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1.  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.
doi:10.2202/1544-6115.1454
PMCID: PMC2861327  PMID: 19572828
nucleosomes; MNase-chip; MNase-Seq; non-homogeneous hidden Markov model; first order differences; smoothing
2.  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.
doi:10.2202/1544-6115.1454
PMCID: PMC2861327  PMID: 19572828
3.  Micrococcal Nuclease Does Not Substantially Bias Nucleosome Mapping 
Journal of Molecular Biology  2012;417-135(3):152-164.
We have mapped sequence-directed nucleosome positioning on genomic DNA molecules using high-throughput sequencing. Chromatins, prepared by reconstitution with either chicken or frog histones, were separately digested to mononucleosomes using either micrococcal nuclease (MNase) or caspase-activated DNase (CAD). Both enzymes preferentially cleave internucleosomal (linker) DNA, although they do so by markedly different mechanisms. MNase has hitherto been very widely used to map nucleosomes, although concerns have been raised over its potential to introduce bias. Having identified the locations and quantified the strength of both the chicken or frog histone octamer binding sites on each DNA, the results obtained with the two enzymes were compared using a variety of criteria. Both enzymes displayed sequence specificity in their preferred cleavage sites, although the nature of this selectivity was distinct for the two enzymes. In addition, nucleosomes produced by CAD nuclease are 8–10 bp longer than those produced with MNase, with the CAD cleavage sites tending to be 4–5 bp further out from the nucleosomal dyad than the corresponding MNase cleavage sites. Despite these notable differences in cleavage behaviour, the two nucleases identified essentially equivalent patterns of nucleosome positioning sites on each of the DNAs tested, an observation that was independent of the histone type. These results indicate that biases in nucleosome positioning data collected using MNase are, under our conditions, not significant.
Graphical Abstract
Highlights
► We measured nucleosome positioning using two distinct nucleases. ► CAD and MNase provided equivalent positioning profiles. ► The results were independent of DNA and histone type used to prepare chromatin. ► Our data are not consistent with the proposal that MNase provides biased nucleosome positioning measurements.
doi:10.1016/j.jmb.2012.01.043
PMCID: PMC3314939  PMID: 22310051
MNase, micrococcal nuclease; CAD, caspase-activated DNase; BLG, β-lactoglobulin; YRO, yeast replication origin; PDB, Protein Data Bank; caspase-activated DNase; nucleosome positioning; β-lactoglobulin; yeast replication origin; micrococcal nuclease
4.  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.
doi:10.1371/journal.pcbi.1000134
PMCID: PMC2515632  PMID: 18725940
5.  Physical properties of naked DNA influence nucleosome positioning and correlate with transcription start and termination sites in yeast 
BMC Genomics  2011;12:489.
Background
In eukaryotic organisms, DNA is packaged into chromatin structure, where most of DNA is wrapped into nucleosomes. DNA compaction and nucleosome positioning have clear functional implications, since they modulate the accessibility of genomic regions to regulatory proteins. Despite the intensive research effort focused in this area, the rules defining nucleosome positioning and the location of DNA regulatory regions still remain elusive.
Results
Naked (histone-free) and nucleosomal DNA from yeast were digested by microccocal nuclease (MNase) and sequenced genome-wide. MNase cutting preferences were determined for both naked and nucleosomal DNAs. Integration of their sequencing profiles with DNA conformational descriptors derived from atomistic molecular dynamic simulations enabled us to extract the physical properties of DNA on a genomic scale and to correlate them with chromatin structure and gene regulation. The local structure of DNA around regulatory regions was found to be unusually flexible and to display a unique pattern of nucleosome positioning. Ab initio physical descriptors derived from molecular dynamics were used to develop a computational method that accurately predicts nucleosome enriched and depleted regions.
