Females are predisposed for developing systemic lupus erythematosus (SLE), but the underlying mechanisms remain obscure. Females have two X-chromosomes and equilibration of X-linked gene dosage to that of males (XY) occurs by X-Chromosome Inactivation (XCI), initiated and maintained by Xist RNA. The X-chromosome is enriched for immunity-related genes and X-linked genes are typically overexpressed in female-biased SLE, suggesting that impaired XCI maintenance contributes to SLE. Unlike other somatic cells, we found that naïve B cells lack enrichment of Xist RNA and heterochromatic marks, and that these modifications return to the inactive X (Xi) upon in vitro stimulation, which we term `dynamic XCI maintenance'. Our published work demonstrates that SLE patient naïve B cells exhibit abnormal expression of X-linked genes, suggesting a dysfunctional Xist- independent mechanism involving DNA methylation for transcriptional silencing prior to autoantigen stimulation could predispose to lupus. DNA methylation, associated with transcriptional repression, is reduced in SLE patient B cells and is an important epigenetic modification for gene silencing on the Xi. XCI is also regulated by various nuclear matrix proteins, such as hnRNPU and SAFB, which bind to Xist RNA and function to anchor Xist to the Xi or the nuclear lamina in somatic cells. Our preliminary data support the novel concept that Xist RNA transcripts are retained at the nuclear lamina by SAFB in naïve B cells, and that Xist RNA binds to hnRNPU upon B cell activation, tethering Xist RNA to the Xi to regulate transcriptional repression. We have developed a novel Xist deletion mouse model to determine how impaired XCI maintenance contributes to lupus disease, and our exciting preliminary data show that increased dsDNA levels in type I interferon induced disease. Our central hypothesis is that Xist-independent DNA methylation maintains transcriptional repression of the Xi and autosomes in naïve B cells and that upon activation, alterations in nuclear matrix protein binding promotes dynamic XCI maintenance, the dysfunction of which exacerbates Type I IFN-driven lupus disease. We will test our hypotheses with the following aims: (1) Are reductions in Xist-independent DNA hypermethylation in naïve B cells associated with aberrant gene expression in lupus? (2) How do interactions between SAFB, hnRNPU protein and Xist RNA maintain XCI in B cells and does lupus disease impair cohesin eviction by hnRNPU-Xist RNA complexes? (3) How does impaired XCI maintenance impact Type I IFN-driven lupus-like disease in female mice? IMPACT: Our novel and innovative genetic and molecular approaches will yield unprecedented mechanistic insight into how biological sex contributes to immune dysregulation of SLE disease, and will enable the identification of new molecular pathways and targets of female-biased autoimmune disease that could be amenable for therapeutic intervention.