The superstructure of chromatin and its condensation mechanism

Solutions of rat liver and chicken erythrocyte chromatin at different ionic strngths were characterized by synchrotron X-ray solution scattering, ultracentrifugation, density and viscosity measurements. Previous observations on nuclei were extended to rat liver, calf thymus and yeast nuclei.It is shown that with monovalent cations condensation is independent of the nature of the cation whereas with divalent cations there are significant differences related to the preference of base binding over phosphate binding. The consistency of hydrodynamic and scattering results confirm the view that chromatin in solution at low ionic strength has a helix-like superstructure. A survey of X-ray and neutron scattering results in the literature shows that previous interpretations, e.g. in terms of a 10 nm filament, are incompatible with the experimental data at low resolution.     

The superstructure of chromatin and its condensation mechanism

Synchroton radiation X-ray scattering experiments have been performed on chicken erythrocyte chromatin fibres over a wide range of ionic conditions and on various states of the fibres (i.e. "native" in solution, in gels and in whole nuclei; chromatin depleted of the H1 (H5) histones and chromatin with bound ethidium bromide). A correlation between the results obtained with the various chromatin preparations provides evidence for a model according to which at low ionic strength the chromatin fibre already possesses a helical superstructure, with a diameter comparable to that of condensed chromatin, held together by the H1(H5) histone. The most significant structural modification undergone upon an increase of the ionic strength is a reduction of the helix pitch, this leads to condensation in a manner similar to the folding of an accordion. The details of this process depend on whether monovalent or divalent cations are used to raise the ionic strength, the latter producing a much higher degree of condensation. Measurements of the relative increase of the mass per unit length indicate that the most condensed state is a helical structure with a pitch around 3.0-4.0 nm. In this paper we give a detailed presentation of the experimental evidence obtained from static and time-resolved scattering experiments, which led to this model.     

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.     

Internucleosome interaction: detection of dinucleosome fragmentation of chromatin by micrococcal nuclease. Analysis of the products of cleavage of chromatin from rat liver nuclei and L cells by micrococcal nuclease

In murine L-cell nuclei micrococcal nuclease causes chromatin fragmentation with predominant liberation of dinucleosomes. Analysis of dynamics of rat liver nuclear chromatin cleavage by micrococcal nuclease revealed that the "dinucleosomal" mode of fragmentation is due to the pretreatment of nuclei with the non-ionic detergent Triton X-100 in the course of the isolation procedure. The set of particles detected in nuclease hydrolysates of nuclear chromatin pretreated with Triton X-100 and those isolated by the standard procedure was shown to be significantly different. In Triton X-100 treated nuclei the dichromatosome is the main hydrolysate component under various experimental conditions of nuclease hydrolysis and the sole component under "mild" conditions, whereas sucrose-treated nuclei contain three types of dinucleosomes. In Triton-treated nuclei prolongation of hydrolysis results in the liberation of the chromatosome which is absent in chromatin hydrolysates of sucrose-treated nuclei. Hydrolysis of Triton-treated nuclear chromatin by micrococcal nuclease is unaccompanied by the liberation (up to the stage of "deep" hydrolysis) of the core particle, the major component of the "sucrose" nuclear hydrolysate under the conditions used. The sharp differences in the accessibility of various types of dinucleosomes observed during pretreatment of nuclei with Triton X-100 are interpreted in terms of the localization of histone H1. The non-random type of the histone H1 molecule orientation along the nucleosome fibril is postulated.     

Chromatin organization in isolated nuclei: flow cytometric characterization employing forward and perpendicular light scatter

Flow cytometric perpendicular and forward light scatters have been employed to evaluate whether the changes in chromatin organization due to ionic strength, Mg++ concentration and pH, visible in electron microscopy, can be monitored by flow cytometry. The average intensity of the perpendicular light scatter signal increased as nuclear chromatin became decondensed by lowering the ionic strength or releasing H1 histone at low pH values. These results indicate that flow cytometry signals and in particular the perpendicular light scatter allow the detection of the conformational transitions in chromatin and may therefore be useful for studying cell cycle associated morphological changes in isolated nuclei.     

