Histone hyperacetylation can induce unfolding of the nucleosome core particle.

A direct correlation exists between the level of histone H4 hyperacetylation induced by sodium butyrate and the extent to which nucleosomes lose their compact shape and become elongated (62.0% of the particles have a length/width ratio over 1.6; overall mean in the length/width ratio = 1.83 +/- 0.48) when bound to electron microscope specimen grids at low ionic strength (1mM EDTA, 10mM Tris, pH 8.0). A marked proportion of elongated core particles is also observed in the naturally occurring hyperacetylated chicken testis chromatin undergoing spermatogenesis when analyzed at low ionic strength (36.8% of the particles have a length/width ratio over 1.6). Core particles of elongated shape (length/width ratio over 1.6) generated under low ionic strength conditions are absent in the hypoacetylated chicken erythrocyte chromatin and represent only 2.3% of the untreated Hela S3 cell core particles containing a low proportion of hyperacetylated histones. The marked differences between control and hyperacetylated core particles are absent if the particles are bound to the carbon support film in the presence of 0.2 M NaCl, 6mM MgCl2 and 10mM Tris pH 8.0, conditions known to stabilize nucleosomes. A survey of the published work on histone hyperacetylation together with the present results indicate that histone hyperacetylation does not produce any marked disruption of the core particle 'per se', but that it decreases intranucleosomal stabilizing forces as judged by the lowered stability of the hyperacetylated core particle under conditions of shearing stress such as cationic competition by the carbon support film of the EM grid for DNA binding.     

Stability of the primary organization of nucleosome core particles upon some conformational transitions.

The sequential arrangement of histones along DNA in nucleosome core particles was determined between 0.5 and 600 mM salt and from 0 to 8 M urea. These concentrations of salt and urea up to 6 M had no significant effect on the linear order of histones along DNA but 8 M urea caused the rearrangement of histones. Conformational changes in cores have been identified within these ranges of conditions by several laboratories 8-21. Also, abrupt structural changes in the cores, apparently their unfolding, were found by gel electrophoresis to occur at urea concentration, between 4 and 5 M. 600 mM salt and 6 M urea were shown to relax the binding of histones to DNA in cores but do not however release histones or some part of their molecules from DNA. It appears therefore that nucleosomal cores can undergo some conformational transitions and unfolding whereas their primary organization remains essentially unaffected. These results are consistent with a model of the core particles in which the histone octamer forms something like a helical "rim" along the superhelical DNA and histone-histone interactions beyond the "rim" are rather weak in comparison with those within the "rim".     

The roles of H1, the histone core and DNA length in the unfolding of nucleosomes at low ionic strength.

Calf thymus nucleosomes exhibit two different and independent hydrodynamic responses to diminishing salt concentration. One change is gradual over the range 40 to 0.2 mM Na+ and is accompanied by decreases in contact-site cross-linking efficiency. The other change is abrupt, being centered between 1 and 2 mM Na+. We found only one abrupt change in sedimentation rate for particles ranging in DNA content fom 144 to 230 base pairs. This response to decreasing ionic strength is similar for particles of both 169 and 230 base pairs. Core particles (144 base pairs) exhibit a somewhat diminished response. The abrupt change is blocked by formaldehyde or dimethylsuberimidate cross-linking. The blockage by dimethylsuberimidate demonstrates that the abrupt conformational change requires the participation of the core histones. H1 completely blocks the abrupt but not the gradual conformational change. Thus H1 uncouples the different responses to low ionic strength and exerts an important constraint on the conformational states available to the nucleosome core.     

