Molecular Mechanisms of the MLL4 Complex in H3K4 Methylation and p53-Dependent Transcription Activation [ChIP-Seq]

MLL4 is a mammalian H3K4 mono-methyltransferase that plays a critical role in enhancer-regulated gene activation and tissue-specific gene expression during development. Here, we utilized a reconstituted MLL4 complex (MLL4C) containing a truncated MLL4 “fusion” protein (MLL4F) that retains its catalytic SET domain and N-terminal functional regions for comprehensive structural and functional analyses. In vitro H3K4 methyltransferase assays demonstrated that MLL4 catalytic activity is notably stimulated by p53 and CBP/p300. Cryo-electron microscopy (cryo-EM) analysis revealed that the MLL4 fusion core complex engages the nucleosome in three major distinct conformational states, providing mechanistic insights into the regulation of MLL4 and other MLL family methyltransferases by two structurally rigid modules: the SET–ASH2L–DPY30 module and the RBBP5–WDR5 module. In addition, we delineated a third structurally rigid module within the functional region upstream of MLL4 SET domain, encompassing the tandem PHD4–6, PHD7, and FYR domains, which is essential for MLL4- and p53-dependent transcription. Comparative structural analyses reveal that this third module is evolutionarily conserved across MLL1 to MLL4. Furthermore, we employed crosslinking mass spectrometry and integrative modeling to generate a complete model of the nine-subunit MLL4FC complex and elucidated its functional interactions with p53. Finally, genomic studies in the human colon cancer cell line HCT116 revealed that MLL4 colocalizes with p53 at active enhancers of p53 target genes. MLL4 knockout led to significant downregulation of p53-regulated genes, coupled with the reduction of p53 binding at active enhancers. These findings provide molecular insights into the catalytic and transcriptional functions of the MLL4 complex in establishing active enhancers and facilitating p53-dependent transcription activation.