Homogeneous reconstituted oligonucleosomes, evidence for salt-dependent folding in the absence of histone H1

Using the method of salt dialysis, we have reconstituted histone octamers onto DNA templates consisting of 12 tandem repeats, each containing a fragment of the sea urchin 5S rRNA gene [Simpson, R.T., Thoma, F., & Brubaker, J.M. (1985) Cell 42, 799-808]. In these templates, each sea urchin repeat contains a sequence for preferred nucleosome positioning. Sedimentation velocity and sedimentation equilibrium studies in the analytical ultracentrifuge indicate that at molar histone/DNA ratios of 1.0-1.1 extremely homogeneous preparations of fully loaded oligonucleosomes (12 nucleosomes/template) can be regularly obtained. Digestion of the oligonucleosomes with micrococcal nuclease, followed by restriction mapping of purified nucleosome-bound DNA sequences, yields a complicated but consistent pattern of nucleosome positioning. Roughly 50% of the nucleosomes appear to be phased at positions 1-146 of each repeat, while the remainder of the nucleosomes occupy a number of other minor discrete positions along the template that differ by multiples of 10 bp. From sedimentation velocity studies of the oligonucleosomes in 0-0.2 M NaCl, we observe a reversible increase in mean sedimentation coefficient by almost 30%, accompanied by development of heterogeneity in sedimentation. These results, in combination with theoretical predictions, indicate that linear stretches of chromatin in the absence of lysine-rich histones exist in solution in a salt-dependent equilibrium between an extended (low salt) conformation and one or more folded (high salt) structures. In addition, by 100 mM NaCl, salt-dependent dissociation of histone octamers from these linear oligonucleosomes is observed.     

Structural studies on Allium cepa L. chromatin: enhanced stability of internucleosome interactions in plant chromatin

The pattern of micrococcal nuclease digestion of chromatin from different organs of Allium cepa has been studied. The DNA from small oligonucleosomes appears to be highly degraded and heterogeneous. In solutions of intermediate ionic strength (0.15 M NaCl) histones H2A and H2B form dimers, however at high salt concentrations (2 M NaCl) they tend to form complexes of higher order, such as tetramers. It is proposed that a correlation exists between the ability of these proteins to form tetramers and the particular stability of internucleosome interactions.     

Fractionation of nucleosomes by salt elution from micrococcal nuclease- digested nuclei

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaCl has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13-- 17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCl. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaCl concentrations in content of nonhistone chromosomal proteins. 40--60% of the nucleosomes were released by 0.3 M NaCl with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaCl. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaCl. These fractions contained the DNA least available for micrococcal nuclease attach. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by 0.2 M NaCl suggest that this population is involved in a special function.     

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)     

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.     

Mobile histone tails in nucleosomes. Assignments of mobile segments and investigations of their role in chromatin folding

The 13C NMR spectrum of isolated nucleosome core particles contains many sharp resonances, including resonances of alpha- and beta-carbons, indicating that certain terminal segments of histones rich in basic residues are highly mobile (Hilliard, R. R., Jr., Smith, R. M., and Rill, R. L. (1986) J. Biol. Chem. 261, 5992-5998). Specific histone termini can be removed sequentially from nucleosome core particles by mild treatment with alpha-chymotrypsin or chymotrypsin plus trypsin (Rosenberg, N. L., Smith. R. M., and Rill, R. L. (1986) J. Biol. Chem. 261, 12375-12383). Comparisons of the 13C NMR spectra of native and several partially proteolyzed core particles indicated that a minimum of residues 1-20 of H3 and 1-11 and 118-128 of H2a are contained in mobile segments of native cores. H4 did not appear to contribute to the resonances from mobile histone segments, but a possible contribution of H2b residues 1-16 could not be ruled out. The 13C NMR spectra of oligonucleosomes containing and lacking lysine-rich histones (H1, H5) were similar to each other and to that of native nucleosome cores both when the oligonucleosomes were in an extended conformation at low ionic strength and when they were in a more compact conformation at higher ionic strength. This similarity suggests that histones H1 and H5 must be largely immobilized upon chromatin binding and that the segments of core histones that are mobile in isolated nucleosome cores are not strongly bound to adjacent linker regions in intact chromatin, and are not immobilized by compaction to the degree achieved in 50 mM phosphate buffer.     

