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

描述(由申请人提供)：CD4辅助T细胞协调免疫反应，高度依赖于CD4基因的表达来实现正常的发育和功能。启动子、增强子和沉默子的功能已经被很好地记录下来，并解释了CD4基因如何在CD4T细胞中被打开，在CD8T细胞中被关闭。然而，一旦启动，在CD4T细胞发育和激活期间，CD4表达的时间和水平会发生变化。这种对CD4表面水平的调节对于正确的谱系规范和T细胞功能是必不可少的。与CD4表达的微小变化会破坏健康免疫系统维持的免疫缺陷和自身免疫之间的微妙平衡的观点一致，最近发现CD4基因调控区的SNPs与自身免疫性疾病的发生有关。然而，CD4表达的微调是如何与TCR信号和其他环境信号调节和协调的，目前还不清楚。这项小规模的研究将集中于在体外识别新的顺式调控元件，并在体内建立小鼠模型，以进一步研究它们对CD4T细胞发育和功能的功能重要性。体外鉴定 鉴定将包括：1)使用UCSC基因组浏览器鉴定CD4中高度保守的序列；2)通过瞬时将EGFP报告质粒瞬时导入CD4+细胞系，评估候选基因在体外促进或增强转录的能力；以及3)进一步鉴定通过截断和定点突变来调节报告基因表达的候选基因，以确定功能所需的最低充分序列和转录因子结合位点。在预赛中 在这个提案中描述的实验中，我们成功地识别了一个新的发育阶段特异的顺式调节元件(NCE)，我们想要在体外和体内详细地表征它。为了进行体内研究，我们将建立两种不同的小鼠模型，分别使用带有或不带有NCE缺失的重组细菌人工染色体(BAC)的CD4基因。这两个模型的目的如下：模型1-利用BAC将EGFP基因插入到CD4的外显子2中，产生绿色荧光报告基因转基因小鼠，在不改变内源性CD4表达模式的情况下，我们可以比较野生型和NCE缺失的CD4-BAC-EGFP的表达模式。模型2-建立野生型CD4-BAC和NCE缺失的CD4-BAC转基因小鼠，同时也缺乏内源性CD4和β2M，以直接确定NCE缺失对MHC-II特异性CD4T细胞的阳性选择、谱系承诺、激活和分化的影响。这些体内模型将是迈向我们长期目标的垫脚石，我们的长期目标是识别和表征CD4基因座中的新转录控制元件，并了解它们如何相互作用来调节不同类型的CD4+细胞在其发育和功能的不同阶段的CD4水平。

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.