Stepwise unfolding of chromatin by urea. A flow linear dichroism and photoaffinity labeling study

The unfolding of chromatin by urea (0-7 M) was studied by means of flow linear dichroism, photoaffinity labeling and nuclease digestion. The linear dichroism results indicate that the unfolding of the DNA is accomplished through two distinct transitions at 1-2 M urea and 6-8 M urea, respectively. The photoaffinity labeling studies indicate that an opening of the nucleosome histone core occurs above 2 M urea, accompanied by general loosening of the structure. Based on the results a model for the unfolding of chromatin fibers by urea is proposed, which includes a stretching of the linker DNA (0-2 M urea) followed by a "loosening" of the nucleosome core, possibly to a one-loop DNA conformation (2-6 M urea), and finally resulting in an almost total stretching of the DNA (greater than 6 M urea).     

Pathway-dependent reconstitution of chromatin structure from separated constituents.

Chicken reticulocyte chromatin can be reassembled from its separated constituents, viz. DNA, H1 plus H5, core histones, and non-histone proteins, to yield a product resembling the native starting material by a series of structural criteria. In particular, it possesses nucleosomes separated by spacer regions; the particles contain DNA with a unit length of approximately 200 base pairs. The recovery of the correctly reassembled product depends critically on the annealing conditions: the components are initially mixed in 2 M NaCl and 5 M urea, and it seems to be important to remove urea at a relatively high salt concentration. The results suggest that the characteristic chromatin structure is formed only when core histones bind to DNA in their native conformation and are followed by the addition of H1 and H5 to the spacer regions.     

Internal architecture of the core nucleosome: fluorescence energy transfer studies at methionine-84 of histone H4

Chicken histone H4, labeled separately at Met-84 with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid and 5-(iodoacetamido)fluorescein, was reassociated with unlabeled histones H2A, H2B, and H3 and 146 base pairs of DNA to produce fluorescently labeled nucleosomes having physical characteristics virtually the same as those of native core particles. Four types of particles were prepared containing respectively unlabeled H4, dansylated H4, fluoresceinated H4, and a mixture of the two labeled H4 molecules. Quantitative singlet-singlet energy-transfer measurements were carried out to determine changes in the distance between the two Met-84 H4 sites within the same nucleosome following conformational transitions which we have reported earlier. In the ionic strength range 0.1-100 mM NaCl, the distance between these sites is less than 2 nm except at 1 mM. Between 100 and 600 mM monovalent salt the distance separating the donor and acceptor fluors at Met-84 H4 increases to 3.8 nm. The conformational change centered around 200 mM NaCl is cooperative. Our results and those of others indicate that there is little unfolding of the histone octamer, at least around Met-84 H4, in the entire ionic strength range studied. A mechanism involving the rotation of the globular portion of H4 is proposed to account for this transition which occurs at physiological ionic strengths.     

Chromatin dynamics of unfolding and refolding controlled by the nucleosome repeat length and the linker and core histones

Chromatin is composed of genomic DNA and histones, forming a hierarchical architecture in the nucleus. The chromatin hierarchy is common among eukaryotes despite different intrinsic properties of the genome. To investigate an effect of the differences in genome organization, chromatin unfolding processes were comparatively analyzed using Schizosaccaromyces pombe, Saccharomyces cerevisiae, and chicken erythrocyte. NaCl titration showed dynamic changes of the chromatin. 400-1000 mM NaCl facilitated beads with approximately 115 nm in diameter in S. pombe chromatin. A similar transition was also observed in S. cerevisiae chromatin. This process did not involve core histone dissociation from the chromatin, and the persistence length after the transition was approximately 26 nm for S. pombe and approximately 28 nm for S. cerevisiae, indicating a salt-induced unfolding to "beads-on-a-string" fibers. Reduced salt concentration recovered the original structure, suggesting that electrostatic interaction would regulate this discrete folding-unfolding process. On the other hand, the linker histone was extracted from chicken chromatin at 400 mM NaCl, and AFM observed the "beads-on-a-string" fibers around a nucleus. Unlike yeast chromatin, therefore, this unfolding was irreversible because of linker histone dissociation. These results indicate that the chromatin unfolding and refolding depend on the presence and absence of the linker histone, and the length of the linker DNA.     

