Specificity of the histone lysine methyltransferases from rat brain chromatin

The histone lysine methyltransferases catalyze the transfer of methyl groups from S-adenosylmethionine to specific epsilon-N-lysine residues in the N-terminal regions of histones H3 and H4. These enzymes are located exclusively within the nucleus and are firmly bound to chromatin. The chromosomal bound enzymes do not methylate free or nonspecifically associated histones, while histones H3 and H4 within newly synthesized chromatin are methylated. These enzymes can be solubilized by limited digestion (10-16%) of chromosomal DNA from rapidly proliferating rat brain chromatin with micrococcal nuclease. Histone H3 lysine methyltransferase remained associated with a short DNA fragment throughout purification. Dissociation of the enzyme from the DNA fragment with DNAase digestion resulted in complete loss of enzyme activity; however, when this enzyme remained associated with DNA it was quite stable. Activity of the dissociated enzyme could not be restored upon the addition of sheared calf thymus or Escherichia coli DNA. Histone H3 lysine methyltransferase was found to methylate lysine residues in chromosomal bound or soluble histone H3, while H3 associated with mature nucleosomes was not methylated. The histone H4 lysine methyltransferase which was detectable in the crude nuclease digest was extremely labile, losing all activity upon further purification. We isolated a methyltransferase by DEAE-cellulose chromatography, which would transfer methyl groups to arginine residues in soluble histone H4. However, this enzyme would not methylate nucleosomal or chromosomal bound histone H4, nor were methylated arginine nucleosomal or chromosomal bound histone H4, nor were methylated arginine residues detectable upon incubating intact nuclei or chromatin with S-adenosylmethionine.     

Partial purification and characterization of a protein lysine methyltransferase from plasmodia of Physarum polycephalum.

Plasmodia of Physarum polycephalum have an active protein lysine methyltransferase (S-adenosylmethionine:protein-lysine methyltransferase, EC 2.1.1.43). This enzyme has been purified 40-fold with a 13% yield, and it catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to the epsilon-amino group of lysine residues with formation of N epsilon-mono-, N epsilon-di-, and N epsilon-trimethyllysines in a molar ratio of 4:1:1 based on [14C]methyl incorporation into the methylated lysines. The ratio remains unchanged at all stages of the partial purification, as well as after fractionation by sucrose density gradient centrifugation and gel electrophoresis. The rate of protein methylation is time dependent, enzyme concentration dependent, and requires the presence of a sulfhydryl reducing agent for optimal activity. The enzyme has optimal activity at pH 8 and is inhibited by S-adenosyl-L-homocysteine and EDTA. Lysine-rich and arginine-rich histones serve as the most effective exogenous protein acceptors; P. polycephalum actomyosin is inactive, and chick skeletal myofibrillar proteins are 25% as effective as exogenous mixed histones as substrates. Lysine, polylysine, ribonuclease A, cytochrome c, and bovine serum albumin are not methylated.     

Protein methylation in animal cells. I. Purification and properties of S-adenosyl-L-methionine:protein (arginine) N-methyltransferase from Krebs II ascites cells.

1. A protein methylase which specifically transfers methyl groups from S-adenosyl-L-methionine to arginine residues of histones has been substantially purified from Krebs II ascites cells. The purified enzyme was obtained free of contamination by other protein methyl transferases specific for carboxyl and lysine residues. This latter activity copurified with the present enzyme until advanced stages of purification. 2. The purified enzyme does not require any divalent cation for maximum activity. It is inhibited by ionic strength, N-ethylmaleimide and S-adenosyl-L-homocysteine. It has an apparent molecular weight on gel filtration of approx. 5 . 10(5). A Km value for S-adenosyl-L-methionine of 2.5 . 10(-6) M was determined, while the dissociation constant Ki for S-adenosyl-L-homocysteine, which acts as a competitor, was 1.4 . 10(-6) M.     

