PT  - JOURNAL ARTICLE
AU  - Mattia Conte
AU  - Luca Fiorillo
AU  - Simona Bianco
AU  - Andrea M. Chiariello
AU  - Andrea Esposito
AU  - Mario Nicodemi
TI  - Chromatin folding variability across single-cells results from state degeneracy in phase-separation
AID  - 10.1101/2020.05.16.099275
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.05.16.099275
4099  - http://biorxiv.org/content/early/2020/05/16/2020.05.16.099275.short
4100  - http://biorxiv.org/content/early/2020/05/16/2020.05.16.099275.full
AB  - Chromosome spatial organization controls functional interactions between genes and regulators, yet the molecular and physical mechanisms underlying folding at the single DNA molecule level remain to be understood. Here we employ models of polymer physics to investigate the conformations of two 2Mb-wide DNA loci in human HCT116 and IMR90 wild-type and cohesin depleted cells. Model predictions on the 3D structure of single-molecules are consistently validated against super-resolution single-cell imaging data, providing evidence that the architecture of the studied loci is controlled by a thermodynamics mechanism of polymer phase separation whereby chromatin self-assembles in segregated globules. The process is driven by interactions between distinct types of cognate binding sites, correlating each with a different combination of chromatin factors, including CTCF, cohesin and histone marks. The intrinsic thermodynamics degeneracy of conformations results in a broad structural and time variability of single-molecules, reflected in their varying TAD-like contact patterns. Globules breathe in time, inducing stochastic unspecific interactions, yet they produce stable, compact environments where specific contacts become highly favored between regions enriched for cognate binding sites, albeit characterized by weak biochemical affinities. Cohesin depletion tends to reverse globule phase separation into a coil, randomly folded state, resulting in much more variable contacts across single-molecules, hence erasing population-averaged patterns. Overall, globule phase separation appears to be a robust, reversible mechanism of chromatin organization, where stochasticity and specificity coexist.Competing Interest StatementThe authors have declared no competing interest.