Nucleosome positioning on yeast plasmids is determined only by the internal signal of DNA sequence occupied by the nucleosome

The possible role of border factors in determining the nucleosome positioning on a DNA sequence was investigated. To this end a family of recombinant plasmids based on Gal10Cyc1 promoter and neomycin phosphotransferase gene NPTII were created. A DNA sequence adjoining the GalCyc promoter was varied in these plasmids. Three nearly equally represented nucleosome positions on the GalCyc promoter were found. In the basal plasmid an FRT sequence adjoins the GalCyc promoter at the right. It contains an internal signal of multiple positioning. Its replacement with different DNA sequences does not affect nucleosome positioning on the GalCyc promoter. The nucleosome positioning on the GalCyc promoter does not depend on nucleosome positioning (or its absence) on adjoining sequences. The same is true for nucleosome positioning on FRT sequence. It was found also that nucleosomes' positioning on the NPTII gene and their mutual disposition, namely the spacing between neighboring nucleosomes (linker length) are determined by the location of positioning signals only. Generally the nucleosome positioning in our experimental model is determined solely by internal DNA sequence occupied by nucleosome. On the other hand, the action of this internal positioning signal does not extend to neighboring DNA sequences.     

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.     

Multiple Aspects of ATP-Dependent Nucleosome Translocation by RSC and Mi-2 Are Directed by the Underlying DNA Sequence

Background

Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the nucleosome positions can be predicted simply by DNA sequence, especially within promoter regions. This seemingly contrasts with remodeler induced nucleosome mobility. The ability of remodeling enzymes to mobilise nucleosomes over short DNA distances is well documented. However, the nucleosome translocation processivity along DNA remains elusive. Furthermore, it is unknown what determines the initial direction of movement and how new nucleosome positions are adopted.

Methodology/Principal Findings

We have used AFM imaging and high resolution PAGE of mononucleosomes on 600 and 2500 bp DNA molecules to analyze ATP-dependent nucleosome repositioning by native and recombinant SNF2-type enzymes. We report that the underlying DNA sequence can control the initial direction of translocation, translocation distance, as well as the new positions adopted by nucleosomes upon enzymatic mobilization. Within a strong nucleosomal positioning sequence both recombinant Drosophila Mi-2 (CHD-type) and native RSC from yeast (SWI/SNF-type) repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalyzed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps.

Conclusions/Significance

Nucleosome repositioning thus appears to be influenced by both remodeler intrinsic and DNA sequence specific properties that interplay to define ATPase-catalyzed repositioning. Here we propose a successive three-step framework consisting of initiation, translocation and release steps to describe SNF2-type enzyme mediated nucleosome translocation along DNA. This conceptual framework helps resolve the apparent paradox between the high abundance of ATP-dependent remodelers per nucleus and the relative success of sequence-based predictions of nucleosome positioning in vivo.     

Sequence-dependent Nucleosome Positioning

Eukaryotic DNA is organized into a macromolecular structure called chromatin. The basic repeating unit of chromatin is the nucleosome, which consists of two copies of each of the four core histones and DNA. The nucleosomal organization and the positions of nucleosomes have profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is therefore of general interest. Among the many determinants of nucleosome positioning, the DNA sequence has been proposed to have a major role. Here, we analyzed more than 860,000 nucleosomal DNA sequences to identify sequence features that guide the formation of nucleosomes in vivo. We found that both a periodic enrichment of AT base pairs and an out-of-phase oscillating enrichment of GC base pairs as well as the overall preference for GC base pairs are determinants of nucleosome positioning. The preference for GC pairs can be related to a lower energetic cost required for deformation of the DNA to wrap around the histones. In line with this idea, we found that only incorporation of both signal components into a sequence model for nucleosome formation results in maximal predictive performance on a genome-wide scale. In this manner, one achieves greater predictive power than published approaches. Our results confirm the hypothesis that the DNA sequence has a major role in nucleosome positioning in vivo.     

