A novel roll-and-slide mechanism of DNA folding in chromatin: implications for nucleosome positioning

How eukaryotic genomes encode the folding of DNA into nucleosomes and how this intrinsic organization of chromatin guides biological function are questions of wide interest. The physical basis of nucleosome positioning lies in the sequence-dependent propensity of DNA to adopt the tightly bent configuration imposed by the binding of the histone proteins. Traditionally, only DNA bending and twisting deformations are considered, while the effects of the lateral displacements of adjacent base pairs are neglected. We demonstrate, however, that these displacements have a much more important structural role than ever imagined. Specifically, the lateral Slide deformations observed at sites of local anisotropic bending of DNA define its superhelical trajectory in chromatin. Furthermore, the computed cost of deforming DNA on the nucleosome is sequence-specific: in optimally positioned sequences the most easily deformed base-pair steps (CA:TG and TA) occur at sites of large positive Slide and negative Roll (where the DNA bends into the minor groove). These conclusions rest upon a treatment of DNA that goes beyond the conventional ribbon model, incorporating all essential degrees of freedom of "real" duplexes in the estimation of DNA deformation energies. Indeed, only after lateral Slide displacements are considered are we able to account for the sequence-specific folding of DNA found in nucleosome structures. The close correspondence between the predicted and observed nucleosome locations demonstrates the potential advantage of our "structural" approach in the computer mapping of nucleosome positioning.     

The helical periodicity of DNA on the nucleosome

The precise number of base pairs per turn of the DNA double helix in the nucleosome core particle has been the subject of controversy. In this paper the positions of nuclease cutting sites are analysed in three dimensions. Using this midpoint of the DNA on the nucleosome dyad as origin, the cutting site locations measured along a strand of DNA are mapped onto models of the nucleosome core containing DNA of different helical periodicities. It is found that a helical periodicity of 10.5 base pairs per turn leads to cutting site positions which are sterically inaccessible. In contrast, a periodicity of 10.0 base pairs per turn leads to cutting site positions which are not only sterically sound, but which fall into a pattern such as would be expected when the access of the nuclease to the DNA is restricted by the presence of the histone core on one side and of the adjacent superhelical turn of DNA on the other. As proposed earlier by us (1), a value for the helical periodicity close to 10 base pairs per turn on the nucleosome, taken together with a periodicity close to 10.5 for DNA in solution - a value now established - resolves the so-called linkage number paradox.     

Nucleosomal locations of dominant DNA sequence motifs for histone-DNA interactions and nucleosome positioning

DNA sequence is an important determinant of the positioning, stability, and activity of nucleosomes, yet the molecular basis of these effects remains elusive. A "consensus DNA sequence" for nucleosome positioning has not been reported and, while certain DNA sequence preferences or motifs for nucleosome positioning have been discovered, how they function is not known. Here, we report that an unexpected observation concerning the reassembly of nucleosomes during salt gradient dialysis has allowed a breakthrough in our efforts to identify the nucleosomal locations of the DNA sequence motifs that dominate histone-DNA interactions and nucleosome positioning. We conclude that a previous selection experiment for high-affinity, nucleosome-forming DNA sequences exerted selective pressure chiefly on the central stretch of the nucleosomal DNA. This observation implies that algorithms for aligning the selected DNA sequences should seek to optimize the alignment over much less than the full 147 bp of nucleosomal DNA. A new alignment calculation implemented these ideas and successfully aligned 19 of the 41 sequences in a non-redundant database of selected high-affinity, nucleosome-positioning sequences. The resulting alignment reveals strong conservation of several stretches within a central 71 bp of the nucleosomal DNA. The alignment further reveals an inherent palindromic symmetry in the selected DNAs; it makes testable predictions of nucleosome positioning on the aligned sequences and for the creation of new positioning sequences, both of which are upheld experimentally; and it suggests new signals that may be important in translational nucleosome positioning.     

Archaeal nucleosome positioning sequence from Methanothermus fervidus.

DNA in Methanothermus fervidus, a hyperthermophilic archaeon, is constrained into archaeal nucleosomes in vivo by the archaeal histones HMfA and HMfB. Here, we document the translational and rotational positioning of archaeal nucleosome assembly in vitro by a sequence from the 7S RNA encoding region of the M. fervidus genome. The minor groove of the DNA at the center of the DNA sequence, protected from micrococcal nuclease digestion by incorporation into a positioned archaeal nucleosome, faces away from the archaeal histone core.     

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.     

