Deshpande, P.; Prentice, E.; Vidal Ceballos, A.; Casaccia, P.; Elbaum-Garfinkle, S.

Biomolecular condensates have emerged as a powerful new paradigm in cell biology with broad implications to human health and disease, particularly in the nucleus where phase separation is thought to underly elements of chromatin organization and regulation. Specifically, it has been recently reported that phase separation of heterochromatin protein 1alpha (HP1) with DNA contributes to the formation of condensed chromatin states. HP1 localization to heterochromatic regions is mediated by its binding to specific repressive marks on the tail of histone H3, such as trimethylated lysine 9 on histone H3 (H3K9me3). However, whether epigenetic marks play an active role in modulating the material properties of HP1 and dictating emergent functions of its condensates, remains only partially understood. Here, we leverage a reductionist system, comprised of modified and unmodified histone H3 peptides, HP1 and DNA to examine the contribution of specific epigenetic marks to phase behavior of HP1. We show that the presence of histone peptides bearing the repressive H3K9me3 is compatible with HP1 condensates, while peptides containing unmodified residues or bearing the transcriptional activation mark H3K4me3 are incompatible with HP1 phase separation. In addition, inspired by the decreased ratio of nuclear H3K9me3 to HP1 detected in cells exposed to uniaxial strain, using fluorescence microscopy and rheological approaches we demonstrate that H3K9me3 histone peptides modulate the dynamics and network properties of HP1 condensates in a concentration dependent manner. These data suggest that HP1-DNA condensates are viscoelastic materials, whose properties may provide an explanation for the dynamic behavior of heterochromatin in cells in response to mechanostimulation.