EXCEED THE SPACE PROVIDED. Commitment of hematopoietic precursors to the T lineage occurs after entry of the cells into the thymus and the onset of expression of a diverse set of T-lineage associated genes. To date, no specific regulatory factor has been identified that carries out T lineage commitment in a way analogous to the role of Pax5 in B lineage cells. In recent work, however, our group has shown two regulatory transitions in T-cell precursors that parallel lineage commitment: the shutoff of PU.1 expression and a marked increase in tolerance for high levels of GATA-3 activity. Although GATA-3 is essential for T-cell development, its effects are dose- and stage-dependent, and high-level expression at an early stage causes a severe block to entry into the T-cell pathway. The pronounced discontinuity between the responses of precursors forced to overexpress GATA- 3 earlier or later in T-cell development suggests that a major regulatory shift in target gene accessibility or target gene function occurs at the time of lineage commitment. This proposal is to determine the nature of this regulatory shift. We have already identified an initial set of target genes that appear to be perturbed by GATA-3 in immature thymocytes, and a mutant form of GATA-3 that retains the severity of the early effects with minimal inhibition after T-lineage commitment. The research we propose to do builds on these initial findings to dissect the mechanisms through which GATA-3 exerts its different actions so that an aspect of T-lineage commitment can be defined at a molecular level.
The specific aims are: 1. Comparison of GATA-3 target genes in pre- and post-commitment T-cell precursors 2. Definition of mechanistic requirements for GATA-3 perturbation of T-cell development 3. Attempted rescue of GATA-3 overexpression effects by cotransduction with targets of specific repression PERFORMANCE SITE ========================================Section End===========================================
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