Mechanism and Function of XIST in X Chromosome Inactivation Maintenance - PROJECT SUMMARY X chromosome inactivation (XCI) is essential in equalizing X-linked gene expression between females (XX) and males (XY). This process is initiated by XIST, a female-specific long non-coding RNA (lncRNA) that recruits diverse co-factors to epigenetically silence the prospective inactive X chromosome (Xi) during embryogenesis. Once established, the Xi “remembers” to maintain silencing in all somatic cells through adulthood, serving as a unique epigenetic memory model. In contrast to the significant progress made to understand XCI initiation, the mechanism and function underlying XCI maintenance remain largely unknown. We recently uncovered an unexpected requirement of XIST in maintaining X-inactivation, a process that is long thought to be independent of XIST. Specifically, we found that XIST prevents the escape of key immune-associated genes from X- inactivation in somatic B cells. Building on our preliminary work, we hypothesize that XIST orchestrates XCI maintenance through evolutionarily conserved domains in a cell-type-specific and signaling-responsive manner, particularly in somatic immune cells. Over the next five years, we will test this hypothesis by answering three fundamental questions: (1) How do XIST's functional domains coordinate to maintain X-inactivation? (2) How do cellular signaling pathways influence XCI maintenance fidelity? (3) When and where is XIST required for XCI maintenance throughout lifespan? We will address these questions using CRISPR perturbation, allele-specific chromatin profiling, RNA mediated proteomics, single-cell genomics and mouse models with clonal XCI tracking ability. This comprehensive strategy will allow us to understand how XIST safeguards Xi silencing against gene escape and female-biased diseases. Completion of this work will reveal fundamental principles of non-coding RNA mediated epigenetic regulation, provide valuable toolkits for broader epigenetic and lncRNA community, and expand the role of X-inactivation maintenance in sex dimorphism of immune compartment.