Mechanism of histone lysine methyl transfer revealed by the structure of SET7/9-AdoMet

The methylation of lysine residues of histones plays a pivotal role in the regulation of chromatin structure and gene expression. Here, we report two crystal structures of SET7/9, a histone methyltransferase (HMTase) that transfers methyl groups to Lys4 of histone H3, in complex with S-adenosyl-L-methionine (AdoMet) determined at 1.7 and 2.3 A resolution. The structures reveal an active site consisting of: (i) a binding pocket between the SET domain and a c-SET helix where an AdoMet molecule in an unusual conformation binds; (ii) a narrow substrate-specific channel that only unmethylated lysine residues can access; and (iii) a catalytic tyrosine residue. The methyl group of AdoMet is directed to the narrow channel where a substrate lysine enters from the opposite side. We demonstrate that SET7/9 can transfer two but not three methyl groups to unmodified Lys4 of H3 without substrate dissociation. The unusual features of the SET domain-containing HMTase discriminate between the un- and methylated lysine substrate, and the methylation sites for the histone H3 tail.     

Properties of soluble rat brain histone lysine methyltransferase.

Histone-lysine methyltransferase has been solubilized from rat brain chromatin by repeated extraction with distilled water. The enzyme was further purified by chromatography on DEAE-cellulose and gel filtration. With chromosomal-bound histones as substrates, the enzyme methylated only the lysyl residues in histones H3 and H4. The ratio of N epsilon-mono-: N epsilon-di-: N epsilon-trimethyllysine in histone H3 was 1.8:1.0:0.45 and the ratio of N epsilon-mono-: N epsilon-dimethyllysine in histone H4 was 0.7:1.0. The enzyme loses specificity with soluble histones as substrates; however, histones H3 and H4 were still the best methyl acceptors. The pH optima for the enzyme with soluble histones H3 and H4 as substrates were 8.2 to 8.7 and 7.2 to 8.0, respectively. S-Adenosyl-L-homocysteine, one of the products of the reaction, was a competitive inhibitor with respect to S-adenosyl-L-methionine.     

Purification and properties of a histone acetyltransferase from Artemia salina, highly efficient with H1 histone.

An histone acetyltransferase has been purified from nuclei of 40-h-old Artemia salina larvae. The enzyme is very unstable at 0 degrees C, requires free -SH groups for activity and is rapidly inactivated at 40 degrees C. The optimal pH for activity is 8.5 and the activity is half inhibited by millimolar concentrations of Mn2+, Ca2+ or Mg2+ or decimolar concentrations of Na+ and K+. The molecular weight of the enzyme, determined by gel filtration chromatography, changed with the ionic strength of the medium (280,000 in 10 mM Tris . HCl, 170,000 in 0.2 M KCl). The very-lysine-rich histone H1 is a better substrate acceptor than the arginine-rich histones H3 or H4. Under proper conditions, the enzyme can modify all the internal lysyl residues in histones H1 and H4. The acetylation of H1 is inhibited when all the other histone fractions are present in the assay mixture.     

Calmodulin N-methyltransferase. Partial purification and characterization

The distribution, properties, and substrate specificity of S-adenosylmethionine:calmodulin (lysine) N-methyltransferse (EC 2.1.1.60, calmodulin N-methyltransferase) of the rat have been studied. This enzyme is cytosolic and is found at high levels in tissues with high levels of calmodulin and at low levels in tissues with little calmodulin. In liver, heart, and skeletal muscle, which have low levels of calmodulin and very low calmodulin N-methyltransferase activity (a low ratio of calmodulin N-methyltransferase to calmodulin), calmodulin was found to be incompletely methylated, as judged by its ability to act as a substrate for purified calmodulin N-methyltransferase. Calmodulin N-methyltransferase was purified 470-fold with a 33% yield from rat testis cytosol, using ammonium sulfate precipitation and chromatography on DEAE-cellulose, CM-Sepharose, and Sephadex G-100. At pH 7.4, calmodulin N-methyltransferase did not bind to DEAE-cellulose, but bound strongly to CM-Sepharose. The enzyme eluted from Sephadex G-100 with an apparent molecular weight of 55,000. Purified calmodulin N-methyltransferase was incubated with extracts of rat tissues, and [methyl-3H]AdoMet and methylated proteins were resolved by electrophoresis in an attempt to discover substances other than calmodulin, but this enzyme only catalyzed the methylation of calmodulin, indicating a high degree of substrate specificity. Conditions were established for the in vitro preparative methylation of des(methyl)-calmodulin from Dictyostelium discoideum. Three moles of methyl/mol of calmodulin were incorporated into lysine 115 of des(methyl)calmodulin, resulting in the formation of 1 mol of trimethyllysine at the site normally methylated in calmodulins from most species. Activation of cyclic nucleotide phosphodiesterase by des(methyl)calmodulin was indistinguishable from activation by in vitro methylated or sham methylated Dictyostelium calmodulin, indicating that methylation does not affect the ability of calmodulin to activate this enzyme.     

