Sequential and directional insulation by conserved CTCF sites underlies the Hox timer in stembryos [ChIP-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: ChIP and ChIPmentation data of wt and mutant stembryos