Genetic Studies of CD4 Expression During Thymopoiesis

Siu, Gerald

Abstract

The experiments in this application are designed to study the molecular mechanisms that mediate the progression of thymopoiesis through two critical checkpoints. The control of CD4 gene expression is an ideal model system to study the downstream targets of signaling pathways that drive these events, as CD4 expression is important in defining each developmental stage. Multiple cis-acting transcriptional control elements are required for the control of CD4 expression; in this grant, we propose to study the two developmental stage-specific enhancers in this locus. The first enhancer referred to as the Thymocyte Enhancer (TE), is first induced to function as the CLP commits to the T cell lineage and is primarily responsible for mediating CD4 expression in DP thymocytes. As the DP T cell passes positive selection, the TE ceases function; at the same time, the second enhancer, referred to as the Mature Enhancer (ME), begins function and is responsible for inducing CD4 expression in mature CD4 SP T cells. Interestingly, the TE cannot induce CD4 promoter function directly; instead, in DP T cells the TE functions on the ME, which in turn induces promoter function. Thus, the factors that bind to these two enhancers are likely to be responsive to the developmental signals that induce the thymocyte to mature past the checkpoints that demarcate the commitment of the CLP to the T cell lineage and the induction and cessation of the repertoire selection process. In this grant, we propose to conduct biochemical and molecular approaches to identify and characterize the factors that are required for ME and TE function. In addition, using both transgenic and targeted-disruption techniques, we will conduct genetic experiments to determine the roles these elements play in the control of CD4 expression. In this manner we will hopefully be able to determine how these factors receive their signals to function during T cell development.