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.     

5-Bromouracil disrupts nucleosome positioning by inducing A-form-like DNA conformation in yeast cells

5-Bromodeoxyuridine (BrdU) modulates expression of particular genes associated with cellular differentiation and senescence. Our previous studies have suggested an involvement of chromatin structure in this phenomenon. Here, we examined the effect of 5-bromouracil on nucleosome positioning in vivo using TALS plasmid in yeast cells. This plasmid can stably and precisely be assembled nucleosomes aided by the α2 repressor complex bound to its α2 operator. Insertion of AT-rich sequences into a site near the operator destabilized nucleosome positioning dependent on their length and sequences. Addition of BrdU almost completely disrupted nucleosome positioning through specific AT-tracts. The effective AT-rich sequences migrated faster on polyacrylamide gel electrophoresis, and their mobility was further accelerated by substitution of thymine with 5-bromouracil. Since this property is indicative of a rigid conformation of DNA, our results suggest that 5-bromouracil disrupts nucleosome positioning by inducing A-form-like DNA.     

基于核小体位置预测的酵母进化印迹研究

在利用核小体定位实验数据训练支持向量机(SVM)对任意酵母DNA序列的核小体形成能力进行预测的过程中,发现染色质结构对基因组DNA分子进化过程有着显著影响。我们观察到核小体DNA比连接DNA的平均预测准确率低15%,这种普遍存在的局部预测准确率差异性代表了酵母核小体定位的进化印迹(Evolutionary footprint),它揭示了核小体组织在基因组的整个进化过程中所具有的保守性。     

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.     

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.     

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.     

Global nucleosome occupancy in yeast

Background  

Although eukaryotic genomes are generally thought to be entirely chromatin-associated, the activated PHO5 promoter in yeast is largely devoid of nucleosomes. We systematically evaluated nucleosome occupancy in yeast promoters by immunoprecipitating nucleosomal DNA and quantifying enrichment by microarrays.     

Evolutionary insights into genome-wide nucleosome positioning

A new study takes an evolutionary approach to investigate to what extent nucleosome positioning is determined by underlying sequence or by trans-acting factors.     

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.     

Predicting nucleosome positioning in genomes: physical and bioinformatic approaches

In eukaryotic genomes, nucleosomes are responsible for packaging DNA and controlling gene expression. For this reason, an increasing interest is arising on computational methods capable of predicting the nucleosome positioning along genomes. In this review we describe and compare bioinformatic and physical approaches adopted to predict nucleosome occupancy along genomes. Computational analyses attempt at decoding the experimental nucleosome maps of genomes in terms of certain dinucleotide step periodicity observed along DNA. Such investigations show that highly significant information about the occurrence of a nucleosome along DNA is intrinsic in certain features of the sequence suggesting that DNA of eukaryotic genomes encodes nucleosome organization. Besides the bioinformatic approaches, physical models were proposed based on the sequence dependent conformational features of the DNA chain, which govern the free energy needed to transform recurrent DNA tracts along the genome into the nucleosomal shape.     

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.