Single-cell absolute contact probability detection reveals that chromosomes are organized by modulated stochasticity

The multi-scale organization of eukaryotic genomes defines and regulates cellular identity and tissue-specific functions. At the kilo-megabase scales, genomes are partitioned into self-interacting modules or topologically associated domains (TADs). TADs are often seen as highly stable over time; however, recent studies have reported different degrees of heterogeneity in TADs organization. Access to single-cell absolute probability contact measurements between loci and efficient detection of low-frequency, long-range interactions is thus essential to unveil the stochastic behaviour of chromatin at different scales. Here, we combined super-resolution microscopy with state-of-the-art DNA labeling methods to reveal the variability in the multi-scale organization of chromosomes in different cell-types and developmental stages in Drosophila. Remarkably, we found that contacts between consecutive TAD borders were infrequent, independently of TAD size, epigenetic state, or cell type. Moreover, long-range contact probabilities between non-consecutive borders, the overall folding of chromosomes, and the clustering of epigenetic domains into active/repressed compartments displayed different degrees of stochasticity that globally depended on cell-type. Overall, our results show that stochasticity is present at all levels of chromosome organization, but can be specifically modulated by sequence and epigenetic state to give rise to different levels of genome organization providing different spatial levels of gene regulation.