WDR5 interacts with mixed lineage leukemia (MLL) protein via the histone H3-binding pocket

WDR5 is a component of the mixed lineage leukemia (MLL) complex, which methylates lysine 4 of histone H3, and was identified as a methylated Lys-4 histone H3-binding protein. Here, we present a crystal structure of WDR5 bound to an MLL peptide. Surprisingly, we find that WDR5 utilizes the same pocket shown to bind histone H3 for this MLL interaction. Furthermore, the WDR5-MLL interaction is disrupted preferentially by mono- and di-methylated Lys-4 histone H3 over unmodified and tri-methylated Lys-4 histone H3. These data implicate a delicate interplay between the effector, WDR5, the catalytic subunit, MLL, and the substrate, histone H3, of the MLL complex. We suggest that the activity of the MLL complex might be regulated through this interplay.     

Structure of WDR5 bound to mixed lineage leukemia protein-1 peptide

The mixed lineage leukemia protein-1 (MLL1) catalyzes histone H3 lysine 4 methylation and is regulated by interaction with WDR5 (WD-repeat protein-5), RbBP5 (retinoblastoma-binding protein-5), and the Ash2L (absent, small, homeotic discs-2-like) oncoprotein. In the accompanying investigation, we describe the identification of a conserved arginine containing motif, called the "Win" or WDR5 interaction motif, that is essential for the assembly and H3K4 dimethylation activity of the MLL1 core complex. Here we present a 1.7-A crystal structure of WDR5 bound to a peptide derived from the MLL1 Win motif. Our results show that Arg-3765 of MLL1 is bound in the same arginine binding pocket on WDR5 that was previously suggested to bind histone H3. Thermodynamic binding experiments show that the MLL1 Win peptide is preferentially recognized by WDR5. These results are consistent with a model in which WDR5 recognizes Arg-3765 of MLL1, which is essential for the assembly and enzymatic activity of the MLL1 core complex.     

The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases

In mammals, the SET1 family of lysine methyltransferases (KMTs), which includes MLL1-5, SET1A and SET1B, catalyzes the methylation of lysine-4 (Lys-4) on histone H3. Recent reports have demonstrated that a three-subunit complex composed of WD-repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5) and absent, small, homeotic disks-2-like (ASH2L) stimulates the methyltransferase activity of MLL1. On the basis of studies showing that this stimulation is in part controlled by an interaction between WDR5 and a small region located in close proximity of the MLL1 catalytic domain [referred to as the WDR5-interacting motif (Win)], it has been suggested that WDR5 might play an analogous role in scaffolding the other SET1 complexes. We herein provide biochemical and structural evidence showing that WDR5 binds the Win motifs of MLL2-4, SET1A and SET1B. Comparative analysis of WDR5-Win complexes reveals that binding of the Win motifs is achieved by the plasticity of WDR5 peptidyl-arginine-binding cleft allowing the C-terminal ends of the Win motifs to be maintained in structurally divergent conformations. Consistently, enzymatic assays reveal that WDR5 plays an important role in the optimal stimulation of MLL2-4, SET1A and SET1B methyltransferase activity by the RbBP5-ASH2L heterodimer. Overall, our findings illustrate the function of WDR5 in scaffolding the SET1 family of KMTs and further emphasize on the important role of WDR5 in regulating global histone H3 Lys-4 methylation.     

WD repeat-containing protein 5, a ubiquitously expressed histone methyltransferase adaptor protein, regulates smooth muscle cell-selective gene activation through interaction with pituitary homeobox 2

WD repeat-containing protein 5 (WDR5) is a common component of mammalian mixed lineage leukemia methyltransferase family members and is important for histone H3 lysine 4 methylation (H3K4me), which has been implicated in control of activation of cell lineage genes during embryogenesis. However, WDR5 has not been considered to play a specific regulatory role in epigenetic programming of cell lineage because it is ubiquitously expressed. Previous work from our laboratory showed the appearance of histone H3K4me within smooth muscle cell (SMC)-marker gene promoters during the early stages of development of SMC from multipotential embryonic cells but did not elucidate the underlying mechanisms that mediate SMC-specific and locus-selective H3K4me. Results presented herein show that knockdown of WDR5 significantly decreased SMC-marker gene expression in cultured SMC differentiation systems and in Xenopus laevis embryos in vivo. In addition, we showed that WDR5 complexes within SMC progenitor cells contained H3K4 methyltransferase enzymatic activity and that knockdown of WDR5 selectively decreased H3K4me1 and H3K4me3 enrichment within SMC-marker gene promoter loci. Moreover, we present evidence that it is recruited to these gene promoter loci through interaction with a SMC-selective pituitary homeobox 2 (Pitx2). Taken together, studies provide evidence for a novel mechanism for epigenetic control of SMC-marker gene expression during development through interaction of WDR5, homeodomain proteins, and chromatin remodeling enzymes.     

