The human histone chaperone sNASP interacts with linker and core histones through distinct mechanisms

Somatic nuclear autoantigenic sperm protein (sNASP) is a human homolog of the N1/N2 family of histone chaperones. sNASP contains the domain structure characteristic of this family, which includes a large acidic patch flanked by several tetratricopeptide repeat (TPR) motifs. sNASP possesses a unique binding specificity in that it forms specific complexes with both histone H1 and histones H3/H4. Based on the binding affinities of sNASP variants to histones H1, H3.3, H4 and H3.3/H4 complexes, sNASP uses distinct structural domains to interact with linker and core histones. For example, one of the acidic patches of sNASP was essential for linker histone binding but not for core histone interactions. The fourth TPR of sNASP played a critical role in interactions with histone H3/H4 complexes, but did not influence histone H1 binding. Finally, analysis of cellular proteins demonstrated that sNASP existed in distinct complexes that contained either linker or core histones.     

Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse

We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins.     

Chromatin organization and basic nuclear proteins in the male germ cells of Rana tigerina

The process of chromatin condensation during spermiogenesis in Rana tigerina is similar to the heterochromatization in somatic cells, where 30 nm fibers are coalesced together into a dense mass in spermatozoa without changing their initial size and nucleosomal organization. This conclusion was supported by the finding that the full set of core histones (H2A, H2B, H3, H4) are still present in sperm chromatin, but histone H1 is replaced by its variant, H1V. Rabbit anti-sera were raised against histone H3, H1, H1V, and H5 (H1 variant in chick erythrocyte). Anti-histone H1 antiserum cross-reacted with histone H1V, which implied the presence of a common epitope. Anti-histone H1V and H5 also showed cross-reaction with each other but not with histone H1, which implied the presence of a common epitope not shared by histone H1. Immunocytochemical studies, using the above antibodies as probes, showed that histones H3 is present in all steps of spermatogenic and spermiogenic cells, and somatic cells including red blood cells, Sertoli cells, and Leydig cells, while histone H1 is present in all of the cells mentioned except in spermatozoa where it is replaced by histone H1V. Histone H1V appears in the early spermatids starting from spermatid 1 (St1), and it persists throughout the course of spermatid differentiation into spermatozoa. Histone H1V is also found in chromosomes of metaphase spermatocyte and red blood cells. Thus histone H1V may cause the final and complete condensation of chromatin in Rana spermatozoa, a process which is similar to the heterochromatization occurring in somatic cells such as metaphase chromosome and chick erythrocyte nucleus.     

Different mechanism for in vitro formation of nucleosome core particles

The interaction of different histone oligomers with nucleosomes has been investigated by using nondenaturing gel electrophoresis. In the presence of 0.2 M NaCl, the addition of the pairs H2A,H2B or H3,H4 or the four core histones to nucleosome core particles produces a decrease in the intensity of the core particle band and the appearance of aggregated material at the top of the gel, indicating that all these histone oligomers are able to associate with nucleosomes. Equivalent results were obtained by using oligonucleosome core particles. Additional electrophoretic results, together with second-dimension analysis of histone composition and fluorescence and solubility studies, indicate that H2A,H2B, H3,H4, and the four core histones can migrate spontaneously from the aggregated nucleosomes containing excess histones to free core DNA. In all cases the estimated yield of histone transfer is very high. Furthermore, the results obtained from electron microscopy, solubility, and supercoiling assays demonstrate the transfer of excess histones from oligonucleosomes to free circular DNA. However, the extent of solubilization obtained in this case is lower than that observed with core DNA as histone acceptor. Our results demonstrate that nucleosome core particles can be formed in 0.2 M NaCl by the following mechanisms: (1) transfer of excess core histones from oligonucleosomes of free DNA, (2) transfer to excess H2A,H2B and H3,H4 associated separately with oligonucleosomes to free DNA, (3) transfer to excess H2A,H2B initially associated with oligonucleosomes to DNA, followed by the reaction of the resulting DNA-(H2A,H2B) complex with oligonucleosomes containing excess H3,H4, and (4) a two-step transfer reaction similar to that indicated in (3), in which excess histones H3,H4 are transferred to DNA before the reaction with oligonucleosomes containing excess H2A,H2B. The possible biological implications of these spontaneous reactions are discussed in the context of the present knowledge of the nucleosome function.     

