Circular dichroism of histone-bound regions in chromatin.

Native, NaCl-treated, trypsin-treated, and polylysine-bound nucleohistones were studied in 2.5 × 10^?4 M EDTA, pH 8.0, using circular dichroism (CD) and thermal denaturation. Removal of histone I by 0.6 M NaCl has a much smaller effect on both Δε₂₂₀ and Δε₂₇₈ than the removal of other histones. This indicates that histone I has less helical content and less conformational effect on the DNA in nucleohistone. By extrapolating to 100% binding by histones other than I, the positive CD band near 275 nm is close to zero. Comparison is also made between the effects of binding by the more basic and the less basic halves of histones by trypsin-digestion and polylysine-binding experiments. Trypsin digestion of nucleohistone reduces melting band IV at 82°C much more than melting band III at 72°C. However, the CD changes of Δε₂₇₈ and Δε₂₂₀ induced by trypsin digestion are small, unless melting band III is also reduced by the use of a higher trypsin level. This implies that the less basic halves of histones, which stabilize DNA to 72°C (melting band III), have more helical structure and are more responsible for conformational change in DNA than are the more basic halves, which stabilize DNA to 82°C (melting band IV). Polylysine binding to nucleohistone diminishes melting band III but has no effect on melting band IV. This binding affects only slightly the Δε₂₂₀ of nucleohistone, indicating that polylysine interferes very little with the structure of the less basic halves of bound histones. The implications of these studies with respect to chromatin structure are discussed.     

Ultraviolet radiation response to the physical properties of the reconstituted nucleohistone

The effect of UV irradiation on the reconstituted nucleohistone have been studied with reference to its (nucleohistone) changes in physical properties, after irradiation at different UV doses. The rate of fall of specific viscosity ratio of the reconstituted nucleohistone as a function of UV dose decreased gradually with the increasing histone to DNA weight ratio (r). This effect, was not observed when the histones remained dissociated from DNA, in high ionic strength (1.5 M NaCl). Histone-DNA complex (r=0.97) irradiated up to a dose of 3.6×10⁴ J/m² had a stable melting temperature unlike free DNA where UV irradiation lowered the melting temperature and the heterogeneous melting profiles were observed. Rate of formaldehyde reaction, with DNA recovered from the irradiated complex, was slower than that with native DNA treated at the same dose. All this suggested that the effect of UV in the DNA of the nucleohistone was less, compared to that in free DNA.     

Studies on interaction between histone V (f2c) and deoxyribonucleic acids.

Histone V (2fc) from chick erythroctes was used in the study of its interaction with DNA from various sources. Complexes between this histone and DNA were formed using the procedure of continuous NaCl gradient dialysis in urea. Two physical methods, namely thermal denaturation and circular dichroism (CD), were used as analytical tools. Thermal denaturation of nucleohistone V with chick or calf thymus DNA shows three melting bands: band I at 45-50 degrees corresponds to free base pairs; band II at 75-79 degrees, and band III at 90-93 degrees correspond to histone-bound base pairs. In histone-bound regions, there are 1.5 amino acid residues/nucleotide in nucleohistone V. In contrast, a value between 2.9 and 3.3 was determined for nucleohistone I (fl) (H. J. Li (1973), Biopolymers 12, 287). Similar melting properties have been observed for histone V complexed with bacterial DNA from Micrococcus luteus. Histone V binding to DNA induces a slight transition from a B-type CD spectrum to a C-type spectrum. Trypsin treatment of nucleohistone V reduces melting band III much more effectively than band II. Such a treatment also restores DNA to B conformation in the free state. Reduction of the melting bands of nucleohistone V by polylysine binding follows the order of I greater than II greater than III, accompanied by the increase of a new band at 100 degrees. When two bacterial DNAs of varied A + T (adenine + thymine) content simultaneously compete for the binding of histone V, the more (A " T)-rich DNA is selectively favored. Under experimental conditions described here, Clostridium perfringens DNA with 69% A + T is bound by histone V in preference to chicken DNA with 56% A + T although the latter has natural sequences for histone V binding.     

A correlation between nucleosome spacer region susceptibility to DNase I and histone acetylation.

