OGT Overexpression in Women with Lupus

描述(由申请人提供)：女性狼疮的发病率是男性的9倍。雌激素有助于狼疮的严重程度，但不能完全解释增加的风险。DNA甲基化受损是一种抑制性的表观遗传修饰，会导致导致狼疮的T细胞基因过度表达。DNA甲基化也会使女性的一条X染色体沉默。CD40LG是一种X连锁基因，已知在狼疮中过表达，并有助于自身抗体的产生。我们发现，非活性X基因上的CD40LG去甲基化，并且在女性狼疮患者中过表达，而在男性患者中不表达，从而使女性容易患上狼疮。其他X连锁基因也可能去甲基化，导致自身免疫。我们调查了甲基化敏感的T细胞基因，并确定O-连锁N-乙酰氨基葡萄糖转移酶(OGT)是另一个在去甲基化的女性T细胞中过度表达的X-连锁基因。OGT将N-乙酰氨基葡萄糖(GlcNAc)与多种蛋白质中的丝氨酸和苏氨酸偶联，包括信号分子，以类似于磷酸化的方式改变功能，被称为氨基己糖信号通路(HSP)。HSP异常与糖尿病和神经退行性变有关，但对其在T细胞中的作用知之甚少。我们假设，OGT在非活性X上的去甲基化导致女性过表达，改变HSP信号，并通过改变信号而促进致病T细胞功能。我们将通过以下方式验证这一假设：1)比较健康男性和女性对照和去甲基化的CD4+和CD8+T细胞中的OGT mRNA和蛋白水平，以及来自静止期狼疮、活动期狼疮和疾病/年龄对照组的男性和女性的CD4+和CD8+T细胞中的OGT水平，并通过亚硫酸氢盐测序确认OGT去甲基化；2)确定OGT过表达是否会损害T细胞ERK、JNK和/或p38途径信号并改变T细胞基因的表达模式；3)使用T细胞特异性OGT转基因小鼠，确定T细胞OGT过度表达在自身免疫发展中的功能意义。这些研究将描述一种新的调节T细胞基因表达的途径，并描述该途径中的异常可能如何使女性更容易产生自身免疫。 公共卫生相关性：狼疮是一种主要影响女性的自身免疫性疾病，会导致残疾，有时还会导致死亡。女性易患狼疮的原因尚不清楚。我们的团队发现，狼疮患者T淋巴细胞中一种名为DNA甲基化的基因表达机制存在缺陷，导致使细胞攻击身体的基因过度表达。这导致了那些拥有适当基因构成的人患上了狼疮。女性有2条X染色体，而男性只有1条。女性的一条X染色体被DNA甲基化灭活，以防止她们表达的基因是男性的两倍。然而，在狼疮患者中，第二个X染色体上的基因去甲基化，导致可能导致她们疾病的基因过度表达。我们发现，OGT是一种重要的X染色体基因，在细胞功能调节中，当DNA甲基化被抑制时，女性T淋巴细胞中OGT的表达会加倍。这表明OGT在狼疮患者体内也可能过度表达，从而导致了她们的疾病。本申请中描述的研究将确定狼疮患者是否过度表达OGT，并确定其功能后果。这一结果可能为了解女性自身免疫的机制提供新的见解，并提出治疗这种疾病的新方法。

DESCRIPTION (provided by applicant): Lupus afflicts women 9 times more often than men. Estrogen contributes to lupus severity, but does not completely explain the increased risk. Impaired DNA methylation, a repressive epigenetic modification, causes overexpression of T cell genes that contribute to lupus. DNA methylation also silences one X chromosome in women. CD40LG is an X-linked gene known to be overexpressed in lupus and contribute to autoantibody production. We found that CD40LG on the inactive X demethylates and is overexpressed in women but not men with lupus, predisposing women to lupus. Other X-linked genes may also demethylate, predisposing to autoimmunity. We surveyed methylation sensitive T cell genes, and identified O-linked N-acetylglucosamine transferase (OGT) as another X-linked gene overexpressed in demethylated female T cells. OGT couples N-acetylglucosamine (GlcNAc) to serines and threonines in a variety of proteins including signaling molecules, modifying function in a manner analogous to phosphorylation and referred to as the hexosamine signaling pathway (HSP). HSP abnormalities are implicated in diabetes and neurodegeneration, but little is known regarding its role in T cells. We hypothesize that demethylation of OGT on the inactive X results in overexpression in women, altering HSP signaling and contributing to pathogenic T cell function by modifying signaling. We will test this hypothesis by: 1) Comparing OGT mRNA and protein levels in control and demethylated CD4+ and CD8+ T cells from healthy men and women with levels in CD4+ and CD8+ T cells from men and women with inactive lupus, active lupus, and disease/age controls, and confirming OGT demethylation by bisulfite sequencing, 2) Determining if OGT overexpression impairs T cell ERK, JNK and/or p38 pathway signaling and modifies T cell gene expression patterns, and 3) Determining the functional significance of T cell OGT overexpression on the development of autoimmunity using transgenic mice with a T cell specific inducible OGT transgene. These studies will characterize a new pathway regulating T cell gene expression, and characterize how abnormalities in the pathway may predispose women to autoimmunity. PUBLIC HEALTH RELEVANCE: Lupus is an autoimmune disease primarily affecting women, causing disability and sometimes death. The reason women are predisposed to lupus is unknown. Our group has found that a mechanism regulating gene expression, called DNA methylation, is defective in T lymphocytes from lupus patients, causing overexpression of genes that make the cells attack the body. This contributes to lupus in those with the appropriate genetic makeup. Women have 2 X chromosomes, while men have only one. One X chromosome is inactivated by DNA methylation in women to prevent them from expressing the genes twice as much as men. However, genes on the second X chromosome demethylate in women with lupus, causing overexpression of genes that may contribute to their disease. We have found that OGT, an X chromosome gene important in regulating cellular function, doubles expression in T lymphocytes from women when DNA methylation is inhibited. This suggests that OGT may also be overexpressed in women with lupus, contributing to their disease. The studies described in this application will determine if women with lupus overexpress OGT, and determine the functional consequences. The results may provide new insights into mechanisms causing autoimmunity in women, and suggest new ways to treat the disease.