Xist RNA dependent and independent mechanisms regulate dynamic X chromosome inactivation in B lymphocytes

X chromosome inactivation (XCI) equalizes X-linked gene expression between the sexes through heterochromatic mark accumulation. 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 naive B cells yet return following stimulation. Here, we asked whether heterochromatic histone marks are present on the Xi in naive B cells and whether Xist RNA is required for their deposition and retention following stimulation. We find that the Xi in naive B cells is depleted for H2AK119Ub and H3K9me3 but enriched for DNA methylation and H3K27me3, which maintain an Xist RNA-dependent 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 Xi gene regulation following female B cell stimulation.