Identification and functional characterization of a novel positive cis-control el

描述（由申请人提供）：CD 4辅助性T细胞协调免疫应答，高度依赖于Cd 4基因的表达以实现正常发育和功能。启动子、增强子和沉默子的功能已经有了很好的记录，并解释了CD 4基因如何在CD 4 T细胞中打开，以及如何在CD 8 T细胞中关闭。然而，一旦打开，CD 4表达的时间和水平在CD 4 T细胞发育和激活过程中会发生变化。这种CD 4表面水平的调节对于适当的谱系特化和T细胞功能是必不可少的。与CD 4表达的微小变化可以破坏健康免疫系统维持的免疫缺陷和自身免疫之间的精细平衡的概念一致，最近已将Cd 4基因座的调节区域中的SNP与自身免疫性疾病的发展相关联。然而，CD 4表达的微调是如何与TCR信号和其他环境线索调节和协调的尚不清楚。这个小规模的调查将集中在确定新的顺式调节元件在体外的Cd 4位点和体内小鼠模型的产生，以进一步研究其功能的重要性，CD 4 T细胞的发展和功能。体外鉴定 和表征将包括1）使用UCSC基因组浏览器鉴定Cd 4中的高度保守序列; 2）通过将EGFP报告质粒瞬时转染到CD 4+细胞系中来评价候选物在体外促进或增强转录的能力;和3）进一步表征通过截短和位点修饰显示出显著调节报道基因表达能力的候选物。定向诱变以确定功能所必需的最小足够序列和转录因子结合位点。在初步 通过本提案中描述的实验，我们成功地鉴定了一种新的发育阶段特异性顺式调节元件（NCE），我们希望在体外和体内详细表征该元件。对于体内表征，我们将使用具有或不具有NCE缺失的Cd 4基因座的重组细菌人工染色体（BAC）开发两个单独的小鼠模型。两种模型的目的如下：模型1 -使用具有插入在Cd 4的外显子2中的EGFP基因的BAC，产生EGFP报告基因转基因小鼠，其中我们可以比较野生型Cd 4-BAC-EGFP与NCE缺失的Cd 4-BAC-EGFP的表达模式，而不改变内源性CD 4表达模式。模型2-产生野生型Cd 4-BAC和NCE缺失的Cd 4-BAC转基因小鼠，也缺乏内源性Cd 4和？2 m，以直接确定NCE缺失对MHC-II特异性CD 4 T细胞的阳性选择、谱系定型、活化和分化的影响。这些体内模型将是我们的长期目标，识别和表征新的转录控制元件在Cd 4基因座，并了解它们如何相互作用，以调节不同的CD 4+细胞类型在其发展和功能的不同阶段的CD 4水平的垫脚石。

DESCRIPTION (provided by applicant): CD4 helper T cells coordinate the immune response and are highly dependent on the expression of the Cd4 gene for proper development and function. The function of a promoter, an enhancer and a silencer have been well documented and explain how the Cd4 gene gets turned on in CD4 T cells and off in CD8 T cells. However, once turned on, the timing and level of Cd4 expression varies during CD4 T cell development and activation. This modulation of CD4 surface levels is essential for proper lineage specification and T cell function. Consistent with the notion that small changes in CD4 expression can disrupt the fine balance between immunodeficiency and autoimmunity that a healthy immune system maintains, SNPs in the regulatory regions of the Cd4 locus have been recently correlated with the development of autoimmune diseases. Yet, how the fine-tuning of CD4 expression is regulated and coordinated with the TCR signal and other environmental cues is not understood. This small-scale investigation will focus on identifying new cis-regulatory elements in the Cd4 locus in vitro and the generation of in vivo mouse models for further studies of their functional importance for CD4 T cell development and function. The in vitro identification and characterization will consist of 1) Identifying highly conserved sequences in Cd4, using the UCSC genome browser; 2) Evaluation of the candidates' ability to promote or enhance transcription in vitro by transient transfection of EGFP reporter plasmids into CD4+ cell lines; and 3) Further characterization of the candidates that exhibit a significant ability to modulate reporter expression by truncation and site-directed mutagenesis to determine the minimum sufficient sequence and transcription factor binding sites necessary for function. In a preliminary experiment described in this proposal, we have successfully identified one new developmental stage-specific cis-regulatory element (NCE) that we would like to characterize in detail both in vitro and in vivo. For the in vivo characterization we will develop two separate mouse models using recombineered bacterial artificial chromosome (BAC) of the Cd4 locus with or without a deletion of NCE. The purpose of the two models will be as follows: Model 1 - Using BAC with the EGFP gene inserted in exon 2 of Cd4, generate EGFP reporter transgenic mice, in which we can compare the expression pattern of the wild type Cd4-BAC-EGFP to that of the NCE-deleted Cd4-BAC-EGFP without changing the endogenous CD4 expression pattern. Model 2- Generate wild type Cd4-BAC and NCE-deleted Cd4-BAC transgenic mice that are also deficient for endogenous Cd4 and ?2m in order to determine directly the effect of NCE deletion on positive selection, lineage commitment, activation and differentiation of MHC-II specific CD4 T cells. These in vivo models will be the stepping stone towards our long-term objective of identifying and characterizing new transcription control elements in the Cd4 locus, and understanding how they interact to modulate CD4 levels in different CD4+ cell types at different stages of their development and function.