Role for nuclear matrix proteins and DNA methylation for XCI maintenance in female lymphocytes

女性易患系统性红斑狼疮（SLE），但其潜在机制 保持模糊。女性有两个X染色体和平衡的X连锁基因剂量的男性 (XY)通过X染色体失活（XCI）发生，由Xist RNA启动和维持。X染色体是 富含免疫相关基因和X-连锁基因通常在女性偏向的SLE中过表达， 提示受损的XCI维持有助于SLE。与其他体细胞不同，我们发现幼稚的B 细胞缺乏Xist RNA和异染色质标记的富集，并且这些修饰返回到失活的 X（Xi），我们称之为“动态XCI维持”。我们发表的工作表明， SLE患者的幼稚B细胞表现出X连锁基因的异常表达，提示功能失调的Xist- 自身抗原刺激前转录沉默的DNA甲基化独立机制 可能会导致狼疮DNA甲基化与转录抑制相关，在SLE患者中减少 B细胞，是一个重要的表观遗传修饰基因沉默的Xi。XCI也受到各种监管 核基质蛋白，如hnRNPU和SAFB，它们与Xist RNA结合，并起锚Xist到细胞核的作用。 Xi或体细胞中的核纤层。我们的初步数据支持Xist RNA转录的新概念， 在幼稚B细胞中，Xist RNA被SAFB保留在核板层，而在B细胞中，Xist RNA与hnRNPU结合 激活，将Xist RNA拴系到Xi以调节转录抑制。我们开发了一种新的Xist 删除小鼠模型，以确定受损的XCI维持如何有助于狼疮疾病，我们令人兴奋的 初步数据显示，I型干扰素诱导的疾病中dsDNA水平增加。我们的核心假设 不依赖于Xist的DNA甲基化维持了幼稚细胞中Xi和常染色体的转录抑制， B细胞和激活后，核基质蛋白结合的改变促进动态XCI 维持，其功能障碍加剧I型IFN驱动的狼疮疾病。我们将测试我们的假设 目的如下：（1）幼稚B细胞中Xist非依赖性DNA高甲基化的减少是否与 与狼疮的异常基因表达有关吗(2)SAFB、hnRNPU蛋白和Xist RNA之间如何相互作用 维持B细胞中的XCI，狼疮疾病是否会损害hnRNPU-Xist RNA复合物对粘着蛋白的驱逐？（三） 受损的XCI维持如何影响雌性小鼠I型IFN驱动的狼疮样疾病？影响：我们的 新的和创新的遗传和分子方法将产生前所未有的机制洞察如何 生物学性别有助于SLE疾病的免疫失调，并将使新的 女性偏好性自身免疫性疾病的分子途径和靶点， 干预

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.