Ultrastructural chromatin dynamics may play a key role in regulating transcriptional activation. Here we have used super-resolution microscopy to study the folding mechanics of the HoxD cluster, as assayed by following the elongation of chromatin in single cells with different status of Hox gene activation. We observed that the spatial separation of Hoxd genes is strongest in those tissues where they are highly expressed. We also document that the opening of chromatin precedes transcription and that the strongest elongations are observed at the location of the boundary between two major topologically associating domains (TADs). These results shed light on how spatial compartmentalization is achieved, likely to accompany efficient chromatin reorganization upon activation of transcriptional switches.