Radioiodination of chicken erythrocyte histones H4 and H5 in chromatin.

The conformational state of histones in isolated chicken erythrocyte chromatin was studied using procedures developed for probing surface proteins on membranes. Under controlled conditions, only exposed tyrosyl residues react with iodide radicals, generated either by the oxidant, chloramine-T (paratoluenesulfonyl chloramide), or the enzyme lactoperoxidase, giving monoidotyrosine. Using 125-iodine, this study compared the reactive tyrosines in free and bound histones H4, and H5. The relative extent of iodination of these histones within (H4) and outside (H5) of the nucleosomes was measured after extraction and gel electrophoresis. Each of the histones was further analyzed for the extent of specific tyrosine iodination by separating the tryptic peptides by high voltage electrophoresis. The identity of the labeled peptide was determined by dansylation of the amino acids present in each hydrolyzed peptide. The results show that there is a difference in the conformational arrangement of these histones on chromatin and in the free forms, since in chromatin not all tyrosine residues are as accessible for iodination as in the denatured state. Residue 53 of histone H5 for instance is more reactive than residues 28 and 58, indicating that the segments containing the latter residues are involved in either protein-DNA or protein-protein interactions. In histone H4, preferential labeling of 2 of the 4 tyrosines present was also observed.     

Linker histone H1 per se can induce three-dimensional folding of chromatin fiber

Higher-order architectures of chromosomes play important roles in the regulation of genome functions. To understand the molecular mechanism of genome packing, an in vitro chromatin reconstitution method and a single-molecule imaging technique (atomic force microscopy) were combined. In 50 mM NaCl, well-stretched beads-on-a-string chromatin fiber was observed. However, in 100 mM NaCl, salt-induced interaction between nucleosomes caused partial aggregation. Addition of histone H1 promoted a further folding of the fiber into thicker fibers 20-30 nm in width. Micrococcal nuclease digestion of these thicker fibers produced an approximately 170 bp fragment of nucleosomal DNA, which was approximately 20 bp longer than in the absence of histone H1 ( approximately 150 bp), indicating that H1 is correctly placed at the linker region. The width of the fiber depended on the ionic strength. Widths of 20 nm in 50 mM NaCl became 30 nm as the ionic strength was changed to 100 mM. On the basis of these results, a flexible model of chromatin fiber formation was proposed, where the mode of the fiber compaction changes depending both on salt environment and linker histone H1. The biological significance of this property of the chromatin architecture will be apparent in the closed segments ( approximately 100 kb) between SAR/MAR regions.     

Internal architecture of the core nucleosome: fluorescence energy transfer studies at methionine-84 of histone H4

Chicken histone H4, labeled separately at Met-84 with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid and 5-(iodoacetamido)fluorescein, was reassociated with unlabeled histones H2A, H2B, and H3 and 146 base pairs of DNA to produce fluorescently labeled nucleosomes having physical characteristics virtually the same as those of native core particles. Four types of particles were prepared containing respectively unlabeled H4, dansylated H4, fluoresceinated H4, and a mixture of the two labeled H4 molecules. Quantitative singlet-singlet energy-transfer measurements were carried out to determine changes in the distance between the two Met-84 H4 sites within the same nucleosome following conformational transitions which we have reported earlier. In the ionic strength range 0.1-100 mM NaCl, the distance between these sites is less than 2 nm except at 1 mM. Between 100 and 600 mM monovalent salt the distance separating the donor and acceptor fluors at Met-84 H4 increases to 3.8 nm. The conformational change centered around 200 mM NaCl is cooperative. Our results and those of others indicate that there is little unfolding of the histone octamer, at least around Met-84 H4, in the entire ionic strength range studied. A mechanism involving the rotation of the globular portion of H4 is proposed to account for this transition which occurs at physiological ionic strengths.     

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

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

Differences in the mode of iodination of H2a variants in chromatin

Modification of H2a variants with radioactive iodine was used to study under different ionic conditions the accessibility of their tyrosine residues in chromatin, in monosomes and when free in solution. The modification of tyrosine 57 in the hydrophobic part of H2a was found responsible for the appearance of new fractions with a reduced electrophoretic mobility in the presence of Trition X 100, detected only by autoradiography (radioactive "ghosts"). At low ionic strength a very small number of molecules were iodinated in chromatin, the modification affecting only their hydrophobic region. At moderate ionic strength the tyrosine residues near the N-terminal region of the molecule were predominantly modified. In chromatin the accessibility of the tyrosine residues of H2a1 was much greater than that of H2a2, a difference not observed with free histones.     

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.     

Preparation of membrane vesicles from isolated myelin: studies on functional and structural properties.

