Primary structure of histone H2A from nucleated erythrocyte of the marine worm Sipunculus nudus. Presence of two forms of H2A in the sipunculid chromatin

The complete amino acid sequence (123 residues) of histone H2A from erythrocytes of the marine worm Sipunculus nudus, has been established from data provided by automated sequence analysis of large fragments generated by V8 staphylococcal protease digestion of histone H2A and by limited hydrolysis of the protein with alpha-chymotrypsin and from structural studies of tryptic peptides of the protein. By comparison with calf homologous histone, the sipunculid histone H2A shows 6 deletions and 13 substitutions. Six of the substitutions are non-conservative. Most of the evolutionary changes are mainly observed in the basic amino-terminal and carboxy-terminal regions of the molecule, which are the primary DNA-binding sites. Few conservative point changes are observed in the central region (residues 18-118) which interacts strongly with histone H2B to form the dimer H2A-H2B. 60% of the H2A molecules were found phosphorylated on the amino-terminal residue, N-acetyl-serine. The high content of phosphorylated histone H2A in the sipunculid erythrocyte chromatin could probably be related to smaller repeat length (177 +/- 5 base pairs) of nucleosomal DNA and to nuclear inactivation and chromatin condensation.     

Methylation of Drosophila histones at proline, lysine, and arginine residues during heat shock

Heat shock or arsenite treatment alter the pattern of histone methylation in Drosophila cells. Both types of stress induce a rapid increase in the methylation level of histone H2B. The methylated amino acid residue of H2B has been identified by thin layer chromatography and electrophoresis as methylproline and is located at the N-terminal end of H2B. Heat shock also induces a decrease in the level of methylation of histone H3. Under normal growth temperature conditions, histone H3 is shown to be methylated on lysine residues. However under heat shock conditions, there is a decrease in the extent of methylation of lysine residues and the appearance of new methylation on arginine residues in H3. These new heat shock-induced methylated residues have been identified as the symmetrical and asymmetrical forms of dimethylarginine. The methylated amino acid residue of histone H4 is lysine with mono-, di-, and trimethyl forms found in both control and heat or chemically stressed cells. These stress-induced changes in the methylation level of the N-terminal proline residue of histone H2B and shift in the methylation sites of histone H3 may be involved in the restructuration of chromatin accompanying the inactivation of normal genes in response to stress. Moreover, we suggest that the hypermethylation of H2B may also be involved in its protection from increased ubiquitin-mediated proteolytic activity under these conditions of cellular stress.     

Multiple forms of histone H2B from the nematode Caenorhabditis elegans.

The complete amino acid sequence of histone H2B from the nematode Caenorhabditis elegans was determined. The protein as obtained by us is a mixture of multiple forms. Approx. 90% of the molecules consist of a polypeptide chain of 122 amino acids with alanine as N-terminal residue and proline at the second position. In the remaining 10% alanine is lacking and the chain starts with proline. In addition to the heterogeneity of chain length, polymorphism occurs at the positions 7 (Ala/Lys), 14 (Ala/Lys) and 72 (Ala/Ser) of the major chain and at position 6 (Ala/Lys) of the shorter chain. In the N-terminal third of the molecule there is a high degree of sequence homology to the corresponding region in H2B from Drosophila (insect), Patella (mollusc) and Asterias (starfish). In contrast, this part of the molecule differs considerably from mammalian histone H2B.     

Synthesis and conformation of the amino-terminal hexapeptide of histone H2B from gonads of the starfish Asterias rubens

This paper describes the synthesis and the physico-chemical characterization of N-(di-CH3)-L-Pro-L-Pro-L-Lys-L-Pro-L-Ser-Gly-OH by conventional methods of peptide chemistry. This peptide corresponds to the amino-terminal moiety of histone H2B from gonads of the starfish Asterias rubens.     

Nucleosomal structure of sea urchin and starfish sperm chromatin. Histone H2B is possibly involved in determining the length of linker DNA.

Comparison has been made between sea urchin and starfish sperm chromatin. The only protein by which chromatins from these sources differ significantly is histone H2B. Sea urchin sperm H2B is known to contain an elongated N-terminal region enriched in Arg. Analysis of the micrococcal nuclease digests of sea urchin and starfish nuclei in one- and two-dimensional electrophoresis has shown that sperm chromatin of both animals consists of repeated units similar in general features to those of rat thymus or liver. However, DNA repeat length in chromatin of sea urchin sperm (237 bp) is higher than that of starfish sperm (224 bp), while the core DNA length does not differ and is the same as in the chromatin of rat liver or thymus. A suggestion has been made that the N-terminal region of histone H2B is associated with the linker DNA and is responsible for the increased length of sea urchin linker DNA.     

