The SET8 H4K20 protein lysine methyltransferase has a long recognition sequence covering seven amino acid residues

The SET8 histone lysine methyltransferase, which monomethylates the histone 4 lysine 20 residue plays important roles in cell cycle control and genomic stability. By employing peptide arrays we have shown that it has a long recognition sequence motif covering seven amino acid residues, viz. R¹⁷–H¹⁸–(R¹⁹KY)–K²⁰–(V²¹ILFY)–(L²²FY)–R²³. Celluspots peptide array methylation studies confirmed specific monomethylation of H4K20 and revealed that the symmetric and asymmetric methylation on R¹⁷ of the H4 tail inhibits methylation on H4K20. Similarly, dimethylation of the R located at the −3 position also reduced methylation of p53 K382 which had been shown previously to be methylated by SET8. Based on the derived specificity profile, we identified 4 potential non-histone substrate proteins. After relaxing the specificity profile, we identified several more candidate substrates and showed efficient methylation of 20 novel non-histone peptides by SET8. However, apart from H4 and p53 none of the identified novel peptide targets was methylated at the protein level. Since H4 and p53 both contain the target lysine in an unstructured part of the protein, we conclude that the long recognition sequence of SET8 makes it difficult to methylate a lysine in a folded region of a protein, because amino acid side chains essential for recognition will be buried.     

Computational characterization of substrate and product specificities,and functionality of S‐adenosylmethionine binding pocket in histone lysine methyltransferases from Arabidopsis,rice and maize

Histone lysine methylation by histone lysine methyltransferases (HKMTs) has been implicated in regulation of gene expression. While significant progress has been made to understand the roles and mechanisms of animal HKMT functions, only a few plant HKMTs are functionally characterized. To unravel histone substrate specificity, degree of methylation and catalytic activity, we analyzed Arabidopsis Trithorax‐like protein (ATX), Su (var)3‐9 h omologs protein (SUVH), Su(var)3‐9 related protein (SUVR), ATXR5, ATXR6, and E(Z) HKMTs of Arabidopsis, maize and rice through sequence and structure comparison. We show that ATXs may exhibit methyltransferase specificity toward histone 3 lysine 4 (H3K4) and might catalyse the trimethylation. Our analyses also indicate that most SUVH proteins of Arabidopsis may bind histone H3 lysine 9 (H3K9). We also predict that SUVH7, SUVH8, SUVR1, SUVR3, ZmSET20 and ZmSET22 catalyse monomethylation or dimethylation of H3K9. Except for SDG728, which may trimethylate H3K9, all SUVH paralogs in rice may catalyse monomethylation or dimethylation. ZmSET11, ZmSET31, SDG713, SDG715, and SDG726 proteins are predicted to be catalytically inactive because of an incomplete S‐adenosylmethionine (SAM) binding pocket and a post‐SET domain. E(Z) homologs can trimethylate H3K27 substrate, which is similar to the Enhancer of Zeste homolog 2 of humans. Our comparative sequence analyses reveal that ATXR5 and ATXR6 lack motifs/domains required for protein‐protein interaction and polycomb repressive complex 2 complex formation. We propose that subtle variations of key residues at substrate or SAM binding pocket, around the catalytic pocket, or presence of pre‐SET and post‐SET domains in HKMTs of the aforementioned plant species lead to variations in class‐specific HKMT functions and further determine their substrate specificity, the degree of methylation and catalytic activity.     

Catalytic properties and kinetic mechanism of human recombinant Lys-9 histone H3 methyltransferase SUV39H1: participation of the chromodomain in enzymatic catalysis

Histone H3 lysine 9 (H3K9) methylation is a major component of gene regulation and chromatin organization. SUV39H1 methylates H3K9 at the pericentric heterochromatin region and participates in the maintenance of genome stability. In this study, a recombinant purified SUV39H1 is used for substrate specificity and steady-state kinetic analysis with peptides representing the un- or dimethylated lysine 9 histone H3 tail or full-length human recombinant H3 (rH3). Recombinant SUV39H1 methylated its substrate via a nonprocessive mechanism. Binding of either peptide or AdoMet first to the enzyme made a catalytically competent binary complex. Product inhibition studies with SUV39H1 showed that S-adenosyl-l-homocysteine is a competitive inhibitor of S-adenosyl-l-methionine and a mixed inhibitor of substrate peptide. Similarly, the methylated peptide was a competitive inhibitor of the unmethylated peptide and a mixed inhibitor of AdoMet, suggesting a random mechanism in a bi-bi reaction for recombinant SUV39H1 in which either substrate can bind to the enzyme first and either product can release first. The turnover numbers (k(cat)) for the H3 tail peptide and rH3 were comparable (12 and 8 h(-)(1), respectively) compared to the value of 1.5 h(-)(1) for an identical dimethylated lysine 9 H3 tail peptide. The Michaelis constant for the methylated peptide (K(m)(pep)) was 13-fold lower compared to that of the unmethylated peptide. The Michaelis constants for AdoMet (K(m)(AdoMet)) were 12 and 6 microM for the unmethylated peptide substrate and rH3, respectively. A reduction in the level of methylation was observed at high concentrations of rH3, implying substrate inhibition. Deletion of the chromodomain or point mutation of the conserved amino acids, W64A or W67A, of SUV39H1 impaired enzyme activity despite the presence of an intact catalytic SET domain. Thus, SUV39H1 utilizes both the chromodomain and the SET domain for catalysis.     

Three-dimensional structures of fibrillar Sm proteins: Hfq and other Sm-like proteins

SET domain lysine methyltransferases are known to catalyze site and state-specific methylation of lysine residues in histones that is fundamental in epigenetic regulation of gene activation and silencing in eukaryotic organisms. Here we report the three-dimensional solution structure of the SET domain histone lysine methyltransferase (vSET) from Paramecium bursaria chlorella virus 1 bound to cofactor S-adenosyl-L-homocysteine and a histone H3 peptide containing mono-methylated lysine 27. The dimeric structure, mimicking an enzyme/cofactor/substrate complex, yields the structural basis of the substrate specificity and methylation multiplicity of the enzyme. Our results from mutagenesis and enzyme kinetics analyses argue that a general base mechanism is less likely for lysine methylation by SET domains; and that the only invariant active site residue tyrosine 105 in vSET facilitates methyl transfer from cofactor to the substrate lysine by aligning intermolecular interactions in the lysine access channel of the enzyme.     

Structure of the conserved core of the yeast Dot1p, a nucleosomal histone H3 lysine 79 methyltransferase

Methylation of Lys79 on histone H3 by Dot1p is important for gene silencing. The elongated structure of the conserved core of yeast Dot1p contains an N-terminal helical domain and a seven-stranded catalytic domain that harbors the binding site for the methyl-donor and an active site pocket sided with conserved hydrophobic residues. The S-adenosyl-L-homocysteine exhibits an extended conformation distinct from the folded conformation observed in structures of SET domain histone lysine methyltransferases. A catalytic asparagine (Asn479), located at the bottom of the active site pocket, suggests a mechanism similar to that employed for amino methylation in DNA and protein glutamine methylation. The acidic, concave cleft between the two domains contains two basic residue binding pockets that could accommodate the outwardly protruding basic side chains around Lys79 of histone H3 on the disk-like nucleosome surface. Biochemical studies suggest that recombinant Dot1 proteins are active on recombinant nucleosomes, free of any modifications.     

Product specificity and mechanism of protein lysine methyltransferases: insights from the histone lysine methyltransferase SET8

Molecular dynamics simulations employing a molecular mechanics (MM) force field and hybrid quantum mechanics (QM) and MM (QM/MM) have been carried out to investigate the product specificity and mechanism of the histone H4 lysine 20 (H4-K20) methylation by human histone lysine methyltransferase SET8. At neutral pH, the target lysine is available to only the enzyme in the protonated state. The first step in the methylation reaction must be deprotonation of the lysine target which is followed by the (+)AdoMet methylation of the neutral lysine [Enz.Lys-CH(2)-NH(3)(+).(+)AdoMet --> H(+) + Enz.Lys-CH(2)-NH(2).(+)AdoMet -->--> Enz.Lys-CH(2)-N(Me)H(2)(+).AdoHcy]. The electrostatic interactions between two positive charges on (+)AdoMet and Lys20-NH(3)(+) decrease the pK(a) of Lys20-NH(3)(+). Upon formation of Enz.Lys-NH(3)(+).(+)AdoMet, a water channel by which the proton escapes to the outer solvent phase is formed. The formation of a water channel for the escape of a proton from Lys20-N(Me)H(2)(+) in Enz.Lys20-N(Me)H(2)(+).(+)AdoMet is not formed because the methyl substituent blocks the starting of the water channel. Thus, a second methylation does not take place. The dependence of the occurrence of methyl transfer on the formation of a water channel in SET8 is in accord with our previous reports on product specificity by histone lysine monomethyltransferase SET7/9, large subunit lysine dimethyltransferase (LSMT), and viral histone lysine trimethyltransferase (vSET). The average value of the experimental DeltaG(E)() for the six lysine methyl transfer reactions catalyzed by vSET, LSMT, and SET7/9 with p53 as a substrate is 22.1 +/- 1.0 kcal/mol, and the computed average (DeltaG(C)()) is 22.2 +/- 0.8 kcal/mol. In this study, the computed free energy barrier of the methyl transfer reaction [Lys20-NH(2) + (+)AdoMet --> Lys20-N(Me)H(2)(+) + AdoHcy] catalyzed by SET8 is 20.8 kcal/mol. This is in agreement with the value of 20.6 kcal/mol calculated from the experimental rate constant (0.43 +/- 0.02 min(-1)). Our bond-order computations establish that the H4-K20 monomethylation in SET8 is a concerted linear S(N)2 displacement reaction.     

Dynamics of epigenetic control in plants via SET domain containing proteins: Structural and functional insights

Plants control expression of their genes in a way that involves manipulating the chromatin structural dynamics in order to adapt to environmental changes and carry out developmental processes. Histone modifications like histone methylation are significant epigenetic marks which profoundly and globally modify chromatin, potentially affecting the expression of several genes. Methylation of histones is catalyzed by histone lysine methyltransferases (HKMTs), that features an evolutionary conserved domain known as SET [Su(var)3–9, E(Z), Trithorax]. This methylation is directed at particular lysine (K) residues on H3 or H4 histone. Plant SET domain group (SDG) proteins are categorized into different classes that have been conserved through evolution, and each class have specificity that influences how the chromatin structure operates. The domains discovered in plant SET domain proteins have typically been linked to protein-protein interactions, suggesting that majority of the SDGs function in complexes. Additionally, SDG-mediated histone mark deposition also affects alternative splicing events. In present review, we discussed the diversity of SDGs in plants including their structural properties. Additionally, we have provided comprehensive summary of the functions of the SDG-domain containing proteins in plant developmental processes and response to environmental stimuli have also been highlighted.     

Catalytic and Functional Roles of Conserved Amino Acids in the SET Domain of the S. cerevisiae Lysine Methyltransferase Set1

In S. cerevisiae, the lysine methyltransferase Set1 is a member of the multiprotein complex COMPASS. Set1 catalyzes mono-, di- and trimethylation of the fourth residue, lysine 4, of histone H3 using methyl groups from S-adenosylmethionine, and requires a subset of COMPASS proteins for this activity. The methylation activity of COMPASS regulates gene expression and chromosome segregation in vivo. To improve understanding of the catalytic mechanism of Set1, single amino acid substitutions were made within the SET domain. These Set1 mutants were evaluated in vivo by determining the levels of K4-methylated H3, assaying the strength of gene silencing at the rDNA and using a genetic assessment of kinetochore function as a proxy for defects in Dam1 methylation. The findings indicate that no single conserved active site base is required for H3K4 methylation by Set1. Instead, our data suggest that a number of aromatic residues in the SET domain contribute to the formation of an active site that facilitates substrate binding and dictates product specificity. Further, the results suggest that the attributes of Set1 required for trimethylation of histone H3 are those required for Pol II gene silencing at the rDNA and kinetochore function.     

The many faces of histone lysine methylation

Diverse post-translational modifications of histone amino termini represent an important epigenetic mechanism for the organisation of chromatin structure and the regulation of gene activity. Within the past two years, great progress has been made in understanding the functional implications of histone methylation; in particular through the characterisation of histone methyltransferases that direct the site-specific methylation of, for example, lysine 9 and lysine 4 positions in the histone H3 amino terminus. All known histone methyltransferases of this type contain the evolutionarily conserved SET domain and appear to be able to stimulate either gene repression or gene activation. Methylation of H3 Lys9 and Lys4 has been visualised in native chromatin, indicating opposite roles in structuring repressive or accessible chromatin domains. For example, at the mating-type loci in Schizosaccharomyces pombe, at pericentric heterochromatin and at the inactive X chromosome in mammals, striking differences between these distinct marks have been observed. H3 Lys9 methylation is also important to direct additional epigenetic signals such as DNA methylation--for example, in Neurospora crassa and in Arabidopsis thaliana. Together, the available data strongly establish histone lysine methylation as a central modification for the epigenetic organisation of eukaryotic genomes.     

