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.     

Evolution of collagen IV genes from a 54-base pair exon: A role for introns in gene evolution

The exon structure of the collagen IV gene provides a striking example for collagen evolution and the role of introns in gene evolution. Collagen IV, a major component of basement membranes, differs from the fibrillar collagens in that it contains numerous interruptions in the triple helical Gly-X-Y repeat domain. We have characterized all 47 exons in the mouse alpha 2(IV) collagen gene and find two 36-, two 45-, and one 54-bp exons as well as one 99- and three 108-bp exons encoding the Gly-X-Y repeat sequence. All these exons sizes are also found in the fibrillar collagen genes. Strikingly, of the 24 interruption sequences present in the alpha 2-chain of mouse collagen IV, 11 are encoded at the exon/intron borders of the gene, part of one interruption sequence is encoded by an exon of its own, and the remaining interruptions are encoded within the body of exons. In such "fusion exons" the Gly-X-Y encoding domain is also derived from 36-, 45-, or 54-bp sequence elements. These data support the idea that collagen IV genes evolved from a primordial 54-bp coding unit. We furthermore interpret these data to suggest that the interruption sequences in collagen IV may have evolved from introns, presumably by inactivation of splice site signals, following which intronic sequences could have been recruited into exons. We speculated that this mechanism could provide a role for introns in gene evolution in general.     

Nucleosome structure and repair of N-methylpurines in the GAL1-10 genes of Saccharomyces cerevisiae

Nucleosome structure and repair of N-methylpurines were analyzed at nucleotide resolution in the divergent GAL1-10 genes of intact yeast cells, encompassing their common upstream-activating sequence. In glucose cultures where genes are repressed, nucleosomes with fixed positions exist in regions adjacent to the upstream-activating sequence, and the variability of nucleosome positioning sharply increases with increasing distance from this sequence. Galactose induction causes nucleosome disruption throughout the region analyzed, with those nucleosomes close to the upstream-activating sequence being most striking. In glucose cultures, a strong correlation between N-methylpurine repair and nucleosome positioning was seen in nucleosomes with fixed positions, where slow and fast repair occurred in nucleosome core and linker DNA, respectively. Galactose induction enhanced N-methylpurine repair in both strands of nucleosome core DNA, being most dramatic in the clearly disrupted, fixed nucleosomes. Furthermore, N-methylpurines are repaired primarily by the Mag1-initiated base excision repair pathway, and nucleotide excision repair contributes little to repair of these lesions. Finally, N-methylpurine repair is significantly affected by nearest-neighbor nucleotides, where fast and slow repair occurred in sites between pyrimidines and purines, respectively. These results indicate that nucleosome positioning and DNA sequence significantly modulate Mag1-initiated base excision repair in intact yeast cells.     

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.     

Structural Mechanics of DNA Wrapping in the Nucleosome

Experimental X-ray crystal structures and a database of calculated structural parameters of DNA octamers were used in combination to analyse the mechanics of DNA bending in the nucleosome core complex. The 1kx5 X-ray crystal structure of the nucleosome core complex was used to determine the relationship between local structure at the base-step level and the global superhelical conformation observed for nucleosome-bound DNA. The superhelix is characterised by a large curvature (597°) in one plane and very little curvature (10°) in the orthogonal plane. Analysis of the curvature at the level of 10-step segments shows that there is a uniform curvature of 30° per helical turn throughout most of the structure but that there are two sharper kinks of 50° at ± 2 helical turns from the central dyad base pair. The curvature is due almost entirely to the base-step parameter roll. There are large periodic variations in roll, which are in phase with the helical twist and account for 500° of the total curvature. Although variations in the other base-step parameters perturb the local path of the DNA, they make minimal contributions to the total curvature. This implies that DNA bending in the nucleosome is achieved using the roll-slide-twist degree of freedom previously identified as the major degree of freedom in naked DNA oligomers. The energetics of bending into a nucleosome-bound conformation were therefore analysed using a database of structural parameters that we have previously developed for naked DNA oligomers. The minimum energy roll, the roll flexibility force constant and the maximum and minimum accessible roll values were obtained for each base step in the relevant octanucleotide context to account for the effects of conformational coupling that vary with sequence context. The distribution of base-step roll values and corresponding strain energy required to bend DNA into the nucleosome-bound conformation defined by the 1kx5 structure were obtained by applying a constant bending moment. When a single bending moment was applied to the entire sequence, the local details of the calculated structure did not match the experiment. However, when local 10-step bending moments were applied separately, the calculated structure showed excellent agreement with experiment. This implies that the protein applies variable bending forces along the DNA to maintain the superhelical path required for nucleosome wrapping. In particular, the 50° kinks are constraints imposed by the protein rather than a feature of the 1kx5 DNA sequence. The kinks coincide with a relatively flexible region of the sequence, and this is probably a prerequisite for high-affinity nucleosome binding, but the bending strain energy is significantly higher at these points than for the rest of the sequence. In the most rigid regions of the sequence, a higher strain energy is also required to achieve the standard 30° curvature per helical turn. We conclude that matching of the DNA sequence to the local roll periodicity required to achieve bending, together with the increased flexibility required at the kinks, determines the sequence selectivity of DNA wrapping in the nucleosome.     

