Epigenetic dysregulation is a prominent feature in cancer, as exemplified by frequent mutations in chromatin regulators, including the MLL/KMT2 family of histone methyltransferases. Although MLL1/KMT2A activity on H3K4 methylation is well documented, their non-canonical activities remain mostly unexplored. Here we show that MLL1/KMT2A methylates Borealin K143 in the intrinsically disordered region essential for liquid–liquid phase separation of the chromosome passenger complex (CPC). The co-crystal structure highlights the distinct binding mode of the MLL1 SET domain with Borealin K143. Inhibiting MLL1 activity or mutating Borealin K143 to arginine perturbs CPC phase separation, reduces Aurora kinase B activity, and impairs the resolution of erroneous kinetochore–microtubule attachments and sister-chromatid cohesion. They significantly increase chromosome instability and aneuploidy in a subset of hepatocellular carcinoma, resulting in growth inhibition. These results demonstrate a non-redundant function of MLL1 in regulating inner centromere liquid condensates and genome stability via a non-canonical enzymatic activity.

表观遗传失调是癌症的一个突出特征，染色质调节因子（包括组蛋白甲基转移酶 MLL/KMT2 家族）的频繁突变就是例证。尽管 MLL1/KMT2A 对 H3K4 甲基化的活性已有充分记录，但它们的非规范活性大多仍未被探索。在这里，我们发现 MLL1/KMT2A 在本质上无序的区域甲基化 Borealin K143，这对于染色体乘客复合物 (CPC) 的液-液相分离至关重要。共晶结构突出了 MLL1 SET 结构域与 Borealin K143 的独特结合模式。抑制 MLL1 活性或将 Borealin K143 突变为精氨酸会扰乱 CPC 相分离，降低极光激酶 B 活性，并损害错误着丝粒-微管附着和姐妹染色单体凝聚力的分辨率。它们显着增加肝细胞癌亚型中的染色体不稳定性和非整倍性，从而导致生长抑制。这些结果证明了 MLL1 通过非规范酶活性调节内部着丝粒液体凝聚物和基因组稳定性的非冗余功能。