Deletion analysis of a DNA sequence that positions itself precisely on the nucleosome core

Exonuclease III digests DNA sequentially from the 3' end. This enzyme is used to analyse the location of nucleosomes on DNA fragments containing a particular 145 base-pair (bp) sequence. When one of these fragments is assembled into chromatin and digested with exonuclease, a strong and persistent pause in digestion is detected at a single location. That this pause is due to the enzyme encountering a nucleosome is suggested, firstly, by its absence from digests of free DNA and, secondly, by the detection of a corresponding pause on the other strand. The two pauses, 146 bp apart, specify the location of a single precisely positioned nucleosome on the DNA fragment. This position corresponds exactly to one of two possible positions of the 145 bp sequence identified previously. A fragment containing only about 80 bp of the original 145 bp continues to position itself in the nucleosome like the parent sequence. Therefore, some of the sequence can be replaced with different DNA without affecting nucleosome positioning. Further exonuclease III analysis of an extensive set of deletions demonstrates that a central region of about 40 bp is essential for positioning the 145 bp sequence. When deletions advance into this region from either side, only a very small proportion of the DNA remains in the original position on the nucleosome. Therefore, the two short lengths of DNA at the edges of the region must each contain all or part of an essential nucleosome-positioning signal. These two critical sequences are symmetrically located across the nucleosome dyad and interact with the same region of histone H3. The sequence TGC occurs at the same place in both sequences; otherwise they are dissimilar.     

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.     

A 145-base pair DNA sequence that positions itself precisely and asymmetrically on the nucleosome core.

A 145-bp DNA sequence, cloned from Escherichia coli, was reconstituted into nucleosome core particles by a number of methods. The behaviour of the resulting complex upon sucrose gradient sedimentation and nucleoprotein gel electrophoresis closely resembled that of control bulk nucleosome core particles. DNase I digestion of the 32P-end-labelled complex revealed the 10-bp periodicity of cleavages expected for DNA bound on a histone surface. The narrow cleavage sites observed (1 bp wide) imply that the sequence occupies a single preferred position on the nucleosome core, accurate to the level of single base pairs. By relating the digestion pattern observed to the pattern of site protection found for random sequence nucleosomes, the DNA position was found to be offset by 17 bp from that in the normal core particle. A number of experiments argue against the involvement of length or end effects and suggest that it is some feature of the DNA sequence itself that determines this precise positioning of DNA on the nucleosome.     

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.     

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.     

Helical repeat of DNA in the nucleosome core particle

Although the crystal structure of nucleosome core particle is essentially symmetrical in the vicinity of the dyad, the linker histone binds asymmetrically in this region to select a single high-affinity site from potentially two equivalent sites. To try to resolve this apparent paradox we mapped to base-pair resolution the dyads and rotational settings of nucleosome core particles reassembled on synthetic tandemly repeating 20 bp DNA sequences. In agreement with previous observations, we observed (1) that the helical repeat on each side of the dyad cluster is 10 bp maintaining register with the sequence repeat and (2) that this register changes by 2 bp in the vicinity of the dyad. The additional 2 bp required to effect the change in the rotational settings is accommodated by an adjustment immediately adjacent to the dyad. At the dyad the hydroxyl radical cleavage is asymmetric and we suggest that the inferred structural asymmetry could direct the binding of the linker histone to a single preferred site.     

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.     

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.     

Using DNA mechanics to predict in vitro nucleosome positions and formation energies

In eukaryotic genomes, nucleosomes function to compact DNA and to regulate access to it both by simple physical occlusion and by providing the substrate for numerous covalent epigenetic tags. While competition with other DNA-binding factors and action of chromatin remodeling enzymes significantly affect nucleosome formation in vivo, nucleosome positions in vitro are determined by steric exclusion and sequence alone. We have developed a biophysical model, DNABEND, for the sequence dependence of DNA bending energies, and validated it against a collection of in vitro free energies of nucleosome formation and a set of in vitro nucleosome positions mapped at high resolution. We have also made a first ab initio prediction of nucleosomal DNA geometries, and checked its accuracy against the nucleosome crystal structure. We have used DNABEND to design both strong and weak histone- binding sequences, and measured the corresponding free energies of nucleosome formation. We find that DNABEND can successfully predict in vitro nucleosome positions and free energies, providing a physical explanation for the intrinsic sequence dependence of histone–DNA interactions.     

Echinomycin and distamycin induce rotation of nucleosome core DNA.

When nucleosome cores reconstituted from chicken erythrocyte histones and a 160 bp DNA molecule are exposed to echinomycin, a bis-intercalating antitumour antibiotic, the DNA appears to rotate with respect to the histone octamer by about half a turn. New bands appear in patterns of DNAase I digestion at positions approximately mid-way between those characteristic of control core samples, while the control pattern is largely suppressed. Similar (but not identical) changes are produced when nucleosome cores are exposed to distamycin, a non-intercalating DNA-binding antibiotic. The effects of both ligands can be explained in terms of a change in rotational orientation of the core DNA, so as to place antibiotic binding sites on the inward-facing (concave) surface of the DNA supercoil. Presumably this serves to optimise non-bonded contacts with the polynucleotide backbone. These results establish that the positioning of DNA about the histone octamer is not absolutely determined by its nucleotide sequence, but may be modified by the binding of such relatively small molecules as antibiotics.     

