Phase separation of the MLL1 core complex catalytic module

The National Institute of General Medical Sciences awarded The Research Foundation For The State University Of New York $562,820 on August 1, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to investigate liquid-liquid phase separation in the MLL1 core complex catalytic module and its role in histone H3 lysine 4 methylation underlying aggressive acute leukemias.

The research defines structural features required for phase separation of the MWRAD2 complex (composed of MLL1, WDR5, RBBP5, ASH2L, and DPY-30), mapping intrinsically disordered regions in three component proteins and validating findings through cell-based experiments measuring nuclear puncta formation and H3K4 methylation at target loci. The project determines composition and mechanism of MWRAD2 condensate assembly using multiwavelength sedimentation velocity analytical ultracentrifugation to quantify scaffold stoichiometry and time-resolved small-angle X-ray scattering to track nucleation kinetics, incorporating MLL1-fusion proteins, WIN motif mutants, and small-molecule inhibitors to clarify how structural disruptions abrogate normal assembly. A third aim employs rapid-quench and stopped-flow kinetic methods supplemented by carbon-13 NMR detection of nucleosome methylation to pinpoint reaction steps accelerated by liquid-liquid phase separation.

Performance is in Syracuse, New York. The award extends through May 31, 2030. The Research Foundation administers sponsored programs across the State University of New York system.

Abstract

Aberrant histone H3 lysine 4 (H3K4) methylation underlies aggressive acute leukemias driven by alterations in the mixed lineage leukemia-1 (MLL1) gene. MLL1 encodes a large, multi-domain protein that forms a catalytic core complex (MWRAD2) with WDR5, RBBP5, ASH2L, and DPY-30 to deposit H3K4 methylation. Our data show that liquid-liquid phase separation (llps) can overcome the thermodynamic barrier to MLL1 core assembly, enhancing its enzymatic activity by up to 60-fold. This discovery reframes MLL1 not as a static enzyme-cofactor complex, but as a dynamic, phase-separating system whose spatial and temporal control is crucial for normal gene regulation. MLL1-fusion proteins and win motif mutations appear to disrupt this phase separation, contributing to leukemogenesis via dominant-negative misregulation of H3K4 methylation. In aim 1, we will define the structural features required for MWRAD2 phase separation, mapping intrinsically disordered regions in WDR5, RBBP5, and ASH2L and validating these findings in cell-based experiments that assess nuclear puncta formation and H3K4 methylation at MLL1 target loci. In aim 2, we will determine the composition and mechanism of MWRAD2 condensate assembly, using multiwavelength sedimentation velocity analytical ultracentrifugation (SV-AUC) to quantify scaffold stoichiometry and time- resolved small-angle x-RAY scattering (TR-saxs) to track nucleation kinetics in real time. By incorporating MLL1-fusions, win motif mutants, and small-molecule inhibitors of MLL1-WDR5 binding, we will clarify how c-terminal disruptions abrogate normal assembly. Finally, in aim 3, we will employ rapid-quench and stopped-flow kinetic methods, supplemented by 13C NMR detection of nucleosome methylation, to pinpoint the reaction steps accelerated by llps. Integrating these data in kintek explorer will reveal the precise mechanism by which llps reconfigures MLL1 for rapid H3K4 methylation. This work will illuminate a fundamental paradigm in epigenetic regulation-how phase separation orchestrates MLL1's catalytic cycle-and provide mechanistic insight into why c-terminal disruptions of MLL1 lead to leukemogenesis. Ultimately, these findings may guide the development of new therapeutic strategies targeting pathological condensates in high-risk leukemias.