Sites of in vivo histone methylation in developing trout testis.

Specific lysyl residues of trout testis histones H3 and H4 are methylated partially during rainbow trout spermatogenesis. Histones H1, H2A, H2B, and protamine are not methylated. The single site (lysine 20) in histone H4 and the two major sites (lysines 9 and 27) in histone H3 are homologous to those determined for other organisms, but an additional minor site (lysine 4) occurs in histone H3. As described for calf thymus, both histones H3 and H4 contain epsilon-N-mono- and dimethyllysine, while histone H3 contains in addition, epsilon-N-trimethyllysine. The trout-specific histone H6, which accounts for 0.5 to 1.0% of total histone, contains a sequence for residues 3 to 5,-Arg-Lys-Ser-, which is the same as one methylated in histones H3, at lysines 9 and 27. However, histone H6 yields only trace amounts of [3H]methyl incorporation and no detectable methyllysines on amino acid analysis.     

ADP-ribosyltransferase from hen liver nuclei. Purification and characterization

Chromatin-bound ADP-ribosyltransferase from adult hen liver nuclei was purified to a homogeneous state through salt extraction, gel filtration, hydroxyapatite, phenyl-Sepharose, Cm-cellulose, and DNA-Sepharose. The ADP-ribosyltransferase has a pH optimum at 9.0 and does not require DNA for reaction. The purified enzyme has a molecular weight of 27,500 +/- 500. Agmatine sulfate, arginine methyl ester, histones, and casein proved to be effective acceptors for the ADP-ribose molecule. Among histones, H3 was most active, followed by H2a, H4, and H2b, in that order, the lowest activity seen with H1. With all the acceptors tested, the rate of nicotinamide release was in excess of the ADP-ribosylation. However, changes in the ratio of nicotinamide release to ADP-ribosylation seemed to depend on concentrations of the acceptor used. ADP-ribose-whole histones X adducts formed by ADP-ribosyltransferase served as initiators for poly(ADP-ribose) synthesis when these adducts were incubated in the presence of NAD, DNA, Mg2+, and the purified poly(ADP-ribose) synthetase, in which poly(ADP-ribose) formation can occur.     

Studies of human kidney gamma-glutamyl transpeptidase. Purification and structural, kinetic and immunological properties.

gamma-Glutamyl transpeptidase, present in various mammalian tissues, transfers the gamma-glutamyl moiety of glutathione to a variety of acceptor amino acids and peptides. This enzyme has been purified from human kidney cortex about 740-fold to a specific activity of 200 units/mg of protein. The purification steps involved incubation of the homogenate at 37 degrees followed by centrifugation and extraction of the sediment with 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate; batchwise absorption on DEAE-cellulose; DEAE-cellulose (DE52) column chromatography; Sephadex G-200 gel filtration; and affinity chromatography using concanavalin A insolubilized on beaded Agarose. Detergents were used throughout the purification of the enzyme. The purified enzyme separated into three protein bands, all of which had enzyme activity, on polyacrylamide disc electrophoresis in the presence of Triton X-100. The enzyme has an apparent molecular weight of about 90,000 as shown by Sephadex G-200 gel filtration, and appears to be a tetramer with subunits of molecular weights of about 21,000. The Km for gamma-glutamyl transpeptidase using the artificial substrate, gamma-glutamyl-p-nitroanilide, with glycylglycine as the acceptor amino acid was found to be about 0.8 mM. The optimum pH for the enzyme activity is 8.2 and the isoelectric point is 4.5. Both GSH and GSSG competitively inhibited the activity of gamma-glutamyl transpeptidase when gamma-glutamyl-p-nitroanilide was used as the substrate. Treatment of the purified enzyme with papain has no effect on the enzyme activity or mobility on polyacrylamide disc electrophoresis. The purified gamma-glutamyl transpeptidase had no phosphate-independent glutaminase activity. The ratio of gamma-glutamyl transpeptidase to phosphate-independent glutaminase changed significantly through the initial steps of gamma-glutamyl transpeptidase purification. These studies indicate that the transpeptidase and phosphate-independent glutaminase activities are not exhibited by the same protein in human kidney.     

