Deposition of newly synthesized histones: new histones H2A and H2B do not deposit in the same nucleosome with new histones H3 and H4

We have developed procedures to study histone-histone interactions during the deposition of histones in replicating cells. Cells are labeled for 60 min with dense amino acids, and subsequently, the histones within the nucleosomes are cross-linked into an octameric complex with formaldehyde. These complexes are sedimented to equilibrium in density gradients and octamer and dioctamer complexes separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With reversal of the cross-link, the distribution of the individual density-labeled histones in the octamer is determined. Newly synthesized H3 and H4 deposit as a tetramer and are associated with old H2A and H2B. Newly synthesized H2A and H2B deposit as a dimer associated with old H2A, H2B, H3, and H4. The significance of these results with respect to the dynamics of histone interactions in the nucleus is discussed. Control experiments are presented to test for artifactual formation of these complexes during preparative procedures. In addition, reconstitution experiments were performed to demonstrate that the composition of these octameric complexes can be determined from their distribution on density gradients.     

Conservative assembly and segregation of nucleosomal histones

The assembly of new histones into nucleosomes and the segregation of old histones during replication were investigated using a density gradient, sedimentation equilibrium analysis of histones labeled in vivo with dense amino acids. After a 1 hr pulse of dense amino acids and 3H-lysine, nucleosomes were isolated from chick myoblast organ cultures, and the histones were cross-linked to octamers. The octamers were purified from DNA and then banded to equilibrium in cesium-formate guanidinium-HCI density gradients. The cross-linked dense octamers have the same density as the noncross-linked dense histones, and both were significantly heavier than histones synthesized in the presence of light amino acids. This experiment shows that new histone does not mix with old histone in the new nucleosomes, since the labeling protocol allows density labeling of only one histone for every seven preexisting unlabeled histones. Thus the assembly of new histone octamers is conservative. Using essentially the same experimental design, but varying the details of the labeling procedures, we also show that the dense histone octamer is stable over 3-4 generations, that neighboring octamers tend to be synthesized at the same time, and that old and new histone octamers segregate conservatively over 2-3 generations.     

Unfolding of nucleosome cores induced by chemical acetylation of histones

Relative accessibility of nucleosomal histones to acetic anhydride during acetylation has been studied as a function of concentration, pH and ionic strength of the solution using high-resolution gel-electrophoresis. It was shown that about 80% of lysine residues in nucleosomal histones and 100% of the same residues in histone complexes without DNA in 2 M NaCl are accessible to the modification, which is proved by the localization of the majority of lysine residues in nucleosomes near the surface of the histone octamer, by their participation in ionic interactions with DNA and, probably, in histone-histone contacts. Gel-electrophoretic experiments with nucleosomes and studies of the histone resistance to mild trypsinolysis indicated that neither nucleosomes themselves nor histone octamers are affected even though 50% of lysine residues in histones have been acetylated. The process of acetylation is accompanied by the growing tendency of histones to participate in mild trypsinolysis and by a gradual decline in electrophoretic mobility and in the value of the sedimentation constant. The circular dichroism spectra and the microscopic appearance of nucleosomes are also markedly changed. These results suggest that a gradual unfolding of nucleosomes occurs when 5 or more lysine residues in the nucleosomal histones have been acetylated.     

Reassembly of nucleosomal histone octamers during replication of chromatin

We have examined critically whether or not new and old histones mix in the octameric units of nucleosomes during chromatin replication. MH-134SC cells were density-labeled by culturing with amino acid mixtures enriched with dense isotopes 2H, 13C, and 15N. Mononucleosomes obtained from labeled cells were fractionated by rate zonal sedimentation through a sucrose gradient in heavy water (Senshu et al. (1985) Eur J. Biochem. 150, 575-580). The fractionation can be performed under conditions that do not destabilize nucleosomes. Density-labeling yielded heterogeneous mononucleosome species which showed higher sedimentation rates than normal mononucleosomes. However, they were indistinguishable with respect to their protein compositions, electrophoretic mobilities, electrophoretic patterns of single-stranded DNA fragments liberated by DNase I digestion, electrophoretic mobilities and sedimentation velocities of the DNA moieties, and metabolic stabilities of the histone moieties. These data suggest that the heterogeneity of density-labeled mononucleosomes resulted from the formation of histone octamers density-substituted to different degrees. This would be an inevitable consequence of mixing of new and old histones in the octameric unit of nucleosomes.     

