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1.  Nucleosome Free Regions in Yeast Promoters Result from Competitive Binding of Transcription Factors That Interact with Chromatin Modifiers 
PLoS Computational Biology  2013;9(8):e1003181.
Because DNA packaging in nucleosomes modulates its accessibility to transcription factors (TFs), unraveling the causal determinants of nucleosome positioning is of great importance to understanding gene regulation. Although there is evidence that intrinsic sequence specificity contributes to nucleosome positioning, the extent to which other factors contribute to nucleosome positioning is currently highly debated. Here we obtained both in vivo and in vitro reference maps of positions that are either consistently covered or free of nucleosomes across multiple experimental data-sets in Saccharomyces cerevisiae. We then systematically quantified the contribution of TF binding to nucleosome positiong using a rigorous statistical mechanics model in which TFs compete with nucleosomes for binding DNA. Our results reconcile previous seemingly conflicting results on the determinants of nucleosome positioning and provide a quantitative explanation for the difference between in vivo and in vitro positioning. On a genome-wide scale, nucleosome positioning is dominated by the phasing of nucleosome arrays over gene bodies, and their positioning is mainly determined by the intrinsic sequence preferences of nucleosomes. In contrast, larger nucleosome free regions in promoters, which likely have a much more significant impact on gene expression, are determined mainly by TF binding. Interestingly, of the 158 yeast TFs included in our modeling, we find that only 10–20 significantly contribute to inducing nucleosome-free regions, and these TFs are highly enriched for having direct interations with chromatin remodelers. Together our results imply that nucleosome free regions in yeast promoters results from the binding of a specific class of TFs that recruit chromatin remodelers.
Author Summary
The DNA of all eukaryotic organisms is packaged into nucleosomes, which cover roughly of the genome. As nucleosome positioning profoundly affects DNA accessibility to other DNA binding proteins such as transcription factors (TFs), it plays an important role in transcription regulation. However, to what extent nucleosome positioning is guided by intrinsic DNA sequence preferences of nucleosomes, and to what extent other DNA binding factors play a role, is currently highly debated. Here we use a rigorous biophysical model to systematically study the relative contributions of intrinsic sequence preferences and competitive binding of TFs to nucleosome positioning in yeast. We find that, on the one hand, the phasing of the many small spacers within dense nucleosome arrays that cover gene bodies are mainly determined by intrinsic sequence preferences. On the other hand, larger nucleosome free regions (NFRs) in promoters are explained predominantly by TF binding. Strikingly, we find that only 10–20 TFs make a significant contribution to explaining NFRs, and these TFs are highly enriched for directly interacting with chromatin modifiers. Thus, the picture that emerges is that binding by a specific class of TFs recruits chromatin modifiers which mediate local nucleosome expulsion.
PMCID: PMC3749953  PMID: 23990766
2.  Quantitative Test of the Barrier Nucleosome Model for Statistical Positioning of Nucleosomes Up- and Downstream of Transcription Start Sites 
PLoS Computational Biology  2010;6(8):e1000891.
The positions of nucleosomes in eukaryotic genomes determine which parts of the DNA sequence are readily accessible for regulatory proteins and which are not. Genome-wide maps of nucleosome positions have revealed a salient pattern around transcription start sites, involving a nucleosome-free region (NFR) flanked by a pronounced periodic pattern in the average nucleosome density. While the periodic pattern clearly reflects well-positioned nucleosomes, the positioning mechanism is less clear. A recent experimental study by Mavrich et al. argued that the pattern observed in Saccharomyces cerevisiae is qualitatively consistent with a “barrier nucleosome model,” in which the oscillatory pattern is created by the statistical positioning mechanism of Kornberg and Stryer. On the other hand, there is clear evidence for intrinsic sequence preferences of nucleosomes, and it is unclear to what extent these sequence preferences affect the observed pattern. To test the barrier nucleosome model, we quantitatively analyze yeast nucleosome positioning data both up- and downstream from NFRs. Our analysis is based on the Tonks model of statistical physics which quantifies the interplay between the excluded-volume interaction of nucleosomes and their positional entropy. We find that although the typical patterns on the two sides of the NFR are different, they are both quantitatively described by the same physical model with the same parameters, but different boundary conditions. The inferred boundary conditions suggest that the first nucleosome downstream from the NFR (the +1 nucleosome) is typically directly positioned while the first nucleosome upstream is statistically positioned via a nucleosome-repelling DNA region. These boundary conditions, which can be locally encoded into the genome sequence, significantly shape the statistical distribution of nucleosomes over a range of up to ∼1,000 bp to each side.
Author Summary
Within the last five years, knowledge about nucleosome organization on the genome has grown dramatically. To a large extent, this has been achieved by an increasing number of experimental studies determining nucleosome positions at high resolution over entire genomes. Particular attention has been paid to promoter regions, where a canonical pattern has been established: a nucleosome-free region with pronounced adjacent oscillations in the nucleosome density. Here we tested to what extent this pattern may be quantitatively described by a minimal physical model, a one-dimensional gas of impenetrable particles, commonly referred to as the “Tonks gas.” In this model, density oscillations occur close to a boundary at dense packing. Our systematic quantitative analysis reveals that, in an average over many promoters, a Tonks gas model can indeed account for the nucleosome organization to both sides of the nucleosome-free region, if one allows for different boundary conditions at the two edges. On the downstream side, a single nucleosome is typically directly positioned such that it forms an obstacle for the neighboring nucleosomes, while such a barrier nucleosome is typically missing on the upstream side.
PMCID: PMC2924246  PMID: 20808881
3.  Predicting Human Nucleosome Occupancy from Primary Sequence 
PLoS Computational Biology  2008;4(8):e1000134.
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.
Author Summary
Inside the nucleus, DNA is wrapped into a complex molecular structure called chromatin, whose fundamental unit is ∼150 bp of DNA organized around the eight-histone protein complex known as the nucleosome. Understanding the local organization of nucleosomes is critical for understanding how chromatin impacts gene regulation. Here, we describe a computational model that predicts nucleosome placement from DNA sequence. We train the model using data derived from human cell lines, and we apply the model systematically to 1% of the human genome. We show that previously described models trained from yeast data correlate strongly with the human-trained model, suggesting a common mechanism for sequence-based determination of nucleosome occupancy. In addition, we observe a striking complementarity between models trained using data from weakly and strongly digested samples: one type of model recognizes nucleosome-free regions, whereas the other identifies well-positioned nucleosomes. Finally, our analysis of predicted nucleosome positions in the human genome allows us to identify common features of nucleosome-forming and inhibitory sequences. Overall, our results are consistent with the classical statistical positioning theory of nucleosome organization.
PMCID: PMC2515632  PMID: 18725940
4.  Diversity of Eukaryotic DNA Replication Origins Revealed by Genome-Wide Analysis of Chromatin Structure 
PLoS Genetics  2010;6(9):e1001092.