Structure of histone octamers in reconstituted polynucleosomes

The salt-dependent structural changes of the histone octamer in complex with high-molecular-weight DNA have been studied by fluorescent spectroscopy. Changes in both the spectra maximum position and anisotropy of the histone tyrosine fluorescence reveal structural transitions in nucleosome within the ranges of 0.5-3 mM and 20-30 mM NaCl. Comparison of the octamer fluorescent parameters in complex with DNA as well as in a free state permits to interpret the revealed structural transitions as a change in degree of contacts stability between (H2A-H2B) dimer and (H3-H4)2 tetramer. More pronounced conformational changes in histone octamer are observed under the conditions of polynucleosome fibers interaction within the range of physiological ionic strength (100-600 mM NaCl). As far as fluorescent parameters are concerned, the aforementioned changes are connected with entire destruction of (H2A-H2B) dimer specific contacts with (H3-H4)2 tetramer. The obtained results suggest the possibility of existence of different structural states of histone octamer in the chromatin composition including those which are quite dissimilar from the octamer structure in the 2M NaCl solution.     

Salt-induced conformational transitions in chromatin. A flow linear dichroism study

The chromatin structure in solution has been studied by the flow linear dichroism method (LD) in a wide range of ionic strengths. It is found that increasing the ionic strength from 0.25 mM Na2EDTA, pH 7.0 to 100 mM NaCl leads to a strong reduction of the LD amplitude of chromatin and inversion of the LD sign from negative to positive at 2 mM NaCl. Chromatin exhibits a positive LD maximum value at 10-20 mM NaCl. These data enable us to conclude that in very low ionic strength (0.25 mM Na2EDTA) the nucleosome discs are oriented with their flat faces more or less parallel to the chromatin filament axis. Increasing ionic strength up to 20 mM NaCl leads to reorientation of the nucleosome discs and to formation of chromatin structures with nucleosome flat faces inclined to the fibril axis. A conformational transition of that kind is not revealed in H1-depleted chromatin. The condensation of the chromatin filaments with increasing concentration of NaCl from 20 mM to 100 mM slightly influences the orientation of the nucleosomes.     

Rat liver HMG1: a physiological nucleosome assembly factor.

Incubation of rat liver single-stranded DNA-binding protein HMG1 with the four core histones at 0.15 M NaCl favors histone association primarily into tetramers and, to a lesser extent, into octamers. The assembly of pre-formed histone-HMG1 complexes with DNA yields nucleosome-like subunits which satisfy most of the criteria defining native core particles: (i) the circular DNA extracted from the complexes is supercoiled indicating that the initially relaxed DNA acquired superhelical turns during complex formation in the presence of topoisomerase I; (ii) the digestion of the complexes with micrococcal nuclease yields a DNA fragment of approximately 140 bp in length; (iii) electron microscopy of the reconstituted complexes shows a beaded structure with the DNA wrapped around the histone cores, leading to a reduction in the contour length of the genome compared with free DNA. Moreover, in the presence of HMG1, nucleosome assembly occurs rapidly at 0.15 M NaCl. Therefore, in addition to its DNA-binding properties, HMG1 mediates the assembly of nucleosomes in vitro under conditions of physiological ionic strength. The possible involvement of these properties in the DNA replication process is discussed.     

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.     