Enzymic acetylation of nucleosome histone.

Rat liver chromatin prepared from purified nuclei catalyzed the acetylation of histones in nucleosomes at the same level as that of nuclei. The activity of histone acetyltransferase in chromatin was destroyed by heat treatment at 65 degrees C for 5 min. Histones in exogenously added nucleosomes also served as substrate for the enzyme. The sites of acetylation in the nucleosomes appeared to be in the trypsin-digestable N-terminal regions of histones H4, H3, and H2A, as has been reported in an in vivo system.     

Mechanism of multiple lysine methylation by the SET domain enzyme Rubisco LSMT

SET domain protein methyltransferases catalyze the transfer of methyl groups from the cofactor S-adenosylmethionine (AdoMet) to specific lysine residues of protein substrates, such as the N-terminal tails of histones H3 and H4 and the large subunit of the Rubisco holoenzyme complex. The crystal structures of pea Rubisco large subunit methyltransferase (LSMT) in ternary complexes with either lysine or epsilon-N-methyllysine (MeLys) and the product S-adenosylhomocysteine (AdoHcy) were determined to resolutions of 2.65 and 2.55 A, respectively. The zeta-methyl group of MeLys is bound to the enzyme via carbon-oxygen hydrogen bonds that play a key role in catalysis. The methyl donor and acceptor are aligned in a linear geometry for S(N)2 nucleophilic transfer of the methyl group during catalysis. Differences in hydrogen bonding between the MeLys epsilon-amino group and Rubisco LSMT and SET7/9 explain why Rubisco LSMT generates multiply methylated Lys, wheras SET7/9 generates only MeLys.     

Purification and properties of S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase from cell suspension cultures of Glycine max L

A soluble enzyme which catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the nitrogen atom of pyridine-3-carboxylic acid (nicotinic acid) could be detected in protein preparations from heterotrophic cell suspension cultures of soybean (Glycine max L.). Enzyme activity was enriched nearly 100-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography to study kinetic properties. S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase (EC 2.1.1.7) showed a pH optimum at pH 8.0 and a temperature optimum between 35 and 40 degrees C. The apparent KM values were determined to be 78 microM for nicotinic acid and 55 microM for the cosubstrate. S-Adenosyl-L-homocysteine was a competitive inhibitor of the methyltransferase with a KI value of 95 microM. The native enzyme had a molecular mass of about 90 kDa. The catalytic activity was inhibited by reagents blocking SH groups, whereas other divalent cations did not significantly influence of the enzyme reaction. The purified methyltransferase revealed a remarkable specificity for nicotinic acid. No other pyridine derivative was a suitable methyl group acceptor. To study a potential methyltransferase activity with nicotinamide as substrate, an additional purification step was necessary to remove nicotinamide amidohydrolase activity from the enzyme preparation. This was achieved by affinity chromatography on S-adenosyl-L-homocysteine-Sepharose thus leading to a 580-fold purified enzyme which showed no methyltransferase activity toward nicotinamide as substrate.     

