Chromatinaccessibility is regulated by DNA sequence and specific modifications(epigenetic marks) that define chromatin types or states. In recentyears a new level of hierarchical organization at the Mb and sub-Mbregions was uncovered (Topologically Associating Domains, TADs). TADsdisplay well-defined epigenetic marks that strongly correlate withchromatin types: HP1 repressed (heterochromatin, green), voidrepressed (black), Polycomb repressed (blue), and active(yellow/red). Herein, we employed multi-color super-resolutionmicroscopy (dSTORM and 3D-SIM) to study the role and mechanism ofepigenetic marks on the nanoscale organization of Drosophilachromatin. We found that active (H3K4me3) and inactive (H3K27me3)chromatin form domains of distinctive sizes displaying an additionallevel of internal organization. As expected, this domains aresegregated from each other but show a high degree of interdigitation.The sizes of domains suggest the presence of one to three TADs perdomain and shows good correlation with the one-dimensional size ofthe epigenetic code. Single-molecule imaging of insulator proteinsdemarking TADs borders show that they locate in the outer interfaceof active and inactive chromatin domains (H3K27me3 and Polycombrespectively), whereas they strongly colocalize with activetranscription sites. By genome wide and pair-wise labelling of TADsbarriers we do not observe long range interaction (e.g. looping)between TADs borders contrasting previous models for TADs assemblyand maintenance. The distances between consecutive andnon-consecutive barriers increased following a power-law scaling withactive and inactive chromatin showing distinct physical behaviour.Our data strongly suggest a model in which active and inactive TADsare dynamically segregated from each other with their spatialpositioning intimately associated with the linear DNA sequence of thechromatin fiber and not necessarily regrouped according to theirepigenetic state.