Conclusions
Our experimental and computational analyses jointly demonstrate a clear correlation between sequence-dependent physical properties of naked DNA and regulatory signals in the chromatin structure. These results demonstrate that nucleosome positioning around TSS (Transcription Start Site) and TTS (Transcription Termination Site) (at least in yeast) is strongly dependent on DNA physical properties, which can define a basal regulatory mechanism of gene expression.
doi:10.1186/1471-2164-12-489
PMCID: PMC3224377  PMID: 21981773
DNA physical properties; Molecular dynamics; MNase digestion; nucleosome positioning; gene regulation; chromatin structure
6.  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.
doi:10.1371/journal.pgen.1003036
PMCID: PMC3499251  PMID: 23166509
7.  In Vivo Effects of Histone H3 Depletion on Nucleosome Occupancy and Position in Saccharomyces cerevisiae 
PLoS Genetics  2012;8(6):e1002771.
Previous studies in Saccharomyces cerevisiae established that depletion of histone H4 results in the genome-wide transcriptional de-repression of hundreds of genes. To probe the mechanism of this transcriptional de-repression, we depleted nucleosomes in vivo by conditional repression of histone H3 transcription. We then measured the resulting changes in transcription by RNA–seq and in chromatin organization by MNase–seq. This experiment also bears on the degree to which trans-acting factors and DNA–encoded elements affect nucleosome position and occupancy in vivo. We identified ∼60,000 nucleosomes genome wide, and we classified ∼2,000 as having preferentially reduced occupancy following H3 depletion and ∼350 as being preferentially retained. We found that the in vivo influence of DNA sequences that favor or disfavor nucleosome occupancy increases following histone H3 depletion, demonstrating that nucleosome density contributes to moderating the influence of DNA sequence on nucleosome formation in vivo. To identify factors important for influencing nucleosome occupancy and position, we compared our data to 40 existing whole-genome data sets. Factors associated with promoters, such as histone acetylation and H2A.z incorporation, were enriched at sites of nucleosome loss. Nucleosome retention was linked to stabilizing marks such as H3K36me2. Notably, the chromatin remodeler Isw2 was uniquely associated with retained occupancy and altered positioning, consistent with Isw2 stabilizing histone–DNA contacts and centering nucleosomes on available DNA in vivo. RNA–seq revealed a greater number of de-repressed genes (∼2,500) than previous studies, and these genes exhibited reduced nucleosome occupancy in their promoters. In summary, we identify factors likely to influence nucleosome stability under normal growth conditions and the specific genomic locations at which they act. We find that DNA–encoded nucleosome stability and chromatin composition dictate which nucleosomes will be lost under conditions of limiting histone protein and that this, in turn, governs which genes are susceptible to a loss of regulatory fidelity.
Author Summary
Chromatin is formed by wrapping 146 bp of DNA around a disc-shaped complex of proteins called histones. These protein–DNA structures are known as nucleosomes. Nucleosomes help to regulate gene transcription, because nucleosomes compete with transcription factors for access to DNA. The precise positioning and level of nucleosome occupancy are known to be vital for transcriptional regulation, but the mechanisms that regulate the position and occupancy of nucleosomes are not fully understood. Recently, many studies have focused on the role of DNA sequence and chromatin remodeling proteins. Here, we manipulate the concentration of histone proteins in the cell to determine which nucleosomes are most susceptible to changes in occupancy and position. We find that the chromatin-associated proteins Sir2 and Tup1, and the chromatin remodelers Isw2 and Rsc8, are associated with stabilized nucleosomes. Histone acetylation and incorporation of the histone variant H2A.z are the factors most highly associated with destabilized nucleosomes. Certain DNA sequence properties also contribute to stability. The data identify factors likely to influence nucleosome stability and show a direct link between changes in chromatin and changes in transcription upon histone depletion.
doi:10.1371/journal.pgen.1002771
PMCID: PMC3380831  PMID: 22737086
8.  The Effect of Micrococcal Nuclease Digestion on Nucleosome Positioning Data 
PLoS ONE  2010;5(12):e15754.
