Crystal structure of the human histone methyltransferase ASH1L catalytic domain and its implications for the regulatory mechanism

Abstract

Absent, small, or homeotic disc1 (Ash1) is a trithorax group histone methyltransferase that is involved in gene activation. Although there are many known histone methyltransferases, their regulatory mechanisms are poorly understood. Here, we present the crystal structure of the human ASH1L catalytic domain, showing its substrate binding pocket blocked by a loop from the post-SET domain. In this configuration, the loop limits substrate access to the active site. Mutagenesis of the loop stimulates ASH1L histone methyltransferase activity, suggesting that ASH1L activity may be regulated through the loop from the post-SET domain. In addition, we show that human ASH1L specifically methylates histone H3 Lys-36. Our data implicate that there may be a regulatory mechanism of ASH1L histone methyltransferases.

FIGURE 1.

Crystal structure of the human histone methyltransferase ASH1L catalytic domain. A, overall structure of the Ash1L-SET catalytic domain. The protein is colored according to panel C. The three zinc ions are shown in orange. The AWS domain shown in yellow is composed of mostly loops, with two short helices interwoven into the SET domain. A loop (shown in magenta) from the post-SET domain is blocking the substrate binding pocket. B, 2F_o − F_c electron density map at 1 σ around the auto-inhibitory loop is shown in magenta. C, schematic representation of the full-length ASH1L and the construct containing the catalytic domain used in this current study (residues 2069–2288). The AWS domain is in yellow, the SET domain is in marine blue, and the post-SET domain is in magenta. BAH, bromo-adjacent homology domain. D, sequence alignment of the catalytic domains of human ASH1L (2069–2288), mouse ASH1L (2056–2275), fly Ash1 (1304–1520), and human NSD2 (991–1209). Hs, Homo sapiens; Mm, Mus musculus; Dm, Drosophila melanogaster.

FIGURE 2.

The auto-inhibitory loop of hASH1L. A, comparison of the hASH1L-SET substrate binding pocket with that of the SET8-histone H4 complex. The auto-inhibitory loop in hASH1L occupies the substrate binding pocket where the histone substrate (colored in pink) binds in the SET8-histone complex structure. B, the auto-inhibitory loop interacts with the SET-I subdomain in the hASH1L SET domain. The carbonyl oxygens of His-2258 and Ser-2259 make hydrogen bonds with Asn-2197. Phe-2260 in the loop interacts with two hydrophobic residues, Val-2203 and Phe-2179. The auto-inhibitory loop also forms a hydrogen bond with AdoMet via Gln-2266. C, the hASH1L SET domain (shown in yellow) is superimposed on the SET8-histone H4 complex structure (SET8 shown in gray and H4 shown in pink). Ser-2259 of hASH1L occupies the substrate Lys position. D, disrupting the interactions between the auto-inhibitory loop and the SET-I subdomain or AdoMet affects the activity of hASH1L. Mutating Asn-2197 or Gln-2265 to alanine (N2197A, Q2265A) increases HMTase activity, whereas F2260A or Q2266A mutations abolish the activity. The HMTase activity was performed with tritium-labeled AdoMet with mononucleosome (Nuc.) or G5E4 nucleosomal array (Nuc. Array) as substrates.

FIGURE 3.

The hASH1L catalytic domain methylates histone H3 Lys-36. A, HMTase assay of the hASH1L catalytic domain was performed with wild-type hASH1L (WT), F2260A, and the hyperactive mutant (Q2265A) (lanes 1, 2, and 3) using recombinant mononucleosomes. Recombinant mononucleosomes (Nuc) and HeLa nucleosomes (Hela Nuc) were used as controls for the antibodies (lanes 4 and 5). The methylation product of the hASH1L catalytic domain is specifically recognized by the anti methyl-H3K36 antibody but not by the methyl-H3K4 antibody. The hyperactive mutant (Q2265A) is able to also produce H3K36 trimethylation. B and C, the wild-type hASH1L SET catalytic domain and the hyperactive mutant methylate the wild-type or H3K4A mutant histone, octamer, and nucleosome (Nuc.), but not the H3K36A mutant histone, octamer, and nucleosome.