BPS2026 – Heterochromatin phase-separated condensates throughout stages of mouse embryonic stem cell differentiation

Heterochromatin condensates support the physical protection against DNA-processing proteins and polymerases in heterochromatin. However, at a given time window and under certain embryonic differentiation stages, genes encoded in heterochromatin become essential and should be expressed. We hypothesize that heterochromatin becomes more accessible and that this temporal accessibility is achieved via changes in the properties of heterochromatin protein 1 (HP1) phase-separated condensates, i.e., phase, density, contents, size, and fragmentation. Using fluorescence lifetime imaging microscopy, fluorescence anisotropy imaging microscopy, and single-molecule fluorescence bursts during photobleaching of knocked-in mCherry-tagged HP1α in mouse embryonic stem cells (mESCs), as well as mass spectrometry and RNA-seq pull-down assays. We show the features of HP1α condensates shifting from heterogeneous dense gel-like features into less heterogeneous and less dense liquid-like features for a few days’ window, before shifting back to more dense, heterogeneous, large gel-like condensates. During mESC differentiation, we also identify clear-cut changes in the biomolecular contents of the heterochromatin condensates. Overall, our results support the suggestion that heterochromatin contains foci of phase-separated condensates that can undergo fusion and compact distant regions into larger clusters, and can undergo fission to facilitate expanding heterochromatin, without loss of phase-separated foci, for a given time window within the stem cell differentiation process.