The regulation of liver glycogen synthetase D phosphatase by ATP and glucose

Cortisol at a concentration of 5×10^?6M induces profound changes in U.V. absorption spectra of isolated nuclei from rat liver and thymus. The changes occur within the first 10 min of cortisol action. In both types of nuclei, a blue shift of 5–10 nm from the normal absorption maximum at 260–270 nm is evident. In addition, liver nuclei exhibit an elevation of the spectrum at 230–270 nm (increased U.V. absorption), while the spectrum of thymus nuclei becomes flattened. No such changes occur in nuclei exposed to a physiologically inactive hormone (pregnenolone). The results are interpreted as evidence for cortisol-induced perturbations in deoxyribonucleoprotein structure with consequent changes in the degree of condensation of nuclear chromatin.     

The Connection between Chromatin Motion on the 100 nm Length Scale and Core Histone Dynamics in Live XTC-2 Cells and Isolated Nuclei

The diffusive motion of DNA-containing chromatin in live cells and isolated nuclei is investigated using a two-photon standing wave fluorescence photobleaching experiment with 100 nm spatial resolution. The chromatin is labeled using the minor groove binding dye Hoechst 33342. In live cells, the mean diffusion rate is 5 × 10⁻⁴ μm²/s, with considerable cell-to-cell variation. This diffusion is highly constrained and cannot be observed in a standard, single beam fluorescence recovery after photobleaching experiment. To determine the chemical origin of the diffusion, we study motion in isolated nuclei and vary the strength of the histone-DNA interactions by changing the ionic strength and using chemical and photocross-linking experiments. At higher NaCl concentrations, we see increased chromatin diffusion as the histone-DNA interaction is weakened due to ionic screening, whereas photocross-linking the core histones to the DNA results in a complete absence of diffusive motion. These trends are consistent with the 100 nm scale motion being correlated with the interactions of histone proteins with the DNA. If chromatin diffusion is connected to the nucleosomal dynamics on much smaller length scales, this may provide a way to assay biochemical activity in vivo based on larger scale macromolecular dynamics observed via fluorescence microscopy.     

Ionic strength-dependent conformational transitions of chromatin. Circular dichroism and thermal denaturation studies

High-molecular-weight chicken erythrocyte chromatin was prepared by mild digestion of nuclei with micrococcal nuclease. Samples of chromatin containing both core (H3, H4, H2A, H2B) and lysine-rich (H1, H5) histone proteins (whole chromatin) or only core histone proteins (core chromatin) were examined by CD and thermal denaturation as a function of ionic strength between 0.75 and 7.0 × 10^?3M Na⁺. CD studies at 21°C revealed a conformational transition over this range of ionic strengths in core chromatin, which indicated a partial unfolding of a segment of the core particle DNA at the lowest ionic strength studied. This transition is prevented by the presence of the lysine-rich histones in whole chromatin. Thermal-denaturation profiles of both whole and core chromatins, recorded by hyperchromicity at 260 nm, reproducibly and systematically varied with the ionic strength of the medium. Both materials displayed three resolvable thermal transitions, which represented the total DNA hyperchromicity on denaturation. The fractions of the total DNA which melted in each of these transitions were extremely sensitive to ionic strength. These effects are considered to result from intra- and/or internucleosomal electrostatic repulsions in chromatin studied at very low ionic strengths. Comparison of the whole and core chromatin melting profiles indicated substantial stabilization of the core-particle DNA by binding sites between the H1/H5 histones and the 140-base-pair core particle.     

Alteration of domain architecture of chicken erythrocyte chromatin

The higher-order organisation of chromatin in chicken erythrocyte nuclei as a function of the ionic strength of the nuclear suspension buffer and also of the time of incubation in this buffer prior to nuclease digestion has been investigated. This organisation is described in terms of a physical parameter called the domain length. The 45-kbp-long domains of control nuclei were unravelled to give rise to domains of length 150 kbp on overnight equilibration at 0 degree C of the nuclei in standard isolation buffer containing 0.135 M NaCl prior to nuclease digestion. However, transition to the equilibrium state was preceded by a metastable and irregular domain architecture when the nuclei were incubated for only 1 h. In contrast, the domain length remained unchanged when nuclei were incubated in the isolation buffer alone for identical periods of time. The proteins dissociated at the higher ionic strength were characterised and their role in stabilising the domain structure is discussed.     

