Molecular Regulation of MS Susceptibility Genes

描述（由申请人提供）：MS易感基因的分子调控。多发性硬化症（MS）是一种自身免疫性疾病，其特征在于中枢神经系统（CNS）中对髓鞘抗原组分的T细胞应答失调。虽然机制导致启动失调的T细胞反应髓鞘衍生的抗原知之甚少，发现一个新的子集的效应CD 4 + T细胞，Th 17细胞，改变了我们的理解MS疾病的发病机制，并承诺新的方法来治疗干预。在最近对Th 17细胞在CNS炎症中的作用的研究中，我们发现了一种新的机制，通过该机制调节免疫调节细胞因子IL-10的产生。具体来说，我们已经确定了一个重要的转录抑制因子，控制生产的IL-10的CD 4 + T细胞-生长因子非依赖性-1（Gfi 1）。我们发现，在EAE模型中，患有Gfi 1疾病的T细胞特异性缺陷的小鼠的临床疾病和CNS炎症显著改善。IL-10信号传导的阻断在很大程度上逆转了Gfi 1缺乏所赋予的保护作用。这强烈表明，疾病的抑制是通过一种机制发生的，该机制将Gfi 1与CD 4 + T细胞中IL-10基因表达的抑制联系起来，或许还有其他可以产生IL-10的免疫细胞。重要的是，在一些MS风险增加的个体中，GFI 1基因周围的扩展位点包含两个单核苷酸多态性（SNP），其纯合性导致激活的外周血细胞表达GFI 1增加。此外，GFI 1表达在后来发展为MS的患者的外周血中升高，这与GFI 1表达失调是MS的重要风险因素的可能性一致。虽然目前将改变的GFI 1表达与MS联系起来的数据仅是相关的，并且尚不清楚该因子的表达升高如何有助于MS易感性的提高，我们的初步研究已经确定了一种机制，可以解释Gfi 1与MS联系的主要组成部分，并将在本提案中进行探讨。具体而言，我们假设GFI 1是IL 10基因表达的主要转录抑制因子，并且是IL 10基因调控的核心。此外，如与GFI 1/IM 5基因座中的MS易感性SNP相关发现的，GFI 1的表达升高赋予免疫细胞受损的IL 10表达，其倾向于CNS炎症或加剧CNS炎症。为了验证这一假设，Gfi 1在调节人IL 10表达中的作用将在CNS炎症的背景下描述，我们将定义Gfi 1靶向的IL 10位点中的顺式调节元件（克雷斯）的功能。这些研究将采用一种新的基因靶向系统，我们最近开发和实施，使位点特异性，单拷贝整合的人IL 10基因位点到小鼠基因组。这代表了一个重大的技术进步，第一次批判性地审查了 远端克雷斯在染色质重塑和基因调控中的作用。使用该模型，我们将能够通过检查这些元件的突变对EAE模型中IL 10基因重塑、表达和保护的影响来定义人IL 10基因座中Gfi 1结合克雷斯的功能。

DESCRIPTION (provided by applicant): Molecular Regulation of MS Susceptibility Genes. Multiple sclerosis (MS) is an autoimmune disorder characterized by dysregulated T cell responses to antigenic components of myelin in the central nervous system (CNS). Although mechanisms leading to initiation of dysregulated T cell responses to myelin-derived antigens are poorly understood, discovery of a new subset of effector CD4+ T cells, Th17 cells, has transformed our understanding of MS disease pathogenesis and promises novel approaches to therapeutic intervention. In recent studies of the role of Th17 cells in CNS inflammation, we have discovered a novel mechanism by which production of the immunomodulatory cytokine, IL-10, is regulated. Specifically, we have identified an important transcriptional repressor that governs the production of IL-10 by CD4+ T cells - growth factor independent-1 (Gfi1). We find that in mice with T cell-specific deficiency of Gfi1 disease clinical disease and CNS inflammation are significantly ameliorated in an EAE model. Blockade of IL-10 signaling largely reverses protection conferred by Gfi1 deficiency. This strongly suggests that suppression of disease occurs via a mechanism linking Gfi1 to repression of IL10 gene expression in CD4+ T cells, and perhaps other immune cells that can produce IL-10. Importantly, in some individuals at increased risk for MS, the extended locus surrounding the GFI1 gene contains two single-nucleotide polymorphisms (SNPs), homozygosity for which leads to increased expression of GFI1 by activated peripheral blood cells. Further, GFI1 expression is elevated in peripheral blood of patients who later develop MS, consistent with the possibility that dysregulated GFI1 expression is an important risk factor for MS. While the data linking altered GFI1 expression to MS are only correlative at present and it is unknown how elevated expression of this factor might contribute to heightened susceptibility to MS, our preliminary studies have identified a mechanism that could explain a major component of Gfi1's linkage to MS and will be explored in this proposal. Specifically, we hypothesize that GFI1 is a major transcriptional repressor of IL10 gene expression and is central to IL10 gene regulation. Further, elevated expression of GFI1, as is found in association with MS susceptibility SNPs in the GFI1/EVI5 locus, confers impaired IL10 expression by immune cells that predisposes to, or exacerbates, CNS inflammation. To test this hypothesis, the role of Gfi1 in regulating human IL10 expression will be delineated in the context of CNS inflammation and we will define the function of cis-regulatory elements (CREs) in the IL10 locus that are targeted by Gfi1. These studies will employ a novel gene-targeting system that we have recently developed and implemented to enable site-specific, single copy integration of the human IL10 gene locus into the mouse genome. This represents a substantial technical advance with which to critically examine, for the first time, the function of distal CREs in chromatin remodeling and gene regulation. Using this model, we will be able to define the function of Gfi1-binding CREs in the human IL10 locus by examining the effects of mutation of these elements on IL10 gene remodeling, expression and protection in the EAE model.