Stability of the conservative mode of nucleosome assembly.

The conservative assembly of nucleosome histone octamer cores has been confirmed by electrophoretic analysis of density labeled histones following equilibrium buoyant density centrifugation. After normal replication, crosslinked octamers are shown not to contain a mixture of new and old core histones. Moreover, when DNA synthesis is inhibited by ara-C nucleosome cores are still assembled exclusively from nascent histone. Similarly, after release from cycloheximide inhibition newly synthesized core histone is conservatively deposited. Thus, a conservative mechanism of histone octamer assembly occurs when nascent histone is present in the normal stoichiometry to nascent DNA and when chromatin is assembled in nascent histone or nascent DNA excess.     

Nucleosome core particles of calf thymus, Tetrahymena, and the reconstituted hybrid. Their structure reflects the nature of the histone octamer

The circular dichroism spectra and the thermal denaturation profiles of the nucleosome core particles isolated by micrococcal nuclease digestion from nuclei of calf thymus and the protozoan Tetrahymena pyriformis were compared with those of the homogeneous and hybrid core particles reconstituted from calf core DNA and either calf or Tetrahymena histone octamer. The core DNA was obtained from the calf core particle, and both the histone octamers were reconstituted from the acid-extracted four core histones of calf thymus or Tetrahymena, whose amino acid sequences show the largest differences hitherto known. The reconstituted homogeneous core particle was identical in both the physical properties with the isolated calf core particle, showing that the correct reconstitution was achieved. The circular dichroism spectra of the calf and Tetrahymena core particles and the hybrid core particle showed no essential differences, indicating that the three core particles have the same overall structure. The derivative thermal-denaturation profiles, however, clearly differed; the calf core particle showed two melting transitions at 60 degrees C and 72 degrees C, while the Tetrahymena and hybrid core particles showed the same three transitions at 48-50 degrees C, 60-61 degrees C, and 72 degrees C. Thus, the thermal denaturation properties of nucleosome core particles do not reflect the nature of DNA, but rather that of the histone octamer bound to the DNA. We conclude that the Tetrahymena histones are more weakly bound to the DNA than the calf thymus histones in the same overall structure of nucleosomes.     

Interactions between core histones and chromatin at physiological ionic strength

Addition of core histones to chromatin or chromatin core particles at physiological ionic strength results in soluble nucleohistone complexes when polyglutamic acid is included in the sample. The interaction between nucleosomes and added core histones is strong enough to inhibit nucleosome formation on a closed circular DNA in the same solution. Complexes consisting of core particles and core histones run as discrete nucleoprotein particles on polyacrylamide gels. Consistent with the electrophoretic properties of these particles, protein cross-linking with dimethyl suberimidate indicates that added core histones are bound as excess octamers. Histones in the excess octamers do not exchange with nucleosomal core histones at an ionic strength of 0.1 M and can be selectively removed from core particles by incubating the complexes in a solution containing sufficient DNA. Under conditions where added histones are confined to the surface of chromatin, the excess histones are mobile and can migrate onto a contiguous extension of naked DNA and form nucleosomes.     

Hydrodynamic studies of the interaction between nucleosome core particles and core histones

We have studied the hydrodynamic properties of the complexes formed by interaction of nucleosome core particles with excess histone octamers containing two each of the four core histones. The results are consistent with tight binding of two to three octamers to the exterior of each core particle. The binding is dependent upon the presence of the H3/H4 histone pair: when H3/H4 alone are added to nucleosome core particles, tight binding is observed, but H2A/H2B alone are bound only weakly. We have also examined the properties of the nucleosome core in solutions containing 0·1 m to 0·7 M-NaCl. We show that in this salt range the core particle undergoes some changes in shape, reflected in a 14% increase in the frictional coefficient. Even at the highest salt concentrations used, however, the nucleosome core is still a compact, folded structure.     

Reconstitution of mononucleosomes: characterization of distinct particles that differ in the position of the histone core

Reconstitution of mononucleosomes from DNA and core histones was carried out to study the positioning of histone octamers on the DNA. Using random DNA molecules in the 200 to 250 bp size range we found that the reconstitution products consisted of a mixture of three different types of particles that could be separated by low ionic strength gel electrophoresis. In one particle, DNA was complexed with histones along its entire length indicating the binding of more than one histone octamer. The second particle contained only one histone core that was always associated, however, with the terminal 145 bp of the DNA regardless of its sequence which can be ascribed to a DNA end effect. Only the third particle consisted of histone octamers bound at internal positions of the DNA and is therefore the only particle suitable for investigating the influence of the DNA sequence on the positioning of the histone cores. A defined 154 bp pBR 322 restriction fragment that contains three BspRI restriction sites was also reconstituted with core histones. The accessibility of these sites to BspRI was measured in order to delineate the utility of restriction nucleases as probes for the structure of chromatin. Two sites located close to the center of the DNA were less susceptible by at least a factor of 1000 as compared to free DNA while the susceptibility of the third site in the terminal section of the DNA decreased about 50 fold after reconstitution.     

