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.     

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.     

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.     

CUL4-DDB1 ubiquitin ligase interacts with multiple WD40-repeat proteins and regulates histone methylation

The CUL4-DDB1-ROC1 ubiquitin E3 ligase regulates cell-cycle progression, replication and DNA damage response. However, the substrate-specific adaptors of this ligase remain uncharacterized. Here, we show that CUL4-DDB1 complexes interact with multiple WD40-repeat proteins (WDRs) including TLE1-3, WDR5, L2DTL (also known as CDT2) and the Polycomb-group protein EED (also known as ESC). WDR5 and EED are core components of histone methylation complexes that are essential for histone H3 methylation and epigenetic control at K4 or K9 and K27, respectively, whereas L2DTL regulates CDT1 proteolysis after DNA damage through CUL4-DDB1 (ref. 8). We found that CUL4A-DDB1 interacts with H3 methylated mononucleosomes and peptides. Inactivation of either CUL4 or DDB1 impairs these histone modifications. However, loss of WDR5 specifically affects histone H3 methylation at K4 but not CDT1 degradation, whereas inactivation of L2DTL prevents CDT1 degradation but not histone methylation. Our studies suggest that CUL4-DDB1 ligases use WDR proteins as molecular adaptors for substrate recognition, and modulate multiple biological processes through ubiquitin-dependent proteolysis.     

组蛋白赖氨酸甲基化在表观遗传调控中的作用

组蛋白赖氨酸的甲基化在表观遗传调控中起着关键作用。组蛋白H3的K4、K9、K27、K36、K79和H4的K20均可被甲基化。组蛋白H3第9位赖氨酸的甲基化与基因的失活相关连; 组蛋白H3第4位赖氨酸和第36位赖氨酸的甲基化与基因的激活相关连; 组蛋白H3第27位赖氨酸的甲基化与同源盒基因沉默、X染色体失活、基因印记等基因沉默现象有关; 组蛋白H3第79位赖氨酸的甲基化与防止基因失活和DNA修复有关。与此同时, 组蛋白的去甲基化也受到更为广泛的关注。 关键词: 组蛋白赖氨酸甲基转移酶; 组蛋白赖氨酸甲基化; 组蛋白去甲基化     

Analysis of histones in Xenopus laevis. I. A distinct index of enriched variants and modifications exists in each cell type and is remodeled during developmental transitions

Histone proteins contain epigenetic information that is encoded both in the relative abundance of core histones and variants and particularly in the post-translational modification of these proteins. We determined the presence of such variants and covalent modifications in seven tissue types of the anuran Xenopus laevis, including oocyte, egg, sperm, early embryo equivalent (pronuclei incubated in egg extract), S3 neurula cells, A6 kidney cells, and erythrocytes. We first developed a new robust method for isolating the stored, predeposition histones from oocytes and eggs via chromatography on heparin-Sepharose, whereas we isolated chromatinized histones via conventional acid extraction. We identified two previously unknown H1 isoforms (H1fx and H1B.Sp) present on sperm chromatin. We immunoblotted this global collection of histones with many specific post-translational modification antibodies, including antibodies against methylated histone H3 on Lys(4), Lys(9), Lys(27), Lys(79), Arg(2), Arg(17), and Arg(26); methylated histone H4 on Lys(20); methylated H2A and H4 on Arg(3); acetylated H4 on Lys(5), Lys(8), Lys(12), and Lys(16) and H3 on Lys(9) and Lys(14); and phosphorylated H3 on Ser(10) and H2A/H4 on Ser(1). Furthermore, we subjected a subset of these histones to two-dimensional gel analysis and subsequent immunoblotting and mass spectrometry to determine the global remodeling of histone modifications that occurs as development proceeds. Overall, our observations suggest that each metazoan cell type may have a unique histone modification signature correlated with its differentiation status.     

A highly specific mechanism of histone H3-K4 recognition by histone demethylase LSD1

Human lysine-specific demethylase (LSD1) is a chromatin-modifying enzyme that specifically removes methyl groups from mono- and dimethylated Lys4 of histone H3 (H3-K4). We used a combination of in vivo and in vitro experiments to characterize the substrate specificity and recognition by LSD1. Biochemical assays on histone peptides show that essentially all epigenetic modifications on the 21 N-terminal amino acids of histone H3 cause a significant reduction in enzymatic activity. Replacement of Lys4 with Arg greatly enhances binding affinity, and a histone peptide incorporating this mutation has a strong inhibitory power. Conversely, a peptide bearing a trimethylated Lys4 is only a weak inhibitor of the enzyme. Rapid kinetics measurements evidence that the enzyme is efficiently reoxidized by molecular oxygen with a second-order rate constant of 9.6x10(3) M-1 s-1, and that the presence of the reaction product does not greatly influence the rate of flavin reoxidation. In vivo experiments provide a correlation between the in vitro inhibitory properties of the tested peptides and their ability of affecting endogenous LSD1 activity. Our results show that epigenetic modifications on histone H3 need to be removed before Lys4 demethylation can efficiently occur. The complex formed by LSD1 with histone deacetylases 1/2 may function as a "double-blade razor" that first eliminates the acetyl groups from acetylated Lys residues and then removes the methyl group from Lys4. We suggest that after H3-K4 demethylation, LSD1 recruits the forthcoming chromatin remodelers leading to the introduction of gene repression marks.     

Structural basis for the methylation state-specific recognition of histone H4-K20 by 53BP1 and Crb2 in DNA repair

Histone lysine methylation has been linked to the recruitment of mammalian DNA repair factor 53BP1 and putative fission yeast homolog Crb2 to DNA double-strand breaks (DSBs), but how histone recognition is achieved has not been established. Here we demonstrate that this link occurs through direct binding of 53BP1 and Crb2 to histone H4. Using X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, we show that, despite low amino acid sequence conservation, both 53BP1 and Crb2 contain tandem tudor domains that interact with histone H4 specifically dimethylated at Lys20 (H4-K20me2). The structure of 53BP1/H4-K20me2 complex uncovers a unique five-residue 53BP1 binding cage, remarkably conserved in the structure of Crb2, that best accommodates a dimethyllysine but excludes a trimethyllysine, thus explaining the methylation state-specific recognition of H4-K20. This study reveals an evolutionarily conserved molecular mechanism of targeting DNA repair proteins to DSBs by direct recognition of H4-K20me2.     

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.     

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.     

Histone methyltransferases direct different degrees of methylation to define distinct chromatin domains

The functional significance of mono-, di-, and trimethylation of lysine residues within histone proteins remains unclear. Antibodies developed to selectively recognize each of these methylated states at histone H3 lysine 9 (H3 Lys9) demonstrated that mono- and dimethylation localized specifically to silent domains within euchromatin. In contrast, trimethylated H3 Lys9 was enriched at pericentric heterochromatin. Enzymes known to methylate H3 Lys9 displayed remarkably different enzymatic properties in vivo. G9a was responsible for all detectable H3 Lys9 dimethylation and a significant amount of monomethylation within silent euchromatin. In contrast, Suv39h1 and Suv39h2 directed H3 Lys9 trimethylation specifically at pericentric heterochromatin. Thus, different methylated states of H3 Lys9 are directed by specific histone methyltransferases to "mark" distinct domains of silent chromatin.