Eukaryotic DNA replication origins differ both in their efficiency and in the characteristic time during S phase when they become active. The biological basis for these differences remains unknown, but they could be a consequence of chromatin structure. The availability of genome-wide maps of nucleosome positions has led to an explosion of information about how nucleosomes are assembled at transcription start sites, but no similar maps exist for DNA replication origins. Here we combine high-resolution genome-wide nucleosome maps with comprehensive annotations of DNA replication origins to identify patterns of nucleosome occupancy at eukaryotic replication origins. On average, replication origins contain a nucleosome depleted region centered next to the ACS element, flanked on both sides by arrays of well-positioned nucleosomes. Our analysis identified DNA sequence properties that correlate with nucleosome occupancy at replication origins genome-wide and that are correlated with the nucleosome-depleted region. Clustering analysis of all annotated replication origins revealed a surprising diversity of nucleosome occupancy patterns. We provide evidence that the origin recognition complex, which binds to the origin, acts as a barrier element to position and phase nucleosomes on both sides of the origin. Finally, analysis of chromatin reconstituted in vitro reveals that origins are inherently nucleosome depleted. Together our data provide a comprehensive, genome-wide view of chromatin structure at replication origins and suggest a model of nucleosome positioning at replication origins in which the underlying sequence occludes nucleosomes to permit binding of the origin recognition complex, which then (likely in concert with nucleosome modifiers and remodelers) positions nucleosomes adjacent to the origin to promote replication origin function.
Author Summary
Eukaryotic DNA replication begins at specific sites in the genome called replication origins, which are bound by the proteins that comprise the origin recognition complex (ORC). In budding yeast, there are more replication origins available than are used in any particular cell division cycle. Each origin has a characteristic time during the cell division cycle when the DNA replication machinery is assembled at a particular origin and begins to replicate DNA. Previous studies have indicated that differences in replication timing and origin use/availability may be a consequence of the chromatin structure surrounding an origin. Here we present a genome-wide analysis of nucleosome architecture of replication origins aligned by their ORC-binding site. We find that origins can be built with a variety of nucleosome occupancy patterns, and that these patterns are influenced by adjacent genomic features. Finally, we determined the genome-wide consequences of ORC depletion on nucleosome architecture at origins. ORC depletion allowed encroachment of flanking nucleosomes towards the origin and changed the nucleosome phasing, indicating that ORC acts as a barrier to position and phase nucleosomes. Our analysis provides a comprehensive, genome-wide view of replication origins that reveals a previously unappreciated diversity in origin structure.
PMCID: PMC2932696  PMID: 20824081
5.  Divergence of nucleosome positioning between two closely related yeast species: genetic basis and functional consequences 
Inter-species hybrids can be used to dissect the relative contribution of cis and trans effects to the evolution of nucleosome positioning. Most (∼70%) differences in nucleosome positioning between two closely related yeast species are due to cis effects.Cis effects are primarily due to divergence of AT-rich nucleosome-disfavoring sequences, but are not associated with divergence of nucleosome-favoring sequences.Differences in nucleosome positioning propagate to multiple adjacent nucleosomes, supporting the statistical positioning hypothesis.Divergence of nucleosome positioning is excluded from regulatory elements and is not correlated with gene expression divergence, suggesting a neutral mode of evolution.
Phenotypic diversity is often due to changes in gene regulation, and recent studies have characterized extensive differences between the gene expression programs of closely related species (Khaitovich et al, 2006; Tirosh et al, 2009). However, very little is known about the mechanisms that drive this divergence. Here, we analyze the evolution of nucleosome positioning, by comparing the patterns of nucleosomes between two yeast species, as well as generating the allele-specific nucleosome profile in their hybrid. We ask two main questions: (1) what is the genetic basis of inter-species differences in nucleosome positioning? and (2) what is the regulatory function of these differences?
Generally speaking, we can classify the genetic basis of the divergence in nucleosome positioning into two mechanisms. First, mutations in the local DNA sequence may influence the ability to bind nucleosomes at this region; we refer to these as cis effects. Second, mutations may affect the activity of various proteins that alter nucleosome positioning either actively (e.g. chromatin-remodeling enzymes) or by simply competing with nucleosomes for binding to the same DNA sequence (e.g. transcription factors); we refer to these as trans effects.
To classify the observed inter-species differences into cis versus trans effects, we measured allele-specific nucleosome positions within the inter-specific hybrid of the two species (Wittkopp et al, 2004; Tirosh et al, 2009). The hybrid contains the alleles of both species; hence, cis effects, which involve mutations that discriminate between the two alleles, will be maintained in the hybrid so that nucleosome positioning will be different between the alleles coming from the different species. Trans effects, in contrast, will not discriminate between the two hybrid alleles from the different species, as these two alleles reside together at the same trans environment (hybrid nucleus) and are thus regulated by the same set of proteins—the combination of proteins from the two species. Using this approach, we found that ∼70% of the inter-species differences in nucleosome positioning are due to cis effects, whereas the rest is due to trans effects.
The local DNA sequence is indeed known to affect nucleosome positions, and many features of DNA sequences were proposed to influence nucleosome binding, either by rejecting nucleosomes, or by being favorable for nucleosome binding (Segal et al, 2006; Lee et al, 2007; Kaplan et al, 2009). We find, however, that nucleosome positions diverged primarily through changes in AT-rich sequences, which exclude nucleosomes, whereas mutations in sequences that correlate with high-nucleosome occupancy do not influence inter-species divergence.
Nucleosomes restrict the access of proteins to the DNA and may thus affect DNA-related processes such as transcription, recombination or replication. Indeed, promoters and regulatory sequences are often depleted of nucleosomes, and highly transcribed genes are associated with low occupancy of nucleosomes at their promoters (Lee et al, 2007). Several earlier studies also suggested that evolutionary divergence of gene expression is driven by changes in chromatin structure (Lee et al, 2006; Choi and Kim, 2008; Tirosh et al, 2008; Field et al, 2009). However, we find that nucleosome positions (or occupancy) at regulatory elements are largely conserved, and furthermore, that the inter-species differences in nucleosome positions do not correlate with gene expression differences. These results suggest that nucleosome positioning is not a central mechanism for evolutionary changes in gene regulation and that most of the observed changes may be due to neutral drift.
Does the apparent low influence of nucleosome positioning on gene expression divergence implies that nucleosome positions do not have a function in gene regulation? To address this, we examined two additional modes of gene regulation: transcriptional response to changes in growth conditions (glucose versus glycerol media), and the expression differences between different cell types (haploid versus diploid cells). Consistent with earlier studies, we found that the response to growth conditions is significantly, albeit weakly, associated with changes in nucleosome positioning. Interestingly, we also found a strikingly strong association between gene expression and nucleosomal changes in the two cell types. Taken together, these results suggest that nucleosome positioning is used preferentially for biological processes in which genes are turned on and off (e.g. different cell type), but less so during divergence of closely related species in which gradual changes accumulate over time.
Gene regulation differs greatly between related species, constituting a major source of phenotypic diversity. Recent studies characterized extensive differences in the gene expression programs of closely related species. In contrast, virtually nothing is known about the evolution of chromatin structure and how it influences the divergence of gene expression. Here, we compare the genome-wide nucleosome positioning of two closely related yeast species and, by profiling their inter-specific hybrid, trace the genetic basis of the observed differences into mutations affecting the local DNA sequences (cis effects) or the upstream regulators (trans effects). The majority (∼70%) of inter-species differences is due to cis effects, leaving a significant contribution (30%) for trans factors. We show that cis effects are well explained by mutations in nucleosome-disfavoring AT-rich sequences, but are not associated with divergence of nucleosome-favoring sequences. Differences in nucleosome positioning propagate to multiple adjacent nucleosomes, supporting the statistical positioning hypothesis, and we provide evidence that nucleosome-free regions, but not the +1 nucleosome, serve as stable border elements. Surprisingly, although we find that differential nucleosome positioning among cell types is strongly correlated with differential expression, this does not seem to be the case for evolutionary changes: divergence of nucleosome positioning is excluded from regulatory elements and is not correlated with gene expression divergence, suggesting a primarily neutral mode of evolution. Our results provide evolutionary insights to the genetic determinants and regulatory function of nucleosome positioning.