Eukaryotic genomes are packed into chromatin, whose basic repeating unit is the nucleosome. Nucleosome positioning is a widely researched area. A common experimental procedure to determine nucleosome positions involves the use of micrococcal nuclease (MNase). Here, we show that the cutting preference of MNase in combination with size selection generates a sequence-dependent bias in the resulting fragments. This strongly affects nucleosome positioning data and especially sequence-dependent models for nucleosome positioning. As a consequence we see a need to re-evaluate whether the DNA sequence is a major determinant of nucleosome positioning in vivo. More generally, our results show that data generated after MNase digestion of chromatin requires a matched control experiment in order to determine nucleosome positions.
doi:10.1371/journal.pone.0015754
PMCID: PMC3012088  PMID: 21206756
9.  Fuzziness and noise in nucleosomal architecture 
Nucleic Acids Research  2014;42(8):4934-4946.
Nucleosome organization plays a key role in the regulation of gene expression. However, despite the striking advances in the accuracy of nucleosome maps, there are still severe discrepancies on individual nucleosome positioning and how this influences gene regulation. The variability among nucleosome maps, which precludes the fine analysis of nucleosome positioning, might emerge from diverse sources. We have carefully inspected the extrinsic factors that may induce diversity by the comparison of microccocal nuclease (MNase)-Seq derived nucleosome maps generated under distinct conditions. Furthermore, we have also explored the variation originated from intrinsic nucleosome dynamics by generating additional maps derived from cell cycle synchronized and asynchronous yeast cultures. Taken together, our study has enabled us to measure the effect of noise in nucleosome occupancy and positioning and provides insights into the underlying determinants. Furthermore, we present a systematic approach that may guide the standardization of MNase-Seq experiments in order to generate reproducible genome-wide nucleosome patterns.
doi:10.1093/nar/gku165
PMCID: PMC4005669  PMID: 24586063
10.  Open chromatin structures regulate the efficiencies of pre-RC formation and replication initiation in Epstein-Barr virus 
The Journal of Cell Biology  2012;198(4):509-528.
Studies of EBV replication origins demonstrate an excess of pre-replication complexes that are formed at flexible MNase-sensitive sites in the genome.
Whether or not metazoan replication initiates at random or specific but flexible sites is an unsolved question. The lack of sequence specificity in origin recognition complex (ORC) DNA binding complicates genome-scale chromatin immunoprecipitation (ChIP)-based studies. Epstein-Barr virus (EBV) persists as chromatinized minichromosomes that are replicated by the host replication machinery. We used EBV to investigate the link between zones of pre-replication complex (pre-RC) assembly, replication initiation, and micrococcal nuclease (MNase) sensitivity at different cell cycle stages in a genome-wide fashion. The dyad symmetry element (DS) of EBV’s latent origin, a well-established and very efficient pre-RC assembly region, served as an internal control. We identified 64 pre-RC zones that correlate spatially with 57 short nascent strand (SNS) zones. MNase experiments revealed that pre-RC and SNS zones were linked to regions of increased MNase sensitivity, which is a marker of origin strength. Interestingly, although spatially correlated, pre-RC and SNS zones were characterized by different features. We propose that pre-RCs are formed at flexible but distinct sites, from which only a few are activated per single genome and cell cycle.
doi:10.1083/jcb.201109105
PMCID: PMC3514025  PMID: 22891264
11.  CENP-A, -B, and -C Chromatin Complex That Contains the I-Type α-Satellite Array Constitutes the Prekinetochore in HeLa Cells 
Molecular and Cellular Biology  2002;22(7):2229-2241.
