Nucleosome Core DNA Research Articles

Prediction of nucleosome dynamic interval based on long-short-term memory network (LSTM).

Nucleosome localization is a dynamic process and consists of nucleosome dynamic intervals (NDIs). We preprocessed nucleosome sequence data as time series data (TSD) and developed a long short-term memory network (LSTM) model for training time series data (TSD; LSTM-TSD model) using iterative training and feature learning that predicts NDIs with high accuracy. Sn, Sp, Acc, and MCC of the obtained LSTM model is 91.88%, 92.72%, 92.30%, and 84.61%, respectively. LSTM model could precisely predict the NDIs of yeast 16 chromosome. The NDIs contain 90.29% of nucleosome core DNA and 91.20% of nucleosome central sites, indicating that NDIs have high confidence. We found that the binding sites of transcriptional proteins and other proteins are outside NDIs, not in NDIs. These results are important for analysis of nucleosome localization and gene transcriptional regulation.

Mesoscale Modeling of Nucleosome-Binding Antibody PL2-6: Mono- versus Bivalent Chromatin Complexes

Interactions of chromatin with bivalent immunoglobin nucleosome-binding antibodies and their monovalent (papain-derived) antigen-binding fragment analogs are useful probes for examining chromatin conformational states. To help interpret antibody-chromatin interactions and explore how antibodies might compete for interactions with chromatin components, we incorporate coarse-grained PL2-6 antibody modeling into our mesoscale chromatin model. We analyze interactions and fiber structures for the antibody-chromatin complexes in open and condensed chromatin, with and without H1 linker histone (LH). Despite minimal and transient interactions at physiological salt, we capture significant differences in antibody-chromatin complex configurations in open fibers, with more intense interactions between the bivalent antibody and chromatin compared to monovalent antigen-binding fragments. For these open chromatin fiber morphologies, antibody binding to histone tails is increased and compaction is greater for bivalent compared to monovalent and antibody-free systems. Differences between monovalent and bivalent binding result from antibody competition with internal chromatin fiber components (nucleosome core and linker DNA) for histone tail (H3, H4, H2A, H2B) interactions. This antibody competition for tail contacts reduces tail-core and tail-linker interactions and increases tail-antibody interactions. Such internal structural changes in open fibers resemble mechanisms of LH condensation, driven by charge screening and entropy changes. For condensed fibers at physiological salt, the three systems are much more similar overall, but some subtle tail interaction differences can be noted. Adding LH results in less-dramatic changes for all systems, except that the bivalent complex at physiological salt shows cooperative effects between LH and the antibodies in condensing chromatin fibers. Such dynamic interactions that depend on the internal structure and complex-stabilizing interactions within the chromatin fiber have implications for gene regulation and other chromatin complexes such as with LH, remodeling proteins, and small molecular chaperones that bind and modulate chromatin structure.

Modulation of cyclobutane thymine photodimer formation in T11-tracts in rotationally phased nucleosome core particles and DNA minicircles.

Cyclobutane pyrimidine dimers (CPDs) are DNA photoproducts linked to skin cancer, whose mutagenicity depends in part on their frequency of formation and deamination. Nucleosomes modulate CPD formation, favoring outside facing sites and disfavoring inward facing sites. A similar pattern of CPD formation in protein-free DNA loops suggests that DNA bending causes the modulation in nucleosomes. To systematically study the cause and effect of nucleosome structure on CPD formation and deamination, we have developed a circular permutation synthesis strategy for positioning a target sequence at different superhelix locations (SHLs) across a nucleosome in which the DNA has been rotationally phased with respect to the histone octamer by TG motifs. We have used this system to show that the nucleosome dramatically modulates CPD formation in a T11-tract that covers one full turn of the nucleosome helix at seven different SHLs, and that the position of maximum CPD formation at all locations is shifted to the 5΄-side of that found in mixed-sequence nucleosomes. We also show that an 80-mer minicircle DNA using the same TG-motifs faithfully reproduces the CPD pattern in the nucleosome, indicating that it is a good model for protein-free rotationally phased bent DNA of the same curvature as in a nucleosome, and that bending is modulating CPD formation.

Nucleosomes determine their own patch size in base excision repair.

Base excision repair (BER) processes non-helix distorting lesions (e.g., uracils and gaps) and is composed of two subpathways that differ in the number of nucleotides (nts) incorporated during the DNA synthesis step: short patch (SP) repair incorporates 1 nt and long patch (LP) repair incorporates 2–12 nts. This choice for either LP or SP repair has not been analyzed in the context of nucleosomes. Initial studies with uracil located in nucleosome core DNA showed a distinct DNA polymerase extension profile in cell-free extracts that specifically limits extension to 1 nt, suggesting a preference for SP BER. Therefore, we developed an assay to differentiate long and short repair patches in ‘designed’ nucleosomes containing a single-nucleotide gap at specific locations relative to the dyad center. Using cell-free extracts or purified enzymes, we found that DNA lesions in the nucleosome core are preferentially repaired by DNA polymerase β and there is a significant reduction in BER polymerase extension beyond 1 nt, creating a striking bias for incorporation of short patches into nucleosomal DNA. These results show that nucleosomes control the patch size used by BER.

Residual Conformational Entropies on the Sugar–Phosphate Backbone of Nucleic Acids: An Analysis of the Nucleosome Core DNA and the Ribosome

A nucleic acid folds according to its free energy, but persistent residual conformational fluctuations remain along its sugar-phosphate backbone even after secondary and tertiary structures have been assembled, and these residual conformational entropies provide a rigorous lower bound for the folding free energy. We extend a recently reported algorithm to calculate the residual backbone entropy along a RNA or DNA given configuration of its bases and apply it to the crystallographic structures of the 23S ribosomal subunit and DNAs in the nucleosome core particle. In the 23S rRNAs, higher entropic strains are concentrated in helices and certain tertiary interaction platforms while residues with high surface accessibility and those not involved in base pairing generally have lower strains. Upon folding, residual backbone entropy in the 23S subunit accounts for an average free energy penalty of +0.47 (kcal/mol)/nt (nt = nucleotide) at 310 K. In nucleosomal DNAs, backbone entropies show periodic oscillations with sequence position correlating with the superhelical twist and shifts in the base-pair-step geometries, and nucleosome positioning on the bound DNA exerts strong influence over where entropic strains are located. In contrast to rRNAs, residual backbone entropies account for a free energy penalty of only +0.09 (kcal/mol)/nt in duplex relative to single-stranded DNAs.

Evaluation of the protective capabilities of nucleosome STRs obtained by large-scale sequencing.

Partial DNA profiles are often obtained from degraded forensic samples and are hard to analyze and interpret. With in-depth studies on degraded DNA, an increasing number of forensic scientists have focused on the intrinsic structural properties of DNA. In theory, nucleosomes offer protection to the bound DNA by limiting access to enzymes. In our study, we performed large-scale DNA sequencing on nucleosome core DNA of human leucocytes. Five nucleosome short tandem repeats (STRs) were selected (including three forensic common STRs (i.e. TPOX, TH01, and D10S1248) and two unpublished STRs (i.e. AC012568.7 and AC007160.3)). We performed a population genetic investigation and forensic genetic statistical analysis of these two unpublished loci on 108 healthy unrelated individuals of the HeBei Han population in China. We estimated the protective capabilities of five selected nucleosome loci and MiniFiler™ loci with artificial degraded DNA and case samples. We also analyzed differences between sequencing results and software predicted results. Our findings showed that nucleosome STRs were more likely to be detected than MiniFiler™ loci. They were well protected from degradation by nucleosomes and could be candidates for further nucleosome multiplex construction, which would increase the chances of obtaining a better balanced profile with fewer allelic drop-outs.

Polymorphism of Histone Tail Interactions in Nucleosome

Histone tails protruding from nucleosome core are known to play important role in gene expression regulation. They affect nucleosome dynamics and remodeling, chromatin compaction, transcription initiation and elongation and serve as targets for multiple posttranslational modifications, known as histone-code. The disordered nature of histone tails makes their structural characterization elusive. To address this question for the first time we perform extensive microsecond molecular dynamics simulations of nucleosome including linker DNA in explicit solvent in order to analyze the evolution of histone tails’ conformation and their interaction patterns with nucleosome core and linker DNA. We show that histone tails readily bind to nucleosomal DNA and become kinetically trapped in a certain number of distinct conformational states at microsecond timescale. Certain bound states of histone tails are associated with secondary structure formation, rearrangement of DNA conformation and protein DNA-interactions in the nucleosome core as well as neutralization of linker DNA. The implications of our findings for nucleosome dynamics, chromatin fiber compaction and histone code functioning are discussed.

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 H1(0) 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 H1(0). 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.

The Influence of Chromatin Structure on DNA Damage Induced by Nitrogen Mustard and Cisplatin Analogues

The interaction of anti-tumour drugs with reconstituted chromatin has been investigated using defined nucleosomal complexes. This allowed the effect of nucleosome cores on drug-induced DNA damage to be assessed for four nitrogen mustard analogues, dimethylsulphate and three cisplatin analogues. A defined nucleosomal complex was employed that contained two precisely positioned nucleosome cores. The construct was then subjected to drug treatment, and the resulting DNA damage was quantitatively analysed using a Taq DNA polymerase stop assay. At the sites of damage, densitometric comparisons between purified and reconstituted DNA were used to evaluate the influence of nucleosomal core proteins on specific drug-DNA interactions. Results were combined with previous data obtained for other DNA-damaging drugs investigated using the same nucleosomal construct. For most of the DNA-damaging agents studied, this method revealed protection at the positioned nucleosome cores and indicated that the preferred site of DNA binding for these compounds was in the linker region of the construct. Statistical analyses confirmed the significant level of damage protection conferred by the nucleosome cores and revealed differences between the examined compounds. Larger compounds generally displayed a greater tendency to target the linker region of the nucleosomal DNA and were impeded from damaging nucleosomal core DNA. In contrast, smaller molecules had greater access to nucleosomal core DNA.

FineStr: a web server for single-base-resolution nucleosome positioning

The DNA in eukaryotic cells is packed into the chromatin that is composed of nucleosomes. Positioning of the nucleosome core particles on the sequence is a problem of great interest because of the role nucleosomes play in different cellular processes including gene regulation. Using the sequence structure of 10.4 base DNA repeat presented in our previous works and nucleosome core DNA sequences database, we have derived the complete nucleosome DNA bendability matrix of Caenorhabditis elegans. We have developed a web server named FineStr that allows users to upload genomic sequences in FASTA format and to perform a single-base-resolution nucleosome mapping on them. FineStr server is freely available for use on the web at http:/www.cs.bgu.ac.il/ approximately nucleom. The site contains a help file with explanation regarding the exact usage. gabdank@cs.bgu.ac.il.

Sequence periodicity in nucleosomal DNA and intrinsic curvature

BackgroundMost eukaryotic DNA contained in the nucleus is packaged by wrapping DNA around histone octamers. Histones are ubiquitous and bind most regions of chromosomal DNA. In order to achieve smooth wrapping of the DNA around the histone octamer, the DNA duplex should be able to deform and should possess intrinsic curvature. The deformability of DNA is a result of the non-parallelness of base pair stacks. The stacking interaction between base pairs is sequence dependent. The higher the stacking energy the more rigid the DNA helix, thus it is natural to expect that sequences that are involved in wrapping around the histone octamer should be unstacked and possess intrinsic curvature. Intrinsic curvature has been shown to be dictated by the periodic recurrence of certain dinucleotides. Several genome-wide studies directed towards mapping of nucleosome positions have revealed periodicity associated with certain stretches of sequences. In the current study, these sequences have been analyzed with a view to understand their sequence-dependent structures.ResultsHigher order DNA structures and the distribution of molecular bend loci associated with 146 base nucleosome core DNA sequence from C. elegans and chicken have been analyzed using the theoretical model for DNA curvature. The curvature dispersion calculated by cyclically permuting the sequences revealed that the molecular bend loci were delocalized throughout the nucleosome core region and had varying degrees of intrinsic curvature.ConclusionsThe higher order structures associated with nucleosomes of C.elegans and chicken calculated from the sequences revealed heterogeneity with respect to the deviation of the DNA axis. The results points to the possibility of context dependent curvature of varying degrees to be associated with nucleosomal DNA.

Nucleosome DNA Bendability Matrix (C. elegans)

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.

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 10–11bp, 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 (∼10.3bp) at the two ends, with larger (∼11.1bp) 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.

Nucleosome positioning in relation to nucleosome spacing and DNA sequence‐specific binding of a protein

Nucleosome positioning is an important mechanism for the regulation of eukaryotic gene expression. Folding of the chromatin fiber can influence nucleosome positioning, whereas similar electrostatic mechanisms govern the nucleosome repeat length and chromatin fiber folding in vitro. The position of the nucleosomes is directed either by the DNA sequence or by the boundaries created due to the binding of certain trans-acting factors to their target sites in the DNA. Increasing ionic strength results in an increase in nucleosome spacing on the chromatin assembled by the S-190 extract of Drosophila embryos. In this study, a mutant lac repressor protein R3 was used to find the mechanisms of nucleosome positioning on a plasmid with three R3-binding sites. With increasing ionic strength in the presence of R3, the number of positioned nucleosomes in the chromatin decreased, whereas the internucleosomal spacings of the positioned nucleosomes in a single register did not change. The number of the positioned nucleosomes in the chromatin assembled in vitro over different plasmid DNAs with 1-3 lac operators changed with the relative position and number of the R3-binding sites. We found that in the presence of R3, nucleosomes were positioned in the salt gradient method of the chromatin assembly, even in the absence of a nucleosome-positioning sequence. Our results show that nucleosome-positioning mechanisms are dominant, as the nucleosomes can be positioned even in the absence of regular spacing mechanisms. The protein-generated boundaries are more effective when more than one binding site is present with a minimum distance of approximately 165 bp, greater than the nucleosome core DNA length, between them.

Histone H2A Ubiquitination Does Not Preclude Histone H1 Binding, but It Facilitates Its Association with the Nucleosome

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.

Dissecting Transcription-coupled and Global Genomic Repair in the Chromatin of Yeast GAL1-10 Genes

Transcription-coupled repair (TCR) and global genomic repair (GGR) of UV-induced cyclobutane pyrimidine dimers were investigated in the yeast GAL1-10 genes. Both Rpb9- and Rad26-mediated TCR are confined to the transcribed strands, initiating at upstream sites approximately 100 nucleotides from the upstream activating sequence shared by the two genes. However, TCR initiation sites do not correlate with either transcription start sites or TATA boxes. Rad16-mediated GGR tightly correlates with nucleosome positioning when the genes are repressed and are slow in the nucleosome core and fast in linker DNA. Induction of transcription enhanced GGR in nucleosome core DNA, especially in the nucleosomes around and upstream of the transcription start sites. Furthermore, when the genes were induced, GGR was slower in the transcribed regions than in the upstream regions. Finally, simultaneous deletion of RAD16, RAD26, and RPB9 resulted in no detectable repair in all sites along the region analyzed. Our results suggest that (a). TCR may be initiated by a transcription activator, presumably through the loading of RNA polymerase II, rather than by transcription initiation or elongation per se; (b). TCR and nucleosome disruption-enhanced GGR are the major causes of rapid repair in regions around and upstream of transcription start sites; (c). transcription machinery may hinder access of NER factors to a DNA lesion in the absence of a transcription-repair coupling factor; and (d). other than GGR mediated by Rad16 and TCR mediated by Rad26 and Rpb9, no other nucleotide excision repair pathway exists in these RNA polymerase II-transcribed genes.

Is a small number of charge neutralizations sufficient to bend nucleosome core DNA onto its superhelical ramp?

X-ray diffraction structures of the nucleosome core particle along with a variety of experiments are consistent with the idea that an important source of the free energy holding DNA to the superhelical ramp on the histone octamer surface is obtained from a relatively small amount of electrostatic neutralization of the DNA phosphate charge by positively charged histone groups, especially arginine residues. Here we present a theoretical analysis of a simple model that emphasizes the competition between the high degree of bending of the stiff DNA molecule required for its tight curvature on the histone octamer and the neutralization of the DNA phosphate charge by basic histone residues. Our calculation accounts for the strong influence of condensed counterions on the electrostatic interactions. We find that the minimum amount of free energy required to bend DNA into axial conformity with the superhelical ramp at physiological salt concentration can be provided by a scant 6% neutralization of the phosphate charge, in close correspondence to the stoichiometric neutralization of phosphate charge by the arginine side chain that intrudes into the inward-facing minor groove of each DNA double helical turn.

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 2N 8A 2N 8A 2 or T 2N 8T 2N 8T 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, 125 nt. 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+∼10× 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.

Association of chromatin with anticancer antibiotics, mithramycin and chromomycin A3

Mithramycin and chromomycin A3 are two anticancer antibiotics, which inhibit protein biosynthesis via transcription inhibition. They bind reversibly to DNA with (G.C) base specificity. At and above physiological pH in the absence of DNA, they form two types of complexes with Mg2+, complex I (1:1 in terms of antibiotic: Mg2+) and complex II (2:1 in terms of antibiotic: Mg2+). These are the DNA binding ligands. In vivo, the antibiotics interact with chromatin, a protein–DNA complex. In order to understand the mode of action of these antibiotics at molecular level, we have carried out spectroscopic, gel electrophoretic and UV melting studies of complex I of these antibiotics with rat liver chromatin, nucleosome core particle and DNA stripped of all chromosomal proteins. Analysis of the results has led us to propose that the antibiotic: Mg2+ complex binds to both nucleosomal and linker DNA in native chromatin. Histone proteins reduce the binding potential and accessibility of the complexes to the minor groove of (G.C) rich regions of chromosomal DNA. The antibiotic: Mg2+ complex stabilizes DNA duplex and histone- DNA contacts in chromatin fiber. It also leads to the aggregation of chromatin fibers. From a comparison of the association of the antibiotic: Mg2+ complexes with different levels of chromatin structure and their effects upon the structure, we suggest that the sugar moieties of the antibiotics play a role in the binding process. Significance of these results to understand the molecular basis of the transcription inhibition potential of the antibiotics in eukaryotes is discussed.

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.