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.     

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.     

Computer simulation of the 30-nanometer chromatin fiber

A new Monte Carlo model for the structure of chromatin is presented here. Based on our previous work on superhelical DNA and polynucleosomes, it reintegrates aspects of the "solenoid" and the "zig-zag" models. The DNA is modeled as a flexible elastic polymer chain, consisting of segments connected by elastic bending, torsional, and stretching springs. The electrostatic interaction between the DNA segments is described by the Debye-Hückel approximation. Nucleosome core particles are represented by oblate ellipsoids; their interaction potential has been parameterized by a comparison with data from liquid crystals of nucleosome solutions. DNA and chromatosomes are linked either at the surface of the chromatosome or through a rigid nucleosome stem. Equilibrium ensembles of 100-nucleosome chains at physiological ionic strength were generated by a Metropolis-Monte Carlo algorithm. For a DNA linked at the nucleosome stem and a nucleosome repeat of 200 bp, the simulated fiber diameter of 32 nm and the mass density of 6.1 nucleosomes per 11 nm fiber length are in excellent agreement with experimental values from the literature. The experimental value of the inclination of DNA and nucleosomes to the fiber axis could also be reproduced. Whereas the linker DNA connects chromatosomes on opposite sides of the fiber, the overall packing of the nucleosomes leads to a helical aspect of the structure. The persistence length of the simulated fibers is 265 nm. For more random fibers where the tilt angles between two nucleosomes are chosen according to a Gaussian distribution along the fiber, the persistence length decreases to 30 nm with increasing width of the distribution, whereas the other observable parameters such as the mass density remain unchanged. Polynucleosomes with repeat lengths of 212 bp also form fibers with the expected experimental properties. Systems with larger repeat length form fibers, but the mass density is significantly lower than the measured value. The theoretical characteristics of a fiber with a repeat length of 192 bp where DNA and nucleosomes are connected at the core particle are in agreement with the experimental values. Systems without a stem and a repeat length of 217 bp do not form fibers.     

The structure of chromatin from the pigeon brain. II. Sedimentation analysis of oligonucleosome density

Compaction of pigeon brain and rat thymus chromatin differing in the length of the linker DNA has been studied by the method of velocity sedimentation. The dependence of sedimentation coefficients of oligonucleosomes on the number of nucleosomes in the chain in solution of different ionic strength (0.005-0.085) has been analyzed. The analyses of these dependences showed that the structure of oligonucleosomes of both cell types at low ionic conditions may be described by the model of a zig-zag-shaped nucleosomal chain. The process of compaction of the oligonucleosomes at higher ionic strength (0.045-0.085) proceeds similarly for brain and thymus chromatin. The formation of a superhelical structure is determined by the interaction of no less than 6 nucleosomes; the compactness of the structure is significantly increased when the number of nucleosomes in the chain exceeds 10. The ability of the brain oligonucleosomes to form a compact structure despite the short linker allow the suggestion that in brain short chromatin the DNA chain does not form two complete turns in the nucleosome. This provides necessary flexibility of brain chromatin.     

Atomic force microscopy sees nucleosome positioning and histone H1-induced compaction in reconstituted chromatin.

We addressed the question of how nuclear histones and DNA interact and form a nucleosome structure by applying atomic force microscopy to an in vitro reconstituted chromatin system. The molecular images obtained by atomic force microscopy demonstrated that oligonucleosomes reconstituted with purified core histones and DNA yielded a 'beads on a string' structure with each nucleosome trapping 158 +/- 27 bp DNA. When dinucleosomes were assembled on a DNA fragment containing two tandem repeats of the positioning sequence of the Xenopus 5S RNA gene, two nucleosomes were located around each positioning sequence. The spacing of the nucleosomes fluctuated in the absence of salt and the nucleosomes were stabilized around the range of the positioning signals in the presence of 50 mM NaCl. An addition of histone H1 to the system resulted in a tight compaction of the dinucleosomal structure.     

The structural organization of dinucleosomes and oligonucleosomes. Electric dichroism and birefringence study.

The spatial organization of nucleosomes and linker DNA in dinucleosomes and oligonucleosomes of various chain lengths has been investigated through electric dichroism, birefringence and relaxation times measurements at low ionic strengths (0.5 to 2.2 mM). From the negative dichroism observed for all the samples, it is concluded that the nucleosome subunits in the oligonucleosome chain must lie with their disc planes closely parallel to the fibre axis. The large increase of the negative dichroism of dinucleosomes upon Hl removal is interpreted by the unwinding of the DNA tails and the internucleosomal segment. All the samples displayed, under bipolar pulses, a predominantly induced orientation mechanism.     

