FIGURE 4.

NSD2-SET domain dimethylates H3K36 on nucleosomes. A, a schematic representation of NSD2 and SET2 domain organization is shown on top, and the sequence alignment of the SET domains of NSD1–3 and SET2 is shown on the bottom. Identical residues are replaced by dots, similar residues are shown in black, and others are in red. The active site residues described in previous SET domain structures are highlighted in yellow. PWWP, PWWP domain containing proline-tryptophan-tryptophan-proline motif in consensus amino acid sequence. B, HKMT assays were performed with NSD2-SET and SET2-C on wild type (WT) and H3K36A nucleosomes using unlabeled SAM, and the product of the reaction was analyzed by Western blot using anti-H3K36me3 (upper panel) and anti-H3K36me2 (lower panel) antibodies. C, recombinant histones bearing methyl analogs mimicking different degrees of methylation on H3K36 were generated by chemical reaction as described under “Experimental Procedures” and analyzed by Western blot using anti-H3K36me2 and anti-H3K36me3 antibodies. D, premethylated histones were assembled into nucleosomes and used as substrates in HKMT assays with NSD2-SET (left panel) and SET2-C (right panel). Me1, monomethyl-H3ϕK36; Me2, dimethyl-H3ϕK36; Me3, trimethyl-H3ϕK36. E, MALDI-TOF mass spectra for chymotyptic digests of H3 (top panel), H3 methylated by NSD2 (middle panel), and H3 methylated by SET2-C (bottom panel). HKMT assays were performed on nucleosomes, and the products of the reaction were separated by SDS-PAGE. The H3 bands were excised and digested in-gel using chymotrypsin, and the resulting peptides were analyzed using MALDI-TOF MS. The calculated protonated monoisotopic masses of peptide 21–41 carrying zero to three methylations are 2195.25, 2209.27, 2223.28, and 2237.30, respectively. The asterisk denotes a proteolyzed form of H3.