CENP-A is a component of centromeric chromatin and defines active centromere regions by forming centromere-specific nucleosomes. We have isolated centromeric chromatin containing the CENP-A nucleosome, CENP-B, and CENP-C from HeLa cells using anti-CENP-A and/or anti-CENP-C antibodies and shown that the CENP-A/B/C complex is predominantly formed on α-satellite DNA that contains the CENP-B box (αI-type array). Mapping of hypersensitive sites for micrococcal nuclease (MNase) digestion indicated that CENP-A nucleosomes were phased on the αI-type array as a result of interactions between CENP-B and CENP-B boxes, implying a repetitive configuration for the CENP-B/CENP-A nucleosome complex. Molecular mass analysis by glycerol gradient sedimentation showed that MNase digestion released a CENP-A/B/C chromatin complex of three to four nucleosomes into the soluble fraction, suggesting that CENP-C is a component of the repetitive CENP-B/CENP-A nucleosome complex. Quantitative analysis by immunodepletion of CENP-A nucleosomes showed that most of the CENP-C and approximately half the CENP-B took part in formation of the CENP-A/B/C chromatin complex. A kinetic study of the solubilization of CENPs showed that MNase digestion first released the CENP-A/B/C chromatin complex into the soluble fraction, and later removed CENP-B and CENP-C from the complex. This result suggests that CENP-A nucleosomes form a complex with CENP-B and CENP-C through interaction with DNA. On the basis of these results, we propose that the CENP-A/B/C chromatin complex is selectively formed on the I-type α-satellite array and constitutes the prekinetochore in HeLa cells.
doi:10.1128/MCB.22.7.2229-2241.2002
PMCID: PMC133672  PMID: 11884609
12.  Factorbook.org: a Wiki-based database for transcription factor-binding data generated by the ENCODE consortium 
Nucleic Acids Research  2012;41(Database issue):D171-D176.
The Encyclopedia of DNA Elements (ENCODE) consortium aims to identify all functional elements in the human genome including transcripts, transcriptional regulatory regions, along with their chromatin states and DNA methylation patterns. The ENCODE project generates data utilizing a variety of techniques that can enrich for regulatory regions, such as chromatin immunoprecipitation (ChIP), micrococcal nuclease (MNase) digestion and DNase I digestion, followed by deeply sequencing the resulting DNA. As part of the ENCODE project, we have developed a Web-accessible repository accessible at http://factorbook.org. In Wiki format, factorbook is a transcription factor (TF)-centric repository of all ENCODE ChIP-seq datasets on TF-binding regions, as well as the rich analysis results of these data. In the first release, factorbook contains 457 ChIP-seq datasets on 119 TFs in a number of human cell lines, the average profiles of histone modifications and nucleosome positioning around the TF-binding regions, sequence motifs enriched in the regions and the distance and orientation preferences between motif sites.
doi:10.1093/nar/gks1221
PMCID: PMC3531197  PMID: 23203885
13.  Nucleosomes are enriched at the boundaries of hypomethylated regions (HMRs) in mouse dermal fibroblasts and keratinocytes 
Epigenetics & Chromatin  2014;7(1):34.
Background
The interplay between epigenetic modifications and chromatin structure are integral to our understanding of genome function. Methylation of cytosine (5mC) at CG dinucleotides, traditionally associated with transcriptional repression, is the most highly studied chemical modification of DNA, occurring at over 70% of all CG dinucleotides in the genome. Hypomethylated regions (HMRs) often occur in CG islands (CGIs), however, they also occur outside of CGIs and function as cell-type specific enhancers. During the process of differentiation, reorganization of chromatin and nucleosome arrangement at regulatory regions is thought to occur in order for the establishment of cell-type specific transcriptional programs. However, the specifics regarding the organization of nucleosomes at HMRs and the potential mechanisms regulating nucleosome occupancy in these regions are unknown. Here, we have investigated nucleosome organization around hypomethylated regions (HMRs) identified in two mouse primary cells.
