On the possibility that H1 histone interaction with DNA occurs through phosphates connecting lysine and arginine side chain groups

Gel filtration and velocity of sedimentation analyses on native and on lysine- and arginine-modified forms of the annelid worm Chaetopterus variopedatus sperm H1 histone indicate that anion-mediated lysine-arginine interactions play a relevant role in the stabilization of the oligomeric states of the molecule. CD spectroscopy shows that phosphate anions are at least an order of magnitude more efficient than chloride as negatively charged groups connecting H1 lysines and arginines. Acetylation of lysines, although not altering grossly the H1 properties, causes a tenfold decrease of the structuring efficiency of phosphates. This suggests that DNA phosphates may be sandwiched between lysine and arginine groups of H1 histone when this molecule binds to chromatin, constituting a relevant parameter for the reciprocal stabilization of the protein and of the chromatin higher order structures.     

Differences in the binding of H1 variants to DNA. Cooperativity and linker-length related distribution

A study of the complexes formed between short linear DNA and three H1 variants, a typical somatic H1, and the extreme variants H5, from chicken erythrocytes, and spH1 from sea urchin sperm, has revealed differences between H1, H5 and spH1 that have implications for chromatin structure and folding. 1. All three histones bind cooperatively to DNA in 35 mM NaCl forming similar, but not identical, rod-like complexes. With sufficiently long DNA the complexes may be circular, circles forming more easily with H5 and spH1 than with H1. 2. The binding of H5 and spH1 to DNA is cooperative even in 5 mM NaCl, resulting in well-defined thin filaments that appear to contain two DNA molecules bridged by histone molecules. In contrast, H1 binds distributively over all the DNA molecules in 5 mM NaCl, but forms short stretches similar in appearance to the thin filaments formed with H5 and spH1. Rods appear to arise from the intertwining of regular thin filaments containing cooperatively bound histone molecules on raising the NaCl concentration to 35 mM. 3. The compositions of the rods correspond to one histone molecule for about every 47 bp (H1), 81 bp (H5) and 112 bp (spH1), suggesting average spacings of 24 bp (H1), 41 bp (H5) and 56 bp (spH1) in the component thin (double) filaments. Strikingly, these values are proportional to the linker lengths of the chromatins in which the particular H1 variant is the main or sole H1.     

The use of DNA-cellulose for analyzing histone-DNA interactions. Discovery of nucleosome-like histone binding to single-stranded DNA.

In this report, we introduce the use of DNA-cellulose chromatography for evaluating the strength of binding of histones to DNA under a variety of conditions. We have found that histones added directly to DNA-cellulose at physiological salt concentrations bind relatively weakly, with all histones eluting together at about 0.5 M NaCl when a salt gradient is applied. However, much tighter binding of the four nucleosomal histones to DNA-cellulose is obtained if gradual histone-DNA reconstitution conditions are used. In this case, the binding of histones H2A, H2B, H3, and H4 to DNA-cellulose closely resembles their binding to native chromatin. The nativeness of the binding is indicated both by the distinctive sodium chloride elution profile of these histones from DNA-cellulose and by their relative resistance to trypsin digestion when DNA-bound. The binding to DNA-cellulose of histones H2A, H2B, H3, and H4, which have had the first 20 to 30 amino acid residues removed from their NH2 termini, is indistinguishable from the binding to DNA-cellulose of the same intact histones, as judged by their salt elution profile. Thus, even though the NH2 termini contain 40 to 50% of the positively charged amino acid residues (thought to interact with the DNA backbone), a major contribution to the DNA binding comes from the remainder of the histone molecule. Finally, we have discovered that histones can form a "nucleosome-like" complex on single-stranded DNA. The same complex does not appear to form on RNA. Histones H3 and H4 play a predominant role in organizing this histone complex on single-stranded DNA, as they do on double-stranded DNA in normal nucleosomes. We suggest that, in the cell nucleus, nucleosomal structures may form transiently on single strands of DNA, as DNA and RNA polymerases traverse DNA packaged by histones.     

