Chromatin topology and the timing of enhancer function at the HoxD locus

Abstract

The HoxD gene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different subgroups of Hoxd genes respond first to a proximal regulatory signal, then to a distal signal that organizes digits. These two regulations are exclusive from one another and emanate from two distinct topologically associating domains (TADs) flanking HoxD, both containing a range of appropriate enhancer sequences. The telomeric TAD (T-DOM) contains several enhancers active in presumptive forearm cells and is divided into two sub-TADs separated by a CTCF-rich boundary, which defines two regulatory submodules. To understand the importance of this particular regulatory topology to control Hoxd gene transcription in time and space, we either deleted or inverted this sub-TAD boundary, eliminated the CTCF binding sites, or inverted the entire T-DOM to exchange the respective positions of the two sub-TADs. The effects of such perturbations on the transcriptional regulation of Hoxd genes illustrate the requirement of this regulatory topology for the precise timing of gene activation. However, the spatial distribution of transcripts was eventually resumed, showing that the presence of enhancer sequences, rather than either their exact topology or a particular chromatin architecture, is the key factor. We also show that the affinity of enhancers to find their natural target genes can overcome the presence of both a strong TAD border and an unfavorable orientation of CTCF sites.

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

“…S3, blue arrowhead). Also, as previously reported (Rodriguez-Carballo et al, 2017;Rodríguez-Carballo et al, 2020;Yakushiji-Kaminatsui et al, 2018), a cluster of CTCF binding sites delimits the boundary between the mouse and the chicken sub-TAD1 and sub-TAD2, thus making the transition between the D4 and D9 regions in both species (Fig. 2; Fig.…”

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

“…Such losses could be explained when looking at variations in chromatin topology. For example, mutations in all five CTCF sites considerably weakened contacts between the cluster and T-DOMb, where most proximal enhancers are located (Andrey et al, 2013;Rodríguez-Carballo et al, 2020). These contacts were redirected towards T-DOMa, which is poor in proximal enhancers, but rich in other regulations, leading to the ectopic expressions described above, concomitantly to a loss of expression in proximal limbs.…”

“…During limb development, various subsets of Hoxd genes respond first to T-DOM-located proximal enhancers, then to C-DOM-located digit enhancers (Andrey et al, 2013). Because the two subgroups of target genes are delimited by CTCF sites, it was suggested that such CBSs may specify, through a 3D structure, particular enhancer-promoter interactions leading to these bimodal transcription patterns (Rodríguez-Carballo et al, 2020). However, the most visible effect observed across the different mutant lines generated in this study was more quantitative than qualitative and no obvious mis-expression of target gene subgroups was scored.…”

Mesomelic dysplasias associated with the HOXD locus are caused by regulatory reallocations

“…For instance, the duplication of the HOXD cluster reported in ref. 1 includes the ELCR2 proximal limb enhancer 63 . In the mutant chromosome, a copy of HOXD13 is in close proximity with this enhancer, regardless of the orientation of the duplicated DNA sequence and will thus receive proximal regulatory inputs (Supplementary Fig.…”