KMT5C displays robust retention and liquid-like behavior in phase separated heterochromatin

The pericentromere exists as a distinct chromatin compartment that is thought to form by a process of phase separation. This reflects the ability of the heterochromatin protein CBX5 ( HP1) to form liquid condensates that encapsulate pericentromeres. In general, phase separation compartmentalizes specific activities within the cell, but unlike membrane-bound organelles, their contents rapidly exchange with their surroundings. Here, we describe a novel state for the lysine methyltransferase KMT5C where it diffuses within condensates of pericentromeric heterochromatin but undergoes strikingly limited nucleoplasmic exchange, revealing a barrier to exit similar to that of biological membranes. This liquid-like behavior maps to a discrete protein segment with a small number of conserved sequence features and containing separable determinants for localization and retention that cooperate to confer strict spatial control. Accordingly, loss of KMT5C retention led to aberrant spreading of its catalytic product (H4K20me3) throughout the nucleus. We further found that KMT5C retention was reversible in response to chromatin state, which differed markedly for CBX5 and the methyl-CpG binding protein MeCP2, revealing considerable plasticity in the control of these phase separated assemblies. Our results establish that KMT5C represents a precedent in the biological phase separation continuum that confers robust spatial constraint of a protein and its catalytic activity without progression to a gel or solid.