Molecular Regulation of MS Susceptibility Genes

描述（申请者提供）：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，其特征是中枢神经系统（CNS）中T细胞对髓磷脂抗原成分的反应失调。尽管导致髓磷脂来源抗原引发失调T细胞反应的机制尚不清楚，但发现一种新的CD4+ T细胞亚群Th17细胞，已经改变了我们对MS疾病发病机制的理解，并有望为治疗干预提供新的方法。在最近对Th17细胞在中枢神经系统炎症中的作用的研究中，我们发现了一种新的机制，通过这种机制，免疫调节细胞因子IL-10的产生受到调节。具体来说，我们已经确定了一个重要的转录抑制因子，控制CD4+ T细胞-生长因子独立-1 （Gfi1）产生IL-10。我们发现，在Gfi1 T细胞特异性缺陷的小鼠中，临床疾病和中枢神经系统炎症在EAE模型中得到显著改善。阻断IL-10信号在很大程度上逆转了Gfi1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，这种机制将Gfi1与CD4+ T细胞中IL-10基因表达的抑制联系起来，可能还有其他可以产生IL-10的免疫细胞。重要的是，在一些MS风险增加的个体中，GFI1基因周围的延伸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增加GFI1的表达。此外，GFI1在后来发展为MS的患者的外周血中表达升高，这与GFI1表达失调是MS的重要危险因素的可能性相一致。而GFI1表达改变与MS的数据目前只是相关的，尚不清楚该因子的表达升高如何导致MS易感性的增加。我们的初步研究已经确定了一种机制，可以解释Gfi1与MS联系的主要组成部分，并将在本提案中进行探索。具体来说，我们假设GFI1是IL10基因表达的主要转录抑制因子，并且是IL10基因调控的核心。此外，GFI1的表达升高，与GFI1/EVI5位点的MS易感性snp相关，导致免疫细胞il - 10表达受损，从而易患或加剧中枢神经系统炎症。为了验证这一假设，Gfi1在调节人类il - 10表达中的作用将在中枢神经系统炎症的背景下进行描述，我们将定义Gfi1靶向的il - 10位点中的顺式调节元件（cre）的功能。这些研究将采用我们最近开发和实施的一种新的基因靶向系统，使人类il - 10基因位点的单拷贝整合到小鼠基因组中。这代表了一项重大的技术进步，我们第一次用它来批判性地研究

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.