Transport of nucleosome core particles in semidilute DNA solutions

We studied the diffusion of native and trypsinized nucleosome core particles (NCPs), in aqueous solution and in concentrated DNA solutions (0.25-100 mg/ml) using fluorescence correlation spectroscopy (FCS). The highest DNA concentrations studied mimic the DNA density inside the cell nucleus. The diffusion coefficient of freely diffusing NCPs depends on the presence or absence of histone tails and is affected by the salt concentration due to the relaxation effect of counterions. NCPs placed in a network of long DNA molecules (30-50 kbp) reveal anomalous diffusion. We demonstrate that NCPs diffusion is in agreement with known particle transport in entangled macromolecular solutions as long as the histone tails are folded onto the particles. In contrast, when these tails are unfolded, the reversible adsorption of NCPs onto the DNA network has to be taken into account. This is confirmed by the fact that removal of the tails leads to reduction of the interaction between NCPs and the DNA network. The findings suggest that histone tail bridging plays an important role in chromatin dynamics.     

31P-NMR studies of DNA in nucleosome core particles

³¹P Nmr parameters (δ, T₁, W_1/2, and NOE) were measured for the DNA in nucleosome core particles at three frequencies and compared with similar data for the histone-free DNA. An essentially linear relationship was found between the frequency of observation and line-width for the single broad envelope of ³¹P resonances of the DNA in the nucleosome cores. We attributed this largely to chemical shift dispersion, with smaller contributions from chemical shift anisotropy and dipolar broadening. These results suggest the presence of different environments for phosphorus atoms in the core particles. However, within the accuracy of the method, no asymmetry in the resonance could be detected, which would tend to rule out any significant degree of DNA “kinking.” To investigate the interactions of the DNA and histones within the core particles we also studied transitions induced by urea and by temperature. Urea caused two stepwise increases in linewidth, which we attributed to conformational changes. A biphasic transition was also observed in the temperature profile, consistent with previous optical studies [Weischet et. al. (1978) Nucleic Acids Res. 5 , 139]. Various models with different types of local mobility were examined by the relaxation theory. A model of isotropic motion having a broad distribution of correlation times gave a fairly good fit to the ³¹P-nmr data.     

Bilayers of nucleosome core particles

Among the multiple effects involved in chromatin condensation and decondensation processes, interactions between nucleosome core particles are suspected to play a crucial role. We analyze them in the absence of linker DNA and added proteins, after the self-assembly of isolated nucleosome core particles under controlled ionic conditions. We describe an original lamellar mesophase forming tubules on the mesoscopic scale. High resolution imaging of cryosections of vitrified samples reveals how nucleosome core particles stack on top of one another into columns which themselves align to form bilayers that repel one another through a solvent layer. We deduce from this structural organization how the particles interact through attractive interactions between top and bottom faces and lateral polar interactions that originate in the heterogeneous charge distribution at the surface of the particle. These interactions, at work under conditions comparable with those found in the living cell, should be of importance in the mechanisms governing chromatin compaction in vivo.     

Phase diagram of nucleosome core particles

We present a phase diagram of the nucleosome core particle (NCP) as a function of the monovalent salt concentration and applied osmotic pressure. Above a critical pressure, NCPs stack on top of each other to form columns that further organize into multiple columnar phases. An isotropic (and in some cases a nematic) phase of columns is observed in the moderate pressure range. Under higher pressure conditions, a lamello-columnar phase and an inverse hexagonal phase form under low salt conditions, whereas a 2D hexagonal phase or a 3D orthorhombic phase is found at higher salt concentration. For intermediate salt concentrations, microphase separation occurs. The richness of the phase diagram originates from the heterogeneous distribution of charges at the surface of the NCP, which makes the particles extremely sensitive to small ionic variations of their environment, with consequences on their interactions and supramolecular organization. We discuss how the polymorphism of NCP supramolecular organization may be involved in chromatin changes in the cellular context.     

DNA base excision repair of uracil residues in reconstituted nucleosome core particles

The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase beta and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase beta on nucleosome cores.     

Primary organization of the nucleosome core particles sequential arrangement of histones along DNA

A high-resolution map for the arrangement of histones along DNA in the nucleosome core particles has been determined by a new sequencing procedure. The lysine groups of histones were crosslinked to partly depurinated DNA at neutral pH. One strand of DNA was split at the points of crosslinking, thus leaving the 5′-terminal DNA fragments bound to histones. The lengths of these crosslinked DNA fragments were measured to determine the position of histones on one strand of the core DNA from its 5′ end.The results demonstrate that histones are bound to regularly arranged, discrete DNA segments about six nucleotides long. These segments are separated by histone-free gaps about four nucleotides wide located at a distance of about 10n nucleotides from the 5′ end of DNA. The first 20 nucleotides from the 5′ ends of DNA seem to be free of histones. Histones appear to be arranged symmetrically and in a similar way on both DNA strands. Any one histone, being bound predominantly to discrete segments on one or other of the strands, can oscillate at the same time between the two strands across the major DNA groove. Two symmetrical models for the arrangement of two molecules of each core histone on linearized and folded DNA are proposed.     

