Bottom-up proteomics of EZH2 mutants in isogenic cells

The Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) is an essential epigenetic modifier able to methylate lysine 27 on histone H3 (H3K27) to induce chromatin compaction, protein complex recruitment and ultimately transcriptional repression. Hematologic malignancies, including Diffuse Large B cell lymphoma (DLBCL) and Acute myeloid leukemia (AML) have shown a high EZH2-mutation frequency (>20%) associated with poor clinical outcomes. Particularly, two distinct oncogenic mutations, so-called gain-of-function (Y641F and A677G) and loss-of-function (H689A and F667I) are found in the catalytic domain of EZH2. In this study, a comprehensive multi-omics approach was employed to characterize downstream effects of H3K27me3 deposition driven by EZH2 mutations. Human embryonic kidney cells (HEK293T) were transfected to generate three stable EZH2 mutants: EZH2(Y641F), EZH2(A677G), and EZH2(H689A/F667I), which were validated via immunoblotting and DIA-MS-based histone profiling assay. Subsequently, constructs were analyzed under a comprehensive multi-omics approach including label-free whole-cell proteomics, acquired with a Bruker timsTOF Pro HPLC-MS/MS with Ion Mobility. Important protein interactors at nuclear level were dysregulated, such as SMYD3 (SET and MYND domain containing 3), NSD2 (nuclear receptor binding SET domain protein 2) and CHD7 (chromodomain helicase DNA binding protein 7), suggesting a cooperative network of chromatin remodelers for gene expression reprogramming.