Abstract
Transcription in eukaryotic genomes depends on enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation. The mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of H3K4 methyltransferases and is frequently rearranged in acute leukemias. Despite sequence comparisons that predict that SET1 family enzymes should only monomethylate their substrates, mono-, di-, and trimethylation of H3K4 has been attributed to SET1 family complexes in vivo and in vitro. To better understand this paradox, we have biochemically reconstituted and characterized a five-component 200-kDa MLL1 core complex containing human MLL1, WDR5, RbBP5, Ash2L, and DPY-30. We demonstrate that the isolated MLL1 SET domain is a slow monomethyltransferase and that tyrosine 3942 of MLL1 prevents di- and trimethylation of H3K4. In contrast, a complex containing the MLL1 SET domain, WDR5, RbBP5, Ash2L, and DPY-30, displays a marked approximately 600-fold increase in enzymatic activity but only to the dimethyl form of H3K4. Single turnover kinetic experiments reveal that the reaction leading to H3K4 dimethylation involves the transient accumulation of a monomethylated species, suggesting that the MLL1 core complex uses a non-processive mechanism to catalyze multiple lysine methylation. We have also discovered that the non-SET domain components of the MLL1 core complex possess a previously unrecognized methyltransferase activity that catalyzes H3K4 dimethylation within the MLL1 core complex. Our results suggest that the mechanism of multiple lysine methylation by the MLL1 core complex involves the sequential addition of two methyl groups at two distinct active sites within the complex.
Highlights
A direct interaction between RbBP5 and MLL1 could not be detected in our sedimentation velocity assays, our results suggest that RbBP5 may have some interaction with the MLL1 SET domain in the context of the MLL3745-WDR5 complex (M-W-R) complex, which is manifested by a small increase in the enzymatic activity of MLL1
Canaani and colleagues [23] reported the isolation of a 29-component mammalian MLL1 supercomplex that is enzymatically active with an unmodified histone H3 peptide but not with a peptide previously dimethylated at H3K4
A similar result was observed with a recombinant MLL1 SET domain fragment containing residues 3745–3969 [23, 32], suggesting that the product specificity of the MLL SET domain is that of either a mono- or dimethyltransferase
Summary
Protein Expression and Purification—A human MLL1 construct consisting of residues 3745–3969 (MLL3745) as well as full-length human WDR5, RbBP5, and ASH2L proteins were individually expressed in Escherichia coli (Rosetta II, Novagen) and purified as described previously [45]. To prevent enzyme inactivation over the duration of the experiment, the reactions were incubated at 15 °C for 24 h, and at various time points, aliquots were removed and quenched by the addition of trifluoroacetic acid to 0.5%. H3K4 methyltransferase assays were conducted by combining 2 g of MLL3745-WDR5-RbBP5-Ash2L complex with 250 M histone H3 peptide and 1 Ci of [3H]methyl-S-adenosyl-methionine ([3H]AdoMet, GE Healthcare) in 50 mM Tris, pH 8.5, 200 mM NaCl, 3 mM dithiothreitol, 5 mM MgCl2, and 5% glycerol. MALDI-TOF mass spectrometry was used to determine the relative distribution of unmodified, mono-, di-, and trimethylated species in each reaction using a procedure similar to that described by Frey and colleagues [51]. Standard curves were constructed by collecting MALDI-TOF spectra on peptide mixtures containing various ratios of synthetic histone H3 peptides that were either unmodified or previously mono- or dimethylated at H3K4. For reactions with two irreversible consecutive steps, the program DynaFit (BioKin, Ltd.) [54] was used to globally fit Equations 1–3 to the data
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