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

本研究的目的是了解CD4沉默的分子机制以及RUNX转录因子在淋巴细胞发育过程中的作用。由于主要的实验方法是对基因操纵的小鼠进行体内分析，我们已经产生了几个突变小鼠品系，包括基因靶向小鼠和转基因小鼠。通过在ES细胞和Cre-loxP系统中的基因打靶，我们已经产生了RUNX1、Runx3或Cbfβ基因的FLOX突变等位基因，通过组织和阶段特异的方式诱导Cre重组酶条件失活。我们还获得了Runx3转基因小鼠，其中Myc表位标记的Runx3在所有αβT系细胞中特异表达。另一种缺失RUNX1蛋白VWRPY氨基酸序列的突变品系RUNX1^；Δ446&gt；小鼠。通过对这些突变小鼠的分析，我们得到了以下结果：1.iNKT细胞来源于DP胸腺细胞，需要RUNX1功能才能发育。2.Runx1 a…更多的ND Runx3在CD4沉默和胸腺细胞成熟过程中具有冗余功能。在RUNX1和Runx3的双重敲除中，阳性选择和胸腺细胞的成熟受到严重损害。3.RUNX1的C端VWRPY序列对于未成熟的糖尿病肾病胸腺细胞和成熟的CD8系胸腺细胞的CD4沉默是必不可少的。然而，VWRPY非依赖的转录抑制作用于RUNX1的另一个靶基因。此外，VWRPY基序对于RUNX1在iNKT细胞发育中的功能是必不可少的。4.转基因表达Runx3不足以启动CD4沉默。5.CBFβ在胸腺细胞分化过程中对RunX复合体的功能起着至关重要的作用。6.RunX1与E-β增强子上的三个Runx识别基序结合在TcRβ位点是激活TcRβ基因所必需的。然而，三个RUNX基序中的一个突变会导致糖尿病肾病胸腺细胞TcRβ活性的部分受损，但不会影响成熟T细胞TcRβ活性的维持

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