PMCID: PMC2890324  PMID: 20461072
evolution; gene regulation; nucleosome positioning
6.  A unique nucleosome arrangement, maintained actively by chromatin remodelers facilitates transcription of yeast tRNA genes 
BMC Genomics  2013;14:402.
RNA polymerase (pol) III transcribes a unique class of genes with intra-genic promoters and high transcriptional activity. The major contributors to the pol III transcriptome, tRNAs genes are found scattered on all chromosomes of yeast. A prototype tDNA of <150 bp length, is generally considered nucleosome-free while some pol III-transcribed genes have been shown to have nucleosome-positioning properties.
Using high resolution ChIP-chip and ChIP-seq methods, we found several unique features associated with nucleosome profiles on all tRNA genes of budding yeast, not seen on nucleosome-dense counterparts in fission yeast and resting human CD4+ T cells. The nucleosome-free region (NFR) on all but three yeast tDNAs is found bordered by an upstream (US) nucleosome strongly positioned at −140 bp position and a downstream (DS) nucleosome at variable positions with respect to the gene terminator. Perturbation in this nucleosomal arrangement interferes with the tRNA production. Three different chromatin remodelers generate and maintain the NFR by targeting different gene regions. Isw1 localizes to the gene body and makes it nucleosome-depleted, Isw2 maintains periodicity in the upstream nucleosomal array, while RSC targets the downstream nucleosome. Direct communication of pol III with RSC serves as a stress-sensory mechanism for these genes. In its absence, the downstream nucleosome moves towards the gene terminator. Levels of tRNAs from different families are found to vary considerably as different pol III levels are seen even on isogenes within a family. Pol III levels show negative correlation with the nucleosome occupancies on different genes.
Budding yeast tRNA genes maintain an open chromatin structure, which is not due to sequence-directed nucleosome positioning or high transcription activity of genes. Unlike 5′ NFR on pol II-transcribed genes, the tDNA NFR, which facilitates tDNA transcription, results from action of chromatin remodeler Isw1, aided by Isw2 and RSC. The RSC-regulated nucleosome dynamics at the 3′ gene-end serves as a novel regulatory mechanism for pol III transcription in vivo, probably by controlling terminator-dependent facilitated recycling of pol III. Salient features of yeast tDNA chromatin structure reported in this study can explain the basis of the novel non-transcriptional roles ascribed to tDNAs.
PMCID: PMC3698015  PMID: 23767421
Chromatin; Isw1; Isw2; Nucleosomes; RNA polymerase III; RSC; Transcription; Yeast tRNA
7.  Genomic Sequence Is Highly Predictive of Local Nucleosome Depletion 
PLoS Computational Biology  2008;4(1):e13.
The regulation of DNA accessibility through nucleosome positioning is important for transcription control. Computational models have been developed to predict genome-wide nucleosome positions from DNA sequences, but these models consider only nucleosome sequences, which may have limited their power. We developed a statistical multi-resolution approach to identify a sequence signature, called the N-score, that distinguishes nucleosome binding DNA from non-nucleosome DNA. This new approach has significantly improved the prediction accuracy. The sequence information is highly predictive for local nucleosome enrichment or depletion, whereas predictions of the exact positions are only modestly more accurate than a null model, suggesting the importance of other regulatory factors in fine-tuning the nucleosome positions. The N-score in promoter regions is negatively correlated with gene expression levels. Regulatory elements are enriched in low N-score regions. While our model is derived from yeast data, the N-score pattern computed from this model agrees well with recent high-resolution protein-binding data in human.
Author Summary
A eukaryotic genome is packaged into chromatin. The chromatin not only makes it possible to fit the relatively long genome into a tiny nucleus, but also plays an important regulatory role. The nucleosome is the fundamental repeating unit of chromatin. High-resolution tiling array experiments have shown that many nucleosomes are well-positioned in vivo, consistent with an important regulatory role. However, the mechanisms that determine nucleosome positioning are still poorly understood. We have developed a novel computational method for predicting nucleosome positions using only the genomic sequence information. The method detects periodic sequence signatures that discriminate nucleosome sequences from linker sequences. We show that this approach has significantly improved predictive power compared to previous studies. Interestingly, the most predictable regions tend to be located where stringent regulations are needed, i.e., the neighborhood of a transcription start site. This model predicts that nucleosome occupancy is not strongly controlled by short DNA sequence motifs but rather progressively controlled by regular organization of short elements into periodic patterns. We also provide evidence that sequence specificity for nucleosome binding is conserved from yeast to human.
PMCID: PMC2211532  PMID: 18225943
8.  The Insulator Binding Protein CTCF Positions 20 Nucleosomes around Its Binding Sites across the Human Genome 
PLoS Genetics  2008;4(7):e1000138.
Chromatin structure plays an important role in modulating the accessibility of genomic DNA to regulatory proteins in eukaryotic cells. We performed an integrative analysis on dozens of recent datasets generated by deep-sequencing and high-density tiling arrays, and we discovered an array of well-positioned nucleosomes flanking sites occupied by the insulator binding protein CTCF across the human genome. These nucleosomes are highly enriched for the histone variant H2A.Z and 11 histone modifications. The distances between the center positions of the neighboring nucleosomes are largely invariant, and we estimate them to be 185 bp on average. Surprisingly, subsets of nucleosomes that are enriched in different histone modifications vary greatly in the lengths of DNA protected from micrococcal nuclease cleavage (106–164 bp). The nucleosomes enriched in those histone modifications previously implicated to be correlated with active transcription tend to contain less protected DNA, indicating that these modifications are correlated with greater DNA accessibility. Another striking result obtained from our analysis is that nucleosomes flanking CTCF sites are much better positioned than those downstream of transcription start sites, the only genomic feature previously known to position nucleosomes genome-wide. This nucleosome-positioning phenomenon is not observed for other transcriptional factors for which we had genome-wide binding data. We suggest that binding of CTCF provides an anchor point for positioning nucleosomes, and chromatin remodeling is an important component of CTCF function.
Author Summary
The accessibility of genomic DNA to regulatory proteins and to the transcriptional machinery plays an important role in eukaryotic transcription regulation. Some regulatory proteins alter chromatin structures by evicting histones in selected loci. Nonetheless, no regulatory proteins have been reported to position nucleosomes genome-wide. The only genomic landmark that has been associated with well-positioned nucleosomes is the transcriptional start site (TSS)—several well-positioned nucleosomes are observed downstream of TSS genome-wide. Here we report that the CCCTC-binding factor (CTCF), a protein that binds insulator elements to prevent the spreading of heterochromatin and restricting transcriptional enhancers from activating unrelated promoters, possesses greater ability to position nucleosomes across the human genome than does the TSS. These well-positioned nucleosomes are highly enriched in a histone variant H2A.Z and 11 histone modifications. The nucleosomes enriched in the histone modifications previously implicated to correlate with active transcription tend to have less protected DNA against digestion by micrococcal nuclease, or greater DNA accessibility. This nucleosome-positioning ability is likely unique to CTCF, because it was not found in the other transcriptional factors we investigated. Thus we suggest that the binding of CTCF provides an anchor for positioning nucleosomes, and chromatin remodeling is an important aspect of CTCF function.
PMCID: PMC2453330  PMID: 18654629
9.  Distinct Modes of Regulation by Chromatin Encoded through Nucleosome Positioning Signals 
PLoS Computational Biology  2008;4(11):e1000216.