Two complexes that contain histones are required for nucleosome assembly in vitro: role of nucleoplasmin and N1 in Xenopus egg extracts

The composition and function of histone storage complexes of Xenopus eggs have been investigated using monoclonal antibodies. We show that core histones are contained in two distinct complexes: H2A and H2B are associated with nucleoplasmin, and H3 and H4 are associated with nuclear protein N1. Immunodepletion analyses demonstrate that both complexes are required for nucleosome core assembly by extracts in vitro, the product being a simple sum of the histones from each complex. In addition, the majority of the stored H2A is shown to be an unusual form that migrates close to the position of H3 by SDS-polyacrylamide gel electrophoresis and resembles a variant synthesized in a cell-cycle-independent manner in mammalian cells.     

Interaction of short peptides with FITC-labeled wheat histones and their complexes with deoxyribooligonucleotides

Judging from fluorescence modulation (quenching), short peptides (Ala-Glu-Asp-Gly, Glu-Asp-Arg, Ala-Glu-Asp-Leu, Lys-Glu-Asp-Gly, Ala-Glu-Asp-Arg, and Lys-Glu-Asp-Trp) bind with FITC-labeled wheat histones H1, H2в, H3, and H4. This results from the interaction of the peptides with the N-terminal histone regions that contain respective and seemingly homologous peptide-binding motifs. Because homologous amino acid sequences in wheat core histones were not found, the peptides seem to bind with some core histone regions having specific conformational structure. Peptide binding with histones and histone-deoxyribooligonucleotide complexes depends on the nature of the histone and the primary structures of the peptides and oligonucleotides; thus, it is site specific. Histones H1 bind preferentially with single-stranded oligonucleotides by homologous sites in the C-terminal region of the protein. Unlike histone H1, the core histones bind pre-dominantly with double-stranded methylated oligonucleotides and methylated DNA. Stern-Volmer constants of interaction of histone H1 and core histones with double-stranded hemimethylated oligonucleotides are higher compared with that of binding with unmethylated ones. DNA or deoxyribooligonucleotides in a complex with histones can enhance or inhibit peptide binding. It is suggested that site-specific interactions of short biologically active peptides with histone tails can serve in chromatin as control epigenetic mechanisms of regulation of gene activity and cellular differentiation.     

Isolation, identification, and characterization of histones from plasmodia of the true slime mold Physarum polycephalum using extraction with guanidine hydrochloride

Histones from plasmodia of the true slime mold Physarum polycephalum have been prepared free of slime by an approach to histone isolation that uses extraction of nuclei with 40% guanidine hydrochloride and chromatography of the extract on Bio-Rex 70. This procedure followed by chromatography or electrophoresis has been used to obtain pure fractions of histones from Physarum microplasmodia. Physarum microplasmodia have five major histone fractions, and we show by amino acid analysis, apparent molecular weight on three gel systems containing sodium dodecyl sulfate, mobility on gels containing Triton X-100, and other characterizations that these fractions are analogous to mammalian histones H1, H2A, H2B, H3, and H4. Significant differences between Physarum and mammalian histones are noted, with histone H1 showing by far the greatest variation. Histones H1 and H4 from Physarum microplasmodia have similar, but not identical, products of partial chymotryptic digestion compared with those of calf thymus histones H1 and H4. Labeling experiments, in vivo, showed that histone H1 is the major phosphorylated histone and approximately 15 separate phosphopeptides are present in a tryptic digest of Physarum histone H1. The core histones from Physarum, histones H2A, H2B, H3, and H4, are rapidly acetylated; histone H4 shows five subfractions, analogous to the five subfractions of mammalian histone H4 (containing zero to four acetyllysine residues per molecule); histone H3 has a more complex pattern that we interpret as zero to four acetyllysine residues on each of two sequence variants of histone H3; histones H2A and H2B show less heterogeneity. Overall, the data show that Physarum microplasmodia have a set of histones that is closely analogous to mammalian histones.     

Nucleosomes from normal and regenerating rat liver

Micrococcal-nuclease digestion of rat liver nuclei selectively released mononucleosomes associated with ADP-ribosylated [Caplan, Ord & Stocken (1978) Biochem. J.174, 475-483] histone H1. Two classes of mononucleosome were detected, those that leaked out during digestion and those that were subsequently released by 5mm-sodium phosphate buffer (pH6.8)/0.2mm-NaEDTA. The former, from which histone H1 had been dissociated, contained 140-base-pair-length DNA and core histones;the latter contained core particles and mononucleosomes with histone H1 and 200-base-pair-length DNA. When normal liver nuclei were phosphorylated with [gamma-(32)P]ATP, dissociated histone H1, which could be separated from core particles with Sephadex G-200, showed (32)P uptake. (32)P uptake into histones H2A and poly(ADP-ribosyl)ated H3 was appreciable in core particles, but was less evident in nucleosomes still containing histone H1. When [(3)H]-thymidine was given to partially hepatectomized rats in S-phase, 5-10min pulses in animals of over 300g body wt. showed the presence of high-specific-radioactivity DNA in released core particles and mononucleosomes compared with DNA retained in the nuclear pellets. Mononucleosomes from rat livers in S-phase with new, [(3)H]lysine-containing histones, had higher (32)P incorporation in histones H1 and their core histones, than for di- or tri-nucleosomes. Thermal-denaturation properties of control and phosphorylated mononucleosomes and core particles were very similar; removal of histone H1 and non-histone chromosomal proteins in 0.5m-NaCl markedly increased the proportion of DNA ;melting' below 70 degrees C.     

