Eucaryotic chromatin is organized into loop domains, which might have both structural and functional roles such as differential gene expression and replication. It is believed that chromatin loops are anchored to the nuclear matrix at specific DNA regions (Matrix attachment regions, or MARs) that exhibit high affinity to the nuclear matrix. SATB1 is a cell-type specific MAR-binding protein which is predominantly expressed in thymocytes. The principal investigator has recently isolated a set of genomic DNA sequences that bind to SATB1 in vivo in thymocytes. By in situ hybridization, it was shown that these in vivo SATB1-binding sequences tightly associated with the nuclear matrix. SATB1 knockout mice were created and it was found that SATB1 is required for the regulation of certain genes, proper T cell development and resistance to apoptosis. It was also shown that SATB1 is site- specifically cleaved in early apoptosis, and concomitantly to this cleavage, SATB1 rapidly dissociates from chromatin in vivo. In the current proposal, the principal investigator proposes to 1) determine whether SATB1 can actively tether MAR sequences of chromatin onto the nuclear matrix, 2) study the effect of SATB1 binding to particular MAR on the transcriptional activity of nearby genes, 3) study the effects in SATB1 knockout mice, focusing on non-lymphoid cells, 4) examine the mechanism for the defect of SATB1-deficient T cells to respond to TCR/CD3 stimulation, and 5) clone cDNAs for SATB1-associating proteins. These experiments using both SATB1 knockout mice and in vivo SATB1- binding sequences, will provide important information on not only the biological function of SATB1, but also how higher-order chromatin structure is organized to exhibit its function through MARs.
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