Xenbase Image ID: 124469

FIGURE 7. NSD2-SET domain can methylate H4K44 on octamers. A, schematic of tail-less histones (left panel). ΔH3 lacks amino acid 1–26, ΔH4 lacks amino acid 1–19, ΔH2A lacks amino acid 1–12, and ΔH2B lacks amino acid 1–23. The tail-less histones were assembled into octamers, and the HKMT assay was carried out on wild type (WT) or tail-less octamers (right panel). 41-bp dsDNA was added in some reactions as indicated. The reactions without enzyme were included as negative controls. B, mapping of the H4 methylation site by mass spectrometry. MALDI-TOF mass spectra for Arg-C digests (upper panel) and Asp-N digests (lower panel) of H4 methylated by NSD2-SET. The peptide identified as H4 (amino acids 42–54) exhibited mono- and dimethylation (upper panel), whereas the peptide identified as H4 (amino acids 1–23) was unmethylated (lower panel). The calculated protonated monoisotopic masses of peptide 41–55 carrying zero to two methylations are 1677.92, 1691.94, and 1705.95, respectively. Tandem mass spectra of precursor ions with m/z of 1691.94 and 1705.95 confirmed the mono- and dimethylation on Lys44 (data not shown). Unmodified peptide 1–23 has a calculated protonated monoisotopic mass of 2360.43. C, HKMT assay with octamers composed of WT H4 or H4K44A (left panel) or H4K44Q (middle panel). The enzyme was titrated at two different amounts, and 41-bp dsDNA was added to the assays as indicated. HKMT assays performed with WT H4, H4K44A, H4K44Q, or H4K44E octamers assembled onto nucleosomes (right panel). Image published in: Li Y et al. (2009) © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license Larger Image Printer Friendly View

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