Automethylation Activities within the Mixed Lineage Leukemia-1 (MLL1) Core Complex Reveal Evidence Supporting a “Two-active Site” Model for Multiple Histone H3 Lysine 4 Methylation

The mixed lineage leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. In this investigation, we report that the suppressor of variegation, enhancer of zeste, trithorax (SET) domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I subdomain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. We also report that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex with WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, we observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di-, or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, we demonstrate that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a “two-active site” model for multiple H3K4 methylation by the MLL1 core complex.     

Subunit Composition and Substrate Specificity of a MOF-containing Histone Acetyltransferase Distinct from the Male-specific Lethal (MSL) Complex

Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811–823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8.     

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.     

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.     

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.     

A Non-Active-Site SET Domain Surface Crucial for the Interaction of MLL1 and the RbBP5/Ash2L Heterodimer within MLL Family Core Complexes

The mixed lineage leukemia-1 (MLL1) enzyme is a histone H3 lysine 4 (H3K4) monomethyltransferase and has served as a paradigm for understanding the mechanism of action of the human SET1 family of enzymes that include MLL1–MLL4 and SETd1a,b. Dimethylation of H3K4 requires a sub-complex including WRAD (WDR5, RbBP5, Ash2L, and DPY-30), which binds to each SET1 family member forming a minimal core complex that is required for multiple lysine methylation. We recently demonstrated that WRAD is a novel histone methyltransferase that preferentially catalyzes H3K4 dimethylation in a manner that is dependent on an unknown non-active-site surface from the MLL1 SET domain. Recent genome sequencing studies have identified a number of human disease-associated missense mutations that localize to the SET domains of several MLL family members. In this investigation, we mapped many of these mutations onto the three-dimensional structure of the SET domain and noticed that a subset of MLL2 (KMT2D, ALR, MLL4)-associated Kabuki syndrome missense mutations map to a common solvent-exposed surface that is not expected to alter enzymatic activity. We introduced these mutations into the MLL1 SET domain and observed that all are defective for H3K4 dimethylation by the MLL1 core complex, which is associated with a loss of the ability of MLL1 to interact with WRAD or with the RbBP5/Ash2L heterodimer. Our results suggest that amino acids from this surface, which we term the Kabuki interaction surface or KIS, are required for formation of a second active site within SET1 family core complexes.     

WRAD: enabler of the SET1-family of H3K4 methyltransferases

Methylation of histone H3 at lysine 4 (H3K4) is a conserved feature of active chromatin catalyzed by methyltransferases of the SET1-family (SET1A, SET1B, MLL1, MLL2, MLL3 and MLL4 in humans). These enzymes participate in diverse gene regulatory networks with a multitude of known biological functions, including direct involvement in several human disease states. Unlike most lysine methyltransferases, SET1-family enzymes are only fully active in the context of a multi-subunit complex, which includes a protein module comprised of WDR5, RbBP5, ASH2L and DPY-30 (WRAD). These proteins bind in close proximity to the catalytic SET domain of SET1-family enzymes and stimulate H3K4 methyltransferase activity. The mechanism by which WRAD promotes catalysis involves elements of allosteric control and possibly the utilization of a second H3K4 methyltransferase active site present within WRAD itself. WRAD components also engage in physical interactions that recruit SET1-family proteins to target sites on chromatin. Here, the known molecular mechanisms through which WRAD enables the function of SET1-related enzymes will be reviewed.     

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.     

Spectrin Domain of Eukaryotic Initiation Factor 3a Is the Docking Site for Formation of the a:b:i:g Subcomplex

eIF3a (eukaryotic translation initiation factor 3a), one of the core subunits of the eIF3 complex, has been implicated in regulating translation of different mRNAs and in tumorigenesis. A subcomplex consisting of eIF3a, eIF3b, eIF3g, and eIF3i (eIF3(a:b:i:g)) has also been identified. However, how eIF3a participates in translational regulation and in formation of the eIF3(a:b:i:g) subcomplex remain to be solved. In this study, we used the tandem affinity purification approach in combination with tandem MS/MS and identified the spectrin domain of eIF3a as the docking site for the formation of eIF3(a:b:i:g) subcomplex. Although eIF3b and eIF3i bind concurrently to the spectrin domain of eIF3a within ∼10–15 amino acids apart, eIF3g binds to eIF3a indirectly via binding to the carboxyl-terminal domain of eIF3b. The binding of eIF3b to the spectrin domain of eIF3a occurs in its RNA recognition motif domain where eIF3j also binds in a mutually exclusive manner. Together, we conclude that the spectrin domain of eIF3a is responsible for the formation of eIF3(a:b:i:g) subcomplex and, because of mutually exclusive nature of bindings of eIF3a and eIF3j to eIF3b, different subcomplexes of eIF3 likely exist and may perform noncanonical functions in translational regulation.     

