The mechanism of nucleosome assembly onto oligomers of the sea urchin 5 S DNA positioning sequence

We have used a model system composed of tandem repeats of Lytechinus variegatus 5 S rDNA (Simpson, R. T., Thoma, F., and Brubaker, J. M. (1985) Cell 42, 799-808) reconstituted into chromatin with chicken erythrocyte core histones to investigate the mechanism of chromatin assembly. Nucleosomes are assembled onto the DNA template by mixing histone octamers and DNA in 2 M NaCl followed by stepwise dialysis into very low ionic strength buffer over a 24-h period. By 1.0 M NaCl, a defined intermediate composed of arrays of H3.H4 tetramers has formed, as shown by analytical and preparative ultracentrifugation. Digestion with methidium propyl EDTA.Fe(II) indicates that these tetramers are spaced at 207 base pair intervals, i.e. one/repeat length of the DNA positioning sequence. In 0.8 M NaCl, some H2A.H2B has become associated with the H3.H4 tetramers and DNA. Surprisingly, under these conditions DNA is protected from methidium propyl EDTA.Fe(II) digestion almost as well as in the complete nucleosome, even though these structures are quite deficient in H2A.H2B. By 0.6 M NaCl, nucleosome assembly is complete, and the MPE digestion pattern is indistinguishable from that observed for oligonucleosomes at very low ionic strength. Below 0.6 M NaCl, the oligonucleosomes are involved in various salt-dependent conformational equilibria: at approximately 0.6 M, a 15% reduction in S20,w that mimics a conformational change observed previously with nucleosome core particles; at and above 0.1 M, folding into a more compact structure(s); at and above 0.1 M NaCl, a reaction involving varying amounts of dissociation of histone octamers from a small fraction of the DNA templates. In low ionic strength buffer (less than 1 mM NaCl), oligonucleosomes are present as fully loaded templates in the extended beads-on-a-string structure.     

The structure of SV 40 chromatin.

Simian virus 40 (SV40) nucleoprotein complexes were studied with the electron microscope. Depending on the isolation procedure, SV40 chromatin has two different conformations: complexes isolated in the presence of 0.15 M NaCl appeared as very compact globular structures, while those isolated in the presence of 0.6 M NaCl had the typical 'beads-on-a-string' appearance of the primary nucleofilament. Concomitant with this structural change was a variation in the histone pattern and sedimentation behaviour of the complexes: with NaCl at 0.15 mol 1(-1) the isolated complexes contained both the nucleosomal histones and histone H1, and sedimented in sucrose gradients at 70S. Increasing the ionic strength to 0.6 M NaCl resulted in the removal of histone H1 from the complexes and in a decrease of the sedimentation coefficient to 40S. DNA relaxing enzyme is associated with the SV40 nucleoprotein complexes. The numbers of superhelical turns in DNA from compact and open types of complexes were found to be the same. Therefore the transition from the condensed to the open structure of viral chromatin does not require a change in the topological winding number of its DNA.     

Specific interaction of histone H1 with eukaryotic DNA.

The interaction of calf thymus histone H1 with homologous and heterologous DNA has been studied at different ionic strengths. It has been found that about 0.5 M NaCl histone H1, and its fragments N-H1 (residues 1-72) and C-H1 (residues 73-C terminal), precipitate selectively a small fraction of calf thymus DNA. This selective precipitation is preserved up to very high values (less than 2.0) of the input histone H1/DNA ratio. The percentage of DNA insolubilized by histone H1 under these ionic conditions is dependent upon the molecular weight of the nucleic acid, diminishing from 18% fro a Mw equals 1.0 x 10(7) daltons to 5% for a Mw equals 8.0 x 10(4) daltons. The base composition of the precipitated DNA is similar to that of the bulk DNA. Calf thymus histone H1 also selectively precipitates a fraction of DNA from other eukaryotes (herring, trout), but not from some prokaryotes (E. coli, phage gamma. On the other hand, at 0.5 M NaCl, the whole calf thymus DNA (but not E. coli DNA) presents a limited number of binding sites for histone H1, the saturation ratio histone H1 bound/total DNA being similar to that found in chromatin. A similar behavior is observed from the histone H1 fragments, N-H1 and C-H1, which bind to DNA in complementary saturation ratios. It is suggested that in eukaryotic organisms histone H1 molecules maintain specific interactions with certain DNA sequences. A fraction of such specific complexes could act as nucleation points for the high-order levels of chromatin organization.     

