The control of CD4 gene expression during thymopoiesis is tightly linked to critical T cell development and selection processes. The CD4 silencer is the critical transcriptional control element that mediates CD4 expression; the factors that bind to it and mediate its function are thus likely to be responsive to signals that drive T cell development. The investigator has identified a novel transcription factor, referred to as Silencer Associated Factor (SAF), which binds to a critical functional site in this silencer. Although expressed in all T cells, SAF is restricted to the cytoplasm of T cells that express CD4, including the precursor CD4+CD8+ DP thymocytes, and to the nucleus of cells that do not express CD4, including the CD8 SP Tc cell. It is hypothesized that the translocation of SAF from the cytoplasm to the nucleus mediates both down-regulation of CD4 expression and the lineage decision process as the DP thymocyte matures into the CD8 SP T cell. This hypothesis further predicts that SAF is a target of the signaling events that drive T cell development. The investigator has determined that the translocation of SAF to the nucleus in DP T cells is mediated by signaling from the Mek1 branch of the MAPK pathway, which has been previously shown to convey positive selection signals in developing thymocytes. These observations are consistent with the hypotheses and suggest that SAF is playing a critical role in the control of T cell selection and fate lineage decisions. In this grant, the investigator will take a multistep molecular, biochemical, and genetic approach to determine the mechanism in which SAF is stimulated to translocate to both the nucleus and the cytoplasm during the T cell repertoire selection process. Dominant negative and constitutively active mutant forms of Mek1 and Mek6 will be used to demonstrate that altering the activity of specific branches of the MAPK pathway affect SAF nuclear translocation. It will be determined how developmental signals induce SAF translocation, first by identifying the domains on SAF that are required for this process and subsequently by determining if these domains are either posttranslationally modified or bind to other factors in a subclass-specific manner. These experiments will allow identification of the components of the signaling pathway through which signaling from Mek1 mediates SAF nuclear localization, and will allow the generation of a panel of SAF mutants for use in genetic analyses outlined in Aim #2. A mouse genetic approach will be used to study the role of SAF during the T cell selection and fate decision process. The investigator will first generate transgenic mice that initiate the overexpression of either wild type or different mutants of SAF at different stages of T cell development, including the DN, DP and post-selection mature SP stages. In addition, a targeted disruption of the endogenous SAF gene will be generated. These genetically manipulated mice will be analyzed for alterations in the phenotype of the thymic and peripheral T cell populations. Using these approaches, the investigator will determine the role of SAF in the TCR-mediated positive and negative selection processes that drive T cell development.
