Systematic Mutational Analysis of Mixed Lineage Leukemia 3 (MLL3) Histone Methyltransferase Active Site Suggests Single Phe/Tyr Switch Position to Regulate Product Specificity

Post‐translational modifications of histones have significant effects on transcription and development in metazoans. One group of histone modifying enzymes is the human SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, consisting of Mixed Lineage Leukemia 1–4 (MLL1–4) and SETd1A/B. These enzymes include a SET domain, which catalyzes the transfer of one, two, or three methyl groups from S‐adenosylmethionine to H3K4. Isolated SET domains in this family have slow mono‐methyltransferase activity and are able to interact with a sub‐complex called WRAD (WDR5, RbBP5, ASH2L, and DPY‐30) that stabilizes the SET domain and regulates the degree of H3K4 methylation. SET1 family members in complex with WRAD catalyze mono‐ (MLL2/3), di‐ (MLL1/4), or tri‐ (SETd1A/B) methylation of H3K4. Within the SET domain superfamily of methyltransferases, there is thought to be one amino acid position that dictates a SET domain's ability to catalyze mono‐ or multiple methylation, a phenomenon known as product specificity. This position, known as the “Phe/Tyr switch”, limits activity to monomethylation when a bulky tyrosine residue occupies this position, but allows for multiple methylation when the position is occupied by a smaller phenylalanine. While this hypothesis has been validated for some SET domains, it is still not clear if an active site can have more than one Phe/Tyr switch position, or if the position relative to the substrate is conserved. We hypothesize that the degree of methylation catalyzed by SET domain enzymes is controlled by active site volume. To test this, we developed a unique Active Site High Throughput Assay (ASHTA) to rapidly screen active site variants for changes in enzymatic activity and product specificity. We substituted all five tyrosine residues in the MLL3 active site with phenylalanine, cysteine, or alanine, and found that MLL3 has only one Phe/Tyr switch (Y4884). In addition, we found that substitution of tyrosines with phenylalanine is sufficient for normal activity, whereas substitution with smaller residues is more detrimental. These results suggest volume at the Phe/Tyr switch position is critical for product specificity in the MLL3 active site. However, volume at non‐Phe/Tyr switch positions is more important for optimal monomethylation and not for multiple methylation. We next further characterized the Y4884C substitution in MLL3 because it has been shown to be a missense variant linked to colorectal cancer. Substitution of Y4884 with cysteine results in the loss of H3K4 monomethylation activity, but a gain of di‐ and trimethylation activity. These effects were not explained by a change in the hydrodynamic properties, as sedimentation coefficients between wild‐type (1.74±0.05s) and mutant MLL3 proteins (1.72±0.04s) are similar. These results suggest that despite conservation of active site residues within the SET1 family, amino acid variation outside of the active site can have profound effects on the product specificity of SET domain enzymes. In addition, these findings suggest that Y4884C is a gain‐of‐function and loss‐of‐function mutation in MLL3, and drives tumorigenesis by epigenetic changes in gene expression. Support or Funding Information This study was supported by National Institutes of Health grant 2RO1CA140522 to M.C.Unpublished results. The MLL3 active has a single amino acid position that regulates product specificity, known as the Phe/Tyr switch position.