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

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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.