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.     

Effects of salt concentration and H1 histone removal on the differential scanning calorimetry of nuclei.

The effects of increasing NaCl concentrations on the melting profiles of chromatin in isolated nuclei contradicted published claims that structural transitions near 76 degrees C (Tn-7), near 89 degrees C (Tn-8), and near 105 degrees C (Tn-10) were respectively the melting of linker DNA, the melting of extended nucleosomal strands, and the collapse of nucleosomes in the 300-A fiber. Contrary to expectations of such an interpretation, decreases in salt concentration stabilized Tn-7 and failed to eliminate Tn-10. Moreover, nuclei depleted of H1 histone, which is known to be essential for the formation of the 300-A fiber, gave the same melting profile as intact nuclei with regard to the relative magnitudes of Tn-8 and Tn-10. The effect of salt concentration on the melting profiles and the insensitivity of Tn-8 and Tn-10 to H1 histone removal supports the notion that Tn-7 is the collapse of the nucleosome while Tn-8 and Tn-10 are respectively the unstacking of nucleotide bases in relaxed chromatin and supercoiled chromatin. The identification of Tn-8 as the unstacking of bases in relaxed DNA, and Tn-10 as unstacking in supercoiled DNA, shows that scanning calorimetry can be used to measure the state of repair of DNA in the nucleus. The gain in Tn-8 at the expense of Tn-10 that is seen as the mitotic index drops and differentiation occurs suggests that nicks accumulate in the DNA, perhaps because the gross aggregation of the inactive majority of the chromatin makes it inaccessible to repair enzymes.     

Quantitative analysis of chromatin structure by circular dichroism

Quantitative analysis of the circular dichroism of nucleohistones and protein-free DNA was carried out in order to determine the structure and the role of the linker region DNA in chromatin, in terms of the conformational change of chromatin as a function of the ionic strength. It is shown clearly that the circular dichroism of Hl-depleted chromatin isolated from calf thymus is determined only by the ratio of the core region to the linker region and demonstrated by the linear combination of the spectrum of protein-free DNA and that of the nucleosome core in 5 mm-Tris · HCl, 1 mm-EDTA (pH 7.8). The calculated spectrum for the linker region in the H1-depleted chromatin was in good agreement with that of protein-free DNA. From the difference spectra between nucleohistones and protein-free DNA, it is suggested that the chromatin has an additional winding of DNA other than 146 base-pairs of DNA around the histone core. By decreasing the ionic strength to values lower than 5 mm-Tris · HCl, 1 mm-EDTA, the ellipticity of H1-depleted chromatin increased greatly between 250 nm and 300 nm while the increase was small in the case of chromatin and the nucleosome core. Nucleosomes with linker region DNA but without histone H1 also show great increase in ellipticity in this range of wavelengths as the ionic strength is decreased. Therefore, the linker region in H1-depleted chromatin plays an important role in the conformational changes brought about by changes in the ionic strength, and the conformational changes caused in the DNA of chromatin by decreasing the ionic strength are suppressed by the presence of histone H1.     

The condensation of chromatin and histone H1-depleted chromatin by spermine

At low ionic strength, spermine induces aggregation of native and H1-depleted chromatin at spermine/phosphate (Sp/P) ratios of 0.15 and 0.3, respectively. Physico-chemical methods (electric dichroism, circular dichroism and thermal denaturation) show that spermine, at Sp/P less than 0.15, does not appreciably alter the conformation of native chromatin and interacts unspecifically with all parts of chromatin DNA (linker as well as regions slightly or tightly bound to histones). In chromatin, the role of spermine could be more important in the stabilization of higher-order structure than in the condensation of the 30 nm solenoid. The addition of spermine to H1-depleted chromatin revealed two important features: (i) spermine can partially mimic the role of histone H1 in the condensation of chromatin; (ii) the core histone octamer does not appear to play any role in the aggregation process by spermine as DNA and H1-depleted chromatin aggregate at the same Sp/P ratio.     

The structure and dynamics of H1-depleted chromatin

The size of DNA involved in the interaction with a histone octamer in H1-depleted chromatin was re-examined. We compared the thermal untwisting of chromatin DNA and naked DNA using CD and electrophoretic topoisomer analysis, and found that DNA of 175 +/- 10 base pairs (bp) in length interacted with the histone core under physiological conditions. The decrease of ionic strength below 20 mM NaCl reduced this length down to 145 bp: apparently, an extra 30 bp DNA dissociated from the histone core to yield well-known 145-bp core particle. Histone cores partly dissociate within the temperature range of 25 to 40 degrees C. Quantitative analysis of histone thermal dissociation from DNA shows that the size of DNA protected against thermal untwisting would be significantly overestimated if this effect is neglected. The results presented in this paper also suggest that the dimers (H2A, H2B) act as a lock, which prevents transmission of conformational alterations from a linker to nucleosome core DNA. The histone core dissociation as well as (H2A, H2B) dimer displacement are discussed in the light of their possible participation in the eukaryotic genome activation.     

Thermal denaturation of nucleosomal core particles.