Characterization of post-translational modifications of histone H2B-variants isolated from Arabidopsis thaliana

Eukaryotic DNA is structurally packed into chromatin by the basic histone proteins H2A, H2B, H3, and H4. There is increasing evidence that incorporation and post-translational modifications of histone variants have a fundamental role in gene regulation. While modifications of H3 and H4 histones are now well-established, considerably less is known about H2B modifications. Here, we present the first detailed characterization of H2B-variants isolated from the model plant Arabidopsis thaliana. We combined reversed-phase chromatography with tandem mass spectrometry to identify post-translational modifications of the H2B-variants HTB1, HTB2, HTB4, HTB9, and HTB11, isolated from total chromatin and euchromatin-enriched fractions. The HTB9-variant has acetylation sites at lysines 6, 11, 27, 32, 38, and 39, while Lys-145 can be ubiquitinated. Analogous modifications and an additional methylation of Lys-3 were identified for HTB11. HTB2 shows similar acetylation and ubiquitination sites and an additional methylation at Lys-11. Furthermore, the N-terminal alanine residues of HTB9 and HTB11 were found to be mono-, di-, or trimethylated or unmodified. No methylation of arginine residues was detected. The data suggest that most of these modification sites are only partially occupied. Our study significantly expands the map of covalent Arabidopsis histone modifications and is the first step to unraveling the histone code in higher plants.     

In vivo cross-linking of nucleosomal histones catalyzed by nuclear transglutaminase in starfish sperm and its induction by egg jelly triggering the acrosome reaction.

A histone heterodimer, designated as p28, which contains an Nepsilon(gamma-glutamyl)lysine cross-link between Gln9 of histone H2B and Lys5 or Lys12 of histone H4, is present in starfish (Asterina pectinifera) sperm. Treatment of sperm nuclei with micrococcal nuclease produced soluble chromatin, which was size-fractionated by sucrose-gradient centrifugation to give p28-containing oligonucleosome and p28-free mononucleosome fractions, indicating that the cross-link is internucleosomal. When sperm nuclei were incubated with monodansylcadaverine, a fluorescent amine, in the presence or absence of Ca(2+), histone H2B was modified only in the presence of Ca(2+). Gln9, in the N-terminal region, was modified, but the other Gln residues located in the internal region were not, suggesting that the modification takes place on the surface of the nucleosome core by the in situ action of a Ca(2+)-dependent nuclear transglutaminase. Treatment of sperm with the egg jelly, which activates Ca(2+) influx to induce the acrosome reaction, resulted in a significant elevation of the p28 content in the nucleus. This is the first demonstration of an in vivo activation of transglutaminase leading to the formation of a cross-link in intracellular proteins.     

Sperm histones and chromatin structure of the "primitive" sea urchin Eucidaris tribuloides

The "primitive" sea urchin Eucidaris tribuloides resembles the advanced sea urchins (euechinoids) in many respects, yet some features of its biochemistry and morphogenesis are more similar to other echinoderms such as starfish or sea cucumbers. Two unique characteristics of the sperm chromatin of all known euechinoids are an extremely long average nucleosomal repeat length and the presence of two male germ-line-specific histone variants, Sp H1 and Sp H2B. Histone composition and nucleosomal repeat length of the sperm chromatin of Eucidaris were compared to those of several euechinoids and a starfish. Eucidaris sperm chromatin contained large H1 and H2B histone variants typical of euechinoids. The H1 was about nine amino acids smaller than Sp H1 of the advanced urchin Strongylocentrotus purpuratus. Its Sp H2B molecules were the same size as in the euechinoids. Peptide maps showed that N-terminal regions of Sp H1 and Sp H2B contained repeating basic amino acid motifs characteristic of euechinoids. The smaller size of Eucidaris H1 is accounted for by a smaller C-terminal region. The repeat length of Eucidaris sperm chromatin was slightly shorter than that of two euechinoids, but significantly larger than starfish, which lacks a large H2B. The Sp H2B gene of Eucidaris was expressed during spermatogenesis in the same cell types as for S. purpuratus. Thus Sp histone subtype expression and chromatin structure in this distantly related echinoid closely resemble the euechinoids. The presence of an Sp H2B and a very long repeat length appear to be characteristic of the echinoids only.     