Results
Microccocal nuclease (MNase) digested mononucleosomes from primary cultures of new-born female mouse dermal fibroblasts and keratinocytes were mapped and compared to the HMRs obtained from single base-pair resolution methylomes. In both cell types, we find that nucleosomes are enriched at HMR boundaries. In contrast to the nucleosomes found at boundaries of HMRs in CGIs, HMRs outside of CGIs are calculated to be preferentially bound by nucleosomes, with phased nucleosomes propagating into the methylated region. Nucleosomes are enriched at the tissue-specific HMRs (TS-HMR) boundaries in both cell types suggesting that nucleosome organization surrounding HMR boundaries is independent of methylation status. In addition, we find potential transcription factor (TF) binding sites (E-box motifs) enriched in non-CGI TS-HMR boundaries.
Conclusions
Our results show that intrinsic nucleosome occupancy score (INOS) positively correlate with the nucleosome organization surrounding non-CGI TS-HMRs, suggesting that DNA sequence plays a role in the establishment of HMRs in the genome. Since nucleosomes impact all processes involving the genome, our results provide a link between epigenetic modifications, chromatin structure, and regulatory function.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-8935-7-34) contains supplementary material, which is available to authorized users.
doi:10.1186/1756-8935-7-34
PMCID: PMC4265496  PMID: 25506399
CG methylation; Hypomethylated regions; HMR; Nucleosomes; Epigenomics; Keratinocytes; Fibroblasts
14.  PING 2.0: an R/Bioconductor package for nucleosome positioning using next-generation sequencing data 
Bioinformatics  2013;29(16):2049-2050.
Summary: MNase-Seq and ChIP-Seq have evolved as popular techniques to study chromatin and histone modification. Although many tools have been developed to identify enriched regions, software tools for nucleosome positioning are still limited. We introduce a flexible and powerful open-source R package, PING 2.0, for nucleosome positioning using MNase-Seq data or MNase– or sonicated– ChIP-Seq data combined with either single-end or paired-end sequencing. PING uses a model-based approach, which enables nucleosome predictions even in the presence of low read counts. We illustrate PING using two paired-end datasets from Saccharomyces cerevisiae and compare its performance with nucleR and ChIPseqR.
Availability: PING 2.0 is available from the Bioconductor website at http://bioconductor.org. It can run on Linux, Mac and Windows.
Contact: rgottard@fhcrc.org
Supplementary Information: Supplementary material is available at Bioinformatics online.
doi:10.1093/bioinformatics/btt348
PMCID: PMC3722530  PMID: 23786769
15.  Presence of nucleosomes within irregularly cleaved fragments of newly replicated chromatin. 
Nucleic Acids Research  1984;12(15):6179-6196.
In previous reports (Annunziato et al., J. Biol. Chem., 256:11880-11886 [1981]; Annunziato and Seale, Biochemistry 21:5431-5438 [1982]) we have described two classes of newly replicated chromatin which differ in structure, solubility properties, and requirements for maturation. One class is nucleosomal, soluble at low to intermediate ionic strengths, and acquires mature nucleosomal composition and normal repeat length in the absence of concurrent protein synthesis. In contrast, the other class is cleaved irregularly by MNase (appearing as a smear in DNA gels), is insoluble at moderate ionic strengths, requires protein synthesis to gain normal subunit structure, and comprises approximately 60% of total new chromatin DNA after mild nuclease digestion. It is now demonstrated that this heterogeneous component (produced by the action of either MNase or Hae III on chromatin replicated in cycloheximide) yields nucleosomes when redigested with MNase. The presence of nucleosomes within heterogeneous chromatin fragments suggests that nucleosomal and non-nucleosomal regions may be juxtaposed during chromatin replication. These findings are discussed with respect to current models of nucleosome segregation.
Images
PMCID: PMC320066  PMID: 6089109
16.  NucleoFinder: a statistical approach for the detection of nucleosome positions 
Bioinformatics  2013;29(6):711-716.
