Antigenic structure of histone H2B

Antigenic determinants of histone H2B were localized using a series of 23 overlapping fragments of H2B obtained either by chemical and enzymatic cleavage of the histone or by solid-phase peptide synthesis. The ability of peptides to bind H2B antibodies was measured in an enzyme-linked immunosorbent assay, using antisera directed against calf thymus and chicken erythrocyte H2B as well as four anti H2B monoclonal antibodies obtained from autoimmune mice. Seven antigenic determinants were localized in the H2B molecule in the vicinity of residues 1-11, 6-18, 15-25, 26-35, 50-65, 94-113 and 114-125. Two of these determinants (residues 6-18 and 26-35) were revealed only through the binding properties of antibodies isolated from autoimmune mice. The usual correlation between hydrophilicity and antigenicity was found to hold for four of the epitopes, and the N- and C-termini of H2B were both antigenically active.     

Nonenzymatic biotinylation of histone H2A

Holocarboxylase synthetase (HCS, eukaryotic enzyme) and BirA (prokaryotic) are biotin protein ligases that catalyze the ATP-dependent attachment of biotin to apocarboxylases via the reactive intermediate, bio-5′-AMP. In this study, we examined the in vitro mechanism of biotin attachment to histone H2A in the presence of HCS and BirA. The experiment derives from our observations that HCS is found in the nucleus of cells in addition to the cytoplasm, and it has the ability to attach biotin to histones in vitro (Narang et al., Hum Mol Genet 2004; 13:15–23). Using recombinant HCS or BirA, the rate of biotin attachment was considerably slower with histone H2A than with the biotin binding domain of an apocarboxylase. However, on incubation of recombinant H2A with chemically synthesized bio-5′-AMP, H2A was observed to be rapidly labeled with biotin in the absence of enzyme. Nonenzymatic biotinylation of a truncated apocarboxylase (BCCP87) has been previously reported (Streaker and Beckett, Protein Sci 2006; 15:1928–1935), though at a much slower rate than we observe for H2A. The specific attachment sites of nonenzymatically biotinylated recombinant H2A at different time points were identified using mass spectrometry, and were found to consist of a similar pattern of biotin attachment as seen in the presence of HCS, with preference for lysines in the highly basic N-terminal region of the histone. None of the lysine sites within H2A resembles the biotin attachment consensus sequence seen in carboxylases, suggesting a novel mechanism for histone biotinylation.     

Structure of polyubiquitinated histone H2A

We have recently demonstrated that trout liver histones H2A, H2B, and H2A.Z can be polyubiquitinated [Davie, J.R., Delcuve, G.P., Nickel, B.E., Moyer, R., & Bailey, G. (1987) Cancer Res. 47, 5407-5410]. In the present study we determined the arrangement of the ubiquitin molecules in polyubiquitinated histone H2A. Trout liver chromatin fragments. which had histone H1 removed, were digested with Staphylococcus aureus (V8 strain) protease which cleaves specifically on the carboxyl side of glutamic acid residues under the conditions used. The V8 protease readily degraded histone H2A and ubiquitinated (u) H2A at equivalent rates. One site in H2A and uH2A, the peptide bond between Glu 121 and Lys 122, was cleaved, yielding protein species cH2A and cuH2A, respectively. None of the other nucleosomal histones (H2B, H2A.Z, H3, and H4) including uH2B and uH2A.Z were sensitive to digestion. Trout liver histones cleaved with either V8 protease, histone H2A specific protease, or cyanogen bromide were resolved by two-dimensional gel electrophoresis and ubiquitinated peptides detected with anti-ubiquitin IgG. The results suggest that the major arrangement of ubiquitin in polyubiquitinated H2A is a chain of ubiquitin molecules joined to each other by isopeptide bonds to a ubiquitin molecule that is attached to the epsilon-amino group of lysine 119 of histone H2A.     

