Maintenance DNA methylation of nucleosome core particles

The enzyme responsible for maintenance methylation of CpG dinucleotides in vertebrates is DNMT1. The presence of DNMT1 in DNA replication foci raises the issue of whether this enzyme needs to gain access to nascent DNA before its packaging into nucleosomes, which occurs very rapidly behind the replication fork. Using nucleosomes positioned along the 5 S rRNA gene, we find that DNMT1 is able to methylate a number of CpG sites even when the DNA major groove is oriented toward the histone surface. However, we also find that the ability of DNMT1 to methylate nucleosomal sites is highly dependent on the nature of the DNA substrate. Although nucleosomes containing the Air promoter are refractory to methylation irrespective of target cytosine location, nucleosomes reconstituted onto the H19 imprinting control region are more accessible. These results argue that although DNMT1 is intrinsically capable of methylating some DNA sequences even after their packaging into nucleosomes, this is not the case for at least a fraction of DNA sequences whose function is regulated by DNA methylation.     

Stability of the primary organization of nucleosome core particles upon some conformational transitions.

The sequential arrangement of histones along DNA in nucleosome core particles was determined between 0.5 and 600 mM salt and from 0 to 8 M urea. These concentrations of salt and urea up to 6 M had no significant effect on the linear order of histones along DNA but 8 M urea caused the rearrangement of histones. Conformational changes in cores have been identified within these ranges of conditions by several laboratories 8-21. Also, abrupt structural changes in the cores, apparently their unfolding, were found by gel electrophoresis to occur at urea concentration, between 4 and 5 M. 600 mM salt and 6 M urea were shown to relax the binding of histones to DNA in cores but do not however release histones or some part of their molecules from DNA. It appears therefore that nucleosomal cores can undergo some conformational transitions and unfolding whereas their primary organization remains essentially unaffected. These results are consistent with a model of the core particles in which the histone octamer forms something like a helical "rim" along the superhelical DNA and histone-histone interactions beyond the "rim" are rather weak in comparison with those within the "rim".     

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.     

H3.H4 tetramer directs DNA and core histone octamer assembly in the nucleosome core particle.

The way in which histones interact with DNA during in vitro assembly of nucleohistone has been examined. Chicken erythrocyte core histones H2A, H2B, H3, and H4 and lambdaDNA in 2 M NaCl were allowed to interact by stepwise decrease in the salt concentration. Binding, although weak, was first observed at 1.4 M NaCl and was essentially completed at 0.6 M NaCl. Analysis of the DNA-bound histones revealed that each of the histones in the pairs H2A,H2B and H3,H4 was always present in equimolar amounts and that the relative proportion of each pair was constant between 1.4 and 0.8 M NaCl. Evidence is presented suggesting that binding occurred via complexes of the four histones, the nature of which is likely to reflect the equilibrium among the octamer and its products of dissociation (Ruiz-Carrillo, A., & Jorcano, J.L. (1979) Biochemistry (preceding paper in this issue)). The presence of complexes of the four core histones is, however not required for the correct assembly of the nucleosome core particle. Nucleohistones obtained by adding at progressively lower ionic strengths the dimer H2A.H2B to the H3.H4-DNA complex (split reconstitutions) had the same characteristics as those assembled with the core histone complexes.     

Comparative filter binding study of H5 to nucleosome core particles,H1, H5 depleted chromatosomes and DNA fragments

The filter-binding technique with PEI treated glass fiber is used to study the interaction of histone H5 to core particles, chromatosomes and DNA derived from it. By working at very low concentrations of interacting particles we are able to study the effective binding process independent of interfering insoluble complexes. The interactions are characterized by a very high affinity. An intrinsically higher affinity of H5 for cores and chromatosomes versus chromatosome derived DNA is demonstrated. Both chromatosomes and DNA derived from these bind about twice the amount as compared to core particles, which saturate at about one H5 per core particle.Abbreviations GH5 globular domain of histone H5 - PEI polyethyleneimine     

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.     

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.     

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.     

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.     

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.     