Sequence-dependent Nucleosome Positioning

Eukaryotic DNA is organized into a macromolecular structure called chromatin. The basic repeating unit of chromatin is the nucleosome, which consists of two copies of each of the four core histones and DNA. The nucleosomal organization and the positions of nucleosomes have profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is therefore of general interest. Among the many determinants of nucleosome positioning, the DNA sequence has been proposed to have a major role. Here, we analyzed more than 860,000 nucleosomal DNA sequences to identify sequence features that guide the formation of nucleosomes in vivo. We found that both a periodic enrichment of AT base pairs and an out-of-phase oscillating enrichment of GC base pairs as well as the overall preference for GC base pairs are determinants of nucleosome positioning. The preference for GC pairs can be related to a lower energetic cost required for deformation of the DNA to wrap around the histones. In line with this idea, we found that only incorporation of both signal components into a sequence model for nucleosome formation results in maximal predictive performance on a genome-wide scale. In this manner, one achieves greater predictive power than published approaches. Our results confirm the hypothesis that the DNA sequence has a major role in nucleosome positioning in vivo.     

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.     

A conserved nucleotide sequence, coding for a segment of the C-propeptide, is found at the same location in different collagen genes.

The nucleotide sequence of a segment of the chick alpha 1 type III collagen gene which codes for the C-propeptide was determined and compared with the corresponding sequence in the alpha 1 type I and alpha 2 type I collagen genes. As in the alpha 2 type I gene the coding information for the C-propeptide of the type III collagen gene is subdivided in four exons. Similarly, the amino proximal exon contains sequences for both the carboxy terminal end of the alpha-helical segment of collagen and for the beginning of the C-propeptide in both genes. Therefore, this organization of exons must have been established before these two collagen genes arose by duplication of a common ancestor. In several subsegments the deduced amino acid sequence for the C-propeptide of type III collagen shows a strong homology with the corresponding amino acid sequence in alpha 1 and alpha 2 type I. For one of these homologous amino acid sequences, however, the nucleotide sequence is much better conserved than for the others. It is possible that a mechanism of gene conversion has maintained the homogeneity of this nucleotide sequence among the interstitial collagen genes. Alternatively, the conserved nucleotide sequence may represent a regulatory signal which could function either in the DNA or in the RNA.     

Inferring natural selection on fine-scale chromatin organization in yeast

Despite its potential role in the evolution of complex phenotypes, the detection of negative (purifying) and positive selection on noncoding regulatory sequence has been elusive because of the inherent difficulty in predicting the functional consequences of mutations on noncoding sequence. Because the functioning of regulatory sequence depends upon both chromatin configuration and cis-regulatory factor binding, we investigate the idea that the functional conservation of regulatory regions should be associated with the conservation of sequence-dependent bending properties of DNA that determine its affinity for the nucleosome. Recent advances in the computational prediction of sequence-dependent affinity to nucleosomes provide an opportunity to distinguish between neutral and nonneutral evolution of fine-scale chromatin organization. Here, a statistical test is presented for detecting evolutionary conservation and/or adaptive evolution of nucleosome affinity from interspecies comparisons of DNA sequences. Local nucleosome affinities of homologous sequences were calculated using 2 recently published methods. A randomization test was applied to sites of mutation to evaluate the similarity of DNA-nucleosome affinity between several closely related species of Saccharomyces yeast. For most of the genes we analyzed, the conservation of local nucleosome affinity was detected at a few distinct locations in the upstream noncoding region. Our results also demonstrate that different patterns of chromatin evolution have shaped DNA-nucleosome interaction at the core promoters of TATA-containing and TATA-less genes and that elevated purifying selection has maintained low affinity for nucleosome in the core promoters of the latter group. Across the entire yeast genome, DNA-nucleosome interaction was also discovered to be significantly more conserved in TATA-less genes compared with TATA-containing genes.     