Alkylation of duplex DNA in nucleosome core particles by duocarmycin SA and yatakemycin

(+)-Yatakemycin (1, Fig. 1) and (+)-duocarmycin SA (2) are exceptionally potent, naturally occurring antitumor agents that derive their biological properties through a characteristic sequence-selective DNA-alkylation reaction. Studies have shown that both the AT-rich binding selectivity (shape-selective recognition) and the alkylation catalysis (shape-dependent catalysis) that contribute to the alkylation selectivity are dependent on the DNA minor groove shape and size characteristics of an AT-rich sequence (ref. 6 and references therein; refs. 7,8). Here we report the alkylation properties of yatakemycin and duocarmycin SA on free DNA (alpha-satellite DNA) and the same sequence bound in a nucleosome core particle (NCP) modeling the state of DNA in eukaryotic cells. Both compounds showed a clear, relatively unaltered ability to alkylate DNA packaged in NCPs in terms of both alkylating efficiency and sequence selectivity, despite the steric and conformational perturbations imposed by NCP packaging. These findings highlight the dynamic nature of NCP-bound DNA and illustrate that cell- and protein-free DNA-alkylation studies of members of this class of antitumor drugs provide valuable insights into their properties.     

Salt-induced structural changes of nucleosome core particles.

The effects of sodium chloride concentration on the structure of chicken erythrocyte nucleosome core particles have been studied by the use of fluorescently labelled histones. Histone H3 was modified with two sulfhydryl-specific dyes and reconstituted into core nucleosomes. Between 10^?4 m and 0.6 M-NaCl four different states were observed by the fluorescent techniques of collisional quenching, polarization and energy transfer. Below 5 × 10^?4 m-NaCl the nucleosome is flexible, with the single cysteine residues of the two H3 species about 48 Å apart and somewhat exposed. Between 5 × 10^?3 m and 10^?1 m-NaCl the nucleosome is rigid and non-spherical. The cysteine residues are close together and buried. Between 10^?1 m and 4 × 10^?1 m-NaCl, the cysteines become slightly more exposed but remain close together. At 6 × 10^?1 m-NaCl the nucleosome is very flexible. The cysteines are more than 70 Å apart and are quite exposed. The dramatic structural changes that are observed in core nucleosomes are consistent with the variety of functions in which they must participate in the cell.     

Chiral discotic columnar germs of nucleosome core particles

In concentrated solution and in the presence of high concentrations of monovalent cations, nucleosome core particles order into a discotic columnar mesophase. This phase is limited to finite-sized hexagonal germs that further divide into six coiled branches, following an iterative process. We show how the structure of the germs comes from the competition between hexagonal packing and chirality with a combination of dendritic facetting and double-twist configurations. Geometrical considerations lead us to suspect that the chirality of the eukaryotic chromosomes may originate from the same competition.     

1H NMR investigation of the conformational states of DNA in nucleosome core particles.

In this study 1H NMR has been used to investigate the conformational state of DNA in nucleosome core particles. The nucleosome core particles exhibit partially resolved low field (10-15 ppm) spectra due to imino protons in Watson-Crick base pairs (one resonance per GC or AT base pair). To a first approximation, the spectrum is virtually identical with that of protein-free 140 base pair DNA, and from this observation we draw two important conclusions: (i) Since the low field spectra of DNA are known to be sensitive to conformation, the conformation of DNA in the core particles is essentially the same as that of free DNA (presumably B-form), (ii) since kinks occurring at a frequency at 1 in 10 or 1 in 20 base pairs would result in a core particle spectrum different from that of free DNA we find no NMR evidence supporting either the Crick-Klug or the Sobell models for kinking DNA around the core histones. Linewidth considerations indicate that the rotational correlation time for the core particles is approximately 1.5 X 10(-7) sec, whereas the end-over-end tumbling time of the free 140 base pair DNA is 3 X 10(-7) sec.     

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.     

Sequence periodicities in chicken nucleosome core DNA

The rotational positioning of DNA about the histone octamer appears to be determined by certain sequence-dependent modulations of DNA structure. To establish the detailed nature of these interactions, we have analysed the sequences of 177 different DNA molecules from chicken erythrocyte core particles. All variations in the sequence content of these molecules, which may be attributed to sequence-dependent preferences for DNA bending, correlate well with the detailed path of the DNA as it wraps around the histone octamer in the crystal structure of the nucleosome core. The sequence-dependent preferences that correlate most closely with the rotational orientation of the DNA, relative to the surface of the protein, are of two kinds: ApApA/TpTpT and ApApT/ApTpT, the minor grooves of which face predominantly in towards the protein; and also GpGpC/GpCpC and ApGpC/GpCpT, whose minor grooves face outward. Fourier analysis has been used to obtain fractional variations in occurrence for all ten dinucleotide and all 32 trinucleotide arrangements. These sequence preferences should apply generally to many other cases of protein-DNA recognition, where the DNA wraps around a protein. In addition, it is observed that long runs of homopolymer (dA) X (dT) prefer to occupy the ends of core DNA, five to six turns away from the dyad. These same sequences are apparently excluded from the near-centre of core DNA, two to three turns from the dyad. Hence, the translational positioning of any single histone octamer along a DNA molecule of defined sequence may be strongly influenced by the placement of (dA) X (dT) sequences. It may also be influenced by any aversion of the protein for sequences in the "linker" region, the sequence content of which remains to be determined.     

