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.     

Electric dichroism of chromatin

The linear dichroism of sheared calf thymus chromatin, oriented in solution by a pulsed electric field, has been measured. The limiting value of this dichroism is considerably less negative than that of calf thymus DNA, but does not approach the positive values predicted if chromatin were uniformly supercoiled in the manner suggested by X-ray studies. The decay of the dichroism of chromatin, after termination of the electric pulse, is similar to that of DNA, indicating that chromatin retains a high degree of chain flexibility. These data, as well as previous flow dichroism studies (Ohba, 1966; Smart &; Bonner, 1971), suggest that chromatin is not predominantly supercoiled in solutions of low ionic strength. Evidence for a structurally heterogeneous model for chromatin is discussed.     

Modification of histone binding in calf thymus chromatin by protamine.

When calf thymus chromatin is incubated with protamine, the protein binds to DNA, forming a chromatin-protamine complex. The binding reaches a saturating level at the weight ratio of protamine to DNA of approximately 0.5. Although the saturated binding of protamine to DNA does not cause major displacement of histones from calf thymus chromatin, examination of the dissociation profiles by salt in combination with urea of protamine-treated chromatin shows that the histone-DNA interactions are markedly altered by such binding. The dissociation of histones from the chromatin-protamine complex requires less NaCl but the same concentration of urea as that for untreated chromatin, suggesting that the electorstatic interactions between the histones and DNA are decreased as a result of protamine binding. When protamine concentration is increased beyond that required for saturated binding to DNA during in vitro exposure of calf thymus chromatin to protamine, lysine-rich histone is completely displaced.     

Magnesium binding and conformational change of DNA in chromatin

The structure of chromatin in the presence of Mg2+ ions was examined by circular dichroism and equilibrium dialysis. Circular dichroism (CD) shows that above 260 nm the intensity of the spectrum of DNA in nucleoproteins decreases as the Mg2+ concentration increases. This change is an intrinsic characteristic of DNA since it is also observed in protein-free DNA and has been attributed to a change in the winding angle of base pairs around the DNA axis. Some structural elements of the DNA in the nucleosome core, therefore, are as movable as those of protein-free DNA. The basic organization of H1-depleted chromatin, 146 base pairs (bp) of DNA wound around core histones and a residual 49 bp in the linker region in the repeating unit, is maintained both in the presence and in the absence of Mg2+ ions, as shown by the fact that the CD spectrum of H1-depleted chromatin has the same type of linear combination between the spectrum of protein-free DNA and that of the nucleosome core in 0.2 mM MgCl2-10 mM triethanolamine (pH 7.8) as it has in 1 mM ethylenediaminetetraacetic acid-10 mM tris(hydroxymethyl) aminomethane (pH 7.8). The ellipticity of chromatin shows a smaller decrease relative to the other nucleoproteins and protein-free DNA upon the addition of Mg2+ ions. Therefore, some structural elements of chromatin are apparently somewhat protected against the conformational change induced by these ions. The spectrum of chromatin becomes almost indistinguishable from that of H1-depleted chromatin in 0.2 mM MgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mercury-induced DNA polymorphism: probing the conformation of Hg(II)-DNA via staphylococcal nuclease digestion and circular dichroism measurements

Exposing native calf thymus DNA to increasing concentrations of Hg(ClO4)2 not only produces dramatic changes in its circular dichroism (CD) but results also in the decrease, and ultimate cessation, of endonucleolytic DNA cleavage by staphylococcal nuclease. Let r = [moles of added Hg(II)]/[mole of DNA base]: the conservative CD spectrum of the DNA B-form becomes nonconservative in appearance at 0.01 less than r less than 0.12 (resembling DNA in C-form geometry) and assumes the spectral characteristics of a left-handed DNA double helix at 0.12 less than r less than or equal to 1.0. DNA cleavage proceeds at or near the rates exhibited by untreated DNA at 0 less than r less than 0.08. At Hg(II) levels of 0.08 less than r less than 0.5, the rate of DNA hydrolysis decreases monotonically with increasing Hg(II) concentrations, and at r greater than 0.4, DNA cleavage ceases. Both the CD changes and the changes in the rate of DNA digestion are totally reversible upon the removal of Hg(II), at least up to r = 1.0, demonstrating that Hg(II) keeps all base pairs in register. For comparison purposes, native calf thymus DNA was also treated with methylmercury [CH3Hg(II)], an agent known to disrupt the secondary structure of DNA. The treatment yielded single-stranded methylmercurated DNA with preserved right-handed helix screwness. In addition, this DNA is digested by staphylococcal nuclease much more rapidly than double-stranded control DNA. Lastly, neither the CD nor the cleavage rate changes are reversible upon the removal of methylmercury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential scanning calorimetry of chromatin at different levels of condensation

