Sequence structure of hidden 10.4-base repeat in the nucleosomes of C. elegans

By measuring prevailing distances between YY, YR, RR, and RY dinucleotides in the large database of the nucleosome DNA fragments from C. elegans, the consensus sequence structure of the nucleosome DNA repeat of C. elegans was reconstructed: (YYYYYRRRRR)n. An actual period was estimated to be 10.4 bases. The pattern is fully consistent with the nucleosome DNA patterns of other eukaryotes, as established earlier, and, thus, the YYYYYRRRRR repeat can be considered as consensus nucleosome DNA sequence repeat across eukaryotic species. Similar distance analysis for [A, T] dinucleotides suggested the related pattern (TTTYTARAAA)n where the TT and AA dinucleotides display rather out of phase behavior, contrary to the "AA or TT" in-phase periodicity, considered in some publications. A weak 5-base periodicity in the distribution of TA dinucleotides was detected.     

Sequence structure of human nucleosome DNA

Positional distributions of various dinucleotides in experimentally derived human nucleosome DNA sequences are analyzed. Nucleosome positioning in this species is found to depend largely on GG and CC dinucleotides periodically distributed along the nucleosome DNA sequence, with the period of 10.4 bases. The GG and CC dinucleotides oscillate counterphase, i.e., their respective preferred positions are shifted about a half-period from one another, as it was observed earlier for AA and TT dinucleotides. Other purine-purine and pyrimidine-pyrimidine dinucleotides (RR and YY) display the same periodical and counterphase pattern. The dominance of oscillating GG and CC dinucleotides in human nucleosomes and the contribution of AG(CT), GA(TC), and AA(TT) suggest a general nucleosome DNA sequence pattern - counterphase oscillation of RR and YY dinucleotides. AA and TT dinucleotides, commonly accepted as major players, are only weak contributors in the case of human nucleosomes.     

Sequence Structure of Hidden 10.4-base Repeat in the Nucleosomes of C. elegans

Abstract

By measuring prevailing distances between YY, YR, RR, and RY dinucleotides in the large database of the nucleosome DNA fragments from C. elegans, the consensus sequence structure of the nucleosome DNA repeat of C. elegans was reconstructed: (YYYYYRRRRR)_n. An actual period was estimated to be 10.4 bases. The pattern is fully consistent with the nucleosome DNA patterns of other eukaryotes, as established earlier, and, thus, the YYYYYRRRRR repeat can be considered as consensus nucleosome DNA sequence repeat across eukaryotic species. Similar distance analysis for [A, T] dinucleotides suggested the related pattern (TTTYTARAAA)_n where the TT and AA dinucleotides display rather out of phase behavior, contrary to the “AA or TT” in-phase periodicity, considered in some publications. A weak 5-base periodicity in the distribution of TA dinucleotides was detected.     

Biochemical screening of stable dinucleosomes using DNA fragments from a dinucleosome DNA library

The dinucleosome is an informative unit for analysis of the higher-order chromatin structure. DNA fragments forming stable dinucleosomes were screened from a dinucleosome DNA library after the reconstitution of nucleosomes in vitro and digestion with micrococcal nuclease. Reconstituted dinucleosomes showed a diversity of sensitivity to micrococcal nuclease, suggesting that the biochemical stability of a dinucleosome depends, in part, on the DNA fragments. The DNA fragments after the screening were classified into three groups represented by clones bf10, af14 and af32 according to the sensitivity to micrococcal nuclease. Mapping of the nucleosome boundaries by Southern blotting of the DNA after restriction digestion and by primer extension analysis showed that each nucleosome position of clone af32 was fixed. Analysis of reconstituted dinucleosomes using mutant DNA fragments of clone af32 revealed a unique property characteristic of a key nucleosome, given that the replacement of a DNA fragment corresponding to the right nucleosome position resulted in marked sensitivity to micrococcal nuclease, whereas the replacement of the other nucleosome fragment had almost no effect on sensitivity as compared to the original af32 construct. The mutant construct in which the right nucleosome was removed showed multiple nucleosome phases, suggesting that the right nucleosome stabilized first each mononucleosome and then the dinucleosome. An oligonucleotide bending assay revealed that the DNA fragment in the right nucleosome included curved DNA, suggesting that the positioning activity of the nucleosome was attributed to its DNA structure. These results suggest that information for forming stable dinucleosome is embedded in the genomic DNA and that a further characterization of the key nucleosome is useful for understanding the building up of the chromatin structure.     

