Nucleosome Positioning,Nucleosome Spacing and the Nucleosome Code

Abstract

Nucleosome positioning has been the subject of intense study for many years. The properties of micrococcal nuclease, the enzyme central to these studies, are discussed. The various methods used to determine nucleosome positions in vitro and in vivo are reviewed critically. These include the traditional low resolution method of indirect end-labelling, high resolution methods such as primer extension, monomer extension and nucleosome sequencing, and the high throughput methods for genome-wide analysis (microarray hybridisation and parallel sequencing). It is established that low resolution mapping yields an averaged chromatin structure, whereas high resolution mapping reveals the weighted superposition of all the chromatin states in a cell population. Mapping studies suggest that yeast DNA contains information specifying the positions of nucleosomes and that this code is made use of by the cell. It is proposed that the positioning code facilitates nucleosome spacing by encoding information for multiple alternative overlapping nucleosomal arrays. Such a code might facilitate the shunting of nucleosomes from one array to another by ATP-dependent chromatin remodelling machines.     

Sequence Signatures of Nucleosome Positioning in Caenorhabditis elegans

Our recent investigation in the protist Trichomonas vaginalis suggested a DNA sequence periodicity with a unit length of 120.9 nt, which represents a sequence signature for nucleosome positioning. We now extended our observation in higher eukaryotes and identified a similar periodicity of 175 nt in length in Caenorhabditis elegans. In the process of defining the sequence compositional characteristics, we found that the 10.5-nt periodicity, the sequence signature of DNA double helix, may not be sufficient for cross-nucleosome positioning but provides essential guiding rails to facilitate positioning. We further dissected nucleosome-protected sequences and identified a strong positive purine (AG) gradient from the 5′-end to the 3′-end, and also learnt that the nucleosome-enriched regions are GC-rich as compared to the nucleosome-free sequences as purine content is positively correlated with GC content. Sequence characterization allowed us to develop a hidden Markov model (HMM) algorithm for decoding nucleosome positioning computationally, and based on a set of training data from the fifth chromosome of C. elegans, our algorithm predicted 60%-70% of the well-positioned nucleosomes, which is 15%-20% higher than random positioning. We concluded that nucleosomes are not randomly positioned on DNA sequences and yet bind to different genome regions with variable stability, well-positioned nucleosomes leave sequence signatures on DNA, and statistical positioning of nucleosomes across genome can be decoded computationally based on these sequence signatures.     

Structure-based Analysis of DNA Sequence Patterns Guiding Nucleosome Positioning in vitro

Abstract

Recent studies of genome-wide nucleosomal organization suggest that the DNA sequence is one of the major determinants of nucleosome positioning. Although the search for underlying patterns encoded in nucleosomal DNA has been going on for about 30 years, our knowledge of these patterns still remains limited. Based on our evaluations of DNA deformation energy, we developed new scoring functions to predict nucleosome positioning. There are three principal differences between our approach and earlier studies: (i) we assume that the length of nucleosomal DNA varies from 146 to 147 bp; (ii) we consider the anisotropic flexibility of pyrimidine-purine (YR) dimeric steps in the context of their neighbors (e.g., YYRR versus RYRY); (iii) we postulate that alternating AT-rich and GC-rich motifs reflect sequence-dependent interactions between histone arginines and DNA in the minor groove. Using these functions, we analyzed 20 nucleosome positions mapped in vitro at single nucleotide resolution (including clones 601, 603, 605, the pGUB plasmid, chicken β-globin and three 5S rDNA genes). We predicted 15 of the 20 positions with 1-bp precision, and two positions with 2-bp precision. The predicted position of the ‘601’ nucleosome (i.e., the optimum of the computed score) deviates from the experimentally determined unique position by no more than 1 bp—an accuracy exceeding that of earlier predictions.

Our analysis reveals a clear heterogeneity of the nucleosomal sequences which can be divided into two groups based on the positioning ‘rules’ they follow. The sequences of one group are enriched by highly deformable YR/YYRR motifs at the minor-groove bending sites SHL ±3.5 and ±5.5, which is similar to the α-satellite sequence used in most crystallized nucleosomes. Apparently, the positioning of these nucleosomes is determined by the interactions between histones H2A/H2B and the terminal parts of nucleosomal DNA. In the other group (that includes the ‘601’ clone) the same YR/YYRR motifs occur predominantly at the sites SHL ±1.5. The interaction between the H3/H4 tetramer and the central part of the nucleosomal DNA is likely to be responsible for the positioning of nucleosomes of this group, and the DNA trajectory in these nucleosomes may differ in detail from the published structures.