The thermal denaturation of calf thymus total chromatin and of fractions enriched in heterochromatin or euchromatin, has been investigated by differential scanning calorimetry and compared to that of calf thymus DNA and DNA-histone complexes. In our experimental conditions, chromatin melts in three thermal transitions: the main one, assigned to separation of the DNA double helix, occurs at 83 °C, while the other two occur at 63 °C and 74 °C. The data show that: (a) the transition enthalpy for denaturation of DNA in the total chromatin and in DNA-histone complexes is nearly the same as that of DNA in solution; (b) the transition at 63 °C is present in the thermogram of the heterocromatin enriched fraction, while it is completely absent in that of the euchromatin enriched one. The results suggest that this transition can be attributed to the higher order structures of heterochromatin.     

Characteristics of the structural state of DNA in chromatin with short linker

Various fragments of pigeon brain neuron chromatin with very short linker DNA have been studied by circular dichroism (CD). The DNA structure in core particles of the brain and thymus chromatins is very similar. Linker DNA and a part of core DNA in the mononucleosomes of brain chromatin is extended. This conclusion follows from increasing CD amplitude of the brain mononucleosomes as compared with the corresponding value for thymus mononucleosomes. The internucleosome interactions stabilized the compactness of core DNA in the brain oligonucleosomes. The whole linker DNA of the brain chromatin unlike thymus chromatin is extended at low ionic strength. This fact can explain the brain chromatin ability to form a compact structure with increasing ionic strength.     

The conformation of proteins in chromatin. A circular dichroism study below 250 nm.

This paper is an investigation of the circular dichroism (CD) spectra of DNA and protein in chromatin. The circular dichroism (CD) of chromatin below 250 nm is due to DNA and protein peptide chromophores. The spectrum in this region is resolved into contributions from salt-extractable proteins (histone and non-histone proteins extractable with sodium chloride), residual non-histone proteins (not extractable with 3 M sodium chloride), and DNA. Below 250 nm, DNA in chromatin has the same CD spectrum as DNA free in solution, in contrast to the CD of DNA above 250 nm (Hjelm, R. and Huang, R. C., (1974), Biochemistry 13, 5275). Histones and salt-extractable non-histone proteins in chromatin are seen to have an average CD like those observed for globular proteins. The average CD of the residual non-histone proteins is consistent with a population of proteins with more extended conformation. The CD of each of these components is found to be the same in chromatins isolated from tissues having different nuclear synthetic activities: chick embryo brain, pig cerebellum, myeloma K41, calf thymus, and chicken erythrocyte.     

Low molecular weight peptides controlling transcription are present in the calf thymus chromatin structure

A calf thymus peptide fraction controlling DNA and chromatin template has been purified by DNA-cellulose and Dowex 50 WX2 chromatography and its amino acid composition determined. The active peptide fraction can be extracted in high pH buffer from calf thymus native chromatin previously deproteinized by chloroform-isamyl alcohol and phenol. These data demonstrate that the thymic peptide(s) is (are) a chromatin protein constituent strongly linked to DNA. The specificity in association of the peptide(s) to DNA has also been considered.     

Structural heterogeneity of chromatin preparations at the level of DNA topology

The structural heterogeneity of calf thymus chromatin preparations was studied at the level of DNA topology by analysing the influence of ethidium bromide on the chromatin viscosity in deproteinizing medium. In 0.7 M NaCl the chromatin was separated into the fractions with linear DNA (3--36% in various preparations) and with supercoiled circular DNA (scc DNA), which differ from each other in their adhesive properties. Reduction of disulfide bonds in residual chromatin protein with 5% mercaptoethanol linearized scc DNA, present in chromatin preparations as nuclear matrix subunits containing some loops of scc DNA on the protein globule.     