Small angle X-ray scattering of intact and lysing cell nuclei

X-ray scattering at very low angles has been applied to the study of chromatin structure in intact and lying nuclei. From maxima and minima in the very low angle range, higher order or very large structures could be deduced, the existence of which has not been found in isolated chromatin; up to now, such structures could be demonstrated only be electron-micrographs of whole nuclei. A strong maximum at 0.12 nm^?1 or a shoulder at 0.08 nm^?1 is interpreted as a hollow or solid cylinder with diameter ～ 70 nm; however, another possible explanation, a side by side packing of 30 nm solenoids^1–4 with a distance of 52 nm, cannot be excluded. A shoulder at 0.033 nm^?1 leads to the conclusion that an even larger structure exist in interphase nuclei. A slight minimum at 0.2 nm^?1 is in the range where mildly isolated chromatin has its first order minimum. This accounts for a coil diameter ～ 30 nm. While in intact nuclei these characteristics of the scattering curve have only a low intensity (except the maximum at 0.12 nm^?1 lysing nuclei exhibit much more pronounced maxima.     

X-ray small angle scattering study of chromatin as a function of fiber length

This work investigates the structure of native calf thymus chromatin as a function of fiber length and isolation procedures by using X-ray small angle scattering technique. Two methods of chromatin isolation have been compared in order to better understand the differences reported by various authors in terms of chromatin high order structure. In addition to these experimental results the effects of shearing have also been studied. In order to explain the differences among these chromatin preparations we built several models of chromatin fibers (represented as a chain of spherical subunits) assuming increasing level of condensation at increasing salt concentrations. For all these fiber models the corresponding theoretical X-ray scattering curves have been calculated and these results have been used to explain the influence of fiber length on the scattering profiles of chromatin. The comparison between experimental and theoretical curves confirms that the high molecular weight chromatin-DNA prepared by hypotonic swelling of nuclei (without enzymatic digestion) displays a partially folded structure even at low ionic strength, whereas the low molecular weight chromatin-DNA prepared by a brief nuclease digestion appears very weakly folded at the same ionic conditions.     

Effect of chromatin decondensation on the intranuclear matrix

We have studied the effect of chromatin condensation on the morphology of the residual structures isolated from rat liver nuclei. DNAse I digestion followed by high salt extraction of nuclei in the presence of Mg++ yields residual structures consisting of a dense peripheral layer surrounding an internal network, similar to those described by Berezney and Coffey [6]. These structures are stable at low ionic strength in the presence of EDTA. When nuclei swollen in EDTA are digested with DNAse II in the presence of EDTA, structures devoid of internal network are obtained even without subsequent treatment with high salt. When swollen nuclei are exposed to Mg++ a specific recondensation of chromatin takes place. The residual structures from recondensed nuclei are similar to those isolated from control nuclei in the presence of Mg++. The results suggest that the integrity and stability of the intranuclear matrix are acquired in the course of the isolation procedure and this is favoured by chromatin condensation.     

The effects of sodium and magnesium-ion interactions on chromatin structure and solubility

