Sequence Signatures of Nucleosome Positioning in Caenorhabditis elegans

Our recent investigation in the protist Trichomonas vaginalis suggested a DNA sequence periodicity with a unit length of 120.9 nt, which represents a sequence signature for nucleosome positioning. We now extended our observation in higher eukaryotes and identified a similar periodicity of 175 nt in length in Caenorhabditis elegans. In the process of defining the sequence compositional characteristics, we found that the 10.5-nt periodicity, the sequence signature of DNA double helix, may not be sufficient for cross-nucleosome positioning but provides essential guiding rails to facilitate positioning. We further dissected nucleosome-protected sequences and identified a strong positive purine (AG) gradient from the 5′-end to the 3′-end, and also learnt that the nucleosome-enriched regions are GC-rich as compared to the nucleosome-free sequences as purine content is positively correlated with GC content. Sequence characterization allowed us to develop a hidden Markov model (HMM) algorithm for decoding nucleosome positioning computationally, and based on a set of training data from the fifth chromosome of C. elegans, our algorithm predicted 60%-70% of the well-positioned nucleosomes, which is 15%-20% higher than random positioning. We concluded that nucleosomes are not randomly positioned on DNA sequences and yet bind to different genome regions with variable stability, well-positioned nucleosomes leave sequence signatures on DNA, and statistical positioning of nucleosomes across genome can be decoded computationally based on these sequence signatures.     

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.     

The affinity of DNA sequences containing R5Y5 motif and TA repeats with 10.5-bp periodicity to histone octamer in vitro

Nucleosome positioning along the genome is partially determined by the intrinsic DNA sequence preferences on histone. RRRRRYYYYY (R5Y5, R?=?Purine and Y?=?Pyrimidine) motif in nucleosome DNA, which was presented based on several theoretical models by Trifonov et al., might be a facilitating sequence pattern for nucleosome assembly. However, there is not a high conformity experimental evidence to support the concept that R5Y5 motif is a key element for the determination of nucleosome positioning. In this work, the ability of the canonical, H2A.Z- and H3.3-containing octamers to assemble nucleosome on DNA templates containing R5Y5 motif and TA repeats within 10.5-bp periodicity was investigated by using salt-dialysis method in vitro. The results showed that the10.5-bp periodical distributions of both R5Y5 motif and TA repeats along DNA templates can significantly promote canonical nucleosome assembly and may be key sequence factors for canonical nucleosome assembly. Compared with TA repeats within 10.5-bp periodicity, R5Y5 motif in DNA templates did not elevate H2A.Z- and H3.3-containing nucleosome formation efficiency in vitro. This result indicates that R5Y5 motif probably isn’t a pivotal factor to regulate nucleosome assembly on histone variants. It is speculated that the regulatory mechanism of nucleosome assembly is different between canonical and variant histone. These conclusions can provide a deeper insight on the mechanism of nucleosome positioning.

Communicated by Ramaswamy H. Sarma     

Yeast nucleosome DNA pattern: deconvolution from genome sequences of S. cerevisiae

Positional correlation analysis for the complete genome of Saccharomyces cerevisiae is performed with the aim to reveal possible chromatin-related sequence features. A strong periodicity with the period 10.4 bases is detected in the distance histograms for the dinucleotides AA and TT, with the characteristic decay distance of approximately 50 base pairs. The oscillations are observed as well in the distributions of other dinucleotides. However, the respective amplitudes are small, consistent with secondary effects, due to dominant periodicity of AA and TT. The observations are in accord with earlier data on the chromatin sequence periodicities and nucleosome DNA sequence patterns. The autocorrelations of AA and TT dinucleotides in yeast include also a counter-phase component. A tentative DNA sequence pattern for the yeast nucleosomes is suggested and verified by comparison of its autocorrelation plots with the respective natural autocorrelations. The nucleosome mapping guided by the pattern is in accord with experimental data on the linker length distribution in yeast.     

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.     