Thermal denaturation of very homogeneous preparations of core particles from chicken erythrocyte chromatin is studied by several techniques. The change in absorbance, which is very closely paralleled by changes in heat capacity, which is very closely paralleled by changes in heat capacity, is a biphasic process with inflexions at 60 degrees C and 74 degrees C. In contrast, isolated DNA of the same length denatures in a single transition around 44 degrees C. Monitoring the circular dichroism of the cores during thermal denaturation reveals biphasic changes in the secondary structure of the DNA, preceding the base unstacking by 10 degrees C in the first and 3 degrees C in the second phase. However, measurable alterations in the secondary structure of the histones are confined to the second phase with a melting temperature at 71 degrees C. Increase in the ionic strength of the buffer from 1 mM to 10 mM leads to almost monophasic melting curves as measured by absorbance and CD, while not causing any measurable conformational changes at room temperature. The melting of core particles is interpreted as a denaturation of about 40 base pairs in the first phase, followed by a massive breakdown of the native structure of a tight histone-DNA complex, which frees the remaining 100 base pairs for unstacking.     

Chromatin ionic atmosphere analyzed by a mesoscale electrostatic approach

Characterizing the ionic distribution around chromatin is important for understanding the electrostatic forces governing chromatin structure and function. Here we develop an electrostatic model to handle multivalent ions and compute the ionic distribution around a mesoscale chromatin model as a function of conformation, number of nucleosome cores, and ionic strength and species using Poisson-Boltzmann theory. This approach enables us to visualize and measure the complex patterns of counterion condensation around chromatin by examining ionic densities, free energies, shielding charges, and correlations of shielding charges around the nucleosome core and various oligonucleosome conformations. We show that: counterions, especially divalent cations, predominantly condense around the nucleosomal and linker DNA, unburied regions of histone tails, and exposed chromatin surfaces; ionic screening is sensitively influenced by local and global conformations, with a wide ranging net nucleosome core screening charge (56-100e); and screening charge correlations reveal conformational flexibility and interactions among chromatin subunits, especially between the histone tails and parental nucleosome cores. These results provide complementary and detailed views of ionic effects on chromatin structure for modest computational resources. The electrostatic model developed here is applicable to other coarse-grained macromolecular complexes.     

Structural domains and conformational changes in nuclear chromatin: a quantitative thermodynamic approach by differential scanning calorimetry

A good deal of information on the thermodynamic properties of chromatin was derived in the last few years from optical melting experiments. The structural domains of the polynucleosomal chain, the linker, and the core particle denature as independent units. The differential scanning calorimetry profile of isolated chromatin is made up of three endotherms, at approximately 74, 90, and 107 degrees C, having an almost Gaussian shape. Previous work on this matter, however, was mainly concerned with the dependence of the transition enthalpy on external parameters, such as the ionic strength, or with the melting of nuclei from different sources. In this paper we report the structural assignment of the transitions of rat liver nuclei, observed at 58, 66, 75, 92, and 107 degrees C. They are representative of the quiescent state of the cell. The strategy adopted in this work builds on the method developed for the investigation of complex biological macromolecules. The heat absorption profile of the nucleus was related to the denaturation of isolated nuclear components; electron microscopy and electrophoretic techniques were used for their morphological and molecular characterization. The digestion of chromatin by endogenous nuclease mimics perfectly the decondensation of the higher order structure and represented the source of several misinterpretations. This point was carefully examined in order to define unambiguously the thermal profile of native nuclei. The low-temperature transitions, centered around 58 and 66 degrees C, arise from the melting of scaffolding structures and of the proteins associated with heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic strength dependence of the binding of methylene blue to chromatin and calf thymus DNA.

The binding of the intercalating dye methylene blue (MB) to chromatin and to free DNA has been studied as a function of ionic strength at very low binding ratios (1 MB/400 DNA bases) using absorption spectroscopy. With increasing salt concentration MB is displaced from chromatin to a higher extent than from DNA. The free energy change for MB binding to chromatin is found to be approximately 5 kJ/mole lower than for binding to DNA. This difference can be explained by the reduced number of high affinity binding sites in chromatin due to the presence of histone proteins. The difference in binding energy is virtually independent of the degree of chromatin condensation and also of the valence of counter ions, suggesting that neither the affinity for, nor the number of intercalation sites in the linker DNA is markedly changed upon the salt-induced condensation. The unaffected thermodynamics of the linker binding suggests that factors such as DNA superhelicity and the electrostatic influence from the chromatosomes remain unchanged during chromatin condensation.     

Ethidium bromide binding to core particle: comparison with native chromatin.

Ethidium bromide intercalation into DNA of nuclease digested erythrocyte chromatin and core particle, was followed at low ionic strength by fluorescence measurements, equilibrium dialysis using 14C labelled dye, circular dichroism and electron microscopy. High affinity binding sites in the chromatin are no more present in the core particle, i.e. when the linker is removed. In the case of core particle, a cooperative process occurs, accompanied by a partial stripping of the DNA from the core histone. Finally two populations of core particles can be detected by electron microscopy as far as their binding properties are concerned.     

Linker DNA destabilizes condensed chromatin.

The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.     

