A complex regulatory landscape involved in the development of mammalian external genitals

Developmental genes are often controlled by large regulatory landscapes matching topologically associating domains (TADs). In various contexts, the associated chromatin backbone is modified by specific enhancer–enhancer and enhancer–promoter interactions. We used a TAD flanking the mouse HoxD cluster to study how these regulatory architectures are formed and deconstructed once their function achieved. We describe this TAD as a functional unit, with several regulatory sequences acting together to elicit a transcriptional response. With one exception, deletion of these sequences didn’t modify the transcriptional outcome, a result at odds with a conventional view of enhancer function. The deletion and inversion of a CTCF site located near these regulatory sequences did not affect transcription of the target gene. Slight modifications were nevertheless observed, in agreement with the loop extrusion model. We discuss these unexpected results considering both conventional and alternative explanations relying on the accumulation of poorly specific factors within the TAD backbone.