Sequence-directed mapping of nucleosome positions

A nucleosome DNA sequence probe is designed that combines recently derived RR/YY counter-phase and AA/TT in-phase periodical patterns. A simple nucleosome mapping procedure is introduced for prediction of the nucleosome positions in the sequence of interest, to serve as a guide for experimental studies of the chromatin structure.     

DNA conformation and energy in nucleosome core: a theoretical approach

DNA conformation in complex with proteins is far from its canonical B-form. The affinity of complex formation and structure of DNA depend on its attachment configuration and sequence. In this article, we develop a mechanical model to address the problem of DNA structure and energy under deformation. DNA in nucleosome core particle is described as an example. The structure and energy of nucleosomal DNA is calculated based on its sequence and positioning state. The inferred structure has remarkable similarity with X-ray data. Although there is no sequence-specific interaction of bases and the histone core, we found considerable sequence dependency for the nucleosomal DNA positioning. The affinity of nucleosome formation for several sequences is examined and the differences are compatible with observations. We argue that structural energy determines the natural state of nucleosomal DNA and is the main reason for affinity differences in vitro. This theory can be utilized for the DNA structure and energy determination in protein–DNA complexes in general.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:17     

DNA accessibility to minor groove ligands in core nucleosome and chromatosome

Using the circular dichroism spectra induced in the visible by the binding to the minor groove of DNA, we found that Hoechst 33258 is able to occupy its specific sites even when these are located inside the nucleosome structure. This high accessibility of the DNA in the nucleosome is not modified by the removal of the amino-terminal domains of the octamer histones and is not reduced by the presence of linker histone. Interesting and reasonable differences were found in the association constants.     

A nonuniform distribution of excision repair synthesis in nucleosome core DNA

We have investigated the distribution in nucleosome core DNA of nucleotides incorporated by excision repair synthesis occurring immediately after UV irradiation in human cells. We show that the differences previously observed for whole nuclei between the DNase I digestion profiles of repaired DNA (following its refolding into a nucleosome structure) and bulk DNA are obtained for isolated nucleosome core particles. Analysis of the differences obtained indicates that they could reflect a significant difference in the level of repair-incorporated nucleotides at different sites within the core DNA region. To test this possibility directly, we have used exonuclease III digestion of very homogeneous sized core particle DNA to "map" the distribution of repair synthesis in these regions. Our results indicate that in a significant fraction of the nucleosomes the 5' and 3' ends of the core DNA are markedly enhanced in repair-incorporated nucleotides relative to the central region of the core particle. A best fit analysis indicates that a good approximation of the data is obtained for a distribution where the core DNA is uniformly labeled from the 5' end to position 62 and from position 114 to the 3' end, with the 52-base central region being devoid of repair-incorporated nucleotides. This distribution accounts for all of the quantitative differences observed previously between repaired DNA and bulk DNA following the rapid phase of nucleosome rearrangement when it is assumed that linker DNA and the core DNA ends are repaired with equal efficiency and the nucleosome structure of newly repaired DNA is identical with that of bulk chromatin. Furthermore, the 52-base central region that is devoid of repair synthesis contains the lowest frequency cutting sites for DNase I in vitro, as well as the only "internal" locations where two (rather than one) histones interact with a 10-base segment of each DNA strand.     

Evolutionary footprints of nucleosome positions in yeast

Using genome-wide maps of nucleosome positions in yeast, we have analyzed the influence of chromatin structure on the molecular evolution of genomic DNA. We have observed, on average, 10-15% lower substitution rates in linker regions than in nucleosomal DNA. This widespread local rate heterogeneity represents an evolutionary footprint of nucleosome positions and reveals that nucleosome organization is a genomic feature conserved over evolutionary timescales.     

Crystals of a nucleosome core particle containing defined sequence DNA

Nucleosome core particles were reconstituted from a DNA restriction fragment and histone octamers, crystallized, and the crystals examined by X-ray diffraction. A DNA fragment was engineered by site-directed mutagenesis to obtain a 146 base-pair sequence that takes up a symmetrical arrangement in the core particle. The resulting DNA sequence was cloned in multiple copies into pUC9 and excised as monomer via EcoRV to produce it in milligram quantities. Nucleosome core particles incorporating the DNA were reconstituted by salt gradient dialysis and purified by anion-exchange high-pressure liquid chromatography. DNase I digestion was used to demonstrate that the termini of the restriction fragment are located 73 base-pairs from the molecular dyad axis of the particle. The diffraction limits of crystals of defined sequence core particles extend along the principal direction to a approximately equal to 4 A, b approximately equal to 5 A and c approximately equal to 3 A, giving about a twofold increase in the number of measurable X-ray reflections over previous crystals containing mixed sequence DNA. The methods developed here should be useful in the study of other large protein-DNA complexes.     

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.     

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.