Nucleosome phasing in Tetrahymena macronuclei

Core-protected DNA can drive only 60% of the Tetrahymena thermophila macronuclear genome into duplexes in hybridization experiments. This core-protected DNA therefore contains only a subset of the genome complexity. We interpret this to mean that a large fraction, if not all, of the genome is phased with respect to nucleosome placement. Among the sequences present in total DNA and absent from core-protected DNA are most of the sequences containing N6-methyladenine (MeAde) residues, consistent with our previous demonstration that most of these residues lie in linker DNA. We show that these results are not due to artifacts resulting from the small size of the DNA driver, nor are they due to any sequence preferences exhibited by staphylococcal (staph) nuclease. This is the first evidence that nucleosome phasing may be a bulk genome characteristic.     

G+C content dominates intrinsic nucleosome occupancy

Background  

The relative preference of nucleosomes to form on individual DNA sequences plays a major role in genome packaging. A wide variety of DNA sequence features are believed to influence nucleosome formation, including periodic dinucleotide signals, poly-A stretches and other short motifs, and sequence properties that influence DNA structure, including base content. It was recently shown by Kaplan et al. that a probabilistic model using composition of all 5-mers within a nucleosome-sized tiling window accurately predicts intrinsic nucleosome occupancy across an entire genome in vitro. However, the model is complicated, and it is not clear which specific DNA sequence properties are most important for intrinsic nucleosome-forming preferences.     

The Molecular Evolution of Nucleosome Positioning Through Sequence-Dependent Deformation of the DNA Polymer

Abstract

The computational prediction of nucleosome positioning from DNA sequence now allows for in silico investigation of the molecular evolution of biophysical properties of the DNA molecule responsible for primary chromatin organization in the genome. To discern what signal components driving nucleosome positioning in the yeast genome are potentially targeted by natural selection, we compare the performance of various models predictive of nucleosome positioning within the context of a simple statistical test, the repositioned mutation test. We demonstrate that while nucleosome occupancy is driven largely by translational exclusion in response to AT content, there is also a strong signature of evolutionary conservation of regular patterns within nucleosomal DNA sequence related to the structural organization of the nucleosome core (e.g., 10-bp dinucleotide periodicity). We also use computer simulations to investigate hypothetical coding and regulatory constraints on the ability of sequence properties affecting nucleosome formation to adaptively evolve. Our results demonstrate that natural selection may act independently on different DNA sequence properties responsible for local chromatin organization. Furthermore, at least with respect to the deformation energy of the DNA molecule in the nucleosome, the presence of the genetic code has greatly restricted the ability of sequences to evolve the dynamic nucleosome organization typically observed in promoter regions.     

Nucleosome Interaction Surface of Linker Histone H1c Is Distinct from That of H10

The fully organized structure of the eukaryotic nucleosome remains unsolved, in part due to limited information regarding the binding site of the H1 or linker histone. The central globular domain of H1 is believed to interact with the nucleosome core at or near the dyad and to bind at least two strands of DNA. We utilized site-directed mutagenesis and in vivo photobleaching to identify residues that contribute to the binding of the globular domain of the somatic H1 subtype H1c to the nucleosome. As was previously observed for the H1⁰ subtype, the binding residues for H1c are clustered on the surface of one face of the domain. Despite considerable structural conservation between the globular domains of these two subtypes, the locations of the binding sites identified for H1c are distinct from those of H1⁰. We suggest that the globular domains of these two linker histone subtypes will bind to the nucleosome with distinct orientations that may contribute to higher order chromatin structure heterogeneity or to differences in dynamic interactions with other DNA or chromatin-binding proteins.     

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.     

DNA sequence organization in the genome of Petroselinum sativum (Umbelliferae)

The genome of parsley was studied by DNA/DNA reassociation to reveal its spectrum of DNA reiteration frequencies and sequence organization. The reassociation of 300 nucleotide DNA fragments indicates the presence of four classes of DNA differing in repetition frequency. These classes are: highly repetitive sequences, fast intermediate repetitive sequences, slow intermediate repetitive sequences, and unique sequences. The repeated classes are reiterated on average 136,000, 3000, and 42 times respectively. A minor part of the genome is made up of palindromes. — The organization of DNA sequences in the P. sativum genome was determined by the reassociation kinetics of DNA fragments of varying length. Further information was derived from S1 nuclease resistance and from hyperchromicity measurements on DNA fragments reassociated to defined C₀t values. — The portion of the genome organized in a short period interspersion pattern amounts to 47%, with the unique sequences on an average 1000 nucleotides long, and most of the repetitive sequences about 300 nucleotides in length, whereas the weight average length may be up to 600 nucleotides. — About 5% unique DNA and 11% slow intermediate repetitive DNA consist of sequences from 10³ up to 10⁴ nucleotides long; these are interspersed with repetitive sequences of unknown length. Long repetitive sequences constitute 33% of the genome, 13% are satellite-like organized, and 20% in long stretches of intermediate repetitive DNA in which highly divergent sequences alternate with sequences that show only minimal divergence. — The results presented indicate remarkable similarities with the genomes of most animal species on which information is available. The most intriguing pecularity of the plant genome derives from its high content of repetitive DNA and the presumed organization of the latter.     

