Salt-and histone H1-induced structural changes of reconstituted minichromosomes.

Structural changes of reconstituted SV 40 minichromosomes have been studied in relation to the salt concentration and addition of histone H1 by sedimentation and electron microscopy. Sedimentation data are represented as functions of the NaCl concentration and the Debye-Hückel electrostatic screening radius 1/alpha. The latter representation which proved to provide more information revealed three structural states of the SV 40 reconstitutes which can be additionally characterized by electron microscopy as follows: Expanded or relaxed conformation including free DNA spacers between the nucleosomes at low salt concentration (approx. 0.001 M-0.05 M NaCl), increasing condensation at moderate salt concentration (approx. 0.05 M-0.3 M NaCl) and expansion of this condensed state above approx. 0.3 M NaCl. The condensation of the reconstitutes at moderate salt concentration does not require the presence of histone H1. H1 seems to stabilize the condensed state against electrostatic expansion. The condensation might be promoted by salt-dependent conformational changes of naked superhelical DNA as revealed by sedimentation measurements.     

Structural analysis of a reconstituted DNA containing three histone octamers and histone H5

Previous work has shown that DNA and the histone proteins will combine to form structures of a complex, yet definite nature. Here, we describe three experiments aimed at a better understanding of the interactions of DNA with the histone octamer and with histone H5. First, there has been some question as to whether the methylation of DNA could influence its folding about the histone octamer. To address this point, we reconstituted the histone octamer onto a 440 base-pair DNA of defined sequence at various levels of cytosine methylation, and also onto the unmethylated DNA. The reconstituted structures were probed by digestion with two different enzymes, micrococcal nuclease and DNase I. All samples were found to contain what appear to be three histone octamers, bound in close proximity on the 440 base-pair DNA. The cutting patterns of micrococcal nuclease and DNase I remain the same in all cases, even if the DNA has been extensively methylated. The results show, therefore, that methylation has little, or no, influence on the folding of this particular DNA about the histone octamer. Second, there has been concern as to whether the base sequence of DNA could determine its folding in a long molecule containing several nucleosomes, just as it does within any single, isolated nucleosome core. In order to deal with this problem, we cut the 440 base-pair DNA into three short fragments, each of nucleosomal length; we reconstituted each separately with the histone octamer; and then we digested the reconstituted complexes with DNase I for comparison with similar data from the intact 440 base-pair molecule. The results show that the folding of this DNA is influenced strongly by its base sequence, both in the three short fragments and in the long molecule. The rotational setting of the DNA within each of the three short fragments is as predicted from a computer algorithm, which measures its homology to 177 known examples of nucleosome core DNA. The rotational setting of the DNA in the 440 base-pair molecule remains the same as in two of the three short fragments, but changes slightly in a third case, apparently because of steric requirements when the nucleosomes pack closely against one another. Finally, there has been little direct evidence of where histone H5 binds within a DNA-octamer complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of histone H2B from sea urchin sperm in the association of reconstituted minichromosomes

A comparative study of the condensation of reconstituted complexes of circular SV40 DNA with core histones from calf thymus and sea urchin sperm was performed using sedimentation and electron microscopic techniques. It is shown that in low ionic strength solutions both types of complexes are similar to native minichromosomes. In the region from 0.08 to 0.16 M NaCl the complexes of SV40 DNA with thymus histones form small compact particles. By contrast, the compaction of the SV40 DNA complexes with sperm histones results in the formation of giant intermolecular associates. The results obtained may mean that histone H2B of sea urchin sperm participates in the formation of a higher order structure in sperm chromatin.     

Influence of DNA topology and histone tails in nucleosome organization on pBR322 DNA.

Recently, we have found that the assembly of nucleosomes reconstituted on negatively supercoiled DNA is cooperative. In the present paper the role of DNA topology and of histone tails in nucleosome assembly was explored. Reconstituted minichromosomes on relaxed DNA at different histone/DNA ratios (R) were assayed by topological analysis and electron microscopy visualization. Both methods show a linear relationship between average nucleosome number (N) and R. This suggests that in the case of relaxed DNA, cooperative internucleosomal interactions are small or absent. The influence of histone tails in nucleosome assembly was studied on minichromosomes reconstituted with trypsinized histone octamer on negatively supercoiled DNA by topological analysis. The topoisomers distribution, after trypsinization, dramatically changes, indicating that nucleosome-nucleosome interactions are remarkably decreased. These results show that, in chromatin folding, in addition to the well known role of histone H1, the interactions between histone octamer tails and DNA are also of importance.     

