Eukaryotic chromatin fibers are folded into topologically independent loop domains in interphase nuclei. This is thought to be accomplished by chromatin making periodic attachments to the residual framework of nuclei, called the nuclear matrix, at specialized DNA sequences called matrix attachment regions, or MARs. Such higher order chromatin structure might have both structural and functional significance in gene regulation. We have previously cloned a cell-type specific MAR-binding protein, SATB 1, which is expressed in thymocytes and activated T cells. SATBI coordinates multiple genes in the T cell lineage. Using SATB1 as a tool to isolate MARS, we have identified a series of SATB1-binding fragments from the -1 megabase mouse cytokine gene cluster region at chomosome 11 as putative MARs. In this proposal, we propose to 1) identify and map all potential MARs within this locus by both biochemical and computational methods, and characterize each MAR by delineating the key structural element (base unpairing region: BUR), which is typically found in MARs, 2) similarly screen for MARs in the human cytokine gene cluster region at chromosome 5q31 and identify BURs that are found in the syntenic region of the mouse chromosome 11, 3) determine which of these BURs make attachment to the nuclear matrix in T cells at different differentiation stages and examine their in vivo binding status to SATB1, and 4) examine the biological function of selected BURs by establishing BUR-deleted mice. In these mice, we will examine the effects of BUR deletion on the specific loop organization of chromatin, T cell differentiation-specific gene expression, and local chromatin structure including the methylation status. The research proposed here will test whether certain BURs can act as long-range regulatory elements by forming a specific loop domain structure during T cell differentiation.
|Balamotis, Michael A; Tamberg, Nele; Woo, Young Jae et al. (2012) Satb1 ablation alters temporal expression of immediate early genes and reduces dendritic spine density during postnatal brain development. Mol Cell Biol 32:333-47|