Specificity of protein lysine methyltransferases and methods for detection of lysine methylation of non-histone proteins Philipp Rathert, a Arunkumar Dhayalan, a Huimin Ma ab and Albert Jeltsch* a DOI: 10.1039/b811673c Post translational modification of histone proteins including lysine methylation is an important epigenetic mark, essential for gene regulation and development. Recently, several examples of lysine methylation of non-histone proteins have been discovered suggesting that this is a common post-translational modification for regulation of protein activity. Here, we review assays for the detection of protein methylation based on mass spectrometry, radiolabel and immunological approaches using protein and peptide substrates including application of SPOT peptide arrays. Candidates for new methylation targets of protein methyltransferases can be predicted using the specificity of the enzyme and protein interaction data. Epigenetic regulation of gene expres- sion by covalent modification of histone proteins and methylation of DNA plays an important role during development and disease processes (reviews 1–4). Post-translational modifications of his- tone proteins include acetylation, phos- phorylation, methylation, ubiquitylation and sumoylation (reviews 5,6). Many of these modifications occur on the N-terminal tails of the histone proteins that protrude from the nucleosomes. Methylation of lysine residues in histone tails has been identified in histone H3 K4, K9, K27, and K36, histone H4 K20, and histone H1b K26 which all have different biological functions (reviews 6,7). For example, H3K9 methylation leads to condensation of the chromatin and inhibition of gene expression. Similarly, H3K27 methylation has been linked to gene silencing, whereas H3K4 methylation is marking active chromatin (review 8). In 2000, Rea et al. identified Suv39H1 which was the first histone lysine methyl- transferase (HKMT). 9 Today about 30 different enzymes of this class are known in different species (review 10). Most HKMTs contain a SET domain of approxi- mately 130 amino acids as catalytic part and use the coenzyme S-adenosyl-L- methionine (AdoMet) as the donor for an activated methyl group (review 11). HKMTs not only differ in their substrate specificity but also in product pattern, because one, two or three methyl groups can be added to a single lysine residue, which can have different biological meanings (review 11). The first non-histone substrate identi- fied for an HKMT was the tumor sup- pressor p53 which is methylated by Set7/9, a SET domain containing enzyme initially identified as histone H3 K4 methyltransferase. 12 Methylation of p53 by Set7/9 at a specific residue within the C-terminal regulatory domain activates p53, 12 because it is required for the bind- ing of the acetyltransferase Tip60 and for the subsequent acetylation of p53. 13 In contrast, methylation of p53 at two other lysine residues by SMYD2 or Set8 leads to the inactivation of p53. 14,15 The tran- scription factor TAF10 was the next non-histone protein found to be methyl- ated by Set7/9. 16,17 Methylated TAF10 has an increased affinity for RNA poly- merase II, pointing to a direct role of this modification in pre-initiation complex formation. 16 The S. cerevisia COMPASS complex methylates a kinetochore com- ponent called Dam1 during chromosome segregation. 18 In addition, amongst others, cytochrome C and Rubisco are methylated at lysine residues as well (reviews 19,20). Recently it was demon- strated, that the histone methyltransfer- ase G9a (which was previously shown to methylate Histone H3 lysine 9 and 27 21 ) catalyses automethylation and that it acts on a variety of different non-histone proteins. 22,23 The new targets include not only transcription factors, but also enzymes, which themselves are involved in transcriptional regulation by changing the epigenetic information. 23 The mod- ification of non-histone proteins by the addition of methyl groups to lysines was demonstrated to have two different pos- sible downstream signaling pathways. The first one has been shown for the G9a automethylation and the methyla- tion of the transcription factors WIZ and ACINUS 22,23 where the post-transla- tional modification of the protein attracts reading domains that specifically bind the methylated lysine similarly as established for reading the histone methylation code (review 24). Therefore, the methylation could direct the non- histone proteins to specific sites inside the cell. The second mechanism was demon- strated for the CDYL1 histone acetyltransferase: 23 the methylation of CDYL1 affects the binding of its chromo- domain to H3K9 methylated peptides, because the methylated internal peptide competes with the external methylated H3K9 peptide for binding to the chromodomain. a Biochemistry Laboratory, School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany. E-mail: a.jeltsch@jacobs-university.de b State Key Laboratory of Organic Geochemistry, Guangzhou Research Center of Mass Spectrometry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou, 510640, China 1186 | Mol. BioSyst., 2008, 4, 1186–1190 This journal is  c The Royal Society of Chemistry 2008 HIGHLIGHT www.rsc.org/molecularbiosystems | Molecular BioSystems