Identifying the Xist interactome during dynamic X inactivation in healthy and lupus-derived B cells

项目总结 我目前是宾夕法尼亚大学(宾夕法尼亚大学)的博士后研究员； 蒙特塞拉特·安格拉我正在接受培训，准备成为一名独立的科学家，着眼于开创自己的事业 实验室，并寻求与人类免疫健康和疾病相关的问题。在安格拉博士的实验室里，我们 感兴趣的是淋巴细胞的X失活机制及其对人类自身免疫性疾病的影响。 一条X染色体的失活通常发生在发育的早期，并在整个过程中保持不变 为了在XX和XY个体之间保持相同的基因表达，细胞的寿命。X染色体 失活(XCI)是通过表达一种称为Xist的RNA转录本来启动和维持的，该转录本是 从未来不活跃的X(Xi)转录而来，然后在染色体上上下传播，覆盖在一团 Xist RNA，它在细胞和任何子代细胞的生命周期中一直留在XI上。与几乎所有其他XX单元格不同 在女性体内，B和T免疫细胞，也被称为淋巴细胞，有一个动态的X染色体 失活机制。习上的Xist云在幼稚的淋巴细胞中消散，并返回覆盖Xi一次 细胞被激活了。在狼疮患者中，XIST成功地清除了幼稚淋巴细胞中的XI，但失败了 激活后完全返回。我的项目更仔细地研究了Xist离开和返回的机制 在B淋巴细胞活化过程中对XI的影响。已证明有80到200种蛋白质与Xist和Xist结合 更多的实验已经阐明了它们在XCI中的作用，然而，之前的高通量分析都没有 已经在展示了这种新的X失活方法的细胞中进行了实验，到目前为止，这种方法似乎是特异的 到淋巴细胞。基于我们实验室之前的发现和公开可用的基因表达数据 其他实验室，我假设有两种蛋白质，粘附素(SMC3)和Lamin B受体(LBR)在 这一过程发生在健康人和狼疮B细胞中。为了验证这一假设，我提议进行一个生物素探测器 基于Xist RNA下拉的RNA结合蛋白质的质谱学综合鉴定 (chirp-MS)在小鼠和人类的B细胞中。Chirp-MS方法收集细胞中的所有Xist RNA并 携带任何与Xist RNA结合的蛋白质，然后使用质谱仪对这些蛋白质进行鉴定 用于同时鉴定每种蛋白质。我会在健康的幼稚和活化的B细胞中进行chirp-MS 以及来自狼疮小鼠模型和人类狼疮患者的激活的B细胞。后质谱学 蛋白质的鉴定，我将比较与Xist相关的蛋白质在幼稚和活化的B细胞和健康 去狼疮B细胞，并寻找那些在任一亚群中富含的蛋白质。这些实验的结果不会 仅确认或修正我对LBR和SMC3在X失活中的作用的预测，但将允许 在人类和小鼠中与Xist相关的所有淋巴细胞特异性蛋白的比较，我们最 在测试狼疮的病理或治疗时常用的模型生物。

PROJECT SUMMARY I am presently a postdoctoral researcher at the University of Pennsylvania (Penn); under the supervision of Dr. Montserrat Anguera I am training to become an independent scientist with an eye toward starting my own laboratory and pursuing questions relevant to human immune health and disease. In Dr. Anguera’s lab, we are interested in X inactivation mechanisms in lymphocytes and their implications for human autoimmune disease. Inactivation of one X chromosome usually happens very early in development and remains constant throughout the life of the cell in order to keep gene expression equal between XX and XY individuals. X Chromosome Inactivation (XCI) is initiated and maintained by expression of an RNA transcript known as Xist, which is transcribed from the future inactive X (Xi) then spreads up and down the chromosome to cover it in a “cloud” of Xist RNA, which remains on the Xi for the life of the cell and any daughter cells. Unlike nearly all other XX cells in a woman’s body, B- and T- immune cells, also known as lymphocytes, have a dynamic X chromosome inactivation mechanism. The Xist cloud on the Xi dissipates in naïve lymphocytes and returns to coat the Xi once the cells are activated. In patients with lupus, Xist successfully evacuates the Xi in naïve lymphocytes but fails to fully return after activation. My project takes a closer look at the mechanisms by which Xist leaves and returns to the Xi during B lymphocyte activation. Between 80 and 200 proteins have been shown to bind to Xist and additional experiments have elucidated their role in XCI, however, none of the previous high-throughput analyses have been performed in cells that exhibit this novel approach to X inactivation, which so far appears to be specific to lymphocytes. Based on previous findings from our laboratory and publicly available gene expression data from other labs, I hypothesize that two proteins, cohesin (SMC3) and Lamin B Receptor (LBR) play critical roles in this process in healthy and lupus B cells. In order to test this hypothesis, I propose to perform a biotin-probe based Xist RNA pull-down called Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) in B cells from mice and humans. The ChIRP-MS approach collects all the Xist RNA in the cell and brings with it any proteins bound to the Xist RNA, those proteins are then identified using a mass spectrometer for simultaneous identification of every protein. I’ll perform ChIRP-MS in healthy naïve and activated B cells as well as activated B cells from mouse models of lupus and human lupus patients. After mass spectrometry identification of proteins, I will compare the proteins associated with Xist in naïve to activated B cells and healthy to lupus B cells and look for those proteins enriched in either subset. Results from these experiments will not only confirm or modify my predictions about the role of LBR and SMC3 in X inactivation but will allow for comparisons between all lymphocyte-specific proteins associated with Xist in both humans and mice, our most commonly used model organism when testing pathology or treatments for lupus.