Sequential and directional insulation by conserved CTCF sites underlies the Hox timer in stembryos [scRNA-seq]. Sequential and directional insulation by conserved CTCF sites underlies the Hox timer in stembryos [scRNA-seq]

During development, Hox genes are activated in a time sequence following their relative positions on their clusters, leading to the proper identities of structures along the rostral to caudal axis. To understand the mechanism operating this Hox timer, we used ES-cells derived stembryos and show that the core of the process involves the start of transcription at the anterior part of the cluster, following Wnt signaling, and a concomitant loading of cohesin complexes enriched on the transcribed DNA segments, i.e., with an asymmetric distribution in favor of the anterior part the gene cluster. Chromatin extrusion then occurs with, along with time, successively more posterior CTCF sites acting as transient insulators, thus generating a progressive time-delay in the activation of more posterior-located genes due to long-range contacts with a flanking TAD. Mutant stembryos support this model and reveal that the iterated presence of evolutionary conserved and regularly spaced intergenic CTCF sites control the precision and the pace of this temporal mechanism. Overall design: Single Cell RNA-seq data of stembryos

在发育过程中，同源框（Hox）基因会按照其在基因簇上的相对位置以时间顺序被激活，进而赋予颅尾轴上各结构以正确的身份特征。为解析调控这一Hox时序程序的分子机制，本研究采用胚胎干细胞（ES）来源的类胚胎模型，研究发现该过程的核心机制为：在Wnt信号通路（Wnt signaling）的调控下，基因簇的前端区域起始转录，同时伴随黏连蛋白复合物在转录的DNA区段上富集，即该复合物在基因簇上呈现偏向前端的不对称分布。随后随着时间推移，染色质环挤出过程启动，依次由更靠后的CCCTC结合因子（CTCF）位点充当瞬时绝缘子，进而通过与侧翼拓扑关联结构域（TAD）的远程相互作用，使位置更靠后的基因的激活产生渐进式的时间延迟。突变体类胚胎实验验证了该模型，并揭示：进化上保守且间隔规律的基因间CTCF位点的重复存在，可调控这一时间程序的精确性与运行速率。整体实验设计：类胚胎的单细胞RNA测序数据。