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.     

Extended C-terminal tail of wheat histone H2A interacts with DNA of the "linker" region

The preparation of hybrid histone octamers with wheat histone H2A variants replacing chicken H2A in the chicken octamer is described. The fidelity of the reconstituted hybrid octamers was confirmed by dimethyl suberimidate cross-linking. Polyglutamic-acid-mediated assembly of these octamers on long DNA and subsequent micrococcal nuclease (MNase) digestion demonstrated that, whereas chicken octamers protected 167 base-pairs (representing 2 full turns of DNA), hybrid histone octamers containing wheat histone H2A(1) with its 19 amino acid residue C-terminal extension protected an additional 16 base pairs of DNA against nuclease digestion. The protection observed by hybrid histone octamers containing wheat histone H2A(3) with both a 15 residue N-terminal and a 19 residue C-terminal extension was identical with that observed with H2A(1)-containing hybrid histone octamers with only the 19 residue C-terminal extension. These results suggest that the role of the C-terminal extension is to bind to DNA of the "linker" region. The thermal denaturation of chicken and hybrid core particles was identical in 10 mM-Tris.HCl.20 mM-NaCl, 0.1 mM-EDTA, confirming that there was no interaction between the basic C-terminal extension and DNA of the core particle. Denaturation in EDTA, however, showed that hybrid core particles had enhanced stability, suggesting that the known conformational change of core particles at very low ionic strength allows the C-terminal extension to bind to core particle DNA under these conditions. A model accounting for the observed MNase protection is presented.     

Chromatosome positioning on assembled long chromatin. Linker histones affect nucleosome placement on 5 S rDNA

Long chromatin containing linker histones H1 or H5 was assembled on tandemly repeated 172 or 207 base-pair nucleosome positioning sequences from a sea urchin 5 S RNA gene. The effects of H1 and H5 on spacing and positioning of nucleosomes were assessed. In the absence of linker histones, precise determinations of core particle boundaries showed that, although a large proportion of the histone octamers occupy a unique position, there is a small group of other, less populated sites located around this major site. The dominant position was found 10 to 15 base-pairs upstream from the unique position previously reported for the histone octamer on the monomer 260 base-pair sequence. Linker histones do not override the underlying DNA signals that induce the very regular spacing of nucleosomes in chromatins assembled on these strongly positioning multimer DNA sequences. They were nevertheless found to be decisive in determining the chromatosome positions and their distributions, and as such define the chromatosome as a positioning entity.     

Structural changes of nucleosomal particles and isolated core-histone octamers induced by chemical modification of lysine residues

Treatment of nucleosomal particles and isolated core-histone octamers with dimethylmaleic anhydride, but not with acetic anhydride, is accompanied by a biphasic release of the two H2A.H2B dimers, the first dimer being more easily released than the second. With both kinds of particles, 50% of histones H2A and H2B are released for modification of approximately 35% of the histone amino groups. The similar behavior of nucleosomal particles and isolated core-histone octamers is consistent with the same structure of the histone octamer in the nucleosomal particle and in the free octamer in 2 M NaCl. The described release of H2A.H2B dimers allows the preparation of nucleosomal particles deficient in one H2A.H2B dimer and of the histone hexamers H2A.H2B.(H3.H4)2. For more extensive modifications, both reagents, acetic and dimethylmaleic anhydrides, cause the dissociation of nucleosomal particles with liberation of double-stranded DNA, which suggests that lysine amino groups are involved in the binding of histones to DNA. The modified nucleosomal particles are more sensitive to ionic strength than those untreated, and the presence of salt (NaCl) increases the extent of DNA release. The histones corresponding to the liberated DNA, except H2A and H2B released with dimethylmaleic anhydride, are apparently bound to the DNA-containing particles as extra histones.     

Limitations of the poly(glutamic acid) reconstitution method in the reassembly of mono- and dinucleosomes

Reconstitution of mononucleosomes and dinucleosomes at physiological ionic strength by means of poly(glutamic acid) is not efficient at physiological histone octamer:DNA ratios, unlike that with the salt dialysis method. The shorter the DNA is, the less transfer of octamers from poly(glutamic acid) to DNA occurs. By increasing the octamer:DNA ratio it is possible to involve all the DNA in the assembly, but for DNA longer than core particle length, nucleoprotein particles containing extra histones are concomitantly generated. Except for core particle and chromatosome lengths of DNA reassembled at 0.6:1 or 1:1 octamer:DNA ratio (and thus with low yield), reconstituted nucleoprotein particles proved to be different from native nucleosomes by their insolubility upon isolation. In the aggregates, DNA ends seemed to be sufficiently loose to allow exonuclease III digestion up to a certain limit. This resulted in patterns that for some cloned DNA fragments could give the impression, without knowledge of the above, of resulting from a unique octamer position. In view of the small range of length of DNA and the low yield of faithful reconstitution, the assembly method using poly(glutamic acid) is only of limited use in mono- or dinucleosome reconstitution experiments, at least in our hands.     