Aggregation equilibria of Escherichia coli RNA polymerase: evidence for anion-linked conformational transitions in the protomers of core and holoenzyme

The aggregation equilibria of Escherichia coli RNA polymerase core and holoenzyme have been studied by velocity sedimentation as a function of [NaCl] both in the presence and in the absence of MgCl2. Effects of other anions (F- and I-), pH, and temperature have also been examined. Diffusion coefficients obtained by quasi-elastic light scattering (QLS) at high and low salt concentrations were used in conjunction with sedimentation coefficients under these conditions to obtain molecular weights of the protomer and aggregates of the core enzyme. At low salt concentration, core aggregates to a tetramer in the absence of MgCl2 and to an octamer in the presence of MgCl2. Some ambiguity exists in the interpretation of the sedimentation and QLS data for holoenzyme. The sedimentation results are consistent with the formation of dimers at low salt, both in the presence and in the absence of MgCl2. In all cases, equilibrium constants were calculated assuming a simple monomer--j-mer stoichiometry. These equilibrium constants are extremely sensitive functions of the concentration and type of monovalent anion. In Cl-, aggregation of both core and holoenzyme begins abruptly when the salt concentration is reduced below approximately 0.2 M (at a protein concentration of approximately 0.30 mg/mL); for core, substitution of I- for Cl- suppresses aggregation while F- enhances aggregation at a fixed anion concentration. No specific effect of monovalent cations (Na+, NH4+) is observed; Mg2+ has no effect on holoenzyme dimerization and has little effect on the salt range of core aggregation, though the stoichiometries of the core aggregates in the presence and absence of Mg2+ differ. Anion effects on these equilibria were modeled by assuming that a class of anion-binding sites on the protomer is not present in the aggregate, so that anion release accompanies aggregation. Analytical expressions for several models of the effect of anions on the aggregation equilibria were derived by using the method of binding polynomials. The salt dependence of the aggregation equilibria in the absence of Mg2+ appears inconsistent with a model in which the anion-binding sites on the protomer are independent (noncooperative), but it is well described by a model in which anion binding to the protomers occurs in a completely cooperative manner. The molecular basis of this apparent cooperative effect of anions on the aggregation equilibria is proposed to be an allosteric effect of anions on conformational equilibria of the protomers of core polymerase and the holoenzyme. Implications of such a salt-dependent conformational transition for the DNA-binding interactions of the enzyme are considered.     

Reversibility of the low-salt transition of chromatin core particles.

The low-salt transition of chromatin core particles is reversible if the monovalent cation concentration is kept above 0.2 mM. Exposure of the particles to salt concentrations below this value results in a nonreversible secondary transition. The nonreversible changes are relatively slow with a half-time of about 15 minutes. Once exposed to such low ionic strength, the particles then begin to refold with increasing salt in at least two steps over a much higher ionic strength range than is required for the usual low-salt transition. The refolding is very fast, with a half-time less than a minute. Small differences between particles which had or had not been exposed to very low salt persist even when the particles are returned to near physiological ionic strengths.     

Analysis of chromatin reconstitutiion.

The ability of high molecular weight chicken erythrocyte chromatin to spontaneously self-assemble into native-like material, after dissociation by high ionic strength and reassociation by salt gradient dialysis, was critically examined. The native conformational state of the reassembled nucleoprotein complex was regenerated to the extent reflected by circular dichroism spectra and thermally induced helix--coil transition of the nucleoprotein DNA. However, internucleosomal packing of approximately 205 base pairs of DNA per repeating unit, as probed by digestion with micrococcal nuclease, was not regenerated upon reassembly and was replaced by a packing of approximately 160 base pairs per repeating unit. Thus, high molecular weight chromatin containing only lysine-rich histones (H1 and H5) and core histones (H2A, H2B, H3, and H4) is not a true self-assembling system in vitro using the salt gradient dialysis system used herein. Circular dichroism and thermal denaturation studies on core chromatin (lysine-rich histones removed) showed that core histones alone are not capable of reassembling high molecular weight DNA into native-like core particles at low temperature (4 degree C). Reassembly at 21 degree C restored the circular dichroism but not the thermal denaturation properties to those characteristic of undissociated core chromatin. Nonetheless, micrococcal nuclease digestions of both reassembled core chromatin products were identical with undissociated native core chromatin. Ressembly in the presence of the complete complement of histones, followed by removal of the lysine-rich histones, did regenerate the thermal denaturation properties of undissociated native core particles. These results indicated multiple functions of the lysine-rich histones in the in vitro assembly of high molecular weight chromatin.     

Immunochemical detection of changes in chromatin subunits induced by histone H4 acetylation.

