Are nucleosome positions in vivo primarily determined by histone–DNA sequence preferences?

Large-scale and genome-wide studies have concluded that ∼80% of the yeast (Saccharomyces cerevisiae) genome is occupied by positioned nucleosomes. In vivo this nucleosome organization can result from a variety of mechanisms, including the intrinsic DNA sequence preferences for wrapping the DNA around the histone core. Recently, a genome-wide study was reported using massively parallel sequencing to directly compare in vivo and in vitro nucleosome positions. It was concluded that intrinsic DNA sequence preferences indeed have a dominant role in determining the in vivo nucleosome organization of the genome, consistent with a genomic code for nucleosome positioning. Some other studies disagree with this view. Using the large amount of data now available from several sources, we have attempted to clarify a fundamental question concerning the packaging of genomic DNA: to what extent are nucleosome positions in vivo determined by histone-DNA sequence preferences? We have analyzed data obtained from different laboratories in the same way, and have directly compared these data. We also identify possible problems with some of the experimental designs used and with the data analysis. Our findings suggest that DNA sequence preferences have only small effects on the positioning of individual nucleosomes throughout the genome in vivo.     

High-Resolution Mapping of Sequence-Directed Nucleosome Positioning on Genomic DNA

We have mapped in vitro nucleosome positioning on the sheep β-lactoglobulin gene using high-throughput sequencing to characterise the DNA sequences recovered from reconstituted nucleosomes. This methodology surpasses previous approaches for coverage, accuracy and resolution and, most importantly, offers a simple yet rapid and relatively inexpensive method to characterise genomic DNA sequences in terms of nucleosome positioning capacity. We demonstrate an unambiguous correspondence between in vitro and in vivo nucleosome positioning around the promoter of the gene; identify discrete, sequence-specific nucleosomal structures above the level of the canonical core particle—a feature that has implications for regulatory protein access and higher-order chromatin packing; and reveal new insights into the involvement of periodically organised dinucleotide sequence motifs of the type GG and CC and not AA and TT, as determinants of nucleosome positioning—an observation that supports the idea that the core histone octamer can exploit different patterns of sequence organisation, or structural potential, in the DNA to bring about nucleosome positioning.     

Telomeric repeats act as nucleosome-disfavouring sequences in vivo

Telomeric DNAs consist of tandem repeats of G-clusters such as TTAGGG and TG_1-3, which are the human and yeast repeat sequences, respectively. In the yeast Saccharomyces cerevisiae, the telomeric repeats are non-nucleosomal, whereas in humans, they are organized in tightly packaged nucleosomes. However, previous in vitro studies revealed that the binding affinities of human and yeast telomeric repeat sequences to histone octamers in vitro were similar, which is apparently inconsistent with the differences in the human and yeast telomeric chromatin structures. To further investigate the relationship between telomeric sequences and chromatin structure, we examined the effect of telomeric repeats on the formation of positioned nucleosomes in vivo by indirect end-label mapping, primer extension mapping and nucleosome repeat analyses, using a defined minichromosome in yeast cells. We found that the human and yeast telomeric repeat sequences both disfavour nucleosome assembly and alter nucleosome positioning in the yeast minichromosome. We further demonstrated that the G-clusters in the telomeric repeats are required for the nucleosome-disfavouring properties. Thus, our results suggest that this inherent structural feature of the telomeric repeat sequences is involved in the functional dynamics of the telomeric chromatin structure.     

DNA sequence patterns in precisely positioned nucleosomes

Several investigators have recognized the importance of non-periodic DNA sequence information in determining the translational position of precisely positioned nucleosomes. The purpose of this study is to determine the extent of such information, in addition to the character of periodic information present. This is accomplished by examining the half-nucleosome DNA sequences of a considerable number of precisely positioned nucleosomes, and determining the probability of occurrence of each dinucleotide type as a function of position from the nucleosome center to the terminus (positions 0 to 72). By the nature of this procedure, no assumptions of periodicity are made. The results show the importance of several DNA sequence periodicities including 6-7, 10, and 21 base pairs, in addition to significant nonperiodic information. The results demonstrate that each dinucleotide type is unique in terms of its positional preference in precisely positioned nucleosomes (for example AA not equal to TT). The probabilities of occurrence for the dinucleotide types can be used to predict the translational positions of a number of observed nucleosomes.     

