Epigenetic compartmentalization of mitotic chromosomes by phase-separation-driven repulsion between WDR5 and the chromosomal passenger complex

Histone modifications recruit chromatin regulators, but how their spatial patterns are established on mitotic chromosomes remains unclear. Spatial separation between active chromatin and centromeric signaling is essential for accurate chromosome segregation, yet the physical principles linking epigenetic marks to mitotic protein organization are poorly defined. Here, we show that liquid-liquid phase separation (LLPS) of WDR5, a core component of H3K4 methyltransferase complexes, acts as a spatial organizer of mitotic chromosomes. WDR5 LLPS is driven by multivalent charged and hydrophobic interactions in its N-terminal region. Phase-separated WDR5 repels the chromosomal passenger complex (CPC), segregating H3K4-trimethylated (H3K4me3) chromatin from H3T3-phosphorylated (H3T3ph) centromeric regions. Disrupting WDR5 condensation causes Aurora B mislocalization and chromosome misalignment, and restoring H3K4me3 alone does not rescue these defects. Computational modeling and rescue experiments support that WDR5-driven LLPS and histone modification signals cooperate to concentrate CPC at centromeres, revealing how LLPS and epigenetic information organize chromatin compartments during mitosis.