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1.  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
2.  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
3.  Using DNA mechanics to predict in vitro nucleosome positions and formation energies 
Nucleic Acids Research  2009;37(14):4707-4722.
In eukaryotic genomes, nucleosomes function to compact DNA and to regulate access to it both by simple physical occlusion and by providing the substrate for numerous covalent epigenetic tags. While competition with other DNA-binding factors and action of chromatin remodeling enzymes significantly affect nucleosome formation in vivo, nucleosome positions in vitro are determined by steric exclusion and sequence alone. We have developed a biophysical model, DNABEND, for the sequence dependence of DNA bending energies, and validated it against a collection of in vitro free energies of nucleosome formation and a set of in vitro nucleosome positions mapped at high resolution. We have also made a first ab initio prediction of nucleosomal DNA geometries, and checked its accuracy against the nucleosome crystal structure. We have used DNABEND to design both strong and weak histone- binding sequences, and measured the corresponding free energies of nucleosome formation. We find that DNABEND can successfully predict in vitro nucleosome positions and free energies, providing a physical explanation for the intrinsic sequence dependence of histone–DNA interactions.
PMCID: PMC2724288  PMID: 19509309
4.  Statistical Mechanics of Nucleosomes Constrained by Higher-Order Chromatin Structure 
Journal of statistical physics  2011;144(2):379-404.
Eukaryotic DNA is packaged into chromatin: one-dimensional arrays of nucleosomes separated by stretches of linker DNA are folded into 30-nm chromatin fibers which in turn form higher-order structures (Felsenfeld and Groudine in Nature 421:448, 2003). Each nucleosome, the fundamental unit of chromatin, has 147 base pairs (bp) of DNA wrapped around a histone octamer (Richmond and Davey in Nature 423:145, 2003). In order to describe how chromatin fiber formation affects nucleosome positioning and energetics, we have developed a thermodynamic model of finite-size particles with effective nearest-neighbor interactions and arbitrary DNA-binding energies. We show that both one-and two-body interactions can be extracted from one-particle density profiles based on high-throughput maps of in vitro or in vivo nucleosome positions. Although a simpler approach that neglects two-body interactions (even if they are in fact present in the system) can be used to predict sequence determinants of nucleosome positions, the full theory is required to disentangle one- and two-body effects. Finally, we construct a minimal model in which nucleosomes are positioned primarily by steric exclusion and two-body interactions rather than intrinsic histone-DNA sequence preferences. The model reproduces nucleosome occupancy patterns observed over transcribed regions in living cells.
PMCID: PMC3156456  PMID: 21857746
Chromatin structure; Nucleosome positioning; One-dimensional classical fluid of interacting particles
5.  Preferential Nucleosome Occupancy at High Values of DNA Helical Rise 
Nucleosomes are the basic structural units of eukaryotic chromatin and play a key role in the regulation of gene expression. Nucleosome formation depends on several factors, including properties of the sequence itself, but also physical constraints and epigenetic factors such as chromatin-remodelling enzymes. In this view, a sequence-dependent approach is able to capture a general tendency of a region to bind a histone octamer. A reference data set of positioned nucleosomes of Saccharomyces cerevisiae was used to study the role of DNA helical rise in histone–DNA interaction. Genomic sequences were transformed into arrays of helical rise values by a tetranucleotide code and then turned into profiles of mean helical rise values. These profiles resemble maps of nucleosome occupancy, suggesting that intrinsic histone–DNA interactions are linked to helical rise. The obtained results show that preferential nucleosome occupancy occurs where the mean helical rise reaches its largest values. Mean helical rise profiles obtained by using maps of positioned nucleosomes of the Drosophila melanogaster and Plasmodium falciparum genomes, as well as Homo sapiens chromosome 20 confirm that nucleosomes are mainly located where the mean helical rise reaches its largest values.
PMCID: PMC3276262  PMID: 22233711
nucleosome; tetranucleotide; helical rise
6.  What controls nucleosome positions? 
Trends in genetics : TIG  2009;25(8):335-343.