Prediction of nucleosome occupancy in Saccharomyces cerevisiae using position-correlation scoring function

Knowledge of the detailed organization of nucleosomes across genomes and the mechanisms of nucleosome positioning is critical for the understanding of gene regulation and expression. In the present work, the bias of 4-mer frequency in nucleosome and linker sequences of the S. cerevisiae genome was analyzed statistically. A novel position-correlation scoring function algorithm based on the bias of 4-mer frequency in linker sequences was presented to distinguish nucleosome vs linker sequences. Five-fold cross-validation demonstrated that the algorithm achieved a good performance with mean area under the receiver operator characteristics curve of 0.981. Next, the algorithm was used to predict nucleosome occupancy throughout the S. cerevisiae genome and relatively high correlation coefficients with experiment maps of nucleosome positioning were obtained. Besides, the distinct nucleosome depleted regions in the vicinity of regulatory sites were confirmed. The results suggest that intrinsic DNA sequence preferences in linker regions have a significant impact on the nucleosome occupancy.     

Evaluation of Elastic Rod Models with Long Range Interactions for Predicting Nucleosome Stability

Abstract

The ability of a dinucleotide-step based elastic-rod model of DNA to predict nucleosome binding free energies is investigated using four available sets of elastic parameters. We compare the predicted free energies to experimental values derived from nucleosome reconstitution experiments for 84 DNA sequences. Elastic parameters (conformation and stiffnessess) obtained from MD simulations are shown to be the most reliable predictors, as compared to those obtained from analysis of base-pair step melting temperatures, or from analysis of x-ray structures. We have also studied the effect of varying the folded conformation of nucleosomal DNA by means of our Fourier filtering knock-out and knock-in procedure. This study confirmed the above ranking of elastic parameters, and helped to reveal problems inherent in models using only a local elastic energy function. Long-range interactions were added to the elastic-rod model in an effort to improve its predictive ability. For this purpose a Debye-Huckel energy term with a single, homogenous point charge per base- pair was introduced. This term contains only three parameters,—its weight relative to the elastic energy, the Debye screening length, and a minimum sequence distance for including pairwise interactions between charges. After optimization of these parameters, our Debye-Huckel term is attractive, and yields the same level of correlation with experiment (R = 0.75) as was achieved merely by varying the nucleosomal shape in the elastic-rod model. We suggest this result indicates a linker DNA—histone attraction or, possibly, entropic effects, that lead to a stabilization of a nucleosome away from the ends of DNA segments longer than 147 bp. Such effects are not accounted for by a localized elastic energy model.     

Nucleosome DNA Bendability Matrix (C. elegans)

Abstract

An original signal extraction procedure is applied to database of 146 base nucleosome core DNA sequences from C. elegans (S. M. Johnson et al. Genome Research 16, 1505–1516, 2006). The positional preferences of various dinucleotides within the 10.4 base nucleosome DNA repeat are calculated, resulting in derivation of the nucleosome DNA bendability matrix of 16x10 elements. A simplified one-line presentation of the matrix (“consensus” repeat) is…A(TTTCCGGAAA)T…. All 6 chromosomes of C. elegans conform to the bendability pattern. The strongest affinity to their respective positions is displayed by dinucleotides AT and CG, separated within the repeat by 5 bases. The derived pattern makes a basis for sequence-directed mapping of nucleosome positions in the genome of C. elegans. As the first complete matrix of bendability available the pattern may serve for iterative calculations of the species-specific matrices of bendability applicable to other genomic sequences.     

The most frequent short sequences in non-coding DNA

The purpose of this work is to determine the most frequent short sequences in non-coding DNA. They may play a role in maintaining the structure and function of eukaryotic chromosomes. We present a simple method for the detection and analysis of such sequences in several genomes, including Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens. We also study two chromosomes of man and mouse with a length similar to the whole genomes of the other species. We provide a list of the most common sequences of 9–14 bases in each genome. As expected, they are present in human Alu sequences. Our programs may also give a graph and a list of their position in the genome. Detection of clusters is also possible. In most cases, these sequences contain few alternating regions. Their intrinsic structure and their influence on nucleosome formation are not known. In particular, we have found new features of short sequences in C. elegans, which are distributed in heterogeneous clusters. They appear as punctuation marks in the chromosomes. Such clusters are not found in either A. thaliana or D. melanogaster. We discuss the possibility that they play a role in centromere function and homolog recognition in meiosis.     