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.     

Impact of Histone H1 on the Progression of Allergic Rhinitis and Its Suppression by Neutralizing Antibody in Mice

Nuclear antigens are known to trigger off innate and adaptive immune responses. Recent studies have found that the complex of nucleic acids and core histones that are derived from damaged cells may regulate allergic responses. However, no fundamental study has been performed concerning the role of linker histone H1 in mast cell-mediated type I hyperreactivity. In this study, we explored the impact of histone H1 on mast cell-mediated allergic responses both in vitro and in vivo. In the course of a bona-fide experimental allergen sensitization model upon co-injection with alum adjuvant, ovalbumin (OVA), but not PBS, induced elevated levels of circulating histone H1. Intranasal challenge with histone H1 to OVA/alum- (but not PBS/alum)-sensitized mice induced significantly severer symptoms of allergic rhinitis than those in mice sensitized and challenged with OVA. A monoclonal antibody against histone H1 not only suppressed mast cell degranulation, but also ameliorated OVA-induced nasal hyperreactivity and IgE-mediated passive cutaneous anaphylaxis. Our present data suggest that nuclear histone H1 represents an alarmin-like endogenous mediator acting on mast cells, and that its blockage has a therapeutic potential for mast cell-mediated type I hyperreactivity.     

Binding of linker histones to the core nucleosome

Binding of chicken erythrocyte linker histones H1/H5 to the core nucleosome has been studied. Histones H1/H5 bind very efficiently to the isolated core nucleosome in vitro. The binding of linker histones to the core nucleosome is associated with aggregation of the particles. Approximately one molecule of linker histone binds per core nucleosome in the aggregates, irrespective of the concentration of the linker histones and the salt used. Histone H5 shows greater binding affinity to the core nucleosome as compared to H1. The carboxyl-terminal fragment of the linker histones binds strongly to the core nucleosome while the binding of the central globular domain is weak. Each core nucleosome is capable of binding two molecules of carboxyl-terminal fragment of linker histone. The core nucleosome containing one molecule of carboxyl-terminal fragment of linker histone requires higher salt concentration for aggregation while the core nucleosome containing two molecules of carboxyl-terminal fragment of linker histone can self-associate even at lower salt concentrations. On the basis of these results we are proposing a novel mechanism for the condensation of chromatin by linker histones and other related phenomena.     

Novobiocin precipitates histones at concentrations normally used to inhibit eukaryotic type II topoisomerase.

At concentrations normally used to inhibit eukaryotic type II topoisomerase activity (100-1000 micrograms/ml) novobiocin binds core histones. Approximately 15 moles of novobiocin bind per mole of histone resulting in histone precipitation from solution in either 0.15 M or 2 M NaCl. The interaction between novobiocin and proteins appears to involve arginine residues: histones H3 and H4 (13.5 and 14 mole percent arginine) are precipitated at lower novobiocin concentrations than histones H2A and H2B (9.5 and 6.5 mole percent arginine). Furthermore, polyarginine but not polyornithine competes for novobiocin in histone precipitation. Moreover, histones with arginine residues modified with 1,2-cyclohexanedione are soluble in 1000 micrograms/ml novobiocin. Because novobiocin can remove histones from solution as well as inhibit topoisomerase activity, and because both of these events can alter DNA topology, novobiocin should be used with caution in experiments designed to implicate topoisomerase activity in chromatin dynamics.     