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.     

Three sequence rules for chromatin

Extensive DNA sequence analysis of three eukaryotes, S. cerevisiae, C. elegans, and D. melanogaster, reveals two different AA/TT periodical patterns associated with the nucleosome positioning. The first pattern is the counter-phase oscillation of AA and TT dinucleotides, which has been frequently considered as the nucleosome DNA pattern. This represents the sequence rule I for chromatin structure. The second pattern is the in-phase oscillation of the AA and TT dinucleotides with the same nucleosome DNA period, 10.4 bases. This pattern apparently corresponds to curved DNA, that also participates in the nucleosome formation, and represents the sequence rule II for chromatin. The positional correlations of AA and TT dinucleotides also indicate that the nucleosomes are separated by specific linker sizes (preferably 8, 18, ... bases), dictated by the steric exclusion rules. Thus, the sequence positions of the neighboring nucleosomes are correlated, and this represents the sequence rule III.     

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.     

Three Sequence Rules for Chromatin

Abstract

Extensive DNA sequence analysis of three eukaryotes, S. cerevisiae, C. elegans, and D. melanogaster, reveals two different AA/TT periodical patterns associated with the nucleosome positioning. The first pattern is the counter-phase oscillation of AA and TT dinucleotides, which has been frequently considered as the nucleosome DNA pattern. This represents the sequence rule I for chromatin structure. The second pattern is the in-phase oscillation of the AA and TT dinucleotides with the same nucleosome DNA period, 10.4 bases. This pattern apparently corresponds to curved DNA, that also participates in the nucleosome formation, and represents the sequence rule II for chromatin. The positional correlations of AA and TT dinucleotides also indicate that the nucleosomes are separated by specific linker sizes (preferably 8, 18,…bases), dictated by the steric exclusion rules. Thus, the sequence positions of the neighboring nucleosomes are correlated, and this represents the sequence rule III.     

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.     

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.     

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.     

Universal full-length nucleosome mapping sequence probe

For the computational sequence-directed mapping of the nucleosomes, the knowledge of the nucleosome positioning motifs – 10–11 base long sequences – and respective matrices of bendability, is not sufficient, since there is no justified way to fuse these motifs in one continuous nucleosome DNA sequence. Discovery of the strong nucleosome (SN) DNA sequences, with visible sequence periodicity allows derivation of the full-length nucleosome DNA bendability pattern as matrix or consensus sequence. The SN sequences of three species (A. thaliana, C. elegans, and H. sapiens) are aligned (512 sequences for each species), and long (115 dinucleotides) matrices of bendability derived for the species. The matrices have strong common property – alternation of runs of purine–purine (RR) and pyrimidine–pyrimidine (YY) dinucleotides, with average period 10.4 bases. On this basis the universal [R,Y] consensus of the nucleosome DNA sequence is derived, with exactly defined positions of respective penta- and hexamers RRRRR, RRRRRR, YYYYY, and YYYYYY.     

A Possible Role of DNA Superstructures in Genome Evolution

The concept of DNA as a simple repository of the gene information has changed in that of a polymorphic macromolecule, which plays a relevant part in the management of the complex biochemical transformations in living matter. As a consequence of the slight stereochemical differences between base pairs, the direction of the DNA double helix axis undergoes deterministic writhing. A useful representation of such sequence-dependent structural distortions is the curvature diagram. Here, it is reported as an evolution simulation obtained by extensive point mutations along a biologically important DNA tract. The curvature changes, consequence of the point mutations. were compared to the related experimental gel electrophoresis mobility. The curvature of most mutants decreases and the mobility increases accordingly, suggesting the curvature of that tract is genetically selected. Moreover, DNA images by scanning force microscopy, show evidence of a sequence-dependent adhesion of curved DNA tracts to inorganic crystal surfaces. In particular, mica shows a large affinity towards the TT-rich dinucleotide sequences. This suggests a possible mechanism of selection of curved DNA regions, characterized by AA.TT dinucleotides in phase with double-helical periodicity, in the very early evolution steps.     