Nucleosomes Shape DNA Polymorphism and Divergence

An estimated 80% of genomic DNA in eukaryotes is packaged as nucleosomes, which, together with the remaining interstitial linker regions, generate higher order chromatin structures [1]. Nucleosome sequences isolated from diverse organisms exhibit ∼10 bp periodic variations in AA, TT and GC dinucleotide frequencies. These sequence elements generate intrinsically curved DNA and help establish the histone-DNA interface. We investigated an important unanswered question concerning the interplay between chromatin organization and genome evolution: do the DNA sequence preferences inherent to the highly conserved histone core exert detectable natural selection on genomic divergence and polymorphism? To address this hypothesis, we isolated nucleosomal DNA sequences from Drosophila melanogaster embryos and examined the underlying genomic variation within and between species. We found that divergence along the D. melanogaster lineage is periodic across nucleosome regions with base changes following preferred nucleotides, providing new evidence for systematic evolutionary forces in the generation and maintenance of nucleosome-associated dinucleotide periodicities. Further, Single Nucleotide Polymorphism (SNP) frequency spectra show striking periodicities across nucleosomal regions, paralleling divergence patterns. Preferred alleles occur at higher frequencies in natural populations, consistent with a central role for natural selection. These patterns are stronger for nucleosomes in introns than in intergenic regions, suggesting selection is stronger in transcribed regions where nucleosomes undergo more displacement, remodeling and functional modification. In addition, we observe a large-scale (∼180 bp) periodic enrichment of AA/TT dinucleotides associated with nucleosome occupancy, while GC dinucleotide frequency peaks in linker regions. Divergence and polymorphism data also support a role for natural selection in the generation and maintenance of these super-nucleosomal patterns. Our results demonstrate that nucleosome-associated sequence periodicities are under selective pressure, implying that structural interactions between nucleosomes and DNA sequence shape sequence evolution, particularly in introns.     

Re-cracking the nucleosome positioning code

Nucleosomes, the fundamental repeating subunits of all eukaryotic chromatin, are responsible for packaging DNA into chromosomes inside the cell nucleus and controlling gene expression. While it has been well established that nucleosomes exhibit higher affinity for select DNA sequences, until recently it was unclear whether such preferences exerted a significant, genome-wide effect on nucleosome positioning in vivo. This question was seemingly and recently resolved in the affirmative: a wide-ranging series of experimental and computational analyses provided extensive evidence that the instructions for wrapping DNA around nucleosomes are contained in the DNA itself. This subsequently labeled second genetic code was based on data-driven, structural, and biophysical considerations. It was subjected to an extensive suite of validation procedures, with one conclusion being that intrinsic, genome-encoded, nucleosome organization explains approximately 50% of in vivo nucleosome positioning. Here, we revisit both the nature of the underlying sequence preferences, and the performance of the proposed code. A series of new analyses, employing spectral envelope (Fourier transform) methods for assessing key sequence periodicities, classification techniques for evaluating predictive performance, and discriminatory motif finding methods for devising alternate models, are applied. The findings from the respective analyses indicate that signature dinucleotide periodicities are absent from the bulk of the high affinity nucleosome-bound sequences, and that the predictive performance of the code is modest. We conclude that further exploration of the role of sequence-based preferences in genome-wide nucleosome positioning is warranted. This work offers a methodologic counterpart to a recent, high resolution determination of nucleosome positioning that also questions the accuracy of the proposed code and, further, provides illustrations of techniques useful in assessing sequence periodicity and predictive performance.     

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.     

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.     

Sequence-dependent Nucleosome Positioning

Eukaryotic DNA is organized into a macromolecular structure called chromatin. The basic repeating unit of chromatin is the nucleosome, which consists of two copies of each of the four core histones and DNA. The nucleosomal organization and the positions of nucleosomes have profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is therefore of general interest. Among the many determinants of nucleosome positioning, the DNA sequence has been proposed to have a major role. Here, we analyzed more than 860,000 nucleosomal DNA sequences to identify sequence features that guide the formation of nucleosomes in vivo. We found that both a periodic enrichment of AT base pairs and an out-of-phase oscillating enrichment of GC base pairs as well as the overall preference for GC base pairs are determinants of nucleosome positioning. The preference for GC pairs can be related to a lower energetic cost required for deformation of the DNA to wrap around the histones. In line with this idea, we found that only incorporation of both signal components into a sequence model for nucleosome formation results in maximal predictive performance on a genome-wide scale. In this manner, one achieves greater predictive power than published approaches. Our results confirm the hypothesis that the DNA sequence has a major role in nucleosome positioning in vivo.     

Poly-dA:dT Tracts Form an In Vivo Nucleosomal Turnstile

Nucleosomes regulate many DNA-dependent processes by controlling the accessibility of DNA, and DNA sequences such as the poly-dA:dT element are known to affect nucleosome binding. We demonstrate that poly-dA:dT tracts form an asymmetric barrier to nucleosome movement in vivo, mediated by ATP-dependent chromatin remodelers. We theorize that nucleosome transit over poly-A elements is more energetically favourable in one direction, leading to an asymmetric arrangement of nucleosomes around these sequences. We demonstrate that different arrangements of poly-A and poly-T tracts result in very different outcomes for nucleosome occupancy in yeast, mouse, and human, and show that yeast takes advantage of this phenomenon in its promoter architecture.     

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.     

G+C content dominates intrinsic nucleosome occupancy

Background  

The relative preference of nucleosomes to form on individual DNA sequences plays a major role in genome packaging. A wide variety of DNA sequence features are believed to influence nucleosome formation, including periodic dinucleotide signals, poly-A stretches and other short motifs, and sequence properties that influence DNA structure, including base content. It was recently shown by Kaplan et al. that a probabilistic model using composition of all 5-mers within a nucleosome-sized tiling window accurately predicts intrinsic nucleosome occupancy across an entire genome in vitro. However, the model is complicated, and it is not clear which specific DNA sequence properties are most important for intrinsic nucleosome-forming preferences.