DNA stretching and extreme kinking in the nucleosome core

DNA stretching in chromatin may facilitate its compaction and influence site recognition by nuclear factors. In vivo, stretching has been estimated to occur at the equivalent of one to two base-pairs (bp) per nucleosome. We have determined the crystal structure of a nucleosome core particle containing 145 bp of DNA (NCP145). Compared to the structure with 147 bp, the NCP145 displays two incidences of stretching one to two double-helical turns from the particle dyad axis. The stretching illustrates clearly a mechanism for shifting DNA position by displacement of a single base-pair while maintaining nearly identical histone-DNA interactions. Increased DNA twist localized to a short section between adjacent histone-DNA binding sites advances the rotational setting, while a translational component involves DNA kinking at a flanking region that initiates elongation by unstacking bases. Furthermore, one stretched region of the NCP145 displays an extraordinary 55° kink into the minor groove situated 1.5 double-helical turns from the particle dyad axis, a hot spot for gene insertion by HIV-integrase, which prefers highly distorted substrate. This suggests that nucleosome position and context within chromatin could promote extreme DNA kinking that may influence genomic processes.     

Are nucleosome positions in vivo primarily determined by histone–DNA sequence preferences?

Large-scale and genome-wide studies have concluded that ∼80% of the yeast (Saccharomyces cerevisiae) genome is occupied by positioned nucleosomes. In vivo this nucleosome organization can result from a variety of mechanisms, including the intrinsic DNA sequence preferences for wrapping the DNA around the histone core. Recently, a genome-wide study was reported using massively parallel sequencing to directly compare in vivo and in vitro nucleosome positions. It was concluded that intrinsic DNA sequence preferences indeed have a dominant role in determining the in vivo nucleosome organization of the genome, consistent with a genomic code for nucleosome positioning. Some other studies disagree with this view. Using the large amount of data now available from several sources, we have attempted to clarify a fundamental question concerning the packaging of genomic DNA: to what extent are nucleosome positions in vivo determined by histone-DNA sequence preferences? We have analyzed data obtained from different laboratories in the same way, and have directly compared these data. We also identify possible problems with some of the experimental designs used and with the data analysis. Our findings suggest that DNA sequence preferences have only small effects on the positioning of individual nucleosomes throughout the genome in vivo.     

Nucleosome structural studies

Chromatin plays a fundamental role in eukaryotic genomic regulation, and the increasing awareness of the importance of epigenetic processes in human health and disease emphasizes the need for understanding the structure and function of the nucleosome. Recent advances in chromatin structural studies, including the first structures of nucleosomes containing the Widom 601 sequence and the structure of a chromatin protein-nucleosome assembly, have provided new insight into stretching of nucleosomal DNA, nucleosome positioning, binding of metal ions, drugs and therapeutic candidates to nucleosomes, and nucleosome recognition by nuclear proteins. These discoveries ensure promising future prospects for unravelling structural attributes of chromatin.     

High-Resolution Mapping of Sequence-Directed Nucleosome Positioning on Genomic DNA

We have mapped in vitro nucleosome positioning on the sheep β-lactoglobulin gene using high-throughput sequencing to characterise the DNA sequences recovered from reconstituted nucleosomes. This methodology surpasses previous approaches for coverage, accuracy and resolution and, most importantly, offers a simple yet rapid and relatively inexpensive method to characterise genomic DNA sequences in terms of nucleosome positioning capacity. We demonstrate an unambiguous correspondence between in vitro and in vivo nucleosome positioning around the promoter of the gene; identify discrete, sequence-specific nucleosomal structures above the level of the canonical core particle—a feature that has implications for regulatory protein access and higher-order chromatin packing; and reveal new insights into the involvement of periodically organised dinucleotide sequence motifs of the type GG and CC and not AA and TT, as determinants of nucleosome positioning—an observation that supports the idea that the core histone octamer can exploit different patterns of sequence organisation, or structural potential, in the DNA to bring about nucleosome positioning.     

Nucleosomal locations of dominant DNA sequence motifs for histone-DNA interactions and nucleosome positioning

DNA sequence is an important determinant of the positioning, stability, and activity of nucleosomes, yet the molecular basis of these effects remains elusive. A "consensus DNA sequence" for nucleosome positioning has not been reported and, while certain DNA sequence preferences or motifs for nucleosome positioning have been discovered, how they function is not known. Here, we report that an unexpected observation concerning the reassembly of nucleosomes during salt gradient dialysis has allowed a breakthrough in our efforts to identify the nucleosomal locations of the DNA sequence motifs that dominate histone-DNA interactions and nucleosome positioning. We conclude that a previous selection experiment for high-affinity, nucleosome-forming DNA sequences exerted selective pressure chiefly on the central stretch of the nucleosomal DNA. This observation implies that algorithms for aligning the selected DNA sequences should seek to optimize the alignment over much less than the full 147 bp of nucleosomal DNA. A new alignment calculation implemented these ideas and successfully aligned 19 of the 41 sequences in a non-redundant database of selected high-affinity, nucleosome-positioning sequences. The resulting alignment reveals strong conservation of several stretches within a central 71 bp of the nucleosomal DNA. The alignment further reveals an inherent palindromic symmetry in the selected DNAs; it makes testable predictions of nucleosome positioning on the aligned sequences and for the creation of new positioning sequences, both of which are upheld experimentally; and it suggests new signals that may be important in translational nucleosome positioning.     

AFM imaging and theoretical modeling studies of sequence-dependent nucleosome positioning

Telomeric chromatin has different features with respect to bulk chromatin, since nucleosomal repeat along the chain is unusually short. We studied the role of telomeric DNA sequences on nucleosomal spacing in a model system. Nucleosomal arrays, assembled on a 1500-bp-long human telomeric DNA and on a DNA fragment containing 8 copies of the 601 strong nucleosome positioning sequence, have been studied at the single molecule level, by atomic force microscopy imaging. Random nucleosome positioning was found in the case of human telomeric DNA. On the contrary, nucleosome positioning on 601 DNA is characterized by preferential positions of nucleosome dyad axis each 200 bp. The AFM-derived nucleosome organization is in satisfactory agreement with that predicted by theoretical modeling, based on sequence-dependent DNA curvature and flexibility. The reported results show that DNA sequence has a main role, not only in mononucleosome thermodynamic stability, but also in the organization of nucleosomal arrays.     

Alteration of the nucleosomal DNA path in the crystal structure of a human nucleosome core particle

Gene expression in eukaryotes depends upon positioning, mobility and packaging of nucleosomes; thus, we need the detailed information of the human nucleosome core particle (NCP) structure, which could clarify chromatin properties. Here, we report the 2.5 Å crystal structure of a human NCP. The overall structure is similar to those of other NCPs reported previously. However, the DNA path of human NCP is remarkably different from that taken within other NCPs with an identical DNA sequence. A comparison of the structural parameters between human and Xenopus laevis DNA reveals that the DNA path of human NCP consecutively shifts by 1 bp in the regions of superhelix axis location −5.0 to −2.0 and 5.0 to 7.0. This alteration of the human DNA path is caused predominantly by tight DNA–DNA contacts within the crystal. It is also likely that the conformational change in the human H2B tail induces the local alteration of the DNA path. In human NCP, the region with the altered DNA path lacks Mn²⁺ ions and the B-factors of the DNA phosphate groups are substantially high. Therefore, in contrast to the histone octamer, the nucleosomal DNA is sufficiently flexible and mobile and can undergo drastic conformational changes, depending upon the environment.     

Measurement of histone-DNA interaction free energy in nucleosomes

Nucleosome positioning DNA sequences are of increasing interest because of their proposed roles in gene regulation and other chromosome functions in vivo, and because they have revealed new insights into the sequence-dependent structures and mechanics of DNA itself. Here, we describe methods to quantify the relative affinities of histone-DNA interactions in nucleosomes, i.e., the nucleosome positioning power of differing DNA sequences. We review methods developed by others and then discuss in detail our own approach to measurement of histone-DNA interaction free energies. Compared to earlier methods, our dialysis-based approach reduces the possibility that non-equilibrium or irreproducible results could be obtained. It facilitates a direct comparison of free energies for many sequences at the same time and it allows analysis of DNAs having a wide range of relative affinities.     

Nucleosome positioning signals in the DNA sequence of the human and mouse H19 imprinting control regions

We have investigated the sequences of the mouse and human H19 imprinting control regions (ICRs) to see whether they contain nucleosome positioning information pertinent to their function as a methylation-regulated chromatin boundary. Positioning signals were identified by an in vitro approach that employs reconstituted chromatin to comprehensively describe the contribution of the DNA to the most basic, underlying level of chromatin structure. Signals in the DNA sequence of both ICRs directed nucleosomes to flank and encompass the short conserved sequences that constitute the binding sites for the zinc finger protein CTCF, an essential mediator of insulator activity. The repeat structure of the human ICR presented a conserved array of strong positioning signals that would preferentially flank these CTCF binding sites with positioned nucleosomes, a chromatin structure that would tend to maintain their accessibility. Conversely, all four CTCF binding sites in the mouse sequence were located close to the centre of positioning signals that were stronger than those in their flanks; these binding sites might therefore be expected to be more readily incorporated into positioned nucleosomes. We found that CpG methylation did not effect widespread repositioning of nucleosomes on either ICR, indicating that allelic methylation patterns were unlikely to establish allele-specific chromatin structures for H19 by operating directly upon the underlying DNA-histone interactions; instead, epigenetic modulation of ICR chromatin structure is likely to be mediated principally at higher levels of control. DNA methylation did, however, both promote and inhibit nucleosome positioning at several sites in both ICRs and substantially negated one of the strongest nucleosome positioning signals in the human sequence, observations that underline the fact that this epigenetic modification can, nevertheless, directly and decisively modulate core histone-DNA interactions within the nucleosome.     

DNA Architecture,Deformability, and Nucleosome Positioning

Abstract

The positioning of DNA on nucleosomes is critical to both the organization and expression of the genetic message. Here we focus on DNA conformational signals found in the growing library of known high-resolution core-particle structures and the ways in which these features may contribute to the positioning of nucleosomes on specific DNA sequences. We survey the chemical composition of the protein-DNA assemblies and extract features along the DNA superhelical pathway—the minor-groove width and the deformations of successive base pairs—determined with reasonable accuracy in the structures. We also examine the extent to which the various nucleosome core-particle structures accommodate the observed settings of the crystallized sequences and the known positioning of the high-affinity synthetic ‘601’ sequence on DNA. We ‘thread’ these sequences on the different structural templates and estimate the cost of each setting with knowledge-based potentials that reflect the conformational properties of the DNA base-pair steps in other high-resolution protein-bound complexes.     

The mechanics behind DNA sequence-dependent properties of the nucleosome

Chromatin organization and composition impart sophisticated regulatory features critical to eukaryotic genomic function. Although DNA sequence-dependent histone octamer binding is important for nucleosome activity, many aspects of this phenomenon have remained elusive. We studied nucleosome structure and stability with diverse DNA sequences, including Widom 601 derivatives with the highest known octamer affinities, to establish a simple model behind the mechanics of sequence dependency. This uncovers the unique but unexpected role of TA dinucleotides and a propensity for G|C-rich sequence elements to conform energetically favourably at most locations around the histone octamer, which rationalizes G|C% as the most predictive factor for nucleosome occupancy in vivo. In addition, our findings reveal dominant constraints on double helix conformation by H3-H4 relative to H2A-H2B binding and DNA sequence context-dependency underlying nucleosome structure, positioning and stability. This provides a basis for improved prediction of nucleosomal properties and the design of tailored DNA constructs for chromatin investigations.     

The DNA Sequence-dependence of Nucleosome Positioning in vivo and in vitro

Abstract

The contribution of histone-DNA interactions to nucleosome positioning in vivo is currently a matter of debate. We argue here that certain nucleosome positions, often in promoter regions, in yeast may be, at least in part, specified by the DNA sequence. In contrast other positions may be poorly specified. Positioning thus has both statistical and DNA-determined components. We further argue that the relative affinity of the octamer for different DNA sequences can vary and therefore the interaction of histones with the DNA is a ‘tunable’ property.     

Probing the effects of DNA-protein interactions on DNA hole transport: the N-terminal histone tails modulate the distribution of oxidative damage and chemical lesions in the nucleosome core particle

The ability of DNA to transport positive charges, or holes, over long distances is well-established, but the mechanistic details of how this process is influenced by packaging into DNA-protein complexes have not been fully delineated. In eukaryotes, genomic DNA is packaged into chromatin through its association with the core histone octamer to form the nucleosome core particle (NCP), a complex whose structure can be modulated through changes in the local environment and the histone proteins. Because (i) varying the salt concentration and removing the histone tails influence the structure of the NCP in known ways and (ii) previous studies have shown that DNA hole transport (HT) occurs in the nucleosome, we have used our previously described 601 sequence NCPs to test the hypothesis that DNA HT dynamics can be modulated by structural changes in a DNA-protein complex. We show that at low salt concentrations there is a sharp increase in long-range DNA HT efficiency in the NCP as compared to naked DNA. This enhancement of HT can be negated by either removal of the histone tails at low salt concentrations or disruption of the interaction of the packaged DNA and the histone tails by increasing the buffer's ionic strength. Association of the histone tails with 601 DNA at low salt concentrations shifts the guanine damage spectrum to favor lesions like 8-oxoguanine in the NCP, most likely through modulation of the rate of the reaction of the guanine radical cation with oxygen. These experimental results indicate that for most genomic DNA, the influence of DNA-protein interactions on DNA HT will depend strongly on the level of protection of the DNA nucleobases from oxygen. Further, these results suggest that the oxidative damage arising from DNA HT may vary in different genomic regions depending on the presence of either euchromatin or heterochromatin.     

Sequence motifs and free energies of selected natural and non-natural nucleosome positioning DNA sequences.

Our laboratories recently completed SELEX experiments to isolate DNA sequences that most-strongly favor or disfavor nucleosome formation and positioning, from the entire mouse genome or from even more diverse pools of chemically synthetic random sequence DNA. Here we directly compare these selected natural and non-natural sequences. We find that the strongest natural positioning sequences have affinities for histone binding and nucleosome formation that are sixfold or more lower than those possessed by many of the selected non-natural sequences. We conclude that even the highest-affinity sequence regions of eukaryotic genomes are not evolved for the highest affinity or nucleosome positioning power. Fourier transform calculations on the selected natural sequences reveal a special significance for nucleosome positioning of a motif consisting of approximately 10 bp periodic placement of TA dinucleotide steps. Contributions to histone binding and nucleosome formation from periodic TA steps are more significant than those from other periodic steps such as AA (=TT), CC (=GG) and more important than those from the other YR steps (CA (=TG) and CG), which are reported to have greater conformational flexibility in protein-DNA complexes even than TA. We report the development of improved procedures for measuring the free energies of even stronger positioning sequences that may be isolated in the future, and show that when the favorable free energy of histone-DNA interactions becomes sufficiently large, measurements based on the widely used exchange method become unreliable.     

The kinetic landscape of nucleosome assembly: A coarse-grained molecular dynamics study

The organization of nucleosomes along the Eukaryotic genome is maintained over time despite disruptive events such as replication. During this complex process, histones and DNA can form a variety of non-canonical nucleosome conformations, but their precise molecular details and roles during nucleosome assembly remain unclear. In this study, employing coarse-grained molecular dynamics simulations and Markov state modeling, we characterized the complete kinetics of nucleosome assembly. On the nucleosome-positioning 601 DNA sequence, we observe a rich transition network among various canonical and non-canonical tetrasome, hexasome, and nucleosome conformations. A low salt environment makes nucleosomes stable, but the kinetic landscape becomes more rugged, so that the system is more likely to be trapped in off-pathway partially assembled intermediates. Finally, we find that the co-operativity between DNA bending and histone association enables positioning sequence motifs to direct the assembly process, with potential implications for the dynamic organization of nucleosomes on real genomic sequences.     

Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 a resolution

Solvent binding in the nucleosome core particle containing a 147 base pair, defined-sequence DNA is characterized from the X-ray crystal structure at 1.9 Å resolution. A single-base-pair increase in DNA length over that used previously results in substantially improved clarity of the electron density and accuracy for the histone protein and DNA atomic coordinates. The reduced disorder has allowed for the first time extensive modeling of water molecules and ions.Over 3000 water molecules and 18 ions have been identified. Water molecules acting as hydrogen-bond bridges between protein and DNA are approximately equal in number to the direct hydrogen bonds between these components. Bridging water molecules have a dual role in promoting histone-DNA association not only by providing further stability to direct protein-DNA interactions, but also by enabling formation of many additional interactions between more distantly related elements. Water molecules residing in the minor groove play an important role in facilitating insertion of arginine side-chains. Water structure at the interface of the histones and DNA provides a means of accommodating intrinsic DNA conformational variation, thus limiting the sequence dependency of nucleosome positioning while enhancing mobility.Monovalent anions are bound near the N termini of histone α-helices that are not occluded by DNA phosphate groups. Their location in proximity to the DNA phosphodiester backbone suggests that they damp the electrostatic interaction between the histone proteins and the DNA. Divalent cations are bound at specific sites in the nucleosome core particle and contribute to histone-histone and histone-DNA interparticle interactions. These interactions may be relevant to nucleosome association in arrays.     

Nucleosome positioning based on the sequence word composition

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