Structural basis for molecular recognition and presentation of histone H3 by WDR5

Histone methylation at specific lysine residues brings about various downstream events that are mediated by different effector proteins. The WD40 domain of WDR5 represents a new class of histone methyl-lysine recognition domains that is important for recruiting H3K4 methyltransferases to K4-dimethylated histone H3 tail as well as for global and gene-specific K4 trimethylation. Here we report the crystal structures of full-length WDR5, WDR5Delta23 and its complexes with unmodified, mono-, di- and trimethylated histone H3K4 peptides. The structures reveal that WDR5 is able to bind all of these histone H3 peptides, but only H3K4me2 peptide forms extra interactions with WDR5 by use of both water-mediated hydrogen bonding and the altered hydrophilicity of the modified lysine 4. We propose a mechanism for the involvement of WDR5 in binding and presenting histone H3K4 for further methylation as a component of MLL complexes.     

A conserved arginine-containing motif crucial for the assembly and enzymatic activity of the mixed lineage leukemia protein-1 core complex

The mixed lineage leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3 lysine 4 methyltransferases. Recent studies indicate that the catalytic subunits of SET1 family members are regulated by interaction with a conserved core group of proteins that include the WD repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5), and the absent small homeotic-2-like protein (Ash2L). It has been suggested that WDR5 functions to bridge the interactions between the catalytic and regulatory subunits of SET1 family complexes. However, the molecular details of these interactions are unknown. To gain insight into the interactions among these proteins, we have determined the biophysical basis for the interaction between the human WDR5 and MLL1. Our studies reveal that WDR5 preferentially recognizes a previously unidentified and conserved arginine-containing motif, called the "Win" or WDR5 interaction motif, which is located in the N-SET region of MLL1 and other SET1 family members. Surprisingly, our structural and functional studies show that WDR5 recognizes arginine 3765 of the MLL1 Win motif using the same arginine binding pocket on WDR5 that was previously shown to bind histone H3. We demonstrate that WDR5's recognition of arginine 3765 of MLL1 is essential for the assembly and enzymatic activity of the MLL1 core complex in vitro.     

A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex

Gene expression within the context of eukaryotic chromatin is regulated by enzymes that catalyze histone lysine methylation. Histone lysine methyltransferases that have been identified to date possess the evolutionarily conserved SET or Dot1-like domains. We previously reported the identification of a new multi-subunit histone H3 lysine 4 methyltransferase lacking homology to the SET or Dot1 family of histone lysine methyltransferases. This enzymatic activity requires a complex that includes WRAD (WDR5, RbBP5, Ash2L, and DPY-30), a complex that is part of the MLL1 (mixed lineage leukemia protein-1) core complex but that also exists independently of MLL1 in the cell. Here, we report that the minimal complex required for WRAD enzymatic activity includes WDR5, RbBP5, and Ash2L and that DPY-30, although not required for enzymatic activity, increases the histone substrate specificity of the WRAD complex. We also show that WRAD requires zinc for catalytic activity, displays Michaelis-Menten kinetics, and is inhibited by S-adenosyl-homocysteine. In addition, we demonstrate that WRAD preferentially methylates lysine 4 of histone H3 within the context of the H3/H4 tetramer but does not methylate nucleosomal histone H3 on its own. In contrast, we find that MLL1 and WRAD are required for nucleosomal histone H3 methylation, and we provide evidence suggesting that each plays distinct structural and catalytic roles in the recognition and methylation of a nucleosome substrate. Our results indicate that WRAD is a new H3K4 methyltransferase with functions that include regulating the substrate and product specificities of the MLL1 core complex.     