The DNA of eukaryotic genomes is wrapped in nucleosomes, which strongly distort and occlude the DNA from access to most DNA-binding proteins. An understanding of the mechanisms that control nucleosome positioning along the DNA is thus essential to understanding the binding and action of proteins that carry out essential genetic functions. New genome-wide data on in vivo and in vitro nucleosome positioning greatly advance our understanding of several factors that can influence nucleosome positioning, including DNA sequence preferences, DNA methylation, histone variants and post-translational modifications, higher order chromatin structure, and the actions of transcription factors, chromatin remodelers and other DNA-binding proteins. We discuss how these factors function and ways in which they might be integrated into a unified framework that accounts for both the preservation of nucleosome positioning and the dynamic nucleosome repositioning that occur across biological conditions, cell types, developmental processes and disease.
PMCID: PMC2810357  PMID: 19596482
7.  A genomic code for nucleosome positioning 
Nature  2006;442(7104):772-778.
Eukaryotic genomes are packaged into nucleosome particles that occlude the DNA from interacting with most DNA binding proteins. Nucleosomes have higher affinity for particular DNA sequences, reflecting the ability of the sequence to bend sharply, as required by the nucleosome structure. However, it is not known whether these sequence preferences have a significant influence on nucleosome position in vivo, and thus regulate the access of other proteins to DNA. Here we isolated nucleosome-bound sequences at high resolution from yeast and used these sequences in a new computational approach to construct and validate experimentally a nucleosome-DNA interaction model, and to predict the genome-wide organization of nucleosomes. Our results demonstrate that genomes encode an intrinsic nucleosome organization and that this intrinsic organization can explain ∼50% of the in vivo nucleosome positions. This nucleosome positioning code may facilitate specific chromosome functions including transcription factor binding, transcription initiation, and even remodelling of the nucleosomes themselves.
PMCID: PMC2623244  PMID: 16862119
8.  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
9.  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
10.  Relationship between nucleosome positioning and DNA methylation 
Nature  2010;466(7304):388-392.
Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer1, 2. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana utilizing massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified ten base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results suggest that nucleosome positioning strongly influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA suggesting that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron-exon and exon-intron boundaries. RNA Pol II was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition.
PMCID: PMC2964354  PMID: 20512117
11.  Nucleosome structural changes induced by binding of non-histone chromosomal proteins HMGN1 and HMGN2☆ 
FEBS Open Bio  2013;3:184-191.
Interactions between the nucleosome and the non-histone chromosomal proteins (HMGN1 and HMGN2) were studied by circular dichroism (CD) spectroscopy to elucidate structural changes in the nucleosome induced by HMGN binding. Unlike previous studies that used a nucleosome extracted from living cells, in this study we utilized a nucleosome reconstituted from unmodified recombinant histones synthesized in Escherichia coli and a 189-bp synthetic DNA fragment harboring a nucleosome positioning sequence. This DNA fragment consists of 5′-TATAAACGCC-3′ repeats that has a high affinity to the histone octamer. A nucleosome containing a unique octamer-binding sequence at a specific location on the DNA was produced at sufficiently high yield for spectroscopic analysis. CD data have indicated that both HMGN1 and HMGN2 can increase the winding angle of the nucleosome DNA, but the extent of the structural changes induced by these proteins differs significantly. This suggests HMGN1 and HMGN2 would have different abilities to facilitate nucleosome remodeling.
•A nucleosome was reconstituted from recombinant histones and a synthetic DNA.•Nucleosomes were produced at sufficiently high yield for spectroscopic analysis.•A nucleosome with and without HMGN proteins was analyzed using CD spectroscopy.•CD data indicate that HMGN proteins increase the winding angle of the nucleosome DNA.•HMGN1 and HMGN2 may have different abilities to facilitate nucleosome remodeling.
PMCID: PMC3668530  PMID: 23772392
CD; Nucleosome; HMGN; Unmodified recombinant histones; Reconstitution; HMG, high mobility group; HMGN1 HMGN2, non-histone chromosomal proteins; CD, circular dichroism; PCR, polymerase chain reaction; phH2A, phH2B, phH3, and phH4, vectors for the gene expression of all four recombinant human core histones H2A, H2B, H3, and H4, respectively; LB, Luria–Bertani; IPTG, isopropyl-β-d-galactopyranoside; RP-HPLC, reverse phase high performance liquid chromatography; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; MNase, micrococcal nuclease; NMR, nuclear magnetic resonance
12.  Prediction of Nucleosome Positioning Based on Transcription Factor Binding Sites 
PLoS ONE  2010;5(9):e12495.