Myelin membranes purified from bovine brain are shown to form membrane vesicles when incubated in hypotonic buffer. Following restoration of isotonicity a resealing of the membrane occurs as judged by a significant decrease in 22Na+ permeability. Electron spin resonance measurements using stearic acid spin label I indicate a small decrease in membrane fluidity with increasing ionic strength between 50 and 80 mM NaCl. Iodination of myelin membrane vesicles by lactoperoxidase shows a four-fold increase in the amount of iodine incorporation into the myeline basic protein from 0--150 mM NaCl, while the iodination of the proteolipid protein remains essentially unaffected by the change in ionic strength. This dependence of the iodination of the myelin basic protein on the ionic strength can be explained by the electrostatic interactions of this protein with membrane lipids. In view of striking analogies with studies on model membranes correlating protein binding with membrane permeability changes, we suggest a similar structure-function relationship for the myelin basic protein.     

Role of individual histone tyrosines in the formation of the nucleosome complex.

We have determined the accessibility of histone tyrosine residues to react with p-nitrobenzenesulfonyl fluoride (NBSF) in intact nuclei, salt-dissociated nucleosomes, isolated histone complexes, and individual core histones. Of the 15 core histone tyrosine residues, 13 are inaccessible in native nucleosomes; only Tyr121 near the C-terminus of H2B is fully accessible, and Tyr54 of H3 is partially accessible under near-physiological conditions. When H1 and the basic N-terminal tails of the core histones are dissociated from the DNA by treating nuclei with 0.4 and 0.8 M NaCl, the two tyrosines which are adjacent to the basic regions of H2B and H3 become accessible as well. This indicates that these tyrosine residues may be involved in histone-DNA interactions, either directly or indirectly. When the H2A-H2B dimers are dissociated from the chromatin by raising the NaCl concentration to 1.2 M, three to four tyrosines located in the structured regions of H2B and H4 are exposed, suggesting that these tyrosine residues may be located at the dimer-tetramer interface. Dissociating all the histones from the DNA at an even higher ionic strength as a mixture of dimers, tetramers, and octamers does not change the pattern of Tyr exposure, but reduces the reactivity of the tyrosines at the dimer-tetramer interface as would be expected from the reassociation of H2A-H2B dimers and H3-H4 tetramers.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Preparation of membrane vesicles from isolated myelin. Studies on functional and structural properties

Myelin membranes purified from bovine brain are shown to form membrane vesicles when incubated in hypotonic buffer. Following restoration of isotonicity a resealing of the membrane occurs as judged by a significant decrease in ²²Na⁺ permeability. Electron spin resonance measurements using stearic acid spin label I indicate a small decrease in membrane fluidity with increasing ionic strength between 50 and 80 mM NaCl. Iodination of myelin membrane vesicles by lactoperoxidase shows a four-fold increase in the amount of iodine incorporation into the myelin basic protein from 0–150 mM NaCl, while the iodination of the proteolipid protein remains essentially unaffected by the change in ionic strength. This dependence of the iodination of the myelin basic protein on the ionic strength can be explained by the electrostatic interactions of this protein with membrane lipids. In view of striking analogies with studies on model membranes correlating protein binding with membrane permeability changes, we suggest a similar structure-function relationship for the myelin basic protein.     

Comparative study of nucleosome particles in chromatin from normal and tumor cells. II. Reconstitution, compaction and association induced by ionic strength of a solution

The method of circular dichroism (CD) has been used to investigate the reconstitution of mononucleosomes from C3HA mice liver and ascitic hepatoma 22A cells chromatin. It has been revealed that the more unfolding state of DNA in ascitic nucleosomes (discovered earlier) is determined by the peculiarities of the interactions between DNA and the dimers H2A-H2B, as well as by the linker histones of the H1 group. The investigation of the DNA folding in the oligonucleosome chains with increasing ionic strength has shown complete invariability of the DNA compactness in the ascitic chromatin up to 100 mM NaCl, while in liver nucleosomes an additional folding of the linker portion of the DNA was observed within the range of 20-40 mM NaCl. Oligonucleosomes from ascitic chromatin are less inclined to association upon increasing ionic strength, as compared with those from liver chromatin.     

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.     

Structure of histone octamers in reconstituted polynucleosomes

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

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.     

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.     