Fractionation of nucleosomes by salt elution from micrococcal nuclease- digested nuclei

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaCl has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13-- 17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCl. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaCl concentrations in content of nonhistone chromosomal proteins. 40--60% of the nucleosomes were released by 0.3 M NaCl with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaCl. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaCl. These fractions contained the DNA least available for micrococcal nuclease attach. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by 0.2 M NaCl suggest that this population is involved in a special function.     

Structural transition in chromatin induced by ions in solution

Structural transition in chromatin was measured as a function of counter ions in solution (NaCl or MgCl(2)) and of histones bound on the DNA. The addition of counter ions to aqueous solutions of chromatin, partially dehistonized chromatin, and DNA caused a drastic reduction in viscosity and a significant increase in sedimentation coefficient. Transitions occurred primarily at about 2 x 10(-3) M NaCl and 1 x 10(-5) M MgCl(2) and are interpreted as a change in structure of chromatin induced by tight binding of cations (Na(+) or Mg(++)) to DNA, either free or bound by histones, and is an intrinsic property of DNA rather than of the type of histone bound. At a given ionic condition, removal of histone H1 from chromatin had only a minor effect on the hydrodynamic properties of chromatin while removal of other histones caused a drastic change in these properties. An increase in the sedimentation coefficient of DNA was observed also for protamine. DNA complexes wherein the bound protein contains only unordered coil rather than the alpha-helices found in histones.     

Isolation and characterization of a spacerless dinucleosome from H1-deleted chromatin.

Calf thymus chromatin, depleted in histone H1, was digested with micrococcal nuclease and fractionated by column chromatography. 140 base pair nucleosome core particles were isolated along with an unusual particle containing 2 histone octamers and 240 base pairs of DNA. Evidence is presented that the spacer DNA region is absent from these modified dinucleosomes, which appear as stable products of the digestion process. The physical properties of both particles are presented along with brief speculation on their possible origin and function.     

The role of histone H2B from sea urchin sperm in the association of reconstituted minichromosomes

A comparative study of the condensation of reconstituted complexes of circular SV40 DNA with core histones from calf thymus and sea urchin sperm was performed using sedimentation and electron microscopic techniques. It is shown that in low ionic strength solutions both types of complexes are similar to native minichromosomes. In the region from 0.08 to 0.16 M NaCl the complexes of SV40 DNA with thymus histones form small compact particles. By contrast, the compaction of the SV40 DNA complexes with sperm histones results in the formation of giant intermolecular associates. The results obtained may mean that histone H2B of sea urchin sperm participates in the formation of a higher order structure in sperm chromatin.     

Rearrangement of chromatin structure induced by increasing ionic strength and temperature.

Native rat liver chromatin fragments exposed to 600 mM NaCl at 37 degrees C for 45 min exhibit substantial modification of their original (approximately 200 base pairs) repeating subunit structure: a new repeat of 140 base pairs, superimposed on a high background, is observed after micrococcal nuclease digestion. The same material appears, in the electron microscope, as clusters of tightly packed beads connected by stretches of 'free' DNA. These modifications are not observed when the native chromatin is incubated at 37 degrees C at NaCl concentrations up to 400 mM. When native rat liver chromatin depleted of histone H1 by tRNA extraction is exposed to ionic strengths up to 600 mM NaCl at 4 degrees C, almost no modifications of the original native repeating structure are observed. However, when the incubation is carried out at 37 degrees C in 150, 300 or 400 mM NaCl, rearrangements of the native structure occur as indicated by micrococcal nuclease digestion and electron microscopic studies. Incubation of H1-depleted chromatin at 600 mM NaCl for 45 min at 37 degrees C induces, as for the native chromatin, a complete rearrangement characterized by the appearance of a 140-base-pair repeat superimposed on a high background upon digestion by micrococcal nuclease. It is suggested that these rearrangements are mediated by hydrophobic interactions between the histone cores and are prevented at ionic strengths lower than 500 mM by the presence of histone H1.     

Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks

The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion. The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated. However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30-40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate-dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair.     

Histone hyperacetylation can induce unfolding of the nucleosome core particle.

A direct correlation exists between the level of histone H4 hyperacetylation induced by sodium butyrate and the extent to which nucleosomes lose their compact shape and become elongated (62.0% of the particles have a length/width ratio over 1.6; overall mean in the length/width ratio = 1.83 +/- 0.48) when bound to electron microscope specimen grids at low ionic strength (1mM EDTA, 10mM Tris, pH 8.0). A marked proportion of elongated core particles is also observed in the naturally occurring hyperacetylated chicken testis chromatin undergoing spermatogenesis when analyzed at low ionic strength (36.8% of the particles have a length/width ratio over 1.6). Core particles of elongated shape (length/width ratio over 1.6) generated under low ionic strength conditions are absent in the hypoacetylated chicken erythrocyte chromatin and represent only 2.3% of the untreated Hela S3 cell core particles containing a low proportion of hyperacetylated histones. The marked differences between control and hyperacetylated core particles are absent if the particles are bound to the carbon support film in the presence of 0.2 M NaCl, 6mM MgCl2 and 10mM Tris pH 8.0, conditions known to stabilize nucleosomes. A survey of the published work on histone hyperacetylation together with the present results indicate that histone hyperacetylation does not produce any marked disruption of the core particle 'per se', but that it decreases intranucleosomal stabilizing forces as judged by the lowered stability of the hyperacetylated core particle under conditions of shearing stress such as cationic competition by the carbon support film of the EM grid for DNA binding.     

Chromatin structure from the marine sponge Geodia cydonium

The histones isolated from the siliceous sponge Geodia cydonium have been separated using two electrophoretic techniques. A comparison of their mobilities with those of calf thymus and rat liver show that some Geodia histone species (H3, H1 and H1(0) exhibit electrophoretic variance. The results show, that as in other eukaryotic systems the sponge chromatin contains the core histones (H2A, H2B, H3 and H4) and the linker histone (H1). ADP-ribosylation of Geodia histones and separation of the individual histones by electrophoresis resulted in four histones being radiolabeled. Digestion of Geodia chromatin with endogenous endonuclease is shown to result in the formation of nucleosome particles containing approximately 200 base pairs of DNA. A major product of endogenous endonuclease digestion is a relatively stable 110 base pair intermediate. Incubation of chromatin with DNase II and separation of the products under denaturing conditions reveals 20 bands migrating at 10 base intervals.     

Structure of subnucleosomal particles. Tetrameric (H3/H4)2 146 base pair DNA and hexameric (H3/H4)2(H2A/H2B)1 146 base pair DNA complexes

The tetrameric (H3/H4)2 146 base pair (bp) DNA and hexameric (H3/H4)2(H2A/H2B)1 146 bp DNA subnucleosomal particles have been prepared by depletion of chicken erythrocyte core particles using 3 or 4 M urea, 250 mM sodium chloride, and a cation-exchange resin. The particles have been characterized by cross-linking and sedimentation equilibrium. The structures of the particles, particularly the tetrameric, have been studied by sedimentation velocity, low-angle neutron scattering, circular dichroism, optical melting, and nuclease digestion with DNase I, micrococcal nuclease, and exonuclease III. It is concluded that since the radius of gyration of the DNA in the tetramer particle and its maximum dimension are very close to those of the core particle, no expansion occurs on removal of all the H2A and H2B. Nuclease digestion results indicate that histones H3/H4 in the tetramer particle protect a total of 70 bp of DNA that are centrally located within the 146 bp. Within the 70 bp DNA length, the two terminal regions of 10 bp are, however, not strongly protected from digestion. The optical melting profile of both particles can be resolved into three components and is consistent with the model of histone protection of DNA proposed from nuclease digestion. The structure proposed for the tetrameric histone complex bound to DNA is that of a compact particle containing 1.75 superhelical turns of DNA, in which the H3 and H4 histone location is the same as found for the core particle in chromatin by histone/DNA cross-linking [Shick, V. V., Belyavsky, A. V., Bavykin, S. G., & Mirzabekov, A. D. (1980) J. Mol. Biol. 139, 491-517]. Optical melting of the hexamer particle shows that each (H2A/H2B)1 dimer of the core particle protects about 22 base pairs of DNA.     