Native, reassociated, and reconstituted core particles from chicken erythrocytes were compared by both biophysical and immunochemical methods. No significant difference between the three types of core particles could be demonstrated by electron microscopy, circular dichroism, or immunochemical analysis with antisera to histone H2B, H2A, and H3. Core particles were also reconstituted with calf thymus non-acetylated H3, H2A, and H2B with either mono-, di-, or tri-acetylated H4 isolated from cuttle -fish testes. The hyperacetylation of H4 did not significantly alter the biophysical characteristics of core particles but it induced several changes in their immunochemical reactivity. Binding to core particles of antibodies specific for H2A, H3, and for the IRGERA (synthetic C-terminal) peptide of H3 was considerably decreased when di- or tri-acetylated H4 was used for reconstitution, whereas binding of H2B antibodies remained the same. Our results suggest that the presence of hyperacetylated H4 within core particles leads to conformational changes that alter the antigenic determinants of several of the histones present at the surface of chromatin subunits. Since histone acetylation is correlated with the open structure of active chromatin, it may become possible to monitor the activity of chromatin by immunochemical methods.     

Changes in the structural organization of chromatin during spermatogenesis in the rat

In this study, histone H4 was shown to be extensively hyperacetylated in mid-spermatids of the rat during the time period when the entire complement of histones is replaced by basic spermatidal transition proteins. The degree of hyperacetylation of histone H4 was minimal in pachytene spermatocytes. Therefore, the hyperacetylation appears to be directly involved in the histone replacement process late in spermatogenesis in mid-spermatids. In order to investigate further the possible effects of histone H4 hyperacetylation and the other dramatic changes in the nuclear proteins on the structure of chromatin in germinal cells, we examined the thermal denaturation profiles of chromatin from various purified germinal cell types. Our analyses revealed that chromatins from pachytene spermatocytes and early spermatids have similar thermal denaturation profiles, with their major thermal transitions slightly lower than those for rat liver. However, the major thermal transitions for chromatin from mid-spermatids are much lower than those from pachytene spermatocytes and early-spermatids. We propose that the greatly lowered thermal stability of mid-spermatid chromatin represents a dramatic relaxation or decondensation of the chromatin in this cell type in preparation for the replacement of histone by the basic spermatidal transition proteins and that the decondensation is due in large part to the extensive histones hyperacetylation which occurs in these cells.     

Effects of Net and Local Charges on the Interaction between Chemically-Modified Horse Heart Cytochrome c and P700 in Photosystem 1-Enriched Subchloroplast Particles

Effects of salt and pH on the re-reduction of P700 by chemically-modifiedhorse heart cytochrome c after a flash illumination were examinedin Triton-treated P700- enriched subchloroplast particles (TSF-1particles). At low salt concentrations net charges on the membrane surfaceand native, guanidinated or succinylated cytochrome c were majorfactors that determined the reaction rates, as in the reactionbetween plastocyanin and P700 [Tamura et al. (1981) Plant &Cell Physiol. 22: 603]. The reaction rates also depended onreactant-specific factors, particularly the localized distributionof charges on macromolecules and their interaction over shortdistances, as well as on long-range Coulombic interaction. Theeffect of this type became clearer at high salt concentrations. (Received October 7, 1982; Accepted December 20, 1982)     

lac Operator nucleosomes. 2. lac Nucleosomes can change conformation to strengthen binding by lac repressor

