Impact of Genetic Variation on Th Cell Differentiation

描述(由申请人提供)：越来越明显的是，在过敏性炎症基础上的Th2反应的严重失调具有很强的遗传成分。我们的团队对自然变异对控制人类Th2细胞分化和/或效应器功能的关键基因的表达和功能的影响特别感兴趣。 GATA-3作为小鼠Th2反应的中央调节因子，可能是决定Th2细胞命运的真正的主开关。相比之下，人们对GATA-3在人类中的调节和功能知之甚少。GATA-3单核苷酸多态(SNP)已被保存在现有的数据库中，但尚未有关联或功能研究的报道。我们最近在人类GA TA-3基因座的一个区域发现了两个SNPs，-7135T/C和-7018C/G，该区域似乎包含一个在非T细胞中活跃的沉默因子。这两个SNPs高度连锁；在一个大的图森儿童群体(n=453)中，罕见的-7018G等位基因的频率为42%。值得注意的是，-7018G或-7135C的纯合子与Th1细胞因子干扰素(干扰素)-伽马表达的高度显著增加、对哮喘的保护和低血清IgE水平相关。这些结果提示，GATA-3基因变异可能影响体内Th2分化和Th2介导性疾病的发生。我们的长期目标是确定GATA-3基因变异调节基因表达和GATA-3依赖事件的机制。在这项应用中，我们将检验这一新的假设，即自然变异可能会影响GATA-3基因座在分化CD4+人类Th细胞过程中的表观遗传控制。这将导致GATA-3表达和GATA-3依赖事件的改变，首先是Th2分化和Th2效应功能。 鉴于分配的时间有限，我们将专门关注核酸酶超敏和DNA甲基化模式中等位基因特异性的变化。我们的工作将分为三个相互依赖的阶段：(1)我们将首先使用全基因座的Southern印迹分析来定位从GATA-3/-7018等位基因纯合的捐赠者分离的人CD4+Th细胞的DNase I超敏反应和GATA-3基因座(=30kb)的DNA甲基化。在Th1和Th2极化条件下，细胞将在体外分化。我们将通过测量GATA-3、T-bet、IL-4、IL-13、IL-5和干扰素-γ的表达水平来评估每种情况下的TH细胞分化。(2)然后，我们将开发高分辨率方法，以量化Southern分析检测到的每个位置的核酸酶超敏和/或DNA甲基化的基因型和/或极化特异性差异。(3)最后，我们将对-7018/GG和-7018/CC纯合子的基因组DNA进行测序，包括差异调控的HS和/或DNA甲基化位点，以便将染色质结构和序列的数据合并到单一的高分辨率图谱中，并将染色质结构的变化与局部序列变化相关联。 所确定的SNPs对GATA-3表达的表观遗传调控的功能影响将在后续研究中分析。分析关键调控基因的自然变异和表观遗传变化之间的关系可能会建立一种新的范式，并最终突出过敏性炎症发病机制的重要机制。

DESCRIPTION (provided by applicant): It is becoming increasingly clear that the profound dysregulation of Th2 responses underlying allergic inflammation has a strong genetic component. Our group is particularly interested in the impact that natural variation has on the expression and function of key genes that control the differentiation and/or the effector functions of human Th2 cells. GATA-3 acts as a central regulator of murine Th2 responses, and possibly as a bona fide master switch for Th2 cell fate determination. By contrast, very little is known about GATA-3 regulation and function in humans. Numerous GATA-3 single nucleotide polymorphisms (SNP) have been deposited in the existing databases, but no association or functional studies have been reported. We recently identified two SNPs, -7135T/C and -7018C/G, in a region of the human GA TA-3 locus which appears to contain a silencer active in non-T cells. The two SNPs are highly linked; the frequency of the rare - 7018G allele in a large Tucson children population (n=453) was 42%. Notably, homozygosity for -7018G or -7135 C was associated with highly significant increases in the expression of the Th1 cytokine interferon (IFN)-gamma, protection against asthma, and low serum IgE levels. These results suggest that genetic variation in GATA-3 may affect Th2 differentiation and incidence of Th2-mediated disease in vivo. Our long-term goal is to identify the mechanisms whereby genetic variation in GATA-3 modulates the expression of the gene and GATA-3-dependent events. In this application we will test the novel hypothesis that natural variation may affect the epigenetic control of the GATA-3 locus in differentiating CD4+human Th cells. This would result in alterations of GATA-3 expression and GATA-3-dependent events, first and foremost Th2 differentiation and Th2 effector functions. Given the limited time allotted, we will focus exclusively on allele-specific changes in patterns of nuclease hypersensitivity and DNA methylation. Our work will be articulated in three interdependent phases: (1) We will initially use locus-wide Southern blot analysis to map DNase I hypersensitivity and DNA methylation at the GATA-3 locus ( =30 kb) in human CD4+ Th cells isolated from donors homozygous for the GATA-3/-7018 alleles. Cells will be differentiated in vitro under Th1 and Th2 polarizing conditions. Th cell differentiation for each condition will be assessed by measuring expression levels of GATA-3, T-bet, IL-4, IL-13, IL-5, and IFN-gamma, (2) We will then develop high resolution methods to quantify genotype- and/or polarization-specific differences in nuclease hypersensitivity and/or DNA methylation at each site detected by Southern analysis. (3) Finally, we will sequence genomic DNA from -7018/GG and -7018/CC homozygotes encompassing differentially regulated HS and/or DNA methylation sites, in order to merge data from chromatin structure and sequence into a single high resolution map, and correlate variation in chromatin structure with local sequence variation. The functional impact on the epigenetic control of GATA-3 expression of the SNPs thus identified will be analyzed in subsequent studies. The analysis of the relationship between natural variation and epigenetic changes in a critical regulatory gene may establish a novel paradigm and ultimately highlight important mechanisms underlying the pathogenesis of allergic inflammation.