![]() The quantification of the H3K27ac signals within the colored vertical columns is in Extended Data Fig. Genomic coordinates are on top, and the positions of C-DOM and T-DOM are shown in red and green, respectively, at the bottom. b, Timecourse of H3K27ac ChIP–seq over the entire HoxD locus. We challenged this model by using mutant stembryos and further showed that, while the first phase is sufficient to introduce a 5′-to-3′ asymmetry in transcription, CTCF sites organize and secure the sequence and the pace of this timer.Ī, Various stembryonic stages were used for the timecourse with the pulse of Wnt agonist in red. This is rapidly followed by the stepwise transcriptional activation of genes in the CTCF-rich region after a 3′-to-5′ progression in loop-extrusion, along with progressive changes in the chromatin architecture of the locus. We show that the Hox timer starts with a Wnt-dependent transcription of the CTCF-free part of the cluster, which triggers an increased asymmetric loading of cohesin complexes over this domain. After activating Wnt signaling for 24 h, such ‘stembryos’ 33 start to elongate a protrusion that resembles the outgrowth of the tail bud 34. Here we revisit this question by using gastruloids 32 derived from aggregated mouse embryonic stem (mES) cells cultivated in vitro for several days. This organization of CTCF sites is highly conserved either between species 29 or between paralogous gene clusters 28, that is, over several hundred million years of evolution, raising the hypothesis that they may act as checkpoints in the temporal activation of interspersed Hox genes, due to their involvement in the making and stabilization of large loops along with the cohesin complex 30, 31. Coincidentally, the mapping of CTCF-binding sites (CBSs) within Hox clusters revealed the following three sub-domains: an ‘anterior’ domain devoid of CTCF sites, a centrally located domain where a series of CTCF sites are orientated toward the 3′ end of the clusters and a posterior domain where several CTCF sites display the opposite orientation 28. In subsequent phases, Cdx transcription factors were reported to activate more centrally located Hox genes 23, 24, 25, while Gdf11 signaling might regulate more 5′-located (posterior) genes 26, 27. Although the function of this timer has been discussed before 14, 15, 16, its mechanism has remained poorly characterized due to the difficulties of analyzing the few neuro-mesodermal progenitor cells that feed the elongating axis with new mesoderm and neurectoderm tissue 17, 18 and where most Hox genes are activated during axial extension.Ī model was proposed whereby a progressive and directional opening of a closed chromatin configuration would parallel a stepwise accessibility of neighboring genes to activating factors 19, 20, with the onset of activation depending on Wnt signaling 21, a signaling pathway active at the most posterior part of the developing embryo 22. In vertebrates, this mechanism is linked to a time sequence in transcriptional activation, initially observed in mammals 10, 11 and subsequently generalized 12, 13. The spatial activation of any Hox gene is largely fixed by its relative position within its genomic cluster 4, an unusual property described in flies 5, 6 and in most animals with an AP axis 7, 8, 9. Consequently, cells at various anterior–posterior (AP) body levels express distinct combinations of HOX proteins, which may genetically instruct cellular populations as to which morphologies they should produce 2, 3. By the end of gastrulation, the embryo displays the classical distribution of Hox mRNAs, with progressively overlapping domains. In mammals, Hox genes are transcribed during gastrulation, when the embryo produces and organizes its major body axis 1. Mutant stembryos support this model and reveal that the presence of evolutionary conserved and regularly spaced intergenic CTCF sites controls the precision and the pace of this temporal mechanism. Chromatin extrusion then occurs with 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 topologically associating domain. Following Wnt signaling, the process involves transcriptional initiation at the anterior part of the cluster and a concomitant loading of cohesin complexes enriched on the transcribed DNA segments, that is, with an asymmetric distribution favoring the anterior part of the cluster. To understand the mechanism underlying this Hox timer, we used mouse embryonic stem cell-derived stembryos. ![]() During development, Hox genes are temporally activated according to their relative positions on their clusters, contributing to the proper identities of structures along the rostrocaudal axis. ![]()
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