The effect of histone H1 on the compaction of oligonucleosomes. A quasielastic light scattering study

The structural properties of H1-depleted oligonucleosomes are investigated by the use of quasielastic laser light scattering, thermal denaturation and circular dichroism and compared to those of H1-containing oligomers. To obtain information on the role of histone H1 in compaction of nucleosomes, translational diffusion coefficients (D) are determined for mono-to octanucleosomes over a range of ionic strength. The linear dependences of D on the number of nucleosomes show that the conformation of stripped oligomers is very extended and does not change drastically with increasing the ionic strength while the rigidness of the chain decreases due to the folding of linker DNA. The results prove that the salt-induced condensation is much smaller for H1-depleted than for H1-containing oligomers and that histone H1 is necessary for the formation of a supercoiled structure of oligonucleosomes, already present at low ionic strength.     

Nucleosome core particles of calf thymus, Tetrahymena, and the reconstituted hybrid. Their structure reflects the nature of the histone octamer

The circular dichroism spectra and the thermal denaturation profiles of the nucleosome core particles isolated by micrococcal nuclease digestion from nuclei of calf thymus and the protozoan Tetrahymena pyriformis were compared with those of the homogeneous and hybrid core particles reconstituted from calf core DNA and either calf or Tetrahymena histone octamer. The core DNA was obtained from the calf core particle, and both the histone octamers were reconstituted from the acid-extracted four core histones of calf thymus or Tetrahymena, whose amino acid sequences show the largest differences hitherto known. The reconstituted homogeneous core particle was identical in both the physical properties with the isolated calf core particle, showing that the correct reconstitution was achieved. The circular dichroism spectra of the calf and Tetrahymena core particles and the hybrid core particle showed no essential differences, indicating that the three core particles have the same overall structure. The derivative thermal-denaturation profiles, however, clearly differed; the calf core particle showed two melting transitions at 60 degrees C and 72 degrees C, while the Tetrahymena and hybrid core particles showed the same three transitions at 48-50 degrees C, 60-61 degrees C, and 72 degrees C. Thus, the thermal denaturation properties of nucleosome core particles do not reflect the nature of DNA, but rather that of the histone octamer bound to the DNA. We conclude that the Tetrahymena histones are more weakly bound to the DNA than the calf thymus histones in the same overall structure of nucleosomes.     

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.     

Linker histone subtype composition and affinity for chromatin in situ in nucleated mature erythrocytes

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.     

Extended C-terminal tail of wheat histone H2A interacts with DNA of the "linker" region

The preparation of hybrid histone octamers with wheat histone H2A variants replacing chicken H2A in the chicken octamer is described. The fidelity of the reconstituted hybrid octamers was confirmed by dimethyl suberimidate cross-linking. Polyglutamic-acid-mediated assembly of these octamers on long DNA and subsequent micrococcal nuclease (MNase) digestion demonstrated that, whereas chicken octamers protected 167 base-pairs (representing 2 full turns of DNA), hybrid histone octamers containing wheat histone H2A(1) with its 19 amino acid residue C-terminal extension protected an additional 16 base pairs of DNA against nuclease digestion. The protection observed by hybrid histone octamers containing wheat histone H2A(3) with both a 15 residue N-terminal and a 19 residue C-terminal extension was identical with that observed with H2A(1)-containing hybrid histone octamers with only the 19 residue C-terminal extension. These results suggest that the role of the C-terminal extension is to bind to DNA of the "linker" region. The thermal denaturation of chicken and hybrid core particles was identical in 10 mM-Tris.HCl.20 mM-NaCl, 0.1 mM-EDTA, confirming that there was no interaction between the basic C-terminal extension and DNA of the core particle. Denaturation in EDTA, however, showed that hybrid core particles had enhanced stability, suggesting that the known conformational change of core particles at very low ionic strength allows the C-terminal extension to bind to core particle DNA under these conditions. A model accounting for the observed MNase protection is presented.     

Binding Dynamics of Disordered Linker Histone H1 with a Nucleosomal Particle

Linker histone H1 is an essential regulatory protein for many critical biological processes, such as eukaryotic chromatin packaging and gene expression. Mis-regulation of H1s is commonly observed in tumor cells, where the balance between different H1 subtypes has been shown to alter the cancer phenotype. Consisting of a rigid globular domain and two highly charged terminal domains, H1 can bind to multiple sites on a nucleosomal particle to alter chromatin hierarchical condensation levels. In particular, the disordered H1 amino- and carboxyl-terminal domains (NTD/CTD) are believed to enhance this binding affinity, but their detailed dynamics and functions remain unclear. In this work, we used a coarse-grained computational model, AWSEM-DNA, to simulate the H1.0b-nucleosome complex, namely chromatosome. Our results demonstrate that H1 disordered domains restrict the dynamics and conformation of both globular H1 and linker DNA arms, resulting in a more compact and rigid chromatosome particle. Furthermore, we identified regions of H1 disordered domains that are tightly tethered to DNA near the entry-exit site. Overall, our study elucidates at near-atomic resolution the way the disordered linker histone H1 modulates nucleosome’s structural preferences and conformational dynamics.