Point of
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MLL Core Components Give the Green Light to Histone Methylation Brendan D. Crawford and Jay L. Hess* Department of Pathology, University of Michigan Medical School, 1301 Catherine Road, M5240 MS1, Ann Arbor, Michigan 48109-0602
O
ver the past decade, considerable progress has been made in understanding how transcription is regulated through covalent modifications of histones. It is now clear that in addition to acetylation, phosphorylation, and ubiquitination, lysine methylation of histone tails plays a fundamental role in transcriptional regulation (1). Defining how the “readers, writers, and erasers” of this epigenetic code interact and how their activity is regulated is currently a major area of interest for many in the transcription field. A recent paper by Dou et al. (2) provides important insights into regulation of histone H3 Lys4 (H3K4) methylation. Methylation of lysines on histone H3 and H4 tails confers either activating or silencing effects on transcription, depending on the specific modified residue (1). Dimethylation and trimethylation of H3K4 are associated with the coding region of actively transcribed genes (3). In contrast, histone H3 Lys9 (H3K9) and Lys27 (H3K27) methylation is associated with transcriptional repression. With the exception of Dot1, an H3 Lys79 methyltransferase (MT), all of the lysine MTs share an evolutionarily conserved catalytic domain, the SET domain, whose name is derived from the MTs Su(var)3-9, Enhancer of Zeste (EZH), and Trithorax. Only one H3K4 MT, Set1, has been identified in yeast; however, many human Set1 homologues have been identified, including Set1a, Set1b, and four members of the Mixed-Lineage Leukemia (MLL) family www.acschemicalbiology.org
(4–9). Of these, MLL1, which is homologous to the Drosophila protein Trithorax, has been the most intensively studied because of its involvement by chromosomal translocations in a variety of acute lymphoid and myeloid leukemias (10). Several years ago, the bacterially expressed MLL SET domain was shown to have modest MT activity itself (11). However, like other Set1 family members, MLL1 exists as part of an MT complex with enhanced MT activity (6) that includes a number of other proteins, such as the histone acetyltransferase MOF (males absent on the first) and three core components, WDR5, RbBP5, and Ash2L (12, Figure 1). The finding that these core components are evolutionarily conserved from yeast to humans and are shared among the different human Set1 family members suggests that they play important roles in regulating MT activity. Indeed, recent work by Wysocka and colleagues (13) shows that WDR5 is required for histone H3K4 trimethylation. In this report, WDR5 was found to bind preferentially to dimethylated H3K4, and its knockdown resulted in decreased expression of MLL1 target genes without affecting binding of MLL1 complexes to these targets. Several recent manuscripts (14–18), notably the study by Dou and colleagues (2), provide a clearer picture of the roles that not only WDR5 but also RbBP5 and Ash2L play in regulating MT activity. An MT Structural “Presentation” Platform. Dou and colleagues (2) used baculovirus expression in insect cells and
A B S T R A C T Trimethylation of histone H3 Lys4 (H3K4) is associated with transcriptional activation. One of the chief effectors of H3K4 methylation is mixed-lineage leukemia 1 (MLL1), a gene that is disrupted by chromosomal translocation in acute leukemia and a master regulator of Hox and other genes. In a recent paper, core components of the human MLL histone methyltransferase (MT) complex were found to form a structural platform, with one component (WDR5) mediating association between the specific histone H3K4 substrate and the MT. This novel regulatory mechanism, which is conserved from yeast to human, is required for both methylation and downstream target gene transcription.
*Corresponding author,
[email protected].
Published online September 15, 2006 10.1021/cb600367v CCC: $33.50 © 2006 by American Chemical Society
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Subsequent in vivo studies based on RNAi on transfected cells by Shilatifard and colleagues (14) show very similar findings. The WDR5 Link. The above results show that MLL1 requires the WDR5-RbBP5Ash2L structural platform for full catalytic activity and indicate a central role for WDR5 in not only Figure 1. MT structural platform. WDR5, RbBP5, and Ash2L, core MLL1 MT activity components of the MLL1 MT complex, form a structural platform for the different Set and MLL-family MTs that are required for trimethylation of but also likely for histone H3K4. the other H3K4 MTs as well. Four immunoaffinity purification to reconstitute a groups have reported the crystal structure of functional MT complex composed of WDR5, WDR5, which contains seven WD40 repeats RbBP5, Ash2L, and the catalytic C-terminus organized in a whorl of -“propeller blades”, each composed of four antiparallel pleated of MLL1 (MLL-C) in vitro. Various combinasheets (15–18). These studies show that a tions of the three subunits were expressed central depression formed by the WD40 through the baculovirus system, and interactions were examined through immunopre- repeats specifically recognizes N-terminal cipitation assays to determine the structural H3 peptides via interactions primarily with organization of the core complex. WDR5 and residues A1, R2, and T3. Mutations of any RbBP5 were shown to jointly mediate asso- of these three residues inhibit binding. All studies show the H3K4 is solvent-exposed ciation with MLL-C. Interestingly, WDR5, and thus available for further methylation. RbBP5, and Ash2L formed an independent Most studies that analyzed binding of complex in the absence of MLL-C, an indication that together they form a structural plat- WDR5 to histone tails report higher affinity of WDR5 for dimethylated H3K4 than the form for association with the different Set1 or MLL-family H3K4 MTs (Figure 1). After Dou unmodified peptide (15, 18), although and colleagues (2) determined the structural Couture et al. (16) report that WDR5 has virtually identical affinities for mono-, di-, or tricontributions to the core complex, they methylated H3K4. Subtle kinetic differences examined the functional contribution of each core component to H3K4 MT activity. In may account for why dimethylated H3K4 is a vitro histone MT (HMT) assays revealed that preferred binding partner in in vitro assays (15, 18), which are likely to be magnified in the absence of RbBP5 or Ash2L in the complex significantly reduces H3K4 trimeth- more in vivo situations. In support of this, pull-down studies in the context of intact ylation. In contrast, the absence of WDR5 MLL complexes show preferential interaccompletely abolishes methylation activity. 496
