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1.
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.  相似文献   

2.
Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.  相似文献   

3.
Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.  相似文献   

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The ATPase ISWI can be considered the catalytic core of several multiprotein nucleosome remodeling machines. Alone or in the context of nucleosome remodeling factor, the chromatin accessibility complex (CHRAC), or ACF, ISWI catalyzes a number of ATP-dependent transitions of chromatin structure that are currently best explained by its ability to induce nucleosome sliding. In addition, ISWI can function as a nucleosome spacing factor during chromatin assembly, where it will trigger the ordering of newly assembled nucleosomes into regular arrays. Both nucleosome remodeling and nucleosome spacing reactions are mechanistically unexplained. As a step toward defining the interaction of ISWI with its substrate during nucleosome remodeling and chromatin assembly we generated a set of nucleosomes lacking individual histone N termini from recombinant histones. We found the conserved N termini (the N-terminal tails) of histone H4 essential to stimulate ISWI ATPase activity, in contrast to other histone tails. Remarkably, the H4 N terminus, but none of the other tails, was critical for CHRAC-induced nucleosome sliding and for the generation of regularity in nucleosomal arrays by ISWI. Direct nucleosome binding studies did not reflect a dependence on the H4 tail for ISWI-nucleosome interactions. We conclude that the H4 tail is critically required for nucleosome remodeling and spacing at a step subsequent to interaction with the substrate.  相似文献   

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Nucleosomes are important for gene regulation because their arrangement on the genome can control which proteins bind to DNA. Currently, few human nucleosomes are thought to be consistently positioned across cells; however, this has been difficult to assess due to the limited resolution of existing data. We performed paired-end sequencing of micrococcal nuclease-digested chromatin (MNase–seq) from seven lymphoblastoid cell lines and mapped over 3.6 billion MNase–seq fragments to the human genome to create the highest-resolution map of nucleosome occupancy to date in a human cell type. In contrast to previous results, we find that most nucleosomes have more consistent positioning than expected by chance and a substantial fraction (8.7%) of nucleosomes have moderate to strong positioning. In aggregate, nucleosome sequences have 10 bp periodic patterns in dinucleotide frequency and DNase I sensitivity; and, across cells, nucleosomes frequently have translational offsets that are multiples of 10 bp. We estimate that almost half of the genome contains regularly spaced arrays of nucleosomes, which are enriched in active chromatin domains. Single nucleotide polymorphisms that reduce DNase I sensitivity can disrupt the phasing of nucleosome arrays, which indicates that they often result from positioning against a barrier formed by other proteins. However, nucleosome arrays can also be created by DNA sequence alone. The most striking example is an array of over 400 nucleosomes on chromosome 12 that is created by tandem repetition of sequences with strong positioning properties. In summary, a large fraction of nucleosomes are consistently positioned—in some regions because they adopt favored sequence positions, and in other regions because they are forced into specific arrangements by chromatin remodeling or DNA binding proteins.  相似文献   

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Although poorly positioned nucleosomes are ubiquitous in the eukaryotic genome, they are difficult to identify with existing nucleosome identification methods. Recently available enhanced high-throughput chromatin conformation capture techniques such as Micro-C, DNase Hi-C, and Hi-CO characterize nucleosome-level chromatin proximity, probing the positions of mono-nucleosomes and the spacing between nucleosome pairs at the same time, enabling nucleosome profiling in poorly positioned regions. Here we develop a novel computational approach, NucleoMap, to identify nucleosome positioning from ultra-high resolution chromatin contact maps. By integrating nucleosome read density, contact distances, and binding preferences, NucleoMap precisely locates nucleosomes in both prokaryotic and eukaryotic genomes and outperforms existing nucleosome identification methods in both precision and recall. We rigorously characterize genome-wide association in eukaryotes between the spatial organization of mono-nucleosomes and their corresponding histone modifications, protein binding activities, and higher-order chromatin functions. We also find evidence of two tetra-nucleosome folding structures in human embryonic stem cells and analyze their association with multiple structural and functional regions. Based on the identified nucleosomes, nucleosome contact maps are constructed, reflecting the inter-nucleosome distances and preserving the contact distance profiles in original contact maps.  相似文献   

11.
Nucleosome positions on the DNA are determined by the intrinsic affinities of histone proteins to a given DNA sequence and by the ATP-dependent activities of chromatin remodeling complexes that can translocate nucleosomes with respect to the DNA. Here, we report a theoretical approach that takes into account both contributions. In the theoretical analysis two types of experiments have been considered: in vitro experiments with a single reconstituted nucleosome and in vivo genome-scale mapping of nucleosome positions. The effect of chromatin remodelers was described by iteratively redistributing the nucleosomes according to certain rules until a new steady state was reached. Three major classes of remodeler activities were identified: (i) the establishment of a regular nucleosome spacing in the vicinity of a strong positioning signal acting as a boundary, (ii) the enrichment/depletion of nucleosomes through amplification of intrinsic DNA-sequence-encoded signals and (iii) the removal of nucleosomes from high-affinity binding sites. From an analysis of data for nucleosome positions in resting and activated human CD4+ T cells [Schones et al., Cell 132, p. 887] it was concluded that the redistribution of a nucleosome map to a new state is greatly facilitated if the remodeler complex translocates the nucleosome with a preferred directionality.  相似文献   