DNA bending and its relation to nucleosome positioning

X-ray and solution studies have shown that the conformation of a DNA double helix depends strongly on its base sequence. Here we show that certain sequence-dependent modulations in structure appear to determine the rotational positioning of DNA about the nucleosome. Three different experiments are described. First, a piece of DNA of defined sequence (169 base-pairs long) is closed into a circle, and its structure examined by digestion with DNAase I: the helix adopts a highly preferred configuration, with short runs of (A, T) facing in and runs of (G, C) facing out. Secondly, the same sequence is reconstituted with a histone octamer: the angular orientation around the histone core remains conserved, apart from a small uniform increase in helix twist. Finally, it is shown that the average sequence content of DNA molecules isolated from chicken nucleosome cores is non-random, as in a reconstituted nucleosome: short runs of (A, T) are preferentially positioned with minor grooves facing in, while runs of (G, C) tend to have their minor grooves facing out. The periodicity of this modulation in sequence content (10.17 base-pairs) corresponds to the helix twist in a local frame of reference (a result that bears on the change in linking number upon nucleosome formation). The determinants of translational positioning have not been identified, but one possibility is that long runs of homopolymer (dA) X (dT) or (dG) X (dC) will be excluded from the central region of the supercoil on account of their resistance to curvature.     

Nucleosome positioning and periodicity of satellite DNA in the liver of aging rats – Nucleosome positioning and periodicity of satellite DNA

The positioning of nucleosomes has been analysed by comparing the pattern of cutting sites of a probing reagent on chromatin and naked DNA. For this purpose, high molecular weight DNA and nuclei from the liver of young (18±2 weeks) and old (100±5 weeks) Wistar male rats were digested with micrococcal nuclease (MNase) and hybridized with ³²P-labelled rat satellite DNA probe. A comparison of the ladder generated by MNase with chromatin and nuclei indicates long range organization of the satellite chromatin fiber with distinct non-random positioning of nucleosomes. However, the positioning of nucleosomes on satellite DNA does not vary with age. For studying the periodicity and subunit structure of satellite DNA, high molecular weight DNA from the liver of young and old rats were digested with different restriction enzymes. Surprisingly, no noteworthy age-related change is visible in the periodicity and subunit structural organization of the satellite DNA. These results suggest that the nucleosome positioning and the periodicity of liver satellite DNA do not vary with age.     

The mechanism of nucleosome assembly onto oligomers of the sea urchin 5 S DNA positioning sequence

We have used a model system composed of tandem repeats of Lytechinus variegatus 5 S rDNA (Simpson, R. T., Thoma, F., and Brubaker, J. M. (1985) Cell 42, 799-808) reconstituted into chromatin with chicken erythrocyte core histones to investigate the mechanism of chromatin assembly. Nucleosomes are assembled onto the DNA template by mixing histone octamers and DNA in 2 M NaCl followed by stepwise dialysis into very low ionic strength buffer over a 24-h period. By 1.0 M NaCl, a defined intermediate composed of arrays of H3.H4 tetramers has formed, as shown by analytical and preparative ultracentrifugation. Digestion with methidium propyl EDTA.Fe(II) indicates that these tetramers are spaced at 207 base pair intervals, i.e. one/repeat length of the DNA positioning sequence. In 0.8 M NaCl, some H2A.H2B has become associated with the H3.H4 tetramers and DNA. Surprisingly, under these conditions DNA is protected from methidium propyl EDTA.Fe(II) digestion almost as well as in the complete nucleosome, even though these structures are quite deficient in H2A.H2B. By 0.6 M NaCl, nucleosome assembly is complete, and the MPE digestion pattern is indistinguishable from that observed for oligonucleosomes at very low ionic strength. Below 0.6 M NaCl, the oligonucleosomes are involved in various salt-dependent conformational equilibria: at approximately 0.6 M, a 15% reduction in S20,w that mimics a conformational change observed previously with nucleosome core particles; at and above 0.1 M, folding into a more compact structure(s); at and above 0.1 M NaCl, a reaction involving varying amounts of dissociation of histone octamers from a small fraction of the DNA templates. In low ionic strength buffer (less than 1 mM NaCl), oligonucleosomes are present as fully loaded templates in the extended beads-on-a-string structure.     

DNA distortion as a factor in nucleosome positioning.

In a previous report we constructed a synthetic DNA sequence that directed the deposition of histone octamers to a single site, and it was proposed that DNA distortion was involved in the positioning effect. In the present study we utilized the chemical probe potassium permanganate to identify sites of DNA distortion in the synthetic positioning sequence. A permanganate hypersite was identified 15 bp from the nucleosome pseudo-dyad at a site known to display DNA distortion in the mature nucleosome. The sequence of the site contained a TA step flanked by an oligo-pyrimidine tract. A series of substitutions were made in the region of the permanganate hypersite and the resulting constructs tested for affinity for histone octamers and translational positioning in in vitro studies. The results revealed that either a single base substitution at the TA step or in the adjacent homopolymeric tract dramatically affected affinity and positioning activity. The rotational orientation of the permanganate-sensitive sequence was shown to be important for functions, since altering the orientation of the site in a positioning fragment reduced positioning activity and octamer affinity, while altering the rotational orientation of the sequence in a non-positioning fragment had the opposite effects. A reconstituted 5 S rDNA positioning sequence from Lytechinus variegatus was also shown to display a permanganate hypersite 16 bp from its pseudo-dyad.     

