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

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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