Total kinetic analysis reveals how combinatorial methylation patterns are established on lysines 27 and 36 of histone H3

Abstract

We have developed a targeted method to quantify all combinations of methylation on an H3 peptide containing lysines 27 and 36 (H3K27-K36). By using stable isotopes that separately label the histone backbone and its methylations, we tracked the rates of methylation and demethylation in myeloma cells expressing high vs. low levels of the methyltransferase MMSET/WHSC1/NSD2. Following quantification of 99 labeled H3K27-K36 methylation states across time, a kinetic model converged to yield 44 effective rate constants qualifying each methylation and demethylation step as a function of the methylation state on the neighboring lysine. We call this approach MS-based measurement and modeling of histone methylation kinetics (M4K). M4K revealed that, when dimethylation states are reached on H3K27 or H3K36, rates of further methylation on the other site are reduced as much as 100-fold. Overall, cells with high MMSET have as much as 33-fold increases in the effective rate constants for formation of H3K36 mono-and dimethylation. At H3K27, cells with high MMSET have elevated formation of K27me1, but even higher increases in the effective rate constants for its reversal by demethylation. These quantitative studies lay bare a bidirectional antagonism between H3K27 and H3K36 that controls the writing and erasing of these methylation marks. Additionally, the integrated kinetic model was used to correctly predict observed abundances of H3K27-K36 methylation states within 5% of that actually established in perturbed cells. Such predictive power for how histone methylations are established should have major value as this family of methyltransferases matures as drug targets.epigenetics | EZH2 | multiple myeloma | mass spectrometry | histone code S everal lysine residues in the tails of histones can be mono-, di-, or trimethylated from yeast to human. These position-and state-specific modifications have been implicated in many chromatin template activities (1). The critical roles of these modifications are further supported by their association with many physiological alterations and diseases (2-4). Genome-wide ChIP studies have provided extensive information regarding the localization of known histone methylation marks (5-7). However, this approach mainly captures snapshots of histone methylation. Therefore, it is unable to answer how methylation patterns are faithfully reestablished and maintained on newly synthesized or old histones to understand mechanisms of epigenetic inheritance (8, 9).There are eight known histone methyltransferases (HMTs) targeting H3K36, including NSD1/2/3, SETD2, ASH1L, SET-MAR, SMYD2, and SETD3 (10). Among them, MMSET (NSD2/ WHSC1) preferentially methylates nucleosomal H3K36 and is capable of catalyzing the addition of as many as two methyl groups at this site (i.e., an H3K36 dimethylase) (11). By contrast, polycomb repressive complex 2 (PRC2) is the only known HMT for H3K27, consisting of four core subunits: EZH2 (catalytic subunit), EED, SUZ12, and RbAp48. Unlike the constitutively active H3K36 HMT, the ca...

Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns*

“…We found that K27me3 rarely co-exist with K36me1/me2 (except for the H3⌬G12 tail). This result conforms to previous studies suggesting antagonism between high methylation states of H3K27 and H3K36 (23,24). K14ac and K23ac were found to be mutually exclusive by interplay analysis, in agreement with a previous report (15).…”

Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression

“…By contrast, combinatorial modifications define the co-existence of any number of modifications that is greater than two on proteolytic peptides or intact proteins. For example, we have identified 15 combinatorial modifications of H3K27 and H3K36 methylation in our previous Bottom-Up study of peptides from H3.1 and H3.2 containing residues 27 to 40, without knowing the modification status beyond this region (29). In this study, we were able to achieve a clean isolation of each Methyl-Eq by virtue of the 0.6 Th isolation window, which ensured the MS 2 spectra were derived from a single isolated precursor.…”

Phosphorylation-mediated EZH2 inactivation promotes drug resistance in multiple myeloma

“…These results underscore the importance of EZH2-mediated H3K27 trimethylation in drug sensitivity in MM cells. Finally, we asked whether MMSET exerted drug resistance via the same mechanisms because MMSET overexpression downregulates H3K27me3 and has been identified as a marker of worse prognosis in MM patients (7,8). To this end, we overexpressed MMSET in t(4;14)-negative RPMI8226 cells and knocked it down in t(4;14)-positive KMS-11 cells.…”

“…For example, the SET domain-containing histone methyltransferase MMSET/KMT3G is overexpressed in an aggressive form of MM carrying t (4;14), leading to altered chromatin states by inducing an increase in H3K36me2 and decrease in H3K27me3 (7,8). The oncogenic function of MMSET is closely associated with its catalytic activity in MM (9)(10)(11), making it amenable to therapeutic interventions.…”