Department of Biochemistry and Purdue University Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, USA
Address correspondence to: Dept. of Biochemistry, Purdue University, 175 S. University, West Lafayette, IN 47907. Tel.: 765-494-0112; Fax: 765-494-7897; E-mail: sdbriggs@purdue.edu.
June 2011
Introduction
ASH2L (Absent, Small, or Homeotic-Like) encodes the protein ASH2L which was named after the Drosophila protein Ash2 a known regulator of HOX genes (Ikegawa et al., 1999). ASH2L is known to be a component of histone H3 lysine 4 (H3K4) methyltransferase complexes and H3K4 methylation is commonly associated with active gene transcription (Ikegawa et al., 1999; Hughes et al., 2004; Dou et al., 2006; Steward et al., 2006; Cho et al., 2007). Previous studies have shown that disruption of ASH2L leads to a decrease in H3K4 trimethylation, which negatively affects gene expression (Dou et al., 2006; Steward et al., 2006). Furthermore, disruption of ASH2L or the methyltransferases involved in H3K4 methylation can lead to oncogenesis mostly through the regulation of HOX gene expression (Hughes et al., 2004; Lüscher-Firzlaff et al., 2008). Interestingly, overexpression of ASH2L leads to tumor proliferation and knock-down of ASH2L inhibits tumorigenesis, which is the reason why ASH2L is thought to be an oncoprotein (Lüscher-Firzlaff et al., 2008). Understanding the role that ASH2L plays in facilitating proper H3K4 methylation may provide insight into how disruption of ASH2L can lead to abnormal cell proliferation and oncogenesis.
ASH2L function
Genetic information and sequence alignments identified ASH2L to be homologous to the transcriptional activator Drosophila Ash2 (Wang et al., 2001; Ikegawa et al., 1999). Drosophila Ash2 (Absent, small , and homeotic discs) is a member of the Trithorax family, known regulators of developmental homeotic genes (LaJeunesse and Shearn, 1995). Mammalian ASH2L is known to be important for development because ASH2L-null mice exhibit an embryonic lethal phenotype (Stoller et al., 2010). Work has established ASH2L as a core component of the H3K4 methyltransferase complexes MLL1-4 and SET1A and SET1B. Furthermore, ASH2L containing methyltransferase complexes are shown to be important for the maintenance of HOX gene expression by binding to HOX gene promoters and by adding H3K4 di- and trimethylation (Fig. 1) (Hughes et al., 2004; Tan et al., 2008; Yates et al., 2010). HOX gene expression is important for proper development and differentiation, and disruption in H3K4 methylation leads to defects in HOX gene expression and the development of cancer (Tan et al., 2008; Hess, 2006; Rampalli et al., 2007; MacConaill et al., 2006; Hughes et al., 2004).
Figure 1. ASH2L functions in a histone methyltransferase complex. The role of ASH2L within the MLL histone H3K4 methyltransferase complex. ASH2L interacts with RBBP5 and DPY-30 increasing the activity of the MLL complex. Histone H3K4 methylation in mammals peaks at the start sight of open reading frames and is important in active transcription. Knock-down of ASH2L in mammalian cells results in a decrease in H3K4 trimethylation and changes in gene expression.
Biochemical data has shown that ASH2L is found in a methyltransferase core complex composed of ASH2L, RBBP5, DPY30, WDR5, and the catalytic SET domain containing protein (Fig. 1). This core complex is highly conserved and similar to the budding yeast Set1 complex that consists of Set1 (MLL/SET1), Bre2 (ASH2L), Swd1 (RBBP5), Swd3 (WDR5), Swd2 (WDR82), Sdc1 (DPY-30), Spp1 (CFP1/CGBP). ASH2L is also known to associate with numerous additional factors listed in Table 1. Many of these additional factors are thought to associate with ASH2L and the H3K4 methyltransferase complexes to target the complex to specific sites within the genome (Stoller et al., 2010; Cho et al., 2007; Steward et al., 2006; Dou et al., 2006; Hughes et al., 2004). ASH2L and Bre2 subunits are important for proper histone methylation. Studies done in yeast show that deletion of the ASH2L homolog BRE2 leads to a complete loss of H3K4 trimethylation and reductions in mono- and dimethylation (Dehé et al., 2006; South et al., 2010; Roguev et al., 2001). In addition, knock-down of ASH2L using siRNA globally decreases H3K4 trimethylation (Steward et al., 2006; Dou et al., 2006). These data suggest that ASH2L may act in a similar manner to yeast Bre2. From these studies it is clear that ASH2L is playing an important role in histone methyltransferase complexes in order to maintain proper H3K4 methylation and gene expression (Patel et al., 2009; Roguev et al., 2001).
Catalytic core Histone methyltransferase (HMT)
Table 1.