Interaction of sperm histone variants and linker DNA during spermiogenesis in the sea urchin

Several physical properties of sea urchin spermatid chromatin, which contains phosphorylated Sp H1 and Sp H2B histone variants, and mature sperm chromatin, in which these histones are dephosphorylated, were compared. Density, thermal stability, average nucleosomal repeat length, and resistance to micrococcal nuclease digestion are all increased in mature sperm relative to spermatid chromatin. Since the chromatins are identical in histone variant subtypes, the altered physical properties are not a consequence of changes in histone primary structure during spermiogenesis. The data are interpreted to mean that dephosphorylation of the N-terminal regions of Sp H1 and Sp H2B in late spermatid nuclei permits strong ionic binding of these highly basic regions to the extended linker, stabilizing the highly condensed structure of sperm chromatin.     

Linker DNA destabilizes condensed chromatin.

The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.     

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.     

A rapid and gentle method for the salt extraction of chromatin core histones H2A,H2B,H3 and H4 from rat liver nuclei

A complex of histones H2A, H2B, H3 and H4 has been isolated from purified rat liver nuclei by a method which is both gentle and rapid. Nuclei were homogenised in 0.25 M sucrose and the residual nuclear material obtained after centrifligation was adsorbed on calcium phosphate gel. After removing histone H1 from the adsorbed material by washing with 1M NaCl in 25 mM sodium phosphate buffer, pH 6.0, histones H2A, H2B, H3 and H4 were eluted together, with 2 M NaCl in 25 mM sodium phosphate buffer, pH 7.0. The core histones so obtained migrated as a single sharp band on polyacrylamide gel electrophoresis under non-denaturing conditions. Fractionation of the freshly prepared core histones on a Sephadex G-100 column yielded two major protein peaks. The peak having the larger elution volume contained histones H2A and H2B in equal amounts while the peak with the smaller elution volume contained all the four histones. Histones H3 and H4 were present in larger proportions in the second peak.     

Patterns of sperm-specific histone variation in sea stars and sea urchins: primary structural homologies in the N-terminal region of spermatogenic H1.

An electrophoretic characterization of histones from pyloric caeca, testes, and sperm of Asterias vulgaris revealed a sperm/testes-specific variant of histone H1 significantly larger than its somatic counterpart from pyloric caeca. Additional proteins were observed in H1 regions of acetic acid-urea polyacrylamide gels in testicular extracts. Sperm or testis-specific variants of H2B observed in sea urchins were not found in the sea star. Evidence presented suggests that sperm- or testes-specific H1 species of intermediate mobility may arise from a single, slow-migrating H1 species (SpH1). Although an increase in nonspecific DNA binding by nuclear proteins must occur during the process of spermatogenesis, different organisms exhibit various patterns of sperm-specific protein mediating differential binding during the process. Sperm-specific variants of both H1 and H2B histones are observed in sea urchins, while the only variant observed in sea stars during spermatogenesis is SpH1. Sequencing of the N-terminus of SpH1 from A. vulgaris revealed a repeating tetrapeptide in residues 3-6 and 8-11 (Ser-Pro-Arg-Lys and Ser-Pro-Lys-Lys, respectively), homologous to repeats in the N-termini of sperm-specific H1s from sea urchins. Primary structure within critical, variable regions of molecules responsible for nonspecific DNA binding appear conserved in many organisms. The occurrence of repeating tetrapeptides in SpH1 and other DNA binding proteins suggests that such domains may function similarly in various chromatins undergoing regulated or reversible condensation.     