Reconstitution of nucleosomes containing dinitrophenylated histones.

Histone octamers of purified monomer nucleosomes were labelled with [3H]dinitrofluorobenzene. Authentic 11 S nucleosomes were reconstituted in vitro from a mixture of [3H]dinitrophenylated histones and excess unlabelled monomer nucleosomes. The reconstituted nucleosomes were found to contain [3H]dinitrophenylated histones H2a and H2b but not [3H]dinitrophenylated histones H3 and H4. Approx. 83% of [3H]dinitrophenylated nucleosomes were immunoprecipitable with anti-dinitrophenyl immunoglobulin and Staphylococcus aureus. These results demonstrate that histones H2a and H2b contain dinitrofluorobenzene-reactive groups that can be modified without destroying their ability to participate in nucleosome formation in vitro.     

Distribution of the core histones H2A.H2B.H3 and H4 during cell replication.

The distribution of newly synthesized core histones H2A, H2B, H3 and H4 relative to the DNA strand synthesized in the same generation has been examined in replicating Chinese Hamster ovary cells. Cells are grown for one generation in [14C]-lysine and thymidine, and then for one generation in [3H]-lysine and 5-bromodeoxyuridine (BrUdRib) and a further generation in unlabeled lysine and thymidine. This protocol produces equal amounts of unifilarly substituted and unsubstituted DNA. Monomer nucleosomes isolated from chromatin containing these two types of DNA can be distinguished by crosslinking with formaldehyde and banding to equilibrium in CsCl density gradients. The results indicate that the core histones are equally distributed between the two types of DNA. These findings are discussed in terms of current models for chromatin replication; they do not support any long term association of newly replicated histones with either the leading or lagging side of the replication fork.     

Studies on histone organization in the nucleosome using formaldehyde as a reversible cross-linking agent

A new procedure is described which allows selective reversal of formaldehyde cross-linking in both histone-histone and histone-DNA of nuclei isolated from calf thymus. All ten possible dimers of the four non-H1 histones, H3, H2B, H2A and H4, are observed, the major dimers being H3-H3, H3-H2A, H2B-H2A, H2a-H2A and two separate dimers of H2B-H4. Although oligomers of the non-H1 histones are formed by prolonged treatment with this reagent, 50% of the histones continue to remain resistant to cross-linking with each other. For those histones which cross-linking with each other. For those histones which cross-link, the site of cross-linking within the molecules is located in the "core" (trysin-resistant) regionand therfore indicates proximities for these molecules within the nucleosome. The core region also cross-links to DNA, indicating intimate interactions between this region in all the non-H1 histones with DNA.     

Histone H1 and HMG 14/17 are deposited nonrandomly in the nucleus.

We have studied the assembly of histone H1 and the high mobility group nonhistones 14/17 by isopycnic analysis after crosslinking density labeled MSB cell nuclei or chromatin. Carbodiimide crosslinking produces dense poly-H1 and hybrid density H1-H2A histone dimers, indicating that new H1 is deposited nonrandomly, albeit nonconservatively relative to new core histones. Core histone-HMG crosslinking with succinimidyl propionate yields dense HMG 14 in uniformly dense particles and new HMG 17 crosslinked to both dense and light protein, implying that HMG 14 and 17 each deposit nonrandomly; but differently with respect to new core octamers. Propionimidate crosslinking yields dense H1-HMG 17 dimers, suggesting that the interactions of new 14/17 with H1 (new HMG 14-old H1, new HMG 17-new H1) are reciprocal to their interactions with the core histones.     

