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 the concomitant loading of cohesin complexes on the transcribed DNA segments, i.e., with an asymmetric distribution along the gene cluster. 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 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.

In the course of evolution, pecorans (i.e. higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as ‘headgear’, which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.

The expression of some genes depends on large, adjacent regions of the genome that contain multiple enhancers. These regulatory landscapes frequently align with Topologically Associating Domains (TADs), where they integrate the function of multiple similar enhancers to produce a global, TAD-specific regulation. We asked if an individual enhancer could overcome the influence of one of these landscapes, to drive gene transcription. To test this, we transferred an enhancer from its native location, into a nearby TAD with a related yet different functional specificity. We used the biphasic regulation of Hoxd genes during limb development as a paradigm. These genes are first activated in proximal limb cells by enhancers located in one TAD, which is then silenced when the neighboring TAD activates its enhancers in distal limb cells. We transferred a distal limb enhancer into the proximal limb TAD and found that its new context suppresses its normal distal specificity, even though it is bound by HOX13 transcription factors, which are responsible for the distal activity. This activity can be rescued only when a large portion of the surrounding environment is removed. These results indicate that, at least in some cases, the functioning of enhancer elements is subordinated to the host chromatin context, which can exert a dominant control over its activity.

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. To understand the mechanism underlying this Hox timer, we used mouse embryonic stem cell-derived stembryos. 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. 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. 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.

Modifications in gene regulation during development are considered to be a driving force in the evolution of organisms. Part of these changes involve rapidly evolving cis-regulatory elements (CREs), which interact with their target genes through higher-order 3D chromatin structures. How such 3D architectures and variations in CREs contribute to transcriptional evolvability nevertheless remains elusive. During vertebrate evolution, Hox genes were redeployed in different organs in a class-specific manner, while maintaining the same basic function in organizing the primary body axis. Since a large part of the relevant enhancers are located in a conserved regulatory landscape, this gene cluster represents an interesting paradigm to study the emergence of regulatory innovations. In this work, we analyzed Hoxd gene regulation in both murine vibrissae and chicken feather primordia, two mammalian- and avian-specific skin appendages which express different subsets of Hoxd genes, and compared their regulatory modalities with the regulations at work during the elongation of the posterior trunk, a mechanism highly conserved in amniotes. We show that in the former two structures, distinct subsets of Hoxd genes are contacted by different lineage-specific enhancers, likely as a result of using an ancestral chromatin topology as an evolutionary playground, whereas the regulations implemented in the mouse and chicken embryonic trunk rely more on conserved CREs. Nevertheless, a high proportion of these non-coding sequences active in the trunk appear to have functionally diverged between the two species, suggesting that transcriptional robustness is maintained despite a considerable divergence in CRE sequences, an observation supported by a genome-wide comparative approach.

Modifications in gene regulation are driving forces in the evolution of organisms. Part of these changes involve cis-regulatory elements (CREs), which contact their target genes through higher-order chromatin structures. However, how such architectures and variations in CREs contribute to transcriptional evolvability remains elusive. We use Hoxd genes as a paradigm for the emergence of regulatory innovations, as many relevant enhancers are located in a regulatory landscape highly conserved in amniotes. Here, we analysed their regulation in murine vibrissae and chicken feather primordia, two skin appendages expressing different Hoxd gene subsets, and compared the regulation of these genes in these appendages with that in the elongation of the posterior trunk. In the two former structures, distinct subsets of Hoxd genes are contacted by different lineage-specific enhancers, probably as a result of using an ancestral chromatin topology as an evolutionary playground, whereas the gene regulation that occurs in the mouse and chicken embryonic trunk partially relies on conserved CREs. A high proportion of these non-coding sequences active in the trunk have functionally diverged between species, suggesting that transcriptional robustness is maintained, despite considerable divergence in enhancer sequences.

The expression of genes with a key function during development is frequently controlled by large regulatory landscapes containing multiple enhancer elements. These landscapes often match Topologically Associating Domains (TADs) and sometimes integrate range of similar enhancers, thus leading to TADs having a global regulatory specificity. To assess the relative functional importance of enhancer sequences versus the regulatory domain they are included in, we set out to transfer one particular enhancer sequence from its native domain into a TAD with a closely related, yet different functional specificity. We used Hoxd genes and their biphasic regulation during limb development as a paradigm, since they are first activated in proximal limb cells by enhancers located in one TAD, which is then silenced at the time when the neighboring TAD starts to activate its enhancers in distal limb cells. We introduced a strong distal limb enhancer into the proximal limb TAD and found that its new context strongly suppresses its distal specificity, even though it continues to be bound by HOX13 transcription factors, which normally are responsible for this activity. Using local genetic alterations and chromatin conformation measurements, we see that the enhancer is capable of interacting with target genes, with a pattern comparable to its adoptive neighborhood of enhancers. Its activity in distal limb cells can be rescued only when a large portion of the surrounding environment is removed. These results indicate that, at least in some cases, the functioning of enhancer elements is subordinated to the local chromatin context, which can exert a dominant control over its activity.

In the course of evolution, pecorans (i.e. higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as ‘headgear’, which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats, are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.