The detailed positions of nucleosomes profoundly impact gene regulation and are partly encoded by the genomic DNA sequence. However, less is known about the functional consequences of this encoding. Here, we address this question using a genome-wide map of ∼380,000 yeast nucleosomes that we sequenced in their entirety. Utilizing the high resolution of our map, we refine our understanding of how nucleosome organizations are encoded by the DNA sequence and demonstrate that the genomic sequence is highly predictive of the in vivo nucleosome organization, even across new nucleosome-bound sequences that we isolated from fly and human. We find that Poly(dA:dT) tracts are an important component of these nucleosome positioning signals and that their nucleosome-disfavoring action results in large nucleosome depletion over them and over their flanking regions and enhances the accessibility of transcription factors to their cognate sites. Our results suggest that the yeast genome may utilize these nucleosome positioning signals to regulate gene expression with different transcriptional noise and activation kinetics and DNA replication with different origin efficiency. These distinct functions may be achieved by encoding both relatively closed (nucleosome-covered) chromatin organizations over some factor binding sites, where factors must compete with nucleosomes for DNA access, and relatively open (nucleosome-depleted) organizations over other factor sites, where factors bind without competition.
Author Summary
The detailed positions of nucleosomes along genomes have critical roles in transcriptional regulation. Consequently, it is important to understand the principles that govern the organization of nucleosomes in vivo and the functional consequences of this organization. Here we report on progress in identifying the functional consequences of nucleosome organization, in understanding the way in which nucleosome organization is encoded in the DNA, and in linking the two, by suggesting that distinct transcriptional behaviors are encoded through the genome's intrinsic nucleosome organization. Our results thus provide insight on the broader question of understanding how transcriptional programs are encoded in the DNA sequence. These new insights were enabled by individually sequencing ∼380,000 nucleosomes from yeast in their entirety. Using this map, we refine our previous model for predicting nucleosome positions and demonstrate that our new model predicts nucleosome organizations in yeast with high accuracy and that its nucleosome positioning signals are predictive across eukaryotes. We show that the yeast genome may utilize these nucleosome positioning signals to encode regions with both relatively open (nucleosome-depleted) chromatin organizations and relatively closed (nucleosome-covered) chromatin organizations and that this encoding can partly explain aspects of transcription factor binding, gene expression, transcriptional noise, and DNA replication.
PMCID: PMC2570626  PMID: 18989395
10.  The Role of Nucleosome Positioning in the Evolution of Gene Regulation 
PLoS Biology  2010;8(7):e1000414.
A comparative genomics study maps nucleosomes across the entire genomes of 12 fungal species, identifying multiple distinct mechanisms linking changes in chromatin architecture to evolution of gene regulation.
Chromatin organization plays a major role in gene regulation and can affect the function and evolution of new transcriptional programs. However, it can be difficult to decipher the basis of changes in chromatin organization and their functional effect on gene expression. Here, we present a large-scale comparative genomic analysis of the relationship between chromatin organization and gene expression, by measuring mRNA abundance and nucleosome positions genome-wide in 12 Hemiascomycota yeast species. We found substantial conservation of global and functional chromatin organization in all species, including prominent nucleosome-free regions (NFRs) at gene promoters, and distinct chromatin architecture in growth and stress genes. Chromatin organization has also substantially diverged in both global quantitative features, such as spacing between adjacent nucleosomes, and in functional groups of genes. Expression levels, intrinsic anti-nucleosomal sequences, and trans-acting chromatin modifiers all play important, complementary, and evolvable roles in determining NFRs. We identify five mechanisms that couple chromatin organization to evolution of gene regulation and have contributed to the evolution of respiro-fermentation and other key systems, including (1) compensatory evolution of alternative modifiers associated with conserved chromatin organization, (2) a gradual transition from constitutive to trans-regulated NFRs, (3) a loss of intrinsic anti-nucleosomal sequences accompanying changes in chromatin organization and gene expression, (4) re-positioning of motifs from NFRs to nucleosome-occluded regions, and (5) the expanded use of NFRs by paralogous activator-repressor pairs. Our study sheds light on the molecular basis of chromatin organization, and on the role of chromatin organization in the evolution of gene regulation.
Author Summary
Divergence in gene regulation plays a major role in organismal evolution. Evidence suggests that changes in the packaging of eukaryotic genomes into chromatin can underlie the evolution of divergent gene expression patterns. Here, we explore the role of chromatin structure in regulatory evolution by whole-genome measurements of nucleosome positions and mRNA levels in 12 yeast species spanning ∼250 million years of evolution. We find several distinct ways in which changes in chromatin structure are associated with changes in gene expression. These include changes in promoter accessibility, changes in promoter chromatin architecture, and changes in the accessibility of specific transcription factor binding sites. In many cases, changes in chromatin architecture are coupled to physiological diversity, including the evolution of a respiration- or fermentation-based lifestyle, mating behavior, salt tolerance, and broad aspects of genomic structure. Together, our data will provide a rich resource for future investigations into the interplay between chromatin structure, gene regulation, and evolution.
PMCID: PMC2897762  PMID: 20625544
11.  Controls of Nucleosome Positioning in the Human Genome 
PLoS Genetics  2012;8(11):e1003036.
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.
Author Summary
Within the nucleus of the cell, the genome of eukaryotic organisms is tightly packaged into chromatin. Chromatin is composed of a repeating series of bead-like nucleosomes, each of which is encircled 1.7 times by a string of DNA. The organization of nucleosomes on the genome is fundamentally important because they can prevent other proteins from accessing the DNA. Previous studies of human nucleosomes concluded that most nucleosomes have fuzzy positioning and tend to occupy different locations in different cells. This interpretation, however, may be a consequence of the low resolution of existing data. Here we revisit the question of nucleosome positioning by generating the most precise map of nucleosome positions that has ever been created for a human cell line. We find that 8.7% of nucleosomes have very consistent positioning, and most nucleosomes are more consistently positioned than expected by chance. Additionally, we estimate that almost half of the genome contains regularly spaced arrays of nucleosomes. Much of this positioning is due to the intrinsic preference of nucleosomes for some DNA sequences over others; but in some regions of the genome, the sequence preferences of nucleosomes are overridden by proteins that out-compete them for binding or displace them using energy from ATP.
PMCID: PMC3499251  PMID: 23166509
12.  Chromatin is an ancient innovation conserved between Archaea and Eukarya 
eLife  2012;1:e00078.
The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ∼147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved −1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient.
eLife digest
Single-celled microorganisms called archaea are one of the three domains of cellular life, along with bacteria and eukaryotes. Archaea are similar to bacteria in that they do not have nuclei, but genetically they have more in common with eukaryotes. Archaea are found in a wide range of habitats including the human colon, marshlands, the ocean and extreme environments such as hot springs and salt lakes.
It has been known since the 1990s that the DNA of archaea is wrapped around histones to form complexes that closely resemble the nucleosomes found in eukaryotes, albeit with four rather than eight histone subunits. Nucleosomes are the fundamental units of chromatin, the highly-ordered and compact structure that all the DNA in a cell is packed into. Now we know exactly how many nucleosomes are present in a given cell for some eukaryotes, notably yeast, and to a good approximation we know the position of each nucleosome during a variety of metabolic states and physiological conditions. We can also quantify the nucleosome occupancy, which is measure of the length of time that the nucleosomes spend in contact with the DNA: this is a critical piece of information because it determines the level of access that other proteins, including those that regulate gene expression, have to the DNA. These advances have been driven in large part by advances in technology, notably high-density microarrays for genome wide-studies of nucleosome occupancy, and massively parallel sequencing for direct nucleosome sequencing.