The pool of histones in the nucleosol and cytosol of proliferating Friend cells is small, uneven and chasable.

This study examines the histones pools in the nucleosol and cytosol of proliferating Friend cells. By using the conventional approach, detectable amounts of these molecules were found in both compartments; however, only H3 and H2B were identified in nucleosol, and H3, H2B and H4 in cytosol. The authenticity of each of these histones was verified by two independent methods, migration in SDS/polyacrylamide gels and peptide mapping. When the sensitivity of the approach was increased by radiolabelling with 125I, two additional proteins, migrating as H2A and H4, were observed in nucleosol. Even by this approach, however, H1 was not detected. Direct quantitative measurements of the histones in both compartments indicated that these pools are uneven and small. This was found also in experiments involving inhibition of protein synthesis by cycloheximide. Considered together, our data do not support the idea of the existence of preformed histone heterocomplexes or octamers. Instead the assembly of nucleosomes during replication occurs by a successive deposition of individual core histones.     

Role of individual histone tyrosines in the formation of the nucleosome complex.

We have determined the accessibility of histone tyrosine residues to react with p-nitrobenzenesulfonyl fluoride (NBSF) in intact nuclei, salt-dissociated nucleosomes, isolated histone complexes, and individual core histones. Of the 15 core histone tyrosine residues, 13 are inaccessible in native nucleosomes; only Tyr121 near the C-terminus of H2B is fully accessible, and Tyr54 of H3 is partially accessible under near-physiological conditions. When H1 and the basic N-terminal tails of the core histones are dissociated from the DNA by treating nuclei with 0.4 and 0.8 M NaCl, the two tyrosines which are adjacent to the basic regions of H2B and H3 become accessible as well. This indicates that these tyrosine residues may be involved in histone-DNA interactions, either directly or indirectly. When the H2A-H2B dimers are dissociated from the chromatin by raising the NaCl concentration to 1.2 M, three to four tyrosines located in the structured regions of H2B and H4 are exposed, suggesting that these tyrosine residues may be located at the dimer-tetramer interface. Dissociating all the histones from the DNA at an even higher ionic strength as a mixture of dimers, tetramers, and octamers does not change the pattern of Tyr exposure, but reduces the reactivity of the tyrosines at the dimer-tetramer interface as would be expected from the reassociation of H2A-H2B dimers and H3-H4 tetramers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of nucleosomes in native and reconstituted chromatins.

The stability of nucleosomes of SV40 minichromosomes extracted from infected cells or reconstituted by association of SV40 DNA and the four histones H2A, H2B, H3 and H4 was studied as a function of the ionic strength. As a measure of the stability of the nucleosome, we followed the disappearance of the nucleosomes from the original chromatin and their appearance on a "competing" DNA. We show here that the DNA and the histone components of the nucleosomes do not apprecially dissociate below 800 mM NaCl. At 800 mM and above, the histone moiety of the nucleosomes can dissociate from the DNA and efficiently participate to the formation of nucleosomes on a "competing" DNA.     

The N-terminus of histone H2B, but not that of histone H3 or its phosphorylation, is essential for chromosome condensation.

We have studied the role of individual histone N-termini and the phosphorylation of histone H3 in chromosome condensation. Nucleosomes, reconstituted with histone octamers containing different combinations of recombinant full-length and tailless histones, were used as competitors for chromosome assembly in Xenopus egg extracts. Nucleosomes reconstituted with intact octamers inhibited chromosome condensation as efficiently as the native ones, while tailless nucleosomes were unable to affect this process. Importantly, the addition to the extract of particles containing only intact histone H2B strongly interfered with chromosome formation while such an effect was not observed with particles lacking the N-terminal tail of H2B. This demonstrates that the inhibition effect observed in the presence of competitor nucleosomes is mainly due to the N-terminus of this histone, which, therefore, is essential for chromosome condensation. Nucleosomes in which all histones but H3 were tailless did not impede chromosome formation. In addition, when competitor nucleosome particles were reconstituted with full-length H2A, H2B and H4 and histone H3 mutated at the phosphorylable serine 10 or serine 28, their inhibiting efficiency was identical to that of the native particles. Hence, the tail of H3, whether intact or phosphorylated, is not important for chromosome condensation. A novel hypothesis, termed 'the ready production label' was suggested to explain the role of histone H3 phosphorylation during cell division.     