NMR studies of conformational states and dynamics of DNA

The application of high resolution NMR techniques to the investigation of DNA double helices in solution is currently in a rapid state of change as a result of advances in three different fields. First, new methods (cloning, enzymatic degradation, sonication, and chemical synthesis) have been developed for producing large quantities of short DNA suitable for NMR studies. Second, there have been major advances in the field of NMR in terms of the introduction of new pulse techniques and improvements in instrumentation. Finally, as a result of recent X-ray diffraction studies on short DNA helices and the discovery of left-handed Z-DNA there is heightened interest in the study of DNA structures in solution and the effect of sequence on structure. In the present review, we discuss the way in which NMR techniques have been used to probe various aspects of the DNA properties, including base pairing structure, dynamics of breathing, effect of sequence on DNA structure, internal molecular motions, the effect of environment on the DNA, and the interaction of DNA with small ligands.     

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.     

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.     

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.     

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.     

Two new photoaffinity polyamines appear to alter the helical twist of DNA in nucleosome core particles

Two new photoaffinity derivatives of polyamines have been synthesized by the reaction of spermine or spermidine with methyl 4-azidobenzimidate. The new compounds were purified chromatographically and characterized by several methods including proton magnetic resonance spectroscopy. The spermine derivative is N1-ABA-spermine [(azidobenzamidino)spermine], and the spermidine derivative is a mixture of N1- and N8-ABA-spermidine. ABA-spermine stabilizes nucleosome core particles in thermal denaturation experiments, with similar but not identical effects when compared with the parent polyamine, spermine. In circular dichroism experiments, ABA-spermine was capable of producing a B----Z transition in poly(dG-m5dC) at a concentration of 30 microM, compared with 5 microM required to produce the same effect with spermine. On the other hand, ANB-spermine [(azidonitrobenzoyl)spermine; Morgan, J. E., Calkins, C. C., & Matthews, H. R. (1989) Biochemistry 28, 5095-5106] stabilized the B form of poly(dG-br5dC). ABA-spermine is a potent inhibitor of ornithine decarboxylase from Escherichia coli, giving 50% inhibition at 0.12 mM, while ANB-spermine is a modest inhibitor, comparable to spermine or spermidine. Under conditions of nitrogen-limited growth, yeast take up ABA-spermine and ABA-spermidine at approximately one-third to half the rate of spermidine or spermine. In contrast, ANB-spermine was not significantly taken up. The photoaffinity polyamines were used to photoaffinity label the DNA in nucleosome core particles, and the sites of labeling were determined by exonuclease protection. All photoaffinity reagents showed both nonspecific labeling and specific sites of higher occupancy. However, the positions of the sites varied: the ANB-spermine sites confirmed those previously reported (Morgan et al., 1989); the ABA-spermine and ABA-spermidine sites were spaced at 9.8 bp intervals from the 3' end of each DNA strand. This observation, together with the effect of spermine on the circular dichroism of DNA in nucleosome core particles, implies that polyamines alter the helical twist of DNA in nucleosome core particles. The ABA-polyamines are offered as general-purpose photoaffinity polyamine reagents.     

Proton NMR investigation of the nucleosome core particle: evidence for regions of altered hydrogen bonding

Novel 1H nuclear magnetic resonance (NMR) resonances, arising from exchangeable protons and centered at approximately 11.2 and 10.1 parts per million (ppm), have been observed in the low-field spectrum (10-15 ppm) of the chicken erythrocyte core particle [145 +/- 2 base pairs (bp)]. These peaks are located upfield from the normal adenine-thymine (A-T) and guanine-cytosine (G-C) imino peaks characteristic of B-form deoxyribonucleic acid (DNA) and are not observed in free DNA under identical conditions. The appearance of the new peaks is ionic strength dependent and temperature-reversible below 75 degrees C. At 25 degrees C, the upfield peak area represents 5% of the DNA base pairs (7 bp), while between 45 and 55 degrees C, the area increases to 18%, affecting approximately 25 bp. Area increases in the upfield resonances result in a complementary decrease in the A-T and G-C imino peaks found between 12 and 14 ppm. We believe these novel proton signals represent a histone-induced DNA conformational change which involves localized alteration of base pairing in the core particle.