Assembly of oligonucleosomes into a limit series of multimeric higher-order chromatin structures

Chicken erythrocyte chromatin, obtained after fragmentation with micrococcal nuclease, appears to remain folded in a stable distribution of supranucleosomal structures in buffers containing 80 mM NaCl. These supranucleosomal particles are composed of on average 25 nucleosomes. However, the integrity of the linker DNA within these particles is not required. The supranucleosomal particles have been interpreted by others as superbeads cut out of a preexisting granular nominal 30-nm chromatin fibre. We show that the same distribution of supranucleosomal structures (even those containing internal DNA scissions) can be reconstituted from unfolded nuclear chromatin extracts as present in 10 mM or 600 mM NaCl. Moreover, fractions of oligonucleosomes with mean lengths between 6 and 15 nucleosomes reassemble or aggregate into a limit series of multimeric species. The existence of an assembly barrier could be inferred as we were unable to observe a stable and soluble assembly product containing more than about 25 nucleosomes. We propose an alternative explanation for the generation and observation of a constant distribution of supranucleosomal structures in nuclear extracts, based on the assembly or aggregation property of oligonucleosomes and on the existence of an assembly barrier.     

Nucleosomal histone protein protects DNA from iron-mediated damage.

Iron promotes DNA damage by catalyzing hydroxyl radical formation. We examined the effect of chromatin structure on DNA susceptibility to oxidant damage. Oxygen radicals generated by H2O2, ascorbate and iron-ADP (1:2 ratio of Fe2+:ADP) extensively and randomly fragmented protein-free DNA, with double-strand breaks demonstrable even at 1 microM iron. In contrast, polynucleosomes from chicken erythrocytes were converted to nucleosome-sized fragments by iron-ADP even up to 250 microM iron. Cleavage occurred only in bare areas where DNA is unassociated with histone. In confirmation, reassembly of nucleosomes from calf thymus DNA and chicken erythrocyte histone also yielded nucleosomes resistant to fragmentation. Protection of DNA by histone was dependent on nucleosome assembly and did not simply reflect presence of scavenging protein. In contrast to this specific cleavage of internucleosomal linker DNA by iron-ADP, iron-EDTA cleaved polynucleosomes indiscriminately at all sites. The hydroxyl radical scavenger thiourea completely inhibited the random cleavage of polynucleosomes by iron-EDTA but inhibited the nonrandom cleavage of polynucleosomes by iron-ADP less completely, suggesting the possibility that the lower affinity iron-ADP chelate may allow association of free iron with DNA. Thus, oxygen radicals generated by iron-ADP indiscriminately cleaved naked DNA but cleaved chromatin preferentially at internucleosomal bare linker sites, perhaps because of nonrandom iron binding by DNA. These findings suggest that the DNA-damaging effects of iron may be nonrandom, site-directed and modified by histone protein.     

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.     

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.     

Nucleosomes stacked with aligned dyad axes are found in native compact chromatin in vitro

In this study, electron tomograms of plunge-frozen isolated chromatin in both open and compacted form were recorded. We have resolved individual nucleosomes in these tomograms in order to provide a 3D view of the arrangement of nucleosomes within chromatin fibers at different compaction states. With an optimized template matching procedure we obtained accurate positions and orientations of nucleosomes in open chromatin in "low-salt" conditions (5 mM NaCl). The mean value of the planar angle between three consecutive nucleosomes is 70°, and the mean center-to-center distance between consecutive nucleosomes is 22.3 nm. Since the template matching approach was not effective in crowded conditions, for nucleosome detection in compact fibers (40 mM NaCl and 1 mM MgCl(2)) we developed the nucleosome detection procedure based on the watershed algorithm, followed by sub-tomogram alignment, averaging, and classification by Principal Components Analysis. We find that in compact chromatin the nucleosomes are arranged with a predominant face-to-face stacking organization, which has not been previously shown for native isolated chromatin. Although the path of the DNA cannot be directly seen in compact conditions, it is evident that the nucleosomes stack with their dyad axis aligned in forming a "double track" conformation which is a consequence of DNA joining adjacent nucleosome stacks. Our data suggests that nucleosome stacking is an important mechanism for generating chromatin compaction in vivo.     

Changing Chromatin Fiber Conformation by Nucleosome Repositioning

Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.     

