Phase separation of chromatin and its link to nuclear mechanotransduction

Organization of the chromatin in the nucleus and its partition into active and inactive domains is critical for transcription regulation. We investigated chromatin mesoscale 3D structure in live muscle cell within a living organism (Drosophila larvae), and demonstrated that chromatin arranges at the periphery of the nucleus, close to the nuclear lamina, and leaving about two-third of the nucleus free of chromatin. Modeling of chromatin interactions suggested that chromatin peripheral organization depends on the extent of chromatin self-attraction, as well as on the degree of chromatin interaction with the nuclear lamina via Lamina Associated Domains (LAD). Experimental manipulation of the association of chromatin with the nuclear lamina abrogated the peripheral chromatin organization, supporting a theoretical model of chromatin phase-separation from the nucleoplasm. Further investigation addressed the contribution of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, which mediates a mechanical linkage between cytoskeletal networks, nuclear lamina, and chromatin. Lack of functional LINC complex led to an increased binding of the transcription repressors Polycomb, and heterochromatin Protein 1 (HP1) to an array of muscle and non-muscle genes, and to the formation of large epigenetic repressive clusters at the nuclear periphery. In addition, RNA Pol II binding to a set of muscle genes decreased significantly, suggesting a basis for the phenotype of thinner muscle fibers in the LINC mutants. Taken together, our experiments demonstrate the essential contribution of nuclear envelope properties to chromatin mesoscale organization, chromatin phase separation, and chromatin transcription regulation.