Nucleosome cores containing H2B,H3, H4 and HMG2 are reconstituted

Nucleosome cores mixed with the high mobility group proteins, HMG1 and HMG2, in 2 M NaCl, 5 M urea, 0.2 mM EDTA and 10 mM Tris pH 7.0, have been reconstituted by salt gradient dialysis. The reconstituted material, in 10 mM Tris pH 7.0, had a sedimentation peak at the same position as that of control nucleosome cores in sucrose density gradient ultracentrifugation. The SDS polyacrylamide gel electrophoresis of the reconstituted nucleosome cores demonstrated that they contain H2B, H3, H4 and HMG2 and are selectively deficient in H2A. The circular dichroism of DNA of the reconstituted cores was indistinguishable from that of control nucleosome cores. The results suggest that HMG2 replaces H2A as a component of the nucleosome histone core during reconstitution.     

Nucleosome core particle self-assembly kinetics and stability at physiological ionic strength

Micrococcal nuclease, DNase I, and trypsin have been employed to study the kinetics of core particle self-assembly by salt jump from 2.0 to 0.2 M NaCl. A few seconds after the initiation of the reassociation reaction, the bulk of core particle DNA becomes protected from digestion by micrococcal nuclease, whereas free DNA, under the same conditions, is completely hydrolyzed. The central and C-terminal regions of core histones are also protected from trypsin digestion immediately after the 2.0-0.2 M NaCl salt jump. Moreover, the extent of degradation produced by trypsin is the same for samples digested a few seconds after the salt jump and for samples digested 20 min after the salt jump. With DNase I, minor structural differences have been detected between samples obtained at different times during the reaction. However, even in this case our results indicate that many of the characteristic histone-DNA contacts within the core particle are made a few seconds after the initiation of the self-assembly reaction. Furthermore, core particles have been labeled with the fluorescent reagent N-(1-pyrenyl)maleimide (NPM), which was previously used as a sensitive probe for nucleosome conformation. Extensive DNase I or trypsin digestion of NPM-labeled core particles in 0.2 M NaCl does not produce significant changes in excimer fluorescence. This allows us to conclude that the covalent continuity of DNA is not required for the maintenance of the folded conformation of the core particle and that the trypsin-resistant domains of core histones play a fundamental role in the stabilization of this structure.     

Role of histone tyrosines in nucleosome formation and histone-histone interaction

We have studied the functional properties of iodinated histones. Isolated, denatured histones were iodinated at trace levels and then renatured together with carrier histones and high molecular weight DNA to form nucleohistone. Nucleosomes were prepared from the reconstitute using micrococcal nuclease, and the relative representations of the individual iodinated tyrosines of the histones in the reconstituted nucleosomes were determined. Our principal findings are 1) that denatured histones can be iodinated at any tyrosine without interfering in subsequent nucleosome reconstitution and 2) that the resulting reconstituted nucleosomes nevertheless possess histone cores of altered stability, being either more or less stable depending on the particular tyrosine which is iodinated. We show that tyrosines 37, 40, and 42 of H2B are protected from iodination in intact core particles, as expected since these tyrosines lie within the H2B-H2A binding site. Yet iodination of these tyrosines in denatured H2B does not interfere with nucleosome assembly. However, the histone cores isolated from these reconstituted nucleosomes are of diminished stability as assayed by Sephadex column chromatography in 2 M salt. In contrast, iodination of tyrosines 83 and 121 of H2B, as well as iodination of the tyrosines of H2A, increases the stability of the histone octamer core. Iodination of H4 tyrosine 72 is without effect on histone octamer stability. Tyrosine iodination constitutes a profound amino acid alteration in the context of the absolute evolutionary conservation of most histone tyrosines. For example, all H2Bs sequenced to date, from fungi to mammals, possess tyrosines at positions 37, 40, and 42. Our results suggest that the immutability of these tyrosines reflects some sophisticated function of the nucleosome histone core beyond the assembly and mere maintenance of a compact structure.     

