The role of MLL4 in the chromatin framework: from the epigenome to 3D genome organization

MLL4 is a histone methyltransferase of the Trithorax (TrxG) group, crucial for maintaining active gene expression by antagonizing Polycomb (PcG) proteins, which enforce repressive states. Proper TrxG-PcG crosstalk is critical for various cellular processes and for determining cell fate. Although the balance between Trithorax and Polycomb has been recognized for decades, the mechanisms achieving and sustaining this equilibrium, and whether it is perpetuated at the topological level of chromatin, remains not fully understood. This issue is particularly relevant given that mutations in the gene coding for MLL4 (KMT2D) are responsible for a rare multi-systemic disorder named Kabuki syndrome, which currently has inadequate therapeutic options. In this thesis, we present an integrated study of the epigenetic and topological changes in the chromatin state associated with MLL4 Loss of Function (MLL4LoF) in a cellular model analogous to Kabuki syndrome. Using sequencing technologies (Cut&Run, ChIP-seq, ATAC-seq), we found that in MLL4 haploinsufficiency, TrxG members show reduced genome binding, while PcG strengthens its enrichment and genome occupancy. This chromatin state rearrangement is also reflected on a three-dimensional scale. Genome Architecture Mapping (GAM) experiments reveal distinct changes in 3D chromatin organization between MLL4WT and MLL4LoF conditions. Specifically, the 3D contacts within PcG compartments are reinforced and lengthened in MLL4LoF, where the MLL4 binding site loss occurs. These findings highlight a possible novel role for MLL4 in fine-tuning 3D genome organization. The study advances understanding of the complex relationship between genome structure and function and deepens insights into the role of 3D genome organization in Kabuki syndrome, a topic that remains underexplored.