Peculiarities of the amino acid composition, spatial organization and interaction with DNA of histones H1 from calf thymus and carp spermatozoa

The amino acid composition of the H1-like histone isolated from carp spermatozoa (H1carp) is characterized by a high content of lysine (34.6%) and a low content of glycine (4.5%) as compared to that of its calf counterpart (H1calf). The Lys/Arg ratio is 21.6, which is much higher than that for the H1-like histones from other species spermatozoa (cf. echinodermata). It was shown that the fluorescence anisotropy and excitation spectra of histones H1carp and H1calf change synchronically. At the same time the final folding of the polypeptide chains of these histones within their ternary structure is different. These differences manifest themselves in a distinct quantum yield of both histones and different accessibility of the single tyrosine residue for fluorescence quenchers. In histone--DNA complexes the tyrosine fluorescence is quenched. An increase in the ionic strength gives rise to a formation of large-sized aggregates in a histone H1--DNA solution which contain structurally heterogenous histones H1 from different sources. Histone H1carp causes DNA aggregation at lower ionic strength values than its calf counterpart. The complexes are dissociated at 0.6 M NaCl.     

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.     

Preparation and characterization of histone H1 from the sperm of the sea-urchin Sphaerechinus granularis.

The separation and purification of histone H1 from the sperm of the sea-urchin Sphaerechinus granularis is described. Physical studies were used to compare this histone H1 molecule with H1 histones from other species. C.d. and 270 MHz n.m.r. spectroscopy indicate that, despite significant compositional differences from other sea-urchin sperm H1 histones, their secondary and tertiary structures are very similar. A large difference in helicity was, however, found between S. granularis histone H1 and calf thymus histone H1, and their n.m.r. and fluorescence spectra also differ considerably. It is concluded that secondary structure and tertiary structure have not been conserved in the evolution of the H1 histone family.     

Sequential assembly of newly synthesized histone on replicating SV40 DNA

Studies on the assembly of histones with newly replicated SV40 DNA show that the four core histones do not associate simultaneously with the DNA. The arginine rich histones H3 and probably H4 associate first, followed by the association of H2a and H2b. Rapid exchange of histone H1 that occurs between cellular and viral chromatins during the extraction hampers studies on the specific association of H1 with newly replicated viral chromatin.     

The histones of the sperm of Spisula solidissima include a novel, cysteine-containing H-1 histone

The histones remaining at the end of the spermiogenic differentiation, which are found associated with a highly basic protamine-like component [Ausio, J. and K.E. Van Holde (1987) Eur. J. Biochem. 165, 363-371] in the mature sperm of Spisula solidissima, have been isolated and characterized for the first time. All four core histones H2A, H2B, H3, H4, and the lysine-rich histone H1 are present. The core histones are found in equal stoichiometric amounts. As has been observed in other bivalve molluscs, the amino acid compositions of the core histones of S. solidissima sperm are very close to those of their counterparts in the calf thymus somatic histones. The spermatic histone H1 exhibits an amino acid composition and structural features similar to other histones of the histone H1 family. Yet this latter histone seems to be sperm-specific, and it contains at least two cysteine residues per molecule, which makes it unique in its class.     

Lysine-rich histone H1 kinase from soybean hypocotyl.

A protein kinase (ATP: histone phosphotransferase) with high specificity for the phosphorylation of the very lysine-rich histone H1 has been partially purified and characterized from soybean hypocotyl. The enzyme has a molecular weight of about 48,500. Its activity and sedimentation behavior are refractory to cyclic nucleoside monophosphates. No significant amount of cyclic AMP or cyclic GMP binding activity could be detected in the crude or partially purified enzyme preparations. Km for ATP and histone H1 are 0.4 μM and 0.7 μM, respectively. The enzyme requires Mg²⁺ or Mn²⁺ for activity, while addition of 0.5 mM Ca²⁺, Zn²⁺ or Hg²⁺ results in 50% inhibition. Arginine-rich histones H3 and H4 are inhibitory to histone H1 phosphorylation; these histones affect the Vmax of the enzyme, but not the Km for histone H1.     

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.     

Influence of partial deproteinization on the cytochemical properties of DNP of lymphocyte nuclei

Fixed lymphocytes from mouse lymphatic nodules were treated on histological preparations with sodium chloride solutions of varying molarity. The extracts were investigated electrophoretically and were found to contain all histone fractions after treatment with 1.5 M NaCl solution and only the histone F1 after treatment with 0.6 M solution. The cytochemical properties of the cells with histones removed were investigated. The experiments showed that histone removal resulted in a marked increase of nucleic capacity to bind AO, and in a significant decrease of DNP thermal stability of these cells in denaturation test. To establish the role played by histone F1 in the course of cell activation this histone was removed from lymphocytes in states of different activity. The experiments showed that after treatment with 0.6 M sodium chloride the difference in AO-binding of cells in states of different activity disappeared. The data obtained confirm the hypothesis about histone-DNA separation at early stages of chromatin activation and suggest that the early change in physico-chemical properties of chromatin are connected with the separation of lysine-rich histone F1.     