Viscosity of chromatin solutions increases with increasing ionic strength

Increasing the ionic strength of rat liver chromatin solutions above 0.4 M causes increasing viscosity. This indicates transformation of the compact chromatin molecules to more elongated forms. In the range of 0.4–0.5 M ionic strength histone H1 is dissociating continuously from the chromatin and the quaternary structure chromatin unravels. At ionic strength higher than 0.5 M the viscosities of chromatin solutions are furthermore increasing due to structural deformation. Near 0.7 M ionic strength the core histones H2A and H2B begin to dissociate from the chromatin, and the opening of the nucleosome cores leads to increasing elongation of the chromatin molecules.     

Porcine follitropin. The amino-acid sequence of the beta subunit

By treatment with tRNA in the presence of 1 mM MgCl2, a chromatin preparation was obtained containing all five major histone fractions but lacking a considerable portion of non-histone proteins. This chromatin preparation as well as chromatin extracted with 0.6 M NaCl (depleted of H1 histone and some non-histone proteins) were characterized in respect of solubility and chromatin DNA accessibility. Both samples possessed practically the same solubility in the presence of 0.15 M NaCl and 1 mM MgCl2. The solubility of tRNA-treated chromatin in 5 and 10 mM MgCl2 was higher than that of salt-extracted chromation. The accessibility of the DNA of these chromatin preparations was tested with DNA-dependent RNA polymerase of Escherichia coli as a probe, using procedure that permits measurement of binding site frequency. Both tRNA-treated and salt-extracted chromatin contained as many as 33% and untreated chromatin as few as 4% of the number of binding sites found on protein-free DNA. These results demonstrate that at least in part the non-histone proteins are responsible for salt-induced insolubility and low DNA accessibility of chromatin, thus revealing the importance of non-histone proteins in the maintenance of an overall chromatin structure.     

Human SWI/SNF nucleosome remodeling activity is partially inhibited by linker histone H1

The physical structure and the compact nature of the eukaryotic genome present a functional barrier for any cellular process that requires access to the DNA. The linker histone H1 is intrinsically involved in both the determination of and the stability of higher order chromatin structure. Because histone H1 plays a pivotal role in the structure of chromatin, we investigated the effect of histone H1 on the nucleosome remodeling activity of human SWI/SNF, an ATP-dependent chromatin remodeling complex. The results from both DNase I digestion and restriction endonuclease accessibility assays indicate that the presence of H1 partially inhibits the nucleosome remodeling activity of hSWI/SNF. Neither H1 bound to the nucleosome nor free H1 affected the ATPase activity of hSWI/SNF, suggesting that the observed inhibition of hSWI/SNF nucleosome remodeling activity depends on the structure formed by the addition of H1 to nucleosomes.     

Histones H2a, H2b, H3, and H4 form a tetrameric complex in solutions of high salt.

In 2 M NaCl, histones H2b, H2a, H3, and H4 form a heterotypic tetrameric complex made up of one chain of each histone. This complex has been analyzed by hydrodynamic techniques. It is indistinguishable from histones in chromatin by its resistance to trypsin, pattern of reactivity with 125I. and ability to form specific crosslinked products after treatment with formaldehyde. It is proposed that this complex is responsible for protecting the small DNA fragments produced by exhausting nuclease digestion of nuclei and that on the average two of these complexes protect the larger 180-200 base pair unit produced by partial treatment of nuclei with nuclease.     

Structural changes of the chromatin DNA in situ in partial deproteinization of DNP by a 0.6 M NaCl solution. A polarization-fluorescence microscopic study

By means of polarization fluorescence microscopy changes in polarization degree of fluorescence of DNA-bound of Acridine orange in situ were observed. A marked decrease in polarization degree of chromatin fluorescence was found after the treatment of cell nuclei with 0.6 M NaCl. It shows that the removal of histone H1 from the chromatin results in destabilization of its structure. The data obtained show that the measurements of polarization degree of fluorescence allow to obtain relevant information about structural changes in DNA chromatin.     

Influence of histone H1 on chromatin structure

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