Dependency of ISW1a chromatin remodeling on extranucleosomal DNA

The nucleosome remodeling activity of ISW1a was dependent on whether ISW1a was bound to one or both extranucleosomal DNAs. ISW1a preferentially bound nucleosomes with an optimal length of approximately 33 to 35 bp of extranucleosomal DNA at both the entry and exit sites over nucleosomes with extranucleosomal DNA at only one entry or exit site. Nucleosomes with extranucleosomal DNA at one of the entry/exit sites were readily remodeled by ISW1a and stimulated the ATPase activity of ISW1a, while conversely, nucleosomes with extranucleosomal DNA at both entry/exit sites were unable either to stimulate the ATPase activity of ISW1a or to be mobilized. DNA footprinting revealed that a major conformational difference between the nucleosomes was the lack of ISW1a binding to nucleosomal DNA two helical turns from the dyad axis in nucleosomes with extranucleosomal DNA at both entry/exit sites. The Ioc3 subunit of ISW1a was found to be the predominant subunit associated with extranucleosomal DNA when ISW1a is bound either to one or to both extranucleosomal DNAs. These two conformations of the ISW1a-nucleosome complex are suggested to be the molecular basis for the nucleosome spacing activity of ISW1a on nucleosomal arrays. ISW1b, the other isoform of ISW1, does not have the same dependency for extranucleosomal DNA as ISW1a and, likewise, is not able to space nucleosomes.     

Higher-order structures of chromatin in solution.

Neutron scatter studies have been made on gently prepared chicken erythrocyte chromatin over a range of ionic strength. At low ionic strength the mass per unit length of the '10 nm nucleofilament corresponds to one nucleosome per 8--12 nm and a DNA packing ratio of between 6 and 9. From the contrast dependence of the cross-section radius of gyration of the nucleofilament the following parameters have been obtained; RgDNA' the cross-section radius of gyration (Rg) when DNA dominates the scatter; RgP, the cross-section Rg when protein dominates the scatter; Rc, the cross-section Rg at infinite contrast and alpha, the constant which describes the dependence of the cross-section Rg on contrast variation. From our understanding of the structure of the core particle, various arrangement of core particles in the nucleofilament have been tested. In models consistent with the above parameters the core particles are arranged edge-to-edge or with the faces of the core particles inclined to within 20 degrees to the axis of the nucleofilament. With increase of ionic strength the transition to the second-order chromatin structure has been followed. This gave the interesting result that above 20 microM NaCL or 0.4 mM MgCL2 the cross-section Rg increases abruptly to about 9 nm with a packing ratio of 0.2 nucleosome/mn and with further increase of ionic strength the Rg increases to 9.5 nm while the packing ratio increases threefold to 0.6 nucleosome/nm. This suggests a family of supercoils of nucleosomes which contract with increasing ionic strength. In its most contracted form the diameter of the hydrated supercoil has been found from the radial distribution function to be 34 nm. Models for the arrangements of core particles in the 34-nm supercoil are discussed.     

A model for chromatin based upon two symmetrically paired half-nucleosomes

We propose that the basic unit of chromatin is constructed of two isologously paired heterotypic protein tetramers each containing one molecule of H2A, H2B, H3, and H4 histone. These proteins form a core that holds 140 base pairs (bp) of DNA in a single left-handed, non-interwound DNA supercoil approximately 95 bp in circumference, creating A nucleosome particle (DNA and protein) organized about a dyad axis of symmetry. Such a nucleosome can open up into its separate half-nucleosomes to allow genetic readout without requiring histone displacement     

The structure of the nucleosome core particle of chromatin in chicken erythrocytes visualized by using atomic force microscopy

The structure of the nuclosome core particle of chromatin in chicken erythrocytes has been examined by using AFM.The 146 bp of DNA wrapped twice around the core histone octamer are clearly visualized.Both the ends of entry/exit of linker DNA are also demonstrated.The dimension of the nucleosome core particles is - 1-4 nm in height and - 13-22 nm in width.In addition,superbeads (width of - 48-57 nm,height of - 2-3 nm )are occasionally revealed,two turns of DNA around the core particles are also detected.     

