Mixed-lineage leukemia (MLL), along with multisubunit (WDR5, RbBP5, ASH2L, and DPY30) complex catalyzes the trimethylation of H3K4, leading to gene activation. Here, we characterize a chromatin-independent role for MLL during mitosis. MLL and WDR5 localize to the mitotic spindle apparatus, and loss of function of MLL complex by RNAi results in defects in chromosome congression and compromised spindle formation. We report interaction of MLL complex with several kinesin and dynein motors. We further show that the MLL complex associates with Kif2A, a member of the Kinesin-13 family of microtubule depolymerase, and regulates the spindle localization of Kif2A during mitosis. We have identified a conserved WDR5 interaction (Win) motif, so far unique to the MLL family, in Kif2A. The Win motif of Kif2A engages in direct interactions with WDR5 for its spindle localization. Our findings highlight a non-canonical mitotic function of MLL complex, which may have a direct impact on chromosomal stability, frequently compromised in cancer.

Attainment of proper cell shape and the regulation of cell migration are essential processes in the development of an organism. The mixed lineage leukemia (MLL or KMT2A) protein, a histone 3 lysine 4 (H3K4) methyltransferase, plays a critical role in cell-fate decisions during skeletal development and haematopoiesis in higher vertebrates. Rho GTPases – RhoA, Rac1 and CDC42 – are small G proteins that regulate various key cellular processes, such as actin cytoskeleton formation, the maintenance of cell shape and cell migration. Here, we report that MLL regulates the homeostasis of these small Rho GTPases. Loss of MLL resulted in an abnormal cell shape and a disrupted actin cytoskeleton, which lead to diminished cell spreading and migration. MLL depletion affected the stability and activity of Rho GTPases in a SET domain-dependent manner, but these Rho GTPases were not direct transcriptional targets of MLL. Instead, MLL regulated the transcript levels of their chaperone protein RhoGDI1 (also known as ARHGDIA). Using MDA-MB-231, a triple-negative breast cancer cell line with high RhoGDI1 expression, we show that MLL depletion or inhibition by small molecules reduces tumour progression in nude mice. Our studies highlight the central regulatory role of MLL in Rho/Rac/CDC42 signalling pathways. This article has an associated First Person interview with the first author of the paper.