A General Molecular Affinity Strategy for Global Detection and Proteomic Analysis of Lysine Methylation

Abstract

Summary Lysine methylation of histone proteins regulates chromatin dynamics and plays important roles in diverse physiological and pathological processes. However, beyond histone proteins, the proteome-wide extent of lysine methylation remains largely unknown. We have engineered the naturally occurring MBT domain repeats of L3MBTL1 to serve as a universal affinity reagent for detecting, enriching, and identifying proteins carrying a mono- or di-methylated lysine. The domain is broadly specific for methylated lysine (“pan-specific”) and can be applied to any biological system. We have used our approach to demonstrate that SIRT1 is a substrate of the methyltransferase G9a both in vitro and in cells, to perform proteome-wide detection and enrichment of novel methylated proteins, and to identify candidate in-cell substrates of G9a and the related methyltransferse GLP. Together, our results demonstrate a powerful new approach for global and quantitative analysis of methylated lysine, and they represent the first systems biology understanding of lysine methylation.

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

“…2D-2E), including previously observed sites in CAPRIN1 (K675) (25) and PDAP1 (K126) (6, 7) that had not been ascribed to SMYD2 activity. Interestingly, multiple novel Kme1 sites in AHNAK (K2589, K1205, and K1791) and AHNAK2 (K939, K932, and K3414) were significantly downregulated by SMYD2 knockdown and inhibition, indicating that multiple sites within these proteins are regulated SMYD2 activity.…”

“…Immunoaffinity enrichment is particularly critical in the case of methyl-lysine peptides, as the methyl moiety itself imparts sufficiently little physiochemical change to the side chain, rendering the enrichment of these peptides refractory to conventional separation methods. Other approaches that exploit the naturally-occurring binding specificity of methyl-lysine recognition protein domains, such as chromodomains and MBT domains, have also shown some promise as enrichment reagents (24,25). Nevertheless, the enrichment of non-histone methyl-lysine peptides has not yet reached the success that other types of PTMs (i.e.…”

Application of histone modification-specific interaction domains as an alternative to antibodies

“…This approach has several distinct advantages over antibodies, such as the ease and cost-effectiveness of recombinant production of HMIDs in Escherichia coli, the amenability of HMIDs to protein engineering, and the possibility of producing them at constant quality, eliminating lot-to-lot variability. In support of this concept, affinity methods based on protein domains have been successfully employed for the enrichment of methylated or unmethylated CpG islands in the analysis of DNA methylation (Cross et al 1994;Blackledge et al 2012) or in proteome-wide analyses of non-histone lysine methylation (Liu et al 2013;Moore et al 2013). In this proof-of-principle study, we started by characterizing the specificity of several HMIDs and compared them with ENCODE-validated antibodies for the same PTM using peptide arrays.…”

Functional Crosstalk Between Lysine Methyltransferases on Histone Substrates: The Case of G9A/GLP and Polycomb Repressive Complex 2

“…Beyond histones, G9a/GLP have been shown to methylate a plethora of non-histone targets, including the tumor suppressor p53, SIRT1, Reptin, MyoD, Wiz, CDYL and several chromatin regulators (50,67,77,88,120). Moreover, G9a methylates itself and this modification mediates its interaction with HP1 and CDYL (127).…”