The solubility of calf thymus chromatin in sodium chloride

The solubility of calf thymus chromatin and chromatin depleted of F1-histone has been examined under various conditions in sodium chloride. F1-depleted DNH was more soluble than native DNH at low concentrations but this difference became small at high concentrations (1mg/ml). Both exhibited minimum solubility in 0.15M -NaCl. The effect of pH and of maleylation of the mino acid side chains on the solubility implied that electrostatic interactions dominated the precipitation reaction. Urea had no effect on the solubility of either complex. N.m.r. studies showed that the chromatin behaved as a rigid complex at all salt concentrations less than 0.6 molar.     

Accessibility of the minor groove of DNA in chromatin to the binding of antibiotics netropsin and distamycin A

The interaction of the antibiotics distamycin A, distamycin analogue and netropsin with chromatin of calf thymus has been studied by circular dichroism measurements and by gel filtration. The minor groove of DNA in chromatin is accessible by 83–89% to the binding of these antibiotics as compared with that of free DNA. The present results combined with our data on the methylation of chromatin with dimethylsulphate [3] strongly suggest that the minor groove of DNA in chromatin is not occupied by chromatin proteins.Abbreviations DM distamycin A - DM₂ analogue of distamycin - Nt netropsin - CD spectra circular dichroism spectra     

Iodination of Xenopus laevis histone F2a1 in chromatin.

The reaction of calf thymus and Xenopus laevis histones with radioactive iodine has been studied under various conditions that affect chromatin structure. All histones from both species contain at least one tyrosine residue and, in a denaturing solvent, all the the histones react with iodine. Histone F2a1 has been studied in detail. Calf thymus F2a1 is known to contain four tyrosyls and all four react with iodine. In high voltage paper electrophoresis, the tyrosine-containing peptides from calf co-migrate with those from Xenopus F2a1, suggesting that the amino acid sequence is strongly conserved between these two species. Therefore, the published calf thymus F2a1 sequence has been used to order the Xenopus F2a1 peptides within the molecules. When gently isolated native chromatin is iodinated in a low ionic strength medium 60% of the radioactivity in F2a1 is in tyrosyl 88, 30% in tyrosyl 51, and tyrosyl 72 and 98 have almost no radioactivity. Reagents which remove the protein from the DNA (2 M NaCl) or partially disrupt protein tertiary structure (5 M urea) increase the reactivity of each of the four tyrosyls five- to tenfold, suggesting that all four are protected about equally by the overall folding of the chromatin. Isolated F2a1 iodinated in the presence of 10 M urea shows uniform labeling in each of the four peptides, suggesting that tyrosyl 72 and 98 are afforded some protection solely by protein-protein interactions. The stepwise removal of histones in increasing NaCl concentrations differentially increases the availability of each F2a1 tyrosyl. The preferential exposure of tyrosyl 88 coincides with the removal of the majority of F1 histones at 0.5 M NaCl while the gradual and stepwise increase in reactivity of tyrosyl 51, 72, and 98 correlates with the gradual removal of histones other than F1. Radioactive iodination of chromatin has been shown to be a sensitive probe for detecting changes in the association state (or conformation) of histone F2a1.     

Sea urchin sperm DNP. I. Chemical composition and template properties of DNP

Electrophoretic mobility, amino acid composition and salt dissociation of histones isolated from sperm of sea urchin Strongylocentrotus intermedius and calf thymus cells were studied. The special arginine-rich histone fraction (I) has been observed in sea urchin sperm chromatin, this fraction being absent in calf thymus chromatin. Dissociation of lysine-containing histone fractions from sea urchin chromatin occured in the range of 0.7 to 1.0 M NaCl concentrations. H1 of calf thymus chromatin was totally extracted with 0.6 M NaCl. In the course of a further increase of salt concentrations (up to 1.5 M NaCl) a practically total extraction of histones from sperm chromatin was observed, while about 20% of proteins remained bound to DNA in thymus chromatin after extraction with 2.0 M NaCl. The template activity of non-extracted DNP preparations from urchin sperm was equal to 2-3% of that of totally deproteinized DNA. The template activity of DNP gradually increased at protein extraction from DNP preparations. The hybridization capacity of RNA transcribed on partially dehistonized DNP templates in vitro also increased.     

