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.

26At the kilo-to mega-base pair scales, eukaryotic genomes are partitioned into 27 self-interacting modules or topologically associated domains (TADs) that associate to 28 form nuclear compartments. Here, we combined high-content super-resolution 29 microscopies with state-of-the-art DNA labeling methods to reveal the variability in 30 the multiscale organization of the Drosophila genome. We found that association 31 frequencies within TADs and between TAD borders are below ~10%, independently 32 of TAD size, epigenetic state, or cell type. Critically, despite this large heterogeneity, 33 we were able to visualize nanometer-sized epigenetic domains at the single-cell 34 level. In addition, absolute contact frequencies within and between TADs were to a 35 large extent defined by genomic distance, higher-order chromosome architecture, 36 and epigenetic identity. We propose that TADs and compartments are organized by 37 multiple, small frequency, yet specific interactions that are regulated by epigenetics 38 and transcriptional state. 39 40 41 42 43The multi-scale organization of eukaryotic genomes defines and regulates 44 cellular identity and tissue-specific functions [1][2][3] . At the kilo-megabase scales, 45 genomes are partitioned into self-interacting modules or topologically associated 46 domains (TADs) 4-6 . TAD formation seems to require specific looping interactions 47 between TAD borders 7,8 , while the association of TADs can lead to the formation of 48 active/repressed compartments 9 . These structural levels were often seen as highly 49 stable over time, however, recent single-cell Hi-C studies have reported different 50 degrees of heterogeneity 10,11 . Other studies have reported that genomes also display 51 stochasticity in their association with the nuclear lamina 12 , in the formation of 52 chromosome territory neighborhoods 13 , and in gene kissing 14 . However, access to 53 single-cell absolute probability contact measurements between loci and efficient 54 detection of low-frequency, long-range interactions are essential to quantify the 55 stochastic behaviour of chromatin at different scales. 56Here, we combined high-content super-resolution microscopy with state-of-57 the-art DNA labeling methods to reveal the variability in the multiscale organization of 58 chromosomes in different cell-types and developmental stages in Drosophila. 59 Remarkably, we found that stochasticity is present at all levels of chromosome 60 architecture, but is locally modulated by sequence and epigenetic state. Contacts 61 between consecutive TAD borders were infrequent, independently of TAD size, 62 epigenetic state, or cell type. Moreover, long-range contact probabilities between 63 non-consecutive borders, the overall folding of chromosomes, and the clustering of 64 epigenetic domains into active/repressed compartments displayed different degrees 65 of stochasticity that globally depended on cell-type. Overall, our results show that 66 contacts between and within TADs are rare, but can be epigenetical...