Nucleosomes are assembled into discrete size structures by histone H1 in vitro

The involvement of histone H1 in the formation and maintenance of higher order chromatin structures in vitro was investigated biochemically. Addition of exogenous histone H1 to isolated calf thymus mononucleosomes in low ionic strength buffer resulted in the formation of electrophoretically distinct mononucleosome assemblies (supernucleosomes). The smaller supernucleosomes were composed of about 12, 18, 24, or 30 nucleosomes and one to two molecules of histone H1 per nucleosome. It was difficult to determine accurately the size of the larger supernucleosomes, but their bands from native gels contained probably between 60 and 300 nucleosomes or more. Similar supernucleosome size classes were also obtained when oligonucleosomes instead of mononucleosomes were employed. When the assembly of mono- and oligo-nucleosomes with histone H1 took place in 0.15 M NaCl, discrete supernucleosomes containing only mono- or di-nucleosomes, but not a mixture of both, were formed. It is proposed that the small supernucleosomes containing oligomers of 6 nucleosomes may represent integral multiples of the second-order chromatin structural subunit, whereas the larger supernucleosomes containing about 60 to 300 or more nucleosomes may correspond to chromatin domains or third-order chromatin structures observed by other techniques.     

Structure of the chromatin with long linker DNA

The method of velocity sedimentation have been used to investigate ionic-strength-induced compaction of sea urchin sperm chromatin characterized by extremely long linker DNA (100 b.p.). The dependence of sedimentation coefficients of oligonucleosomes on the number of nucleosomes in the chain have been studied in the range of ionic strength from 0.005 to 0.085. Analysis of these data indicates that such structural parameters of sea urchin sperm chromatin fibre as the diameter of the chain and the length of the chain per nucleosome are quite similar to those of chromatin with shorter linker DNA, but the DNA packing ratio is higher. The structure of sea urchin sperm oligonucleosomes agrees well with the model of three-dimensional zig-zag-shaped chain with linker DNA forming a loop. The possible role of alpha-helical regions of the C-terminal domain of sea urchin sperm histone H1 in the long linker DNA folding is discussed.     

Energetics and affinity of the histone octamer for defined DNA sequences

Previous studies have compared the relative free energies for histone octamer binding to various DNA sequences; however, no reports of the equilibrium binding affinity of the octamer for unique sequences have been presented. It has been shown that nucleosome core particles (NCPs) dissociate into free DNA and histone octamers (or free histones) on dilution without generation of stable intermediates. Dissociation is reversible, and an equilibrium distribution of NCPs and DNA is rapidly attained. Under low ionic strength conditions (<400 mM NaCl), NCP dissociation obeys the law of mass action, making it possible to calculate apparent equilibrium dissociation constants (K(d)s) for NCPs reconstituted on defined DNA sequences. We have used two DNA sequences that have previously served as model systems for nucleosome reconstitution studies, human alpha-satellite DNA and Lytechinus variegatus 5S DNA, and find that the octamer exhibits K(d)s of 0.03 and 0.06 nM, respectively, for these sequences at 50 mM NaCl. These DNAs form NCPs that are approximately 2 kcal/mol more stable than total NCPs isolated from cellular chromatin. As for mixed-sequence NCPs, increasing ionic strength or temperature promotes dissociation. van't Hoff plots of K(a)s versus temperature reveal that the difference in binding free energy for alpha-satellite and 5S NCPs compared to bulk NCPs is due almost entirely to a more favorable entropic component for NCPs formed on the unique sequences compared to mixed-sequence NCPs. Additionally, we address the contribution of the amino-terminal tail domains of histones H3 and H4 to octamer affinity through the use of recombinant tailless histones.     

Thiol reactivity of histone H3 in soluble and DNA-associated histone complexes: evidence for allosteric and torsional regulation