Nucleosome positioning based on the sequence word composition

The DNA of all eukaryotic organisms is packaged into nucleosomes (a basic repeating unit of chromatin). A nucleosome consists of histone octamer wrapped by core DNA and linker histone H1 associated with linker DNA. It has profound effects on all DNA-dependent processes by affecting sequence accessibility. Understanding the factors that influence nucleosome positioning has great help to the study of genomic control mechanism. Among many determinants, the inherent DNA sequence has been suggested to have a dominant role in nucleosome positioning in vivo. Here, we used the method of minimum redundancy maximum relevance (mRMR) feature selection and the nearest neighbor algorithm (NNA) combined with the incremental feature selection (IFS) method to identify the most important sequence features that either favor or inhibit nucleosome positioning. We analyzed the words of 53,021 nucleosome DNA sequences and 50,299 linker DNA sequences of Saccharomyces cerevisiae. 32 important features were abstracted from 5,460 features, and the overall prediction accuracy through jackknife cross-validation test was 76.5%. Our results support that sequence-dependent DNA flexibility plays an important role in positioning nucleosome core particles and that genome sequence facilitates the rapid nucleosome reassembly instead of nucleosome depletion. Besides, our results suggest that there exist some additional features playing a considerable role in discriminating nucleosome forming and inhibiting sequences. These results confirmed that the underlying DNA sequence plays a major role in nucleosome positioning.     

Unwrapping of nucleosomal DNA ends: a multiscale molecular dynamics study

To permit access to DNA-binding proteins involved in the control and expression of the genome, the nucleosome undergoes structural remodeling including unwrapping of nucleosomal DNA segments from the nucleosome core. Here we examine the mechanism of DNA dissociation from the nucleosome using microsecond timescale coarse-grained molecular dynamics simulations. The simulations exhibit short-lived, reversible DNA detachments from the nucleosome and long-lived DNA detachments not reversible on the timescale of the simulation. During the short-lived DNA detachments, 9 bp dissociate at one extremity of the nucleosome core and the H3 tail occupies the space freed by the detached DNA. The long-lived DNA detachments are characterized by structural rearrangements of the H3 tail including the formation of a turn-like structure at the base of the tail that sterically impedes the rewrapping of DNA on the nucleosome surface. Removal of the H3 tails causes the long-lived detachments to disappear. The physical consistency of the CG long-lived open state was verified by mapping a CG structure representative of this state back to atomic resolution and performing molecular dynamics as well as by comparing conformation-dependent free energies. Our results suggest that the H3 tail may stabilize the nucleosome in the open state during the initial stages of the nucleosome remodeling process.     

Regular location of exon starts in collagen I and VII genes with periods comparable to nucleosome repeat lengths

Wavelet transform analysis revealed quasiperiodicity of exon starts in the genes for collagens of types I and VII. In the regions coding for the fibrillar part of the protein, the average sum length of an exon and the following intron was ～165 nt, i.e. close to the minimal nucleosome repeat length. Such quasiperiodic segments comprising 2–5 contiguous exon+intron pairs of similar length encompassed more than 50% of exons making the fibrillar part. We also observed regular disposition of exon starts in the nonfibrillar part of collagen VII, but with a different period of 227 nt.     

A phase relationship associates tRNA structural gene sequences with nucleosome cores

DNA (760 bp) isolated from nucleosome tetramers of staphylococcal nuclease-digested chicken embryo chromatin was highly enriched for tRNA genes and subsequently cloned in E. coli chi 1776. The location of genes coding for chicken embryo tRNALys, tRNAPhe and tRNAiMet within the cloned nucleosome tetramer DNA was determined using restriction endonucleases for which single cleavage sites could be predicted from the respective tRNA base sequence. All our tRNA genes reside nonrandomly at four locations on nucleosome tetramer DNA. The spacing between the tRNA gene locations is approximately 190 bp, similar to the DNA repeat length of chicken embryo chromatin. The four tRNA gene locations were also defined in noncloned nucleosome tetramer DNA highly enriched for tRNA genes. The majority of genes coding for tRNALys, tRNAPhe and tRNAiMet, respectively, are located in equal proportion 40-45, 230, 420 and 610 bp distant from the 5' end of the tRNA-identical strand. Thus the tRNA structural gene sequences all appear to begin about 20 bp "inside" the nucleosome core. As observed with nucleosomal DNA not enriched for tRNA genes, the phase relationship between tRNA genes and nucleosome location is maintained over a distance of 4-6 subsequent nucleosomes. A cloned molecule of nucleosomal DNA containing both a tRNALys gene and a tRNAiMet gene in the same polarity reveals that a phase adjustment might be necessary for the nucleosomes between these two tRNA genes in chicken embryo chromatin.