Alternative to ASH2L's function in H3K4 methylation ASH2L may also be playing a role in endosomal trafficking (Xu et al., 2009). ASH2L, DPY-30 and WDR5 were originally implicated in endosomal trafficking when siRNA knock-down of these genes increased the amount of internalized CD8-CIMPR and overexpression increased the amount of cells displaying a altered CIMPR distribution (Xu et al., 2009). This affect was limited to components of H3K4 methyltransferases and not to other methyl marks such as lysine 9 (Xu et al., 2009). The mechanism in which ASH2L and other components of H3K4 methyltransferase complexes modulate endosomal trafficking remains unclear. However, two possible mechanisms have been suggested, one is that the H3K4 methyltransferase components are part of an unknown complex that regulates trafficking, or that changes in H3K4 methylation lead to changes in expression of another regulating factor (Xu et al., 2009).
ASH2L structure
One way to better understand the function of ASH2L is to determine the role of specific domains within ASH2L in facilitating H3K4 methylation. There are three known isoforms of ASH2L (Wang et al., 2001). Isoform 1 is considered the canonical sequence and consists of 628 amino acids (Wang et al., 2001). Isoform 2 is missing amino acids 1-94 and 541-573 from isoform 1 (Wang et al., 2001). Isoform 3 is missing the amino acids 1-94 from isoform 1 (Fig. 2) (Wang et al., 2001). There are four identified domains within ASH2L which include a N-terminus containing a PHD finger and a winged helix motif (WH) and the C-terminus containing a SPRY domain and a newly identified Sdc1 DPY-30 Interacting domain (SDI) (Fig. 2) (Wang et al., 2001; Roguev et al., 2001; South et al., 2010; Sarvan et al., 2011; Chen et al., 2011). Interestingly, the domains with known biological function are the C-terminal SDI domain, which is responsible for the interaction with another histone methyltransferase component DPY-30 and the winged helix motif which binds to DNA (South et al., 2010; Sarvan et al., 2011; Chen et al., 2011).
Figure 2. ASH2L has three known isoforms. Schematic model of the three known isoforms of ASH2L and the amino acid sequence changes compared to the canonical isoform 1 (aa 1-628). The positions of known domains within ASH2L are displayed. PHD finger (aa 95-161), WH motif (aa 162-273), SPRY domain (aa 360-583), and SDI domain (aa 602-628). Isoform 2 and 3 are numbered according to isoform 1.
The function of the SDI domain was determined using in vitro binding experiments. ASH2L was shown to directly interact with DPY-30 without any additional MLL or Set1 complex components (South et al., 2010). The function of the SDI domain is conserved from yeast to humans because the yeast ASH2L homolog Bre2 was also shown to interact with the DPY-30 homolog Sdc1 (South et al., 2010). There are conserved hydrophobic residues in both the SDI domain of ASH2L and the Dpy-30 domain of DPY-30 that are important for binding, which suggests that the interaction between the SDI domain of ASH2L and the DPY-30 domain of DPY-30 is through hydrophobic interactions (South et al., 2010). In addition, binding affinities between ASH2L and DPY-30, as well as ASH2L and RBBP5 have been determined by sedimentation velocity analytical ultracentrifugation showing dissociation constants of 0.1 μM and 0.75 μM respectively (Patel et al., 2009). Interestingly, in yeast the ASH2L homolog Bre2 must interact with Sdc1 through the SDI domain to interact with the yeast Set1 histone methyltransferase complex (South et al., 2010). In contrast, in vitro experiments have shown ASH2L does not require DPY-30 to interact with MLL complex. To better understand how ASH2L interacts with MLL, in vivo studies must be done to determine if DPY-30 is required for ASH2L interaction. However, it is quite possible that the yeast and human complexes assemble differently.
The N-terminal winged helix (WH) motif was recently discovered when the crystal structure of the N-terminus of ASH2L was solved (Sarvan et al., 2011; Chen et al., 2011). Using in vitro DNA binding analyses as well as chromatin immunoprecipitation, it was determined that ASH2L can bind DNA at the HS2 promoter region and the β-globin locus as well as non-specific DNA sequence (Sarvan et al., 2011; Chen et al., 2011). The DNA binding activity of ASH2L promotes H3K4 methylation and gene expression at the β-globin locus by 50% when overexpressed in a cell line where ASH2L is knocked-down by siRNA (Sarvan et al., 2011). In addition, chromatin immunoprecipitation followed by a tiling array (ChIP-chip) analysis shows that disruption of the winged helix motif causes mis-localization of ASH2L (Chen et al., 2011). It was also shown that the DNA binding activity of the N-terminus of ASH2L increases when the C-terminal SPRY and SDI domains are present (Chen et al., 2011). Altogether, these data suggests that multiple domains in ASH2L may contribute to its ability to bind chromatin. However, more work will be needed to clearly establish the function of each domain.