Sequence specificity and role of proximal amino acids of the histone H3 tail on catalysis of murine G9A lysine 9 histone H3 methyltransferase

The activity of recombinant murine G9a toward lysine 9 of histone H3 was investigated. GST fusion proteins containing various lengths of the histone H3 amino-terminal tail were used as substrates in the presence of recombinant G9a enzyme and AdoMet cosubstrate. The minimal substrate methylated by G9a contained seven amino acids (TARKSTG) of the histone H3 tail. Furthermore, mutational analysis of the minimal substrate was performed to identify the amino acids essential for G9a-mediated methylation. All amino acids except Thr-11 were indispensable for the methylation reaction. Steady-state kinetic analysis of the wild-type and histone H3 point mutants, lysine 4 changed to alanine (K4A) or lysine 27 changed to alanine (K27A), with purified G9a revealed similar catalytic efficiency but a reduction in turnover number (k(cat)) from 78 to 58 h(-)(1). G9a methylated synthetic peptide substrates containing the first 13 amino acids of histone H3 efficiently, although methylation, acetylation, or mutation of proximal Lys-4 amino acids reduced Lys-9 methylation. The k(cat) for wild-type peptide substrate vs Lys-4 acetyl- or trimethyl-modified peptide were 88 and 32 h(-)(1), respectively, and the K(m) for the peptides varied from 0.6 to 2.2 muM, resulting in a large difference (15-91) in catalytic efficiency. Ser-10 or Thr-11 phosphorylation resulted in poor methylation by G9a. Immunoprecipitation of unmodified and Ser-10 and Thr-11 phosphorylated histone H3 displayed mostly Lys-4 dimethylation. Dimethylated Lys-9 was reduced in Ser-10 and Thr-11 immunoprecipitated phosphorylated histones as compared to nonphosphorylated H3. In an immunocytochemical assay, GFP fusion SUV39H1 or G9a did not colocalize with phosphorylated histone H3. Thus, Ser-10/Thr-11 phosphorylation impairs Lys-9 methylation. These data suggest that the sequence context of the modified residue affects G9a activity and the modification in the proximal amino acids influences methylation.     

Methylation of histone H4 lysine 20 controls recruitment of Crb2 to sites of DNA damage

Histone lysine methylation is a key regulator of gene expression and heterochromatin function, but little is known as to how this modification impinges on other chromatin activities. Here we demonstrate that a previously uncharacterized SET domain protein, Set9, is responsible for H4-K20 methylation in the fission yeast Schizosaccharomyces pombe. Surprisingly, H4-K20 methylation does not have any apparent role in the regulation of gene expression or heterochromatin function. Rather, we find the modification has a role in DNA damage response. Loss of Set9 activity or mutation of H4-K20 markedly impairs cell survival after genotoxic challenge and compromises the ability of cells to maintain checkpoint mediated cell cycle arrest. Genetic experiments link Set9 to Crb2, a homolog of the mammalian checkpoint protein 53BP1, and the enzyme is required for Crb2 localization to sites of DNA damage. These results argue that H4-K20 methylation functions as a "histone mark" required for the recruitment of the checkpoint protein Crb2.     

Dominant alleles identify SET domain residues required for histone methyltransferase of Polycomb repressive complex 2

Polycomb gene silencing requires histone methyltransferase activity of Polycomb repressive complex 2 (PRC2), which methylates lysine 27 of histone H3. Information on how PRC2 works is limited by lack of structural data on the catalytic subunit, Enhancer of zeste (E(Z)), and the paucity of E(z) mutant alleles that alter its SET domain. Here we analyze missense alleles of Drosophila E(z), selected for molecular study because of their dominant genetic effects. Four missense alleles identify key E(Z) SET domain residues, and a fifth is located in the adjacent CXC domain. Analysis of mutant PRC2 complexes in vitro, and H3-K27 methylation in vivo, shows that each SET domain mutation disrupts PRC2 histone methyltransferase. Based on known SET domain structures, the mutations likely affect either the lysine-substrate binding pocket, the binding site for the adenosylmethionine methyl donor, or a critical tyrosine predicted to interact with the substrate lysine epsilon-amino group. In contrast, the CXC mutant retains catalytic activity, Lys-27 specificity, and trimethylation capacity. Deletion analysis also reveals a functional requirement for a conserved E(Z) domain N-terminal to CXC and SET. These results identify critical SET domain residues needed for PRC2 enzyme function, and they also emphasize functional inputs from outside the SET domain.     

Crystal structure and functional analysis of the histone methyltransferase SET7/9

Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure. The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity. We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta-sheet domain as well as the conserved SET domain. Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3. Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate.