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.     

Reconstitution experiments show that sequence-specific histone-DNA interactions are the basis for nucleosome phasing on mouse satellite DNA

The molecular basis underlying the sequence-specific positioning of nucleosomes on DNA was investigated. We previously showed that histone octamers occupy multiple specific positions on mouse satellite DNA in vivo and have now reconstituted the 234 bp mouse satellite repeat unit with pure core histones into mononucleosomes. Histones from mouse liver or chicken erythrocytes bind to the DNA in multiple precisely defined frames in perfect phase with a diverged 9 bp subrepeat of the satellite DNA. This is the first time that nucleosome positions on a DNA in vivo have been compared to those found on the same DNA by in vitro reconstitution. Most of the nucleosomes occupy identical positions in vivo and in vitro. There are, however, some characteristic differences. We conclude that sequence-dependent histone-DNA interactions play a decisive role in the positioning of nucleosomes in vivo, but that the nucleosome locations in native chromatin are subject to additional constraints.     

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.     

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.     

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.     

Improved alignment of nucleosome DNA sequences using a mixture model

DNA sequences that are present in nucleosomes have a preferential approximately 10 bp periodicity of certain dinucleotide signals, but the overall sequence similarity of the nucleosomal DNA is weak, and traditional multiple sequence alignment tools fail to yield meaningful alignments. We develop a mixture model that characterizes the known dinucleotide periodicity probabilistically to improve the alignment of nucleosomal DNAs. We assume that a periodic dinucleotide signal of any type emits according to a probability distribution around a series of 'hot spots' that are equally spaced along nucleosomal DNA with 10 bp period, but with a 1 bp phase shift across the middle of the nucleosome. We model the three statistically most significant dinucleotide signals, AA/TT, GC and TA, simultaneously, while allowing phase shifts between the signals. The alignment is obtained by maximizing the likelihood of both Watson and Crick strands simultaneously. The resulting alignment of 177 chicken nucleosomal DNA sequences revealed that all 10 distinct dinucleotides are periodic, however, with only two distinct phases and varying intensity. By Fourier analysis, we show that our new alignment has enhanced periodicity and sequence identity compared with center alignment. The significance of the nucleosomal DNA sequence alignment is evaluated by comparing it with that obtained using the same model on non-nucleosomal sequences.     

Regular arrangement of nucleosomes on 5S rRNA genes in Xenopus laevis.

The chromatin structure of the oocyte-type 5S RNA genes in Xenopus laevis was investigated. Blot hybridization analysis of DNA from micrococcal nuclease digests of erythrocyte nuclei showed that 5S DNA has the same average nucleosome repeat length, 192 +/- 4 base pairs, as two Xenopus satellite DNAs and bulk erythrocyte chromatin. The positions of nuclease-sensitive regions in the 5S DNA repeats of purified DNA and chromatin from erythrocytes were mapped by using an indirect end-labeling technique. Although most of the sites cleaved in purified DNA were also cleaved in chromatin, the patterns of intensities were strikingly different in the two cases. In 5S chromatin, three nuclease-sensitive regions were spaced approximately a nucleosome length apart, suggesting a single, regular arrangement of nucleosomes on most of the 5S DNA repeats. The observed nucleosome locations are discussed with respect to nucleotide sequences known to be important for expression of 5S RNA. Because the preferred locations appear to be reestablished in each repeating unit, despite spacer length heterogeneity, we suggest that the regular chromatin structure reflects the presence of a sequence-specific DNA-binding component on inactive 5S RNA genes.     

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.     

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.     

Eight different highly specific nucleosome phases on alpha-satellite DNA in the African green monkey.

The question of nucleosome phasing on African Green Monkey (AGM) alpha-satellite DNA has been addressed by employing a new approach. Nucleosome cores were prepared from AGM nuclei with micrococcal nuclease, exonuclease III and nuclease S1. The core DNA population derived from alpha-satellite DNA containing chromatin was purified from total core DNA by denaturation of the DNA, reassociation to a low Cot value, and hydroxyapatite chromatography to separate the renatured satellite fraction. After end-labeling the termini of the alpha-satellite containing core DNA fragments were mapped by high resolution gel electrophoresis relative to known restriction sites along the 172 bp repeat unit of the satellite DNA. The results show that nucleosomes occupy eight strictly defined positions on the alpha-satellite DNA which could be determined with an accuracy of +/- 1 base pair. Approximately 35% of all nucleosomes are organized in one of these frames while the other seven registers contribute about 10% each.     

Positioning of nucleosomes in satellite I-containing chromatin of rat liver

The location of nucleosomes on rat satellite I DNA has been investigated using a new approach. Nucleosome cores were prepared from rat liver nuclei with micrococcal nuclease, exonuclease III and nucleases S1. From the total population of core DNA fragments the satellite-containing fragments were isolated by molecular cloning and the complete sequence of 50 clones was determined. The location of nucleosomes along the satellite sequence was found to be non-random. Our results show that nucleosomes occupy a number of positions on satellite I DNA. About 35 to 50% of all nucleosomes are positioned in two corresponding major sites, the remainder in about 16 less preferred sites. The major nucleosome positions are apparently strictly defined with the precision of a single base-pair. These results were confirmed by other approaches, including restriction nuclease digestion experiments. There are good indications of a defined long-range organization of the satellite chromatin fiber in two or more oligonucleosomal arrays with distinct nucleosome configurations.     

Sequence-specific positioning of core histones on an 860 base-pair DNA. Experiment and theory

Previous experiments have shown that the locations of the histone octamer on DNA molecules of 140 to 240 base-pairs (bp) are influenced strongly by the nucleotide sequence. Here we have studied the locations of the histone octamer on a relatively long DNA molecule of 860 bp, using two different nucleases, micrococcal and DNAase I. Data were obtained from both the protein--DNA complexes and from the naked DNA at single-bond resolution, and then were analyzed by densitometry to yield plots of differential cleavage, which show clearly the changes in cutting due to the addition of protein. Our results show that the placement of core histones on the 860 bp molecule is definitely non-random. The digestion data provide evidence for five nucleosome cores, the centers of which lie in defined locations. In all but one of these protein--DNA complexes, the DNA adopts a unique, highly preferred rotational setting with respect to the protein surface. Another protein--DNA complex is unusual in that it protects 200 bp from digestion, yet is cut in its very center as if it were split into two parts. The apparent average twist of the DNA within all of these protein--DNA complexes is 10.2(+/- 0.1) bp, as measured by the periodicity of DNAase I digestion. This value is in excellent agreement with the twist of 10.21(+/- 0.05) bp deduced from the periodicity of sequence content in chicken nucleosome core DNA. In addition, we observe a discontinuity in the periodic cutting by DNAase I of about -1 to -3 bonds in going from any nucleosome core to the next. The most plausible interpretation of this discontinuity is that it reflects the angle by which adjacent protein--DNA complexes are aligned. Thus, any nucleosome may be related to its neighbor by a left-handed rotation in space of -1/10.2 to -3/10.2 helix turns, or -35 degrees to -105 degrees. Repeated many times, this operation would build a long, left-handed helix of nucleosomes similar to that described by many workers for the packing of nucleosomes in chromatin. In order to look for any long-range influences on the positioning of the histone octamer in the 860 bp molecule (as would be expected if the nucleosomes have to fit into some higher-order structure), we have examined the locations of the histone octamer on five different isolated short fragments of the 860-mer, all of nucleosomal length.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Deoxyribonuclease II as a probe for chromatin structure. I. Location of cleavage sites

DNAase II has been shown to cleave condensed mouse liver chromatin at 100-bp² intervals while chromatin in the extended form is cleaved at 200-bp intervals (Altenburger et al., 1976). Evidence is presented here that DNA digestion patterns of a half-nucleosomal periodicity are also obtained upon DNAase II digestion of chicken erythrocyte nuclei and yeast nuclei, both of which differ in their repeat lengths (210 and 165 bp) from mouse liver chromatin. In the digestion of mouse liver nuclei a shift from the 100-bp to the 200-bp cleavage mode takes place when the concentration of monovalent cations present during digestion is decreased below 1 mM. When soluble chromatin prepared by micrococcal nuclease is digested with DNAase II the same type of shift occurs, albeit at higher ionic strength.In order to map the positions of the DNAase II cleavage sites on the DNA relative to the positions of the nucleosome cores, the susceptibility of DNAase II-derived DNA termini to exonuclease III was investigated. In addition, oligonucleosome fractions from HaeIII and micrococcal nuclease digests were end-labelled with polynucleotide kinase and digested with DNAase II under conditions leading to 100 and 200-bp digestion patterns. Analysis of the chain lengths of the resulting radioactively labelled fragments together with the results of the exonuclease assay allow the following conclusions. In the 200-bp digestion mode, DNAase II cleaves exclusively in the internucleosomal linker region. Also in the 100-bp mode cleavage occurs initially in the linker region. Subsequently, DNAase II cleaves at intranucleosomal locations, which are not, however, in the centre of the nucleosome but instead around positions 20 and 125 of the DNA associated with the nucleosome core. At late stages of digestion intranucleosomal cuts predominate and linkers that are still intact are largely resistant to DNAase II due to interactions between adjacent nucleosomes. These findings offer an explanation for the sensitivity of DNAase II to the higher-order structure of chromatin.     

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.     

The core histone tail domains contribute to sequence-dependent nucleosome positioning

The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin.     

On the occurrence of nucleosome phasing in chromatin.

We have found that DNAase I digestion of yeast, HeLa and chicken erythrocyte nuclei produces a pattern of DNA fragments spaced 10 bases apart and extending to at least 300 bases. This "extended ladder" of DNA fragments is most clearly seen with yeast, and least clearly with chicken erythrocytes. The appearance of regular and discrete bands at sizes much larger than the repeat size shows that the core particles (140 bp of DNA + H2A, H2B, H3 H4) in at least some fraction of chromatin are spaced in a particular fashion, by discrete lengths of spacer DNA, and not randomly. Based on the abundance of small repeats in yeast and from experiments with nucleosome oligomers, we conclude that the extended ladder and nucleosomal phasing probably arise mainly from regions in the chromatin in which nucleosome cores are closely packed or closely spaced (140-160 bp X n). Contributions from less closely packed but still accurately phased nucleosomes, however, cannot be entirely excluded.     

Identification of sequence elements contributing to the intrinsic curvature of the mouse satellite DNA repeat.

In this paper, the contribution of different sequence elements to the intrisic curvature of the mouse satellite DNA repeat was investigated. This DNA fragment contains nineteen groups of three or more consecutive adenines which are only poorly phased with respect to the helical repeat. The mouse satellite DNA repeat shows a sinusoidal pattern of cleavage by the hydroxyl radical; the waves of reactivity are phased with respect to the A-tracts. Some interesting observations arise from a detailed analysis of these cleavage patterns: a) the maxima of hydroxyl radical cleavage are more periodically spaced along the DNA sequence than the A-tracts themselves. As a consequence, the position of each maximum with respect to the A-tract is variable; b) the sequence 5' TGGAATATG/AA 3' shows a sinusoidal pattern of hydroxyl radical cleavage. This sequence shows a retarded migration in polyacrylamide gels indicating that it is actually intrinsically curved. These results are discussed in view of the current models for DNA curvature.