In this grant we propose a number of experiments aimed at understanding in detail the role of the transcription factor GATA-3 during CD4/CD8 lineage commitment in the thymus. We have recently shown that GATA-3 is differentially expressed in immature thymocytes that develop into the CD4 lineage, and that by modifying GATA-3 activity in the thymus we can bias lineage commitment. We will now: Dissect the mechanisms that regulate TCR-induced GATA-3 upregulation in the thymus, using a gain-of function/loss-of-function genetic approach to identify the signal transduction pathways involved in this process in normal DP thymocytes. Use reaggregate fetal thymic organ culture (rFTOC), lentiviral transgenesis and different GATA-3 mutants to map the structural regions of GATA-3 required for its effect during CD4/CD8 lineage commitment. Analyze the interactions of GATA-3 with two transcription factors, Egr-2 and Lmo-4, that we have identified as preferentially expressed in CD4 lineage intermediates in a genome-wide screen. These studies should provide us with a better understanding of the mechanisms that control CD4/CD8 lineage commitment, and, more widely, will provide clues about the genetic networks that establish cell fate determination and developmental programs in the thymus.
