X-Chromosome Inactivation (XCI) is an epigenetic process where females will randomly select one of their X-chromosomes for silencing to achieve gene dosage compensation between sexes. This is done through a large RNA molecule known as Xist, which binds to the inactive X (Xi), and recruits silencing compounds. Recent work found that female lymphocytes have an unusual form of XCI maintenance: The Xi in naïve quiescent B and T cells is missing Xist RNA and heterochromatic marks. Upon stimulation, Xist RNA and heterochromatic marks return to the Xi. Naïve lymphocytes are quiescent: the cells reversibly exit the cell cycle to reside in G0. This leads to the question of whether this dynamic form of XCI maintenance could be specific to the immune system, where quiescent cells re-enter the cell cycle in response to antigen stimulation, or perhaps be a feature of all quiescent cells. If the absence of Xist RNA from the Xi is a general feature of quiescent cells, then the mechanisms governing quiescence and this dynamic XCI may be interconnected.
With the support of a College Alumni Society Grant, I spent the semester attempting to address this question. We were interested in two quiescent populations: Hopx+ intestinal stem cells and B4± lung epithelial cells. The initial goal was to profile the inactive X in these populations; however, there was concern that the process of isolating, handling and sorting these cells may perturb them to re-enter the cell cycle. I decided to attempt to gain an in situ perspective by developing small intestine sections and performing RNA FISH on them. This involves incubating the cells with a fluorescent probe to visualize the Xist RNA within the nuclei. The small intestine consists of tendril-like villi with crypts separating them; at the bottom of the crypts are quiescent intestinal stem cells which replenish and maintain the intestines. My goal was to successfully perform RNA FISH on the sections to label Xist RNA on the inactive X and characterize the status of the inactive X in various crypt cells. I spent the semester optimizing the RNA FISH protocol on the tissue sections. Our lab (and I for that matter) hadn’t done tissue sectioning outside of the core facilities, so I started with a pre-established protocol from Nature and adjusted certain aspects to achieve successful staining. These steps were to ensure the sectioning was done cleanly and reduce background signal under the scope.
It was a very tedious and lengthy process for me. My first attempt was a complete failure, the features of the intestine were clearly visible, but the fluorescent probe hadn’t penetrated the cells and my view was obstructed by background signal. Over several more attempts, I slowly reduced the background on the slides until I began to observe signals. I haven’t completely optimized the protocol to the point where I can begin quantifying XCI in the cells, but I’m excited to finally be observing RNA FISH signals and optimistic that the next attempt will be successful.