LINEAGE COMMITMENT, GENE SILENCING, AND EPIGENETIC REGULATION IN THYMOCYTES The distinct stages of T cell development in the thymus can be readily followed by the pattern of expression of various cell surface markers, particularly the CD4 and CD8 glycoproteins, whose expression is coupled to the functional specification of helper and cytotoxic T cells, respectively. We have characterized transcriptional regulators of CD4 and CD8 gene expression at different stages of T cell development to gain insight into the mechanism of thymocyte lineage commitment. By using gene targeting analyses, we have shown that CD4 gene expression is repressed in both immature thymocytes and in cytotoxic T cells by a stage-specific silencer element and by Runx family transcription factors that bind to it. By using Cre-Lox-mediated deletion of the silencer or the Runx factors, we have shown that these are required for establishment, but not maintenance, of silencing in CD8+ cytotoxic T cells, while a different silencing mechanism operates in immature thymocytes, in which silencing needs to be reversed. The roles of Runx1 and Runx3 and other silencer-binding transcription factors in recruitment of epigenetic machinery involved in remodeling of chromatin, as well as in lineage specification, are being studied using a series of recently-generated mutant mice. We have found that the transcription factor ThPOK, which is required for commitment of thymocytes to the CD4+ helper lineage, is up-regulated early after specification of this lineage and acts to repress expression of Runx3, thus preventing mis-direction of cells towards the cytotoxic lineage. We are following up on this finding by using gene expression profiling combined with chromatin immunoprecipitation studies to identify gene targets of transcriptional specification and commitment factors. A key goal is to characterize the mechanism by which epigenetic modifiers (e.g. histone methyltransferases) are recruited to specific genes for establishment of heritable chromatin modification. In this context, we have identified an enhancer that is essential for expression of CD4 in immature "double positive" thymocytes but becomes dispensable once T helper cells have completed their development and migrated to the periphery. Thus, the CD4 gene locus offers both positive and negative epigenetically regulated states of gene expression that can be exploited experimentally to better understand how stable lineages are generated during metazoan development. |