Ammar et al. have used these techniques to explore how the DNA of Haloferax volcanii, a species of archaea that thrives in the hyper-salty waters of the Dead Sea, is organized on a genome-wide basis. Despite some clear differences between the genomes of archaea and eukaryotes—for example, genomic DNA is typically circular in archaea and linear in eukaryotes—they found that the genome of Hfx. volcanii is organized into chromatin in a way that is remarkably similar to that seen in all eukaryotic genomes studied to date. This is surprising given that the chromatin in eukaryotes is confined to the nucleus, whereas there are no such constraints in archaea. In particular, Ammar et al. found that those regions of the DNA near the ends of genes that mark where the transcription of the DNA into RNA should begin and end contain have lower nucleosome occupancy than other regions. Moreover, the overall level of occupancy in Hfx. volcanii was twice that of eukaryotes, which is what one would expect given that nucleosomes in archaea contain half as many histone subunits as nucleosomes in eukaryotes. Ammar et al. also confirmed that that the degree of nucleosome occupancy is correlated with gene expression.
These two findings—the similarities between the chromatin in archaea and eukaryotes, and the correlation between nucleosome occupancy and gene expression in archaea—raise an interesting evolutionary possibility: the initial function of nucleosomes and chromatin formation might have been for the regulation of gene expression rather than the packaging of DNA. This is consistent with two decades of research that has shown that there is an extraordinary and complex relationship between the structure of chromatin and the process of gene expression. It is possible, therefore, that as the early eukaryotes evolved, nucleosomes and chromatin started to package DNA into compact structures that, among other things, helped to prevent DNA damage, and that this subsequently enabled the early eukaryotes to flourish.
PMCID: PMC3510453  PMID: 23240084
Haloferax volcanii; Nucleosome; Chromatin; Transcriptome; RNA-seq; Archaea; Other
13.  Dissecting Nucleosome Free Regions by a Segmental Semi-Markov Model 
PLoS ONE  2009;4(3):e4721.
Nucleosome free regions (NFRs) play important roles in diverse biological processes including gene regulation. A genome-wide quantitative portrait of each individual NFR, with their starting and ending positions, lengths, and degrees of nucleosome depletion is critical for revealing the heterogeneity of gene regulation and chromatin organization. By averaging nucleosome occupancy levels, previous studies have identified the presence of NFRs in the promoter regions across many genes. However, evaluation of the quantitative characteristics of individual NFRs requires an NFR calling method.
In this study, we propose a statistical method to identify the patterns of NFRs from a genome-wide measurement of nucleosome occupancy. This method is based on an appropriately designed segmental semi-Markov model, which can capture each NFR pattern and output its quantitative characterizations. Our results show that the majority of the NFRs are located in intergenic regions or promoters with a length of about 400–600bp and varying degrees of nucleosome depletion. Our quantitative NFR mapping allows for an investigation of the relative impacts of transcription machinery and DNA sequence in evicting histones from NFRs. We show that while both factors have significant overall effects, their specific contributions vary across different subtypes of NFRs.
The emphasis of our approach on the variation rather than the consensus of nucleosome free regions sets the tone for enabling the exploration of many subtler dynamic aspects of chromatin biology.
PMCID: PMC2648986  PMID: 19266098
14.  Blurring of High-Resolution Data Shows that the Effect of Intrinsic Nucleosome Occupancy on Transcription Factor Binding is Mostly Regional, Not Local 
PLoS Computational Biology  2010;6(1):e1000649.
Genome wide maps of nucleosome occupancy in yeast have recently been produced through deep sequencing of nuclease-protected DNA. These maps have been obtained from both crosslinked and uncrosslinked chromatin in vivo, and from chromatin assembled from genomic DNA and nucleosomes in vitro. Here, we analyze these maps in combination with existing ChIP-chip data, and with new ChIP-qPCR experiments reported here. We show that the apparent nucleosome density in crosslinked chromatin, when compared to uncrosslinked chromatin, is preferentially increased at transcription factor (TF) binding sites, suggesting a strategy for mapping generic transcription factor binding sites that would not require immunoprecipitation of a particular factor. We also confirm previous conclusions that the intrinsic, sequence dependent binding of nucleosomes helps determine the localization of TF binding sites. However, we find that the association between low nucleosome occupancy and TF binding is typically greater if occupancy at a site is averaged over a 600bp window, rather than using the occupancy at the binding site itself. We have also incorporated intrinsic nucleosome binding occupancies as weights in a computational model for TF binding, and by this measure as well we find better prediction if the high resolution nucleosome occupancy data is averaged over 600bp. We suggest that the intrinsic DNA binding specificity of nucleosomes plays a role in TF binding site selection not so much through the specification of precise nucleosome positions that permit or occlude binding, but rather through the creation of low occupancy regions that can accommodate competition from TFs through rearrangement of nucleosomes.
Author Summary
Genomic DNA is largely covered by proteins that compete with one another for binding to regulatory sequences. Most of these proteins are in the form of nucleosomes. How nucleosomes come to occupy particular sites and thereby compete with sequence specific transcription factors is a central problem in developing a systems-level understanding of gene regulation. Here, we performed a series of computational analyses using high-resolution nucleosome position data that has recently become available in yeast, thanks to advances in DNA sequencing technology. Analysis of these data, combined with data on the location and occupancy of transcription factors genome-wide, shows that the precise location of nucleosomes as determined by nucleosome sequence specificity is often less important to transcription factor binding than the broader, regional occupancy of nucleosomes that is encoded in genomic DNA. This result has implications for the evolution of DNA regulatory elements.
PMCID: PMC2799660  PMID: 20098497
15.  Substantial Histone Reduction Modulates Genomewide Nucleosomal Occupancy and Global Transcriptional Output 
PLoS Biology  2011;9(6):e1001086.
The basic unit of genome packaging is the nucleosome, and nucleosomes have long been proposed to restrict DNA accessibility both to damage and to transcription. Nucleosome number in cells was considered fixed, but recently aging yeast and mammalian cells were shown to contain fewer nucleosomes. We show here that mammalian cells lacking High Mobility Group Box 1 protein (HMGB1) contain a reduced amount of core, linker, and variant histones, and a correspondingly reduced number of nucleosomes, possibly because HMGB1 facilitates nucleosome assembly. Yeast nhp6 mutants lacking Nhp6a and -b proteins, which are related to HMGB1, also have a reduced amount of histones and fewer nucleosomes. Nucleosome limitation in both mammalian and yeast cells increases the sensitivity of DNA to damage, increases transcription globally, and affects the relative expression of about 10% of genes. In yeast nhp6 cells the loss of more than one nucleosome in four does not affect the location of nucleosomes and their spacing, but nucleosomal occupancy. The decrease in nucleosomal occupancy is non-uniform and can be modelled assuming that different nucleosomal sites compete for available histones. Sites with a high propensity to occupation are almost always packaged into nucleosomes both in wild type and nucleosome-depleted cells; nucleosomes on sites with low propensity to occupation are disproportionately lost in nucleosome-depleted cells. We suggest that variation in nucleosome number, by affecting nucleosomal occupancy both genomewide and gene-specifically, constitutes a novel layer of epigenetic regulation.
