During T cell development, T-cell receptor (TCR) genes are rearranged and expressed in a T-cell specific manner. Chromatin accessibility plays a crucial role in this process but its molecular mechanism is poorly understood. Locus control region (LCR) is a set of cis-acting elements that can confer copy-number dependent, positional independent, high level and tissue-specific expression to a linked gene,and is thought to control the chromatin accessibility of a locus. In the TCR alpha/delta region, elements with a T-cell specific LCR activity have been found. This LCR consists of a T-cell specific enhancer, a ubiquitously active chromatin-opening region and some yet to be identified negative elements. In contrast to other LCRs, the chromatin structure of this TCRalpha LCR is open in all tissues. This correlates with the presence of a nearby anti-apoptotic gene Dad1, which is expressed ubiquitously. As T cells undergo extensive apoptosis during development and the close proximity of Dad1 to TCRalpha gene is conserved evolutionarily, Dad1 may play an important role in T cell development. Experiments in this proposal are designed to explore this possibility and to characterize the molecular mechanisms of the chromatin-opening activity by the TCRalpha LCR.
In aim 1, transgenic mice carrying a reporter gene and various parts of the TCRalpha LCR will be characterized. In vivo footprinting analysis of the TCRalpha LCR will be conducted. These experiments should yield considerable accessibility.
In aim 2, the link between TCRalpha, delta and Dad1 gene regulation will be studied. Mutant mice with deletion of the entire or part of the TCRalpha LCR will be generated and analyzed.
In aim 3, the effect of Dad1 over expression to T cell development will be examined. The possible anti-apoptic effect of Dad1 and its possiblde role in negative and positive selections during T cell development will be assessed using transtgenic approach. As DAD1- deficient mice die in utero, T-cell specific Dad1-/- mice will be generated in aim 4. The functional role of Dad1 in T cell development will be examined.
