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.     

Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.     

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.     

Sequence-directed Mapping of Nucleosome Positions

Abstract

A nucleosome DNA sequence probe is designed that combines recently derived RR/YY counter-phase and AA/TT in-phase periodical patterns. A simple nucleosome mapping procedure is introduced for prediction of the nucleosome positions in the sequence of interest, to serve as a guide for experimental studies of the chromatin structure.     

Sequence-directed mapping of nucleosome positions

A nucleosome DNA sequence probe is designed that combines recently derived RR/YY counter-phase and AA/TT in-phase periodical patterns. A simple nucleosome mapping procedure is introduced for prediction of the nucleosome positions in the sequence of interest, to serve as a guide for experimental studies of the chromatin structure.     

Quantifying the role of steric constraints in nucleosome positioning

Statistical positioning, the localization of nucleosomes packed against a fixed barrier, is conjectured to explain the array of well-positioned nucleosomes at the 5′ end of genes, but the extent and precise implications of statistical positioning in vivo are unclear. We examine this hypothesis quantitatively and generalize the idea to include moving barriers as well as nucleosomes actively packed against a barrier. Early experiments noted a similarity between the nucleosome profile aligned and averaged across genes and that predicted by statistical positioning; however, we demonstrate that aligning random nucleosomes also generates the same profile, calling the previous interpretation into question. New rigorous results reformulate statistical positioning as predictions on the variance structure of nucleosome locations in individual genes. In particular, a quantity termed the variance gradient, describing the change in variance between adjacent nucleosomes, is tested against recent high-throughput nucleosome sequencing data. Constant variance gradients provide support for generalized statistical positioning in ∼50% of long genes. Genes that deviate from predictions have high nucleosome turnover and cell-to-cell gene expression variability. The observed variance gradient suggests an effective nucleosome size of 158 bp, instead of the commonly perceived 147 bp. Our analyses thus clarify the role of statistical positioning 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.     

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.     

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.     

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.     

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.     

Dinucleosome DNA of human K562 cells: experimental and computational characterizations

Dinucleosome formation is the first step in the organization of the higher order chromatin structure. With the ultimate aim of elucidating the dinucleosome structure, we constructed a library of human dinucleosome DNA. The library consists of PCR-amplifiable DNA fragments obtained by treatment of nuclei of erythroid K562 cells with micrococcal nuclease followed by extraction of DNA and adaptor ligation to the blunt-ended DNA fragments. The library was then cloned using a plasmid vector and the sequences of the clones were determined. The dominating clones containing the Alu elements were removed. A total of 1002 clones, which comprised a dinucleosome database, contained 84 and 918 clones from the clones before and after removing Alu elements, respectively. Approximately 70% of the clones were between 300 and 400 bp in size and they were distributed to various locations of all chromosomes except the Y chromosome. The clones containing A(2)N(8)A(2)N(8)A(2) or T(2)N(8)T(2)N(8)T(2) sequences were classified into three types, Type I (N shape), Type II (V shape) and Type III (M shape) according to DNA curvature plots. The locations of experimentally determined curved DNA segments matched well with the calculated ones though the clones of Types I and III showed additional curved DNA segments as revealed by the curvature plots. The distributions of complementary dinucleotides in the nucleosome DNA, at the ends of the dinucleosome DNA clones, allowed us to predict the positions of the nucleosome dyad axis, and estimate the size of the nucleosome core DNA, 125nt. The distributions of AA and TT dinucleotides, as well as other RR and YY dinucleotides, showed a periodicity with an average period of 10.4 bases, close to the values observed before. Mapping of nucleosome positions in the dinucleosome database based on the observed periodicity revealed that the nucleosomes were separated by a linker of 7.5+ approximately 10 x n nt. This indicates that the nucleosome-nucleosome orientations are, typically, halfway between parallel and antiparallel. Also an important finding is that the distributions of AA/TT and other RR/YY dinucleotides, apparently, reflect both DNA curvature and DNA bendability, cooperatively contributing to the nucleosome formation.     

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.     

Polymorphisms of <Emphasis Type="Italic">STAT5A</Emphasis> gene and their association with milk production traits in Holstein cows

