Xist RNA Dependent and Independent Mechanisms Regulate Dynamic X Chromosome Inactivation in B Lymphocytes

X-Chromosome Inactivation (XCI) involves epigenetic pathways to equalize X-linked gene expression between female and male mammals. XCI is dynamic in female B cells, as cytological enrichment of Xist RNA and heterochromatic marks on the inactive X-chromosome (Xi) are absent in naïve B cells yet return following mitogenic stimulation. Here, we asked whether any heterochromatic histone marks are present on the Xi in naïve B cells, and whether Xist RNA is required for their deposition and retention following stimulation. We find that the Xi in naïve B cells is depleted for H2AK119Ub and H3K9me3 but enriched for DNA methylation and H3K27me3, which maintain an Xist RNA-dependent epigenetic memory of XCI. Upon stimulation, Xist-independent H3K27me3 and Xist-dependent H2AK119Ub modifications accumulate across the Xi with temporal and spatial specificity. Our findings reveal the importance of Xist RNA, H3K27me3, and H2AK119Ub marks for the epigenetic integrity of X-linked genes across the Xi following female B cell stimulation.