The complete amino-acid sequence of histone H2B from the mollusc Patella granatina.

1. From the marine mollusc, Patella granatina, a histone has been isolated. Its primary structure has been established and it has been designated histone H2Bpatella. It consists of a polypeptide chain of 121 amino acids. 2. In the carboxy-terminal two thirds of the molecule there is a highly degree of sequence homology to the corresponding region in calf histone H2B with identical residues in 95% of the positions. 3. In the N-terminal 22 amino acids histone H2Bpatella differs considerably from the mammalian histone H2B and it is shorter by four residues.     

Histones of Chlamydomonas reinhardtii. Synthesis, acetylation, and methylation.

Histones of the green alga Chlamydomonas reinhardtii were prepared by a new method and fractionated by reversed-phase high-performance liquid chromatography. Acid-urea-Triton gel analysis and tritiated acetate labeling demonstrated high levels of steady-state acetylation for the single histone H3 protein, in contrast to low levels on histones H4 and H2B. Twenty percent of histone H3 is subject to dynamic acetylation with, on average, three acetylated lysine residues per protein molecule. Histone synthesis in light-dark-synchronized cultures was biphasic with pattern differences between two histone H1 variants, between two H2A variants, and between H2B and ubiquitinated H2B. Automated protein sequence analysis of histone H3 demonstrated a site-specific pattern of steady-state acetylation between 7 and 17% at five of the six amino-terminal lysines and of monomethylation between 5 and 81% at five of the eight amino-terminal lysines in a pattern that may limit dynamic acetylation. An algal histone H3 sequence was confirmed by protein sequencing with a single threonine as residue 28 instead of the serine28-alanine29 sequence, present in all other known plant and animal H3 histones.     

Histone autoantigens in murine lupus. Definition of a major epitope within an accessible region of chromatin.

In SLE and in the (NZB x NZW)F1 murine model of this disease, IgG autoantibodies are frequently produced to DNA and histones. In the present study, we define a linear epitope on histone H2B that is recognized by (NZB x NZW)F1 mice. IgG antibodies from anti-H2B positive (but not anti-H2B negative) mice bound strongly to a peptide containing the first 15 N-terminal amino acids, a region that is exposed in chromatin. Competitive inhibition studies showed that the binding of autoantibodies to H2B in ELISA as well as the binding to soluble H2B was substantially blocked by this peptide. Studies with smaller peptides mapped the epitope to residues 3-12. Individual mice recognized different residues within this region, and a sequence search did not reveal proteins other than H2B that could elicit this spectrum of antibodies. Interestingly, these autoantibody specificities were not a component of those induced in preautoimmune mice by immunization with H2B/RNA complexes or with H2B peptide 1-30 containing the autoantigenic sequence. These findings argue that recognition of a specific N-terminal region of self histone contributes to the anti-H2B autoantibody response in lupus. Autoreactive B cells with specificity for this sequence seem to develop only after the autoimmune process has been initiated.     

Histone H4 K16Q mutation, an acetylation mimic, causes structural disorder of its N-terminal basic patch in the nucleosome

Histone tails and their posttranslational modifications play important roles in regulating the structure and dynamics of chromatin. For histone H4, the basic patch K(16)R(17)H(18)R(19) in the N-terminal tail modulates chromatin compaction and nucleosome sliding catalyzed by ATP-dependent ISWI chromatin remodeling enzymes while acetylation of H4 K16 affects both functions. The structural basis for the effects of this acetylation is unknown. Here, we investigated the conformation of histone tails in the nucleosome by solution NMR. We found that backbone amides of the N-terminal tails of histones H2A, H2B, and H3 are largely observable due to their conformational disorder. However, only residues 1-15 in H4 can be detected, indicating that residues 16-22 in the tails of both H4 histones fold onto the nucleosome core. Surprisingly, we found that K16Q mutation in H4, a mimic of K16 acetylation, leads to a structural disorder of the basic patch. Thus, our study suggests that the folded structure of the H4 basic patch in the nucleosome is important for chromatin compaction and nucleosome remodeling by ISWI enzymes while K16 acetylation affects both functions by causing structural disorder of the basic patch K(16)R(17)H(18)R(19).     

