Multimodal interactions drive chromatin phase separation and compaction

Gene silencing is intimately connected to DNA condensation and the formation of transcriptionally inactive heterochromatin by Heterochromatin Protein 1 alpha (HP1 alpha). Because heterochromatin foci are dynamic and HP1 alpha can promote liquid-liquid phase separation, HP1 alpha-mediated phase separation has been proposed as a mechanism of chromatin compaction. The molecular basis of HP1 alpha-driven phase separation and chromatin compaction and the associated regulation by trimethylation of lysine 9 in histone 3 (H3K9me3), which is the hallmark of constitutive heterochromatin, is however largely unknown. Using a combination of chromatin compaction and phase separation assays, site- directed mutagenesis, and NMR-based interaction analysis, we show that human HP1 alpha can compact chromatin in the absence of liquid-liquid phase separation. We further demonstrate that H3K9- trimethylation promotes compaction of chromatin arrays through multimodal interactions. The results provide molecular insights into HP1 alpha-mediated chromatin compaction and thus into the role of human HP1 alpha in the regulation of gene silencing.