Long-range oscillation in a periodic DNA sequence motif may influence nucleosome array formation

We have experimentally examined the characteristics of nucleosome array formation in different regions of mouse liver chromatin, and have computationally analyzed the corresponding genomic DNA sequences. We have shown that the mouse adenosine deaminase (MADA) gene locus is packaged into an exceptionally regular nucleosome array with a shortened repeat, consistent with our computational prediction based on the DNA sequence. A survey of the mouse genome indicates that <10% of 70 kb windows possess a nucleosome-ordering signal, consisting of regular long-range oscillations in the period-10 triplet motif non-T, A/T, G (VWG), which is as strong as the signal in the MADA locus. A strong signal in the center of this locus, confirmed by in vitro chromatin assembly experiments, appears to cooperate with weaker, in-phase signals throughout the locus. In contrast, the mouse odorant receptor (MOR) locus, which lacks locus-wide signals, was representative of ~40% of the mouse genomic DNA surveyed. Within this locus, nucleosome arrays were similar to those of bulk chromatin. Genomic DNA sequences which were computationally similar to MADA or MOR resulted in MADA- or MOR-like nucleosome ladders experimentally. Overall, we provide evidence that computationally predictable information in the DNA sequence may affect nucleosome array formation in animal tissue.     

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.     

Effects of DNA sequence and conformation on nucleosome formation

A simple theoretical analysis of the free energy balance controlling nucleosome formation shows that the specific effects of different DNA sequences and/or conformations observed in vitro are mainly due to their different elastic properties. A calculation of the elastic free energy required to fold DNA on histone octamers yields quantitative results rationalizing the experimental findings provided that: (i) the average helical repeat of DNA on nucleosomes is greater than 10.2 bp per turn, and (ii) poly[dG.dC] adopts an A-type conformation.     

Noncoding DNA, isochores and gene expression: nucleosome formation potential

The nucleosome formation potential of introns, intergenic spacers and exons of human genes is shown here to negatively correlate with among-tissues breadth of gene expression. The nucleosome formation potential is also found to negatively correlate with the GC content of genomic sequences; the slope of regression line is steeper in exons compared with noncoding DNA (introns and intergenic spacers). The correlation with GC content is independent of sequence length; in turn, the nucleosome formation potential of introns and intergenic spacers positively (albeit weakly) correlates with sequence length independently of GC content. These findings help explain the functional significance of the isochores (regions differing in GC content) in the human genome as a result of optimization of genomic structure for epigenetic complexity and support the notion that noncoding DNA is important for orderly chromatin condensation and chromatin-mediated suppression of tissue-specific genes.     

DNA sequence alterations affect nucleosome array formation of the chicken ovalbumin gene

The role of the large amount (more than half of the genome) of noncoding DNA in higher organisms is not well understood. DNA evolved to function in the context of chromatin, and the possibility exists that some of the noncoding DNA serves to influence chromatin structure and function. In this age of genomics and bioinformatics, genomic DNA sequences are being searched for informational content beyond the known genetic code. The discovery that period-10 non-T, A/T, G (VWG) triplets are among the most abundant motifs in human genomic DNA suggests that they may serve some function in higher organisms. In this paper, we provide direct evidence that the regular oscillation of period-10 VWG that occurs in the chicken ovalbumin gene sequence with a dinucleosome-like period facilitates nucleosome array formation. Using a linker histone-dependent in vitro chromatin assembly system that spontaneously aligns nucleosomes into a physiological array, we show that nucleosomes tend to avoid DNA regions with low period-10 VWG counts. This avoidance leads to the formation of an array with a nucleosome repeat equal to half the period value of the oscillation in period-10 VWG, as determined by Fourier analysis. Two different half-period deletions in the wild-type DNA sequence altered the nucleosome array, as predicted computationally. In contrast, a full-period deletion had an insignificant effect on the nucleosome array formed, also consistent with the prediction. An inversion mutation, with no DNA sequences deleted, again altered the nucleosome array formed, as predicted computationally. Hence, a VWG dinucleosome signal is plausible.     

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.     

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 impact of the HIRA histone chaperone upon global nucleosome architecture

HIRA is an evolutionarily conserved histone chaperone that mediates replication-independent nucleosome assembly and is important for a variety of processes such as cell cycle progression, development, and senescence. Here we have used a chromatin sequencing approach to determine the genome-wide contribution of HIRA to nucleosome organization in Schizosaccharomyces pombe. Cells lacking HIRA experience a global reduction in nucleosome occupancy at gene sequences, consistent with the proposed role for HIRA in chromatin reassembly behind elongating RNA polymerase II. In addition, we find that at its target promoters, HIRA commonly maintains the full occupancy of the ?1 nucleosome. HIRA does not affect global chromatin structure at replication origins or in rDNA repeats but is required for nucleosome occupancy in silent regions of the genome. Nucleosome organization associated with the heterochromatic (dg-dh) repeats located at the centromere is perturbed by loss of HIRA function and furthermore HIRA is required for normal nucleosome occupancy at Tf2 LTR retrotransposons. Overall, our data indicate that HIRA plays an important role in maintaining nucleosome architecture at both euchromatic and heterochromatic loci.     

