NFkB Signaling is Required for Xist RNA Localization at the Inactive X and for X-Chromosome Inactivation Maintenance Following T cell Activation [CUT&Run]

X Chromosome Inactivation (XCI) is a female-specific process which balances X-linked gene dosage between sexes. Unstimulated T cells lack cytological enrichment of Xist RNA and heterochromatic modifications on the inactive X chromosome (Xi), and these modifications become enriched at the Xi after cell stimulation. Here, we examined allele-specific gene expression and epigenomic profiling of the Xi following T cell stimulation. We found that the Xi in unstimulated T cells is dosage compensated, and is enriched with the repressive H3K27me3 modification, but not H2AK119-ubiquitin (Ub) mark, including at promoters of XCI escape genes. Upon CD3/CD28 mediated T cell stimulation, the Xi accumulates H2AK119-Ub and H3K27me3 across the Xi. Next, we examined the T cell stimulation pathways responsible for Xist RNA localization to the Xi and found that T cell receptor (TCR) engagement, specifically NFkB signaling downstream of TCR, is required. Disruption of NFkB signaling, using inhibitors or genetic deletions, in mice and patients with immunodeficiencies prevents Xist/XIST RNA accumulation at the Xi and alters expression of some X-linked genes. Our findings reveal a novel connection between NFkB signaling pathways which impact XCI maintenance in female T cells. CUT&RUN analysis of Ring1b enrichment from F1 mus x cast CD3+ T cells that were unstimulated or stimulated with anti-CD3/anti-CD28 for 48 hours (n=3 per condition). F1 mus x cast is a hybrid mouse model of skewed XCI generated through mating female C57BL/6 mice harboring a heterozygous Xist deletion to male M. m. castaneous mice.