Yeast Nucleosome DNA Pattern: Deconvolution from Genome Sequences of S. cerevisiae

Abstract

Positional correlation analysis for the complete genome of Saccharomyces cerevisiae is performed with the aim to reveal possible chromatin-related sequence features. A strong periodicity with the period 10.4 bases is detected in the distance histograms for the dinucleotides AA and TT, with the characteristic decay distance of approximately 50 base pairs. The oscillations are observed as well in the distributions of other dinucleotides. However, the respective amplitudes are small, consistent with secondary effects, due to dominant periodicity of AA and TT. The observations are in accord with earlier data on the chromatin sequence periodicities and nucleosome DNA sequence patterns. The autocorrelations of AA and TT dinucleotides in yeast include also a counter-phase component. A tentative DNA sequence pattern for the yeast nucleosomes is suggested and verified by comparison of its autocorrelation plots with the respective natural autocorrelations. The nucleosome mapping guided by the pattern is in accord with experimental data on the linker length distribution in yeast.     

Periodicity in prokaryotic and eukaryotic genomes identified by power spectrum analysis

We used a power spectrum method to identify periodic patterns in nucleotide sequence, and characterized nucleotide sequences that confer periodicities to prokaryotic and eukaryotic genomes and genomes. A 10-bp periodicity was prevalent in hyperthermophilic bacteria and archaebacteria, and an 11-bp periodicity was prevalent in eubacteria. The 10-bp periodicity was also prevalent in the eukaryotes such as the worm Caenorhabditis elegans. Additionally, in the worm genome, a 68-bp periodicity in chromosome I, a 59-bp periodicity in chromosome II, and a 94-bp periodicity in chromosome III were found. In human chromosomes 21 and 22, approximately 167- or 84-bp periodicity was detected along the entire length of these chromosomes. Because the 167-bp is identical to the length of DNA that forms two complete helical turns in nucleosome organization, we speculated that the respective sequences may correspond to arrays of a special compact form of nucleosomes clustered in specific regions of the human chromosomes. This periodic element contained a high frequency of TGG. TGG-rich sequences are known to form a specific subset of folded DNA structures, and therefore, the sequences might have potential to form specific higher order structures related to the clustered occurrence of a specific form of the speculated nucleosomes.     

Periodicity of exonuclease III digestion of chromatin and the pitch of deoxyribonucleic acid on the nucleosome

Exonuclease III has previously been shown to pause about every 10 nucleotides along the 3' strands while it invades the nucleosome core. Here, the exact periodicity of this digestion, i.e., the spacing of the pauses, was determined. Results showed that the exonuclease digests the first 20 nucleotides at the edge of the nucleosome core with a periodicity of approximately 11 nucleotides; in contrast, DNA closer to the center of the particle is digested with a smaller periodicity of about 10 nucleotides. These figures differ from the known periodicity of DNase I digestion, approximately 10 and 10.5 nucleotides at the edge and in the center of the nucleosome, respectively. Moreover, as shown by sedimentations in sucrose gradients, the structure of the nucleosome does not appear to be significantly altered by the gradual destruction of its DNA moiety by the exonuclease. Such stability of the nucleosome, along with other complementary observations, indicates that the transition in the digestion periodicity of the exonuclease may not be the consequence of a structural rearrangement of the particle upon trimming. This transition may rather be ascribed to the properties of the native nucleosome and to the intrinsic mechanism of action of the enzyme. Finally, evidence is presented which suggests that the exonuclease 10-nucleotide periodicity of digestion of the inner region of the nucleosome reflects a 10 base pair/turn pitch of the DNA in that region.     

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.     

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.     

