A Novel “Dual Substrate” Kinetics Assay Suggests the Presence of Two Active Sites in the MLL1 Core Complex

Mixed Lineage Leukemia 1 (MLL1) protein is a 3,969 amino acid histone H3 lysine 4 (H3K4) methyltransferase with multiple conserved domains involved in mediating protein‐protein interactions, gene targeting and H3K4 reading and writing. MLL1 is known to regulate expression of homeotic genes through its methyltransferase activity. Deregulation of MLL through reciprocal chromosomal translocations is associated with acute lymphocytic and myeloid leukemias. MLL catalyzes H3K4 methylation, the degree of which is regulated by interaction with W DR5, R bBP5, A sh2L and D PY30 (WRAD), forming the MLL1 core complex. The molecular mechanisms for how the MLL1 core complex regulates the degree of H3K4 methylation are still not well understood, but are crucial for understanding the role of MLL1 in transcription and disease. In our lab, we have developed a biochemical reconstitution system to study the structure and enzymology of MLL family complexes. Our lab has previously determined that the MLL1 SET domain alone performs slow monomethylation, but the addition of WRAD significantly increases the rate of monomethylation and also introduces H3K4 dimethylation activity. The molecular mechanisms for these WRAD‐dependent changes are unknown. It was suggested, based on the presence of a cryptic methyltransferase activity within the WRAD sub‐complex, that two distinct active sites within the MLL1 core complex are responsible for the mono‐ and dimethylation reactions. Consistent with this hypothesis, single turnover kinetics experiments suggest that the MLL1 core complex uses a non‐processive (distributive) kinetic mechanism, where the monomethylated product of the first reaction is released from the complex before rebinding for the second methylation reaction. However, it is still unclear if the monomethylated product from the first reaction rebinds the SET domain or to the cryptic WRAD active site for the dimethylation reaction. In the current study, pulse‐chase experiments confirm that the monomethylated product of the first reaction is released from the complex before rebinding for the dimethylation reaction. These results are consistent with a distributive kinetic mechanism. To determine if the complex uses two active sites, we developed a novel “Dual‐Substrate Assay”, in which the rates of methylation of unmethylated (H3K4me0) and monomethylated (H3K4me1) histone H3 peptide substrates are monitored simultaneously using quantitative MALDI‐TOF mass spectrometry. The results show that the rates of methylation of each peptide are similar in the presence or absence of the other peptide, suggesting that both reactions can occur simultaneously. Furthermore, we show that an amino acid substitution that prevents peptide binding to the MLL1 SET domain abolishes the monomethylation reaction, but leaves the dimethylation reaction unaltered. Taken together, these results are consistent with a model in which the MLL1 SET domain and WRAD each contain a distinct active site that catalyzes the mono‐ and dimethylation reactions, respectively. Support or Funding Information National Institutes of Health Grant R01CA140522 (to M. S. C.)