Isolation of yeast histone genes H2A and H2B

Analysis of cloned sequences for yeast histone genes H2A and H2B reveals that there are only two copies of this pair of genes within the haploid yeast genome. Within each copy, the genes for H2A and H2B are separated by approximately 700 bp of spacer DNA. The two copies are separated from one another in the yeast genome by a minimum distance of 35-60 kb. Sequence homology between the two copies is restricted to the genes for H2A and H2B; the spacer DNA between the genes is nonhomologous. In both copies, the genes for H2A and H2B are divergently transcribed. In addition, both plasmids code for other nonhistone proteins. Sequences coding for histones H3 and H4 have not been detected in the immediate vicinity of the genes for H2A and H2B.     

The human H2A and H2B histone gene complement

Sequences and expression patterns of newly isolated human histone H2A and H2B genes and the respective proteins were compared with previously isolated human H2A and H2B genes and proteins. Altogether, 15 human H2A genes and 17 human H2B genes have been identified. 14 of these are organized as H2A/H2B gene pairs, while one H2A gene and three H2B genes are solitary genes. Two H2A genes and two H2B genes turned outto be pseudogenes. The 13 H2A genes code for at least 6 different amino acid sequences, and the 15 H2B genes encode 11 different H2B isoforms. Each H2A/H2B gene pair is controlled by a divergent promoter spanning 300 to 330 nucleotides between the coding regions of the two genes. The highly conserved divergent H2A/H2B promoters can be classified in two groups based on the patterns of consensus sequence elements. Group I promoters contain a TATA box for each gene, two Oct-1 factor binding sites, and three CCAAT boxes. Group II promoters contain the same elements as group I promoters and an additional CCAAT box, a binding motif for E2F and adjacent a highly conserved octanucleotide (CACAGCTT) that has not been described so far. Five of the 6 gene pairs and 4 solitary genes with group I promoters are localized in the large histone gene cluster at 6p21.3-6p22, and one gene pair is located at 1q21. All group II promoter associated genes are contained within the histone gene subcluster at D6S105, which is located at a distance of about 2 Mb from the major subcluster at 6p21.3-6p22 containing histone genes with group I promoters. Almost all group II H2A genes encode identical amino acid sequences, whereas group I H2A gene products vary at several positions. Using human cell lines, we have analyzed the expression patterns of functional human H2A/H2B gene pairs organized within the two histone gene clusters on the short arm of chromosome 6. The genes show varying expression patterns in different tumor cell lines.     

Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6

The DNA of eukaryotes is wrapped around nucleosomes and packaged into chromatin. Covalent modifications of the histone proteins that comprise the nucleosome alter chromatin structure and have major effects on gene expression. Methylation of lysine 4 of histone H3 by COMPASS is required for silencing of genes located near chromosome telomeres and within the rDNA (Krogan, N. J, Dover, J., Khorrami, S., Greenblatt, J. F., Schneider, J., Johnston, M., and Shilatifard, A. (2002) J. Biol. Chem. 277, 10753-10755; Briggs, S. D., Bryk, M., Strahl, B. D., Cheung, W. L., Davie, J. K., Dent, S. Y., Winston, F., and Allis, C. D. (2001) Genes. Dev. 15, 3286-3295). To learn about the mechanism of histone methylation, we surveyed the genome of the yeast Saccharomyces cerevisiae for genes necessary for this process. By analyzing approximately 4800 mutant strains, each deleted for a different non-essential gene, we discovered that the ubiquitin-conjugating enzyme Rad6 is required for methylation of lysine 4 of histone H3. Ubiquitination of histone H2B on lysine 123 is the signal for the methylation of histone H3, which leads to silencing of genes located near telomeres.     

