At the kilo- to megabase pair scales, eukaryotic genomes are partitioned into self-interacting modules or topologically associated domains (TADs) that associate to form nuclear compartments. Here, we combine high-content super-resolution microscopies with state-of-the-art DNA-labeling methods to reveal the variability in the multiscale organization of the Drosophila genome. We find that association frequencies within TADs and between TAD borders are below ~10%, independently of TAD size, epigenetic state, or cell type. Critically, despite this large heterogeneity, we are able to visualize nanometer-sized epigenetic domains at the single-cell level. In addition, absolute contact frequencies within and between TADs are to a large extent defined by genomic distance, higher-order chromosome architecture, and epigenetic identity. We propose that TADs and compartments are organized by multiple, small-frequency, yet specific interactions that are regulated by epigenetics and transcriptional state.

This research was supported by funding from the European Research Council under the 7th Framework Program (FP7/2010-2015, ERC grant agreement 260787 to M.N. and FP7/2007-2013, and ERC grant agreement 609989 to M.A.M.-R.). M.A.M.-R. and G.C. acknowledge support from the European Union's Horizon 2020 research and innovation program under grant agreement 676556. This work has also benefited from support by the Labex EpiGenMed, an «Investments for the future» program, reference ANR-10-LABX-12-01, the Spanish Ministry of Economy and Competitiveness (BFU2013-47736-P to M.A.M.-R.), and from “Centro de Excelencia Severo Ochoa 2013-2017”, SEV-2012-0208 to the CRG. 3D-SIM experiments were performed at Montpellier Resource Imaging. We acknowledge the France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»).