Functional analysis of Th-POK in T cell development

锌指转录因子Th-POK已被鉴定为胸腺中CD 4/CD 8谱系定型的主要调节因子。它是CD 4单阳性T细胞分化所必需的，其表达将谱系命运从CD 8-SP转换为CD 4-SP。尽管Th-POK突变小鼠的表型很有趣，但其在胸腺细胞谱系定型中的功能完全未知。为了研究Th-POK各结构域的功能，我们将不同突变的Th-POK cDNA导入Pax 5缺陷的proB细胞，并用这些细胞重建胸腺。我们发现Th-POK的谱系转换活性需要其BTB结构域和Zn指结构域。为了鉴定Th-POK的下游靶标，我们接下来使用抗Th-POK抗体进行染色质免疫沉淀实验。通过这种方法，我们发现Th-POK直接结合到Runx 3基因的远端启动子区。因此，Th-POK可以直接抑制DP胸腺细胞中CD 4阻遏所需的Runx 3的表达。此外，我们发现钙离子载体以及一些凋亡诱导剂诱导转录的Th-POK在无信号的DP胸腺细胞。DP胸腺细胞中凋亡的诱导由强TCR信号诱导，并且分化为CD 4-SP谱系需要比CD 8-SP谱系所需的TCR信号更强（或更长）的TCR信号。因此，这将是有趣的，如果诱导的Th-POK和诱导凋亡共享某些部分相同的信号通路在DP胸腺细胞。

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.