DNA sequence directs placement of histone cores on restriction fragments during nucleosome formation.

Restriction fragments, 203 and 144 base pairs in length, bearing the Escherichia coli lac control region have been reconstituted with the core histones from calf thymus to form nucleosomes. By several criteria the reconstituted nucleosomes are similar to native nucleosomes obtained by micrococcal nuclease digestion of calf thymus nuclei. However, sensitive nuclease digestion studies reveal subtle and important differences between native monosomes and the lac reconstitutes. Each reconstitute consists mainly of nucleosomes containing histone cores placed nonrandomly with respect to the DNA sequence. The shorter reconstitute forms asymmetric nucleosomes as evidenced by the DNase I digestion pattern. Exonuclease III digestion followed by 5'-end analysis of the larger reconstitute suggests that, of the many possible arrangements of histone core with DNA sequence, only two are highly favored.     

The mechanics behind DNA sequence-dependent properties of the nucleosome

Chromatin organization and composition impart sophisticated regulatory features critical to eukaryotic genomic function. Although DNA sequence-dependent histone octamer binding is important for nucleosome activity, many aspects of this phenomenon have remained elusive. We studied nucleosome structure and stability with diverse DNA sequences, including Widom 601 derivatives with the highest known octamer affinities, to establish a simple model behind the mechanics of sequence dependency. This uncovers the unique but unexpected role of TA dinucleotides and a propensity for G|C-rich sequence elements to conform energetically favourably at most locations around the histone octamer, which rationalizes G|C% as the most predictive factor for nucleosome occupancy in vivo. In addition, our findings reveal dominant constraints on double helix conformation by H3-H4 relative to H2A-H2B binding and DNA sequence context-dependency underlying nucleosome structure, positioning and stability. This provides a basis for improved prediction of nucleosomal properties and the design of tailored DNA constructs for chromatin investigations.     

DNA folding in the nucleosome

Digestion of chromatin with a number of nucleases shows that the DNA is regularly folded in the nucleosome. Particularly cleavage by pancreatic DNase (DNase I) in the 140 base-pair nucleosome has been examined. This nuclease nicks the DNA every ten bases on each strand as demonstrated by labeling the 5′-ends of the 140 base-pair nucleosome. Cleavage sites on opposite strands are staggered by two bases. This proves that the DNA is arranged on the outside of the histone core in a regular way. The probability distribution of nicking might indicate a 2-fold symmetry of the 140 base-pair nucleosome. In particular it is shown that the predominant band of 80 bases is derived from several regions within the 140 base-pairs and suggested to reflect the pitch of the DNA superhelix surrounding the histone core of the nucleosome. Its possible significance with respect to chromatin structure is discussed.     

DNA recognition and nucleosome organization

The affinity of a DNA sequence for the histone octamer in a core nucleosome depends on the intrinsic flexibility of the DNA. This parameter can be affected both by the sequence-dependent conformational preferences of individual base steps and by the nature and location of the exocyclic groups of the DNA bases. By adopting highly preferred conformations particular types of base step can influence the rotational positioning of the DNA on the surface of the histone octamer. The asymmetry of the next higher order of chromatin structure is determined in part by the asymmetric binding of the globular domain of histone H5 to the core nucleosome. © 1998 John Wiley & Sons, Inc. Biopoly 44: 423–433 1997     

Replication-guided nucleosome packing and nucleosome breathing expedite the formation of dense arrays

The first level of genome packaging in eukaryotic cells involves the formation of dense nucleosome arrays, with DNA coverage near 90% in yeasts. How cells achieve such high coverage within a short time, e.g. after DNA replication, remains poorly understood. It is known that random sequential adsorption of impenetrable particles on a line reaches high density extremely slowly, due to a jamming phenomenon. The nucleosome-shifting action of remodeling enzymes has been proposed as a mechanism to resolve such jams. Here, we suggest two biophysical mechanisms which assist rapid filling of DNA with nucleosomes, and we quantitatively characterize these mechanisms within mathematical models. First, we show that the ‘softness’ of nucleosomes, due to nucleosome breathing and stepwise nucleosome assembly, significantly alters the filling behavior, speeding up the process relative to ‘hard’ particles with fixed, mutually exclusive DNA footprints. Second, we explore model scenarios in which the progression of the replication fork could eliminate nucleosome jamming, either by rapid filling in its wake or via memory of the parental nucleosome positions. Taken together, our results suggest that biophysical effects promote rapid nucleosome filling, making the reassembly of densely packed nucleosomes after DNA replication a simpler task for cells than was previously thought.     

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.