Gene silencing is intimately connected to DNA condensation and the formation of transcriptionally inactive heterochromatin by Heterochromatin Protein 1α (HP1α). Because heterochromatin foci are dynamic and HP1α can promote liquid-liquid phase separation, HP1α-mediated phase separation has been proposed as a mechanism of chromatin compaction. The molecular basis of HP1α-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 HP1α 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 HP1α-mediated chromatin compaction and thus into the role of human HP1α in the regulation of gene silencing.
Keywords: H3K9-trimethylation; Heterochromatin Protein 1α (HP1α); chromatin compaction; phase separation.