DNA stretching and extreme kinking in the nucleosome core

DNA stretching in chromatin may facilitate its compaction and influence site recognition by nuclear factors. In vivo, stretching has been estimated to occur at the equivalent of one to two base-pairs (bp) per nucleosome. We have determined the crystal structure of a nucleosome core particle containing 145 bp of DNA (NCP145). Compared to the structure with 147 bp, the NCP145 displays two incidences of stretching one to two double-helical turns from the particle dyad axis. The stretching illustrates clearly a mechanism for shifting DNA position by displacement of a single base-pair while maintaining nearly identical histone-DNA interactions. Increased DNA twist localized to a short section between adjacent histone-DNA binding sites advances the rotational setting, while a translational component involves DNA kinking at a flanking region that initiates elongation by unstacking bases. Furthermore, one stretched region of the NCP145 displays an extraordinary 55° kink into the minor groove situated 1.5 double-helical turns from the particle dyad axis, a hot spot for gene insertion by HIV-integrase, which prefers highly distorted substrate. This suggests that nucleosome position and context within chromatin could promote extreme DNA kinking that may influence genomic processes.     

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.     

Conformation of the HMG 14 nucleosome core complex from flow birefringence.

Flow birefringence and extinction angles have been measured for HMG 14 complexes with nucleosome core particles from chicken erythrocytes under cooperative "tight" binding conditions, and for the uncomplexed core particles used in the preparations. Results are interpreted using optical models for the observed DNA anisotropy, and are compared to recent small angle neutron scattering results. (19) The studies effectively rule out highly distorted DNA conformations and configurations in which DNA ends are unwound and extended. It is concluded that the most likely conformation of the complex is one in which the DNA superhelix is radially increased, either uniformly or bilaterally, with the DNA ends remaining tightly bound to the particle. This conformation does not require large changes in spatial relationships between the DNA ends compared to the uncomplexed core as would accompany, for example, significant unwinding of the ends. However, it may lead to more subtle but possibly highly significant differences in the angles at which the DNA exits the core particle.     

Self-assembly of single and closely spaced nucleosome core particles.

Self-assembly of DNA with the four core histones but in the absence of H1 generates nucleosome core particles which are spaced randomly over large distances. Closely spaced core particles, however, exhibit a preferred short linkage which is not a multiple of 10 base pairs. They bind about 140 base pairs whereas apparently shorter DNA lengths per nucleosome observed after digestion with micrococcal nuclease are the result of degradation from the ends. The DNA length of one superhelical turn in the core particle is 83 +/- 4 base pairs. Single core particles may bind more DNA than closely spaced core particles but probably less than two full turns of 168 base pairs. The internal structures of single and of native core particles are very similar as judged by their amount of DNA, sedimentation coefficient, appearance in the electron microscope, and digestion with DNase I. In addition to core particles, a particle is described which sediments at 9 S and consists of 108 base pairs of DNA bound to the histone octamer. It appears to be the smallest stable "core particle" but it is not a degradation product of the 146-base-pair core particle. Digestion of end-labeled 9 S and nucleosome core particles with DNase I shows distinct differences.     

Linker histone protection of chromatosomes reconstituted on 5S rDNA from Xenopus borealis:a reinvestigation.

The location of the linker histone (LH) on the nucleosome has been the subject of recent controversy. Although previous evidence had supported a location over the dyad axis, some recent experiments suggest an asymmetric, off-axis position. In this paper we show that the DNA sequence used to reconstitute chromatosomes in these experiments is prone to artifacts in nuclease digestion: results interpreted as 'protection' by LHs can be obtained with either naked DNA or with reconstituted core nucleosomes, in the absence of LHs. Consequently, we feel that general interpretation or extrapolation of such results must be regarded with the utmost caution. In addition, we show that the protection data on an alternative, previously unreported major core position on this same DNA sequence support a model of asymmetric, off-axis position of the LH, with linker DNA protection on only one side of the core particle.     

Neutron scattering studies of nucleosome structure at low ionic strength

Ionic strength studies using homogeneous preparations of chicken erythrocyte nucleosomes containing either 146 or 175 base pairs of DNA show a single unfolding transition at about 1.5 mM ionic strength as determined by small-angle neutron scattering. The transition seen by some investigators at between 2.9 and 7.5 mM ionic strength is not observed by small-angle neutron scattering in either type of nucleosome particle. The two contrasts measured (H2O and D2O) indicate that only small conformational changes occur in the protein core, but the DNA is partially unfolded below the transition point. Patterson inversion of the data and analysis of models indicate that the DNA in both types of particle is unwinding from the ends, leaving about one turn of supercoiled DNA bound to the histone core in approximately its normal (compact) conformation. The mechanism of unfolding appears to be similar for both types of particles and in both cases occurs at the same ionic strength. The unfolding observed for nucleosomes in this study is in definite disagreement with extended superhelical models for the DNA and also disagrees with models incorporating an unfolded histone core.     