Nucleosome positioning in the <Emphasis Type="Italic">NPTII</Emphasis> neomycin phosphotransferase gene on a yeast plasmid in the repressed and active states

A yeast plasmid was constructed to contain a hybrid GAL-CYC promoter, the NPTII neomycin phosphotransferase gene, and the FRT sequence between them. The CYC part of the GAL-CYC promoter harbored four upstream activating sequences (UASs) and two close TATA boxes. NPTII was efficiently expressed upon induction with galactose, conferring G418 resistance on yeast cells. Nucleosome positioning was studied in repressed and induced NPTII in transformed cells. A stable positioning of three nucleosomes was detected under repressive conditions (growth on glucose). Two nucleosomes were on the CYC part of the promoter, one including both of the TATA boxes. The third nucleosome overlapped the FRT sequence and the start of the NPTII coding region. Each of the three nucleosomes displayed multiple positions, suggesting their sliding along DNA. After induction of NPTII expression with galactose, a sliding of two nucleosomes was detected, exposing the TATA box and a long promoter segment. The 5′-distal nucleosome moved closer to the UASs, bringing them closer to the TATA box, which was assumed to facilitate the assembly of the preinitiation complex. The two nucleosomes slid independently of each other. The second nucleosome moved towards the FRT sequence and repositioned at its nucleosome positioning signal. Galactose-induced expression did not affect the nucleosome positioning in the coding region of NPTII. Unidirectional sliding and repositioning were detected without induction after deacetylase inhibition with trichostatin A. Basal NPTII expression was observed without activation of the GAL-CYC promoter and after a spatial uncoupling of the coding sequence and promoter via gene inversion and was probably driven by the FRT TATA-like element, which is in the region permanently exposed in vivo.     

Nucleosome positioning and periodicity of satellite DNA in the liver of aging rats – Nucleosome positioning and periodicity of satellite DNA

The positioning of nucleosomes has been analysed by comparing the pattern of cutting sites of a probing reagent on chromatin and naked DNA. For this purpose, high molecular weight DNA and nuclei from the liver of young (18±2 weeks) and old (100±5 weeks) Wistar male rats were digested with micrococcal nuclease (MNase) and hybridized with ³²P-labelled rat satellite DNA probe. A comparison of the ladder generated by MNase with chromatin and nuclei indicates long range organization of the satellite chromatin fiber with distinct non-random positioning of nucleosomes. However, the positioning of nucleosomes on satellite DNA does not vary with age. For studying the periodicity and subunit structure of satellite DNA, high molecular weight DNA from the liver of young and old rats were digested with different restriction enzymes. Surprisingly, no noteworthy age-related change is visible in the periodicity and subunit structural organization of the satellite DNA. These results suggest that the nucleosome positioning and the periodicity of liver satellite DNA do not vary with age.     

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.     

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.     

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.     

The Molecular Evolution of Nucleosome Positioning Through Sequence-Dependent Deformation of the DNA Polymer

Abstract

The computational prediction of nucleosome positioning from DNA sequence now allows for in silico investigation of the molecular evolution of biophysical properties of the DNA molecule responsible for primary chromatin organization in the genome. To discern what signal components driving nucleosome positioning in the yeast genome are potentially targeted by natural selection, we compare the performance of various models predictive of nucleosome positioning within the context of a simple statistical test, the repositioned mutation test. We demonstrate that while nucleosome occupancy is driven largely by translational exclusion in response to AT content, there is also a strong signature of evolutionary conservation of regular patterns within nucleosomal DNA sequence related to the structural organization of the nucleosome core (e.g., 10-bp dinucleotide periodicity). We also use computer simulations to investigate hypothetical coding and regulatory constraints on the ability of sequence properties affecting nucleosome formation to adaptively evolve. Our results demonstrate that natural selection may act independently on different DNA sequence properties responsible for local chromatin organization. Furthermore, at least with respect to the deformation energy of the DNA molecule in the nucleosome, the presence of the genetic code has greatly restricted the ability of sequences to evolve the dynamic nucleosome organization typically observed in promoter regions.     

Controls of Nucleosome Positioning in the Human Genome

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.     

DNA sequence plays a major role in determining nucleosome positions in yeast CUP1 chromatin.