The largest of the three identified domains within ASH2L is the SPRY domain, which is also conserved from yeast to humans. SPRY domains were originally named after the SPIa kinase and the RYanodine receptor proteins in which it was first identified (Rhodes et al., 2005). Multiple crystal structures have been solved for proteins that contain an SPRY domain. Crystal structures of SPRY domain containing proteins show primarily a β-sandwich structure with extending loops (Woo et al., 2006b; Kuang et al., 2009; Filippakopoulos et al., 2010; Simonet et al., 2007). The SPRY domain is thought to be a specific protein-protein interaction domain with specific partners, but instead of recognizing a particular motif or interaction domain the SPRY domain binds to interaction partners using non-conserved binding loops (Filippakopoulos et al., 2010; Woo et al., 2006b; Woo et al., 2006a). SPRY domain-containing proteins are involved in a wide array of functions including RNA metabolism, calcium release, and developmental processes (Woo et al., 2006b; Kuang et al., 2009; Filippakopoulos et al., 2010; Simonet et al., 2007; Woo et al., 2006a). Recent work has shown that the C-terminus of ASH2L that contains the SPRY domain and the SDI domain are able to interact with the other MLL complex member RBBP5 in vitro (Avdic et al., 2011). This interaction is most likely through the SPRY domain and not the SDI domain, though further work would need to be done to better map this interaction.
ASH2L also contains a putative Plant Homeo Domain (PHD) finger in its N-terminus (Wang et al., 2001). PHD fingers are a family of zinc finger domains that are known to bind to both modified and unmodified histone tails (Bienz, 2006; Mellor, 2006). The structure of PHD fingers shows that conserved cysteine and histidine residues bind to Zn2+ ions (Champagne et al., 2008; van Ingen et al., 2008; Champagne and Kutateladze, 2009). PHD fingers generally form a globular fold, consisting of a two-stranded beta-sheet and an alpha-helix. Loop regions of PHD fingers tend to vary giving rise to specificity of the domain. Some PHD fingers are considered to be readers of epigenetic marks by binding to specific modifications or sites on histones to stabilize or localize an interaction (Mellor, 2006). Primarily, PHD fingers have been shown to interact with trimethylated histone residues such as trimethylated histone H3 lysine 4 and lysine 9 (Mellor, 2006). There is no known function attributed to the PHD finger in ASH2L, though in conjunction with the winged helix motif it may be necessary for DNA binding. However, the PHD finger may also be needed in binding to MLL, other MLL/SET1 components, or recognizing a specific histone modification or for binding to a histone tail. Additional studies are needed to determine how the PHD finger of ASH2L and the SPRY domain may help the MLL and Set1 methyltransferase complexes interact and catalyze H3K4 methylation.
Conclusion
Currently, relatively little is known about the contribution of ASH2L to facilitate and or regulate the degree of methylation along the eukaryotic genome, but disruption of ASH2L and H3K4 methylation both appear to play a key role in oncogenesis (Lüscher-Firzlaff et al., 2008; Hess, 2006). Interestingly, recent work has suggested that ASH2L in combination with WDR5 and RBBP5 exhibits H3K4 methyltransferase activity (Cao et al., 2010; Patel et al., 2009; Patel et al., 2011). In addition, this catalytic activity is not dependent on the SET domain containing proteins such as MLL1 (Patel et al., 2009; Cao et al., 2010; Patel et al., 2011). One report shows the catalytic activity of the ASH2L, WDR5, RBBP5, DPY-30 complex in an in vitro histone methyltransferase assay is observed but only after eight hours of incubation (Patel et al., 2009; Patel et al., 2011). In contrast, more methyltransferase activity and much shorter incubation times are required when these components are incubated with the MLL1 SET domain containing methyltransferase (Patel et al., 2009; Patel et al., 2011). This indicates the sub-complex has poor catalytic activity when the main catalytic SET domain-containing subunit is not present in the reaction. However, Cao et al. shows that only ASH2L/RBBP5 heterodimer is needed for weak H3K4 methyltransferase activity (Cao et al., 2010). Because ASH2L, WDR5, RBBP5, and DPY-30 complex does not contain a known methyltransferase domain, more work needs to be done to determine if a new class of methyltransferase has been identified and whether or not this methyltransferase activity is biologically relevant.
ASH2L is found to be over abundant in many cancer cell lines and knock-down of ASH2L by siRNA can prevent tumorigenesis (Lüscher-Firzlaff et al., 2008). ASH2L is important for proper H3K4 methylation but how ASH2L contributes to the distribution and degree of methylation and its role in gene expression remains unclear. To better understand the role of ASH2L in methylation and gene expression several questions need to be addressed. What is the mechanism of interaction that contributes to ASH2L's interaction with histone methyltransferase complexes? What is ASH2L's role in regulating the degree of methylation along genes and what genes are affected by changes in ASH2L? Additional structural studies will help address the mechanism of how ASH2L interacts with other methyltransferase complex members and microarray experiments will be needed to determine the genes that are affected by changes in ASH2L expression levels. Addressing these questions could provide valuable information for the development specific inhibitors for the treatment of various cancers.
Atlas of Genetics and Cytogenetics in Oncology and Haematology
Understanding the structure and function of ASH2L
Online version: http://atlasgeneticsoncology.org/deep-insight/20097/understanding-the-structure-and-function-of-ash2l