The DNA of all eukaryotic organisms is packaged into nucleosomes, the basic repeating units of chromatin. The nucleosome consists of a histone octamer around which a DNA core is wrapped and the linker histone H1, which is associated with linker DNA. By altering the accessibility of DNA sequences, the nucleosome has profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is of great importance for the study of genomic control mechanisms. Transcription factors (TFs) have been suggested to play a role in nucleosome positioning in vivo.
Principal Findings
Here, the minimum redundancy maximum relevance (mRMR) feature selection algorithm, the nearest neighbor algorithm (NNA), and the incremental feature selection (IFS) method were used to identify the most important TFs that either favor or inhibit nucleosome positioning by analyzing the numbers of transcription factor binding sites (TFBSs) in 53,021 nucleosomal DNA sequences and 50,299 linker DNA sequences. A total of nine important families of TFs were extracted from 35 families, and the overall prediction accuracy was 87.4% as evaluated by the jackknife cross-validation test.
Our results are consistent with the notion that TFs are more likely to bind linker DNA sequences than the sequences in the nucleosomes. In addition, our results imply that there may be some TFs that are important for nucleosome positioning but that play an insignificant role in discriminating nucleosome-forming DNA sequences from nucleosome-inhibiting DNA sequences. The hypothesis that TFs play a role in nucleosome positioning is, thus, confirmed by the results of this study.
PMCID: PMC2931695  PMID: 20824131
13.  Predicting nucleosome positioning using a duration Hidden Markov Model 
BMC Bioinformatics  2010;11:346.
The nucleosome is the fundamental packing unit of DNAs in eukaryotic cells. Its detailed positioning on the genome is closely related to chromosome functions. Increasing evidence has shown that genomic DNA sequence itself is highly predictive of nucleosome positioning genome-wide. Therefore a fast software tool for predicting nucleosome positioning can help understanding how a genome's nucleosome organization may facilitate genome function.
We present a duration Hidden Markov model for nucleosome positioning prediction by explicitly modeling the linker DNA length. The nucleosome and linker models trained from yeast data are re-scaled when making predictions for other species to adjust for differences in base composition. A software tool named NuPoP is developed in three formats for free download.
Simulation studies show that modeling the linker length distribution and utilizing a base composition re-scaling method both improve the prediction of nucleosome positioning regarding sensitivity and false discovery rate. NuPoP provides a user-friendly software tool for predicting the nucleosome occupancy and the most probable nucleosome positioning map for genomic sequences of any size. When compared with two existing methods, NuPoP shows improved performance in sensitivity.
PMCID: PMC2900280  PMID: 20576140
14.  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
15.  Global remodeling of nucleosome positions in C. elegans 
BMC Genomics  2013;14:284.
Eukaryotic chromatin architecture is affected by intrinsic histone-DNA sequence preferences, steric exclusion between nucleosome particles, formation of higher-order structures, and in vivo activity of chromatin remodeling enzymes.
To disentangle sequence-dependent nucleosome positioning from the other factors, we have created two high-throughput maps of nucleosomes assembled in vitro on genomic DNA from the nematode worm Caenorhabditis elegans. A comparison of in vitro nucleosome positions with those observed in a mixed-stage, mixed-tissue population of C. elegans cells reveals that in vivo sequence preferences are modified on the genomic scale. Indeed, G/C dinucleotides are predicted to be most favorable for nucleosome formation in vitro but not in vivo. Nucleosome sequence read coverage in vivo is distinctly lower in chromosome arms than in central regions; the observed changes in apparent nucleosome sequence specificity, likely due to genome-wide chromatin remodeler activity, contribute to the formation of these megabase-scale chromatin domains. We also observe that the majority of well-positioned in vivo nucleosomes do not occupy thermodynamically favorable sequences observed in vitro. Finally, we find that exons are intrinsically more amenable to nucleosome formation compared to introns. Nucleosome occupancy of introns and exons consistently increases with G/C content in vitro but not in vivo, in agreement with our observation that G/C dinucleotide enrichment does not strongly promote in vivo nucleosome formation.
Our findings highlight the importance of both sequence specificity and active nucleosome repositioning in creating large-scale chromatin domains, and the antagonistic roles of intrinsic sequence preferences and chromatin remodelers in C. elegans.
