Transcription in eukaryotic genomes depends on enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation. The mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of H3K4 methyltransferases and is frequently rearranged in acute leukemias. Despite sequence comparisons that predict that SET1 family enzymes should only monomethylate their substrates, mono-, di-, and trimethylation of H3K4 has been attributed to SET1 family complexes in vivo and in vitro. To better understand this paradox, we have biochemically reconstituted and characterized a five-component 200-kDa MLL1 core complex containing human MLL1, WDR5, RbBP5, Ash2L, and DPY-30. We demonstrate that the isolated MLL1 SET domain is a slow monomethyltransferase and that tyrosine 3942 of MLL1 prevents di-and trimethylation of H3K4. In contrast, a complex containing the MLL1 SET domain, WDR5, RbBP5, Ash2L, and DPY-30, displays a marked ϳ600-fold increase in enzymatic activity but only to the dimethyl form of H3K4. Single turnover kinetic experiments reveal that the reaction leading to H3K4 dimethylation involves the transient accumulation of a monomethylated species, suggesting that the MLL1 core complex uses a non-processive mechanism to catalyze multiple lysine methylation. We have also discovered that the non-SET domain components of the MLL1 core complex possess a previously unrecognized methyltransferase activity that catalyzes H3K4 dimethylation within the MLL1 core complex. Our results suggest that the mechanism of multiple lysine methylation by the MLL1 core complex involves the sequential addition of two methyl groups at two distinct active sites within the complex.Lysine methylation of histones is an important epigenetic indexing system for transcriptionally active and inactive chromatin domains in eukaryotic genomes (1). Lysine residues can be mono-, di-, or trimethylated at the ⑀-amino group, with each state correlating with a distinct functional outcome (2). For example, trimethylation of histone H3 lysine 4 (H3K4me3) is enriched at the 5Ј ends of actively transcribed genes in a wide range of eukaryotes (3, 4) and is thought to regulate transcription through the recruitment of proteins that activate (5-7) or repress transcription (8). In contrast, H3K4 monomethylation (H3K4me1) is enriched in nucleosomes at the 3Ј-ends of genes (2, 4, 9) or in the distal enhancer sequences of active genes (10). In addition, H3K4 monomethylation in Saccharomyces cerevisiae (11-13) and Chlamydomonas reinhardtii (14) is associated with gene silencing. These results suggest that distinct strategies have evolved to regulate the degree of H3K4 methylation in eukaryotic genomes. Although numerous histone lysine methyltransferases and demethylases have been identified in recent years (15, 16), relatively little is understood about how the different states of lysine methylation are achieved and regulated.The majority of histone lysine methyltransferases that have been identified share a conserved SET domain motif, named for its presence in...

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.Eukaryotes have evolved a complex system to regulate access to genomic information by packaging DNA into chromatin. Chromatin can adopt various levels of organization that regulate essential cellular activities such as transcription, DNA replication, recombination, and repair (1). The fundamental repeating unit of chromatin is the nucleosome, in which 147 base pairs of genomic DNA is wrapped around a disc-shaped octamer of histone proteins H2A, H2B, H3, and H4 (2, 3). Nucleosome positioning on DNA is fundamentally involved in controlling gene access and is regulated in part by a diverse array of enzymes that introduce covalent post-translational modifications on histone proteins (4, 5). An extensive array of histone modifications have been characterized, including lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitylation (6, 7). The large number of potential histone modification patterns provides cells with an enormous combinatorial potential for the precise regulation of gene function. This potential is summarized by the histone code hypothesis (8 -10), which predicts that patterns of histone modifications are recognized by specialized "effector" domains found in numerous chromatin regulators. It is thought that effector domains help recruit the activities of proteins that either stabilize or remodel specific chromatin states (5,11,12).The combinatorial complexity of histone modifications is further increased by histone lysine residues that can be mono-, di-, or trimethylated, which are correlated with distinct functional outcomes. For example, chromatin immunoprecipitation experiments suggest that nucleosomes occupying...

Post-translational modifications of the histone tails are correlated with distinct chromatin states that regulate access to DNA. Recent proteomic analyses have revealed several new modifications in the globular nucleosome core, many of which lie at the histone-DNA interface. We interpret these modifications in light of previously published data and propose a new and testable model for how cells implement the histone code by modulating nucleosome dynamics.

Sir2 proteins are NAD(+)-dependent protein deacetylases that play key roles in transcriptional regulation, DNA repair, and life span regulation. The structure of an archaeal Sir2 enzyme, Sir2-Af2, bound to an acetylated p53 peptide reveals that the substrate binds in a cleft in the enzyme, forming an enzyme-substrate beta sheet with two flanking strands in Sir2-Af2. The acetyl-lysine inserts into a conserved hydrophobic tunnel that contains the active site histidine. Comparison with other structures of Sir2 enzymes suggests that the apoenzyme undergoes a conformational change upon substrate binding. Based on the Sir2-Af2 substrate complex structure, mutations were made in the other A. fulgidus sirtuin, Sir2-Af1, that increased its affinity for the p53 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 (retinoblastomabinding 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-Å 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.Histone H3 lysine 4 methylation catalyzed by the mixed lineage leukemia protein-1 (MLL1) is important for the regulation of hox genes in hematopoiesis and development (1-3). MLL1 belongs to a family of SET domain histone methyltransferases that are regulated by a core complex of proteins that are conserved from yeast to humans (4 -13). It has previously been demonstrated that WDR5, RbBP5, and Ash2L form a stable structural platform that can interact interchangeably with the different catalytic subunits from the SET1 family (12). However, the structural basis for this interaction is unknown.Previous studies have shown that WDR5 interacts directly with the catalytic subunits of MLL1, MLL3, and MLL4 and mediates interactions with the other components of SET1 family complexes (6,12,14). In addition, previous crystal structures have shown that WDR5 recognizes arginine in a histone H3R2 context (15-18) (PDB 3 codes 2h13, 2g99, 2co0, 2h9n, 2cnx, 2o9k, 2g9a, 2h6k, 2h6n, 2h9p, 2h9m, and 2h6q), which has been suggested to mediate histone methylation by SET1 family complexes (12,17). In the accompanying investigation (22), we describe the identification of a WDR5 interaction motif, or "Win" motif, which is located in the N-SET region of MLL1. We demonstrate that the MLL1 Win motif is highly conserved among metazoan SET1 family members and is recognized by WDR5 in a mechanism that is crucial for the assembly and enzymatic activity of the MLL1 core complex in vitro. Intriguingly, our functional results suggest that WDR5 preferentially recognizes the conserved Arg-3765 within the MLL1 Win motif using the same arginine binding site as suggested previously for histone H3. To test this hypothesis, we determined the crystal structure of WDR5 bound to a peptide derived from the MLL1 Win motif. Our results are consistent with a model in which WDR5 preferentially recognizes Arg-3765 of MLL1 using the same binding site previously suggested for histone H3. These results suggest that the previously observed histone H3-WDR5 interaction m...