The assembly of an H2A2,H2B2,H3,H4 hexamer onto DNA under conditions of physiological ionic strength

A novel nucleohistone particle is generated in high yield when a complex of DNA with the four core histones formed under conditions that are close to physiological (0.15 M NaCl, pH 8) is treated with micrococcal nuclease. The particle was found to contain 102 base pairs of DNA in association with six molecules of histones in the ratio 2H2A:2H2B:1H3:1H4 after relatively brief nuclease treatment. Prolonged nuclease digestion resulted in a reduction in the DNA length to a sharply defined 92-base pair fragment that was resistant to further degradation. Apparently normal nucleosome core particles containing two molecules each of the four core histones in association with 145 base pairs of DNA and a particle containing one molecule each of histones H2A and H2B in association with approximately 40 base pairs of DNA were also generated during nuclease treatment of the histone-DNA complexes formed under physiological ionic strength conditions. Kinetic studies have shown that the hexamer particle is not a subnucleosomal fragment produced by the degradation of nucleosome core particles. Furthermore, the hexamer particle was not found among the products of nuclease digestion when histones and DNA were previously assembled in 0.6 M NaCl. The high sedimentation coefficient of the hexameric complex (8 S) suggests that the DNA component of the particle has a folded conformation.     

Generation of a third-order folded structure for chromatin

A model for the initiation of the diffuse-condensed transition of chromatin induced by a change in the conformation of lysine-rich histones is proposed. Three levels of folded structures are discussed. The first-order folded structure refers to the structure of the repeat unit of chromatin, which is called the nucleosome. The nucleosome contains a nuclease resistant region in which 140 base pairs of DNA are wrapped around the surface of a histone aggregated of two copies each of the histones H2A, H2B, H3 and H4. This DNA-histone aggregate is called a core particle. The nuclease accessible region of the nucleosome is approximately 60 base pairs of DNA which link the core particle, hence the terminology “linker DNA.” The lysine-rich histones, (Hl, H5), which are more loosely bound than the core histones, are associated with the linker DNA. The second-order folded structure refers to the conformation of a polynucleosome. Based on neutron scattering and quasielastic light scattering studies the second-order folded structure is assumed to be an extended helix in solution with 5–7 nucleosome units per turn. The third-order folded structure is defined as that structure resulting from the first stage in the condensation process induced by a conformational change in the lysine-rich histones. Generation of the third-order folded structure in the proposed model is effected by an increased affinity of the lysine-rich histones for super-helical DNA in the core particles in adjacent turns of the second-order folded structure. Since the lysine-rich histones preferentially bind to A-T rich regions in DNA, the distribution of these regions would determine the third-order folded structure. The net effect of a non-random distribution of A-T rich regions as in the proposed model is the generation of a helix for the third-order folded structure. The assumption of a non-random distribution of A-T rich regions is indirectly supported by proflavine binding studies reported herein and by the existence of repetitive and non-repetitive DNA regions inferred from renaturation studies. One consequence of the proposed mechanism is that the majority of the A-T rich regions are in the interior of the third-order folded structure. Promoter sites of high A-T content would then be inaccessible to polymerases. The proposed model also suggests a role for spacer DNA in the genome. Higher order folded structures must also be present in the final state of condensed chromatin since the three orders of folded structures considered in this communication accounts for only 2% of that required in the diffuse-condensed transition.     

Photofootprint of nucleosome core DNA in intact chromatin having different structural states

Recently, we reported that the distribution of ultraviolet light (u.v.) induced pyrimidine dimers in nucleosome core DNA has a striking 10.3(+/- 0.1) base periodicity and the regions of enhanced quantum yield map to positions where DNA strands are farthest from the core histone surface. Improvement of the mapping procedure has allowed us to analyze this distribution in more detail, and compare the distribution pattern for nucleosome cores from intact chromatin having different higher-order structures (from the 10 nm filament to the 30 nm fiber). At all levels of chromatin compaction, we observed the following. (1) The average periodicity in pyrimidine dimer yield is 10.3 bases. (2) The peak-to-peak spacing in this distribution is significantly different from 10.3 bases in the region covering three helix turns immediately 5' of the dyad axis. (3) There is a suppression of photoproduct formation in the region of the dyad axis, especially at position 84 from the 5' end. (4) The approximately 10 base ensembles have alternating peak intensities throughout core DNA. Furthermore, peak deconvolution analysis of the pyrimidine dimer pattern yielded a striking similarity in photoproduct yield for the different levels of chromatin compaction. Irradiation of isolated core DNA yields a much more random distribution of photoproducts, although a weak modulation pattern is observed (indicating that there is a non-random alignment of adjacent pyrimidines in our core DNA preparations). This pattern includes a depression in photoproduct yield near position 95, suggesting that the sequence in this region plays a role in nucleosome positioning. These results show that the u.v. photofootprint is a sensitive, diagnostic probe of core histone-DNA interactions in intact chromatin, and these interactions are not significantly altered by changes in the structural state of the chromatin fiber.     

