Structural analyses of the assembly, chromatin recognition and modification of the human KMT2D complex

• Applicant: Dr. Simon Pöpsel
• Subject Area: Structural Biology ; Biochemistry
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 504090354

Enhancers are gene regulatory elements that control cellular differentiation and identity by establishing patterns of gene activity. The human KMT2D (MLL4) complex is a central enhancer co-activator and is essential for development and differentiation by enabling enhancer activation. Commonly, chromatin associated regulators function as multi-subunit complexes. The KMT2D complex (KMT2Dc) comprises nine subunits, two of which – KMT2D and UTX – are enzymes that catalyze histone post-translational modifications (PTMs) that are hallmarks of enhancers: KMT2D is a methyltransferase that mono-methylates lysine 4 of histone H3 (H3K4me1), and UTX demethylates trimethylated lysine 27 of histone H3 (H3K27me3). Genetic alterations of KMT2D and UTX lead to the developmental disorder Kabuki syndrome, and are frequently observed in human cancer. Despite key roles in development and disease, little is known about the mechanisms of KMT2Dc function, its chromatin interactions and activity regulation. Due to large and partially disordered subunits, KMT2Dc has so far resisted recombinant expression and structural analyses. We have established the purification of native KMT2Dc from human cells and obtained cross-linking mass spectrometry (CL/MS) data, a valuable resource of topological information on the endogenous complex, providing insights into the spatial relationship of protein subunits and domains. Based on this information, we have designed complexes for recombinant expression and purification. These are composed of UTX, the WRAD core (WDR5, RBBP5, ASH2L and DPY-30) and parts of the ~600 kDa large KMT2D protein that our CL/MS data suggest to be interaction hubs within endogenous KMT2Dc. Here, we will use recombinant sub-complexes to obtain high-resolution structures by single particle cryo-electron microscopy (cryo-EM). We will focus on the following questions: How are UTX and KMT2D domains integrated into the complex? What is the structural relationship of the catalytically active domains of UTX and KMT2D? Using recombinant nucleosomes, we will visualize chromatin engagement and the recognition of specific histone PTMs at high resolution. We will further study how the recognition of chromatin and histone PTMs affects the histone methylation and demethylation activities by KMT2D and UTX, respectively. Finally, we will optimize cryo-EM sample preparation for structural studies of endogenous KMT2Dc to incorporate our findings into the native, complete complex. Human K562 cells will be used to determine how the disruption of defined interfaces within KMT2Dc or between KMT2Dc and chromatin affects cellular KMT2Dc integrity, its recruitment to chromatin, and the distribution of histone PTMs at enhancers. Taken together, we hope to significantly enhance our understanding of KMT2Dc function by providing mechanistic models of the assembly, chromatin engagement and activity regulation of KMT2Dc based on structural, biochemical and cell biological approaches.

• DFG Programme: Research Grants