Functional analysis of Th-POK in T cell development

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Zinc finger transcription factor Th-POK has been identified as a master regulator of CD4/CD8 lineage commitment in the thymus. It is required for the differentiation of CD4 single positive T cells and its expression converts lineage fate from CD8-SP to CD4-SP. Despite the interesting phenotypes of Th-POK mutant mice, its function in thymocyte lineage commitment is completely unknown. In order to investigate the function of each domain of Th-POK, we introduced various mutant Th-POK cDNAs into Pax5 deficient proB cells and reconstituted the thymus with these cells. We found that the lineage converting activity of Th-POK requires its BTB domain and Zn finger domain. In order to identify downstream targets of Th-POK, we next performed chromatin immunoprecipitation experiments using anti-Th-POK antibody. By this method we found that Th-POK binds directly to the distal promoter region of the Runx3 gene. Therefore Th-POK could directly suppress expression of Runx3, which is required for CD4 repression in DP thymocyte. Furthermore, we found that calcium ionophore as well as some apoptosis inducing agents induced transcription of Th-POK in unsignaled DP thymocytes. Induction of apoptosis in DP thymocytes is induced by strong TCR-signals and differentiation to CD4-SP lineage requires a stronger (or longer) TCR-signal than the one required for the CD8-SP lineage. Therefore, it would be interesting if induction of Th-POK and induction of apoptosis share some part of the same signaling pathway in DP thymocytes.