A single-column chromatographic system for the analysis and preparation of high mobility group proteins 1 and 2 and other chromosomal proteins using nondenaturing solvents

One-step chromatography on a Mono S column allows the purification of high mobility group (HMG) proteins 1 and 2 under nondenaturing conditions. Chromatography of HMG1 and -2 on Mono S can be achieved with three of the most widely employed extraction techniques for chromosomal proteins, 0.35 M sodium chloride, 0.74 M perchloric acid, and 0.4 N sulfuric acid. In each case HMG1 and -2 are purified away from the other chromosomal proteins, histone H1, and core histones, and are resolved into both their reduced and oxidized forms. Additionally histone H1 and the core histones are fractionated on Mono S, thus the entire complement of chromosomal proteins can be analyzed in a single rapid chromatographic step.     

A study of histone-histone interactions by affinity chromatography

Homologous whole histone from calf thymus was adsorbed on Sepharose 4B columns with covalently coupled histone fractions H2a, H2b, H3 or H4 in 0.01 M phosphate buffer, pH 6.7–1 M NaCl. The adsorbed histones were eluted from the columns with 5 M urea in the same buffer. Electrophoretic analysis has shown that the different columns exhibit selective affinity to the histone fractions: the H2b column to histone H2b and H2a (with only weak affinity to histones H3 and H4), the H2a column to histones H2b and H3 (moderate affinity to histones H2a and H4), the H3 column to histones H3, H4, H2a (moderate affinity to histone H2b), and the H4 column to histone H3, H4 and H2b (weak affinity to histone H2a). Histone H1 displayed no fixation by either of the columns tested.     

Protein kinase associated with chromatin and changes in substrate-specificity during germination of wheat seeds

Protein kinase activity was associated with chromatin in wheat ( Triticum aestivum L. cv. Mukakomugi) embryos. The kinase activity did not change significantly during germination, whereas the activity of poly (ADP-ribose) synthetase decreased significantly. The protein kinase activity in chromatin was inhibited by NAD, NADH, and ADP-ribose, and was enhanced by treatment of the chromatin with snake venom phosphodiesterase or soybean trypsin inhibitor. The activity in chromatin was not stimulated by cyclic AMP. Different subfractions of the histones, H1 and H2, were mainly phosphorylated in germ and 3 day-germinated seedling chromatins. The histones, H3 and H4, seemed unable to accept phosphate from ATP in the in vitro reaction system. Different acidic non-histone chromosomal proteins were phosphorylated in germ and 3-day-germinated seedling chromations, and germ-specific and seedling-specific acidic non-histone chromosomal proteins seemed unable to accept phosphate from ATP.     

Preliminary study of sperm chromatin characteristics of the brachyuran crab Maja brachydactyla. Histones and nucleosome-like structures in decapod crustacean sperm nuclei previously described without SNBPs

An interesting characteristic of decapod crustacean sperm nuclei is that they do not contain highly packaged chromatin. In the present study we re-examine the presence of DNA-interacting proteins in sperm nuclei of the brachyuran Maja brachydactyla. Although previous reports have indicated that, unlike the majority of sperm cells, DNA of decapod sperm is not organized by basic proteins, in this work we show that: (1) histones are present in sperm of M. brachydactyla; (2) histones are associated with sperm DNA; (3) histone H3 appears in lower proportions than the other core histones, while histone H2B appears in higher proportions; and (4) histone H3 in sperm nuclei is acetylated. This work complements a previous study of sperm histones of Cancer pagurus and supports the suggestion that decapod crustacean sperm chromatin deserves further attention.