NFkB Signaling is Required for Xist RNA Localization at the Inactive X and for X-Chromosome Inactivation Maintenance Following T cell Activation [RNAseq_geneexpression]

X Chromosome Inactivation (XCI) is a female-specific process which balances X-linked gene dosage between sexes. Unstimulated T cells lack cytological enrichment of Xist RNA and heterochromatic modifications on the inactive X chromosome (Xi), and these modifications become enriched at the Xi after cell stimulation. Here, we examined allele-specific gene expression and epigenomic profiling of the Xi following T cell stimulation. We found that the Xi in unstimulated T cells is dosage compensated, and is enriched with the repressive H3K27me3 modification, but not H2AK119-ubiquitin (Ub) mark, including at promoters of XCI escape genes. Upon CD3/CD28 mediated T cell stimulation, the Xi accumulates H2AK119-Ub and H3K27me3 across the Xi. Next, we examined the T cell stimulation pathways responsible for Xist RNA localization to the Xi and found that T cell receptor (TCR) engagement, specifically NFkB signaling downstream of TCR, is required. Disruption of NFkB signaling, using inhibitors or genetic deletions, in mice and patients with immunodeficiencies prevents Xist/XIST RNA accumulation at the Xi and alters expression of some X-linked genes. Our findings reveal a novel connection between NFkB signaling pathways which impact XCI maintenance in female T cells. Overall design: RNAseq from F1 mus x cast CD3+ T cells that were unstimulated or stimulated with anti-CD3/anti-CD28 for 48 hours or stimulated with anti-CD3/anti-CD28 for 48 hours in the presence of 2.5uM IMD-0354. (n=3 per condition). F1 mus x cast is a hybrid mouse model of skewed XCI generated through mating female C57BL/6 mice harboring a heterozygous Xist deletion to male M. m. castaneous mice.

X染色体失活（X Chromosome Inactivation, XCI）是一种雌性特异性生物学过程，用于平衡两性间X连锁基因的剂量。未受刺激的T细胞中，失活X染色体（inactive X chromosome, Xi）不存在Xist RNA与异染色质修饰的细胞学富集，而此类修饰会在细胞受刺激后于Xi处显著富集。本研究针对T细胞活化后的Xi开展了等位基因特异性基因表达与表观基因组谱分析。研究发现，未受刺激的T细胞内Xi存在剂量补偿效应，且富集有抑制性组蛋白修饰H3K27me3，但不含H2AK119泛素化（ubiquitin, Ub）标记，该现象同样存在于X染色体失活逃逸基因的启动子区域。经CD3/CD28介导的T细胞刺激后，Xi全染色体范围内会同时积累H2AK119-Ub与H3K27me3。随后，本研究探究了介导Xist RNA定位于Xi的T细胞刺激通路，证实T细胞受体（T cell receptor, TCR）交联，尤其是TCR下游的NF-κB信号通路，是该过程必需的。在小鼠与免疫缺陷患者中，通过抑制剂或基因敲除手段阻断NF-κB信号通路，会阻止Xist/XIST RNA在Xi处的积累，并改变部分X连锁基因的表达模式。本研究揭示了影响雌性T细胞中XCI维持的NF-κB信号通路与该过程间的全新关联。总体实验设计：采集未受刺激、或经抗CD3/抗CD28刺激48小时、或在2.5μM IMD-0354存在下经抗CD3/抗CD28刺激48小时的F1 mus x cast CD3+ T细胞进行RNA测序（RNA-seq），每组设置3个生物学重复（n=3）。F1 mus x cast是通过将携带杂合Xist缺失的雌性C57BL/6小鼠与雄性小家鼠卡斯尔亚种（M. m. castaneus）交配获得的偏向性X染色体失活杂交小鼠模型。