Hepatoma tissue culture (HTC) cell nuclei were digested with either DNase I or micrococcal nuclease and the nucleohistone digestion products fractionated by gel electrophoresis or exclusion chromatography. Under appropriate conditions, gel electrophoresis demonstrates that for both nucleases, only cleavages within the nucleosome spacer regions and not within the nucleosome core lead to freely migrating nucleohistone particles. These particles consist of nucleosome cores, nucleosomes and nucleosome oligomers. Following DNase I digestion and fractionation by exclusion chromatography, analysis of the histones indicates a direct relationship between increased spacer region susceptibility to nuclease and increased nucleosomal histone acetylation. Evidently digestion sites outside the regions of DNA protected by core histones can reflect the degree of acetylation of core histones. Such a relationship is not found when micrococcal nuclease is used to digest the samples.     

Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry

Thermal denaturation of nuclear DNA is studied in situ in individual cells or isolated cell nuclei by employing the property of the fluorochrome acridine orange (AO) to differentially stain native and denatured DNA and by using an automated flow-through cytofluorimeter for measurement of cell fluorescence. RNAse-treated cells, or cell nuclei, are heated, stained and measured while in suspension and AO-DNA interaction is studied under equilibrium conditions. Measurements are made rapidly (200 cells/sec); subpopulations of cells from a measured sample can be chosen on the basis of differences in their staining or light-scattering properties and analysed separately. DNA denaturation in situ is rapid; it approaches maximum during the first 5 min of cell heating. Divalent cations stabilize DNA against denaturation. At low pH the transition occurs at lower temperature and the width of the transition curves (‘melting profiles’) is increased. Decrease in ionic strength lowers the DNA melting temperature. This effect is much more pronounced in cells pretreated with acids under conditions known to remove histones. Histones thus appear to stabilize DNA in situ by providing counterions. At least four separate phases can be distinguished in melting profiles of DNA in situ; they are believed to indicate different melting points of DNA in complexes with particular histones. A decrease in cell (nuclear) ability to scatter light coincides with DNA melting in situ, possibly representing altered refractive and/or reflective properties of cell nuclei. Formaldehyde, commonly used to prevent DNA renaturation, is not used in the present method. The heat-induced alterations in nuclear chromatin are adequately stabilized after cell cooling in the absence of this agent. Cells heated at 60–85 °C exhibit increased total fluorescence after AO-staining, which is believed to be due to unmasking of new sites on DNA. This increase is neither correlated with DNA melting, nor with the presence of histones. Possibly, it reflects destruction of DNA superstructure maintained at lower temperatures by DNA associations with other than histone macromolecules (nuclear membrane).     

Role of the histone "tails" in the folding of oligonucleosomes depleted of histone H1.

An oligonucleosome 12-mer was reconstituted in the absence of linker histones, onto a DNA template consisting of 12 tandemly arranged 208-base pair fragments of the 5 S rRNA gene from the sea urchin Ly-techinus variegatus (Simpson, R. T., Thoma, F. S., and Burbaker, J. M. (1985) Cell 42, 799-808). The ionic strength-dependent folding of this nucleohistone complex was compared with that of a native oligonucleosome fraction obtained from chicken erythrocyte chromatin, which had been carefully stripped of linker histones and fractionated in sucrose gradients. The DNA of this native fraction exhibited a narrow size distribution centered around the length of the 208-12 DNA template used in the reconstituted complex. These two complexes displayed very similar hydrodynamic behavior as judged by sedimentation velocity analysis. By combining these data with electron microscopy analysis, it was shown that the salt-dependent folding of oligonucleosomes in the absence of linker histones involves the bending of the linker DNA region connecting adjacent nucleosomes. It was also found that selective removal by trypsin of the N-terminal regions ("tails") of the core histones prevents the oligonucleosome chains from folding. Thus, in the absence of these histone domains, the bending of the linker DNA necessary to bring the nucleosomes in contact is completely abolished. In addition to the complete lack of folding, removal of the histone tails results in an unwinding at low salt of a 20-base pair region at each flanking side of the nucleosome core particle. The possible functional relevance of these results is discussed.     

