Protein dissociation from DNA in model systems and chromatin.

Salt induced dissociation of protamine, poly(L-lysine) and poly(L-arginine) from DNA was measured by relative light scattering at theta = 90 degrees and/or centrifugation. Dissociation of histones from DNA was studied using relative light scattering and intrinsic tyrosine fluorescence. Protamine was dissociated from DNA at 0.15 M MgCl2 (ionic strength mu = 0.45) or 0.53 M NaCl (mu = 0.53) based on light scattering data and at approximately 0.2 M MgCl2 (mu = 0.6) or 0.6 M NaCl based on centrifugation data. NaCl induced dissociation of poly(Lys) or poly(Arg) from natural DNAs measured by light scattering did not depend on the guanine plus cytosine content. To dissociate poly(Arg) from DNA higher ionic strength using NaCl, MgCl2, or CaCl2, similar ionic strength using NaClo4, and lower ionic strength using Na2SO4 was needed then to dissociated poly(Lys). Both the decrease in light scattering and the enhancement of tyrosine fluorescence of chromatin occurred between 0.5 and 1.5 M NaCl when histones were dissociated.     

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.     

Stability of nucleosomes in native and reconstituted chromatins.

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

Nucleosome dissociation and transfer in concentrated salt solutions.

We have examined the dissociation of nucleosomes into histones and free, 4.5S DNA over a range of sodium chloride concentrations between 0.25 and 1 M. We have also studied this dissociation as a function of nucleosome concentration at two salt concentrations, 0.8 M and 0.9 M. In addition, we have measured the kinetics of transfer of histone cores from nucleosomes onto recipient bacteriophage T7 DNA in 0.6, 0.7 and 0.8 M NaCl solutions. Although the mechanism of nucleosome transfer is unknown the data presented here are consistent with either a reversible dissociation of the nucleosome or DNA strand displacement by another DNA.     

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.     

Fluorescence spectra of Hoechst 33258 bound to chromatin

Fluorescence spectra of Hoechst 33258 bound to rat thymocytes were measured by flow cytometry. At low dye concentrations (less than or equal to 2 micrograms/ml) the fluorescence maximum was situated at 460 nm irrespective of solvent composition. With higher dye concentrations the fluorescence maximum was shifted upwards, the intensity decreased and the width of the fluorescence peak increased. Linear combinations of a spectrum obtained at a low dye concentration (0.5 microgram/ml, type 1 binding) and one obtained at a high dye concentration (42.4 micrograms/ml, type 2 binding) failed to reproduce spectra measured at intermediate dye concentrations (0.15 M NaCl). Hence, Hoechst 33258 forms at least three different fluorescing complexes with DNA in chromatin. The shift in the fluorescence maximum of the Hoechst 33258/chromatin complex towards higher wavelengths decreased with ionic strength. 25% ethanol in the 0.15 M NaCl staining buffer reduced the wavelength shift at high dye concentrations, indicating that the strength of type 2 binding depends on DNA conformation in addition to ionic strength. The fluorescence spectrum was independent of whether DNA in chromatin was complexed with histones or not. However, histone-depleted thymocytes fluoresced more intensely than cells in which DNA was complexed with histones, the difference being greater at low concentrations of Hoechst 33258. Hence, type 2 binding to DNA in chromatin appears to be less restricted by histones than type 1 binding.     

Salt-induced structural changes in nucleosomes

Ehrlich ascites tumor (EAT) nucleosomes treated with increasing NaCl concentrations were analyzed by sucrose density gradient centrifugation. Two events were found to take place in the course of the salt treatment: a) increasing amounts of nucleosomes dissociated into free DNA and protein in the interval 0.6M–1.5M NaCl, and b) the sedimentation coefficient of the nucleosomes decreased from 11S to 8S in the interval 0.6M-1M NaCl. This decrease was not caused by loss of protein and was fully reversible upon slow and gradual lowering of the ionic strength. This shows that before dissociation of the protein core from DNA, nucleosomes undergo a structural transition. The electron microscopic observations revealed that it consisted in detachment of the ends of nucleosomal DNA from the protein core. It is suggested that an arginine-rich domain in the protein core exists, which holds more tightly the central part of the nucleosomal DNA, while its ends are relatively loosely bound to lysine-rich domains.     

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.     

Some properties of tobacco protoplast chromatin.

Chromatin was prepared from tobacco-leaf protoplasts. Its solubility in increasing molarities of NaCl was studied and the structure of the soluble fraction observed by electron microscopy. We demonstrate that in plants, the DNA and histones are associated in beaded structures similar to those called omicron-bodies or nucleosomes in animal chromatin. The nucleosomes were associated with DNA in either compact or extended forms. The compact arrangement was predominant in the fraction solubilized between 0.1 and 0.4 M NaCl. The extended form, present at 0.5 and 0.6 M NaCl. showed DNA filaments of various lengths interspacing the nucleosomes. At these ionic strengths ring structures were present, associated with the DNA. At 0.7 M NaCl and above, only DNA filaments were present, occasionally associated with big rings, and nucleosomes were compoetely dissociated. Free DNA molecules were present at all ionic strengths used. The possible origin and significance of the rings are discussed.     