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tion with K4 dimethylated tails (19). One take-home message is clear: WDR5 is required for full MLL1-complex mediated H3K4 trimethylation, possibly through regulating its activity. Dou et al. (2) explored this possibility by constructing WDR5 mutants with a disruption of each of the three residues that make contact with histone H3 peptides, as determined by the structural studies above. Two of the WDR5 mutants, S91K and F133A, disrupted the interaction of MLL1 with the complex. The Y191F mutant was particularly instructive because it preserved the integrity of the MLL1 complex and dramatically reduced both binding of the complex to histone and MLL1 HMT activity. Together with the structural studies, Dou et al.’s (2) work indicates that WDR5 plays a unique role by linking the catalytic subunit of MLL-C and its histone H3K4 substrate in such a way that the complex can bind mono- or dimethylated H3K4 while “presenting” K4 for further methylation (Figure 2), a concept suggested from structural studies (15, 18). Effects on MLL1-Mediated Transcription. After Dou and colleagues (2) identified a functional role for WDR5 in MLL1-mediated HMT activity, they examined the role of
Figure 2. WDR5 link. WDR5 interacts with methylated H3K4, presenting it for further methylation and, with MLL1, linking the catalytic MT domain with its substrate. This interaction is necessary for H3K4 trimethylation and subsequent transcription of MLL1 target Hox genes. www.acschemicalbiology.org
Point of
VIEW WDR5-RbBP5-Ash2L in regulating the transcription of two well-characterized MLL1 target genes, HOXA9 and HOXC8. An analysis of separate small interfering RNA (siRNA) knockdowns of each of the components revealed reduced expression of HOXA9 and HOXC8, compared with the core complex. Chromatin immunoprecipitation experiments revealed that trimethylated H3K4 was reduced at the HOXA9 locus when each of the three components was individually knocked down with siRNA. Trimethylated H3K4 was also reduced at the HOXC8 locus with RbBP5 or WDR5 knockdown and dimethylated H3K4 with RbBP5 knockdown. Of note is a very recent report by Shilatifard and colleagues (14), who used a similar approach and showed that ASH2L is also pivotal for trimethylation of MLL1 targets. These combined siRNA knockdown results demonstrate that all three core components, WDR5, RbBP5, and Ash2L, are needed for full MLL1 target gene expression and that this requirement lies in the regulation of H3K4 dimethylation and/or trimethylation. Notably, in the study by Dou et al. (2), MLL1 recruitment to either the HOXA9 or HOXC8 locus was not disturbed by knockdown of WDR5, RbBP5, or Ash2L, an indication that these core components are not involved in MLL1 recruitment but rather in regulating MLL1 complex MT activity. Looking Ahead: Biological and Chemical Implications. This study provides important insights into how histone methylation is regulated by bringing together specific MTs and histone tails via scaffolding components with WD40 repeats such as WDR5. One area of interest will be to determine whether the expression of the core subunits, or perhaps their post-translational modifications (PTMs), are developmentally regulated and whether, for example, this plays a role in the regulation of target genes such as the Hox genes. It will also be important to identify the specific sites of interaction of the SET domain with core components and whether these interactions are developmenwww.acschemicalbiology.org
tally regulated. The function of many of the other components of the MLL complexes, such as HCF-1 and -2, Dpy-30, and menin, remain to be determined. Using approaches similar to those employed by Dou et al. (2) should be a powerful tool for resolving these issues. It is intriguing that the amino acids in the histone H3 tail recognized by WDR5, in particular R2 and T3, are known to be methylated and phosphorylated in vivo. Couture et al. (16) showed that these PTMs inhibit WDR5 binding, and this lends further credence to the concept of a “phosphomethyl switch” in which the modification status of the histone tail regulates its ability to undergo subsequent PTMs (20). Another important question will be to determine whether similar mechanisms apply to other HMTs involved in transcriptional activation and repression. Other WD40-containing proteins, such as Embryonic ectoderm development (Eed) and RbAp46/48, have been shown to associate with repressive EZH2 complexes that methylate Lys27 (21, 22). Furthermore, Eed knockout mice show global defects in histone H3K27 methylation, an indication that Eed plays a role similar to that of WDR5 in facilitating histone methylation (23). Because considerable amounts of WDR5 are present in lower-molecular-weight complexes compared with those identified for either the MLL family or EZH2 (14), determining what roles WDR5 and related WD40 repeat proteins may play beyond histone methylation will be very interesting. Acknowledgment: We thank C. David Allis, Alexander J. Ruthenburg (Rockefeller University), and Joanna Wysocka (Stanford University) for critical review of this article and Jinron Min (University of Toronto) for permission to communicate results prior to publication.
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