The role of DNA sequence in determining nucleosome positions in vivo was investigated by comparing the positions adopted by nucleosomes reconstituted on a yeast plasmid in vitro using purified core histones with those in native chromatin containing the same DNA, described previously. Nucleosomes were reconstituted on a 2.5 kilobase pair DNA sequence containing the yeast TRP1ARS1 plasmid with CUP1 as an insert (TAC-DNA). Multiple, alternative, overlapping nucleosome positions were mapped on TAC-DNA. For the 58 positioned nucleosomes identified, the relative positioning strengths and the stabilities to salt and temperature were determined. These positions were, with a few exceptions, identical to those observed in native, remodeled TAC chromatin containing an activated CUP1 gene. Only some of these positions are utilized in native, unremodeled chromatin. These observations suggest that DNA sequence is likely to play a very important role in positioning nucleosomes in vivo. We suggest that events occurring in yeast CUP1 chromatin determine which positions are occupied in vivo and when they are occupied.     

Nucleosome organization on Kluyveromyces lactis centromeric DNAs

The preferential assembly of specialized nucleosomes on budding yeast centromeres can be due either to the higher stability of specialized centromeric nucleosomes and/or to the lower stability of canonical centromeric nucleosomes with respect to bulk nucleosomes. We have evaluated the thermodynamic stability of canonical nucleosomes, assembled on Kluyveromyces lactis centromeric DNAs, with a competitive reconstitution assay and a theoretical method recently developed by us. The results, obtained by both methods, show that all five known centromeric DNAs from K. lactis are able to organize canonical nucleosomes, characterized by higher stability with respect those of bulk DNA. With 'footprinting' and theoretical prediction, based on sequence-dependent DNA elasticity, we have found that centromeric canonical nucleosomes are characterized by nucleosome dyad axis multiple positioning, rotationally phased. The isoenergetic nucleosome multiple positions are relevant in understanding the transition from canonical to specialized nucleosomes in interacting with centromere protein complexes. The satisfactory agreement between the results obtained from theoretical and experimental methods shows that sequence-dependent centromeric DNA elasticity has a main role in nucleosome thermodynamic stability and positioning.     

Oligonucleotide Sequence Motifs as Nucleosome Positioning Signals

To gain a better understanding of the sequence patterns that characterize positioned nucleosomes, we first performed an analysis of the periodicities of the 256 tetranucleotides in a yeast genome-wide library of nucleosomal DNA sequences that was prepared by in vitro reconstitution. The approach entailed the identification and analysis of 24 unique tetranucleotides that were defined by 8 consensus sequences. These consensus sequences were shown to be responsible for most if not all of the tetranucleotide and dinucleotide periodicities displayed by the entire library, demonstrating that the periodicities of dinucleotides that characterize the yeast genome are, in actuality, due primarily to the 8 consensus sequences. A novel combination of experimental and bioinformatic approaches was then used to show that these tetranucleotides are important for preferred formation of nucleosomes at specific sites along DNA in vitro. These results were then compared to tetranucleotide patterns in genome-wide in vivo libraries from yeast and C. elegans in order to assess the contributions of DNA sequence in the control of nucleosome residency in the cell. These comparisons revealed striking similarities in the tetranucleotide occurrence profiles that are likely to be involved in nucleosome positioning in both in vitro and in vivo libraries, suggesting that DNA sequence is an important factor in the control of nucleosome placement in vivo. However, the strengths of the tetranucleotide periodicities were 3–4 fold higher in the in vitro as compared to the in vivo libraries, which implies that DNA sequence plays less of a role in dictating nucleosome positions in vivo. The results of this study have important implications for models of sequence-dependent positioning since they suggest that a defined subset of tetranucleotides is involved in preferred nucleosome occupancy and that these tetranucleotides are the major source of the dinucleotide periodicities that are characteristic of positioned nucleosomes.     

Painting a Perspective on the Landscape of Nucleosome Positioning

Abstract

DNA sequence influences the position of nucleosomes and chromatin architecture. The extent to which underlying DNA sequence affects nucleosome positioning is currently a topic of considerable discussion and active experimentation. To contribute to the discussion, I will outline a few of the methods, data and arguments that I find compelling and believe will ultimately resolve the question of what positions nucleosomes. Basically, I will give a portrait of my current perspective on what influences the landscape of nucleosome positioning and chromatin architecture.