Regulation of histone synthesis during early Urechis caupo (Echiura) development

The histones present in mature oocytes and embryos of Urechis caupo and their pattern of synthesis during early development have been characterized. Acid-soluble proteins extracted from mature oocyte germinal vesicles and from embryonic nuclei were analyzed by two-dimensional polyacrylamide gel electrophoresis. Histones are accumulated in the mature oocytes in amounts sufficient to provide for the assembly of chromatin through the 32- to 64-cell stage of embryogenesis. Two H1 histones, which appear to be variants, were found. Germinal vesicles and cleavage-stage nuclei are enriched in H1M (maternal). During late cleavage a faster-migrating H1, H1E (embryonic), appears among the nuclear histones and, as embryogenesis continues, replaces H1M as the predominant H1. No new core histone variants are detected during early development. Examination of [³H]lysine-labeled histones from germinal vesicles and embryonic nuclei reveals stage-specific patterns of histone synthesis. H1M is the major H1 species synthesized in mature oocytes. After fertilization, a switch to the predominant synthesis of H1E occurs. Comparison of the [³H]lysine incorporated into H1E and core histones indicates that H1E synthesis is disproportionately high from midcleavage through the midblastula stage. By the gastrula stage, a balanced synthesis of H1E and each core histone is established. The results indicate that there is noncoordinate regulation of H1 and core histone synthesis during Urechis development.     

Separation of histones by reverse-phase high-performance liquid chromatography: analysis of the binding of carcinogens to histones

Reverse-phase high-performance liquid chromatography (RP-HPLC) has been examined as an approach to the rapid analysis of carcinogen-modified histones. H1 and core histone fractions were prepared by differential acid extraction of 0.35 M NaCl-extracted rat liver nuclei previously exposed to [3H]-7r,8t-dihydroxy-9t, 10t-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene [( 3H]BPDE-I). Using a sodium perchlorate-phosphate (PCP)/acetonitrile solvent system, the H1 histone fraction was eluted from an Aquapore RP-300 column in five peaks (P1-P5). The core histone fraction was resolved into eight peaks (C1-C8) using a PCP/acetonitrile-methanol solvent system. The histones of each peak were identified by sodium dodecyl sulfate and Triton/acid/urea gel electrophoresis or amino acid analysis as follows: P1, H1 degrees; P2-P5, four different H1 variant fractions; C1, H4 + A24; C2, H2B; C3, H2A X 2 + to one H2A variant; C4, H2A.1; C5, H2A.1 + two H2A variants; C6, H3.2; C7, H3.3; C8, H3.1. The bulk of radioactivity was covalently bound to histone H2A, which had higher specific activities of BPDE-I than other histones. Significant amounts of radioactivity were observed in histones H3 and H1, but not in histones H2B and H4. These RP-HPLC systems have the advantages of an analysis time within 60 min, the identification of H1, H2A, and H3 variants, and the quantitative analysis of radioactive histones. These results indicate that these RP-HPLC systems are very useful to analyze the binding of carcinogens to histones.     

The Histone Database: a comprehensive resource for histones and histone fold-containing proteins

The Histone Database is a curated and searchable collection of full-length sequences and structures of histones and nonhistone proteins containing histone-like folds, compiled from major public databases. Several new histone fold-containing proteins have been identified, including the huntingtin-interacting protein HYPM. Additionally, based on the recent crystal structure of the Son of Sevenless protein, an interpretation of the sequence analysis of the histone fold domain is presented. The database contains an updated collection of multiple sequence alignments for the four core histones (H2A, H2B, H3, and H4) and the linker histones (H1/H5) from a total of 975 organisms. The database also contains information on the human histone gene complement and provides links to three-dimensional structures of histone and histone fold-containing proteins. The Histone Database is a comprehensive bioinformatics resource for the study of structure and function of histones and histone fold-containing proteins. The database is available at http://research.nhgri.nih.gov/histones/.     

Nonrandom assembly of chromatin during hydroxyurea inhibition of DNA synthesis

Incubation of MSB-1 chicken lymphoblastoid cells with hydroxyurea leads to a rapid 25-fold decrease in the incorporation of [3H]thymidine into DNA and a 5-fold decrease [3H]lysine into the nucleosome core histones. I have investigated whether the distortion in the normal proportion of histone-DNA synthesis results in alterations in the nucleosome assembly process and find that neither the stoichiometry of new histone synthesis nor the deposition is appreciably changed during hydroxyurea incubation. Protein cross-linking and micrococcal nuclease digestion show that the histones synthesized during hydroxyurea treatment form octamer structures and are assembled into typical nucleosome particles. Minor nucleosome subpopulations are found which exhibit altered sensitivity to nuclease digestion and which are depleted in new histones H3 and H4. When MSB-1 cells incubated in hydroxyurea are pulsed briefly with density-labeled amino acids and [3H]lysine, the radiolabeled core histone octamers formed are as dense as individual monomer histones. These results suggest that the newly synthesized histone octamers are uniformly dense and do not contain mixtures of new and old histones. Thus, histones synthesized during hydroxyurea incubation are deposited nonrandomly and do not exchange with preexisting histones.