t(4;14)-positive cells have a different histone methylation pattern than t(4;14)-negative cells. (A) Nuclear extracts from 9 t(4;14)-positive cells and 7 t(4;14)-negative cells were immunoblotted with the indicated antibodies. MMSET expression correlates with higher H3K36me2 and lower H3K27me3 marks. (B) Intact mass profiles of histones H3.1, H3.2, and H3.3 from 4 multiple myeloma cell lines. H929 and LP1 are t(4;14)+ and show a global increase in H3.1 and H3.2 methylation compared with the t(4;14)− KMS12 and MM.M1 cells. The [M+18H]¹⁸⁺ species are shown. Distinct peaks correspond to different numbers of methyl or acetyl groups (14 and 42 Da). The 6 methyl equivalent is labeled for each histone variant. (C) K36 methylation levels were determined from y₁₇⁴⁺, y₁₈⁴⁺, and y₁₉³⁺ from collision-induced dissociation (CID) of the N-terminal 1-50 [M+9H]⁹⁺ peptide of H3.1. (D) K36 dimethylation is reported on by y₁₉³⁺ ions (717.42 m/z) from CID of the N-terminal 1-50 [M+9H]⁹⁺ peptide of H3.1. Peak intensity (arbitrary units) is shown in the top right corner for each mass spectrum. (E) K27 methylation levels were determined from y₂₄⁴⁺, y₂₇⁴⁺, y₃₅⁶⁺, and y₃₆⁶⁺ for K36+K27 from CID of the N-terminal 1-50 [M+9H]⁹⁺ peptide of H3.1. Values for K27 methylation were deconvoluted from K36+K27 and K36 data using a system of inequalities (Mathematica). The methylation levels, with SEM, are shown in supplemental Table 1.