Sequence-dependent bending of DNA and phasing of nucleosomes

Conformational analysis has revealed anisotropic flexibility of the B-DNA double helix: it bends most easily into the grooves, being the most rigid when bent in a perpendicular direction. This result implies that DNA in a nucleosome is curved by means of relatively sharp bends ("mini-kinks") which are directed into the major and minor grooves alternatively and separated by 5-6 base pairs. The "mini-kink" model proved to be in keeping with the x-ray structure of the B-DNA dodecamer resolved later, which exhibits two "annealed kinks", also directed into the grooves. The anisotropy of B DNA is sequence-dependent: the pyrimidine-purine dimers (YR) favor bending into the minor groove, and the purine-pyrimidine dinucleotides (RY), into the minor one. The RR and YY dimers appear to be the most rigid dinucleotides. Thus, a DNA fragment consisting of the interchanging oligopurine and oligopyrimidine blocks 5-6 base pairs long should manifest a spectacular curvature in solution. Similarly, a nucleotide sequence containing the RY and YR dimers separated by a half-pitch of the double helix is the most suitable for wrapping around the nucleosomal core. Analysis of the numerous examples demonstrating the specific alignment of nucleosomes on DNA confirms this concept. So, the sequence-dependent "mechanical" properties of the double helix influence the spatial arrangement of DNA in chromatin.     

Repertoires of the Nucleosome-Positioning Dinucleotides

It is generally accepted that the organization of eukaryotic DNA into chromatin is strongly governed by a code inherent in the genomic DNA sequence. This code, as well as other codes, is superposed on the triplets coding for amino acids. The history of the chromatin code started three decades ago with the discovery of the periodic appearance of certain dinucleotides, with AA/TT and RR/YY giving the strongest signals, all with a period of 10.4 bases. Every base-pair stack in the DNA duplex has specific deformation properties, thus favoring DNA bending in a specific direction. The appearance of the corresponding dinucleotide at the distance 10.4 xn bases will facilitate DNA bending in that direction, which corresponds to the minimum energy of DNA folding in the nucleosome. We have analyzed the periodic appearances of all 16 dinucleotides in the genomes of thirteen different eukaryotic organisms. Our data show that a large variety of dinucleotides (if not all) are, apparently, contributing to the nucleosome positioning code. The choice of the periodical dinucleotides differs considerably from one organism to another. Among other 10.4 base periodicities, a strong and very regular 10.4 base signal was observed for CG dinucleotides in the genome of the honey bee A. mellifera. Also, the dinucleotide CG appears as the only periodical component in the human genome. This observation seems especially relevant since CpG methylation is well known to modulate chromatin packing and regularity. Thus, the selection of the dinucleotides contributing to the chromatin code is species specific, and may differ from region to region, depending on the sequence context.     

Human nucleosomes: special role of CG dinucleotides and Alu-nucleosomes

Background

The periodical occurrence of dinucleotides with a period of 10.4 bases now is undeniably a hallmark of nucleosome positioning. Whereas many eukaryotic genomes contain visible and even strong signals for periodic distribution of dinucleotides, the human genome is rather featureless in this respect. The exact sequence features in the human genome that govern the nucleosome positioning remain largely unknown.

Results

When analyzing the human genome sequence with the positional autocorrelation method, we found that only the dinucleotide CG shows the 10.4 base periodicity, which is indicative of the presence of nucleosomes. There is a high occurrence of CG dinucleotides that are either 31 (10.4 × 3) or 62 (10.4 × 6) base pairs apart from one another - a sequence bias known to be characteristic of Alu-sequences. In a similar analysis with repetitive sequences removed, peaks of repeating CG motifs can be seen at positions 10, 21 and 31, the nearest integers of multiples of 10.4.

Conclusions

Although the CG dinucleotides are dominant, other elements of the standard nucleosome positioning pattern are present in the human genome as well. The positional autocorrelation analysis of the human genome demonstrates that the CG dinucleotide is, indeed, one visible element of the human nucleosome positioning pattern, which appears both in Alu sequences and in sequences without repeats. The dominant role that CG dinucleotides play in organizing human chromatin is to indicate the involvement of human nucleosomes in tuning the regulation of gene expression and chromatin structure, which is very likely due to cytosine-methylation/-demethylation in CG dinucleotides contained in the human nucleosomes. This is further confirmed by the positions of CG-periodical nucleosomes on Alu sequences. Alu repeats appear as monomers, dimers and trimers, harboring two to six nucleosomes in a run. Considering the exceptional role CG dinucleotides play in the nucleosome positioning, we hypothesize that Alu-nucleosomes, especially, those that form tightly positioned runs, could serve as "anchors" in organizing the chromatin in human cells.     

