Molecular Regulation of MS Susceptibility Genes

描述（由申请人提供）：多发性硬化症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 T 细胞对中枢神经系统 (CNS) 髓磷脂抗原成分的反应失调。尽管导致 T 细胞对髓磷脂衍生抗原反应失调的机制尚不清楚，但效应 CD4 T 细胞的新子集 Th17 细胞的发现改变了我们对多发性硬化症发病机制的理解，并有望提供新的治疗干预方法。在最近关于 Th17 细胞在中枢神经系统炎症中作用的研究中，我们发现了一种调节免疫调节细胞因子 IL-10 产生的新机制。具体来说，我们发现了一种重要的转录抑制因子，它控制 CD4 T 细胞产生 IL-10 - 生长因子独立 1 (Gfi1)。我们发现，在 EAE 模型中，T 细胞特异性 Gfi1 疾病缺陷小鼠的临床疾病和中枢神经系统炎症得到显着改善。 IL-10 信号传导的阻断很大程度上逆转了 Gfi1 缺陷所赋予的保护作用。这有力地表明，疾病的抑制是通过一种机制来实现的，该机制将 Gfi1 与 CD4 T 细胞以及可能产生 IL-10 的其他免疫细胞中 IL10 基因表达的抑制联系起来。重要的是，在一些 MS 风险增加的个体中，GFI1 基因周围的扩展基因座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，后来发展为 MS 的患者外周血中 GFI1 表达升高，这与 GFI1 表达失调是 MS 的重要危险因素的可能性一致。虽然目前将 GFI1 表达改变与 MS 联系起来的数据仅具有相关性，并且尚不清楚该因​​子的表达升高如何导致 MS 易感性增加，但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讨。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 表达升高（与 GFI1/EVI5 基因座中 MS 易感性 SNP 相关）会导致免疫细胞 IL10 表达受损，从而诱发或加剧 CNS 炎症。为了检验这一假设，我们将在中枢神经系统炎症的背景下描述 Gfi1 在调节人 IL10 表达中的作用，我们将定义 Gfi1 靶向的 IL10 位点中顺式调节元件 (CRE) 的功能。这些研究将采用我们最近开发和实施的新型基因靶向系统，以实现人类 IL10 基因位点的位点特异性、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，首次批判性地检验了 染色质重塑和基因调控中的远端 CRE。使用该模型，我们将能够通过在 EAE 模型中检查这些元件的突变对 IL10 基因重塑、表达和保护的影响来定义人类 IL10 位点中 Gfi1 结合 CRE 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.