Xist exerts gene-specific silencing during XCI maintenance and impacts lineage-specific cell differentiation and proliferation during hematopoiesis [RNA-seq]

X chromosome inactivation (XCI) is a dosage compensation phenomenon that occurs in females. Initiation of XCI depends on Xist RNA, which triggers silencing of one of the two X chromosomes, except for XCI escape genes that continue to be biallelically expressed. In the soma XCI is stably maintained with continuous Xist expression. How Xist impacts XCI maintenance remains an open question. Here we conditionally deleted Xist in hematopoietic system of mice and report differentiation and cell cycle defects in female hematopoietic stem and progenitor cells (HPSCs). By utilizing female HSPCs and mouse embryonic fibroblasts, we find that X-linked genes show variable tolerance to Xist loss. Specifically, XCI escape genes exhibit preferential transcriptional upregulation, which associates with low H3K27me3 occupancy, and high chromatin accessibility that accommodates preexisting binding of transcription factors such as Yin Yang 1 (YY1) at the basal state. We conclude that Xist is necessary for gene-specific silencing during XCI maintenance and impacts lineage-specific cell differentiation and proliferation during hematopoiesis. Overall design: Bone marrow Lin- (lineage negative), LSK+ (Lin- SCA-1+ c-KIT+) and LSK- (Lin- SCA-1- c-KIT+) cells in wild type (WT) and Xist knockout (Xist?/?) female mice were sorted by FACS and utiliized for gene expression profiling by RNA-Seq.

X染色体失活（X chromosome inactivation, XCI）是雌性个体中存在的剂量补偿现象。XCI的起始依赖于Xist RNA，该RNA可触发两条X染色体中的一条发生沉默，但XCI逃逸基因除外——此类基因仍可维持双等位基因表达。在体细胞中，XCI可通过持续的Xist表达得以稳定维持。目前，Xist如何影响XCI维持仍是一个悬而未决的科学问题。

本研究在小鼠造血系统中条件性敲除Xist，并报道了雌性造血干细胞与祖细胞（hematopoietic stem and progenitor cells, HPSCs）出现的分化与细胞周期缺陷。通过利用雌性HPSCs与小鼠胚胎成纤维细胞，我们发现X连锁基因对Xist缺失的耐受程度存在显著差异。具体而言，XCI逃逸基因呈现出优先的转录上调，这一现象与低水平的H3K27me3占据、以及高水平的染色质开放性相关——这种开放性可容纳转录因子如阴阳因子1（Yin Yang 1, YY1）在基础状态下预先结合于染色质。

我们得出结论：Xist在XCI维持过程中对于基因特异性沉默是必需的，并可影响造血过程中的谱系特异性细胞分化与增殖。

实验设计：本研究通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS），分选野生型（wild type, WT）与Xist敲除（Xist knockout, Xist?/?）雌性小鼠的骨髓Lin-（lineage negative, 谱系阴性）、LSK+（Lin- SCA-1+ c-KIT+）与LSK-（Lin- SCA-1- c-KIT+）细胞，并通过RNA测序（RNA-Seq）进行基因表达谱分析。