Defining the Role of Dynamic X Chromosome Inactivation in Age-Associated B Cells for Female-Biased Systemic Lupus Erythematosus

项目摘要/摘要 90%被诊断为系统性红斑狼疮(SLE)的人是女性，但潜在的 在自身免疫性疾病中控制性别偏见的机制尚不清楚。系统性红斑狼疮易感性增加 随着个体携带的X染色体的数量和X连锁基因表达的增加， 在患有系统性红斑狼疮的女性身上发现。这些发现表明，来自两条X染色体的基因表达可能 导致女性偏向的系统性红斑狼疮发病。X连锁基因在哺乳动物中表达的剂量 一条X染色体通过X染色体的表观遗传沉默来平衡 失活(XCI)。XCI在体细胞中是通过抑制表观遗传特征与 不活跃的X染色体(Xi)，包括用长长的非编码RNA Xist包被Xi。尽管如此 抑制功能，一些基因已经被证明可以逃脱XI的沉默。我们的实验室发现B 细胞是SLE发病机制中的一种关键细胞类型，具有动态的XCI维持，而幼稚的B细胞则看不到 Xist RNA信号尚未证明Xist RNA在激活时重新定位到XI。动态XCI维护 被破坏，X连锁免疫基因在SLE B细胞中异常表达，表明受损的 抑制表观遗传标记在XI上的定位可能导致免疫基因从XI异常逃逸， 增强B细胞活性。年龄相关的B细胞(ABC)正在成为女性偏见患者的一种关键细胞类型 自身免疫，并通过TLR7通路对信号做出强有力的反应。几个TLR7途径成员 是X连锁的，已被确定为系统性红斑狼疮的风险变异。从以下方面确定贡献的重要性 女性偏向的SLE的X染色体，我将定义ABCs中XI的表观遗传学特征，并阐明 B细胞室中扰动的XCI维持在SLE发病机制中的影响。在目标1中，我假设 ABC有动态的XCI维持，X连锁的免疫调节基因在 ABC。我将分离小鼠脾B细胞，并可视化抑制性表观遗传特征与 XI在ABC的分化和激活过程中，并执行等位基因特异性RNA测序以确定基因 逃脱了ABC的沉默。在目标2中，我将以我的初步数据为基础，证明雌性老鼠 具有B细胞特异性Xist缺失导致的扰乱性XCI(“Xist CKO”)更容易发生自发性和 化学诱导的系统性红斑狼疮样疾病。我推测雌性Xist CKO小鼠的XCI维持能力受损 导致免疫调节X连锁基因上调，产生更容易分化为 效应器种群，并在刺激时产生更多抗体。我将化验转录和功能 雌性Xist CKO小鼠稳态和自发发育过程中B细胞的特征 和化学诱导的系统性红斑狼疮。研究ABC中XCI维持的表观遗传机制将揭示 来自XI的遗传贡献如何对ABC功能至关重要，并将首次阐明X 基于染色体的机制，是女性偏向自身免疫的基础。

Project Summary / Abstract 90% of individuals diagnosed with systemic lupus erythematosus (SLE) are women, but the underlying mechanisms that govern sex bias in autoimmune disease are not understood. Susceptibility to SLE increases with the number of X chromosomes carried by an individual, and increased expression of X-linked genes has been found in women with SLE. These findings suggest that gene expression from two X chromosomes likely contributes to female-biased SLE pathogenesis. The dosage of X-linked gene expression in mammals with more than one X chromosome is balanced by epigenetically silencing one X chromosome through X-chromosome inactivation (XCI). XCI is maintained in somatic cells by association of repressive epigenetic features with the inactive X chromosome (Xi), including coating of the Xi with the long non-coding RNA Xist. Despite these repressive features, some genes have been shown to escape silencing from the Xi. Our lab discovered that B cells, a key cell type in SLE pathogenesis, have “dynamic” maintenance of XCI, where naïve B cells lack visible Xist RNA signal yet demonstrate re-localization of Xist RNA to the Xi upon activation. Dynamic XCI maintenance is disrupted and X-linked immune genes are aberrantly expressed in SLE B cells, suggesting that impaired localization of repressive epigenetic marks on the Xi may cause aberrant escape of immune genes from the Xi, enhancing B cell activity. “Age-associated B cells” (ABCs) are emerging as a critical cell type in female-biased autoimmunity and respond robustly to signaling through the TLR7 pathway. Several TLR7 pathway members are X-linked and have been identified as risk variants in SLE. To determine the importance of contributions from the X chromosome in female-biased SLE, I will define epigenetic features of the Xi in ABCs and elucidate the impact of perturbed XCI maintenance in the B cell compartment on SLE pathogenesis. In Aim 1, I hypothesize that ABCs have dynamic XCI maintenance, and that X-linked immune-regulatory genes escape silencing in ABCs. I will isolate murine splenic B cells and visualize the association of repressive epigenetic features with the Xi during differentiation and activation of ABCs and perform allele-specific RNA sequencing to determine genes that escape silencing in ABCs. In Aim 2, I will build on my preliminary data that demonstrates that female mice with a B cell-specific Xist deletion yielding perturbed XCI (“Xist cKO”) are more susceptible to spontaneous and chemically-induced SLE-like disease. I hypothesize that impaired XCI maintenance in female Xist cKO mice leads to upregulation of immune-regulatory X-linked genes, yielding B cells that differentiate more readily into effector populations and produce more antibodies upon stimulation. I will assay transcriptional and functional characteristics of B cells from female Xist cKO mice at steady state and during the development of spontaneous and chemically induced SLE. Investigating the epigenetic mechanisms of XCI maintenance in ABCs will reveal how genetic contributions from the Xi are critical for ABC function, and for the first time will illuminate an X chromosome-based mechanism that underlies female-biased autoimmunity.