Effect of gamma-irradiation on H3 histone and DNA in solution

Three methods, namely, absorbance of colour by reaction with Folin-Ciocalteau reagent, UV absorbance and fluorescence intensity measurements for detection of H3 histone in 0.15 M standard saline citrate (SSC) solution were compared. Maximum sensitivity was found with the Folin-Ciocalteau method. Effect of varying pH and of gamma- radiation on H3 histone and on interaction of H3 histone with DNA were studied. For this, solutions of H3 histone in SSC, in 0.9% NaCl, H3 histone + DNA in 0.9% NaCl were subjected to varying pH (1-10) and gamma- radiation (dose 10-50 Gy) and lambda(max) and Alambda(max) were monitored. From the molar ratios of histone and DNA in the complex, it was observed that at gamma -radiation dose of 50 Gy and pH 8.54, there was a depletion of 6-8 microg/ml of histone from the histone-DNA complex.     

Kinetic persistence of cruciform structures in reconstituted minichromosomes

Cruciforms persist in reconstituted minichromosomes, as revealed by cleavage with specific nucleases and hybridization with synthetic oligonucleotides. Relaxation by topoisomerase I suggests that cruciforms are located mainly on internucleosomal DNA and that their persistence on minichromosomes may be due to kinetic effects. The analysis of the kinetic behaviour of cruciforms in minichromosomes shows a definite velocity of reabsorption with respect to stable cruciforms in supercoiled naked DNA. An explanation based on suppression of the untwisting of linker DNA due to adjacent nucleosomes is proposed.     

Labelling of histone H5 and its interaction with DNA. 2. Cooperative binding of histone H5 to DNA as probed by steady-state fluorescence and diffusion-enhanced energy transfer

The interaction of histone H5 labelled with fluorescein isothiocyanate (FITC) with DNA has been studied by fluorescence titration, and diffusion-enhanced fluorescence energy transfer (DEFET) measurements with Tb(III) lanthanide chelates as donors. Analysis of the binding data by the model of Schwarz and Watanabe (J.Mol.Biol. 163, 467-484 (1983)) yielded a mean stoichiometry of 60 nucleotides per H5 molecule, independently of ionic strength, in the range of 3 to 300 mM NaCl, at very low DNA concentration (6 microM in mononucleotide). It ensues an approximate electroneutrality of the saturated complexes. Histone H5 molecules appeared to be clustered along the DNA lattice in clusters containing on average 3 to 4 H5 molecules separated by about 79 base pairs, at mid-saturation of the binding sites. The interaction process was found highly cooperative but the cooperativity parameter was also insensitive to ionic strength in the above range. DEFET experiments indicated an important decrease of accessibility of the FITC label to the TbHED3A and TbEDTA- chelates with ionic strength in the 0 to 100 mM NaCl range. In the presence of DNA, H5 appears already folded at low ionic strength so that the FITC probe is also not accessible to the donor chelate. The present study constitutes an indispensable preliminary step to further studies on the localization of histone H5 in condensed chromatin structures.     

Labelling of histone H5 and its interaction with DNA. 1. Histone H5 labelling with fluorescein isothiocyanate

Histone H5 has been labelled with fluorescein isothiocyanate (FITC) with particular attention to the reaction conditions (pH, reaction time and input FITC/H5 molar ratio) and to the complete elimination of non-covalently bound dye. We preferred to use reaction conditions which yielded non-specific uniform labelling rather than specific alpha-NH2 terminal labelling, in order to obtain higher sensitivity in further studies dealing with the detection of perturbation at the binding sites of H5 on DNA. FITC-labelled H5 was further characterized by absorption and circular dichroism spectroscopy, and the fluorescein probe titrated in the 4-8 pH range. The structural integrity of H5 was found to be preserved after labelling. The positive electrostatic potential of the environment in which the FITC probe is embedded in the arginine/lysine-rich tails of H5 is believed to be responsible for the drop of pK of 1 unit found for H5-FITC as compared to free FITC. For the globular part of H5, the pK of covalently-bound FITC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region.     

Co-operative binding of the globular domain of histone H5 to DNA.