The effects of sodium and magnesium-ion interactions on chromatin structure and solubility were examined in isolated mouse liver nuclei. To facilitate this study, a simple assay of chromatin structure was developed, based on the absorbances at 260 nm (A260) and 320 nm (A320) of nuclei in test solutions. By subtracting the A320 from the A260, a single "spectral index" was obtained which served as a useful, but not absolute, indicator of chromatin structure. Electron microscopy verified the validity of this approach. The results indicate that either 200 mM NaCl or 0.5 mM MgCl2 were capable of preserving the native 20 to 30 nm chromatin fiber structure. Below 200 mM NaCl, the native fiber progressively uncoiled to the 10 nm unit fiber. The presence of 0.5 mM MgCl2 inhibited this uncoiling. Only divalent cations stabilized condensed chromatin (heterochromatin) within the nucleus. Monovalent and divalent cations interacted with one another at critical concentrations and modified their individual effects on chromatin structure; e.g., 10 to 25 mM NaCl interfered with the action of 0.5 to 1.5 mM MgCl2, causing a complete loss of condensed chromatin. Maximum solubility of micrococcal nuclease-digested chromatin occurred at 10 mM NaCl, which treatment allowed the chromatin to unfold to the 10 nm fiber. However, ionic conditions that disrupted condensed chromatin but maintained the native chromatin fiber morphology still resulted in relatively high yields of soluble chromatin. Minimum solubility occurred under conditions which preserved the structure of condensed chromatin.     

Radial density distribution of chromatin: evidence that chromatin fibers have solid centers

Fiber diameter, radial distribution of density, and radius of gyration were determined from scanning transmission electron microscopy (STEM) of unstained, frozen-dried chromatin fibers. Chromatin fibers isolated under physiological conditions (ionic strength, 124 mM) from Thyone briareus sperm (DNA linker length, n = 87 bp) and Necturus maculosus erythrocytes (n = 48 bp) were analyzed by objective image-processing techniques. The mean outer diameters were determined to be 38.0 nm (SD = 3.7 nm; SEM = 0.36 nm) and 31.2 nm (SD = 3.6 nm; SEM = 0.32 nm) for Thyone and Necturus, respectively. These data are inconsistent with the twisted-ribbon and solenoid models, which predict constant diameters of approximately 30 nm, independent of DNA linker length. Calculated radial density distributions of chromatin exhibited relatively uniform density with no central hole, although the 4-nm hole in tobacco mosaic virus (TMV) from the same micrographs was visualized clearly. The existence of density at the center of chromatin fibers is in strong disagreement with the hollow-solenoid and hollow-twisted-ribbon models, which predict central holes of 16 and 9 nm for chromatin of 38 and 31 nm diameter, respectively. The cross-sectional radii of gyration were calculated from the radial density distributions and found to be 13.6 nm for Thyone and 11.1 nm for Necturus, in good agreement with x-ray and neutron scattering. The STEM data do not support the solenoid or twisted-ribbon models for chromatin fiber structure. They do, however, support the double-helical crossed-linker models, which exhibit a strong dependence of fiber diameter upon DNA linker length and have linker DNA at the center.     

Role of alkaline endonucleases in the release of soluble chromatin from thymus, spleen and liver nuclei of normal and irradiated mice.

Thymus, spleen and liver nuclei released a large fraction of soluble chromatin in vitro when incubation was carried out in sucrose media containing low concentrations of CaCl2 and/or MgCl2. A significant fraction of deoxyribopolynucleotides (DPN) was also extracted from nuclei. After 30 min of incubation at 37 degrees C, the maximum release of soluble chromatin was observed near a pH of 8, which corresponds to the optimum pH of the alkaline endonuclease activity from thymus, spleen and liver. The soluble chromatin and DPN were precipitated by increasing the bivalent ion concentration of the medium. The protein/DNA ratio and the molecular weight of DNA suggest that the soluble chromatin and DPN represent nucleosome-like particles. The release of soluble chromatin in the first 4 hours of incubation was significantly increased if the nuclear fraction was isolated from the thymus and spleen of whole-body irradiated mice (1000 rad). This effect was absent in the liver nuclei.     

