Abstract
Histones, and their modifications, are critical components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of combinatorial modifications (i.e. methylation and acetylation) on full length histone H3 from human cell lines derived from multiple myeloma patients with overexpression of the histone methyltransferase MMSET as the result of a t(4;14) chromosomal translocation. Using the latest in Orbitrap-based technology for clean isolation of isobaric proteoforms containing up to 10 methylations and/or up to two acetylations, we provide extensive characterization of histone H3.1 and H3.3 proteoforms. Differential analysis of modifications by electron-based dissociation recapitulated antagonistic crosstalk between K27 and K36 methylation in H3.1, validating that full-length histone H3 (15 kDa) can be analyzed with site-specific assignments for multiple modifications. It also revealed K36 methylation in H3.3 was affected less by the overexpression of MMSET because of its higher methylation levels in control cells. The co-occurrence of acetylation with a minimum of three methyl groups in H3K9 and H3K27 suggested a hierarchy in the addition of certain modifications. Comparative analysis showed that high levels of MMSET in the myeloma-like cells drove the formation of hypermethyled proteoforms containing H3K36me2 co-existent with the repressive marks H3K9me2/3 and H3K27me2/3. Unique histone proteoforms with such "trivalent hypermethylation" (K9me2/3-K27me2/3-K36me2) were not discovered when H3.1 peptides were analyzed by Bottom-Up. Such disease-correlated proteoforms could link tightly to aberrant transcription programs driving cellular proliferation, and their precise description demonstrates that Top-Down mass spectrometry can now decode crosstalk involving up to three modified sites.
Highlights
From the ‡Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, 2145 N
Direct Infusion Mass Spectrometry of reverse phase high pressure liquid chromatography (RP-HPLC) Purified Histone H3.1 and H3.3— all three H3 variants are fully separable by RP-HPLC, we focused primarily on H3.1 and H3.3 which differ in only five residues
We observed a ϳ20% increase of H3.3 and a ϳ10% increase of H3.2 counterbalanced by ϳ25% loss of H3.1 in MMSET-High nontargeted knockout (NTKO) cells compared with targeted knockout (TKO)
Summary
From the ‡Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, 2145 N. Histone modifications, including methylation (me), acetylation (ac), monoubiquitylation (ub1), etc., are related to the study of epigenetics [11, 12] These modifications, as well as their different states in case of methylation (i.e. mono-, di-, and trimethylation) and positions on the histone, play important and distinct roles in almost every activity operative on the chromatin template. The significance of these modifications are further underscored by the unexpected identification of many driver mutations underlying cancer biology within histone modifying enzymes [13, 14] and somatic mutations in histone H3.3 [7, 15, 16]. The value of mass spectrometric analysis of histone modifications in the field of cancer epigenetics was further demonstrated by the recent identification of a recurrent point mutation of E1099K in MMSET in lymphoid malignancies [8, 21]
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