H3 phosphorylation-dependent structural changes in chromatin. Implications for the role of very lysine-rich histones

Contrary to native H1/H5-containing chromatin where phosphorylation induces local structural changes affecting chromatin condensation, in stripped fibers phosphorylation of the totality of H3 molecules does not affect significantly chromatin conformation and DNA-protein interactions. Modification of H3 causes only a slight increase of flexibility of nucleosomal chains, despite important changes in histone topography revealed by immunochemical reactivity studies. We suggest that phosphorylation may only induce into the system the potential for dynamic change by modulating histone-histone interactions within and between nucleosomes, probably as a result of conformational change in the H3 protein. The signal for structural change would come from one or other factors (very lysine-rich histones, non-histones) that influence internucleosomal interactions at very specific locations in the chromatin, probably through protein-protein contacts. So, phosphorylation may modify a direct interaction between the N-terminal basic tail of H3 and very lysine-rich histones.     

Formation of hybrid nucleosomes cantaining new and old histones.

5 mM hydroxyurea (HU) inhibits DNA synthesis in mouse P815 cells by 94-97% in less than 1 hr. Nevertheless, histone synthesis continues and newly-synthesised histones are incorporated into non-replicating chromatin at a rate of about 20% of that in control exponentially-growing cells. To study the organization of these histones in chromatin P815 cells were treated with 5 mM HU in medium containing dense (15N, 13C, 2H) - substituted amino acids. After inhibition of DNA synthesis, newly-synthesised histones were labelled with (3H)-arginine. The cells were harvested 90 min later, and mono- and oligonucleosomes were prepared and analysed on metrizamide-triethanolamine (MA-TEA density gradients. Analysis of the distribution of 3H-labelled histones in these gradients shows that they are incorporated into hybrid mononucleosomes containing both new and old histones. It is also shown that these hybrid nucleosomes are not randomly distributed, but show a certain tendency to be clustered in certain chromatin regions.     

Preparation of fluorescently labelled hybrid histone octamers

Labelling hybrid histone octamers (the Cys variant of histone H4 replaced histone H4 in the chicken erythrocyte octamer) with the fluorescent probe 5-(2(iodoacetyl)aminoethyl)aminonapthalene- 1-sulfonic acid, IAEDANS, resulted in significant non-specific incorporation of label. Fluorescently labelled hybrid histone octamers were prepared by reconstitution methodology after labelling the isolated histone Cys-H4 and separation of specifically and non-specifically labelled histone. Core particles prepared from these octamers have identical thermal denaturation to unlabelled core particles demonstrating that the incorporation of a fluorescent probe at this site has no overall effect on either histone-histone or histone-DNA interactions. DNase 1 digestion of 32P end-labelled fluorescent core particles yielded the anticipated asymmetric cutting pattern with a 10 bp interval between fragments. Comparison of the cutting pattern with those previously obtained in these laboratories for both polyglutamic acid reconstituted and 'native' core particles demonstrated that fluorescent core particles had an enhanced susceptibility to digestion at site 7.     

Histon-histone interactions within chromatin. Preliminary location of multiple contact sites between histones 2A, 2B, and 4.

