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

项目摘要 我目前是宾夕法尼亚大学的博士后研究员; 我正在接受培训，成为一名独立的科学家，着眼于开始我自己的研究。 实验室和追求与人类免疫健康和疾病有关的问题。在安格拉博士的实验室里， 对淋巴细胞中的X失活机制及其对人类自身免疫性疾病的影响感兴趣。 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。 细胞的寿命，以保持XX和XY个体之间的基因表达相等。X染色体 失活（XCI）通过称为Xist的RNA转录物的表达启动和维持， 从未来的非活性X（Xi）转录，然后在染色体上上下传播，以一种“云”的形式覆盖它。 Xist RNA，其在细胞和任何子细胞的生命中保留在Xi上。与几乎所有其他XX细胞不同， 在女性体内，B和T免疫细胞，也称为淋巴细胞，具有动态X染色体 失活机制Xi上的Xist云在幼稚淋巴细胞中消散，并再次覆盖Xi 细胞被激活。在狼疮患者中，Xist成功地清除了幼稚淋巴细胞中的Xi， 激活后完全恢复。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与Xi结合。80至200种蛋白质已被证明与Xist结合， 其他实验已经阐明了它们在XCI中的作用，然而，以前的高通量分析都没有 已经在展示这种新的X灭活方法的细胞中进行了，到目前为止，这种方法似乎是特异性的 淋巴细胞。基于我们实验室先前的发现和来自 其他实验室，我假设两种蛋白质，粘蛋白（SMC 3）和核纤层蛋白B受体（LBR）在 在健康和狼疮B细胞中的这一过程。为了验证这一假设，我建议进行生物素探针 基于Xist的RNA下拉，称为通过质谱法全面鉴定RNA结合蛋白 在来自小鼠和人的B细胞中的ChIRP-MS。ChIRP-MS方法收集细胞中的所有Xist RNA， 携带任何与Xist RNA结合的蛋白质，然后使用质谱仪识别这些蛋白质 用于同时鉴定每种蛋白质。我将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活化B细胞。质谱分析后 为了鉴定蛋白质，我将比较与幼稚到活化的B细胞和健康的Xist相关的蛋白质。 到狼疮B细胞中寻找那些在任一亚群中富集的蛋白质。这些实验的结果不会 我只是证实或修改了我关于LBR和SMC 3在X失活中的作用的预测， 在人类和小鼠中与Xist相关的所有淋巴细胞特异性蛋白质之间的比较，我们最 在检测狼疮病理学或治疗时常用的模式生物。

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.