Author Summary
The accurate preservation and correct retrieval of genetic information is crucial for all living organisms. In eukaryotes, whether single-celled yeast or complex mammals, the DNA containing the genetic information is wrapped around beads of histone proteins to form structures called nucleosomes along the length of the DNA; this packaging arrangement helps protect the genome from damage and may restrict access to the genetic information. Until recently, the amount of histones and, consequently, the number of nucleosomes in the cell were considered fixed. Here, we show that in both mammalian and yeast cells that lack a single protein—HMGB1 in mammals or Nhp6a/b in yeast—the abundance of histones and nucleosomes decreases by 20%–30%. Contrary to expectations, we found that in yeast the nucleosomes do not redistribute along DNA when they are fewer: they largely maintain their positions, but the amount of time each specific DNA site spends wrapped in a nucleosome (i.e., its occupancy) decreases. Sequences that are already less frequently occupied than others in normal yeast cells lose disproportionally more nucleosomes in the mutant yeast that lack Nhp6a/b. This gives rise to a global increase in transcription and specific alterations in the expression of certain genes. This study thus contributes to a deeper understanding of how the DNA is packaged and organized. It also suggests that the cell's histone content might contribute in an important way to gene regulation.
PMCID: PMC3125158  PMID: 21738444
16.  Dynamic Remodeling of Individual Nucleosomes Across a Eukaryotic Genome in Response to Transcriptional Perturbation 
PLoS Biology  2008;6(3):e65.
The eukaryotic genome is packaged as chromatin with nucleosomes comprising its basic structural unit, but the detailed structure of chromatin and its dynamic remodeling in terms of individual nucleosome positions has not been completely defined experimentally for any genome. We used ultra-high–throughput sequencing to map the remodeling of individual nucleosomes throughout the yeast genome before and after a physiological perturbation that causes genome-wide transcriptional changes. Nearly 80% of the genome is covered by positioned nucleosomes occurring in a limited number of stereotypical patterns in relation to transcribed regions and transcription factor binding sites. Chromatin remodeling in response to physiological perturbation was typically associated with the eviction, appearance, or repositioning of one or two nucleosomes in the promoter, rather than broader region-wide changes. Dynamic nucleosome remodeling tends to increase the accessibility of binding sites for transcription factors that mediate transcriptional changes. However, specific nucleosomal rearrangements were also evident at promoters even when there was no apparent transcriptional change, indicating that there is no simple, globally applicable relationship between chromatin remodeling and transcriptional activity. Our study provides a detailed, high-resolution, dynamic map of single-nucleosome remodeling across the yeast genome and its relation to global transcriptional changes.
Author Summary
The eukaryotic genome is packed in a systematic hierarchy to accommodate it within the confines of the cell's nucleus. This packing, however, presents an impediment to the transcription machinery when it must access genomic DNA to regulate gene expression. A fundamental aspect of genome packing is the spooling of DNA around nucleosomes—structures formed from histone proteins—which must be dislodged during transcription. In this study, we identified all the nucleosome displacements associated with a physiological perturbation causing genome-wide transcriptional changes in the eukaryote Saccharomyces cerevisiae. We isolated nucleosomal DNA before and after subjecting cells to heat shock, then identified the ends of these DNA fragments and, thereby, the location of nucleosomes along the genome, using ultra-high–throughput sequencing. We identified localized patterns of nucleosome displacement at gene promoters in response to heat shock, and found that nucleosome eviction was generally associated with activation and their appearance with gene repression. Nucleosome remodeling generally improved the accessibility of DNA to transcriptional regulators mediating the response to stresses like heat shock. However, not all nucleosomal remodeling was associated with transcriptional changes, indicating that the relationship between nucleosome repositioning and transcriptional activity is not merely a reflection of competing access to DNA.
Ultra-high-throughput sequencing is used to show that distinct, localized patterns of nucleosome repositioning at promoters underlie the genome-wide transcriptional response to a physiological stimulus.
PMCID: PMC2267817  PMID: 18351804
17.  Structural features based genome-wide characterization and prediction of nucleosome organization 
BMC Bioinformatics  2012;13:49.
Nucleosome distribution along chromatin dictates genomic DNA accessibility and thus profoundly influences gene expression. However, the underlying mechanism of nucleosome formation remains elusive. Here, taking a structural perspective, we systematically explored nucleosome formation potential of genomic sequences and the effect on chromatin organization and gene expression in S. cerevisiae.
We analyzed twelve structural features related to flexibility, curvature and energy of DNA sequences. The results showed that some structural features such as DNA denaturation, DNA-bending stiffness, Stacking energy, Z-DNA, Propeller twist and free energy, were highly correlated with in vitro and in vivo nucleosome occupancy. Specifically, they can be classified into two classes, one positively and the other negatively correlated with nucleosome occupancy. These two kinds of structural features facilitated nucleosome binding in centromere regions and repressed nucleosome formation in the promoter regions of protein-coding genes to mediate transcriptional regulation. Based on these analyses, we integrated all twelve structural features in a model to predict more accurately nucleosome occupancy in vivo than the existing methods that mainly depend on sequence compositional features. Furthermore, we developed a novel approach, named DLaNe, that located nucleosomes by detecting peaks of structural profiles, and built a meta predictor to integrate information from different structural features. As a comparison, we also constructed a hidden Markov model (HMM) to locate nucleosomes based on the profiles of these structural features. The result showed that the meta DLaNe and HMM-based method performed better than the existing methods, demonstrating the power of these structural features in predicting nucleosome positions.
Our analysis revealed that DNA structures significantly contribute to nucleosome organization and influence chromatin structure and gene expression regulation. The results indicated that our proposed methods are effective in predicting nucleosome occupancy and positions and that these structural features are highly predictive of nucleosome organization.
The implementation of our DLaNe method based on structural features is available online.
PMCID: PMC3378464  PMID: 22449207
18.  A Nucleosome-Guided Map of Transcription Factor Binding Sites in Yeast 
PLoS Computational Biology  2007;3(11):e215.
Finding functional DNA binding sites of transcription factors (TFs) throughout the genome is a crucial step in understanding transcriptional regulation. Unfortunately, these binding sites are typically short and degenerate, posing a significant statistical challenge: many more matches to known TF motifs occur in the genome than are actually functional. However, information about chromatin structure may help to identify the functional sites. In particular, it has been shown that active regulatory regions are usually depleted of nucleosomes, thereby enabling TFs to bind DNA in those regions. Here, we describe a novel motif discovery algorithm that employs an informative prior over DNA sequence positions based on a discriminative view of nucleosome occupancy. When a Gibbs sampling algorithm is applied to yeast sequence-sets identified by ChIP-chip, the correct motif is found in 52% more cases with our informative prior than with the commonly used uniform prior. This is the first demonstration that nucleosome occupancy information can be used to improve motif discovery. The improvement is dramatic, even though we are using only a statistical model to predict nucleosome occupancy; we expect our results to improve further as high-resolution genome-wide experimental nucleosome occupancy data becomes increasingly available.
Author Summary
Identifying transcription factor (TF) binding sites across the genome is an important problem in molecular biology. Large-scale discovery of TF binding sites is usually carried out by searching for short DNA patterns that appear often within promoter regions of genes that are known to be co-bound by a TF. In such problems, promoters have traditionally been treated as strings of nucleotide bases in which TF binding sites are assumed to be equally likely to occur at any position. In vivo, however, TFs localize to DNA binding sites as part of a complicated thermodynamic process of cooperativity and competition, both with one another and, importantly, with DNA packaging proteins called nucleosomes. In particular, TFs are more likely to bind DNA at sites that are not occupied by nucleosomes. In this paper, we show that it is possible to incorporate knowledge of the nucleosome landscape across the genome to aid binding site discovery; indeed, our algorithm incorporating nucleosome occupancy information is significantly more accurate than conventional methods. We use our algorithm to generate a condition-dependent, nucleosome-guided map of binding sites for 55 TFs in yeast.