Hyperacetylation of core histones does not cause unfolding of nucleosomes. Neutron scatter data accords with disc shape of the nucleosome

Recent studies report that the frictional resistance of partially acetylated core particles increases when the number of acetyl groups/particle exceeds 10 (Bode, J., Gomez-Lira, M. M. & Schr?ter, H. (1983) Eur. J. Biochem. 130, 437-445). This was attributed to an opening of the core particle though other explanations, e.g. unwinding of the DNA ends were also suggested. Another possible explanation is that release of the core histone N-terminal domains by acetylation increased the frictional resistance of the particle. Neutron scatter studies have been performed on core particles acetylated to different levels up to 2.4 acetates/H4 molecule. Up to this level of acetylation the neutron scatter data show no evidence for unfolding of the core particle. The fundamental scatter functions for the envelope shape and internal structure are identical to those obtained previously for bulk core particles. The structure that gave the best fit to these fundamental scatter functions was a flat disc of diameter 11-11.5 nm and of thickness 5.5-6 nm with 1.7 +/- 0.2 turns of DNA coiled with a pitch of 3.0 nm around a core of the histone octamer. The data analysis emphasizes the changes in pair distance distribution functions at relatively low contrasts, particularly when the protein is contrast matched and DNA dominates the scatter. Under these conditions there is no evidence for the unwinding of long DNA ends in the hyperacetylated core particles. The distance distribution functions go to zero between 11.5 and 12 nm which gives the maximum chord length in a particle of dimension, 11 nm X 5.5 nm. The distance distribution function for the histone octamer contains 85% of the vectors within the 7.0-nm diameter of the histone core. 15% of the histone vectors lie between 7.0 and 12.0 nm, and these are attributed to the N-terminal domains of the core histones which extend out from the central histone core. Histone vectors extending beyond 7.0 nm are necessary to account for the measured radius of gyration of the histone core of 3.3 nm. A similar value of 3.2 nm is calculated for the recent ellipsoidal shape of 11.0 X 6.5 X 6.5 nm from the crystal structure of the octamer. However, the nucleosome model based on this structure is globular, roughly 11 nm in diameter, which does not accord with the flat disc shape core particle obtained from detailed neutron scatter data nor with the cross-section radii of gyration of the histone and DNA found previously for extended chromatin in solution.     

Folding of DNA by histones which lack their NH2-terminal regions

HeLa chromatin core particles were digested with trypsin to excise the NH2-terminal histone regions. The resulting nucleoprotein complexes were dissociated in 2.5 M NaCl; the DNA and polypeptides were then allowed to reassemble by lowering the NaCl concentration. Eighty per cent of the DNA reassociated with the polypeptides. The reassembled nucleoprotein complexes sediment at 9.7 S, have a molecular elipticity at 280 nm of 3000 degrees cm2/dmol of PO4, and contain DNase I-susceptible sites at 10 nucleotide intervals. The pattern of products generated by cross-linking the polypeptides with dimethylsuberimidate is very similar to the pattern generated by cross-linking native core particles. The results indicate that histones which lack their HN2-terminal regions retain both the features necessary for correct protein-protein interactions and the ability to fold DNA into a nucleoprotein complex resembling the chromatin core particle.     

Characterization of nucleosomes consisting of the human testis/sperm-specific histone H2B variant (hTSH2B)

We have reported earlier the occurrence of a specific histone H2B variant in human testis and sperm. Here we have structurally characterized this protein, its association with the rest of the histone octamer, and its effects on the nucleosome structure. We show that a reconstituted octamer consisting of hTSH2B and a stoichiometric complement of histones H2A, H3, and H4 exhibits a lower stability compared to the reconstituted native counterpart consisting of H2B. In contrast, the hTSH2B containing octamers are able to form nucleosome core particles which are structurally and dynamically indistinguishable from those reconstituted with octamers consisting of only native histones. Furthermore, the presence of hTSH2B in the nucleosome does not affect its ability to bind to linker 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.     