The contact-site cross-linkers tetranitromethane, UV light, formaldehyde, and a monofunctional imido ester have been used to generate a collection of histone-histone dimers and trimers from nuclei and chromatin. Four different H2B-H4 dimers have been isolated. Preliminary CNBr peptide mapping has shown that all are cross-linked at different positions that are apparently clustered within the C-terminal regions of these histones. Similarily, two different H2A-H2B dimers and two different H2A-H2B-H4 trimers have been partially characterized. The data suggest a functional map for H2B in which the N-terminal third interacts with DNA, the middle third interacts with H2A, and the C-terminal third interacts with H4. We hope, by pursuing this type of analysis, to develop a detailed understanding of each histone-histone binding interaction through saturation cross-linking of the binding sites.     

Accessibility and structural role of histone domains in chromatin. biophysical and immunochemical studies of progressive digestion with immobilized proteases

The accessibility and role of histone regions in chromatin fibres were investigated using limited proteolysis with enzymes covalently bound to collagen membranes. The changes in chromatin conformation and condensation monitored by various biophysical methods, were correlated to the degradation of the histone proteins revealed by antibodies specific for histones and histone peptides. Upon digestion with trypsin and subtilisin, chromatin undergoes successive structural transitions. The cleavage of the C-terminal domains of H1, H2A and H2B, and of the N-terminal tail of H3 led to a decondensation of chromatin fibres, indicated by increases in electric birefringence and orientational relaxation times. It corresponds to a 15% increase in linear dimensions. The degradation of the other terminal regions of histones H3, H2A and H2B resulted in the appearance of hinge points between nucleosomes without alteration of the overall orientation of polynucleosome chains. Despite the loss of all the basic domains of H1, H3, H2A and H2B, no significant change in DNA-protein interactions occurred, suggesting that most of these protease-accessible regions interact weakly, if at all, with DNA in chromatin. Further proteolysis led to H4 degradation and other additional cleavages of H1, H2B and H3. This caused the relaxation of no more than 8% of the total DNA but resulted in changes in the ability of chromatin to condense at high ionic strength. More extensive digestion resulted in a total unravelling of nucleosomal chains which acquired properties similar to those of H1-depleted chromatin, although the globular part of H1 was still present. The data suggest that histone-histone interactions between H1 and core histone domains play a central role in stabilizing the chromatin fibres, and cuts in H3, H2A and H2B as well as H1, seem necessary for chromatin expansion. On the contrary, H4 might be involved in the stabilization of nucleosomes only.     

Histone synthesis and deposition in the G1 and S phases of hepatoma tissue culture cells

Hepatoma tissue culture cells were synchronized in G1 and in S phase in order to examine the level of synthesis of different histone types and to determine the rate, timing, and location of their deposition onto DNA. We observe a basal level of synthesis in G1 (5% of that seen in S phase) for H2A.1, H2A.2, H3.2, H2B, and H4. The minor histone variants X and Z are synthesized at 30% of the rate observed in S cells. The rate of synthesis of the ubiquinated histones uH2A.1,2 is not as depressed in G1 cells as seen for H2A.1 and H2A.2. Histones synthesized in G1 are not deposited on the DNA of these cells at equivalent rates. Thus, histones H3.2 and H4 are not deposited significantly until S phase begins, at which time deposition occurs selectively on newly synthesized DNA. The deposition of H2A.1, H2A.2, H2B, X, and Z proceeds in G1; however, it occurs to a 2-4-fold lower extent than seen for the deposition of H1, HMG 14, and HMG 17. The deposition of all histones synthesized in S phase occurs rapidly, but there are variations in the sites of deposition. Thus, newly synthesized H3.1, H3.2, and H4 deposit primarily on newly replicated DNA whereas H2A.1, H2A.2, uH2A.1, 2, and H2B deposit only partially on new DNA (30%) and mostly on old. H1, HMG 14, and HMG 17 are deposited in an apparently fully random manner over the chromatin. To interpret these observations, we propose a model which includes a measure of histone exchange on the chromatin fiber. The model emphasizes the dynamics of histone-histone and histone-DNA interactions in regions of active genes and at replication forks.