The reactivity of chick erythrocyte and calf thymus histone H3 thiol groups toward 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) has been investigated both in the soluble, DNA-free state and in various nucleohistone complexes. We have found that the thiol reactivity of both tetramers and octamers decreases continuously as the ionic strength of the assay is increased, up to and beyond 2.0 M NaCl. Upon association of dimers with tetramers, there is loss of labeling by DTNB at one site, suggesting the existence of allosteric regulation [see also Godfrey, J. E., Eickbush, T. H., & Moudrianakis, E. N. (1980) Biochemistry 19, 1339-1346] of dimer-tetramer interfaces emanating from within the tetramer complex. Comparison of the thiol reactivities of chick and calf tetramers indicates that the thiol groups at amino acid positions 96 and 110 are not chemically equivalent. When the histones are associated with DNA, in either reconstituted complexes, core particles, or long soluble chromatin, the thiol reactivity is greatly diminished, and this "DNA effect" overwhelms any influence of dimers. However, if single-strand nicks are introduced into the DNA backbone of core particles and other chromatin-like complexes by the action of DNase I, the influence of the DNA double helix upon thiol reactivity is reduced, and the effect of dimers can be detected once again. We can therefore conclude that the DNA effect derives from intranucleosomal torsional strain of the continuum of the double helix in equilibrium with coupled protein conformational changes. These observations support the concept that the octamer complex is a dynamic tripartite structure whose properties can be modulated through its interactions with DNA and by changes occurring in the dimer-tetramer interfaces.     

The structure and dynamics of H1-depleted chromatin

The size of DNA involved in the interaction with a histone octamer in H1-depleted chromatin was re-examined. We compared the thermal untwisting of chromatin DNA and naked DNA using CD and electrophoretic topoisomer analysis, and found that DNA of 175 +/- 10 base pairs (bp) in length interacted with the histone core under physiological conditions. The decrease of ionic strength below 20 mM NaCl reduced this length down to 145 bp: apparently, an extra 30 bp DNA dissociated from the histone core to yield well-known 145-bp core particle. Histone cores partly dissociate within the temperature range of 25 to 40 degrees C. Quantitative analysis of histone thermal dissociation from DNA shows that the size of DNA protected against thermal untwisting would be significantly overestimated if this effect is neglected. The results presented in this paper also suggest that the dimers (H2A, H2B) act as a lock, which prevents transmission of conformational alterations from a linker to nucleosome core DNA. The histone core dissociation as well as (H2A, H2B) dimer displacement are discussed in the light of their possible participation in the eukaryotic genome activation.     

The structure of SV 40 chromatin.

Simian virus 40 (SV40) nucleoprotein complexes were studied with the electron microscope. Depending on the isolation procedure, SV40 chromatin has two different conformations: complexes isolated in the presence of 0.15 M NaCl appeared as very compact globular structures, while those isolated in the presence of 0.6 M NaCl had the typical 'beads-on-a-string' appearance of the primary nucleofilament. Concomitant with this structural change was a variation in the histone pattern and sedimentation behaviour of the complexes: with NaCl at 0.15 mol 1(-1) the isolated complexes contained both the nucleosomal histones and histone H1, and sedimented in sucrose gradients at 70S. Increasing the ionic strength to 0.6 M NaCl resulted in the removal of histone H1 from the complexes and in a decrease of the sedimentation coefficient to 40S. DNA relaxing enzyme is associated with the SV40 nucleoprotein complexes. The numbers of superhelical turns in DNA from compact and open types of complexes were found to be the same. Therefore the transition from the condensed to the open structure of viral chromatin does not require a change in the topological winding number of its DNA.     

ESR spin label studies of the nucleosome core particle and histone core

An imidazole spin label has been used to study the accessibility and conformational state of tyrosines in both the nucleosome core particles and histone core extracted from chicken erythrocytes. About 40% of the tyrosyl residues in the histone core can be labeled under nondenaturing conditions. However, less than 15% of the tryosyls in the nucleosome core particle can be labeled even at 200- to 300-fold M excess of label. The effect of urea on the conformational state of the spin-labeled tyrosyls in both the nuclesome core particles and the histone core has been studied. Ionic effects on the spin-labeled nucleosome core have been investigated. Several conformational transitions are observed in the range of 1 mM NaCl to 2.5 M NaCl. Three major transitions are found at 0.1 M to 0.6 M, 0.7 M to 1.8 M and 2 M to 2.5 M NaCl, respectively. The observed changes can be interpreted as swelling and conformational change of the inner histone core, gradual separation of DNA from the histone core, and tightening of the histone core.