A family of dispersed repeats in the genome of Vicia faba: structure, chromosomal organization, redundancy modulation, and evolution

A family of repeated DNA sequences of about 1200 bp in length and bordered by well-conserved, 18 bp inverted repeats (VfB family) was found in the nuclear genome of Vicia faba. The structure, chromosomal organization, redundancy modulation and evolution of these sequences were investigated. They are enriched in A+T base pairs (about 40% G+C) and lack any obvious internally repeated motif. A 64%–73% nucleotide sequence identity was found when pairwise comparisons between VfB sequences were carried out (average 69%). Direct repeats were not found to flank the inverted repeats that border these DNA sequences. The results obtained by hybridizing VfB repeats to Southern blots of V. faba genomic DNA digested with EcoRI indicated that these DNA elements are interspersed in the genome. The appearance of bands in these Southern blots and comparison of the structure of the sequences that flank different VfB elements showed that these repeats might be part of other, longer repeated DNA sequences. A high degree of dispersion throughout the genome was confirmed by cytological hybridization, which showed VfB sequences to be scattered along the length of all chromosomes and to be absent or rare only at heterochromatic chromosomal regions. These sequences contribute to intraspecific alterations of genomic size. Indeed, dot-blot hybridizations proved that their redundancy, which is positively correlated with the overall amount of nuclear DNA in each accession, varies between V. faba land races (27×10³–230×10³ copies per 1C DNA). Southern blot hybridization of VfB repeats to restriction endonuclease-digested genomic DNAs of V. faba, V. narbonensis, V. sativa, Phaseolus coccineus, Populus deltoides, and Triticum durum revealed nucleotide sequence homology of these DNA elements, whatever the stringency conditions, only to the DNAs of Vicia species, and to a reduced extent to the DNAs of V. narbonensis and V. sativa compared with that of V. faba. It is concluded that VfB repeats might be descended from mobile DNA elements and contribute to change genomic size and organization during evolution. Received: 10 September 1998; in revised form: 12 May 1999 / Accepted: 19 May 1999     

Strong nucleosomes of mouse genome including recovered centromeric sequences

Recently discovered strong nucleosomes (SNs) characterized by visibly periodical DNA sequences have been found to concentrate in centromeres of Arabidopsis thaliana and in transient meiotic centromeres of Caenorhabditis elegans. To find out whether such affiliation of SNs to centromeres is a more general phenomenon, we studied SNs of the Mus musculus. The publicly available genome sequences of mouse, as well as of practically all other eukaryotes do not include the centromere regions which are difficult to assemble because of a large amount of repeat sequences in the centromeres and pericentromeric regions. We recovered those missing sequences using the data from MNase-seq experiments in mouse embryonic stem cells, where the sequence of DNA inside nucleosomes, including missing regions, was determined by 100-bp paired-end sequencing. Those nucleosome sequences, which are not matching to the published genome sequence, would largely belong to the centromeres. By evaluating SN densities in centromeres and in non-centromeric regions, we conclude that mouse SNs concentrate in the centromeres of telocentric mouse chromosomes, with ~3.9 times excess compared to their density in the rest of the genome. The remaining non-centromeric SNs are harbored mainly by introns and intergenic regions, by retro-transposons, in particular. The centromeric involvement of the SNs opens new horizons for the chromosome and centromere structure studies.     

Organization of the Euplotes crassus Micronuclear Genome1

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

The possible involvement of CHI sequences in adaptive mutagenesis: Evidence from sequence analysis

Recently we proposed that sequences in the immediate neighbourhood of cytosine residues whose sequence context permits their methylation by DNA cytosine methyltransferase (Dcm) experience hypermutagenesis in cells exposed to nonlethal stresses. This hypothesis could explain the peculiar spectrum of the late-arising Lac⁺ mutants seen in theE. coli strain FC40. Here we present results of computer analysis which show that Dcm substrate sequences are overrepresented in theE. coli genome. Interestingly, certain noncanonical Dcm sequences are more overrepresented than the canonical one. The most overabundant of these, DCM-III (5’ GCTGG3’), forms the 5’ end of the recombinogenic octamer CHI (5’ GCTGGTGG3’). CHI is even more overrepresented than DCM-III. We propose that the overabundance of the DCM and CHI sequences is due to their ability to enhance adaptive fitness of the host by inducing hypermutagenesis in cells exposed to nonlethal, growth-blocking stresses. The CHI context seems to stimulate the adaptive activity of DCM-III by a mechanism which may not directly involve its recombinogenic activity.     

Nucleosomes affect local transformation efficiency

Genetic transformation is a natural process during which foreign DNA enters a cell and integrates into the genome. Apart from its relevance for horizontal gene transfer in nature, transformation is also the cornerstone of today''s recombinant gene technology. Despite its importance, relatively little is known about the factors that determine transformation efficiency. We hypothesize that differences in DNA accessibility associated with nucleosome positioning may affect local transformation efficiency. We investigated the landscape of transformation efficiency at various positions in the Saccharomyces cerevisiae genome and correlated these measurements with nucleosome positioning. We find that transformation efficiency shows a highly significant inverse correlation with relative nucleosome density. This correlation was lost when the nucleosome pattern, but not the underlying sequence was changed. Together, our results demonstrate a novel role for nucleosomes and also allow researchers to predict transformation efficiency of a target region and select spots in the genome that are likely to yield higher transformation efficiency.     

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.