Urea perturbation and the reversibility of nucleohistone conformation

Urea effect on conformation and thermal stabilities in nucleohistone and NaCl-treated partially dehistonized nucleohistones has been studied by circular dichroism (CD) and thermal denaturation. Urea imposes a CD change at 278mm of DNA base pairs in native and NaCl-treated nucleohistones which can be decomposed into two parts: a decrease in Δε₂₇₈ for histone-free base pairs and an increase for histone-bound base pairs. The reduction by urea of Δε₂₂₀ of bound histones is approximately proportional to the increase of Δε₂₇₈ of histone-bound base pairs. Urea also lowers the melting temperatures of base pairs both free and bound by histones. The presence of urea indeed destroys the secondary structure of bound histones, causing changes in the conformation and thermal stabilities of histone-bound base pairs in nucleohistone. Such a urea perturbation on nucleohistone conformation is reversible.     

Comparison between histones FV and F2a2 of chicken erythrocyte. II. Interaction with homologous DNA.

The conformation and stability of artificial complexes between chicken erythrocyte DNA and homologous histones FV and F2a2 was studied by circular dichroism (CD) and thermal denaturation followed by both absorbance and CD measurements. The complexes are made after a stepwise potassium fluoride gradient dialysis without urea and studied at low ionic strength (10-minus 3 M). 1) No structural changes of the DNA can be detected up to r equals 0.2 with FV and r equals 0.6 for F2a2. With FV at higher values of r the CD spectrum is altered, indicating the organization of DNA and histones in some kind of aggregate. 2) The conformation of histone molecules inside the complexes is not related to the ionic strength of the medium but to an effective ionic environment close to 0.1 M. This ionic strength would also correspond to the melting temperature of histone-covered DNA. 3) From the analysis of the absorbance melting profile the length of DNA covered with an histone molecule can be estimated. A good agreement is found between the negative charge of this piece of DNA and the net positive charge of the histone. 4) Since the CD transition at 227 nm occurs before the second absorbance transition at 280 nm, the DNA is stabilized no longer by native histone but partially or fully denatured histones. The helical regions of the histone molecule are not involved in the binding process, which appears to be almost purely coulombian and most likely related to some structural fit between the pattern of negative charges in the DNA helix and that of positive charges along the peptide chain.     

Isopycnic banding of chromatin in chloral hydrate gradients

Chromatin from Ehrlich ascites tumor cells bands in chloral hydrate gradients at densities ranging from 1.40 to 1.60 g/cm³. The total chromatin after recovery from the gradient has a composition similar to that of native chromatin. Chloral hydrate does not dissociate the chromatin into its nucleic acid and protein components. The chloral hydrate is inert toward both the DNA and the histone, as indicated by the lack of influence either on the melting curves of the DNA, or on the electrophoretic patterns of the histones, when these substances are recovered from chromatin treated with chloral hydrate. The resolution of chromatin on the gradients reflects different buoyant densities of the chromatin, and this in turn, different protein to DNA ratios. Extraction of the histones from chromatin obtained from different regions of the gradients reveals differences in prevalence of at least two of the histone species, and provides evidence suggesting that different stretches of DNA in the genome may associate with different histones.     

Cytofluorometric studies on conformation of nucleic acids in situ. II. Denaturation of deoxyribonucleic acid.

Thermal denaturation of deoxyribonucleic acid (DNA) in situ in individual unbroken cells is studied by a cytofluorometric method. This method allows us to investigate DNA denaturation in the presence of divalent cations at concentrations reported to be necessary to maintain native structure of nuclear chromatin. Under these conditions the pattern of DNA denaturation is very different than when studied in the presence of ethylenediaminetetraacetate or citrate. The results suggest that with divalent cations present, the histone basic charges are more uniformly distributed along whole nuclear DNA. Various cell types exhibit great differences in sensitivity to DNA denaturation when assayed in the presence of 1 mM MgCl2. Human lymphocytes, monocytes and certain kinds of human leukemic cells show differences large enough to be used as a parameter for their recognition in mixed samples. Possible applications of the method in basic research on chromatin conformation and as a tool for cell recognition in diagnostic cytology or in the classification of human leukemia are proposed.     

