Tracking DOT1L methyltransferase activity by stable isotope labelling using a selective synthetic co-factor

Abstract

Epigenetic processes influence health and disease through mechanisms which alter gene expression. In contrast to genetic changes which affect DNA sequences, epigenetic marks include DNA base modifications or post-translational modification (PTM) of proteins. Histone methylation is a prominent and versatile example of an epigenetic marker: gene expression or silencing is dependent on the location and extent of the methylation. Protein methyltransferases exhibit functional redundancy and broad preferences for multiple histone residues, which presents a challenge for the study of their individual activities. We developed an isotopically labelled analogue of co-factor S-adenosyl-L-methionine (¹³CD₃-BrSAM), with selectivity for the histone lysine methyltransferase DOT1L, permitting tracking of methylation activity by mass spectrometry (MS). This concept could be applied to other methyltransferases, linking PTM discovery to enzymatic mediators.

Fig. 1. Overview of previous work in the field and the approach developed in this study to track HKMT activity.

a HKMTs catalyse turnover of the SAM co-factor to generate mono-, di- or trimethylated lysine residues on protein substrates; (b) application of the bump-and-hole approach to track lysine labelling by the HKMT G9a. A co-factor analogue bearing an alkyl “bump” was designed to selectively bind an engineered form of G9a featuring a “hole” created by a Y1154A mutation; (c) MS method of detecting protein methylation by DOT1L developed in this work. The selective co-factor analogue, ¹³CD₃-BrSAM, binds to DOT1L to transfer a heavy methyl group to a protein substrate, which is digested to peptides and analysed by MS. The ¹³CD₃-labelled peptide appears with a + 4 isotopic shift relative to the corresponding endogenous light methyl peptide; (d) X-ray co-crystal structures of SAH:DOT1L (left, green, PDB 3QOX) and bromo-deaza-SAH (5):DOT1L (middle, turquoise, PDB 3SX0). An overlay of the two inhibitors is shown on the right, RMSD = 0.092 Å.

Fig. 2. Synthesis of ¹³CD₃-BrSAM, 7.

Bromo-deaza-SAH 5 was synthesized using modification of previously reported conditions (see Supplementary Methods provided in the SI), then converted to 7 using a heavy methyl triflate reagent.

Fig. 3. Titration response curves for the treatment of nucleosomes with heavy labelled co-factor analogues in the presence of DOT1L.

The heavy mono-methyl peptide signal was quantified relative to the K79un-containing peptide standard (see also Supplementary Fig. 2 in the SI) and calculated as a proportion over the total unmodified, light mono-methyl and heavy mono-methyl signals, as carried out for the time course assays. Error = s.d. between three replicates. See Supplementary Methods provided in the SI.

Fig. 4. MS detection of heavy-labelled nucleosomes incubated with methyltransferases and heavy co-factors.

a Representative mass spectra of H3K79 showing masses corresponding to mono and di-methyl K79 with “light” (CH₃ – from endogenous/pre-existing methylation) and “heavy” (¹³CD₃) labelling arising from transfer of isotopically labelled methyl-groups from ¹³CD₃-BrSAM. b The relative abundance (100 x abundance / sum of all abundances for each condition) of each H3K79 methyl peptide, calculated from the area of the extracted ion chromatograph for each peptidoforms. c An annotated fragmentation spectra for H3K79 heavy demethylation (2[¹³CD₃]), orange = a/b-ion series, pink = y-ion series, green = parent ion. The mass difference from the theoretical mass is given by each peak in Da (see Supplementary Table 4 for peak lists). rt = retention time. d–g Detection of heavy-methyl peaks H3K9, H3K36, H3K4, SUV420H2 after incubation with their respective modifying enzyme and heavy-labelled SAM (¹³CD₃). There was no heavy labelling observed following the same reaction conditions in the presence of heavy labelled BrSAM. The number of heavy labels in the methyl-moiety is indicated (u = unmodified, m/d/t = mono/di/tri-methylation, a = acetylation). h, i Representative mass spectra showing heavy methyl-peaks observed for H3K9 in the presence of ¹³CD₃SAM but not ¹³CD₃-BrSAM.