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. 2011 Mar 11;286(10):8361-8368.
doi: 10.1074/jbc.M110.204115. Epub 2010 Dec 31.

The structure of NSD1 reveals an autoregulatory mechanism underlying histone H3K36 methylation

Qi Qiao  1 , Yan Li  2 , Zhi Chen  1 , Mingzhu Wang  1 , Danny Reinberg  3 , Rui-Ming Xu  4
Affiliations

The structure of NSD1 reveals an autoregulatory mechanism underlying histone H3K36 methylation

Qi Qiao et al. J Biol Chem. .

Abstract

The Sotos syndrome gene product, NSD1, is a SET domain histone methyltransferase that primarily dimethylates nucleosomal histone H3 lysine 36 (H3K36). To date, the intrinsic properties of NSD1 that determine its nucleosomal substrate selectivity and dimethyl H3K36 product specificity remain unknown. The 1.7 Å structure of the catalytic domain of NSD1 presented here shows that a regulatory loop adopts a conformation that prevents free access of H3K36 to the bound S-adenosyl-L-methionine. Molecular dynamics simulation and computational docking revealed that this normally inhibitory loop can adopt an active conformation, allowing H3K36 access to the active site, and that the nucleosome may stabilize the active conformation of the regulatory loop. Hence, our study reveals an autoregulatory mechanism of NSD1 and provides insight into the molecular mechanism of the nucleosomal substrate selectivity of this disease-related H3K36 methyltransferase.

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Figures

FIGURE 1.
FIGURE 1.
Domain structure and sequence alignment of NSD1-CD. A, the domain structure of NSD1-CD. Each domain is represented by a colored box with the domain name indicated (N, N-terminal domain; P, post-SET domain). The numbers above and below the colored boxes indicate the numbers of the first and last residues of the domains, respectively. The red box indicates the post-SET loop connecting the SET and post-SET domains. B, structure-guided sequence alignment of the catalytic domains of human NSD1, SET2, ASH1, MLL1, and Neurospora Dim-5. Identical residues are indicated with white letters over a blue background, similar residues are highlighted in yellow, and those with all but one identical residue are shown in red. Different domains are enclosed in boxes colored as in A. A schematic diagram of the secondary structure elements of NSD1-CD is shown above the sequences, and every 10 residues is indicated with a + sign. Numbers to the left or right of the sequence indicate the numbering of the end residues. Note that the numbering for Dim-5 follows that of the PDB entry (1PEG). A red triangle below the sequences indicates the positions of missense mutations associated with Sotos syndrome.
FIGURE 2.
FIGURE 2.
HKMT assay of NSD1-CD. A, nucleosomes assembled with recombinant, unmethylated wild-type or mutant histones (H3K18A, H3K27A, H3K36A, and H4K20A) or nucleosomes bearing methyl analogs mimicking different degrees of methylation on H3K36 (H3K36Me1, H3K36Me2 and H3K36Me3). Top panel, 3H fluorography using [3H]AdoMet as the methyl donor. Bottom panel, Coomassie Brilliant Blue (CBB) staining of histones. The concentration of NSD1-CD used for the assay was 0.1 μm, and that of nucleosomes in the reactions was 0.35 μm. B, HKMT activity on recombinant histone octamers and nucleosomes. Increasing concentrations (0.03, 0.1, and 0.3 μm) of NSD1-CD were titrated in the assay. Top panel, 3H fluorography shows that histones H3, H2A/2B, and H4 were methylated using histone octamers, whereas only H3K36 was methylated when nucleosomes were used as substrates. Middle panel, Coomassie Brilliant Blue staining of histones. Bottom panel, Coomassie Brilliant Blue staining of NSD1-CD.
FIGURE 3.
FIGURE 3.
Structure of NSD1-CD. A, a ribbon representation of the overall structure. Different segments of the protein are colored following the coloring scheme in Fig. 1A. The AdoMet molecule is shown as a stick model (green, carbon; blue, nitrogen; red, oxygen). Zinc ions are shown as gray spheres. Secondary structure elements and the protein termini are labeled. B, superposition of NSD1 (cyan), SET2 (yellow; PDB id: 3H6L) and Dim-5 (magenta; PDB id: 1PEG) structures. The SET domains are highly similar, and for clarity, only the NSD1 SET domain is shown. Dashed lines indicate structurally disordered regions. C, superposition of the post-SET domains of NSD1, SET2, Dim-5, and MLL1 (PDB id: 2W5Y). The magenta and green dashed lines indicate the disordered loop segments of Dim-5 and MLL1, respectively.
FIGURE 4.
FIGURE 4.
Structure of the active site and modeling of substrate binding. A, the post-SET loop of NSD1 occludes substrate binding. The H3K9 peptide (magenta) bound to Dim-5 and the H4K20me1 peptide (yellow) bound to Pr-Set7/Set8 (gray; PDB id: 2BQZ) are superimposed with the NSD1-CD structure. For clarity, the structure of Dim-5 is not shown, and only the post-SET domain of Pr-Set7/Set8 is shown. The loop colored in red represents the post-SET loop of NSD1-CD, and the rest of the protein structure is colored cyan. AdoMet is shown as a stick model (green, carbon; blue, nitrogen; red, oxygen), as are H3K9 (magenta, carbon) and H4K20me1 (yellow, carbon). B, amino acids surrounding the AdoMet molecule and their interactions with AdoMet. C, conformational dynamics of the post-SET loop. The post-SET loops from the crystal structure (cyan), from the molecular dynamics simulation at 276 ps (yellow), and from an energy-minimized model of nucleosome docking (red) are superimposed. The docked H3 tail and nucleosomal DNA are shown in magenta and light blue, respectively. H3K36 is shown as a stick model. Note that in the docked model, the post-SET domain contacts DNA. D, a global view of the modeled NSD1-CD complex with a nucleosome core particle. NSD1-CD is shown in red; two H3 molecules are shown in magenta and salmon; two H4 molecules are shown in green and light green; and H2A and H2B are shown in blue, and DNA is shown in light blue.
FIGURE 5.
FIGURE 5.
Sotos syndrome missense mutations in NSD1-CD. A, amino acids at positions where the missense mutations of Sotos syndrome occur are shown as stick models (red and magenta), superimposed on a ribbon diagram of the NSD1-CD structure. AdoMet is shown as a stick model (yellow, carbon; blue, nitrogen; red, oxygen), and the spheres represent zinc ions. The residues shown in red correspond to arginines selected for in vitro mutagenesis and HKMTase assays. B, five arginine residues were individually changed to amino acids found in Sotos syndrome patients, and their HKMTase activities toward recombinant nucleosomes (Nucs) and histone octamers (Octs) were assayed using [3H]AdoMet as the methyl donor (top panel). The concentrations of NSD1-CD used in the assays were 0.04 and 0.2 μm, and those of nucleosomes and histone octamers were both 0.35 μm. Coomassie Brilliant Blue (CBB) staining of histones and the enzyme are shown in the middle and bottom panels, respectively.
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