Chromatin topology and the timing of enhancer function at thehoxdlocus

Abstract

The HoxD gene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different sub-groups 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 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 sub-modules. 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 enhancers 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 unfavourable orientation of CTCF sites.

A gene desert required for regulatory control of pleiotropicShox2expression and embryonic survival

“…The region shared between the +325kb and +326kb SV enhancer elements is delineated by red lines, shows reduced conservation and ATAC-seq signal, and harbors a significant Tbx5 motif (green line) (Fig. S6) (Ihh) gene, or the HoxD cluster genes, in multiple tissues and due to additively acting enhancers with partially overlapping activities 45,46 . While many developmental enhancers with overlapping counterparts are known to exert specific tasks, they also exhibit partially redundant functions serving as a regulatory buffer to ensure phenotypic robustness 24,47,48 .…”

“…In addition, our 4C-seq chromatin conformation capture from limb in combination with subsequent transgenic analysis starts to delineate the likely critical cluster of limb enhancers orchestrating Shox2 -mediated stylopod formation. This cluster is reminiscent of a multipartite enhancer ensemble, such as the one regulating the Indian Hedgehog ( Ihh ) gene, or the HoxD cluster genes, in multiple tissues and due to additively acting enhancers with partially overlapping activities 45,46 . While many developmental enhancers with overlapping counterparts are known to exert specific tasks, they also exhibit partially redundant functions serving as a regulatory buffer to ensure phenotypic robustness 24,47,48 .…”

MESOMELIC DYSPLASIAS ASSOCIATED WITH THEHOXDLOCUS ARE CAUSED BY REGULATORY REALLOCATIONS

“…For instance, the duplication of the HOXD cluster reported in Kantaputra et al (2010) includes the ELCR2 proximal limb enhancer (Rodríguez-Carballo et al, 2020). 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 Figure 5b, blue arrows), a fortiori since one of the duplicated copies of HOXD13 is now located far from its digits-specific enhancers (red arrows), a situation which was shown in mice to de-sequester Hoxd13 and re-allocate it towards proximal enhancers on the other side of the cluster (Tschopp and Duboule, 2011).…”

CONTEXT-INDEPENDENT FUNCTION OF A CHROMATIN BOUNDARYIN VIVO

“…(15)). In this context, it was proposed that chromosome topology may refine the action and timing of distant enhancers on their target genes during development (16)(17)(18), implying that the importance of such structures should be considered on a case-by-case basis, rather than drawing too global conclusions.…”

“…The T-DOM TAD is further divided into two sub-TADs at the level of a DNA segment called CS38-40, which contains a limb enhancer, a CpG island in close proximity to the transcription start site (TSS) of the Hog and Tog long non-coding RNAs (lncRNAs) and three occupied CTCF sites, all oriented towards the gene cluster (21,22,28). This region helps to properly implement the timing of limb enhancer action and constitutes a bona fide topological boundary, for its deletion abrogates the observed contact segregation, whereas its inversion reinforces it (18). We wondered whether this boundary region would by itself carry the capacity to create a topological boundary when positioned into a different genomic context in vivo, and hence we generated transgenic mouse models with random ectopic integrations of this region.…”