Dissociation of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach by urea

The dissociation of D-ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach, which consists of eight large subunits (L, 53 kDa) and eight small subunits (S, 14 kDa) and thus has a quarternary structure L8S8, has been investigated using a variety of physical techniques. Gel chromatography using Sephadex G-100 indicates the quantitative dissociation of the small subunit S from the complex at 3-4 M urea (50 mM Tris/Cl pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol and 5 mM 2-mercaptoethanol). The dissociated S is monomeric. Analytical ultracentrifuge studies show that the core of large subunits, L, remaining at 3-4 M urea sediments with S20, w = 15.0 S, whereas the intact enzyme (L8S8) sediments with S20, w = 17.7S. The observed value is consistent with a quarternary structure L8. The dissociation reaction in 3-4 M urea can thus be represented by L8S8----L8 + 8S. At urea concentrations c greater than 5 M the L8 core dissociates into monomeric, unfolded large subunits. A large decrease in fluorescence emission intensity accompanies the dissociation of the small subunit S. This change is completed at 4 M urea. No changes are observed upon dissociating the L8 core. The kinetics of dissociation of the small subunit, as monitored by fluorescence spectroscopy, closely follow the kinetics of loss of carboxylase activity of the enzyme. Studies of the circular dichroism of D-ribulose-1,5-bisphosphate carboxylase in the wavelength region 200-260 nm indicate two conformational transitions. The first one ([0]220 from -8000 to -3500 deg cm2 dmol-1) is completed at 4 M urea and corresponds to the dissociation of the small subunit and coupled conformational changes. The second one ([0]220 from -3500 to -1200 deg cm2 dmol-1) is completed at 6 M urea and reflects the dissociation and unfolding of large subunits from the core. The effect of activation of the enzyme by addition of MgCl2 (10 mM) and NaHCO3 (10 mM) on these conformational transitions was investigated. The first conformational transition is then shifted to higher urea concentrations: a single transition ([0]220 from -8000 to -1200 deg cm2 dmol-1) is observed for the activated enzyme. From the urea dissociation experiments we conclude that both large (L) and small (S) subunits are important for carboxylase activity of spinach D-ribulose-1,5-bisphosphate carboxylase: the L-S subunit interactions tighten upon activation and dissociation of S leads to a coupled, proportional loss of enzyme activity.     

Physical properties of nucleoprotein cores from adenovirus type 5.

Analytical ultracentrifugation, thermal denaturation, and electron microscopy have been used to study nucleoprotein core particles, obtained from disrupted type 5 adenovirus and partially purified on glycerol density gradients. Electron microscopy at low salt concentrations has shown that the cores are homogeneous particles with characteristic structures, which vary with conditions of observation from a fairly loose network of fibers to a highly condensed, compact particle. Sedimentation measurements in the analytical ultracentrifuge, both by boundary and by band techniques, show that the cores are relatively homogeneous in solution and have sedimentation coefficients near 185 S at low salt concentrations, about 243 S in 1 or 2 M NaCl, and 376 S in 1 mM MgCl2. Correlation of sedimentation data with electron microscopic observations suggests that the 185 S particle has a loose, fibrous structure, while the faster species are more highly condensed particles. The melting temperature of the cores in 5 mM Tris/HCl is 79 degrees C, which is 10 degrees C higher than the Tm for purified, viral DNA. This indicates that the protein enhances the stability of DNA in the nucleoprotein complex.     

Ionic effects on the structure of nucleoprotein cores from adenovirus

Nucleoprotein cores, prepared from adenovirus type 5 with a deoxycholate/heat treatment, consist of the viral DNA and two major internal proteins. The core particles exhibit structural characteristics that are highly reproducible and dependent on their ionic environment. In low-ionic-strength buffer, the cores had a sedimentation coefficient of 180 S and appeared in the electron microscope as homogeneous particles with distinct centers from which numerous arms and loops radiated. Condensation of the cores was induced by Mg2+ or Ca2+ over the range 0 to 1 mM. The sedimentation coefficient increased monotonically with divalent cation concentration, reaching a maximum of 405 S in 1 mM Mg2+. A corresponding condensation in the core structure was observed by electron microscopy. Increasing concentrations of NaCl also produced a conformational change in the cores, with an almost linear increase in sedimentation velocity up to 274 S in 0.04 M NaCl. Between 0.05 and 1.0 M NaCl, the cores were insoluble. In 2.0 M NaCl, the cores were again soluble with an s20,w of 228 S. Under all ionic strength conditions in which the cores were soluble, both core proteins remained bound to the DNA.     