The higher order repeat structure of chromatin is built up of globular particles containing eight nucleosomes

Mild nuclease digestion of rat liver chromatin generates particles with sedimentation coefficients of about 33S, 60S, and 90S (in 50 mM NaCl). The kinetics of appearance and disappearance of these particles with progressive digestion suggest that they are produced by cleavage from a higher order repeat structure, the 33S particle representing the monomer. At an intermediate stage of digestion, about 75 % of the nuclear chromatin can be recovered as monomers to trimers of this higher order structure. Sedimentation profiles indicate that monomer particles containing 7–8 nucleosomes occur at the highest frequency. The DNA fragments in monomers have a size corresponding to hepta- and octanucleosomes, and those in dimers have a size corresponding to chains of sixteen nucleosomes. The higher order repeat structure is only stable between 30 and 200 mM NaCl; the particles unfold below 30 and above 200 mM NaCl. When examined by electron microscopy, monomers and dimers appear as compact globular structures. Relaxation by lowering the salt concentration results in the appearance of polynucleosomes with a chain length of eight beads in the monomer and sixteen in the dimer particle. These results indicate that the unit particle of the higher order repeat structure of rat liver chromatin contains eight nucleosomes.     

Distribution of H1 histone in chromatin digested by micrococcal nuclease.

The relative amount of H1 histone associated with isolated nucleosomes from calf thymus was determined as a function of the extent of DNA digestion by micrococcal nuclease. Generally the amount of H1 histone associated with mononucleosomes decreases with increasing digestion until 60% of the original H1 remains associated with DNA 150 base pirs or less in size. Coincidentally, H1 histone increases relative to the other histones in aggregated material that sediments through sucrose gradients to form a pellet. However, the level of H1 histone remains at control values for oligonucleosomes (dimer to hexamer) over the 30% digestion range studied. An increase in ionic strength to 0.3 M NaCl in the density gradient reveals a different pattern of H1 binding, whereby the amount of H1 reflects the average size of the DNA fragments with which it is associated. Although there is significant binding to nucleosomes per se, it appears that the major ionic involvement of H1 is with internucleosomal spacer DNA.     

Psoralen-crosslinking of soluble and of H1-depleted soluble rat liver chromatin

We purified soluble rat liver chromatin and H1-depleted chromatin and photocrosslinked its DNA with psoralen at pH 7. Digestion of this chromatin with micrococcal nuclease produced a normal nucleosomal repeat. Chromatin was photoreacted in the presence of 0 to 700 mM-NaCl and was fractionated in sucrose gradients containing the same NaCl concentrations. The dissociation of H1 occurred as in the non-crosslinked controls and no preferential dissociation of core histones was observed. The samples between 100 and 500 mM-NaCl showed precipitation. In the electron microscope, the fibers appeared indistinguishable from the controls at low ionic strength. In the presence of 40 mM-NaCl, the fibers of the photoreacted chromatin were slightly more compact than the controls, and at 500 mM-NaCl, despite the complete dissociation of H1, there were still apparently intact fibers at this ionic strength. The disruption of the psoralen-treated chromatin fibers occurred only in 600 mM-NaCl, as opposed to 500 mM-NaCl in controls. The DNA of all the photoreacted samples was spread for electron microscopy under denaturing conditions. They revealed, for all the samples, single-stranded bubbles corresponding to 200 to 400 base-pairs in size. H1-depleted chromatin containing stoichiometric amounts of core histones was photoreacted at pH 10 and very low ionic strength. Under these conditions many of the nucleosomes appeared to be unraveled, although to a variable extent. In the electron microscope, the purified DNA from these samples showed extensive crosslinking when spread under denaturing conditions. These observations show that histone-DNA interactions different from those in intact nucleosomes may be created, which allow extensive access of psoralen to the DNA.     

Thermal stability of nucleosomes studied in situ by flow cytometry: effect of ionic strength and n-butyrate

Heating of cells permeabilized with ethanol and resuspended in aqueous media increases accessibility of DNA to intercalating dyes such as acridine orange (AO). The curves, representing increase in binding of AO as a function of rise in temperature, indicate that the transitions are cooperative. The transitions are sensitive to ionic strength and occur at lower temperatures when cells are suspended in media of increasing ionic strength. Extraction of histones raises accessibility of DNA to intercalators at room temperature, and heating has little effect on additional binding. The results are interpreted as indicating thermal destruction of nucleosomal structure in nuclear chromatin; dissociation of DNA from core histones results in its increasing ability to intercalate AO, most likely due to increased topological freedom to undergo unwinding and elongation following binding of the intercalator. Preincubation of cells with n-butyrate, known to induce histone hyperacetylation, lowers the heat stability of nucleosomes by about 5 degrees C. On the other hand, no differences are observed between chromatin of mitotic vs interphase cells tested over a wide range of ionic strengths (0.1-0.7 N NaCl). The method appears to be useful as a probe of chromatin structure at the nucleosomal level.     

