Dual interactions of K27M mutant nucleosomes with PRC2 and MLL1 shape the cancer epigenetic landscape [CUT&RUN, HEK293]

Cancer associated mutations in genes encoding histones dramatically reshape chromatin patterns and support tumorigenesis, despite their low prevalence. Lysine to Methionine substitution of residue 27 on histone H3 (K27M) is a driver mutation in high-grade pediatric gliomas, known to abrogate Polycomb activity. We applied single-molecule systems to image individual nucleosomes and delineate the combinatorial chromatin patterns associated with K27M expression. We find that K27M-mutant nucleosomes not only sequester PRC2, but also bind preferentially to MLL1, thus leading to genome-wide redistribution of H3K4me3. K27M-mediated deregulation of both repressive and activating chromatin marks maintains ‘bivalent’ chromatin, supporting a poorly differentiated cellular state. This study provides evidence for widespread effects of the K27M oncohistone on multiple core epigenetic pathways, and highlights the use of single-molecule tools to reveal mechanisms of chromatin deregulation in cacner. Cut&Run analysis of HEK293 cell line. CUT&RUN assay was done as described in Skene et al (2017) with slight modifications as follows. Cells were harvested and counted with 200000 cells per reaction. Permeabilized cells, bound to Concanavalin A-coated beads were mixed with individual primary antibody and incubated overnight at 4°C while rotating. Secondary antibody, anti-rabbit HRP was used as a negative control.