Evidence for the formation of nucleosome-like histone complexes on single-stranded DNA.

Using histones reconstituted with RNA and DNA celluloses, we have shown elsewhere that histones elute identically with salt from single- and double-stranded DNA, but differently from RNA (Palter and Alberts, 1979). In this paper we characterize further the suspected specific binding interactions between histones and single-stranded DNA. Nuclease digestion of complexes of histone reconstituted with single-stranded DNA generates only a small yield of discrete (approximately 9S) particles. We can, however, efficiently obtain such 9S "nucleosome-like" complexes when nuclease treatment is avoided and histones are reconstituted directly with short single-stranded DNA pieces. Strikingly, these 9S subunits contain an equimolar composition of the four nucleosomal histones. When these subunits are visualized in the electron microscope, they appear as globular particles which are morphologically indistinguishable from normal mononucleosomes. Based on their sedimentation properties, histone-to-DNA ratio, histone composition and particle diameter, we conclude that they represent an octamer of the four histones (containing two molecules of each histone) associated with single-stranded DNA. These data, viewed in the context of other information concerning chromatin, suggest that nucleosome cores may become transiently bound to single strands of DNA as DNA and RNA polymerases pass.     

Natural abundance carbon-13 nuclear magnetic resonance studies of histone and DNA dynamics in nucleosome cores

Natural abundance carbon-13 nuclear magnetic resonance spectra (67.9 MHz) were obtained for native nucleosome cores: cores dissociated in 2 M NaCl and 2 M NaCl, 6 M urea; and cores degraded with DNase I plus proteinase K. Phosphorus-31 NMR spectra of native and dissociated cores and core length DNA were also obtained at 60.7 MHz. The 31P resonance and spin-lattice relaxation time (T1) of DNA were only slightly affected by packaging in nucleosome cores, in agreement with other reports, but 13C resonances of DNA were essentially unobservable. The loss of DNA spectral intensity suggests that rapid internal motions of DNA sugar carbons in protein-free DNA previously demonstrated by 13C NMR methods are partly restricted in nucleosomes. The 13C spectrum of native cores contains many narrow intense resonances assigned to lysine side chain and alpha-carbons, glycine alpha-carbons, alanine alpha- and beta- carbons, and arginine side chain carbons. Several weaker resonances were also assigned. The narrow line widths, short T1 values, and non-minimal nuclear Overhauser enhancements of these resonances, including alpha- and beta-carbons, show that some terminal chain segments of histones in nucleosomes are as mobile as small random coil polypeptides. The mobile segments include about 9% of all histone residues and 25% of all lysines, but only 10% of all arginines. The compositions of these segments indicate that mobile regions are located in amino- or carboxyl-terminal sequences of two or more histones. In addition, high mobility was observed for side chain carbons of 45-50% of all lysines (delta and epsilon carbons) and about 25% of all arginines (zeta carbon) in histones (including those in mobile segments), suggesting that basic residues in terminal histone sequences are not strongly involved in nucleosome structure and may instead help stabilize higher order chromatin structure.     

The in vitro reconstitution of nucleosome and its binding patterns with HMG1／2 and HMG14／17 proteins

Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissociatedfrom DNA at 1M NaC1. When the salt concentration was slowly reduced to 650 mM and 300 mM, the core histones bound to the naked DNA gradually. Once the salt concentration was reduced to 50 mM the classic “beads-on-a-string“ structure was clearly visualized. Furthermore, using the technique of the in vitro reconstitution of nucleosome,the mono- and di- nucleosomes were assembled in vitro with both HS2core (-10681 to -10970 bp) and NCR2 (-372to -194 bp) DNA sequences in the 5‘flanking sequence of human b-globin gene. Data revealed that HMG 1/2 and HMG 14/17 proteins binding to both DNA sequences are changeable following the assembly and disassembly of nucleosomes. We suggest that the changeable binding patterns of HMG 14/17 and HMG1/2 proteins with these regulatory elements may be critical in the process of nucleosome assembly, recruitment of chromatin-modifying activities, and the regulation of human b-globin gene expression.     