Spectroscopic studies on histone-DNA interactions. II. Three transitions in nucleosomes resolved by salt-titration

The multiple-step transitions in DNA-histone interactions in chicken erythrocyte nucleosomes with increasing ionic strength are resolved by salt-titration spectroscopy. Both the circular dichroism of the DNA and the fluorescence of the histones in nucleosomes change during the titration process with concentrations of NaCl from 0.1 M to 2.5 M. By differentiating the titration curves, three distinct peaks corresponding to three structural transitions are observed. The two peaks near 0.95 M and 1.45 M-NaCl are common to the circular dichroism and fluorescence curves. The circular dichroism curve has another peak near 0.55 M-NaCl. Because the derivative of the fluorescence titration curve for the DNA-(H3, H4) complex has only one peak near 1.45 M-NaCl, that peak is attributed to the dissociation of the histone dimer (H3, H4). The peak near 0.95 M-NaCl corresponds to the dissociation of the dimer (H2A, H2B) from the DNA-(H3, H4) complex, as shown by binding experiments of (H2A, H2B) to the DNA-(H3, H4) complex at the salt concentration near this peak. The peak near 0.55 M-NaCl reflects some inner-core structural change. As the change of the circular dichroism signal is reversible, salt-titration spectroscopy is applicable to equilibrium studies of the physical chemical properties of DNA-histone interactions. By the assumption of a non-co-operative model, the binding constant for the chicken erythrocyte (H2A, H2B) dimer to the DNA-(H3, H4) complex is calculated as 2.8 X 10(6) M-1 at 1.0 M-NaCl (20 degrees C, pH 7.6). The DNA sequence dependence of the stability of the DNA-(H3, H4) interaction is observed in the salt-titration profiles of reconstituted material. Decreasing stability of the interaction of (H3, H4) is observed following the order: poly[(dG)-(dC)] much greater than chicken erythrocyte DNA greater than poly[(dA)-(dT)]. It is concluded that histones (H3, H4) have a different DNA sequence dependence from histones (H2A, H2B).     

The distribution of nuclear proteins and transcriptionally-active sequences in rat liver chromatin fractions

Chromatin fractions from rat liver nuclei digested by nucleases were separated by differential solubility into several fractions. Material solubilized during digestion (predominantly monomer nucleosomes and polynucleosomes) had the highest HMG14 + 17/DNA ratios but were not enriched in active gene sequences (albumin and c-Ha-ras1 genes). Material soluble in a low ionic strength buffer containing 0.2 mM MgCl2 (monomer nucleosomes and polynucleosomes) contained in addition to the histones, HMG14 and 17 plus a 41K non-histone protein. This fraction was depleted in active gene sequences and enriched in inactive sequences. The insoluble material was highly enriched in active sequences and had the lowest HMG14 + 17/DNA ratio. This fraction could be further fractionated into a histone-containing 2 M NaCl-soluble fraction and a 2 M NaCl-insoluble matrix-bound fraction, both of which were enriched in active sequences. The results show that the HMG proteins do not partition with active sequences during fractionation of chromatin. The 41K protein may be associated with inactive chromatin fraction.     

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.     

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.     

Dissociation of single-stranded DNA from nucleosomes following modification with acetic anhydride

Modification with acetic anhydride of nucleosomes from chicken erythrocytes at low ionic strength (less than 0.1 M NaCl) is accompanied by the formation of residual particles and the release of free DNA. This DNA has been identified as single-stranded by thermal denaturation, digestion with nuclease S1, and elution from hydroxyapatite. In contrast, if modification takes place at 0.6 M NaCl, the liberated DNA is mainly double-stranded. The release of the free energy stored in folded nucleosomal DNA, triggered by the weakening of lysine-DNA interactions which takes place upon modification, might be responsible for the observed denaturation of DNA at low ionic strength.     

Nucleosome structure and conformational changes.

We have used a variety of chemical probes to measure the accessibility of DNA on the surface of the nucleosome. We review these results, and describe new experiments which show that T4 phage DNA can form complexes with the core histones, possessing the properties of normal nucleosomes. Since T4 DNA is largely occupied by glucose residues in the major groove, this suggests (as did earlier probe experiments) that the major groove is not filled with histone amino acid side chains. We also report results of recent measurements which appear to show that only a few strong charge interactions are involved in the attachment of the terminal 20 nucleotide pairs at each end of nucleosome core DNA. We speculate on the possible functional significance of the accessibility of DNA revealed by all of these experiments. We have also examined conformational changes induced in nucleosomes at high ionic strength (0.5-0.7M NaCl). The frictional coefficient is found to undergo a small increase in this region, not consistent with models in which the nucleosome is completely unfolded, but possibly reflecting the dissociation of terminal DNA from the nucleosome surface.