12.
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.  相似文献   

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Nucleosomes are the fundamental repeating unit of chromatin and comprise the structural building blocks of the living eukaryotic genome. Micrococcal nuclease (MNase) has long been used to delineate nucleosomal organization. Microarray-based nucleosome mapping experiments in yeast chromatin have revealed regularly-spaced translational phasing of nucleosomes. These data have been used to train computational models of sequence-directed nuclesosome positioning, which have identified ubiquitous strong intrinsic nucleosome positioning signals. Here, we successfully apply this approach to nucleosome positioning experiments from human chromatin. The predictions made by the human-trained and yeast-trained models are strongly correlated, suggesting a shared mechanism for sequence-based determination of nucleosome occupancy. In addition, we observed striking complementarity between classifiers trained on experimental data from weakly versus heavily digested MNase samples. In the former case, the resulting model accurately identifies nucleosome-forming sequences; in the latter, the classifier excels at identifying nucleosome-free regions. Using this model we are able to identify several characteristics of nucleosome-forming and nucleosome-disfavoring sequences. First, by combining results from each classifier applied de novo across the human ENCODE regions, the classifier reveals distinct sequence composition and periodicity features of nucleosome-forming and nucleosome-disfavoring sequences. Short runs of dinucleotide repeat appear as a hallmark of nucleosome-disfavoring sequences, while nucleosome-forming sequences contain short periodic runs of GC base pairs. Second, we show that nucleosome phasing is most frequently predicted flanking nucleosome-free regions. The results suggest that the major mechanism of nucleosome positioning in vivo is boundary-event-driven and affirm the classical statistical positioning theory of nucleosome organization.  相似文献   

15.
Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.  相似文献   

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Nucleosome positioning signals embedded within the DNA sequence have the potential to influence the detailed structure of the higher-order chromatin fibre. In two previous studies of long stretches of DNA, encompassing the chicken beta-globin and ovine beta-lactoglobulin genes, respectively, we mapped the relative affinity of every site for the core histone octamer. In both cases a periodic arrangement of the in vitro positioning sites suggests that they might influence the folding of a nucleosome chain into higher-order structure; this hypothesis was borne out in the case of the beta-lactoglobulin gene, where the distribution of the in vitro positioning sites is related to the positions nucleosomes actually occupy in sheep liver cells. Here, we have exploited the in vitro nucleosome positioning datasets to simulate nucleosomal organisation using in silico approaches. We use the high-resolution, quantitative positioning maps to define a one-dimensional positioning energy lattice, which can be populated with a defined number of nucleosomes. Monte Carlo techniques are employed to simulate the behaviour of the model at equilibrium to produce a set of configurations, which provide a probability-based occupancy map. Employing a variety of techniques we show that the occupancy maps are a sensitive function of the histone octamer density (nucleosome repeat length) and find that a minimal change in this property can produce dramatic localised changes in structure. Although simulations generally give rise to regular periodic nucleosomal arrangements, they often show octamer density-dependent discontinuities, which tend to co-localise with sequences that adopt distinctive chromatin structure in vivo. Furthermore, the overall organisation of simulated chromatin structures are more closely related to the situation in vivo than is the original in vitro positioning data, particularly at a nucleosome density corresponding to the in vivo state. Although our model is simplified, we argue that it provides a unique insight into the influence that DNA sequence can have in determining chromatin structure and could serve as a useful basis for the incorporation of other parameters.  相似文献   

18.
Some models of in vitro chromatin assembly suggest a biphasic molecular mechanism. The first phase, nucleosome formation, is comprised of the formation of histone-DNA complexes which mature into a canonical nucleosome structure. The second phase represents the process by which these nucleosomes become properly spaced with a regular periodicity on the DNA. In this report, we examine the role of DNA topoisomerases in the latter phase of chromatin assembly. To study this process, we use a Xenopus laevis cell-free extract, which assembles quantitative amounts of chromatin on circular DNA templates, and the type II topoisomerase-specific antitumor drugs VM-26 and endrofloxicin. Our results suggest that nucleosome formation is unaffected by the presence of VM-26 or endrofloxicin. However, periodic spacing of nucleosomes is inhibited significantly by these drugs. In the absence of proper chromatin assembly, circular DNA molecules are processed into nucleoprotein complexes which are transcribed poorly. Taken together, these results indicate that the antitumor drugs VM-26 and endrofloxicin influence gene expression indirectly by blocking the periodic spacing of nucleosomes.  相似文献   

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