The STAT5A gene was studied as a candidate gene for five milk production traits (milk yield at 305 days, protein percentage, fat percentage, lactose percentage and dry matter percentage) in Holstein cows. According to the sequence of bovine STAT5A gene, two pairs of primers (P1 and P2) were designed to detect polymorphisms of STAT5A gene in 401 Holstein cows by PCR-RFLP and PCR-SSCP. The results showed that the products amplified by primers P1 and P2 displayed polymorphisms. For P1, three genotypes (AA, AG, and GG) were detected, and the frequency of AA/AG/GG was 0.252/0.486/0.262, respectively. Sequence analysis revealed a single nucleotide substitution A–G at 14217 bp (GenBank NC_007317) of bovine STAT5A gene while compared GG genotype with AA genotype. The differences of the least squares means for the four milk production traits (milk yield at 305 days, fat percentage, lactose percentage and dry matter percentage) between AA, AG and GG were not significant (P > 0.05). Least squares mean of protein percentage for AG or GG was significantly higher than that for AA (P < 0.05); the difference of the least squares mean for protein percentage was not significant between AG and GG (P > 0.05). For P2, three genotypes (CC, CT, and TT) were detected in Holstein cows, and the frequency of CC/CT/TT was 0.751/0.234/0.015, respectively. Sequencing revealed an insertion CCT at 17266 (NC_007317) of bovine STAT5A gene while compared CC genotype with TT genotype. The differences of the least squares means for the three milk production traits (protein percentage, lactose percentage and dry matter percentage) between CC, CT and TT were not significant (P > 0.05). Least squares mean of milk yield at 305 days for TT or CT was significantly higher than that for CC (P < 0.05); the difference of the least squares mean for milk yield at 305 days was not significant between TT and CT (P > 0.05). Least squares mean of fat percentage for CC or CT was significantly higher than that for TT (P < 0.05); the difference of the least squares mean for fat percentage was not significant between CC and CT (P > 0.05). The results preliminarily indicated that allele G of A14217G polymorphic site of STAT5A gene is a potential DNA marker for improving protein percentage in dairy cattle, 17266indelCCT polymorphic site of STAT5A gene is a potential DNA marker for improving milk yield at 305 days and fat percentage in dairy cattle.     

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.     

The Intramolecular Impact to the Sequence Specificity of B→A Transition: Low Energy Conformational Variations in AA/TT and GG/CC Steps

Abstract It is well known, that local B→A transformation in DNA is involved in several biological processes. In vitro B?A transition is sequence-specific. The physical basis of this specificity is not known yet. Here we analyze the effect of intramolecular interactions on the structural behavior of the GG/CC and AA/TT steps. These steps exemplify sequence specific bias to the B- or A-form structure. Optimization of potential energy of the molecular systems composed of an octanucle-otide, neutralized by Na(+) and solvated with TIP3P water molecules in rectangular box with periodic boundary conditions gives the statistically representative sets of low energy structures for GG/CC and AA/TT steps in the middle of the diverse flanking sequences. Permissible 3D variations of GG/CC and AA/TT, and correlation of the relative motion of base pairs in these steps were analyzed. AA/TT step permits high variability for low energy conformers in the B-form DNA and small variability for low energy conformers in the A-form DNA. In contrast GG/CC step permits high variability for low energy conformers in the A-form DNA and small variability for low energy conformers in the B-form DNA. The relative motion of base pairs in GG/CC step is high correlated, while in AA/TT step this correlation is notably less. Atom-atom interactions inside-the-step always favors the B-form and their component - stacking interactions (atomatom interactions between nucleic bases) is crucial for the duplex stabilization. Formation of the A-form for both steps is a result of interactions with the flanking sequences and water-cation environment in the box. The average energy difference between conformations presenting B-form and A-form for the GG/CC step is high, while for the AA/TT step it is rather low. Thus, intramolecular interactions in GG/CC and AA/TT steps affect the possible conformational diversity ("conformational entropy") of the A- and B- type structures of DNA step. This determines the known bias of the A-form DNA depending on the enrichment of sequences with GG/CC. If structural tuning during the process of protein-DNA complex formation lead to the local B→A transformation of DNA, it is largely directed by high conformational diversity of GG/CC step in the A-form. In such a case the presence in the target site of both kinds of examined steps ensures the reversible character of ligand binding.     

Direct measurements of the nucleosome-forming preferences of periodic DNA motifs challenge established models

Several periodic motifs have been implicated in facilitating the bending of DNA around the histone core of the nucleosome. For example, di-nucleotides AA/TT/TA and GC at ∼10-bp periods, but offset by 5 bp, are found with higher-than-expected occurrences in aligned nucleosomal DNAs in vitro and in vivo. Additionally, regularly oscillating period-10 trinucleotide motifs non-T, A/T, G and their complements have been implicated in the formation of regular nucleosome arrays. The effects of these periodic motifs on nucleosome formation have not been systematically tested directly by competitive reconstitution assays. We show that, in general, none of these period-10 motifs, except TA, in certain sequence contexts, facilitates nucleosome formation. The influence of periodic TAs on nucleosome formation is appreciable; with some of the 200-bp DNAs out-competing bulk nucleosomal DNA by more than 400-fold. Only the nucleotides immediately flanking TA influence its nucleosome-forming ability. Period-10 TA, when flanked by a pair of permissive nucleotides, facilitates DNA bending through compression of the minor groove. The free energy change for nucleosome formation decreases linearly with the number of consecutive TAs, up to eight. We suggest how these data can be reconciled with previous findings.     

Changing Chromatin Fiber Conformation by Nucleosome Repositioning

Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.