Prediction of nucleosome rotational positioning in yeast and human genomes based on sequence-dependent DNA anisotropy

Background

An organism’s DNA sequence is one of the key factors guiding the positioning of nucleosomes within a cell’s nucleus. Sequence-dependent bending anisotropy dictates how DNA is wrapped around a histone octamer. One of the best established sequence patterns consistent with this anisotropy is the periodic occurrence of AT-containing dinucleotides (WW) and GC-containing dinucleotides (SS) in the nucleosomal locations where DNA is bent in the minor and major grooves, respectively. Although this simple pattern has been observed in nucleosomes across eukaryotic genomes, its use for prediction of nucleosome positioning was not systematically tested.

Results

We present a simple computational model, termed the W/S scheme, implementing this pattern, without using any training data. This model accurately predicts the rotational positioning of nucleosomes both in vitro and in vivo, in yeast and human genomes. About 65 – 75% of the experimentally observed nucleosome positions are predicted with the precision of one to two base pairs. The program is freely available at http://people.rit.edu/fxcsbi/WS_scheme/. We also introduce a simple and efficient way to compare the performance of different models predicting the rotational positioning of nucleosomes.

Conclusions

This paper presents the W/S scheme to achieve accurate prediction of rotational positioning of nucleosomes, solely based on the sequence-dependent anisotropic bending of nucleosomal DNA. This method successfully captures DNA features critical for the rotational positioning of nucleosomes, and can be further improved by incorporating additional terms related to the translational positioning of nucleosomes in a species-specific manner.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-313) contains supplementary material, which is available to authorized users.     

The coexistence of the nucleosome positioning code with the genetic code on eukaryotic genomes

It is known that there are several codes residing simultaneously on the DNA double helix. The two best-characterized codes are the genetic code—the code for protein production, and the code for DNA packaging into nucleosomes. Since these codes have to coexist simultaneously on the same DNA region, both must be degenerate to allow this coexistence. A-tracts are homopolymeric stretches of several adjacent deoxyadenosines on one strand of the double helix, having unusual structural properties, which were shown to exclude nucleosomes and as such are instrumental in setting the translational positioning of DNA within nucleosomes. We observe, cross-kingdoms, a strong codon bias toward the avoidance of long A-tracts in exon regions, which enables the formation of high density of nucleosomes in these regions. Moreover, long A-tract avoidance is restricted exclusively to nucleosome-occupied exon regions. We show that this bias in codon usage is sufficient for enabling DNA organization within nucleosomes without constraints on the actual code for proteins. Thus, there is inter-dependency of the two major codes within DNA to allow their coexistence. Furthermore, we show that modulation of A-tract occurrences in exon versus non-exon regions may result in a unique alternation of the diameter of the ‘30-nm’ fiber model.     

Anticipated nucleosome positioning pattern in prokaryotes

Linguistic (word count) analysis of prokaryotic genome sequences, by Shannon N-gram extension, reveals that the dominant hidden motifs in A+T rich genomes are T(A)(T)A and G(A)(T)C with uncertain number of repeating A and T. Since prokaryotic sequences are largely protein-coding, the motifs would correspond to amphipathic alpha-helices with alternating lysine and phenylalanine as preferential polar and non-polar residues. The motifs are also known in eukaryotes, as nucleosome positioning patterns. Their existence in prokaryotes as well may serve for binding of histone-like proteins to DNA. In this case the above patterns in prokaryotes may be considered as anticipated nucleosome positioning patterns which, quite likely, existed in prokaryotic genomes before the evolutionary separation between eukaryotes and prokaryotes.     

nucleR: a package for non-parametric nucleosome positioning

SUMMARY: nucleR is an R/Bioconductor package for a flexible and fast recognition of nucleosome positioning from next generation sequencing and tiling arrays experiments. The software is integrated with standard high-throughput genomics R packages and allows for in situ visualization as well as to export results to common genome browser formats. AVAILABILITY: Additional information and methodological details can be found at http://mmb.pcb.ub.es/nucleR     

Sequencing crop genomes: approaches and applications

Many challenges face plant scientists, in particular those working on crop production, such as a projected increase in population, decrease in water and arable land, changes in weather patterns and predictability. Advances in genome sequencing and resequencing can and should play a role in our response to meeting these challenges. However, several barriers prevent rapid and effective deployment of these tools to a wide variety of crops. Because of the complexity of crop genomes, de novo sequencing with next-generation sequencing technologies is a process fraught with difficulties that then create roadblocks to the utilization of these genome sequences for crop improvement. Collecting rapid and accurate phenotypes in crop plants is a hindrance to integrating genomics with crop improvement, and advances in informatics are needed to put these tools in the hands of the scientists on the ground.     