We have shown previously that lac repressor binds specifically and quantitatively to lac operator restriction fragments which have been complexed with histones to form artificial nucleosomes (203 base pair restriction fragment) or core particles (144 base pair restriction fragment. We describe here a quantitative method for determining the equilibrium binding affinities of repressor for these lac reconstitutes. Quantitative analysis shows that the operator-histone reconstitutes may be grouped into two affinity classes: those with an affinity for repressor close to that of naked DNA and those with an affinity 2 or more orders of magnitude less than that of naked DNA. All particles in the lac nucleosome preparations bind repressor with high affinity, but the lac core particle preparations contain particles of both high and low affinities for repressor. Formaldehyde cross-linking causes all high-affinity species to suffer a 100-fold decrease in binding affinity. In contrast, there is no effect of cross-linking on species of low affinity. Therefore, the ability of a particle to be bound tightly by repressor depends on a property of the particle which is eliminated by cross-linking. Control experiments have shown that chemical damage to the operator does not accompany cross-linking. Therefore, the property sensitive to cross-linking must be the ability of the particle to change conformation. We infer that the particles of low native affinity, like cross-linked particles, are of low affinity because of an inability to facilitate repressor binding by means of this conformational change. Dimethyl suberimidate cross-linking experiments show that histone-histone cross-linking is sufficient to preclude high-affinity binding. Thus, the necessary conformational change involves a nucleosome histone core event. We find that the ability of a particle to undergo a repressor-induced facilitating conformational change appears to depend on the position of the operator along the DNA binding path of the nucleosome core. We present a general model which proposes that nucleosomes are divided into domains which function differentially to initiate conformational changes in response to physiological stimuli.     

Ultrastructural changes in chloroplasts resulting from fluctuations in NaCl concentration: freeze-fracture of thylakoid membranes in Dunaliella salina.

The structure of chloroplasts isolated from Dunaliella salina has been studied with respect to changing concentrations of sodium chloride in the culture medium. Freeze-fracture replicas and thin sections of intact chloroplasts do not exhibit any noticeable changes in structure at concentrations ranging between 3.5 and 25% NaCl. Chloroplasts isolated from algal cells that have been acclimatized to the higher salt concentration show a change in the thylakoid membranes. The thylakoid membranes appear compressed over a major portion of the membrane surface, with only the end of the thylakoid membranes unappressed. The number of particles per unit area on the B face is also altered by the salt concentration. The chloroplasts acclimatized to 25% NaCl have about 3 times the number of particles per unit area on a B face of end-membranes as on a comparable face of thylakoid membranes acclimatized to low (3.5% NaCl) salt concentration. These morphological changes can be reversed if the chloroplasts acclimatized to high or low salt concentrations are returned to a medium of different salt concentration prior to freeze-fracturing.     

Characterization of the structure and melting of DNAs containing backbone nicks and gaps

A DNA molecule containing a gap (a missing phosphate) has been examined and compared to two other molecules of the same sequence, one containing a nick (a phosphorylated gap) and the other a normal duplex containing no break in the backbone. A second gapped sequence was also compared to a normal duplex of the same sequence. The molecules containing nicks or gaps were generated as dumbbell molecules, short helices closed by a loop at each end. The dumbbells were formed by the association of two hairpins with self-complementary dangling 5'-ends. Nuclear magnetic resonance was used to monitor the melting transition and to probe structural differences between molecules. Under the conditions used here no change in stability was observed upon phosphorylation of the gap. Structural changes upon phosphorylation of a gap or closure of a nick were minimal and were localized to the region immediately around the gap or nick. Two transitions can be observed as a gapped or nicked molecule melts, although the resolution of the two transitions varies with the salt concentration. At moderate to high salt (greater than or equal to 30 mM) the molecule melts essentially all at once. At low salt the two transitions occur at temperatures that differ by as much as 15 degrees C. In addition, comparison with other NMR melting studies indicates that the duplex formed by the overlap of the dangling ends of the hairpins is stabilized relative to a free duplex of the same sequence, probably by stacking onto the hairpin stem.     

Physical properties of nucleoprotein cores from adenovirus type 5.

Analytical ultracentrifugation, thermal denaturation, and electron microscopy have been used to study nucleoprotein core particles, obtained from disrupted type 5 adenovirus and partially purified on glycerol density gradients. Electron microscopy at low salt concentrations has shown that the cores are homogeneous particles with characteristic structures, which vary with conditions of observation from a fairly loose network of fibers to a highly condensed, compact particle. Sedimentation measurements in the analytical ultracentrifuge, both by boundary and by band techniques, show that the cores are relatively homogeneous in solution and have sedimentation coefficients near 185 S at low salt concentrations, about 243 S in 1 or 2 M NaCl, and 376 S in 1 mM MgCl2. Correlation of sedimentation data with electron microscopic observations suggests that the 185 S particle has a loose, fibrous structure, while the faster species are more highly condensed particles. The melting temperature of the cores in 5 mM Tris/HCl is 79 degrees C, which is 10 degrees C higher than the Tm for purified, viral DNA. This indicates that the protein enhances the stability of DNA in the nucleoprotein complex.     