PMCID: PMC2065891  PMID: 17997593
19.  The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo 
eLife  2014;3:e01861.
In budding yeast, a single cenH3 (Cse4) nucleosome occupies the ∼120-bp functional centromere, however conflicting structural models for the particle have been proposed. To resolve this controversy, we have applied H4S47C-anchored cleavage mapping, which reveals the precise position of histone H4 in every nucleosome in the genome. We find that cleavage patterns at centromeres are unique within the genome and are incompatible with symmetrical structures, including octameric nucleosomes and (Cse4/H4)2 tetrasomes. Centromere cleavage patterns are compatible with a precisely positioned core structure, one in which each of the 16 yeast centromeres is occupied by oppositely oriented Cse4/H4/H2A/H2B hemisomes in two rotational phases within the population. Centromere-specific hemisomes are also inferred from distances observed between closely-spaced H4 cleavages, as predicted from structural modeling. Our results indicate that the orientation and rotational position of the stable hemisome at each yeast centromere is not specified by the functional centromere sequence.
eLife digest
DNA is tightly packaged in cells for a variety of reasons—to allow it to fit inside the nucleus, to protect it from damage, and to help control the production of proteins from genes. The basic unit of packaged DNA is called a nucleosome, which consists of DNA wrapped around a structure formed by two pairs of four different proteins.
These proteins, which are called histones, have a role that extends beyond providing structural support for DNA. When cells divide, for example, pairs of ‘sister chromosomes’ are pulled apart to ensure that the two daughter cells both have the same chromosomes as the original cell. The sister chromosomes are pulled apart from a single position called a centromere, and the nucleosomes at this position contain a histone that is different from the histones found everywhere else in the cell. However, until recently it was not clear if the nucleosomes that contained these special cenH3 histones had the same structure as other nucleosomes.
Now Henikoff et al. have used a method called H4S47C-anchored cleavage mapping to study every nucleosome in the genome of the yeast S. cerevisiae. This mapping technique uses DNA sequencing to measure the precise distances between fixed points on the DNA in the nucleosome. Knowing these distances tells researchers a great deal about the number and position of the histones within each nucleosome in the genome.
Using this approach, Henikoff et al. found that nucleosomes at centromeres are different from other nucleosomes in histone number and arrangement. In particular, the nucleosome at each yeast centromere contains only one each of the four different histones in an asymmetrical orientation, in contrast to all other yeast nucleosomes, which contain two sets of four histones in a symmetrical arrangement. Furthermore, each nucleosome at a centromere can adopt one of two orientations: these orientations are mirror images of each other, and they occur with equal probability. It should also be possible to use the mapping technique developed by Henikoff et al. to study the larger and more complex centromeres found in other organisms, including humans.
PMCID: PMC3983907  PMID: 24737863
centromeres; nucleosome; chemical cleavage mapping; S. cerevisiae
20.  Preferentially Quantized Linker DNA Lengths in Saccharomyces cerevisiae 
PLoS Computational Biology  2008;4(9):e1000175.
The exact lengths of linker DNAs connecting adjacent nucleosomes specify the intrinsic three-dimensional structures of eukaryotic chromatin fibers. Some studies suggest that linker DNA lengths preferentially occur at certain quantized values, differing one from another by integral multiples of the DNA helical repeat, ∼10 bp; however, studies in the literature are inconsistent. Here, we investigate linker DNA length distributions in the yeast Saccharomyces cerevisiae genome, using two novel methods: a Fourier analysis of genomic dinucleotide periodicities adjacent to experimentally mapped nucleosomes and a duration hidden Markov model applied to experimentally defined dinucleosomes. Both methods reveal that linker DNA lengths in yeast are preferentially periodic at the DNA helical repeat (∼10 bp), obeying the forms 10n+5 bp (integer n). This 10 bp periodicity implies an ordered superhelical intrinsic structure for the average chromatin fiber in yeast.
Author Summary
Eukaryotic genomic DNA exists as chromatin, with the DNA wrapped locally into a repeating array of protein–DNA complexes (“nucleosomes”) separated by short stretches of unwrapped “linker” DNA. Nucleosome arrays further compact into ∼30-nm-wide higher-order chromatin structures. Despite decades of work, there remains no agreement about the structure of the 30 nm fiber, or even if the structure is ordered or random. The helical symmetry of DNA couples the one-dimensional distribution of nucleosomes along the DNA to an intrinsic three-dimensional structure for the chromatin fiber. Random linker length distributions imply random three-dimensional intrinsic fiber structures, whereas different possible nonrandom length distributions imply different ordered structures. Here we use two independent computational methods, with two independent kinds of experimental data, to experimentally define the probability distribution of linker DNA lengths in yeast. Both methods agree that linker DNA lengths in yeast come in a set of preferentially quantized lengths that differ one from another by ∼10 bp, the DNA helical repeat, with a preferred phase offset of 5 bp. The preferential quantization of lengths implies that the intrinsic three-dimensional structure for the average chromatin fiber is ordered, not random. The 5 bp offset implies a particular geometry for this intrinsic structure.
PMCID: PMC2522279  PMID: 18787693
21.  Nucleosome positioning from tiling microarray data 
Bioinformatics  2008;24(13):i139-i146.
Motivation: The packaging of DNA around nucleosomes in eukaryotic cells plays a crucial role in regulation of gene expression, and other DNA-related processes. To better understand the regulatory role of nucleosomes, it is important to pinpoint their position in a high (5–10 bp) resolution. Toward this end, several recent works used dense tiling arrays to map nucleosomes in a high-throughput manner. These data were then parsed and hand-curated, and the positions of nucleosomes were assessed.
Results: In this manuscript, we present a fully automated algorithm to analyze such data and predict the exact location of nucleosomes. We introduce a method, based on a probabilistic graphical model, to increase the resolution of our predictions even beyond that of the microarray used. We show how to build such a model and how to compile it into a simple Hidden Markov Model, allowing for a fast and accurate inference of nucleosome positions.
We applied our model to nucleosomal data from mid-log yeast cells reported by Yuan et al. and compared our predictions to those of the original paper; to a more recent method that uses five times denser tiling arrays as explained by Lee et al.; and to a curated set of literature-based nucleosome positions. Our results suggest that by applying our algorithm to the same data used by Yuan et al. our fully automated model traced 13% more nucleosomes, and increased the overall accuracy by about 20%. We believe that such an improvement opens the way for a better understanding of the regulatory mechanisms controlling gene expression, and how they are encoded in the DNA.
PMCID: PMC2718629  PMID: 18586706
22.  Genome-Wide Nucleosome Positioning Is Orchestrated by Genomic Regions Associated with DNase I Hypersensitivity in Rice 
PLoS Genetics  2014;10(5):e1004378.
Nucleosome positioning dictates the DNA accessibility for regulatory proteins, and thus is critical for gene expression and regulation. It has been well documented that only a subset of nucleosomes are reproducibly positioned in eukaryotic genomes. The most prominent example of phased nucleosomes is the context of genes, where phased nucleosomes flank the transcriptional starts sites (TSSs). It is unclear, however, what factors determine nucleosome positioning in regions that are not close to genes. We mapped both nucleosome positioning and DNase I hypersensitive site (DHS) datasets across the rice genome. We discovered that DHSs located in a variety of contexts, both genic and intergenic, were flanked by strongly phased nucleosome arrays. Phased nucleosomes were also found to flank DHSs in the human genome. Our results suggest the barrier model may represent a general feature of nucleosome organization in eukaryote genomes. Specifically, regions bound with regulatory proteins, including intergenic regions, can serve as barriers that organize phased nucleosome arrays on both sides. Our results also suggest that rice DHSs often span a single, phased nucleosome, similar to the H2A.Z-containing nucleosomes observed in DHSs in the human genome.
