Mutant EZH2 alters the epigenetic network and increases epigenetic heterogeneity in B cell lymphoma (CUT&Run)

Diffuse large B cell lymphomas and follicular lymphomas show recurrent mutations in epigenetic regulators; among these are loss-of-function mutations in KMT2D and gain-of-function mutations in EZH2. To systematically explore the effects of these mutations, alone or in combination, on the epigenetic landscape, we applied a single cell approach, based on Cytometry by Time of Flight (CyTOF), to probe a wide array of histone modifications. We show that mutant-EZH2 elicits extensive effects on the epigenome of lymphomas, beyond alterations to H3K27 methylations, and is dominant over KMT2D mutations. Utilizing the single-cell data, we present computational methods to measure epigenetic heterogeneity. We identify an unexpected characteristic of mutant-EZH2, but not KMT2D, in increasing heterogeneity, shedding light on a novel oncogenic mechanism mediated by this mutation. Finally, we present analysis to reveal potential cross talk between various modifications in the epigenetic network, validated by functional perturbations. Our work highlights novel roles for mutant-EZH2 in lymphomagenesis and establishes new concepts for measuring epigenetic heterogeneity and intra-chromatin connectivity in cancer cells 0.2m of cells were incubated with primary antibody, then pAG-MNase enzyme was added to each sample followed by incubation step of 1 h at 4°C. Targeted digestion was done by 15 min incubation on ice block (0°C) under low salt conditions. DNA purification was done using Nucleospin gel and PCR clean-up kit (Machery-Nagel, 740609 ). Sequencing was done on a Next-Seq 500 instrument (Illumina) using a V2 150 cycles mid output kit, allocating 10M reads per sample (paired end sequencing).