Transcription factor ZNF263 primes human embryonic stem cells for pluripotency dissolution and early differentiation [RNA-seq]

Conventional human embryonic stem cells (hESCs) are capable of self-renewal and simultaneously poised for differentiation, and thus have been considered to resemble a primed pluripotent state. But the underlying regulatory mechanisms remain largely underexplored. Especially, little was known about the pivotal transcription factors (TFs) driving hESCs through the naïve-to-primed transition to prepare for differentiation. Here, we conduct a large-scale in-silico screening and identify ZNF263 as a new key TF in primed hESCs, as evidenced by its extensive binding capacity at the active and poised genes. Genetic and functional assays reveal that ZNF263 directly initiates the incipient expression of early differentiation genes and concurrently dampens the core pluripotency circuitry in hESCs, greatly tilting the balance from pluripotency maintenance to lineage priming. Importantly, ZNF263 deficiency in hESCs further led to clear defects in pluripotency dissolution and lineage commitment, suggesting it's indispensably required for the whole process. Together, our results demonstrate a pivotal role of ZNF263 in coordinating the establishment of primed pluripotency state in hESCs and facilitating the differentiation into primary germ layer lineages, which opens a new gate for understanding the central regulatory network governing human early development. Overall design: To investigate the role of ZNF263 in the regulation of hESC pluripotency, we established H1 and H9 KO hESCs in which ZNF263 has been deleted using cas9 technology. We then performed gene expression profiling using RNA-seq data of two cell lines. Comparative analysis was conducted between wildtype hESCs and its KO derivatives (1-5, 1-10, 9-1, 9-D).

传统人类胚胎干细胞（human embryonic stem cells, hESCs）兼具自我更新能力与分化潜能，因此被认为对应于始发态多能状态。然而其深层调控机制仍有待深入解析。尤为关键的是，此前对于驱动人类胚胎干细胞经由初始态-始发态转变以启动分化的核心转录因子（transcription factors, TFs）仍知之甚少。本研究通过大规模计算机（in silico）筛选，鉴定出ZNF263是始发态人类胚胎干细胞中的新型关键转录因子，其在活跃基因与预激活基因（poised genes）上广泛结合的特性可为佐证。遗传与功能实验显示，ZNF263可直接启动早期分化基因的初始表达，同时抑制人类胚胎干细胞的核心多能调控网络，大幅推动细胞平衡从多能性维持向谱系始发态偏移。尤为重要的是，人类胚胎干细胞中ZNF263缺失会进一步导致多能性消退与谱系定向分化的明显缺陷，表明其在整个过程中不可或缺。综上，本研究结果证实ZNF263在协调人类胚胎干细胞始发态多能状态建立、推动其向初级胚层谱系分化中发挥关键作用，为解析调控人类早期发育的核心调控网络提供了全新视角。实验设计概要：为探究ZNF263在人类胚胎干细胞多能性调控中的作用，本研究利用CRISPR-Cas9技术构建了ZNF263敲除的H1及H9型人类胚胎干细胞系。随后通过RNA测序（RNA-seq）对两种细胞系进行基因表达谱分析，并对野生型人类胚胎干细胞与其ZNF263敲除株（1-5、1-10、9-1、9-D）开展比较分析。