Author Summary
The fundamental unit of chromatin is the nucleosome, which consists of 147 bp of DNA wrapped around a histone octamer containing four core histones (H3, H4, H2A, and H2B). Nucleosome positioning in the genome affects the DNA accessibility for regulatory proteins, and thus is critical for gene expression and regulation. Genomic regions associated with regulatory proteins are associated with a pronounced sensitivity to DNase I digestion, and are thus called DNase I hypersensitive sites (DHSs). It is well known that only a subset of nucleosomes are reproducibly positioned in eukaryotic genomes. However, it is less clear what factors determine genome-wide nucleosome positioning, especially in intergenic regions. We mapped both nucleosome positioning and DHS datasets across the rice genome. We discovered that DHSs located in a variety of contexts, both genic and intergenic, were flanked by strongly phased nucleosome arrays. We confirmed the same association of DHSs with phased nucleosomes in the human genome. We conclude that genomic loci associated with a diverse set of regulatory proteins are major determinants of nucleosome phasing, and this is true in both genic and intergenic regions.
PMCID: PMC4031139  PMID: 24852592
23.  Regulation of the Nucleosome Repeat Length In Vivo by the DNA Sequence, Protein Concentrations and Long-Range Interactions 
PLoS Computational Biology  2014;10(7):e1003698.
The nucleosome repeat length (NRL) is an integral chromatin property important for its biological functions. Recent experiments revealed several conflicting trends of the NRL dependence on the concentrations of histones and other architectural chromatin proteins, both in vitro and in vivo, but a systematic theoretical description of NRL as a function of DNA sequence and epigenetic determinants is currently lacking. To address this problem, we have performed an integrative biophysical and bioinformatics analysis in species ranging from yeast to frog to mouse where NRL was studied as a function of various parameters. We show that in simple eukaryotes such as yeast, a lower limit for the NRL value exists, determined by internucleosome interactions and remodeler action. For higher eukaryotes, also the upper limit exists since NRL is an increasing but saturating function of the linker histone concentration. Counterintuitively, smaller H1 variants or non-histone architectural proteins can initiate larger effects on the NRL due to entropic reasons. Furthermore, we demonstrate that different regimes of the NRL dependence on histone concentrations exist depending on whether DNA sequence-specific effects dominate over boundary effects or vice versa. We consider several classes of genomic regions with apparently different regimes of the NRL variation. As one extreme, our analysis reveals that the period of oscillations of the nucleosome density around bound RNA polymerase coincides with the period of oscillations of positioning sites of the corresponding DNA sequence. At another extreme, we show that although mouse major satellite repeats intrinsically encode well-defined nucleosome preferences, they have no unique nucleosome arrangement and can undergo a switch between two distinct types of nucleosome positioning.
Author Summary
The DNA molecule of a human or mouse can be up to two meters long, if stretched. However, it is stored inside the small volume of the nucleus in the living cell. DNA compaction is achieved at different hierarchical levels with the help of a number of architectural proteins. The elementary unit of compaction is the nucleosome, where DNA is wrapped around the protein octamer core. Each nucleosome contains about 147 DNA base pairs; the length of DNA between the neighboring nucleosomes varies from nearly zero to several hundred of base pairs. This variability determines the biological function of the underlying DNA, since some parts of the genome are less compact, and thus potentially actively transcribed, while others are more compact, and their transcription is limited. The DNA distances between neighboring nucleosomes depend on the interaction with many chromatin-associated proteins. The average distance between two neighboring nucleosomes change for different genomic locations and even for the same genomic region in different cell states of the same organism. Here we study these effects and provide their quantitative biophysical description using available experimental data in a number of organisms, ranging from yeast to frog to mouse.
PMCID: PMC4081033  PMID: 24992723
24.  Evidence of association between Nucleosome Occupancy and the Evolution of Transcription Factor Binding Sites in Yeast 
Divergence of transcription factor binding sites is considered to be an important source of regulatory evolution. The associations between transcription factor binding sites and phenotypic diversity have been investigated in many model organisms. However, the understanding of other factors that contribute to it is still limited. Recent studies have elucidated the effect of chromatin structure on molecular evolution of genomic DNA. Though the profound impact of nucleosome positions on gene regulation has been reported, their influence on transcriptional evolution is still less explored. With the availability of genome-wide nucleosome map in yeast species, it is thus desirable to investigate their impact on transcription factor binding site evolution. Here, we present a comprehensive analysis of the role of nucleosome positioning in the evolution of transcription factor binding sites.
We compared the transcription factor binding site frequency in nucleosome occupied regions and nucleosome depleted regions in promoters of old (orthologs among Saccharomycetaceae) and young (Saccharomyces specific) genes; and in duplicate gene pairs. We demonstrated that nucleosome occupied regions accommodate greater binding site variations than nucleosome depleted regions in young genes and in duplicate genes. This finding was confirmed by measuring the difference in evolutionary rates of binding sites in sensu stricto yeasts at nucleosome occupied regions and nucleosome depleted regions. The binding sites at nucleosome occupied regions exhibited a consistently higher evolution rate than those at nucleosome depleted regions, corroborating the difference in the selection constraints at the two regions. Finally, through site-directed mutagenesis experiment, we found that binding site gain or loss events at nucleosome depleted regions may cause more expression differences than those in nucleosome occupied regions.
Our study indicates the existence of different selection constraint on binding sites at nucleosome occupied regions than at the nucleosome depleted regions. We found that the binding sites have a different rate of evolution at nucleosome occupied and depleted regions. Finally, using transcription factor binding site-directed mutagenesis experiment, we confirmed the difference in the impact of binding site changes on expression at these regions. Thus, our work demonstrates the importance of composite analysis of chromatin and transcriptional evolution.
PMCID: PMC3124427  PMID: 21627806
25.  The DNA-encoded nucleosome organization of a eukaryotic genome 
Nature  2008;458(7236):362-366.
Nucleosome organization is critical for gene regulation1. In living cells this organization is determined by multiple factors, including the action of chromatin remodellers2, competition with site-specific DNA-binding proteins3, and the DNA sequence preferences of the nucleosomes themselves4-8. However, it has been difficult to estimate the relative importance of each of these mechanisms in vivo7,9-11, because in vivo nucleosome maps reflect the combined action of all influencing factors. Here we determine the importance of nucleosome DNA sequence preferences experimentally by measuring the genome-wide occupancy of nucleosomes assembled on purified yeast genomic DNA. The resulting map, in which nucleosome occupancy is governed only by the intrinsic sequence preferences of nucleosomes, is similar to in vivo nucleosome maps generated in three different growth conditions. In vitro, nucleosome depletion is evident at many transcription factor binding sites and around gene start and end sites, indicating that nucleosome depletion at these sites in vivo is partly encoded in the genome. We confirm these results with a micrococcal nuclease-independent experiment that measures the relative affinity of nucleosomes for ∼40,000 double-stranded 150-base-pair oligonucleotides. Using our in vitro data, we devise a computational model of nucleosome sequence preferences that is significantly correlated with in vivo nucleosome occupancy in Caenorhabditis elegans. Our results indicate that the intrinsic DNA sequence preferences of nucleosomes have a central role in determining the organization of nucleosomes in vivo.
PMCID: PMC2658732  PMID: 19092803

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