Critical role of monoubiquitination of histone H2AX protein in histone H2AX phosphorylation and DNA damage response

DNA damage response is an important surveillance mechanism used to maintain the integrity of the human genome in response to genotoxic stress. Histone variant H2AX is a critical sensor that undergoes phosphorylation at serine 139 upon genotoxic stress, which provides a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) and DNA repair protein complex to sites of DNA breaks for DNA repair. Here, we show that monoubiquitination of H2AX is induced upon DNA double strand breaks and plays a critical role in H2AX Ser-139 phosphorylation (γ-H2AX), in turn facilitating the recruitment of MDC1 to DNA damage foci. Mechanistically, we show that monoubiquitination of H2AX induced by RING finger protein 2 (RNF2) is required for the recruitment of active ataxia telangiectasia mutated to DNA damage foci, thus affecting the formation of γ-H2AX. Importantly, a defect in monoubiquitination of H2AX profoundly enhances ionizing radiation sensitivity. Our study therefore suggests that monoubiquitination of H2AX is an important step for DNA damage response and may have important clinical implications for the treatment of cancers.     

Interplay between histone deacetylase SIR-2, linker histone H1 and histone methyltransferases in heterochromatin formation

Maintenance of intact heterochromatin structure through epigenetic mechanisms is essential for cell survival. Defects in heterochromatin formation caused by loss of chromatin-modifying enzymes lead to genomic instability and cellular senescence. The NAD+-dependent histone deacetylase SIR-2 and the H1 linker histone are intriguing chromatin elements that are connected to chromatin regulation and cell viability in the single cellular eukaryotic organism yeast. However, it remains an open question how SIR-2 and H1 mediate heterochromatin formation in simple multi-cellular organisms such as C. elegans and in even more complex organisms such as mammals. Recently we have identified SIR-2.1 and the H1 histone subtype, HIS-24 as factors involved in heterochromatin regulation at subtelomeric regions in C. elegans. In addition we show that SIR-2.1, HIS-24, and MES-2, a ortholog to Enhancer of zeste E(Z) are functionally related in heterochromatin formation contributing to fertility and embryogenesis. Here we discuss the interplay between SIR-2, H1 histone and histone methyltransferases in modulation of chromatin structure in further detail.     

Changes in the histone H2A variant H2A.Z and polyubiquitinated histone species in developing trout testis

The trout histone H2A variant H2A.Z has been identified by its electrophoretic mobility on two-dimensional polyacrylamide gels and its N-terminal amino acid sequence. Similar to bovine H2A.Z and chicken H2A.F (also called H2A.Z and M1), the trout H2A.Z had a two-residue extension when aligned with trout H2A and a 67% sequence homology with the N-terminal portion of trout H2A. The first 29 amino acids of trout H2A.Z were identical with those of chicken H2A.F and differed from those of bovine H2A.Z at only one position. Thus, the N-terminal part of histone H2A.Z appears to be highly conserved. The levels of histone H2A.Z and ubiquitinated species of the histones H2A, H2A.Z, and H2B, which were detected with an anti-ubiquitin antibody, were studied at various stages of trout testis development. At the final stages of spermatogenesis in trout, histones are replaced by protamines. Ubiquitinated and diubiquitinated histone H2A remained at similar levels in early and late stage testis nucleohistone. In the late stage testis chromatin (nucleohistone), ubiquitinated histone H2A.Z was not detected, the level of ubiquitinated histone H2B was reduced, and the amount of diubiquitinated histone H2B increased. There was also a marked reduction in the level of histone H2A.Z. This observation suggests nucleosomes with this histone variant were selectively disassembled during the transition from nucleohistone to nucleoprotamine, indicating that protamine deposition is not a random process in rainbow trout.     

Yeast histone H2A and H2B amino termini have interchangeable functions

The N-terminal ends of histones H2B and H2A have very different sequences and rates of evolution. However, they both extend from the nucleosome core and are positively charged. Short sequences at the C termini of both proteins also differ from each other and appear to be hydrophilic. Deletions at the N and C termini of yeast histones H2B2 and H2A1 do not obviously affect the cell's viability under normal growth conditions. However, deletions at the N termini of both H2B and H2A in the same cell are lethal or result in greatly reduced viability. Even switching portions of the N termini between H2B and H2A to create two chimeric histone proteins within the same cell has no obvious effect on viability. This supports the argument that the N-terminal end of one protein complements the function of the other.     