Sequence read data has been deposited into Sequence Read Archive (; accession number SRA050182). Additional data, software and computational predictions are available on the Nucleosome Explorer website (
PMCID: PMC3663828  PMID: 23622142
Nucleosome; Histone-DNA interactions; Chromatin domains; Nucleosome positioning
16.  Nucleosome Positioning in Saccharomyces cerevisiae 
Summary: The DNA of eukaryotic cells is spooled around large histone protein complexes, forming nucleosomes that make up the basis for a high-order packaging structure called chromatin. Compared to naked DNA, nucleosomal DNA is less accessible to regulatory proteins and regulatory processes. The exact positions of nucleosomes therefore influence several cellular processes, including gene expression, chromosome segregation, recombination, replication, and DNA repair. Here, we review recent technological advances enabling the genome-wide mapping of nucleosome positions in the model eukaryote Saccharomyces cerevisiae. We discuss the various parameters that determine nucleosome positioning in vivo, including cis factors like AT content, variable tandem repeats, and poly(dA:dT) tracts that function as chromatin barriers and trans factors such as chromatin remodeling complexes, transcription factors, histone-modifying enzymes, and RNA polymerases. In the last section, we review the biological role of chromatin in gene transcription, the evolution of gene regulation, and epigenetic phenomena.
PMCID: PMC3122627  PMID: 21646431
17.  Nucleosome occupancy reveals regulatory elements of the CFTR promoter 
Nucleic Acids Research  2011;40(2):625-637.
Access to regulatory elements of the genome can be inhibited by nucleosome core particles arranged along the DNA strand. Hence, sites that are accessible by transcription factors may be located by using nuclease digestion to identify the relative nucleosome occupancy of a genomic region. In order to define novel cis regulatory elements in the ∼2.7-kb promoter region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, we define its nucleosome occupancy. This profile reveals the precise positions of nucleosome-free regions (NFRs), both cell-type specific and others apparently unrelated to CFTR-expression level and offer the first high-resolution map of the chromatin structure of the entire CFTR promoter in relevant cell types. Several of these NFRs are strongly bound by nuclear factors in a sequence-specific manner, and directly influence CFTR promoter activity. Sequences within the NFR1 and NFR4 elements are highly conserved in many human gene promoters. Moreover, NFR1 contributes to promoter activity of another gene, angiopoietin-like 3 (ANGPTL3), while NFR4 is constitutively nucleosome-free in promoters genome wide. Conserved motifs within NFRs of the CFTR promoter also show a high level of protection from DNase I digestion genome-wide, and likely have important roles in the positioning of nucleosome core particles more generally.
PMCID: PMC3258145  PMID: 21948798
18.  Nucleosome positioning: bringing order to the eukaryotic genome 
Trends in Cell Biology  2012;22(5):250-256.
Nucleosomes are an essential component of eukaryotic chromosomes. The impact of nucleosomes is seen not just on processes that directly access the genome such as transcription, but also on an evolutionary timescale. Recent studies in a number of organisms have provided high-resolution maps of nucleosomes throughout the genome. Computational analysis, in conjunction with many other kinds of data, has shed light on several aspects of nucleosome biology. Nucleosomes are positioned by several means, including intrinsic sequence biases, by stacking against a fixed barrier, by DNA-binding proteins and by chromatin remodelers. These studies underscore the critical organizational role of nucleosomes in all eukaryotic genomes. Here, I review recent genomic studies that shed light on the determinants of nucleosome positioning and their impact on the genome.
PMCID: PMC3348441  PMID: 22421062
Nucleosome; chromatin; remodeling; epigenetic; genome packaging
19.  Extensive role of the general regulatory factors, Abf1 and Rap1, in determining genome-wide chromatin structure in budding yeast 
Nucleic Acids Research  2010;39(6):2032-2044.
The packaging of eukaryotic DNA into chromatin has profound consequences for gene regulation, as well as for other DNA transactions such as recombination, replication and repair. Understanding how this packaging is determined is consequently a pressing problem in molecular genetics. DNA sequence, chromatin remodelers and transcription factors affect chromatin structure, but the scope of these influences on genome-wide nucleosome occupancy patterns remains uncertain. Here, we use high resolution tiling arrays to examine the contributions of two general regulatory factors, Abf1 and Rap1, to nucleosome occupancy in Saccharomyces cerevisiae. These factors have each been shown to bind to a few hundred promoters, but we find here that thousands of loci show localized regions of altered nucleosome occupancy within 1 h of loss of Abf1 or Rap1 binding, and that altered chromatin structure can occur via binding sites having a wide range of affinities. These results indicate that DNA-binding transcription factors affect chromatin structure, and probably dynamics, throughout the genome to a much greater extent than previously appreciated.