Hydrodynamic studies of the interaction between nucleosome core particles and core histones

We have studied the hydrodynamic properties of the complexes formed by interaction of nucleosome core particles with excess histone octamers containing two each of the four core histones. The results are consistent with tight binding of two to three octamers to the exterior of each core particle. The binding is dependent upon the presence of the H3/H4 histone pair: when H3/H4 alone are added to nucleosome core particles, tight binding is observed, but H2A/H2B alone are bound only weakly. We have also examined the properties of the nucleosome core in solutions containing 0·1 m to 0·7 M-NaCl. We show that in this salt range the core particle undergoes some changes in shape, reflected in a 14% increase in the frictional coefficient. Even at the highest salt concentrations used, however, the nucleosome core is still a compact, folded structure.     

AFM imaging and theoretical modeling studies of sequence-dependent nucleosome positioning

Telomeric chromatin has different features with respect to bulk chromatin, since nucleosomal repeat along the chain is unusually short. We studied the role of telomeric DNA sequences on nucleosomal spacing in a model system. Nucleosomal arrays, assembled on a 1500-bp-long human telomeric DNA and on a DNA fragment containing 8 copies of the 601 strong nucleosome positioning sequence, have been studied at the single molecule level, by atomic force microscopy imaging. Random nucleosome positioning was found in the case of human telomeric DNA. On the contrary, nucleosome positioning on 601 DNA is characterized by preferential positions of nucleosome dyad axis each 200 bp. The AFM-derived nucleosome organization is in satisfactory agreement with that predicted by theoretical modeling, based on sequence-dependent DNA curvature and flexibility. The reported results show that DNA sequence has a main role, not only in mononucleosome thermodynamic stability, but also in the organization of nucleosomal arrays.     

Structural dynamics of nucleosome core particle: comparison with nucleosomes containing histone variants

The present study provides insights on the dominant mechanisms of motions of the nucleosome core particle and the changes in its functional dynamics in response to histone variants. Comparative analysis of the global dynamics of nucleosomes with native and variant H2A histones, using normal mode analysis revealed that the dynamics of the nucleosome is highly symmetric, and its interaction with the nucleosomal DNA plays a vital role in its regulation. The collective dynamics of nucleosomes are predicted to be dominated by two types of large-scale motions: (1) a global stretching-compression of nucleosome along the dyad axis by which the nucleosome undergoes a breathing motion with a massive distortion of nucleosomal DNA, modulated by histone-DNA interactions; and (2) the flipping (or bending) of both the sides of the nucleosome in an out-of-plane fashion with respect to the dyad axis, originated by the highly dynamic N-termini of H3 and (H2A.Z-H2B) dimer in agreement with the experimentally observed perturbed dynamics of the particular N-terminus under physiological conditions. In general, the nucleosomes with variant histones exhibit higher mobilities and weaker correlations between internal motions compared to the nucleosome containing ordinary histones. The differences are more pronounced at the L1 and L2 loops of the respective monomers H2B and H2A, and at the N-termini of the monomers H3 and H4, all of which closely interact with the wrapping DNA.     

Attraction, phasing and neighbour effects of histone octamers on curved DNA

Nucleosome core particles were reconstituted on various DNA fragments containing a Crithidia fasciculata kinetoplast curved tract. The results show that, on curved DNA, nucleosome core particles form six- to sevenfold preferentially, relative to bulk sequences. The preferential deposition occurs at multiple periodic positions, whose distribution reveals a unique rotational setting of DNA with respect to the histone octamer surface and whose average periodicity is 10.26 +/- 0.04. Evidence is provided for a context effect in histone octamer deposition: octamers bound to a segment of curved DNA influence the positions of neighbour octamers. Taken together, the preferential formation of nucleosome core particles and the influence on the localization of neighbouring particles suggest for intrinsically bent sequences the biologically relevant role of organizers of nucleosomal arrays.