H2AX: tailoring histone H2A for chromatin-dependent genomic integrity.

During the last decade, chromatin research has been focusing on the role of histone variability as a modulator of chromatin structure and function. Histone variability can be the result of either post-translational modifications or intrinsic variation at the primary structure level: histone variants. In this review, we center our attention on one of the most extensively characterized of such histone variants in recent years, histone H2AX. The molecular phylogeny of this variant seems to have run in parallel with that of the major canonical somatic H2A1 in eukaryotes. Functionally, H2AX appears to be mainly associated with maintaining the genome integrity by participating in the repair of the double-stranded DNA breaks exogenously introduced by environmental damage (ionizing radiation, chemicals) or in the process of homologous recombination during meiosis. At the structural level, these processes involve the phosphorylation of serine at the SQE motif, which is present at the very end of the C-terminal domain of H2AX, and possibly other PTMs, some of which have recently started to be defined. We discuss a model to account for how these H2AX PTMs in conjunction with chromatin remodeling complexes (such as INO80 and SWRI) can modify chromatin structure (remodeling) to support the DNA unraveling ultimately required for DNA repair.     

The structure of ubiquitinated histone H2B.

Ubiquitinated histone H2B (uH2B) has been purified from both calf and pig thymus by exclusion chromatography in 7 M urea. Digestion of uH2B with Staphylococcus aureus V8 protease yielded the peptide 114-125 containing the ubiquitin moiety. Further digestion of this peptide with trypsin removed the ubiquitin and three H2B residues from the N-terminus. Edman degradations of both peptides established that ubiquitin is attached to the epsilon-amino group of lysine 120 in both calf and pig uH2B by an iso-peptide bond to the C-terminal glycine 76 of ubiquitin.     

Primary structure of chicken erythrocyte histone H2A

The complete amino acid sequence (128 residues) of the chicken erythrocyte histone H2A was deduced from the data provided by structural studies on the tryptic peptides from the maleylated histone and of the peptides obtained by thermolysin digestion of the native protein. The sequence of chicken histone H2A differs from the calf homologous histone by the deletion of one residue of histidine at position 123 or 124 and three conservative substitutions: a residue of serine replaces a residue of threonine at position 16, a residue of aspartic acid replaces a residue of glutamic acid at position 121 and a residue of alanine replaces a residue of glycine at position 128.     

Phosphorylation of histone H2AX in radiation-induced micronuclei

DNA double-strand breaks are thought to precede the formation of most radiation-induced micronuclei. Phosphorylation of the histone H2AX is an early indicator of DNA double-strand breaks. Here we studied the phosphorylation status of the histone H2AX in micronuclei after exposure of cultured cells to ionizing radiation or treatment with colchicine. In human astrocytoma SF268 cells, after exposure to gamma radiation, the proportion of gamma-H2AX-positive to gamma-H2AX-negative micronuclei increases. The majority of the gamma-H2AX-positive micronuclei are centromere-negative. The number of gamma-H2AX-positive micronuclei continues to increase even 24 h postirradiation when most gamma-H2AX foci in the main nucleus have disappeared. In contrast, in normal human fibroblasts (BJ), the proportion of gamma-H2AX-positive to gamma-H2AX-negative micronuclei remains constant, and the majority of the centromere-negative cells are gamma-H2AX-negative. Treatment of both cell lines with colchicine results in mostly centromere-positive, gamma-H2AX-negative micronuclei. Immunostaining revealed co-localization of MDC1 and ATM with gamma-H2AX foci in both main nuclei and micronuclei; however, other repair proteins, such as Rad50, 53BP1 and Rad17, that co-localized with gamma-H2AX foci in the main nuclei were not found in the micronuclei. Combination of the micronucleus assay with gamma-H2AX immunostaining provides new insights into the mechanisms of the formation and fate of micronuclei.