binds in vivo to specialized genomic DNA sequences at the bases of chromatin loop domains, tightly attached to the nuclear matrix. SATP1-deficient thymocytes are highly prone to apoptosis. SATB1 is site-specifically cleaved early in apoptosis, and concomitantly to this cleavage, SATB1 rapidly dissociates from chromatin in vivo. We have now identified a proteases that cleaves SATB1 in vitro to generate the cleavage product typically detected in apoptotic cells. In the current proposal, we propose to 1) determine whether SATB1-binding sequences are the initial targets of endonuclease cleavage early in apoptosis leading to disassembly of higher order chromatin structure, 2) test if the SATB1 cleavage also occur in thymocytes and T cells induced for apoptosis by environmental agents and during negative selection of CD4+CD8+ double positive (DP) thymocytes, 3) determine the cleavage site of SATB1 and mutate the site to create an uncleavable SATB1 and 4) make transgenic mice that express lck-drive mutated SATB1 in SATB1(+/-) and SATB1 (-/-) background to determine whether mutated SATB1 delay or inhibit apoptosis. Our studies will provide valuable insights into the mechanism(s) as to how SATB1 ablation leads to accelerated apoptosis in SATB1 knockout mice. The experiments described here address a novel question regarding a potential link between higher experiments described here address a novel question regarding a potential link between higher order chromatin structure and the role of SATB1 in thymocyte and T cell apoptosis induced by environmental agents as well as by negative selection.
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