Figure 1: Dnmt3a is dimethylated by GLP in vitro. | Nature Communications

Figure 1: Dnmt3a is dimethylated by GLP in vitro.

From: MPP8 mediates the interactions between DNA methyltransferase Dnmt3a and H3K9 methyltransferase GLP/G9a

Figure 1

(a) Dnmt3a and GLP domain arrangements (h: human; m: mouse). Underlining indicates the shared TARK sequence motif that includes K44 of mDNMT3a, K47 of hDNMT3A, and K9 of histone H3. (b) The N-terminal domain of mDnmt3a (residues 1–274) is methylated (lanes 1 and 3). The methylation reaction was carried out with [3H]-AdoMet and analysed by fluorography. The N-terminal domain of mDnmt3b is not methylated (lane 2). A K44R substitution in mDnmt3a eliminates methylation (lane 4). (c) Mass spectrometry of the products of in vitro methylation assays (0 h, preincubation; 1 and 5 h, after reaction) after isolating the methylated mDnmt3a (residues 1–274) from SDS-gel, performing in-gel digestion with V8 protease, and analysing the peptide fragments by MALDI mass spectrometry. The mass shift of 14 Da represents addition of one methyl group. (d) Gel image (top) and autoradiogram (bottom) of in vitro methylation assays with His-GLP-catalytic SET domain on various GST-tagged hDNMT3A N-terminal fragments (labelled with *) with residue numbers indicated. The N-terminal fragments of hDNMT3A (residues 1–223 and 1–94) are methylated (lanes 2 and 3). (e) K47R substitution in hDNMT3A N-terminal domain (residues 1–223) eliminates methylation (lane 4). (f) K47R substitution in hDNMT3A, in the context of full-length protein, eliminates methylation.

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