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.     

A translational signature for nucleosome positioning in vivo

In vivo nucleosomes often occupy well-defined preferred positions on genomic DNA. An important question is to what extent these preferred positions are directly encoded by the DNA sequence itself. We derive here from in vivo positions, accurately mapped by partial micrococcal nuclease digestion, a translational positioning signal that identifies the approximate midpoint of DNA bound by a histone octamer. This midpoint is, on average, highly A/T rich (∼73%) and, in particular, the dinucleotide TpA occurs preferentially at this and other outward-facing minor grooves. We conclude that in this set of sequences the sequence code for DNA bending and nucleosome positioning differs from the other described sets and we suggest that the enrichment of AT-containing dinucleotides at the centre is required for local untwisting. We show that this signature is preferentially associated with nucleosomes flanking promoter regions and suggest that it contributes to the establishment of gene-specific nucleosome arrays.     

CpG deficiency, dinucleotide distributions and nucleosome positioning

The dinucleotide CpG is deficient in (A + T)-rich regions of vertebrate DNA in both coding and non-coding sequences and there is a corresponding increase above expectation in the occurrence of TpG and CpA. By contrast in (G + C)-rich regions no deficiency of CpG is found. Such (G + C)-rich sequences, containing the expected number of CpG dinucleotides, alternate along the genome with (A + T)-rich sequences which have a lower than expected CpG content. The G + C content of vertebrate DNA can oscillate with a period of 150-200 bp and this may be a factor in positioning nucleosomes. The role of mutagenesis in loss of CpG and increase of A + T, particularly in non-coding regions, is discussed.     

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.     

Chromatin folding modulates nucleosome positioning in yeast minichromosomes

Based on the chromatin structures of the yeast URA3 gene and the TRP1ARS1 circle, we have designed circular minichromosomes of different sizes that should each form a tight tetranucleosome. This structure was assumed to be stiff and bulky and therefore likely to be sensitive to packaging into a three-dimensional structure. The structures of the minichromosomes were determined using micrococcal nuclease. Only one of the minichromosomes showed a protected region of about 570 bp, compatible with the predicted tight tetranucleosome, while all other constructs showed alternative structures. A comparison of the structures revealed that neither histone-DNA interactions nor influences from flanking boundaries are sufficient determinants of nucleosome positions. The data strongly suggest that chromatin folding modulates the nucleosome arrangement along the DNA.     

Epigenetic nucleosomes: Alu sequences and CG as nucleosome positioning element

Alu sequences carry periodical pattern with CG dinucleotides (CpG) repeating every 31-32 bases. Similar distances are observed in distribution of DNA curvature in crystallized nucleosomes, at positions +/-1.5 and +/-4.5 periods of DNA from nucleosome DNA dyad. Since CG elements are also found to impart to nucleosomes higher stability when positioned at +/-1.5 sites, it suggests that CG dinucleotides may play a role in modulation of the nucleosome strength when the CG elements are methylated. Thus, Alu sequences may harbor special epigenetic nucleosomes with methylation-dependent regulatory functions. Nucleosome DNA sequence probe is suggested to detect locations of such regulatory nucleosomes in the sequences.     

Nucleosome positioning and nucleosome stacking: two faces of the same coin

Nucleosomes are regularly spaced along eukaryotic genomes. In the emerging model, known as "statistical positioning", this spacing is due to steric repulsion between nucleosomes and to the presence of nucleosome excluding barriers on the genome. However, new experimental evidence recently challenged the "statistical positioning" model (Z. Zhang et al., Science, 2011, 332(6032), 977-980). We propose here that the regular spacing can be better explained by adding attractive interactions between nucleosomes. In our model those attractions are due to the fact that nucleosomes are stacked in regular chromatin fibers. In a self-reinforcing mechanism, regular nucleosome spacing promotes in turn nucleosome stacking. We first show that this model can precisely account for the nucleosome spacing observed in Saccharomyces cerevisiae. We then use a simple toy model to show that attraction between nucleosomes can fasten the formation of the chromatin fiber.     

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.