Conformation and domain structure of the non-histone chromosomal proteins, HMG 1 and 2. Isolation of two folded fragments from HMG 1 and 2

Proteins HMG 1 and 2 have been digested with trypsin and two major products, stable to further digestion between 8 min and 2 h, have been purified (peptides A and B). Peptide B from HMG 1 has been identified as residues 12-75 and peptide A as residues 94/96-169 by amino acid analyses and Edman degradations. Peptide B spontaneously folds with the formation of 51% helix and exhibits the majority of the perturbed NMR resonances characteristic of folded intact HMG 1. Peptide B is stably folded in the presence of 150 mM NaCl between pH 3 and 10, like intact HMG 1. Peptide A forms 30% alpha-helix and also exhibits tertiary folding but is denatured by pH 10. The 11 N-terminal residues removed by trypsin contain both sites of post-synthetic acetylation (residues 2 and 11), a situation very similar to that found with core histones. It is proposed that HMG 1 and 2 consist of four structural domains, viz: (a) residues 1-11, (b) residues 12 to approximately 75, (c) residues 94-169 and (d) the very acidic region beyond residue 169. The instability of peptide A may mean that it is not a truly independent domain. No structural similarities to histone H1 are therefore observed in HMG 1 and 2.     

Histone H3 N-terminal mutations allow hyperactivation of the yeast GAL1 gene in vivo.

Recent work has shown that the yeast histone H4 N-terminus, while not essential for viability, is required for repression of the silent mating loci and activation of GAL1 and PHO5 promoters. Because histone H3 shares many structural features with histone H4 and is intimately associated with H4 in the assembled nucleosome, we asked whether H3 has similar functions. While the basic N-terminal domain of H3 is found to be non-essential (deletion of residues 4-40 of this 135 amino acid protein allows viability), its removal has only a minor effect on mating. Surprisingly, both deletions (of residues 4-15) and acetylation site substitutions (at residues 9, 14 and 18) within the N-terminus of H3 allow hyperactivation of the GAL1 promoter as well as a number of other GAL4-regulated genes including GAL2, GAL7 and GAL10. To a limited extent glucose repression is also alleviated by H3 N-terminal deletions. Expression of another inducible promoter, PHO5, is shown to be relatively unaffected. We conclude that the H3 and H4 N-termini have different functions in both the repression of the silent mating loci and in the regulation of GAL1.     

Identification of five sites of acetylation in alfalfa histone H4.

Radioactive acetylation in vivo of plant histone H4 of alfalfa, Arabidopsis, tobacco, and carrot revealed five distinct forms of radioactive, acetylated histone. In histone H4 of eukaryotes ranging from fungi to man, acetylation is restricted to four lysines (residues 5, 8, 12, and 16) possibly caused by a quantitative methylation of lysine-20. Chemical and proteolytic fragmentation of the amino terminally blocked alfalfa H4 protein, dynamically acetylated by radioactive acetate in vivo, allowed protein sequencing and identification of selected peptides. Peptide identification was facilitated by analyzing fully characterized calf histone H4 in parallel. Acetylation in vivo of alfalfa histone H4 was restricted to the lysines in the amino-terminal domain of the protein, residues 1-23. Lysine-20 was shown to be free of methylation, as in pea histone H4. This apparently makes lysine-20 accessible as a novel target for histone acetylation. The in vivo pattern of lysine acetylation (16 greater than 12 greater than 8 greater than or equal to 5 = 20) revealed a preference for lysines -16 and -12 without an apparent strict sequential specificity of acetylation.     

Accessibility of tyrosyl residues altered by formation of the histone 2A/2B complex

The availability of tyrosyl residues to surface iodination was analyzed for histone 2A (H2A), histone 2B (H2B), and the H2A/H2B complex. When H2A is free in solution (200 mM NaCl, pH 7.4) tyrosine-39 and one or both tyrosines-50 and -57 were readily iodinated. Tyrosines-83 and -121 of H2B were iodinated, both when the histone was free in solution and when it was associated with H2A, while tyrosines-37, -40, and -42 of H2B were not iodinated under either condition. When H2A and H2B were associated or covalently cross-linked, all tyrosyl residues of H2A were unavailable for iodination. We also found that the iodination of nondenatured H2A and H2B did not inhibit formation of the H2A/H2B complex. These results indicate that the amino-terminal regions of the hydrophobic portions of H2A and H2B undergo significant conformational changes upon formation of the H2A/H2B complex. These conformational shifts occur in the same region of the H2A/H2B complex that contains a contact site between H2A and H2B in the nucleosome, thus indicating an involvement of this region in chromatin assembly.     

