CRISPR activation screen to identify X-chromosomal Xist activators in male embryonic stem cells

As the master regulator of X-chromosome inactivation (XCI), the Xist RNA is expressed nearly ubiquitously in female mice. Xist is only absent in the germ line and at the pluripotent state. Xist is generally assumed to be expressed “by default” in females, while being actively repressed in the few tissues where it is silent. Whether activating mechanisms also contribute remained largely unknown. Through a pooled CRISPR screen we identify the GATA family of transcription factors as potent direct activators of Xist. We describe a GATA-responsive regulatory element (RE79), located ~100 kb upstream of the Xist promoter. In cell lines derived from the two extraembryonic lineages, XEN and TS cells, where imprinted Xist expression is maintained, RE79 is bound by different sets of GATA factors expressed in those tissues. Here we use a CRISPR activation screen targeting X-chromosomal genes on differentiating XO mouse embryonic stem cells to identify putative Xist activators. A CRISPRa-SunTag screen for Xist was performed in differentiating mESCs (E14-STN) carrying a deletion of the Tsix-promoter to sensitize the male cell line for Xist upregulation. The library contained 8773 gDNAs targeting 757 X-chromosomal genes and 23 autosomal controls, as well as 200 non-targeting guides. After 2 days of differentiation via LIF-withdrawal, the cells were stained for Xist RNA using a FlowFISH protocol and the top 15% bins of Xist-expression was sorted.

作为X染色体失活（X-chromosome inactivation, XCI）的核心调控因子，Xist RNA在雌性小鼠中几乎呈泛在表达模式。仅在生殖系与多能状态下，Xist的表达才会缺失。学界普遍认为，雌性个体中Xist的表达处于默认激活状态，而在其沉默的少数组织中则处于主动抑制状态。但激活机制是否也参与调控Xist表达，目前在很大程度上仍未明确。
本研究通过混合CRISPR筛选（pooled CRISPR screen）鉴定出GATA家族转录因子为Xist强效的直接激活因子。我们发现了一个受GATA调控的调控元件（regulatory element, RE79），其位于Xist启动子上游约100 kb处。在维持印记Xist表达的两种胚外谱系细胞系——XEN细胞与TS细胞中，该调控元件会被对应组织中表达的不同GATA因子结合。
本研究针对分化中的XO小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）的X染色体基因开展CRISPR激活筛选，以鉴定潜在的Xist激活因子。我们在携带Tsix启动子缺失的雄性细胞系E14-STN小鼠胚胎干细胞中开展了针对Xist的CRISPR-SunTag筛选，该缺失可使雄性细胞系更易发生Xist上调。该筛选文库包含靶向757个X染色体基因的8773条向导DNA（gDNAs）、23个常染色体对照向导序列，以及200条非靶向向导序列。在通过撤除白血病抑制因子（Leukemia Inhibitory Factor, LIF）诱导分化2天后，我们采用流式荧光原位杂交（FlowFISH）技术对Xist RNA进行染色，并分选出Xist表达量最高的15%细胞群。