Thermal denaturation of nucleohistones--effects of formaldehyde reaction

Thermal denaturation of native or partially dehistonized nucleohistones shows two melting bands at 66 and 81° in 2.5 × 10^?4 M EDTA, pH 8.0. These correspond to the melting of DNA segments bound by the less basic and the more basic half-molecules of histones, respectively. These two melting bands combine into a broad melting band from around 70 to 85° when these nucleohistones are pre-treated with formaldehyde. A formaldehyde reaction which fixes histones on DNA by covalent bonds account for the effect. Formaldehyde fixation also increases the melting temperature of some free DNA segments from around 42 to around 55°. This is interpreted as a result of closed or rigid boundaries between free DNA and formaldehyde-reacted histone-bound DNA segments. MgCl₂ dissociates histones from DNA more effectively and leaves longer free DNA segments than does NaCl. Thermal denaturation of a formaldehyde-reacted nucleoprotein thus provides an effective tool for comparing the relative size of free DNA regions on nucleoproteins. The effect of reversible binding of ligands on helix-coil transition of DNA is descussed and found not adequate for thermal denaturation of nucleohistones.     

Distribution of histone F1 on calf thymus nucleohistone DNA.

A method of large-scale preparation of the histone F1-DNA complex by removing all other proteins from calf thymus nucleohistone was established. This involved gel filtration of nucleohistone through a column containing a band of sodium dodecyl sulfate. The F1-DNA complex obtained had the original amount of F1 and no other. The F1-DNA complex exhibited distinct two-step melting on thermal denaturation. The first step was apparently attributable to naked DNA regions and the second step, about 30 deg. C higher than the first step, to the regions covered with F1. Buoyant density experiments with the complex after fixation with formalin revealed that F1 was distributed fairly evenly over DNA fragments of an average molecular weight of about 4 × 10⁶. Electron microscopic examination of the complex after various degrees of denaturation with formalin indicated that the longest stretch of unbound DNA was about 0·3 μm.     

Circular dichroism as a probe of DNA structure inside reconstituted nucleohistones.

Reconstituted nucleohistones were obtained by mixing in given conditions acid extracted histones and eukaryotic DNA. The histone/DNA ratio (w/w) was in the range 0.35 - 0.95. With the four histones (H2A2B) we have been able to obtain subunits (nucleosomes or upsilon-bodies). The variation of cirsular dichroism signal with temperature at 280 nm was measured to follow structural changes of the DNA inside the complex. The true change of ellipticity (see article) of histone-bound DNA regions, is similar for reconstituted nucleohistone and H1-depleted chromatin, and is therefore a physical probe of the presence of nucleosomes.     

Helix-coil transition in nucleoprotein: renaturation of polylysine-DNA and polylysine-nucleohistone complexes

Thermal denaturation and renaturation of directly mixed and reconstituted polylysine–DNA, directly mixed polylysine–nucleohistone complexes, and NaCl-treated nucleohistones in 2.5 × 10^?4 M EDTA, pH 8.0 have been studied. At the same input ratio of polylysine to DNA, the percent of renaturation of free base pairs in a directly mixed polylysine–DNA complex is higher than that in a reconstituted complex. For a directly mixed complex, the renaturation of free base pairs is proportional to the fraction of DNA bound by polylysine or inversely proportional to the sizes of free DNA loops. A of large amount of renaturation of free base pairs has also been observed for 0.6 M and 1.6 M NaCl-treated nucleohistones. The binding of polylysine to nucleohistone enhances the renaturation of histone-bound base pairs. The percent of renaturation of polylysine–bound base pairs is high and is approximately independent of the extent of binding on DNA by polylysine. This is true in polylysine–DNA complexes prepared either by reconstitution or by directly mixing. It also applies for polylysine–nucleohistone complexes. The model where polylysine-bound base pairs collapse at T_m′ with two complementary strands still bound by polylysine is favored over the model where polylysine is dissociated from DNA during melting. The low renaturation of histone-bound base pairs in nucleo-histone indicates that either histones do not hold two complementary strands of DNA tightly or that histones are fully or partially dissociated from DNA when the nucleo-histone is fully denatured.     

Specific degradation of histones H1 and H3

It is shown that acid treated histones H1 and H3 are susceptible to specific degradation by an associated acid resistant protease. Dialysis against distilled water (pH 5.5–6) of the acid treated histones enhances proteolysis. On the other hand, no degradation is observed in nucleohistone either in the presence of Ca⁺⁺ or Na⁺⁺ ions. The conditions required to avoid degradation during nucleohistone and histone manipulation are described.     

