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.Nucleosomes, the fundamental unit of the highly ordered chromatin structure, are composed of DNA wrapped around histone octamers. Histones have N-terminal tails that are exposed on the outside of nucleosomes. These tails are subjected to several post-translational modifications, including acetylation, phosphorylation, ubiquitination, sumoylation, and methylation (1).The site-specific methylation of histone lysine residues is important for the epigenetic control of gene expression. These marks serve to regulate epigenetically the organization of chromatin structure and to recruit other chromatin modifiers (2, 3). Methylation can occur at multiple lysine residues, including lysines 4, 9, 27, 36, and 79 of histone H3 and lysine 20 of histone H4. Absent, small, or homeotic disc1 (Ash1) is a member of the trithorax group proteins, which are essential for epigenetic gene activation (4,5). Previous studies have shown that Drosophila Ash1 activates homeotic gene ultrabithorax expression in imaginal discs of the third leg (6) and interacts with trithorax to regulate and maintain ultrabithorax expression (7). It has also been reported that the mammalian homolog of Ash1, ASH1L, is a histone methyltransferase (HMTase) 2 that is associated with transcribed regions of active genes (8, 9). ASH1L has several domains, including an associated with SET domain (AWS), a SET domain, a post-SET domain, a bromodomain, a bromoadjacent homology domain (BAH), and a plant homeodomain finger (SMART database (10)). The SET domain in HMTases is responsible for catalyzing the formation of monomethylated, dimethylated, and trimethylated lysine, establishing an additional complex system with respect to methylated lysine recognition in signaling (11). Several crystal structures of SET domain proteins have been solved, and they revealed that the SET domain forms a knot-like structure that constitutes the active site of HMTases (12). Notably, ASH1L contains a SET domain in the middle of the protein, whereas other proteins possess a SET domain at the C terminus. Drosophila Ash1 has been previously shown to affect H3K4 methylation levels genetically and to methylate H3K4, H3K9, and H4K20 in in vitro assays (8, 13). The Tanaka group (14) has found H3K36 ...

dSet1 Is the Main H3K4 Di- and Tri-Methyltransferase ThroughoutDrosophilaDevelopment

“…This would be consistent with their proposed role in human cells, where the Trr and Trx homologs MLL1-2 and MLL3-4 are thought to methylate H3K4 at a limited number of non-overlapping gene targets (Wang et al 2009;Ansari and Mandal 2010;Eissenberg and Shilatifard 2010). We will not discuss the roles of Ash1 further, as recent findings suggest that the predominant function of Ash1 is the catalysis of H3K36, and not H3K4 methylation (Tanaka et al 2007;An et al 2011).…”

H3K36 Methyltransferases as Cancer Drug Targets: Rationale and Perspectives for Inhibitor Development

“…In NSD1, the autoinhibitory loop is stabilized by a β-turn within the loop as well as hydrophobic contacts with SET-I [121]. In contrast, no β-turn exists in the ASH1L autoinhibitory loop, and there are longer range contacts to SET-I [120,127]. In the SETD2 autoinhibitory loop, there is also no β-turn to stabilize the loop, but the interaction of Arg1670 at the beginning of the loop with the substrate lysine-binding channel pulls the loop into contact with SET-I [122] ( Figure 3B).…”

“…For the H3K36-specific KMTases, structural studies of the catalytic SET domain have revealed valuable information to jumpstart inhibitor development. An important feature shared among the NSD, ASH1L and SETD2 SET domains is an autoinhibitory loop blocking access of histone substrate to the active site [120][121][122]. This loop must undergo a conformational change to accommodate substrate binding.…”

Ash1l controls quiescence and self-renewal potential in hematopoietic stem cells

“…However, unlike the much smaller protein ASH2L, ASH1L has not been identified so far as a member of the MLL protein complex. Recently, the ASH1L SET domain was reported to have intrinsic H3K36 dimethyltransferase activity using in vitro biochemical assays (37)(38)(39). Neither the physiological significance of Ash1l nor the idea of cooperativity with Mll1 and other TrxG members has been evaluated in vivo.…”