Factors affecting nucleosome disassembly by protamines in vitro. Histone hyperacetylation and chromatin structure, time dependence, and the size of the sperm nuclear proteins

Histone displaced in vitro from nuclei by protamine competition display a higher degree of hyperacetylation than the residual histones. In addition, hyperacetylated core particle pools are disassembled in vitro with a higher efficiency than control or nonacetylated core particles and when analyzed by electron microscopy display an elongated shape (length/width ratio = 1.52 +/- 0.19) instead of the round compact shape of control nucleosomes (length/width ratio = 1.06 +/- 0.06). In the absence of histone hyperacetylation, the fish protamines, salmine and iridine (32-33 residues), are relatively inefficient in disassembling nucleosomal core particles in vitro as compared to the large (65-70 residues), tyrosine-containing protamines from rooster (galline), squid, and cuttlefish which disassemble nucleosomes in a range of protamine concentrations close to physiological. The fact that an artificially cross-linked salmine dimer acquires the ability of the large protamines from rooster, squid, and cuttlefish to disassemble core particles in vitro and also binds more tightly to the DNA, suggests that the size of the sperm nuclear protamines is a critical factor in this process. Even when the core histones of spermatid chromatin are hyperacetylated in the trout testis, the replacement process by iridine or salmine is slow and time-dependent in vitro. However, since spermiogenesis in trout occurs over several weeks, the slow in vitro nucleosome disassembly process by salmine is sufficient to allow complete displacement, thus supporting the hypothesis that a protamine-mediated displacement of the histones from DNA in vivo may take place in the salmonid fishes by a mechanism similar to that in the rooster, squid, and cuttlefish.     

The lipid core model of lipoproteins

Several aspects of the lipid core model for lipoproteins were examined in a quantitative manner. Detailed consideration was given to a geometric factor relevant to the packing of lipids on curved surfaces, which allowed computation of the maximum amount of lipid which can be accommodated about a sphere of given size. The model was found to be consistent with presently available data for size and shape of very low density lipoproteins of serum and egg yolk low density lipoproteins. In addition, the model led to hypotheses regarding the organization and spatial arrangement of lipids within the core, the factors controlling these arrangements, and the way the lipids may interact with protein and water. The results were consistent also with dynamic equilibria where triglyceride moves from a neutral lipid nucleus to a phospholipid-cholesterol interfacial region, with concomitant changes in the structural arrangement of the interfacial lipids. Also consistent was the possibility of structural transitions or differences in packing behavior of interfacial lipids at fixed interfacial triglyceride concentrations.

Important parameters for characterizing the structure of lipid core particles where shown to be the dimensional parameters of lipids at the core water interface (effective length and surface area) and the composition of the interfacial region, in addition to size, mass and total lipid composition. Three extreme possibilities for the location of the protein moiety were consistent, hence also the various composite or intermediate possibilities. These locations consisted of (1) the protein completely interdigitated between the interfacial lipids, (2) the protein occupying a monolayer completely covering the core and (3) the protein located as islands in a sea of interfacial lipids.     

Effects of pH on the stability of chromatin core particles.

Chromatin core particles near physiological ionic strength undergo a reversible transition induced by changes in pH near neutrality. While sedimentation studies indicate no significant effect on size or shape, changes in tyrosine fluorescence anisotropy and in circular dichroism suggest a somewhat looser structure at high pH. Further support of this suggestion is given by high salt dissociation experiments; at pH 8 core particles begin to show changes at lower salt concentration than at pH 6. The pH transition appears unaffected by the presence of Mg2+ but can be blocked by crosslinking of the histones. A possible relationship is suggested between this transition and increases in intracellular pH which correlate with enhancement in several aspects of cellular activity including DNA replication.