This proposal addresses the question of how pluripotent hematopoietic precursors undergo specification and commitment to the T- lymphocyte lineage. In the past two years, the investigator's group has systematically identified molecular landmarks for this process, that is, specific changes in expression of multiple T-cell and non-T-cell (B-cell or dendritic-cell) genes that are correlated with each developmental transition. The investigator has also correlated these changes in target gene expression with parallel changes in the pattern of expression of particular members of the Ets, bHLH, and CBFa transcription factor families. The investigator has thus identified a discrete set of candidates for factors that might participate in the direct changes of phenotype of differentiating T-cell precursors and/or in the focusing of their developmental potential. Of particular interest is the Ets family transcription factor PU. l, which appears to be shut off abruptly at the stage when precursors become irreversibly committed to the T lineage. Most recently, the investigator has found that forced expression of PU. 1 in T-cell precursors both restricts entry into the T-cell differentiation pathway and blocks progression through the later, critical beta-selection checkpoint of the few cells that do enter this pathway. The goal of this project is to define the underlying mechanisms of T-lineage specification, using as probes the gene expression landmarks the investigator has defined and the ability of PU. 1 overexpression to perturb these mechanisms. The investigator plans: (1). To determine the structural and temporal requirements for PU. 1 inhibition of lymphoid development; ( 2). To establish direct linkages between expression of particular genes by precursor cells and the abilities of those cells to give rise to T, B, NK, dendritic, or mast cells; (3) To test the roles of developmentally regulated transcription factors in the alteration of expression of these indicator genes, and of PU.1 itself.
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