In mammalian cells, distinct H3K4 methylation states are created by deposition of methyl groups by multiple complexes of histone lysine methyltransferase 2 (KMT2) family proteins. For comprehensive analyses that directly compare the catalytic properties of all six human KMT2 complexes, we employed a biochemically defined system reconstituted with recombinant KMT2 core complexes (KMT2CoreCs) containing minimal components required for nucleosomal H3K4 methylation activity. We found that each KMT2CoreC generates distinct states and different levels of H3K4 methylation, and except for MLL3 all are stimulated by H2Bub. Notably, SET1BCoreC exhibited the strongest H3K4 methylation activity and, to our surprise, did not require H2B ubiquitylation (H2Bub); in contrast, H2Bub was required for the H3K4me2/3 activity of the paralog SET1ACoreC. We also found that WDR5, RbBP5, ASH2L and DPY30 are required for efficient H3K4 methyltransferase activities of all KMT2CoreCs except MLL3, which could produce H3K4me1 in the absence of WDR5. Importantly, deletion of the PHD2 domain of CFP1 led to complete loss of the H3K4me2/3 activities of SET1A/BCoreCs in the presence of H2Bub, indicating a critical role for this domain in the H2Bub-stimulated H3K4 methylation. Collectively, our results suggest that each KMT2 complex methylates H3K4 through distinct mechanisms in which individual subunits differentially participate.
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