Thermodynamic Study of Interactions of Distamycin A with Chromatin in Rat Liver Nuclei in the Presence of Polyamines

We studied the thermodynamics of melting of isolated rat liver nuclei with different degrees of chromatin condensation determined by the concentration of polyamines (PA) and the solution ionic strength, as well as the effect of the antibiotic distamycin A (DM) on melting. Differential scanning calorimetry (DSC) profiles of nuclear preparations contained three peaks that reflected melting of three main chromatin domains. The number of peaks did not depend on the degree of condensation; however, nuclei with more condensed chromatin had a higher total enthalpy. DM stabilized peaks II and III corresponding to the melting of relaxed and topologically strained DNA, respectively, but destabilized peak I corresponding to the melting of nucleosome core histones. At the saturating concentration (DM/DNA molar ratio = 0.1), DM increased T_m of peaks II and III by ~5°C and decreased T_m of peak I by ~2.5°C. Based on the dependence of ΔH on DM concentration, we established that at low DM/DNA ratio (?0.03), when DM interacted predominantly with AT-rich DNA regions, the enthalpy of peak II decreased in parallel with the increase in the enthalpy of peak III, which indicated that DM induces structural transitions in the nuclear chromatin associated with the increase in torsional stress in DNA. An increase in free energy under saturation conditions was equal to the change in the free energy of DM interaction with DNA. However, the increase in the enthalpy of melting of the nuclei in the presence of DM was much greater than the enthalpy of titration of nuclei with DM. This indicates a significant increase in the strength of interaction between the two DNA strands apparently due, among other things, to changes in the torsional stress of DNA in the nuclei. Titration of the nuclei with increasing PA concentrations resulted in the decrease in the number of DM-binding sites and the non-monotonous dependence of the enthalpy and entropy contribution to the binding free energy on the PA content. We suggested that the observed differences in the thermodynamic parameters were due to the different width of the minor groove in the nuclear chromatin DNA, which depends on PA concentration.     

Studies by small-angle x-ray scattering of the quaternary structure of dissociation products of the beta-haemocyanin of Helix pomatia.

Helix pomatia beta-haemocyanin was split into dissociation products by varying the pH and the ionic strength. The purity of the solution was checked in an ultracentrifuge. Two defined dissociation products were studied in solution by small-angle X-ray scattering. In Tris-HC1 buffer, pH 8.0 and ionic strength 1 M, the following parameters of the dissociation product (tenths) could be determined: molecular weight = 7 x 10(5), volume = 1350 nm3, radius of gyration = 9.0 nm, maximal distance = 28.3 nm, radius of the spherical subunits about 2.6 nm, number of the subunits approximately 19. Tris-HC1 buffer, pH 8.7 and ionic strength 0.01 M, yielded dissociation products (twentieths) with the following parameters: molecular weight = 3.5 x 10(5), volume = 635 nm3, radius of gyration = 7.5 nm, maximal distance = 21.9 nm, radius of the spherical subunits about 2.5 nm. With this information, the assumption that the larger fragment was double the smaller one and the latest biochemical and morphological results, theoretical scattering curves of models were calculated and compared with the experimental curves. Two models are suggested which argee well with the dissociation products in radius of gyration and scattering.     

Structural organization of the meiotic prophase chromatin in the rat testis

Pachytene nuclei were isolated from rat testes by the unit gravity sedimentation technique and contained histone variants H1a, H1t, TH2A, TH2B, and X2 in addition to the somatic histones H1bde, H1c, H2A, H2B, H3, and H4. The basic organization of the pachytene chromatin namely the nucleosome repeat length and the accessibility to micrococcal nuclease, was similar to that of rat liver interphase chromatin. However, when digested by DNase I, the susceptibility of pachytene chromatin was 25% more than liver chromatin under identical conditions. Nucleosome core particles were isolated from both liver and pachytene nuclei and were characterized for their DNA length and integrity of the nucleoprotein on low ionic strength nucleoprotein gels. While liver core particles contained all the somatic histones H2A, H2B, H3, and H4, in the pachytene core particles, histone variants TH2A, X2, and TH2B had replaced nearly 60% of the respective somatic histones. A comparison of the circular dichroism spectra obtained for pachytene and liver core particles indicated that the pachytene core particles were less compact than the liver core particles. Studies on the thermal denaturation properties of the two types of core particles revealed that the fraction of the pachytene core DNA melting at the premelting temperature region of 55-60 degrees C was significantly higher than that of the liver core DNA.