Increased stability of the higher order structure of chicken erythrocyte chromatin: nanosecond anisotropy studies of intercalated ethidium

Internal motion of the DNA in chicken erythrocyte chromatin fibers was studied by measurement of the fluorescence anisotropy decay of ethidium intercalated in the linker region. A comparison of the decay curves of the dye in chicken erythrocyte chromatin with those of calf thymus chromatin [Ashikawa, I., Kinosita, K., Jr., Ikegami, A., Nishimura, Y., Tsuboi, M., Watanabe, K., Iso, K., & Nakano, T. (1983) Biochemistry 22, 6018-6026] revealed greater suppression of nucleosome movement in chicken erythrocyte chromatin. Furthermore, the transition of this chromatin to the compact (solenoidal) structure occurred at lower solvent concentrations of Na+ or Mg2+ than those for calf thymus chromatin. These results demonstrated increased stability of the higher order structure (the solenoid) of chicken erythrocyte chromatin, which may be related to the reduction of nuclear activity in the chicken erythrocyte cell. In addition to intact chicken erythrocyte chromatin, we studied the structural transitions of H1-depleted and H1,H5-depleted chromatins. The result indicated that histone H5 of this chromatin stabilizes the higher order structure in the presence of magnesium (or divalent) cation and did not induce the transition in the solution containing only sodium cation.     

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.     

Optical anisotropy of chromatin. Flow linear dichroism and electric dichroism studies

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentrations by the use of flow linear dichroism (LD) and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA - 2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA - for chromatins with the linker DNA of 10-30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin. Presented results explain the contradictory data obtained by electrooptical and hydrooptical methods.     

On the arrangement of histones in chromatin.

It was found that nucleoprotein particles formed after DNase I action on calf thymus chromatin contain single-stranded DNA fragments, associated with histones only by ionic linkages. These results suggest that histones in chromatin are bound ionically only to one polynucleotide strand of double-helical DNA, protecting it against nucleolytic attack.     

Dynamic and static chromatin

It was found that the dependence of the viscosity of calf thymus chromatin dispersions and human leukocytes on ethidium bromide concentration had two peaks indicative of domains with circular supercoiled DNA and varying resistance to ultrasound in the cells and isolated chromatin. The hypothesis of V. D. Paponov and P. S. Gromov (Bull. Exp. Biol. Med., N5, 590, 1985) on the transformation of static relations of nucleosome DNA-containing nuclei into dynamic, after chromatin exposure to ultrasound due to DNA linearization in chromatin domains possessing circular supercoiled DNA, has been confirmed.     

Protein-protein and protein-DNA interactions in calf thymus nuclear matrix using cross-linking by ultraviolet irradiation

Nuclear matrices from calf thymus contained 30-50 protein species with one prominent band at 70 kilodaltons tentatively identified by its isoelectric point, apparent molecular weight, charge modification, and abundance as bovine lamin. The amount of DNA present in the matrix fraction was strongly dependent on the extent of digestion of the nuclei by micrococcal nuclease. The size of the DNA was higher than two kilobase pairs, although the chromatin DNA had been digested down to short oligonucleosomes. The lamin band was preferentially dissociated from isolated matrices during repeated treatment by 2 M NaCl or 5 M urea. Irradiation of calf thymus nuclear matrices at 313 nm induced protein-protein and protein-DNA cross-linking, as well as double-strand breaking of DNA, presumably at unprotected, protein-free regions. Lamin protein was more dramatically affected than other protein species by ultraviolet (UV) irradiation. In situ DNA hydrolysis, after the separation of the cross-linked matrix components on polyacrylamide-sodium dodecyl sulfate gels, followed by two-dimensional electrophoresis, showed lamin to be the major protein that was cross-linked to the DNA. Lamin molecules were also cross-linked by UV light to each other to form lamin homo-oligomers. A discrete size DNA fragment of approximately 450 base pairs is protected by lamin homo-oligomers from breakdown during UV irradiation. It is proposed that the direct contact between lamin and DNA found in this study is responsible for anchoring chromatin loops (domains) to a stable, immobile matrix structure.