Chromatin structure. Further evidence against the existence of a beaded subunit for the 30-nm fiber

The size distribution of chromatin fragments released by micrococcal nuclease digestion of liver chromatin at various ionic strengths was examined. Below 20 mM ionic strength, gradient profiles with a peak centered at 6 nucleosomes are generated, whereas between 20 and 50 mM the peak is always centered on 12 nucleosomes, and above 50 mM ionic strength the 30-nm fiber becomes less accessible to the nuclease and there is a corresponding increase in the size distribution of fragments in the gradients. However, extensive digestions always give profiles with a peak of 12 nucleosomes as nuclease-resistant dodecamers accumulate. All of these observations are consistent with the winding of the 10-nm polynucleosome chain into a helical coil commencing at about 20 mM ionic strength. The helical turns are stabilized by histone H1 interactions between 20 and 50 mM ionic strength producing stable dodecamers. Above 50 mM ionic strength the coil condenses longitudinally and the profiles are consistent with a random attack of this fiber by the nuclease. Consequently it is not necessary to invoke the existence of a subunit bead to explain the profiles. We further define the conditions at which specific structural transitions take place and provide methodology for the preparation of chromatin at various levels of condensation.     

Nuclease digestion promotes structural rearrangements in H1-depleted chromatin.

Digestion of H1-depleted chromatin with micrococcal nuclease at an ionic strength of 0.35M gives rise to structural rearrangements indicating nucleosomal sliding. The ionic strength necessary to reveal this effect is significantly lower than that required in the absence of an accompanying digestion. As an explanation, a model is presented in which the progressing terminal degradation of oligomeric nucleosomes is made responsible for promoting structural rearrangements.     

Synchrotron X-ray scattering study of chromatin condensation induced by monovalent salt: analysis of the small-angle scattering data

Small-angle X-ray scattering experiments were carried out on rat thymus chromatin in "native" and "H1-depleted" states at various NaCl concentrations using synchrotron radiation. From the analysis of cross-sectional Guinier plots, the radius of gyration of the cross section (Rc) and the mass per unit length (Mc) of native chromatin were evaluated. In the absence of NaCl, the cross section of chromatin filament has a radius of gyration of 3.44 nm, suggesting the structure corresponding to the "10 nm" filament. With increasing NaCl concentration, the Rc value increases steeply to 6.74 nm at 5 mM NaCl and then gradually to 8.82 nm at 50 mM NaCl, whereas the Mc value, which is determined relative to that of tobacco mosaic virus (TMV), increases steadily from 1.58 nucleosomes per 10 nm in the absence of NaCl to 7.66 nucleosomes per 10 nm at 50 mM NaCl. However, since calibration with TMV tends to overestimate the Mc value, the actual Mc values may be less than those values. Above about 40 mM NaCl, aggregation of chromatin is suggested. Similar analysis of H1-depleted chromatin confirmed that H1-depleted chromatin takes a more disordered structure than native chromatin at low ionic strength and does not undergo a definite structure change upon further addition of NaCl.     

Influence of histone H1 on chromatin structure

Removal of histone H1 produces a transition in the structure of chromatin fibers as observed by electron microscopy. Chromatin containing all histone proteins appears as fibers with a diameter of about 250 A. The nucleosomes within these fibers are closely packed. If histone H1 is selectively removed with 50-100 mM NaCl in 50 mM sodium phosphate buffer (pH 7.0) in the presence of the ion-exchange resin AG 50 W - X2, chromatin appears as "beads-on-a-string" with the nucleosomes separated from each other by distances of about 150-200 A. If chromatin is treated in the presence of the resin with NaCl at concentrations of 650 mM or more, the structural organization of the chromatin is decreased, yielding fibers of irregular appearance.     

Physiologically relevant concentrations of NaCl and KCl increase DNA photocleavage by an N-substituted 9-aminomethylanthracene dye

This paper describes the synthesis of a new 9-aminomethylanthracene dye N-substituted with a pyridinylpolyamine side chain (4). The effects of NaCl and KCl on anthracene/DNA interactions were then studied, with the goal of simulating the conditions of high ionic strength that a DNA photosensitizer might encounter in the cell nucleus (~150 mM of NaCl and 260 mM of KCl). As exemplified by methylene blue (5), the expected effect of increasing ionic strength is to decrease DNA binding and photocleavage yields. In contrast, the addition of 150 mM of NaCl in combination with 260 mM of KCl to photocleavage reactions containing micromolar concentrations of 4 triggers the conversion of supercoiled, nicked, and linear forms of pUC19 plasmid into a highly degraded band of DNA fragments (350 nm hν, pH 7.0). Circular dichroism spectra point to a correlation between salt-induced unwinding of the DNA helix and the increase in DNA photocleavage yields. The results of circular dichroism, UV-vis absorption, fluorescence emission, thermal denaturation, and photocleavage inhibition experiments suggest that the combination of salts causes a change in the DNA binding mode of 4 from intercalation to an external interaction. This in turn leads to an increase in the anthracene-sensitized production of DNA-damaging reactive oxygen species.