The globular domain of histone H5 (GH5) was prepared by trypsin digestion of H5 that was extracted from chicken erythrocyte nuclei with NaCl. Electron microscopy, sucrose gradient centrifugation, native agarose gel electrophoresis and equilibrium density gradient ultracentrifugation show that GH5 binds co-operatively to double-stranded DNA. The electron microscopic images suggest that the GH5-DNA complexes are very similar in structure to co-operative complexes of intact histone H1 (or its variants) with double-stranded DNA, studied previously, which have been proposed to consist of two parallel DNA double helices sandwiching a polymer of the protein. For complexes with GH5 or with intact H1, naked DNA co-sediments with the protein-DNA complexes through sucrose gradients, and DNA also appears to protrude from the ends and sides of the complexes; measurements of the protein-DNA stoichiometry in fractionated samples may not reflect the stoichiometry in the complexes. An estimate of the stoichiometry obtained from the buoyant density of fixed GH5-DNA complexes in CsCl suggests that sufficient GH5 is present in the complexes for the GH5s to be in direct contact, as required by a simple molecular mechanism for the co-operative binding. Chemical crosslinking demonstrates that GH5s are in close proximity in the complexes. In the absence of DNA, GH5-GH5 interactions are weak or non-existent.     

Effect of spontaneous twist on DNA minicircles

Monte Carlo simulations are used to study the effect of spontaneous (intrinsic) twist on the conformation of topologically equilibrated minicircles of dsDNA. The twist, writhe, and radius of gyration distributions and their moments are calculated for different spontaneous twist angles and DNA lengths. The average writhe and twist deviate in an oscillatory fashion (with the period of the double helix) from their spontaneous values, as one spans the range between two neighboring integer values of intrinsic twist. Such deviations vanish in the limit of long DNA plasmids.     

Closely spaced nucleosome cores in reconstituted histone.DNA complexes and histone-H1-depleted chromatin

It has been demonstrated by digestion studies with micrococcal nuclease that reconstitution of complexes from DNA and a mixture of the four small calf thymus histones H2A, H2B, H3, and H4 leads to subunits closely spaced in a 137 +/- 7-nucleotide-pair register. Subunits isolated from the reconstituted complex contain nearly equimolar amounts of the four histones and sediment at 11.6S. On DNase I digestion both the reconstituted complex and the separated subunits gave rise to series of single-stranded DNA fragments with a 10-nucleotide periodicity. This indicates that the reconstitution leads to subunits very similar to nucleosome cores. Nucleosome cores closely spaced in a 140-nucleotide-pair register were also obtained upon removal of histone H1 from chromatin by dissociation with 0.63 M NaCl and subsequent ultracentrifugation. In reconstitution experiments with all five histones (including histone H1) our procedure did not lead to tandemly arranged nucleosomes containing about 200 nucleotide pairs of DNA. In the presence of EDTA, DNase II cleaved calf thymus nuclei and chromatin at about 200-nucleotide-pair intervals whereas in the presence of Mg2+ cleavage at intervals of approximately half this size was observed. The change in the nature of the cleavage pattern, however, was no longer found after removal of histone H1 from chromatin. This indicates that H1 influences the accessibility of DNase II cleavage sites in chromatin. This finding is discussed with respect to the influence of histone H1 on chromatin superstructure.     

Linker histone tails and N-tails of histone H3 are redundant: scanning force microscopy studies of reconstituted fibers.

The mechanisms responsible for organizing linear arrays of nucleosomes into the three-dimensional structure of chromatin are still largely unknown. In a companion paper (Leuba, S. H., et al. 1998. Biophys. J. 74:2823-2829), we study the contributions of linker histone domains and the N-terminal tail of core histone H3 to extended chromatin fiber structure by scanning force microscopy imaging of mildly trypsinized fibers. Here we complement and extend these studies by scanning force microscopy imaging of selectively reconstituted chromatin fibers, which differ in subtle but distinctive ways in their histone composition. We demonstrate an absolute requirement for the globular domain of the linker histones and a structural redundancy of the tails of linker histones and of histone H3 in determining conformational stability.     

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.     

Two DNA-binding sites on the globular domain of histone H5 are required for binding to both bulk and 5 S reconstituted nucleosomes

We have previously shown the existence of two DNA-binding sites on the globular domain of H5 (termed GH5), both of which are required for nucleosome organisation, as judged by the protection of a 166 bp chromatosome intermediate during micrococcal nuclease digestion of chromatin. This supports a model in which GH5 contacts two duplexes on the nucleosome. However, studies of a nucleosome assembled on the 5 S rRNA gene have argued against the requirement for two DNA-binding sites for chromatosome protection, which has implications for the role of linker histones. We have used this proposed difference in the requirement for a second site on the globular domain in the two models as a means of investigating whether bulk and reconstituted 5 S nucleosomes are indeed fundamentally different. GH5 protects a 166 bp chromatosome in both "bulk" and 5 S systems, and in both cases protection is abolished when all four basic residues in site II are replaced by alanine. Binding to four-way DNA junctions, which present a pair of juxtaposed duplexes, is also abolished. Single mutations of the basic residues did not abolish chromatosome protection in either system, or binding to four-way junctions, suggesting that the residues function as a cluster. Both bulk and 5 S nucleosomes thus require a functional second DNA-binding site on GH5 in order to bind properly to the nucleosome. This is likely to reflect a similar mode of binding in each case, in which two DNA duplexes are contacted in the nucleosome. There is no indication from these experiments that linker histones bind fundamentally differently to 5 S and bulk nucleosomes.     

Cooperative interaction of histone H1 with DNA.

The cooperative binding of histone H1 with DNA was studied using a fluorescently labelled histone H1. The titration data were analysed in terms of the large ligand model. The stoichiometric number, n = 65 +/- 10 bases/H1, was independent of NaCl concentration (0.02 - 0.35 M). The nucleation and the cooperative binding constants, K' and K, and the cooperativity parameter q were sensitive to salt concentration; K = 3.6 +/- 0.8 X 10(7) M-1 and q = 1.1 +/- 0.4 X 10(3) at 0.2 M NaCl. The dependence of K' on NaCl concentration revealed that 6 Na+ ions were released from DNA upon complex formation. An extrapolation of K' to 1M NaCl yielded a small value, K' = 5 +/- 2 M-1. Thus the binding of H1 is essentially electrostatic, being compatible with its independence of temperature. A calculation of K' based on the counterion release reproduced the salt concentration dependence of K'. Therefore, the binding of H1 is of an electrostatic territorial type. Thus, H1 may move along the DNA chain to a certain extent, when both salt concentration and the degree of saturation are sufficiently low. The condition is so restricted that the sliding would not play an important role in vivo. It was concluded from the DNA concentration independent binding isotherm that H1 can cooperatively bind onto a single DNA molecule. A simple power law dependence of the cooperativity parameter q upon NaCl concentration was found; q oc[NaCl]h with h = 0.72, though the physical basis of this dependence remains unknown.     

Binding of phosphorylated histone H1 to DNA.

A chromatin associated protein kinase was used to add 3 moles of phosphate to seryl side chains of 1 mole of histone H1. The DNA binding properties of this in vitro phosphorylated H1 were compared with those of unmodified H1. Considerably more radioactive superhelical DNA was retained on nitrocellulose filters at 20mM-40mM NaCl by phosphorylated H1 than by unmodified H1. However, zone velocity sedimentation analysis of histone-DNA complexes indicated that similar amounts of phosphorylated and unmodified H1 are bound to DNA. It is therefore concluded that phosphorylated H1 binds distributively to many or all DNA molecules available (depending on the histone/DNA ratio) while unmodified H1 binds cooperatively to a fraction of the DNA molecules in the reaction mixture.     

Effect of DNA length and H4 acetylation on the thermal stability of reconstituted nucleosome particles

To examine the factors involved with nucleosome stability, we reconstituted nonacetylated particles containing various lengths (192, 162, and 152 base pairs) of DNA onto the Lytechinus variegatus nucleosome positioning sequence in the absence of linker histone. We characterized the particles and examined their thermal stability. DNA of less than chromatosome length (168 base pairs) produces particles with altered denaturation profiles, possibly caused by histone rearrangement in those core-like particles. We also examined the effects of tetra-acetylation of histone H4 on the thermal stability of reconstituted nucleosome particles. Tetra-acetylation of H4 reduces the nucleosome thermal stability by 0.8 degrees C as compared with nonacetylated particles. This difference is close to values published comparing bulk nonacetylated nucleosomes and core particles to ones enriched for core histone acetylation, suggesting that H4 acetylation has a dominant effect on nucleosome particle energetics.