XIST dampens X chromosome activity in a SPEN-dependent manner during early human development [GRO-seq]

XIST long non-coding RNA is responsible for X chromosome inactivation (XCI) in placental mammals, yet it accumulates on both X chromosomes in human female pre-implantation embryos without triggering X chromosome silencing. The long non-coding RNA XACT co-accumulates with XIST on active Xs and may antagonize XIST function. Here we used human ES cells in a naïve state of pluripotency to assess the function of XIST and XACT in shaping the X chromosome chromatin and transcriptional landscapes during pre-implantation development. We show that XIST triggers the deposition of polycomb-mediated repressive histone modifications and attenuates transcription of most X-linked genes in a SPEN-dependent manner, while XACT deficiency does not significantly affect XIST activity or X-linked gene expression. Our study demonstrates that XIST is functional prior to XCI, confirms the existence of a transient process of X chromosome dosage compensation, and reveals that X chromosome inactivation and dampening rely on the same set of factors. Overall design: Low input fastGRO-seq on 3 independent clones of XIST CRISPRi naïve hESCs treated with doxycyclin. Untreated clones were used as controls.

XIST长链非编码RNA（XIST long non-coding RNA）在胎盘类哺乳动物中负责X染色体失活（X chromosome inactivation, XCI），但在人类女性植入前胚胎中，该RNA会在两条X染色体上富集，却不会触发X染色体沉默。长链非编码RNA XACT会与XIST共同富集于活跃X染色体上，可能拮抗XIST的功能。本研究利用处于原始态多能状态的人类胚胎干细胞，探究XIST与XACT在植入前发育过程中塑造X染色体染色质及转录组景观的功能。研究结果显示，XIST可通过SPEN依赖的方式，介导多梳蛋白复合物（polycomb）催化的抑制性组蛋白修饰沉积，并减弱大多数X连锁基因的转录；而XACT缺失并不会显著影响XIST的活性或X连锁基因的表达。本研究证实，XIST在XCI发生前即具备调控功能，验证了X染色体剂量补偿的瞬时过程的存在，并揭示X染色体失活与转录抑制依赖同一组调控因子。整体实验设计：对3株独立的经多西环素（doxycyclin）处理的XIST CRISPR干扰（CRISPR interference, CRISPRi）原始态人类胚胎干细胞系进行低起始量fastGRO-seq测序，以未处理的细胞系作为对照。