A novel post-translational modification of yeast elongation factor 1A. Methylesterification at the C terminus

Protein methylation reactions can play important roles in cell physiology. After labeling intact Saccharomyces cerevisiae cells with S-adenosyl-l-[methyl-(3)H]methionine, we identified a major methylated 49-kDa polypeptide containing [(3)H]methyl groups in two distinct types of linkages. Peptide sequence analysis of the purified methylated protein revealed that it is eukaryotic elongation factor 1A (eEF1A, formerly EF-1alpha), the protein that forms a complex with GTP and aminoacyl-tRNAs for binding to the ribosomal A site during protein translation. Previous studies have shown that eEF1A is methylated on several internal lysine residues to give mono-, di-, and tri-N-epsilon-methyl-lysine derivatives. We confirm this finding but also detect methylation that is released as volatile methyl groups after base hydrolysis, characteristic of ester linkages. In cycloheximide-treated cells, methyl esterified eEF1A was detected largely in the ribosome and polysome fractions; little or no methylated protein was found in the soluble fraction. Because the base-labile, volatile [methyl-(3)H]radioactivity of eEF1A could be released by trypsin treatment but not by carboxypeptidase Y or chymotrypsin treatment, we suggest that the methyl ester is present on the alpha-carboxyl group of its C-terminal lysine residue. From the results of pulse-chase experiments using radiolabeled intact yeast cells, we find that the N-methylated lysine residues of eEF1A are stable over 4 h, whereas the eEF1A carboxyl methyl ester has a half-life of less than 10 min. The rapid turnover of the methyl ester suggests that the methylation/demethylation of eEF1A at the C-terminal carboxyl group may represent a novel mode of regulation of the activity of this protein in yeast.     

S-adenosylmethionine: protein-arginine methyltransferase. Purification and mechanism of the enzyme.

Protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase, EC 2.1.1.23) has been purified from calf brain approximately 120-fold with a 14% yield. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate protein. The enzyme has an optimum pH of 7.2 and pI value of 5.1. The Km values for S-adenosyl-L-methionine, histone H4, and an ancephalitogenic basic protein are 7.6 X 10(-6), 2.5 X 10(-5), and 7.1 X 10(-5) M, respectively, and the Ki value for S-adenosyl-L-homocysteine is 2.62 X 10(-6) M. The enzyme is highly specific for the arginine residues of protein, and the end products after hydrolysis of the methylated protein are NG,NG-di(asymmetric), NG,N'G-di(symmetric), and NG-monomethylarginine. The ratio of [14C]methyl incorporation into these derivatives by enzyme preparation at varying stages of purification remains unchanged at 40:5:55, strongly indicating that a single enzyme is involved in the synthesis of the three arginine derivatives. The kinetic mechanism of the protein methylase I reaction was studied with the purified enzyme. Initial velocity patterns converging at a point on the extended axis of abscissas were obtained with either histone H4 or S-adenosyl-L-methionine as the varied substrate. Product inhibition by S-adenosyl-L-homocysteine with S-adenosyl-L-methionine as the varied substrate was competitive regardless of whether or not the enzyme was saturated with histone H4. On the other hand, when histone H4 is the variable substrate, noncompetitive inhibition was obtained with S-adenosyl-L-homocysteine under conditions where the enzyme is not saturated with the other substrate, S-adenosyl-L-methionine. These results suggest that the mechanism of the protein methylase I reaction is a Sequential Ordered Bi Bi mechanism with S-adenosyl-L-methionine as the first substrate, histone H4 as the second substrate, methylated histone H4 as the first product, and S-adenosyl-L-homocysteine as the second product released.     

S-adenosylmethionine: DNA-cytosine 5-methyltransferase from a Novikoff rat hepatoma cell line.

Partial purification of DNA methylase from Novikoff rat hepatoma cells is described. Contamination with other proteins persists although the enzyme preparation has a high specific activity and is purified 980-fold over homogenate activity. Evidence suggests, but does not prove, that there may be more than one species of DNA methylase in these cells. The enzyme has two broad pH optima at pH 7.0 and 7.5 and most readily methylates heterologous denatured DNAs although complex reaction kinetics indicate that native DNAs may eventually be methylated to an equal or greater level. The preparation of undermethylated DNA from Novikoff cells is also described. Undermethylated homologous DNA is an 85-fold greater acceptor of methyl groups than fully methylated Novikoff cell DNA. In contrast to other DNA substrates, the enzyme preparation methylates native undermethylated homologous DNA at a 3.5-fold greater than denatured undermethylated homologous DNA.     

