Long-range enhancer-promoter interactions in the HoxD complex

Collinear expression of 5′ Hoxd genes during mammalian limb development is required to properly determine digits number and identity. We recently proposed a two-step model to account for the underlying regulatory mechanism, which involves an initial looping and recognition of the cluster by a complex of enhancer sequences, followed by a micro-scanning of nearby-located genes. This model could account for both the wild-type dosage of the various Hoxd transcripts, as well as for their quantitative variations in several mutant strains. Here, using the Chromosome Conformation Capture (3C) technique, we confirm the initial looping step by showing that the 5′ extremity of the HoxD cluster physically interacts with two previously identified, remote regulatory sequences displaying enhancer activity in developing digits. This spatial conformation is specific to presumptive digits and is not observed in tissues devoid of Hox gene transcription. The flexibility of these long-range interactions is illustrated by the ability of the enhancers to activate reporter transgenes inserted at various positions along the locus. To further define this regulatory landscape, we produced a large chromosomal inversion, separating the reporter transgene from the digit enhancers and HoxD cluster. Surprisingly, this configuration fails to abolish the transgene expression in developing digits, but leads to a major down-regulation of Hoxd genes in this domain, and concurrent morphological defects. These results suggest that the transcriptional activation of Hoxd genes requires the activity of additional and distant regulatory sequences, in addition to the enhancers identified so far by transgenic analysis.