Unique Role of the WD-40 Repeat Protein 5 (WDR5) Subunit within the Mixed Lineage Leukemia 3 (MLL3) Histone Methyltransferase Complex

The MLL3 (mixed lineage leukemia 3) protein is a member of the human SET1 family of histone H3 lysine 4 methyltransferases and contains the conserved WDR5 interaction (Win) motif and the catalytic suppressor of variegation, enhancer of zeste, trithorax (SET) domain. The human SET1 family includes MLL1–4 and SETd1A/B, which all interact with a conserved subcomplex containing WDR5, RbBP5, Ash2L, and DPY-30 (WRAD) to form the minimal core complex required for full methyltransferase activity. However, recent evidence suggests that the WDR5 subunit may not be utilized in an identical manner within all SET1 family core complexes. Although the roles of WDR5 within the MLL1 core complex have been extensively studied, not much is known about the roles of WDR5 in other SET1 family core complexes. In this investigation, we set out to characterize the roles of the WDR5 subunit in the MLL3 core complex. We found that unlike MLL1, the MLL3 SET domain assembles with the RbBP5/Ash2L heterodimer independently of the Win motif-WDR5 interaction. Furthermore, we observed that WDR5 inhibits the monomethylation activity of the MLL3 core complex, which is dependent on the Win motif. We also found evidence suggesting that the WRAD subcomplex catalyzes weak H3K4 monomethylation within the context of the MLL3 core complex. Furthermore, solution structures of the MLL3 core complex assembled with and without WDR5 by small angle x-ray scattering show similar overall topologies. Together, this work demonstrates a unique role for WDR5 in modulating the enzymatic activity of the MLL3 core complex.     

Molecular regulation of H3K4 trimethylation by ASH2L, a shared subunit of MLL complexes

MLL complexes are homologs of yeast COMPASS capable of methylating histone H3 Lys4 (H3K4). ASH2L, RbBP5 and WDR5 are conserved subunits of MLL complexes with homology to the Cps40/Cps60, Cps50 and Cps30 subunits of COMPASS, respectively. We report that ASH2L differentially regulates MLL's catalysis of H3K4 trimethylation similarly to Cps40 and Cps60. Furthermore, WDR5 is required to maintain MLL complex integrity, including the stability of ASH2L within the complex. These findings offer insight into the molecular role of ASH2L, and by extension that of WDR5, in proper H3K4 trimethylation.     

MLL1 is regulated by KSHV LANA and is important for virus latency

Mixed lineage leukemia 1 (MLL1) is a histone methyltransferase. Kaposi''s sarcoma-associated herpesvirus (KSHV) is a leading cause of malignancy in AIDS. KSHV latently infects tumor cells and its genome is decorated with epigenetic marks. Here, we show that KSHV latency-associated nuclear antigen (LANA) recruits MLL1 to viral DNA where it establishes H3K4me3 modifications at the extensive KSHV terminal repeat elements during primary infection. LANA interacts with MLL1 complex members, including WDR5, integrates into the MLL1 complex, and regulates MLL1 activity. We describe the 1.5-Å crystal structure of N-terminal LANA peptide complexed with MLL1 complex member WDR5, which reveals a potential regulatory mechanism. Disruption of MLL1 expression rendered KSHV latency establishment highly deficient. This deficiency was rescued by MLL1 but not by catalytically inactive MLL1. Therefore, MLL1 is LANA regulable and exerts a central role in virus infection. These results suggest broad potential for MLL1 regulation, including by non-host factors.     

Molecular recognition of histone H3 by the WD40 protein WDR5

The WD40-repeat protein WDR5 is a conserved subunit of Trithorax (TRX) histone methyltransferase complexes. WDR5 has been reported to selectively bind dimethylated Lys4 (K4me2) in histone H3 to promote K4 trimethylation by TRX. To elucidate the basis of this binding specificity, we have determined the crystal structure of WDR5 bound to a histone H3 peptide bearing K4me2. The structure reveals that the N terminus of histone H3 binds as a 3(10)-helix in the central depression formed by the WD40 repeats. R2 in histone H3 is bound in the acidic channel in the protein's core, whereas K4me2 is solvent exposed and does not engage in direct interactions with WDR5. Functional studies confirm that WDR5 recognizes A1, R2 and T3 in histone H3 but has virtually identical affinities for the unmodified and mono-, di- and trimethylated forms of K4, demonstrating that it does not discriminate among different degrees of methylation of this residue.