Thus, from the stereochemical perspective, the in vitro nucleosomes studied here follow either an X-ray-like pattern (with strong deformations in the terminal parts of nucleosomal DNA), or an alternative pattern (with the deformations occurring predominantly in the central part of the nucleosomal DNA). The results presented here may be useful for genome-wide classification of nucleosomes, linking together structural and thermodynamic characteristics of nucleosomes with the underlying DNA sequence patterns guiding their positions.     

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.     

Positioning of a Nucleosome on Mouse Satellite DNA Inserted into a Yeast Plasmid Is Determined by Its DNA Sequence and an Adjacent Nucleosome

It has earlier been shown that multiple positioning of nucleosomes on mouse satellite DNA is determined by its nucleotide sequence. To clarify whether other factors, such as boundary ones, can affect the positionings, we modified the environment of satellite DNA monomer by inserting it into a yeast plasmid between inducible GalCyc promoter and a structural region of the yeast FLP gene. We have revealed that the positions of nucleosomes on satellite DNA are identical to those detected upon reconstruction in vitro. The positioning signal (GAAAAA sequence) of satellite DNA governs nucleosome location at the adjacent nucleotide sequence as well. Upon promoter induction the nucleosome, translationally positioned on the GalCyc promoter, transfers to the satellite DNA and its location follows the positioning signal of the latter. Thus, the alternatives of positioning of a nucleosome on satellite DNA are controlled by its nucleotide sequence, though the choice of one of them is determined by the adjacent nucleosome.     

DNA Curvature of the Tobacco GRS Repetitive Sequence Family and its Relation to Nucleosome Positioning

Abstract

Recently, a highly repetitive DNA sequence family (GRS) from tobacco was described in our laboratory. These sequences were found to be localized predominantly in the pericen-tromeric heterochromatin of tobacco chromosomes. To test the hypothesis that these sequences play an important role in the formation of heterochromatin, we investigated the DNA curvature of the GRS sequences and its possible impact to the chromatin structure at these loci. Application of the nearest-neighbour wedge model of intrinsic DNA curvature for the GRS1 family member predicted two loci of curvature: a major bend at the 5′ end of the sequence and a minor bend of opposite direction at the centre of the GRS1. The presence of the major and the minor loci of DNA curvature was studied experimentally using permutation analysis and site-directed mutagenesis. The experimental results were consistent with the computer predictions. We gave evidence that the described DNA curvature is also present in the entire GRS family. Genomic statistical sequencing showed the conservation of the major bend sequence determinants in the members of the GRS family. To investigate the chromatin structure at the GRS sequences, we determined the nucleosome positioning in vivo at these sequences using thermal cycle primer extension. A relation between the curvature pattern and the histone octamer position was observed: the major bend is excluded from the nucleosome surface to the linker region, while the minor bend is distributed along the core DNA The suggestion is made that the sequences in the minor locus of curvature define the rotational setting of the nucleosome, and a possible role of the major bend as a factor, which defines the translational setting, is discussed.     

Electrostatic Interactions between Arginines and the Minor Groove in the Nucleosome

Abstract

Proteins rely on a variety of readout mechanisms to preferentially bind specific DNA sequences. The nucleosome offers a prominent example of a shape readout mechanism where arginines insert into narrow minor groove regions that face the histone core. Here we compare DNA shape and arginine recognition of three nucleosome core particle structures, expanding on our previous study by characterizing two additional structures, one with a different protein sequence and one with a different DNA sequence. The electrostatic potential in the minor groove is shown to be largely independent of the underlying sequence but is, however, dominated by groove geometry. Our results extend and generalize our previous observation that the interaction of arginines with narrow minor grooves plays an important role in stabilizing the deformed DNA in the nucleosome.     

Molecular Dynamics Simulations of a Nucleosome and Free DNA

Abstract

All atom molecular dynamics simulations (10ns) of a nucleosome and of its 146 basepairs of DNA free in solution have been conducted. DNA helical parameters (Roll, Tilt, Twist, Shift, Slide, Rise) were extracted from each trajectory to compare the conformation, effective force constants, persistence length measures, and fluctuations of nucleosomal DNA to free DNA. The conformation of DNA in the nucleosome, as determined by helical parameters, is found to be largely within the range of thermally accessible values obtained for free DNA. DNA is found to be less flexible on the nucleosome than when free in solution, however such measures are length scale dependent. A method for disassembling and reconstructing the conformation and dynamics of the nucleosome using Fourier analysis is presented. Long length variations in the conformation of nucleosomal DNA are identified other than those associated with helix repeat. These variations are required to create a proposed tetrasome conformation or to qualitatively reconstruct the 1.75 turns of the nucleosome's superhelix. Reconstruction of free DNA using selected long wavelength variations in conformation can produce either a left-handed or a right-handed superhelix. The long wavelength variations suggest 146 basepairs is a natural length of DNA to wrap around the histone core.     

Sequence determinants of the capping box, a stabilizing motif at the N-termini of alpha-helices.

The capping box, a recurrent hydrogen bonded motif at the N-termini of alpha-helices, caps 2 of the initial 4 backbone amide hydrogen donors of the helix (Harper ET, Rose GD, 1993, Biochemistry 32:7605-7609). In detail, the side chain of the first helical residue forms a hydrogen bond with the backbone of the fourth helical residue and, reciprocally, the side chain of the fourth residue forms a hydrogen bond with the backbone of the first residue. We now enlarge the earlier definition of this motif to include an accompanying hydrophobic interaction between residues that bracket the capping box sequence on either side. The expanded box motif--in which 2 hydrogen bonds and a hydrophobic interaction are localized within 6 consecutive residues--resembles a glycine-based capping motif found at helix C-termini (Aurora R, Srinivasan R, Rose GD, 1994, Science 264:1126-1130).     

Crystal Structures of Nucleosome Core Particles Containing the ‘601’ Strong Positioning Sequence

Nucleosome positioning plays a key role in genomic regulation by defining histone-DNA context and by modulating access to specific sites. Moreover, the histone-DNA register influences the double-helix structure, which in turn can affect the association of small molecules and protein factors. Analysis of genomic and synthetic DNA has revealed sequence motifs that direct nucleosome positioning in vitro; thus, establishing the basis for the DNA sequence dependence of positioning would shed light on the mechanics of the double helix and its contribution to chromatin structure in vivo. However, acquisition of well-diffracting nucleosome core particle (NCP) crystals is extremely dependent on the DNA fragment used for assembly, and all previous NCP crystal structures have been based on human α-satellite sequences. Here, we describe the crystal structures of Xenopus NCPs containing one of the strongest known histone octamer binding and positioning sequences, the so-called ‘601’ DNA.Two distinct 145-bp 601 crystal forms display the same histone-DNA register, which coincides with the occurrence of DNA stretching-overtwisting in both halves of the particle around five double-helical turns from the nucleosome center, giving the DNA an ‘effective length’ of 147 bp. As we have found previously with stretching around two turns from the nucleosome center for a centromere-based sequence, the terminal stretching observed in the 601 constructs is associated with extreme kinking into the minor groove at purine-purine (pyrimidine-pyrimidine) dinucleotide steps. In other contexts, these step types display an overall nonflexible behavior, which raises the possibility that DNA stretching in the nucleosome or extreme distortions in general have unique sequence dependency characteristics. Our findings indicate that DNA stretching is an intrinsically predisposed site-specific property of the nucleosome and suggest how NCP crystal structures with diverse DNA sequences can be obtained.     

核小体定位模式及其与DNA甲基化位点分布的关系

核小体定位是指DNA双螺旋相对于组蛋白八联体的位置.核小体定位通过限制蛋白结合位点参与基因转录调控.本文利用实验检测的人类CD4+ T细胞核小体定位数据,研究了核小体定位在转录因子结合位点(TFBS)和转录起始位点(TSS)附近的分布模式,并分析了在TFBS和TSS周围,核小体定位与DNA甲基化之间的关系.结果表明,在休眠和激活的人类CD4+ T细胞中,部分TFBS和TSS周围的核小体定位在动态改变,即在定位和缺失两种状态之间切换.在TFBS周围,核小体定位和DNA甲基化存在一种互补模式,核小体定位与DNA低甲基化相联系;而在TSS周围,两者呈现同步模式,DNA高甲基化伴随高核小体水平.而且,在TFBS和TSS周围,DNA甲基化位点的分布呈周期模式.CD4+ T细胞被激活时,较少的转录因子启动了较多的基因.     

