Temporal constraints on enhancer usage shape the regulation of limb gene transcription.

Enhancer repertoires orchestrate gene expression during embryonic development, shaping organ structure and function. Individual enhancers can act in overlapping or distinct spatial domains, but their temporal specificity and coordinated action over time remain poorly understood. Here, we identify temporally restricted enhancer repertoires at multiple loci involved in mouse limb development. To capture their dynamic roles, we introduce the regulatory trajectory framework comprising initiation, maintenance, and decommissioning of gene expression. Using a transgenic recorder at the Shox2 locus, we demonstrate that early enhancers initiate transcription, while late enhancers maintain it. Additionally, we found that changes in 3D topology associate with enhancer activities and that loss of enhancer-promoter contacts occurs during decommissioning. Finally, we show that Shox2 regulatory decommissioning can be driven by Hoxd13, a known antagonist of Shox2 expression. Overall, our work uncovers how temporally restricted enhancers generate complex expression patterns and sheds light on the dynamics of enhancer-promoter interactions.