Yeast Hsl7 (histone synthetic lethal 7) catalyses the in vitro formation of omega-N(G)-monomethylarginine in calf thymus histone H2A

The HSL7 (histone synthetic lethal 7) gene in the yeast Saccharomyces cerevisiae encodes a protein with close sequence similarity to the mammalian PRMT5 protein, a member of the class of protein arginine methyltransferases that catalyses the formation of omega-N(G)-monomethylarginine and symmetric omega-N(G),N'(G)-dimethylarginine residues in a number of methyl-accepting species. A full-length HSL7 construct was expressed as a FLAG-tagged protein in Saccharomyces cerevisiae. We found that FLAG-tagged Hsl7 effectively catalyses the transfer of methyl groups from S-adenosyl-[methyl-3H]-L-methionine to calf thymus histone H2A. When the acid-hydrolysed radiolabelled protein products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species that co-migrated with an omega-N(G)-monomethylarginine standard. No radioactivity was observed that co-migrated with either the asymmetric or symmetric dimethylated derivatives. In control experiments, no methylation of histone H2A was found with two mutant constructs of Hsl7. Surprisingly, FLAG-Hsl7 does not appear to effectively catalyse the in vitro methylation of a GST (glutathione S-transferase)-GAR [glycine- and arginine-rich human fibrillarin-(1-148) peptide] fusion protein or bovine brain myelin basic protein, both good methyl-accepting substrates for the human homologue PRMT5. Additionally, FLAG-Hsl7 demonstrates no activity on purified calf thymus histones H1, H2B, H3 or H4. GST-Rmt1, the GST-fusion protein of the major yeast protein arginine methyltransferase, was also found to methylate calf thymus histone H2A. Although we detected Rmt1-dependent arginine methylation in vivo in purified yeast histones H2A, H2B, H3 and H4, we found no evidence for Hsl7-dependent methylation of endogenous yeast histones. The physiological substrates of the Hsl7 enzyme remain to be identified.     

Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor

Dynamic methylations and demethylations of histone lysine residues are important for gene regulation and are facilitated by histone methyltransferases and histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2-specific lysine demethylase belonging to the JmjC domain-containing family of histone demethylases (JHDMs). Several studies have linked KDM5B to breast, prostate and skin cancer, highlighting its potential as a drug target. However, most inhibitor studies have focused on other JHDMs, and inhibitors for KDM5B remain to be explored. Here, we report the expression, purification and characterization of the catalytic core of recombinant KDM5B (ccKDM5B, residues 1-769). We show that ccKDM5B, recombinantly expressed in insect cells, demethylates H3K4me3 and H3K4me2 in vitro. The kinetic characterization showed that ccKDM5B has an apparent Michaelis constant (K(m) (app) ) value of 0.5 μm for its trimethylated substrate H3(1-15)K4me3, a considerably increased apparent substrate affinity than reported for related HDMs. Despite the presence of a PHD domain, the catalytic activity was not affected by additional methylation at the H3K9 position, suggesting that in vitro chromatin cross-talk between H3K4 and H3K9 does not occur for ccKDM5B. Inhibition studies of ccKDM5B showed both in vitro and in cell inhibition of ccKDM5B by 2,4-pyridinedicarboxylic acid (2,4-PDCA) with a potency similar to that reported for the HDM KDM4C. Structure-guided sequence alignment indicated that the binding mode of 2,4-PDCA is conserved between KDM4A/C and KDM5B.     

Organization, primary structure, and evolution of histone H2A and H2B genes of the fission yeast Schizosaccharomyces pombe.

The histone H2A and H2B genes of the fission yeast Schizosaccharomyces pombe were cloned and sequenced. Southern blot and sequence analyses showed that, unlike other eucaryotes, Saccharomyces cerevisiae included, S. pombe has unequal numbers of these genes, containing two histone H2A genes (H2A-alpha and -beta) and only one H2B gene (H2B-alpha) per haploid genome. H2A- and H2B-alpha are adjacent to each other and are divergently transcribed. H2A-beta has no other histone gene in close proximity. Preceding both H2A-alpha and -beta is a highly conserved 19-base-pair sequence (5'-CATCAC/AAACCCTAACCCTG-3'). The H2A DNA sequences encode two histone H2A subtypes differing in amino acid sequence (three residues) and size (H2A-alpha, 131 residues; H2A-beta, 130 residues). H2B-alpha codes for a 125-amino-acid protein. Sequence evolution is extensive between S. pombe and S. cerevisiae and displays unique patterns of divergence. Certain N-terminal sequences normally divergent between eucaryotes are conserved between the two yeasts. In contrast, the normally conserved hydrophobic core of H2A is as divergent between the yeasts as between S. pombe and calf.