PMCID: PMC3064788  PMID: 21081559
20.  NOrMAL: accurate nucleosome positioning using a modified Gaussian mixture model 
Bioinformatics  2012;28(12):i242-i249.
Motivation: Nucleosomes are the basic elements of chromatin structure. They control the packaging of DNA and play a critical role in gene regulation by allowing physical access to transcription factors. The advent of second-generation sequencing has enabled landmark genome-wide studies of nucleosome positions for several model organisms. Current methods to determine nucleosome positioning first compute an occupancy coverage profile by mapping nucleosome-enriched sequenced reads to a reference genome; then, nucleosomes are placed according to the peaks of the coverage profile. These methods are quite accurate on placing isolated nucleosomes, but they do not properly handle more complex configurations. Also, they can only provide the positions of nucleosomes and their occupancy level, whereas it is very beneficial to supply molecular biologists additional information about nucleosomes like the probability of placement, the size of DNA fragments enriched for nucleosomes and/or whether nucleosomes are well positioned or ‘fuzzy’ in the sequenced cell sample.
Results: We address these issues by providing a novel method based on a parametric probabilistic model. An expectation maximization algorithm is used to infer the parameters of the mixture of distributions. We compare the performance of our method on two real datasets against Template Filtering, which is considered the current state-of-the-art. On synthetic data, we show that our method can resolve more accurately complex configurations of nucleosomes, and it is more robust to user-defined parameters. On real data, we show that our method detects a significantly higher number of nucleosomes.
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PMCID: PMC3371838  PMID: 22689767
21.  Regulation of the nucleosome unwrapping rate controls DNA accessibility 
Nucleic Acids Research  2012;40(20):10215-10227.
Eukaryotic genomes are repetitively wrapped into nucleosomes that then regulate access of transcription and DNA repair complexes to DNA. The mechanisms that regulate extrinsic protein interactions within nucleosomes are unresolved. We demonstrate that modulation of the nucleosome unwrapping rate regulates protein binding within nucleosomes. Histone H3 acetyl-lysine 56 [H3(K56ac)] and DNA sequence within the nucleosome entry-exit region additively influence nucleosomal DNA accessibility by increasing the unwrapping rate without impacting rewrapping. These combined epigenetic and genetic factors influence transcription factor (TF) occupancy within the nucleosome by at least one order of magnitude and enhance nucleosome disassembly by the DNA mismatch repair complex, hMSH2–hMSH6. Our results combined with the observation that ∼30% of Saccharomyces cerevisiae TF-binding sites reside in the nucleosome entry–exit region suggest that modulation of nucleosome unwrapping is a mechanism for regulating transcription and DNA repair.
PMCID: PMC3488218  PMID: 22965129
22.  A Non-Homogeneous Hidden-State Model on First Order Differences for Automatic Detection of Nucleosome Positions* 
The ability to map individual nucleosomes accurately across genomes enables the study of relationships between dynamic changes in nucleosome positioning/occupancy and gene regulation. However, the highly heterogeneous nature of nucleosome densities across genomes and short linker regions pose challenges in mapping nucleosome positions based on high-throughput microarray data of micrococcal nuclease (MNase) digested DNA. Previous works rely on additional detrending and careful visual examination to detect low-signal nucleosomes, which may exist in a subpopulation of cells. We propose a non-homogeneous hidden-state model based on first order differences of experimental data along genomic coordinates that bypasses the need for local detrending and can automatically detect nucleosome positions of various occupancy levels. Our proposed approach is applicable to both low and high resolution MNase-Chip and MNase-Seq (high throughput sequencing) data, and is able to map nucleosome-linker boundaries accurately. This automated algorithm is also computationally efficient and only requires a simple preprocessing step. We provide several examples illustrating the pitfalls of existing methods, the difficulties of detrending the observed hybridization signals and demonstrate the advantages of utilizing first order differences in detecting nucleosome occupancies via simulations and case studies involving MNase-Chip and MNase-Seq data of nucleosome occupancy in yeast S. cerevisiae.
