Abstract
Despite advances in the identification of chromatin regulators and genome interactions, the principles of higher-order chromatin structure have remained elusive. Here, we applied FLIM-FRET microscopy to analyse, in living cells, the spatial organisation of nanometre range proximity between nucleosomes, which we called "nanocompaction." Both in naive embryonic stem cells (ESCs) and in ESC-derived epiblast-like cells (EpiLCs), we find that, contrary to expectations, constitutive heterochromatin is much less compacted than bulk chromatin. The opposite was observed in fixed cells. HP1α knockdown increased nanocompaction in living ESCs, but this was overridden by loss of HP1β, indicating the existence of a dynamic HP1-dependent low compaction state in pluripotent cells. Depletion of H4K20me2/3 abrogated nanocompaction, while increased H4K20me3 levels accompanied the nuclear reorganisation during EpiLCs induction. Finally, the knockout of the nuclear cellular-proliferation marker Ki-67 strongly reduced both interphase and mitotic heterochromatin nanocompaction in ESCs. Our data indicate that, contrary to prevailing models, heterochromatin is not highly compacted at the nanoscale but resides in a dynamic low nanocompaction state that depends on H4K20me2/3, the balance between HP1 isoforms, and Ki-67.