Large-scale periodicity of nucleosome positioning signal in pericentric regions of chromosomes (Drosophila melanogaster)

Nucleosome positioning signal (NPS) in heterochromatin is not uniform. We suggest the analysis of its heterogeneity by correlation with periodic function (analog of Furrier analysis). It was established the periodical repetition of the nucleosome clusters of large size in pericentric regions in a discontinuous manner. In the 3L pericentric region, it was revealed the domination of 78–85?kbp wavelength in the correlation coefficient profile and also strong presentation of 50?kbp signal. In further to centromere position, the 69?kbp value strongly dominates as well as the 50?kbp value in the closest proximity. In addition to the long wavelength signals, there are plenty of short wavelengths signals especially in the closest vicinity to centromere. In some positions throughout pericentric region of 2L chromosome, there are two sizes of repeated intermingled correlation signals (50, and 75?kbp) with dominating value of 75?kbp in proximity and 50?kbp distantly to centromere, the situation for 2R is analogous. Some genes with long introns support these quantitative characteristics of NPSs and to some extent their dominating character in each region. The characteristic repeat periods for 3L pericentric region coincide with the distances between heterochromatin epigenetic mark clusters and their distribution throughout this region for fly embryos, larvae, and some cell lines.     

A signal encoded in vertebrate DNA that influences nucleosome positioning and alignment.

Evidence is provided that the nucleotide triplet con-sensus non-T(A/T)G (abbreviated to VWG) influences nucleosome positioning and nucleosome alignment into regular arrays. This triplet consensus has been recently found to exhibit a fairly strong 10 bp periodicity in human DNA, implicating it in anisotropic DNA bendability. It is demonstrated that the experimentally determined preferences for nucleosome positioning in native SV40 chromatin can, to a large extent, be pre-dicted simply by counting the occurrences of the period-10 VWG consensus. Nucleosomes tend to form in regions of the SV40 genome that contain high counts of period-10 VWG and/or avoid regions with low counts. In contrast, periodic occurrences of the dinucleotides AA/TT, implicated in the rotational positioning of DNA in nucleosomes, did not correlate with the preferred nucleosome locations in SV40 chromatin. Periodic occurrences of AA did correlate with preferred nucleosome locations in a region of SV40 DNA where VWG occurrences are low. Regular oscillations in period-10 VWG counts with a dinucleosome period were found in vertebrate DNA regions that aligned nucleosomes into regular arrays in vitro in the presence of linker histone. Escherichia coli and plasmid DNA, which fail to align nucleosomes in vitro, lacked these regular VWG oscillations.     

Correlation between dinucleotide periodicities and nucleosome positioning on mouse satellite DNA

Previous studies demonstrated 16 well-defined nucleosome locations (A-P) on a tandemly repeated prototype 234 base pair (bp) mouse satellite repeat unit. We have aligned the A-P fragments to search for DNA sequence elements that might contribute to nucleosome placement at these positions. Our results demonstrate a strikingly regular, uninterrupted, periodic pattern for the AA dinucleotide occurrences along the entire length of the aligned fragments. The periodicity of the AA occurrences is about 9.7 bp. The pattern exhibits a local minimum at position 74, near the nucleosome dyad axis of symmetry. Other dinucleotides--including AC: GT, CA: TG, and CC: GG--are also placed periodically, but their patterns of occurrence are less regular and less frequent than AA. The calculated spacings between consecutive preferred nucleosome locations on mouse satellite DNA are nearly identical, corresponding to multiples of 9.7 bp. The correlation between the periodicity of dinucleotide occurrences and the average spacing of nucleosome positions suggests that the preferred nucleosome locations recur at intervals that may correspond to the DNA helical repeat in the mouse satellite nucleosomes, and that the histone octamers sample (or slip along) the duplex in steps of 9.7 bp during nucleosome formation on mouse satellite DNA.     

Nucleosome positioning on yeast plasmids is determined only by the internal signal of DNA sequence occupied by the nucleosome

The possible role of border factors in determining the nucleosome positioning on a DNA sequence was investigated. To this end a family of recombinant plasmids based on Gal10Cyc1 promoter and neomycin phosphotransferase gene NPTII were created. A DNA sequence adjoining the GalCyc promoter was varied in these plasmids. Three nearly equally represented nucleosome positions on the GalCyc promoter were found. In the basal plasmid an FRT sequence adjoins the GalCyc promoter at the right. It contains an internal signal of multiple positioning. Its replacement with different DNA sequences does not affect nucleosome positioning on the GalCyc promoter. The nucleosome positioning on the GalCyc promoter does not depend on nucleosome positioning (or its absence) on adjoining sequences. The same is true for nucleosome positioning on FRT sequence. It was found also that nucleosomes' positioning on the NPTII gene and their mutual disposition, namely the spacing between neighboring nucleosomes (linker length) are determined by the location of positioning signals only. Generally the nucleosome positioning in our experimental model is determined solely by internal DNA sequence occupied by nucleosome. On the other hand, the action of this internal positioning signal does not extend to neighboring DNA sequences.