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.     

Changes in chromatin structure induced by EDTA treatment and partial removal of histone H1.

Electron microscopy shows that EDTA treatment or partial removal of histone HI converts 200-250 A chromatin fibres characteristic for native chromatin, isolated in low ionic strength conditions into fibres consisting of nucleosomes connected by segments of DNA. This structural transition is accompanied by an increase in the amplitude of positive band of CD spectra at 280 nm. Comparison of electron microscopic, thermal denaturation and electrophoretic data suggests that multiphasic character of melting curves, observed for chromatin, lacking histone HI is due to the removal of histone HI and destabilisation of the DNA segments, connecting nucleosomes. It is also shown that bivalent cations play an important part both in the stabilisation of 200 A globules and of nucleosomes.     

Mechanics of chromatin template activation. Physical evidence for destabilization of nucleoproteins by polyanions.

Thermal denaturation profiles were used to quantitate the destabilizing activity of five classes of polyanions in an effort to explain previously reported differences in activity among apparently similar polymers. This physical assay for polyanion action on chromatin is free of the biochemical complications associated with template assay systems. Most polyanions tested caused a thermal destabilization of the normal nucleohistone complexes of rat thymus chromatin and exposed free or weakly complexed DNA. Polyphosphates dissociated chromatin even when their net charge was as low as 6, and activity per unit of weight increased with chain length only for polymers containing less than about 20 phosphate units; pyrophosphate was inactive. Two polymers which had no influence on chromatin had a low negative charge density. Some, such as polycytidlate and denatured DNA, possessed a high charge but caused only minor changes in the shape of denaturation profiles and exposed little or no free DNA. The highly active nucleic acids, ribosomal RNA and polyguanylate, were distinguishable from inactive nucleic acids by having more secondary structure and potentially less steric hindrance for anionic interaction with chromatin. There was evidence that some polyanions dissociate certain histones before others. Although data obtained show that some histones may be redistributed from chromatin onto added double-stranded DNA in a medium containing urea, this does not appear to interfere significantly with the interpretation of typical thermal denaturation profiles of chromatin.     

Thermodynamics of condensation of nuclear chromatin. A differential scanning calorimetry study of the salt-dependent structural transitions

We present a detailed thermodynamic investigation of the conformational transitions of chromatin in calf thymus nuclei. Differential scanning calorimetry was used as the leading method, in combination with infrared spectroscopy, electron microscopy, and techniques for the molecular characterization of chromatin components. The conformational transitions were induced by changes in the counterion concentration. In this way, it was possible to discriminate between the interactions responsible for the folding of the higher order structure and for the coiling of nucleosomal DNA. Our experiments confirm that the denaturation of nuclear chromatin at physiological ionic strength occurs at the level of discrete structural domains, the linker and the core particle, and we were able to rule out that the actual denaturation pattern might be determined by dissociation of the nucleohistone complex and successive migration of free histones toward native regions, as recently suggested. The sequence of the denaturation events is (1) the conformational change of the histone complement at 66 degrees C, (2) the unstacking of the linker DNA at 74 degrees C, and (3) the unstacking of the core particle DNA, that can be observed either at 90 or at 107 degrees C, depending on the degree of condensation of chromatin. Nuclear chromatin unfolds in low-salt buffers, and can be refolded by increasing the ionic strength, in accordance with the well-known behavior of short fragments. The process is athermal, therefore showing that the stability of the higher order structure depends on electrostatic interactions. The transition between the folded conformation and the unfolded one proceeds through an intermediate condensation state, revealed by an endotherm at 101 degrees C. The analysis of the thermodynamic parameters of denaturation of the polynucleosomal chain demonstrates that the wrapping of the DNA around the histone octamer involves a large energy change. The most striking observation concerns the linker segment, which melts a few degrees below the peak temperature of naked DNA. This finding is in line with previous thermal denaturation investigations on isolated chromatin at low ionic strength, and suggests that a progressive destabilization of the linker occurs in the course of the salt-induced coiling of DNA in the nucleosome.