FIGURE 1.

Post-translational modifications of MeCP2 from mouse brain identified by mass spectrometry analysis. (A) MeCP2 domain structure comprising N-terminal domain (NTD), methyl-CpG binding domain (MBD), intervening domain (ID), N-CoR/SMRT interacting domain (NID), transcriptional repression domain (TRD) and C-terminal domain (CTD). (B) MeCP2 protein sequence with modifications identified by mass spectrometry. MBD and NID, sequence coverage, sequence motifs, PTMs (acetylation, phosphorylation, methylation, dimethylation) and the number of biological replicates from independent experiments are marked as indicated. Modifications selected for further validation are marked in red. The software-based annotation of spectra and modification sites shown were obtained from Proteome Discoverer and MaxQuant. The arginine methylation sites selected for further validation were additionally manually inspected (see Supplementary Figures S4–6). The location of modifications identified on the same peptide might be uncertain. (C) Western blot analysis of mouse brain nuclei extracts tested for monomethyl arginine (MMA), symmetric dimethyl arginine (SDMA), asymmetric dimethyl arginine (ADMA), tyrosine phosphorylation (phosTyr), S80 and S421 phosphorylation (phos) and reprobed with MeCP2 specific antibodies. The full membranes of the Western blots are shown in Supplementary Figure S7. (D) Western blot analysis of MeCP2-GFP purified from human HEK cells and MeCP2 purified from E. coli probed with the same modification specific antibodies and reprobed with MeCP2 specific antibodies. The Ponceau stain visualizes the total proteins on the membrane and the full membranes are shown in Supplementary Figure S7. (E) Scheme of the arginine methylation reaction: protein arginine methyltransferases (PRMTs) can catalyze the monomethylation of arginine and subsequently the dimethylation, which can occur either on the same nitrogen (ADMA, catalyzed by type I PRMTs) or on the unmodified nitrogen (SDMA, catalyzed by type II PRMTs).