Exploration into the MLL4/WRAD Enzyme-Substrate Network: Systematic In Vitro Identification of CFP1 as a Potential Non-Histone Substrate of the MLL4 Lysine Methyltransferase

Lysine methylation is a critical post-translational modification catalyzed by lysine methyltransferases (KMTs), originally characterized in the regulation of histones. However, the breadth of non-histone targets remains largely unexplored. Here, we used a systematic peptide array-based approach to define a substrate preference motif for the SET-domain-containing KMT MLL4 (KMT2D), a member of the COMPASS complex and a known H3K4 methyltransferase. Using this motif, we identified CXXC finger protein 1 (CFP1), a core component of Setd1A/B complexes, as a putative MLL4 substrate. In vitro methyltransferase assays confirmed robust methylation of CFP1 by an MLL4-WRAD complex. Surprisingly, while initial predictions implicated K328, array-based methylation profiling revealed multiple lysine residues within CFP1's lysine-rich basic domain as methylation targets, including K331, K335, K339, and K340. We further demonstrated that CFP1 methylation likely modulates its interaction with MLL4's PHD cassettes and facilitates binding to Setd1A. Binding preferences of MLL4's PHD1-3 and PHD4-6 domains varied with methylation state and site, suggesting non-histone methyl mark recognition by these cassettes. Pulldown assays confirmed that methylated, but not unmethylated, CFP1 binds Setd1A, supporting a potential methyl-switch mechanism. Together, our findings propose CFP1 as a potential non-histone substrate of MLL4 and suggest that MLL4 may regulate Setd1A/B function indirectly via CFP1 methylation. This study expands the substrate landscape of MLL4 and lays the groundwork for future investigations into non-histone methylation signaling in chromatin regulation.