Study of molecular mechanisms of CD4 silencing and role s of Runx proteins during lymphocyte development

本研究旨在了解淋巴细胞发育过程中CD4沉默的分子机制和Runx转录因子的功能。由于主要的实验方法是基因操作小鼠的体内分析，我们已经产生了几种突变小鼠菌株，包括基因靶向小鼠和转基因小鼠。通过基因靶向Es细胞和Cre- loxp系统，我们通过组织特异性和分期特异性的方式诱导Cre重组酶使Runx1、Runx3或CBFβ基因有条件失活，从而获得了Runx1、Runx3或CBFβ基因的flox突变等位基因。我们还生成了Runx3转基因小鼠，其中myc表位标记的Runx3在所有αβT系细胞中特异性表达。另一突变株Runx1^<Δ446>小鼠，其中Runx1蛋白的VWRPY氨基酸序列被删除。通过对这些突变小鼠的分析，我们得到了以下结果：iNKT细胞起源于DP胸腺细胞，其发育需要Runx1功能。Runx1和Runx3在CD4沉默和胸腺细胞成熟中具有冗余功能。Runx1和Runx3基因的双敲除使阳性选择和胸腺细胞成熟受到严重损害。Runx1的c端VWRPY序列对于未成熟DN胸腺细胞和成熟cd8胸腺细胞的CD4沉默都是必不可少的。然而，VWRPY独立的转录抑制作用于Runx1的另一个靶基因。此外，VWRPY基序对于Runx1在iNKT细胞发育中的功能至关重要。通过转基因表达Runx3并不足以启动CD4沉默。Runx3不会是定义CD4沉默谱系特异性的分子。CBFβ在胸腺细胞分化过程中对Runx复合物的功能起重要作用。TCRβ基因座Eβ增强子Runx1与三个Runx识别基序的结合是TCRβ基因激活的必要条件。然而，三个Runx基序中有一个的突变导致DN胸腺细胞中TCRβ激活的部分损伤，但不影响成熟T细胞中TCRβ激活的维持

The aim of this research is to understand the molecular mechanisms of CD4 silencing and function of Runx transcriptional factors during lymphocyte development. Because a main experimental approach was in vivo analyses of gene manipulated mice, we have generated several mutant mouse strains including gene targeted mice and transgenic mice. By using gene targeting in Es cells and Cre-loxP system, we have generated flox mutant allele for Runx1,Runx3 or CBFβ gene for conditional inactivation by induction of Cre recombinase by tissue and stage specific manner. We have also generated Runx3 transgenic mouse in which myc-epitope tagged Runx3 was specifically expressed in all αβT lineage cells. Another mutant strain, the Runx1^<Δ446> mouse, in which VWRPY amino acid sequence of Runx1 protein was deleted, was kindly provided. By analyzing these mutant mice, we have obtained following results.1.iNKT cells are originated from DP thymocytes and require Runx1 function for their development.2.Runx1 a … More nd Runx3 possess redundant function in CD4 silencing and thymocyte maturation. In the double knock out of Runx1 and Runx3, positive selection and thymocyte maturation were severely impaired.3.The C-terminal VWRPY sequence of Runx1 is essential for CD4 silencing in both immature DN thymocytes and mature CD8-lineage thymocytes. However, VWRPY independent transcriptional repression operate ob another target genes of Runx1. Moreover, the VWRPY motif is essential for Runx1 function in iNKT cells development.4.Expression of Runx3 by transgene is not sufficient to initiate CD4 silencing. Runx3 would not a molecule that defines lineage specificity of CD4 silencing.5.CBFβ plays essential roles for Runx complex function during thymocytes differentiation.6.Binding of Runx1 to three Runx recognition motifs in Eβ enhancer at TCRβ locus is essential for activation of TCRβ gene. However, mutation on one Runx motif out of three result in partial impairment of TCRβ activation in DN thymocytes, but did not affect maintenance of TCRβ activation in mature T cells Less