Dynamics of Xist RNA Localization to the Inactive X in quiescent cell populations



Associate Professor

Project Summary

Numerous autoimmune disorders exhibit a strong female bias, including Systemic Lupus Erythematosus (SLE) where 85% of patients are female. While female cells have two X chromosomes, males have an X and Y. The X-chromosome has been implicated in the development of autoimmunity, as individuals with multiple Xs (XXY; XXX) have greater risk for developing SLE. The X-chromosome contains many genes involved in immune function. Female, but not male, SLE patients exhibit abnormal over-expression of several of these genes in lymphocytes, yet the mechanisms responsible are unknown. Normally female cells will inactivate all but one of their X chromosomes through X chromosome inactivation (XCI). This is done through a large RNA molecule known as Xist, which binds to the inactive X (Xi), and recruits silencing compounds. However, recent work from the Anguera lab found that female lymphocytes have an unusual form of XCI maintenance: naïve (unstimulated) quiescent B and T cells lack Xist RNA and silencing marks, and these features are present on the Xi in activated lymphocytes. From this observation, we questioned whether all quiescent cells would lack Xist RNA on the inactive X. 

Through the Penn Undergraduate Mentorship Program, I had the opportunity to work in the Anguera lab and perform profiling of the inactive X using three distinct quiescent cell populations: the Hopx+ intestinal stem cell population, naive monocytes (a type of immune cell) and the Beta-4± lung airway epithelial cells. To profile cells, I used RNA FISH to visualize Xist RNA and rate the extent of its localization. In addition, I determined the timepoint for peak localization of Xist RNA to the inactive X during T cell activation. 

In order to profile these different cell types, I had to become proficient in isolating and culturing T cells, RT-PCR and RNA FISH.

By the end of the summer, I found that both Beta-4 negative cells exhibit weaker Xist RNA localization than Beta-4 positive cells, despite both producing Xist RNA at similar levels, while Hopx+ cells had weaker Xist RNA localization than the Hopx- fraction. I determined that murine T cells achieve peak Xist RNA localization between two and three days post stimulation. It appeared that different subsets of T cells (Treg and Th1 cells) had different peak timepoints. Profiling of naive monocytes revealed that they also shared the characteristic lack of Xist RNA localization. Upon stimulating the monocytes, I was able to observe Xist RNA localizing to the Xi. These results further reveal a relationship between XCI and quiescent cells that I’ll be doing further research on this fall. 

Throughout the summer I gained valuable experience not only presenting my work and findings at weekly lab meetings, but also collaborating with other labs. Additionally, I had to learn to organize my experiments and time wisely. There were many moving parts to my project and keeping everything running smoothly forced me to become a more disciplined and practical experimentalist. As an aspiring physician-scientist these are skills I hope to utilize in the future.

Dynamics of Xist RNA Localization to the Inactive X in quiescent cell populations