Nucleosomes structure and dynamics: effect of CHAPS

Dynamics of nucleosomes and spontaneous unwrapping of DNA are fundamental property of the chromatin enabling access to nucleosomal DNA for regulatory proteins. Probing of such dynamics of nucleosomes performed by single molecule techniques revealed a large scale dynamics of nucleosomes including their spontaneous unwrapping. Dissociation of nucleosomes at low concentrations is a complicating issue for studies with single molecule techniques. In this paper, we tested the ability of 3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS) to prevent dissociation of nucleosomes. The study was performed with mononucleosome system assembled with human histones H2A, H2B, H3 and H4 on the DNA substrate containing sequence 601 that provides the sequencespecific assembly of nucleosomes. We used Atomic Force Microscopy (AFM) to directly identify nucleosomes and analyze their structure at the nanometer level. These studies showed that in the presence of CHAPS at millimolar concentrations, nucleosomes, even at sub-nanomolar concentrations, remain intact over days compared to a complete dissociation of the same nucleosome sample over 10 min in the absence of CHAPS. Importantly, CHAPS does not change the conformation of nucleosomes as confirmed by the AFM analysis. Moreover, 16 µM CHAPS stabilizes nucleosomes in over one hour incubation in the solution containing as low as 0.4 nM in nucleosomes. The stability of nucleosomes is slightly reduced at physiological conditions (150 mM NaCl), although the nucleosomes dissociate rapidly at 300 mM NaCl. The sequence specificity of the nucleosome in the presence of CHAPS decreased suggesting that the histone core translocates along the DNA substrate utilizing sliding mechanism.     

Changing Chromatin Fiber Conformation by Nucleosome Repositioning

Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.     

The mechanism of nucleosome assembly onto oligomers of the sea urchin 5 S DNA positioning sequence

We have used a model system composed of tandem repeats of Lytechinus variegatus 5 S rDNA (Simpson, R. T., Thoma, F., and Brubaker, J. M. (1985) Cell 42, 799-808) reconstituted into chromatin with chicken erythrocyte core histones to investigate the mechanism of chromatin assembly. Nucleosomes are assembled onto the DNA template by mixing histone octamers and DNA in 2 M NaCl followed by stepwise dialysis into very low ionic strength buffer over a 24-h period. By 1.0 M NaCl, a defined intermediate composed of arrays of H3.H4 tetramers has formed, as shown by analytical and preparative ultracentrifugation. Digestion with methidium propyl EDTA.Fe(II) indicates that these tetramers are spaced at 207 base pair intervals, i.e. one/repeat length of the DNA positioning sequence. In 0.8 M NaCl, some H2A.H2B has become associated with the H3.H4 tetramers and DNA. Surprisingly, under these conditions DNA is protected from methidium propyl EDTA.Fe(II) digestion almost as well as in the complete nucleosome, even though these structures are quite deficient in H2A.H2B. By 0.6 M NaCl, nucleosome assembly is complete, and the MPE digestion pattern is indistinguishable from that observed for oligonucleosomes at very low ionic strength. Below 0.6 M NaCl, the oligonucleosomes are involved in various salt-dependent conformational equilibria: at approximately 0.6 M, a 15% reduction in S20,w that mimics a conformational change observed previously with nucleosome core particles; at and above 0.1 M, folding into a more compact structure(s); at and above 0.1 M NaCl, a reaction involving varying amounts of dissociation of histone octamers from a small fraction of the DNA templates. In low ionic strength buffer (less than 1 mM NaCl), oligonucleosomes are present as fully loaded templates in the extended beads-on-a-string structure.     

Histone H1 can be removed selectively from chicken erythrocyte chromatin at near physiological conditions.

Histone H1 was depleted selectively from chicken erythrocyte polynucleosomes, without any detectable concomitant loss of H5 or core particle histones. The depletion is performed with ion exchange resin at low ionic strength (80 mM NaCl). The nucleosomes did not slide during the procedure. In contrast to the native chromatin, H1 depleted polynucleosomes are completely soluble in the 5--600 mM NaCl range.     

Distribution of linker DNA in relation to core DNA in H1 depleted nucleosomes.

The mode of interaction of histone H1 with the nucleosome is governed by the relative distribution of the linker with respect to the core DNA. Preliminary experiments (Simpson, R.T. 1978, Biochemistry, 17, 5524-5531) and tentative models (Thoma, F. et al. (1979), J. Cell. Biol., 83, 403-427) suggest that part of the linker complete two full turns of DNA around the histone core, probably by adding 10 base pairs at each end of the core DNA. In the present study Exonuclease III has been utilized to digest the 3' ends of H1 depleted nucleosomes. (i.e. the 195 base pair particle). The analysis of the resulting DNA fragments under denaturing conditions shows that the whole linker is distributed symmetrically with respect to the core DNA.