DNA methylation, nucleosome formation and positioning.

Recent mapping of nucleosome positioning on several long gene regions subject to DNA methylation has identified instances of nucleosome repositioning by this base modification. The evidence for an effect of CpG methylation on nucleosome formation and positioning in chromatin is reviewed here in the context of the complex sequence-structure requirements of DNA wrapping around the histone octamer and the role of this epigenetic mark in gene repression.     

Characteristics of nucleosome core DNA and their applications in predicting nucleosome positions

By analyzing dinucleotide position-frequency data of yeast nucleosome-bound DNA sequences, dinucleotide periodicities of core DNA sequences were investigated. Within frequency domains, weakly bound dinucleotides (AA, AT, and the combinations AA-TT-TA and AA-TT-TA-AT) present doublet peaks in a periodicity range of 10-11 bp, and strongly bound dinucleotides present a single peak. A time-frequency analysis, based on wavelet transformation, indicated that weakly bound dinucleotides of core DNA sequences were spaced smaller (∼10.3 bp) at the two ends, with larger (∼11.1 bp) spacing in the middle section. The finding was supported by DNA curvature and was prevalent in all core DNA sequences. Therefore, three approaches were developed to predict nucleosome positions. After analyzing a 2200-bp DNA sequence, results indicated that the predictions were feasible; areas near protein-DNA binding sites resulted in periodicity profiles with irregular signals. The effects of five dinucleotide patterns were evaluated, indicating that the AA-TT pattern exhibited better performance. A chromosome-scale prediction demonstrated that periodicity profiles perform better than previously described, with up to 59% accuracy. Based on predictions, nucleosome distributions near the beginning and end of open reading frames were analyzed. Results indicated that the majority of open reading frames’ start and end sites were occupied by nucleosomes.     

DNA triple-helix formation on nucleosome core particles. Effect of length of the oligopurine tract.

We have used DNase I footprinting to examine the formation of intermolecular triplexes on DNA fragments which have been complexed with nucleosome core particles. We have prepared five DNA fragments, based on the 160-bp tyrT sequence, which contain different length oligopurine tracts (up to 25 bp) at two different positions along the fragment, and have examined their availability for triple-helix formation after reconstituting onto nucleosome core particles. These results are compared with the formation of shorter triplexes in the same regions. In general we find that increasing the length of the complex does not facilitate nucleosomal triplex formation and that the most important factor affecting triplex formation is the position of the target site within the nucleosome-bound fragment. In some instances we find that longer oligonucleotides inhibit triplex formation. Although successful triplex formation was achieved on the longest nucleosome-bound oligopurine tracts, this was accompanied by changes in cleavage pattern that suggest oligonucleotide-induced changes in nucleosome structure.     

Site-specific aflatoxin B1 adduction of sequence-positioned nucleosome core particles

The question of how the presence of nucleosomal packing of DNA modifies carcinogen interaction at specific sites cannot be answered by studies on whole chromatin or bulk nucleosomes because of the heterogeneity of DNA sequences in the particles. We have circumvented this problem by using nucleosomes that are homogenous in DNA sequence and hence in DNA-histone contact points. A cloned DNA fragment containing a sea urchin 5 S gene which precisely positions a histone octamer was employed. By using 32P end-labeled DNA and genotoxins that allow cleavage at sites of attack, the frequency of adduction at every susceptible nucleotide can be determined on sequencing gels. The small methylating agent dimethyl sulfate and the bulky alkylating agent aflatoxin B1-dichloride (AFB1-Cl2) were used to probe the influence of DNA-histone interactions on DNA alkylation patterns in the sequence-positioned core particle. We find dimethyl sulfate to bind with equal preference to naked or nucleosomal DNA. In contrast, AFB1-Cl2 binding is suppressed an average of 2.4-fold at guanyl sites within nucleosomes compared with AFB1-Cl2 affinity at the corresponding site in naked DNA. The DNA is more accessible in regions near the particle boundary. We observe no other histone-imposed localized changes in AFB1-Cl2 sequence specificity. Further, sites of DNase I cleavage or proposed DNA bending show neither enhanced nor reduced AFB1-Cl2 adduction to N7-guanine. Since AFB1-Cl2 binding sites lie in the major groove, nucleosomal DNA appears accessible to AFB1-Cl2 at all points of analysis but with an access which is uniformly restricted in the central 100 nucleotides of the core particle. The data available do not indicate further localized or site-specific perturbations in DNA interactions with the two carcinogens studied.