OGT Overexpression in Women with Lupus

描述（由申请人提供）：女性患狼疮的几率是男性的9倍。雌激素有助于狼疮的严重程度，但不能完全解释风险的增加。DNA甲基化受损是一种抑制表观遗传修饰，会导致T细胞基因过度表达，从而导致狼疮。DNA甲基化也会使女性的一条X染色体沉默。CD40LG是一个已知在狼疮中过度表达的x连锁基因，并有助于自身抗体的产生。我们发现，不活跃的X染色体上的CD40LG去甲基化，并且在患有狼疮的女性中过度表达，而在患有狼疮的男性中则没有，这使女性易患狼疮。其他x连锁基因也可能发生去甲基化，导致自身免疫。我们调查了甲基化敏感的T细胞基因，发现O-linked N-acetylglucosamine transferase （OGT）是另一个在去甲基化的女性T细胞中过表达的X-linked基因。OGT将n -乙酰氨基葡萄糖（GlcNAc）偶联到包括信号分子在内的多种蛋白质中的丝氨酸和苏氨酸上，以类似磷酸化的方式修饰功能，称为己糖胺信号通路（HSP）。热休克蛋白异常与糖尿病和神经变性有关，但对其在T细胞中的作用知之甚少。我们假设，OGT在失活X上的去甲基化导致女性的过表达，改变HSP信号，并通过改变信号传导来促进致病性T细胞功能。我们将通过以下方式检验这一假设：1)比较来自健康男性和女性的对照和去甲基化的CD4+和CD8+ T细胞的OGT mRNA和蛋白水平，与来自非活发狼疮、活发狼疮和疾病/年龄对照的男性和女性的CD4+和CD8+ T细胞的水平，并通过亚硫酸酯测序确认OGT去甲基化。2)确定OGT过表达是否损害T细胞ERK、JNK和/或p38信号通路并改变T细胞基因表达模式。3)利用T细胞特异性诱导的OGT转基因小鼠，确定T细胞OGT过表达对自身免疫发展的功能意义。这些研究将描述一种调节T细胞基因表达的新途径，并描述该途径的异常如何使女性易患自身免疫。

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.