The action of chymotrypsin on nucleosome cores. Histone products and conformational effects of limited digestion

alpha-Chymotrypsin was used to probe accessible hydrophobic amino acid residues in nucleosome cores. Small amounts of chymotrypsin rapidly and selectively cleaved at leucine 20 of histone H3. Cleavage at this site caused partial unfolding of the nucleosome core at low ionic strengths indicated by a small decrease in sedimentation coefficient and increase in circular dichroism in the 265-285-nm range. Unfolding did not occur at moderate ionic strengths, probably because of more effective electrolyte screening of residual negative charge on the nucleosome core. More extensive treatment with chymotrypsin partially degraded other core histones in nucleosome cores at similar rates. The primary sites of cleavage were assigned to Leu115 of H2a, Val18 or Gln22 of H2b, and Leu10 plus Leu22 of H4. We conclude that these primary sites of chymotrypsin cleavage of the four core histones lie on or near the nucleosome core surface, while the large number of other hydrophobic histone residues located in more central sequences must be inaccessible. Extensive chymotrypsin treatment yielded a set of "limit" products approximately 80-100 residues long that were similar to the limit products of trypsin digestion. Sedimentation coefficients and circular dichroism spectra of nucleosome cores treated to near limits with chymotrypsin or chymotrypsin followed by trypsin were not consistent with significant unfolding of the proteolyzed cores at moderate ionic strength. These results indicate that the amino-terminal 20-30 residues of H2b, H3, and H4 and the amino- and carboxyl-terminal approximately 12 residues of H2a, in toto, interact weakly if at all with DNA of isolated nucleosome cores. These histone termini stabilize less than two turns and perhaps only one turn on each DNA terminus.     

Studies on histone oligomers. V. Reconstitution of chromatin from purified DNA and acid-extracted histones

DNA-histone complexes were reconstituted from DNA and acid-extracted core histones and the products were characterized by micrococcal nuclease digestion to examine whether proper nucleosome structure had been reconstituted. No nucleosome structure was produced starting from the mixture of acid-extracted histones and purified DNA in 2 M NaCl-5 M urea, while the reassociation of chromatin by the same procedures was successful. This was due to the inappropriate conformation of acid-extracted histones, which was preserved in 2 M NaCl even in the presence of 5 M urea. If acid-extracted histones were reannealed from the completely denatured state, such as in 5 M urea, 6 M guanidine hydrochloride or 0.6 M NaCl-5 M urea, reconstitution of nucleosome structure was always successful.     

Effects of pH on the stability of chromatin core particles.

Chromatin core particles near physiological ionic strength undergo a reversible transition induced by changes in pH near neutrality. While sedimentation studies indicate no significant effect on size or shape, changes in tyrosine fluorescence anisotropy and in circular dichroism suggest a somewhat looser structure at high pH. Further support of this suggestion is given by high salt dissociation experiments; at pH 8 core particles begin to show changes at lower salt concentration than at pH 6. The pH transition appears unaffected by the presence of Mg2+ but can be blocked by crosslinking of the histones. A possible relationship is suggested between this transition and increases in intracellular pH which correlate with enhancement in several aspects of cellular activity including DNA replication.     

Primary organization of nucleosomes containing all five histories and DNA 175 and 165 base-pairs long

The sequential arrangement of histones along DNA in nucleosomes containing all five histones and DNA about 165 and 175 base-pairs in length has been determined. The data provide evidence that core histones (H2A, H2B, H3 and H4) are arranged in nucleosomes and nucleosome core particles in a largely similar way with the following differences. (1) On nucleosomal DNA about 175 basepairs long core histones are probably shifted by 20 nucleotides on one DNA strand and by 10 nucleotides on the complementary DNA strand from the 5′ end. On nucleosomal DNA 165 base-pairs long, histones appear to be shifted by 10 nucleotides from the 5′ end of DNA on both the DNA strands. (2) Histone H3 is extended beyond core DNA and is bound to the 3′ end of DNA about 175 nucleotides long. Thus, core histones span the whole length of nucleosomal DNA. (3) Histone H2A seems to be absent from the central region of nucleosomal DNA. These results indicate that during the preparation of core particles, some rearrangement of histones or some of their regions occurs.Histone H1 has been shown to be bound mainly to the ends of nucleosomal DNA and, along the whole DNA length, to the gap regions that are free of core histones.