Impact of Genetic Variation on Th Cell Differentiation

描述（由申请人提供）：越来越清楚的是，过敏性炎症背后的Th 2应答的严重失调具有很强的遗传成分。我们的小组特别感兴趣的影响，自然变异的表达和功能的关键基因控制的分化和/或效应功能的人Th 2细胞。 加塔-3作为鼠Th 2应答的中心调节因子，并且可能作为真正的Th 2细胞命运决定的主开关。相比之下，对加塔-3在人类中的调节和功能知之甚少。大量的加塔-3单核苷酸多态性（single nucleotide polymorphism，SNP）已被保存在现有的数据库中，但尚未有相关性或功能性研究的报道。我们最近在人GA TA-3基因座的一个区域中鉴定了两个SNP，-7135T/C和-7018C/G，该区域似乎含有在非T细胞中有活性的沉默物。这两个SNPs是高度连锁的，罕见的-7018G等位基因在一个大型图森儿童人群（n=453）的频率为42%。值得注意的是，-7018 G或-7135 C的纯合性与Th 1细胞因子干扰素（IFN）-γ表达的显著增加、对哮喘的保护和低血清IgE水平相关。这些结果表明，在加塔-3的遗传变异可能会影响Th 2分化和Th 2介导的疾病在体内的发病率。我们的长期目标是确定加塔-3的遗传变异调节基因表达和加塔-3依赖事件的机制。在本申请中，我们将测试新的假设，即自然变异可能影响加塔-3基因座在分化CD 4+人Th细胞中的表观遗传控制。这将导致加塔-3表达和加塔-3依赖性事件的改变，首先是Th 2分化和Th 2效应子功能的改变。 由于时间有限，我们将专门关注核酸酶超敏反应和DNA甲基化模式中的等位基因特异性变化。我们的工作将在三个相互依赖的阶段进行阐述：（1）我们将首先使用基因座范围的Southern印迹分析来定位从加塔-3/-7018等位基因纯合供体分离的人CD 4 + Th细胞中加塔-3基因座（=30 kb）处的DNA酶I超敏性和DNA甲基化。细胞将在Th 1和Th 2极化条件下体外分化。通过测量加塔-3、T-bet、IL-4、IL-13、IL-5和IFN-γ的表达水平来评估每种条件下的Th细胞分化。（2）然后，我们将开发高分辨率方法来量化通过Southern分析检测的每个位点处的核酸酶超敏性和/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，包括差异调节的HS和/或DNA甲基化位点，以便将来自染色质结构和序列的数据合并到单个高分辨率图谱中，并将染色质结构的变化与局部序列变异相关联。 将在后续研究中分析由此鉴定的SNP对加塔-3表达的表观遗传控制的功能影响。分析一个关键调控基因的自然变异和表观遗传变化之间的关系，可能会建立一个新的范式，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.