PMCID: PMC2861327  PMID: 19572828
nucleosomes; MNase-chip; MNase-Seq; non-homogeneous hidden Markov model; first order differences; smoothing
23.  A Non-Homogeneous Hidden-State Model on First Order Differences for Automatic Detection of Nucleosome Positions 
The ability to map individual nucleosomes accurately across genomes enables the study of relationships between dynamic changes in nucleosome positioning/occupancy and gene regulation. However, the highly heterogeneous nature of nucleosome densities across genomes and short linker regions pose challenges in mapping nucleosome positions based on high-throughput microarray data of micrococcal nuclease (MNase) digested DNA. Previous works rely on additional detrending and careful visual examination to detect low-signal nucleosomes, which may exist in a subpopulation of cells. We propose a non-homogeneous hidden-state model based on first order differences of experimental data along genomic coordinates that bypasses the need for local detrending and can automatically detect nucleosome positions of various occupancy levels. Our proposed approach is applicable to both low and high resolution MNase-Chip and MNase-Seq (high throughput sequencing) data, and is able to map nucleosome-linker boundaries accurately. This automated algorithm is also computationally efficient and only requires a simple preprocessing step. We provide several examples illustrating the pitfalls of existing methods, the difficulties of detrending the observed hybridization signals and demonstrate the advantages of utilizing first order differences in detecting nucleosome occupancies via simulations and case studies involving MNase-Chip and MNase-Seq data of nucleosome occupancy in yeast S. cerevisiae.
PMCID: PMC2861327  PMID: 19572828
24.  Nucleosome Positioning, Nucleosome Spacing and the Nucleosome Code 
Nucleosome positioning has been the subject of intense study for many years. The properties of micrococcal nuclease, the enzyme central to these studies, are discussed. The various methods used to determine nucleosome positions in vitro and in vivo are reviewed critically. These include the traditional low resolution method of indirect end-labelling, high resolution methods such as primer extension, monomer extension and nucleosome sequencing, and the high throughput methods for genome-wide analysis (microarray hybridisation and parallel sequencing). It is established that low resolution mapping yields an averaged chromatin structure, whereas high resolution mapping reveals the weighted superposition of all the chromatin states in a cell population. Mapping studies suggest that yeast DNA contains information specifying the positions of nucleosomes and that this code is made use of by the cell. It is proposed that the positioning code facilitates nucleosome spacing by encoding information for multiple alternative overlapping nucleosomal arrays. Such a code might facilitate the shunting of nucleosomes from one array to another by ATP-dependent chromatin remodelling machines.
PMCID: PMC2935628  PMID: 20232933
25.  Weakly Positioned Nucleosomes Enhance the Transcriptional Competency of Chromatin 
PLoS ONE  2010;5(9):e12984.
Transcription is affected by nucleosomal resistance against polymerase passage. In turn, nucleosomal resistance is determined by DNA sequence, histone chaperones and remodeling enzymes. The contributions of these factors are widely debated: one recent title claims “… DNA-encoded nucleosome organization…” while another title states that “histone-DNA interactions are not the major determinant of nucleosome positions.” These opposing conclusions were drawn from similar experiments analyzed by idealized methods. We attempt to resolve this controversy to reveal nucleosomal competency for transcription.
Methodology/Principal Findings
To this end, we analyzed 26 in vivo, nonlinked, and in vitro genome-wide nucleosome maps/replicates by new, rigorous methods. Individual H2A nucleosomes are reconstituted inaccurately by transcription, chaperones and remodeling enzymes. At gene centers, weakly positioned nucleosome arrays facilitate rapid histone eviction and remodeling, easing polymerase passage. Fuzzy positioning is not due to artefacts. At the regional level, transcriptional competency is strongly influenced by intrinsic histone-DNA affinities. This is confirmed by reproducing the high in vivo occupancy of translated regions and the low occupancy of intergenic regions in reconstitutions from purified DNA and histones. Regional level occupancy patterns are protected from invading histones by nucleosome excluding sequences and barrier nucleosomes at gene boundaries and within genes.
Dense arrays of weakly positioned nucleosomes appear to be necessary for transcription. Weak positioning at exons facilitates temporary remodeling, polymerase passage and hence the competency for transcription. At regional levels, the DNA sequence plays a major role in determining these features but positions of individual nucleosomes are typically modified by transcription, chaperones and enzymes. This competency is reduced at intergenic regions by sequence features, barrier nucleosomes, and proteins, preventing accessibility regulation of untargeted genes. This combination of DNA- and protein-influenced positioning regulates DNA accessibility and competence for regulatory protein binding and transcription. Interactive nucleosome displays are offered at
PMCID: PMC2945322  PMID: 20886052

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