B cell stimulation changes the structure and higher-order organization of the inactive X chromosome

X Chromosome Inactivation (XCI) equalizes X-linked gene expression between sexes. B cells exhibit dynamic XCI, with Xist RNA/heterochromatic marks absent on the inactive X (Xi) in naive B cells but returning following mitogenic stimulation. The impact of dynamic XCI on Xi structure and maintenance was previously unknown. Here, we find dosage compensation of the Xi with state-specific XCI escape genes in naive and in vitro activated B cells. Allele-specific OligoPaints indicate similar Xi and Xa territories in B cells that are less compact than in fibroblasts. Allele-specific Hi-C reveals a lack of TAD-like structures on the Xi of naive B cells, and stimulation-induced alterations in TAD-like boundary strength independent of gene expression. Notably, Xist deletion in B cells changes TAD boundaries and large-scale Xi compaction. Altogether, our results uncover B cell-specific Xi plasticity which could underlie sex-biased biological mechanisms. To determine how Xist deletion impacts transcriptional changes in splenic follicluar B cells, we sorted CD23+ splenic B cell subsets from female C57/BL6 WT and Xist cKO mice. We stimulated some follicular B cells with the TLR9 agonist CpG in vitro for 24 hours. We performed RNA sequencing naive and CpG-stimulated splenic follicular B cells with at least n=3 biological replicates per experimental group.