From the Sequence to the Conformation of the Unabridged Transmembrane Domains TM1 and TM2 of the cASIC1a Ion Channel – A Parallel Tempering Approach

This work was devised to unravel, along replica‐exchange molecular‐dynamics (REMD) simulations, the conformation in solution of the TM1 and TM2 transmembrane domains of the homotrimeric cASIC1a ion channel. This includes the head of TM1 and tail of TM2 that had previously defied X‐ray diffraction analysis in the crystal. The structure of the open‐channel complex of cASIC1a with psalmotoxin 1 (PcTx1) was chosen here as a basis, although, to make the simulations affordable, the procedure was limited to the missing portions, including a few adjacent α‐helical turns. The latter were held fixed during the simulations. Reassembling the whole subunit, by superimposition of the fixed portions, resulted in diving of both TM1 and TM2 as continuous α‐helices into the cytoplasm. At completion of this work, it appeared, from similar X‐ray diffraction studies, that TM2 for both the complex of cASIC1a with the coral snake MitTx toxin, and the isolated desensitized ion channel, is discontinuous, with the triad G443‐A444‐S445 taking an extended, belt‐like conformation. In this way, a filter ring against hydrated ions is formed by G443 in the trimer. Our REMD examination of this complex revealed a strong resistance by G443, and only that residue, to take dihedral‐angle values compatible with an α‐helical conformation. This suggests that the flexibility of glycine alone does not explain formation of the extended, belt‐like conformation of the triad G443‐A444‐S445. This also requires cooperation in the trimer.     

Design of Sequence-Specific DNA Binding Ligands That Use a Two-Stranded Peptide Motif for DNA Sequence Recognition

Abstract

The design and DNA binding activity of β-structure-forming peptides and netropsin-peptide conjugates are reported. It is found that a pair of peptides - S,S'-bis(Lys-Gly-Val-Cys-Val- NH-NH-Dns) - bridged by an S-S bond binds at least 10 times more strongly to poly(dG)?poly(dC) than to poly(dA)?poly(dT). This peptide can also discriminate between 5′-GpG-3′ and 5′-GpC-3′ steps in the DNA minor groove. Based on these observations, new synthetic ligands, bis-netropsins, were constructed in which two netropsin-like fragments were attached by means of short linkers to a pair of peptides - Gly-Cys-Gly- or Val-Cys-Val - bridged by S-S bonds. These compounds possess a composite binding specificity: the peptide chains recognize 5′-GpG-3′ steps on DNA, whereas the netropsin-like fragments bind preferentially to tuns of 4 AT base pairs. Our data indicate that combining the AT-base-pair specific properties of the netropsin-type structure with the 5′-GpG-3′-specific properties of certain oligopeptides offers a new approach to the synthesis of ligands capable of recognizing mixed sequences of AT- and GC-base pairs in the DNA minor groove. These compounds are potential models for DNA-binding domains in proteins which specifically recognize base pair sequences in the minor groove of DNA.     

Studying the Structural and Folding Features of Long‐Sequence Trichobrachin Peptides

In this theoretical study, the folding processes of long‐sequence trichobrachin peptides (i.e., TB IIb peptides) were investigated by molecular dynamics methods. The formation of various helical structures (i.e., 3₁₀‐, α‐, and left‐handed α‐helices) was studied with regard to the entire sequence of peptides, as well as to each amino acid. The results pointed out that TB IIb molecules showed a propensity to form helical conformations, and they could be characterized by 3₁₀‐helical structure rather than by α‐helical structure. The formation of local (i.e., i←i+3 and i←i+4) as well as of non‐local (i.e., i←i+n, where n>4; and all i→i+n) H‐bonds was also examined. The results revealed that the occurrence of local, helix‐stabilizing H‐bonds was in agreement with the appearance of helical conformations, and the non‐local H‐bonds did not produce relevant effects on the evolution of helical structures. Based on the data obtained by our structural investigation, differences were observed between the TB IIb peptides, according to the type of amino acid located in the 17th position of their sequences. In summary, the folding processes were explored for TB IIb molecules, and our theoretical study led to the conclusion that these long‐sequence peptaibols showed characteristic structural and folding features.