A Dynamic cpSRP43-Albino3 Interaction Mediates Translocase Regulation of Chloroplast Signal Recognition Particle (cpSRP)-targeting Components

The chloroplast signal recognition particle (cpSRP) and its receptor, chloroplast FtsY (cpFtsY), form an essential complex with the translocase Albino3 (Alb3) during post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCPs). Here, we describe a combination of studies that explore the binding interface and functional role of a previously identified cpSRP43-Alb3 interaction. Using recombinant proteins corresponding to the C terminus of Alb3 (Alb3-Cterm) and various domains of cpSRP43, we identify the ankyrin repeat region of cpSRP43 as the domain primarily responsible for the interaction with Alb3-Cterm. Furthermore, we show Alb3-Cterm dissociates a cpSRP·LHCP targeting complex in vitro and stimulates GTP hydrolysis by cpSRP54 and cpFtsY in a strictly cpSRP43-dependent manner. These results support a model in which interactions between the ankyrin region of cpSRP43 and the C terminus of Alb3 promote distinct membrane-localized events, including LHCP release from cpSRP and release of targeting components from Alb3.     

Assembly of the Biogenesis of Lysosome-related Organelles Complex-3 (BLOC-3) and Its Interaction with Rab9

The Hermansky-Pudlak syndrome (HPS) is a genetic hypopigmentation and bleeding disorder caused by defective biogenesis of lysosome-related organelles (LROs) such as melanosomes and platelet dense bodies. HPS arises from mutations in any of 8 genes in humans and 16 genes in mice. Two of these genes, HPS1 and HPS4, encode components of the biogenesis of lysosome-related organelles complex-3 (BLOC-3). Herein we show that recombinant HPS1-HPS4 produced in insect cells can be efficiently isolated as a 1:1 heterodimer. Analytical ultracentrifugation reveals that this complex has a molecular mass of 146 kDa, equivalent to that of the native complex and to the sum of the predicted molecular masses of HPS1 and HPS4. This indicates that HPS1 and HPS4 interact directly in the absence of any other protein as part of BLOC-3. Limited proteolysis and deletion analyses show that both subunits interact with one another throughout most of their lengths with the sole exception of a long, unstructured loop in the central part of HPS4. An interaction screen reveals a specific and strong interaction of BLOC-3 with the GTP-bound form of the endosomal GTPase, Rab9. This interaction is mediated by HPS4 and the switch I and II regions of Rab9. These characteristics indicate that BLOC-3 might function as a Rab9 effector in the biogenesis of LROs.     

MicroRNA-323-3p Regulates the Activity of Polycomb Repressive Complex 2 (PRC2) via Targeting the mRNA of Embryonic Ectoderm Development (Eed) Gene in Mouse Embryonic Stem Cells

PRC2 (Polycomb repressive complex 2) mediates epigenetic gene silencing by catalyzing the triple methylation of histone H3 Lys-27 (H3K27me3) to establish a repressive epigenetic state. PRC2 is involved in the regulation of many fundamental biological processes and is especially essential for embryonic stem cells. However, how the formation and function of PRC2 are regulated is largely unknown. Here, we show that a microRNA encoded by the imprinted Dlk1-Dio3 region of mouse chromosome 12, miR-323-3p, targets Eed (embryonic ectoderm development) mRNA, which encodes one of the core components of PRC2, the EED protein. Binding of miR-323-3p to Eed mRNA resulted in reduced EED protein abundance and cellular H3K27me3 levels, indicating decreased PRC2 activity. Such regulation seems to be conserved among mammals, at least between mice and humans. We demonstrate that induced pluripotent stem cells with varied developmental abilities had different miR-323-3p as well as EED and H3K27me3 levels, indicating that miR-323-3p may be involved in the regulation of stem cell pluripotency through affecting PRC2 activity. Mouse embryonic fibroblast cells had much higher miR-323-3p expression and nearly undetectable H3K27me3 levels. These findings identify miR-323-3p as a new regulator for PRC2 and provide a new approach for regulating PRC2 activity via microRNAs.     

HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation

C-C chemokine receptor 5 (CCR5) is a receptor for chemokines and a co-receptor for HIV-1 entry into the target CD4+ cells. CCR5 delta 32 deletion is a loss-of-function mutation, resistant to HIV-1 infection. We tried to induce the CCR5 delta 32 mutation harnessing the genome editing technique, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR and CRISPR associated protein 9, Cas9) in the commonly used cell line human embryonic kidney HEK 293T cells. Surprisingly, we found that HEK293T cells are heterozygous for CCR5 delta 32 mutation, in contrast to the wild type CCR5 cells, human acute T cell leukemia cell line Jurkat and human breast adenocarcinoma cell line MDA-MB-231 cells. This finding indicates that at least one human cell line is heterozygous for the CCR5 delta 32 mutation. We also found that in PCR amplification, wild type CCR5 DNA and mutant delta 32 DNA can form mismatched heteroduplex and move slowly in gel electrophoresis.     

Collagen-binding Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMM) of Gram-positive Bacteria Inhibit Complement Activation via the Classical Pathway

Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r₂C1s₂ was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens.