Researchers Reveal Structural Basis for Product Specificities of MLL Family Methyltransferases

The lysine residues in histone proteins can be chemically modified, including methylation, acetylation, and ubiquitination, which are important epigenetic marks to regulate gene expression. Methylation of histone H3 Lys4 (H3K4) is one of the most conserved modifications across species. H3K4 can be methylated by adding one, two, or three methyl groups to the ε-amino group of the lysine side chain. Each methylation status is associated with distinct genome distributions and physiological functions. 

In mammalian cells, H3K4 is mainly methylated by mixed-lineage leukemia (MLL) family methyltransferases (including MLL1, MLL2, MLL3, MLL4, SET1A, and SET1B). Originating from the same ancestor Set1 in yeast, MLL proteins play non-redundant roles in cells because the knockout of any MLL member is embryonic lethal. The non-redundant functions of MLLs are partly associated with their different catalytic ability to generate mono-, di-, or tri-methylated products, termed product specificity. Although the product specificities of MLL family methyltransferases have been extensively studied in vitro and in vivo, the molecular mechanisms remain poorly understood. 

In a study published in Molecular Cell, Dr. CHEN Yong's team from the Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences (CAS), Dr. QUAN Shu's team from East China University of Science and Technology, the teams of Drs. LIU Zhijun and PENG Chao from the National Facility for Protein Science in Shanghai of CAS, and Dr. LI Guohui's team from Dalian Institute of Chemical Physics of CAS, reported the structural basis for product specificities of MLL family methyltransferases. 

The researchers comprehensively compared product specificities of six MLL complexes. To quantify the kinetic differences between each methylation step, they developed three methylation-quantification assays (Methyl-Quant WB, Methyl-Quant LC-MS/MS, and Methyl-Quant MALDI-TOF MS). By comparing the rate constants for the first methylation event (k1), the second methylation event (k2), and the third methylation event (k3) catalyzed by different MLL complexes, they defined MLL1/2 as nonprocessive di-methyltransferases, MLL3/4 as monomethyltransferases, and SET1A/B as nonprocessive tri-methyltransferases. 

To further investigate the molecular mechanism of product specificity, the researchers used X-ray crystallography, 19F-NMR, and molecular dynamics computational simulations to characterize the dynamic of key tyrosine residues in the active pocket of MLL family proteins. They discovered that crucial tyrosine residues in different MLL family proteins have the same conformation but exhibit different dynamics. They determined the role of each F/Y switch residue in cascaded methylation steps and proposed a modified F/Y switch rule that the dynamics of F/Y switch residues fine-tune the product specificity. 

This work suggests that the rate ratio between the cascaded methylation steps (k1/k2 and k2/k3), rather than the absolute reaction rate, is a quantitative criterion to define the product specificity for a histone methyltransferase. Although the F/Y switch rule has been established for a long time, the current F/Y switch rule cannot distinguish between di- and tri-methyltransferases and fails to explain outliers such as MLL-family proteins. This work proposes a modified F/Y switch rule applicable for most SET domain methyltransferases and provides a deeper understanding of dynamic histone methylation.