The intrinsically disordered distal face of nucleoplasmin recognizes distinct oligomerization states of histones

The role of Nucleoplasmin (NP) as a H2A-H2B histone chaperone has been extensively characterized. To understand its putative interaction with other histone ligands, we have characterized its ability to bind H3-H4 and histone octamers. We find that the chaperone forms distinct complexes with histones, which differ in the number of molecules that build the assembly and in their spatial distribution. When complexed with H3-H4 tetramers or histone octamers, two NP pentamers form an ellipsoidal particle with the histones located at the center of the assembly, in stark contrast with the NP/H2A-H2B complex that contains up to five histone dimers bound to one chaperone pentamer. This particular assembly relies on the ability of H3-H4 to form tetramers either in solution or as part of the octamer, and it is not observed when a variant of H3 (H3C110E), unable to form stable tetramers, is used instead of the wild-type protein. Our data also suggest that the distal face of the chaperone is involved in the interaction with distinct types of histones, as supported by electron microscopy analysis of the different NP/histone complexes. The use of the same structural region to accommodate all type of histones could favor histone exchange and nucleosome dynamics.     

Attraction, phasing and neighbour effects of histone octamers on curved DNA

Nucleosome core particles were reconstituted on various DNA fragments containing a Crithidia fasciculata kinetoplast curved tract. The results show that, on curved DNA, nucleosome core particles form six- to sevenfold preferentially, relative to bulk sequences. The preferential deposition occurs at multiple periodic positions, whose distribution reveals a unique rotational setting of DNA with respect to the histone octamer surface and whose average periodicity is 10.26 +/- 0.04. Evidence is provided for a context effect in histone octamer deposition: octamers bound to a segment of curved DNA influence the positions of neighbour octamers. Taken together, the preferential formation of nucleosome core particles and the influence on the localization of neighbouring particles suggest for intrinsically bent sequences the biologically relevant role of organizers of nucleosomal arrays.     

The core histone N termini function independently of linker histones during chromatin condensation

The relationships between the core histone N termini and linker histones during chromatin assembly and salt-dependent chromatin condensation were investigated using defined chromatin model systems reconstituted from tandemly repeated 5 S rDNA, histone H5, and either native "intact" core histone octamers or "tailless" histone octamers lacking their N-terminal domains. Nuclease digestion and sedimentation studies indicate that H5 binding and the resulting constraint of entering and exiting nucleosomal DNA occur to the same extent in both tailless and intact chromatin arrays. However, despite possessing a normal chromatosomal structure, tailless chromatin arrays can neither condense into extensively folded structures nor cooperatively oligomerize in MgCl(2). Tailless nucleosomal arrays lacking linker histones also are unable to either fold extensively or oligomerize, demonstrating that the core histone N termini perform the same functions during salt-dependent condensation regardless of whether linker histones are components of the array. Our results further indicate that disruption of core histone N termini function in vitro allows a linker histone-containing chromatin fiber to exist in a decondensed state under conditions that normally would promote extensive fiber condensation. These findings have key implications for both the mechanism of chromatin condensation, and the regulation of genomic function by chromatin.     

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.     

S(T)PXX motifs promote the interaction between the extended N-terminal tails of histone H2B with "linker" DNA.

The assembly of hybrid core particles onto long chicken DNA with histone H2B in the chicken histone octamer replaced with either wheat histone H2B(2) or sea urchin sperm histone H2B(1) or H2B(2) is described. All these histone H2B variants have N-terminal extensions of between 18 and 20 amino acids, although only those from sea urchin sperm have S(T)PXX motifs present. Whereas chicken histone octamers protected 167 base pairs (bp) (representing two full turns) of DNA against micrococcal nuclease digestion (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813), all the hybrid histone octamers protected an additional 17-bp DNA against nuclease digestion. This protection was more marked in the case of hybrid octamers containing sea urchin sperm histone H2B variants and similar to that described previously (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813) for hybrid histone octamers containing wheat histone H2A variants all of which also have S(T)PXX motifs present. Continued micrococcal nuclease digestion reduced the length of DNA associated with the core particle via 172-, 162-, and 152-bp intermediates until the 146-bp core particle was obtained. These DNA lengths were approximately 5 bp or half a helical turn longer than those reported previously for stripped chicken chromatin and for core particles containing histone octamers reconstituted using "normal" length histone H2B variants. This protection pattern was also found in stripped sea urchin sperm chromatin, demonstrating that the assembly/digestion methodology reflects the in vivo situation. The interaction between the N-terminal histone H2B extension and DNA of the "linker" region was confirmed by demonstrating that stripped sea urchin sperm chromatin precipitated between 120 and 500 mM NaCl in a manner analogous to unstripped chromatin whereas stripped chicken chromatin did not. Tryptic digestion to remove all the histone tails abolished this precipitation as well as the protection of DNA outside of the 167-bp core particle against nuclease digestion.