Oligonucleotide Sequence Motifs as Nucleosome Positioning Signals

To gain a better understanding of the sequence patterns that characterize positioned nucleosomes, we first performed an analysis of the periodicities of the 256 tetranucleotides in a yeast genome-wide library of nucleosomal DNA sequences that was prepared by in vitro reconstitution. The approach entailed the identification and analysis of 24 unique tetranucleotides that were defined by 8 consensus sequences. These consensus sequences were shown to be responsible for most if not all of the tetranucleotide and dinucleotide periodicities displayed by the entire library, demonstrating that the periodicities of dinucleotides that characterize the yeast genome are, in actuality, due primarily to the 8 consensus sequences. A novel combination of experimental and bioinformatic approaches was then used to show that these tetranucleotides are important for preferred formation of nucleosomes at specific sites along DNA in vitro. These results were then compared to tetranucleotide patterns in genome-wide in vivo libraries from yeast and C. elegans in order to assess the contributions of DNA sequence in the control of nucleosome residency in the cell. These comparisons revealed striking similarities in the tetranucleotide occurrence profiles that are likely to be involved in nucleosome positioning in both in vitro and in vivo libraries, suggesting that DNA sequence is an important factor in the control of nucleosome placement in vivo. However, the strengths of the tetranucleotide periodicities were 3–4 fold higher in the in vitro as compared to the in vivo libraries, which implies that DNA sequence plays less of a role in dictating nucleosome positions in vivo. The results of this study have important implications for models of sequence-dependent positioning since they suggest that a defined subset of tetranucleotides is involved in preferred nucleosome occupancy and that these tetranucleotides are the major source of the dinucleotide periodicities that are characteristic of positioned nucleosomes.     

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.     

The histone core exerts a dominant constraint on the structure of DNA in a nucleosome.

We have examined the structures of unique sequence, A/T-rich DNAs that are predicted to be relatively rigid [oligo(dA).oligo(dT)], flexible [oligo[d(A-T)]], and curved, using the hydroxyl radical as a cleavage reagent. A 50-base-pair segment containing each of these distinct DNA sequences was placed adjacent to the T7 RNA polymerase promoter, a sequence that will strongly position nucleosomes. The final length of the DNA fragments was 142 bp, enough DNA to assemble a single nucleosome. Cleavage of DNA in solution, while bound to a calcium phosphate crystal, and after incorporation into a nucleosome is examined. We find that the distinct A/T-rich DNAs have very different structural features in solution and helical periodicities when bound to a calcium phosphate. In contrast, the organization of the different DNA sequences when associated with a histone octamer is very similar. We conclude that the histone core exerts a dominant constraint on the structure of DNA in a nucleosome and that inclusion of these various unique sequences has only a very small effect on overall nucleosome stability and structure.     

Periodicity in DNA coding sequences: implications in gene evolution

In this paper we have employed Fourier analysis of DNA coding and non-coding sequences in an attempt to identify possible patterns in gene sequences. It was found that while intronic sequences show a rather random pattern, coding sequences show periodicities and in particular a periodicity of 3. We were able to reconstruct such patterns by assuming a gene having one codon occurring in about 40% of the sequence. This could indicate that the predominant presence of codons all starting from the same base could confer the observed periodicities. Indeed, it was found that proteins do obey this rule. Implications of this finding in gene evolution are discussed.     

Introns of the chicken ovalbumin gene promote nucleosome alignment in vitro.

A defined in vitro chromatin assembly system was used to examine the nucleosome alignment induced by histone H5 throughout a 12 kilobase pair chicken genomic DNA fragment containing the ovalbumin gene. In contrast with total fragmented chicken DNA and several anonymous cloned fragments, much of the gene permitted histone H5 to space nucleosomes at physiological intervals in an extended array. Nucleosomes at the 3'-end of the gene and on approximately 4 kilobase pairs of 5'-flanking ovalbumin sequence did not become aligned to appreciable extents. Analysis of cloned 2-3 kilobase pair subfragments suggested that a strong nucleosome alignment signal, specifying a 196 +/- 5 base pair repeat exists in intron E. A second discrete region of the gene, which mapped approximately to intron A, exhibited nucleosome alignment with a spacing periodicity of about 200 base pairs. The ovalbumin cDNA did not permit nucleosome alignment. These findings suggest that some of the introns contain signals that direct nucleosome alignment over the ovalbumin gene in a way conducive to its regulation.     

A measure of DNA periodicity

A Fourier transform g(n) of a sequence of bases along a given stretch of DNA is defined. The transform is invariant to the labelling of the bases and can therefore be used as a measure of periodicity for segments of DNA with differing base content. It can also be conveniently used to search for base periodicities within large DNA data bases.