A big thank to @guandrey.bsky.social for giving me the opportunity to lead this wonderful project, all the co-authors Antonella Rauseo, Fabrice Darbellay, Guillaume Sapin, @olimpiabompadre.bsky.social, Lucille Lopez-Delisle, all the UNIGE facilities, and @denisduboule.bsky.social lab for discussions

Overall, our findings, potentially applicable to a dozen developmental loci, describe the presence of temporally restricted enhancer repertoires with distinct initiating or maintaining regulatory functions, and a synchronized shutdown during locus decommissioning.
13/14

Finally, we engineered a Hoxd13 overexpressing model that allowed us to observe that HOXD13 binds Shox2 enhancers in both its ectopic zeugopod and normal distal domains, suggesting it could repress the locus via an enhancer decommissioning mechanism.
12/14

To define how Shox2 transcription is terminated, we examined the changes in locus topology and observed that Shox2 locus decommissioning in the limb associates with rapid 3D chromatin changes featuring enhancer-promoter disconnection.
11/14

A partial deletion of late enhancers showed no impact on the proportion of inactive cells but only led to an increase in decommissioned cells at late stage only, indicating that previously ongoing transcription was halted, entering a premature decommissioning phase.
10/14

Partial removal of early enhancers leads to a transient inability to initiate Shox2 transcription in early limb progenitor cells. Proximal lineages gradually restore expression by the remaining enhancers, while cells destined for the distal posterior limb do not regain Shox2.
9/14

To further functionally assess the role of Shox2 enhancer repertoire, we investigated how partial early and late enhancer deletions induced stage-specific alterations.
8/14

Moreover, the 3D structure of the Shox2 locus associates with active enhancer-promoter interactions and accompanies the shift between early and late enhancer repertoires usage.
7/14

We then asked how Shox2 supports its transcription through these transitions and adapts to this changing environment. We observed that Shox2-expressing cells across development seemed to employ distinct enhancer repertoires.
6/14

This shows that part of the distal limb, digit 4 and 5, originates from progenitor cells that had expressed the Shox2 proximal marker at earlier stages.
5/14

In a nutshell, this tracking system allowed us to observe how Shox2 expression is initiated in limb progenitors and later maintained in proximal cell-types, while it is decommissioning in distal limb progenitors.
4/14

We developed a transgenic recorder system to characterize Shox2 regulatory trajectories. We tracked cells from a poised state that may transition towards a regulatory repression or an initiation phase, eventually followed by a maintenance phase and finally a decommissioning phase.
3/14

We identified that within 90 limb-associated loci, including the proximal limb gene Shox2, most of them presented putative enhancers with restricted time window of activities: early- (E10.5) late-active (E13.5) enhancers.
2/14