Developmental and Evolutionary Comparative Analysis of a Regulatory Landscape in Mammals and Birds

Abstract

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.

Sequential in-cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster

“…1A) (Andrey et al, 2013). In another context, Hoxd1 to Hoxd4 and Hoxd1 to Hoxd9 are expressed in the emerging vibrissae follicles (VFs) and in facial muscle progenitors (FMPs), respectively, driven by enhancers located within the T-DOM (Hintermann et al, 2021)(Fig. 1A-C).…”

“…5A). Likewise, a set of enhancers located across the same T-DOMa drives the expression of Hoxd1 to Hoxd9 into facial migrating muscle progenitors (FMPs) (Hintermann et al, 2021)(Fig. 5A).…”

“…Figure 5: Alterations in Hoxd genes expression in facial structures. (A) Schematic representation of Hoxd gene regulation in both the vibrissae follicles (VFs, light purple) and some facial muscle precursors (FMPs, dark purple) as well as the DNA segments within T-DOM where the corresponding enhancers are located(Hintermann et al, 2021). A magnification of the gene cluster is shown below with the subsets of Hoxd genes expressed in each structure.…”

Sequential in cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster

“…In another context, Hoxd1 to Hoxd4 and Hoxd1 to Hoxd9 are expressed in the emerging vibrissae follicles (VFs) and in facial muscle progenitors (FMPs), respectively, driven by enhancers located within the T-DOM (Fig. 1A-C; Hintermann et al 2021)). Alternatively, a single gene can display one particular functionality such as Hoxd9, which is the only gene expressed in the mesenchymal condensates of the future embryonic mammary glands (Chen and Capecchi 1999), a specificity controlled by an enhancer also located within T-DOM, at the boundary between two sub-TADs: T-DOMa and T-DOMb (Fig.…”

“…Likewise, a set of enhancers located across the same T-DOMa drives the expression of Hoxd1 to Hoxd9 into FMPs (Fig. 5A; Hintermann et al 2021). We asked whether the disruption of intracluster CBSs would change the allocations of target genes for these two regulatory specificities, which concern either genes located within the part of the gene cluster that is devoid of CTCF sites or genes extending slightly behind CBS1 and CBS2.…”