Wrapping of genomic polydA.polydT tracts around nucleosome core particles.

Five human clones containing genomic regions of polydA have been isolated by their ability to form intermolecular triple helices with agarose cross-linked polyU. All of these clones contain Alu repetitive DNA sequences. End-labelled DNA fragments containing these sequences have been successfully reconstituted onto nucleosome core particles by salt exchange. The structure of these has been examined by digesting with DNase I, hydroxyl radicals or diethylpyrocarbonate. DNase I cleavage of the polydA tracts is poor in the free DNA but is markedly enhanced at certain positions when complexed with nucleosome cores. Phased digestion patterns are observed which continue through the (A)n blocks and reveal an average helical periodicity of about 10 base pairs. The distance between adjacent maxima varies between 8-12 base pairs, suggesting that the exact helical repeat is not necessarily constant. One fragment containing the sequence (TA)11T34 reveals a 12 base pair repeat within the (AT)n region. A pUC19 polylinker fragment containing a block of A69.T69 cloned into the Smal site could also be reconstituted onto nucleosome cores and reveals the same phased DNaseI digestion pattern. The DNase I cleavage pattern is not identical at each of the maxima, suggesting that the structural distortions imposed by the core particles are not constant along the DNA.     

Sequence-Dependent Bending of DNA and Phasing of Nucleosomes

Abstract

Conformational analysis has revealed anisotropic flexibility of the B-DNA double helix: it bends most easily into the grooves, being the most rigid when bent in a perpendicular direction. This result implies that DNA in a nucleosome is curved by means of relatively sharp bends (“mini-kinks”) which are directed into the major and minor grooves alternatively and separated by 5–6 base pairs. The “mini-kink” model proved to be in keeping with the x-ray structure of the B-DNA dodecamer resolved later, which exhibits two “annealed kinks”, also directed into the grooves.

The anisotropy of B DNA is sequence-dependent: the pyrimidine-purine dimers (YR) favor bending into the minor groove, and the purine-pyrimidine dinucleotides (RY), into the minor one. The RR and YY dimers appear to be the most rigid dinucleotides. Thus, a DNA fragment consisting of the interchanging oligopurine and oligopyrmidine blocks 5–6 base pairs long should manifest a spectacular curvature in solution.

Similarly, a nucleotide sequence containing the RY and YR dimers separated by a half-pitch of the double helix is the most suitable for wrapping around the nucleosomal core. Analysis of the numerous examples demonstrating the specific alignment of nucleosomes on DNA confirms this concept. So, the sequence-dependent “mechanical” properties of the double helix influence the spatial arrangement of DNA in chromatin.     

Unusual DNA structures associated with germline genetic activity in Caenorhabditis elegans

We describe a surprising long-range periodicity that underlies a substantial fraction of C. elegans genomic sequence. Extended segments (up to several hundred nucleotides) of the C. elegans genome show a strong bias toward occurrence of AA/TT dinucleotides along one face of the helix while little or no such constraint is evident on the opposite helical face. Segments with this characteristic periodicity are highly overrepresented in intron sequences and are associated with a large fraction of genes with known germline expression in C. elegans. In addition to altering the path and flexibility of DNA in vitro, sequences of this character have been shown by others to constrain DNA::nucleosome interactions, potentially producing a structure that could resist the assembly of highly ordered (phased) nucleosome arrays that have been proposed as a precursor to heterochromatin. We propose a number of ways that the periodic occurrence of An/Tn clusters could reflect evolution and function of genes that express in the germ cell lineage of C. elegans.     

Localization and characterization of curved DNA in the human erythropoietin receptor gene by experimental and theoretical approaches.

We report here the locations of curved DNA in the human erythropoietin receptor gene. A total of 13 DNA bend sites were mapped by circular permutation assays, appearing at an average interval of 651.2+/-214.6 (S.D.) in the 8-kb region. The bend centers in these 13 bend sites were confirmed by oligonucleotide-based assays where most of these centers had bend angles higher than that shown by (AAACCGGGCC) x (A)20 and lower than that shown by (AAACCGGGCC)2 x (A)10. DNA curvature mapping by TRIF software, which is based on the distribution of dinucleotides, primarily AA and TT, provided a highly accurate prediction for the locations of the bend sites. They showed approximately 20 degrees to 40 degrees of bend angles demonstrated by the oligonucleotide assays and by computer analysis.