A cytochrome c methyltransferase from Crithidia oncopelti.

The mitochondrial cytochrome c-557 of Crithidia oncopelti contains two lysine residues and an N-terminal proline residue that are methylated in vivo by the methyl group of methionine. The purified cytochrome can act as a methyl acceptor for a methyltransferase activity in the cell extract that uses S-adenosylmethionine as methyl donor. Crithidia cytochrome c-557 is by far the best substrate for this methyltransferase of those tested, in spite of the fact that methylation sites are already almost fully occupied. The radioactive uptake of [14C]methyl groups from S-adenosylmethionine occurred only at a lysine residue (-8) and the N-terminal proline residue. This methyltransferase appears to differ from that of Neurospora and yeast [Durban, Nochumson, Kim, Paik & Chan (1978) J. Biol. Chem. 253, 1427-1435; DiMaria, Polastro, DeLange, Kim & Paik (1979) J. Biol. Chem. 254, 4645-4652] in that lysine-72 of horse cytochrome c is a poor acceptor. Also, the Crithidia methyltransferase appears to be stable to carry lysine methylation much further to completion than do the enzymes from yeast and Neurospora, which produce very low degrees of methylation in native cytochromes c.     

Porcine liver nuclear histone acetyltransferase. Partial purification and basic properties

The major histone acetyltransferase activity from porcine liver nuclei has been isolated and partially purified by a simple, rapid, and reproducible method. Extraction of nuclei in buffered 30% saturated ammonium sulfate and subsequent ammonium sulfate fractionation, chromatography on DEAE-Sephacel and hydroxylapatite, and ultracentrifugation on linear 15-30% glycerol gradients provides an 8650-fold purification (over nuclei) in 42% yield. The molecular weight of the enzyme is approximately 94,000 as determined by glycerol gradient ultracentrifugation and gel filtration on Sephacryl S-200. The optimum pH for the reaction is 7.5 and the activity is inhibited by monovalent and divalent salts and by sulfhydryl blocking reagents. The enzyme activity is substantially protected from thermal denaturation at 37 degrees C by the addition of glycerol to the incubation medium. In the presence of the core histones, the enzyme catalyzes the acetylation reaction in the order H3 greater than H4 greater than H2B greater than H2A; the order for histones bound in nucleosome core particles is H4 greater than H2B greater than H3 greater than H2A. The high mobility group proteins 14 and 17 serve as substrates for the enzyme in vitro, suggesting a possible role for enzymatic high mobility group acetylation in chromatin function.     

Dimethylation of histone H3 lysine 9 is a critical mark for DNA methylation and gene silencing in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The Arabidopsis KRYPTONITE gene encodes a member of the Su(var)3-9 family of histone methyltransferases. Mutations of kryptonite cause a reduction of methylated histone H3 lysine 9, a loss of DNA methylation, and reduced gene silencing. Lysine residues of histones can be either monomethylated, dimethylated or trimethylated and recent evidence suggests that different methylation states are found in different chromatin domains. Here we show that bulk Arabidopsis histones contain high levels of monomethylated and dimethylated, but not trimethylated histone H3 lysine 9. Using both immunostaining of nuclei and chromatin immunoprecipitation assays, we show that monomethyl and dimethyl histone H3 lysine 9 are concentrated in heterochromatin. In kryptonite mutants, dimethyl histone H3 lysine 9 is nearly completely lost, but monomethyl histone H3 lysine 9 levels are only slightly reduced. Recombinant KRYPTONITE can add one or two, but not three, methyl groups to the lysine 9 position of histone H3. Further, we identify a KRYPTONITE-related protein, SUVH6, which displays histone H3 lysine 9 methylation activity with a spectrum similar to that of KRYPTONITE. Our results suggest that multiple Su(var)3-9 family members are active in Arabidopsis and that dimethylation of histone H3 lysine 9 is the critical mark for gene silencing and DNA methylation.