A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus

During vertebrate limb development, Hoxd genes are regulated following a bimodal strategy involving two topologically associating domains (TADs) located on either side of the gene cluster. These regulatory landscapes alternatively control different subsets of Hoxd targets, first into the arm and subsequently into the digits. We studied the transition between these two global regulations, a switch that correlates with the positioning of the wrist, which articulates these two main limb segments. We show that the HOX13 proteins themselves help switch off the telomeric TAD, likely through a global repressive mechanism. At the same time, they directly interact with distal enhancers to sustain the activity of the centromeric TAD, thus explaining both the sequential and exclusive operating processes of these two regulatory domains. We propose a model in which the activation of Hox13 gene expression in distal limb cells both interrupts the proximal Hox gene regulation and re-enforces the distal regulation. In the absence of HOX13 proteins, a proximal limb structure grows without any sign of wrist articulation, likely related to an ancestral fish-like condition.

Regulatory Switches Underlie Hoxd Gene Activation in Tetrapod Limbs

Guillaume Andrey

Accurate spatio-temporal control of Hox cluster genes expression is critical for the proper organization of body structures during vertebrate embryogenesis. In the limbs, Hoxd genes are critical in setting up the proximodistal and anteroposterior axes. Several studies have shown that long-range global regulatory elements are required to establish Hoxd genes expression territories in this tissue, via the co-regulation of several genes at once. Limb buds take their origin from the lateral plate mesoderm where the HoxD cluster is controlled by cues from the main body axis. During limb outgrowth Hoxd genes are then subjected to two subsequent activation waves: an early phase patterning proximal limb structures, arms and forearms and a late one instructing digits. As the late activation of Hoxd genes is known to rely upon a large regulatory archipelago located centromeric to the gene cluster, the control of the early phase remains poorly described (Montavon et al., 2011). In this work we show that the early activation domain requires a different, telomeric-located, regulatory landscape. This one megabase (Mb) large domain holds at least three regulatory regions contributing to Hoxd gene activation in early forelimb and hindlimb buds. To our surprise the abrogation of most of the early telomeric enhancers induced a dramatic decrease of central Hoxd transcription in forelimbs but only removed 50% of the transcripts in hindlimbs. This discrepancy most likely arises from the difference in the epigenetic status of the HoxD cluster in anterior and posterior limb fields where forelimb and hindlimb respectively take their origin. As the cluster does not possess the same fraction of active genes in these progenitors it most likely does not respond to early cues in the same way. In the second part of this work we noticed an unexpected decrease of the late expression phase in all mutants lacking early enhancers. This observation shows that digit progenitors have to implement the early phase in order to gain competence toward the late centromeric regulation. This transition between early and late regulations involves a set of central Hoxd genes, i.e. Hoxd11 to Hoxd9. To implement both regulations, these loci switch between two topological domains containing the centromeric and telomeric regulatory landscapes respectively (Dixon et al., 2012). The ability to switch from one domain to another depends on the relative position of genes towards the boundary between both domains and correlates with their respective expression. Moreover, the switch between both topological domains mechanically induces an intermediate, Hoxd-free zone, in-between the two expression phases that will later produce the mesopodium, the articulation between our arms and our hands.

EPFL2013

Conservation and Divergence of Regulatory Strategies at Hox Loci and the Origin of Tetrapod Digits

Denis Duboule, Marion Leleu, Daniël Noordermeer

The evolution of tetrapod limbs from fish fins enabled the conquest of land by vertebrates and thus represents a key step in evolution. Despite the use of comparative gene expression analyses, critical aspects of this transformation remain controversial, in particular the origin of digits. Hoxa and Hoxd genes are essential for the specification of the different limb segments and their functional abrogation leads to large truncations of the appendages. Here we show that the selective transcription of mouse Hoxa genes in proximal and distal limbs is related to a bimodal higher order chromatin structure, similar to that reported for Hoxd genes, thus revealing a generic regulatory strategy implemented by both gene clusters during limb development. We found the same bimodal chromatin architecture in fish embryos, indicating that the regulatory mechanism used to pattern tetrapod limbs may predate the divergence between fish and tetrapods. However, when assessed in mice, both fish regulatory landscapes triggered transcription in proximal rather than distal limb territories, supporting an evolutionary scenario whereby digits arose as tetrapod novelties through genetic retrofitting of preexisting regulatory landscapes. We discuss the possibility to consider regulatory circuitries, rather than expression patterns, as essential parameters to define evolutionary synapomorphies.

Public Library Science2014

Context-dependent enhancer function revealed by targeted inter-TAD relocation

Guillaume Andrey, Christopher Chase Bolt, Lucille Delisle, Denis Duboule, Aurélie Hintermann

Here the authors show that a strong enhancer sequence can be controlled by the chromatin environment provided by a topologically associated domain (TAD) located nearby. An enhancer relocated by homologous recombination takes all the hallmarks of its new neighboring enhancers located in the recipient TAD. 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.

NATURE PORTFOLIO2022

Regulatory Switches Underlie Hoxd Gene Activation in Tetrapod Limbs

Guillaume Andrey

EPFL2013