Chromatin topology and the timing of enhancer function at the HoxD locus [RNA-seq]
The HoxD gene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different sub-groups of Hoxd genes respond first to a proximal regulatory signal, then to a distal signal that organizes digits. These two regulations are exclusive from one another and emanate separately from two TADs flanking HoxD, both containing a range of appropriate enhancer sequences. The telomeric TAD (T-DOM) contains several enhancers active in presumptive forearm cells and is divided into two sub-TADs separated by a CTCF-rich boundary, which defines two regulatory sub-modules. To understand the importance of this particular regulatory topology to control Hoxd gene transcription in time and space, we either deleted or inverted this sub-TAD boundary, eliminated the CTCF binding sites or inverted the entire T-DOM to exchange the respective positions of the two sub-TADs. The effects of such perturbations on the transcriptional regulation of Hoxd genes illustrates the requirement of this regulatory topology for the precise timing of gene activation. However, the spatial distribution of transcripts is eventually resumed, showing that the presence of enhancers sequences, rather than either their exact topology or a particular chromatin architecture, is the key factor. We also show that the affinity of enhancers to find their natural target genes can overcome the presence of both a strong TAD border and an unfavourable orientation of CTCF sites. Overall design: RNA-seq analysis of wild-type and mutant (del(CS38-40)) in forelimb buds from E9.5 mouse embryos.