Motivation: The identification of nucleosomes along the chromatin is key to understanding their role in the regulation of gene expression and other DNA-related processes. However, current experimental methods (MNase-ChIP, MNase-Seq) sample nucleosome positions from a cell population and contain biases, making thus the precise identification of individual nucleosomes not straightforward. Recent works have only focused on the first point, where noise reduction approaches have been developed to identify nucleosome positions.
Results: In this article, we propose a new approach, termed NucleoFinder, that addresses both the positional heterogeneity across cells and experimental biases by seeking nucleosomes consistently positioned in a cell population and showing a significant enrichment relative to a control sample. Despite the absence of validated dataset, we show that our approach (i) detects fewer false positives than two other nucleosome calling methods and (ii) identifies two important features of the nucleosome organization (the nucleosome spacing downstream of active promoters and the enrichment/depletion of GC/AT dinucleotides at the centre of in vitro nucleosomes) with equal or greater ability than the other two methods.
Availability: The R code of NucleoFinder, an example datafile and instructions are available for download from https://sites.google.com/site/beckerjeremie/
Contact: cholmes@stats.ox.ac.uk
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/bts719
PMCID: PMC3597142  PMID: 23297036
17.  c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts display more decondensed nucleosomal organization than normal fibroblasts 
The Journal of Cell Biology  1990;111(1):9-17.
We have compared the nucleosomal organization of c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts with that of normal fibroblasts by using micrococcal nuclease (MNase) as a probe for the chromatin structure. The bulk chromatin from asynchronously and exponentially growing ras-transformed cells was much more sensitive to MNase digestion than chromatin from the normal cells. Southern hybridization analyses of the MNase digests with probes specific for the ornithine decarboxylase (odc) and c-myc genes showed that the coding and/or 3' end regions of these growth-inducible genes carry a nucleosomal organization both in ras-transformed and normal cells. Studies with cells synchronized by serum starvation showed that in both cell lines the nucleosomal organization of chromatin is relatively condensed at the quiescent state, becomes highly decondensed during the late G1 phase of the cell cycle, and starts again to condense during the S phase. However, in ras-transformed cells the decondensation state stayed much longer than in normal cells. Moreover, irrespective of the phase of the cell cycle the bulk chromatin as well as that of the odc and c-myc genes was more sensitive to MNase digestion in the ras- transformed cell than in the normal fibroblast. Decondensation of the chromatin was also observed in the normal c-Ha-ras protooncogene- transfected cells, but to a lesser extent than in the mutant ras- transformed cells. Whether the increased degree of chromatin decondensation plays a regulatory role in the increased expression of many growth-related genes in the ras-transformed cells remains an interesting object of further study.
PMCID: PMC2116149  PMID: 2195041
18.  Competitive inactivation of a double-strand DNA break site involves parallel suppression of meiosis-induced changes in chromatin configuration. 
Nucleic Acids Research  1999;27(10):2175-2180.
In Saccharomyces cerevisiae, DNA double-strand breaks (DSBs) initiate meiotic recombination at open sites in chromatin, which display a meiosis-specific increase in micrococcal nuclease (MNase) sensitivity. The arg4 promoter contains such a DSB site. When arg4 sequences are placed in a pBR322-derived insert at HIS4 (his4 :: arg4 ), the presence of strong DSB sites in pBR322 sequences leads to an almost complete loss of breaks from the insert-borne arg4 promoter region. Most of the MNase-sensitive sites occurred at similar positions in insert-borne and in normal ARG4 sequences, indicating that hotspot inactivation is not a consequence of changes in nucleosome positioning. However, a meiosis-specific increase in MNase hypersensitivity was no longer detected at the inactive insert-borne arg4 DSB site. Elimination of pBR322 sequences restored DSBs to the insert-borne arg4 promoter region and also restored the meiotic induction of MNase hypersensitivity. Thus, the meiotic induction of MNase hypersensitivity at the DSB sites is suppressed and activated in parallel to DSBs themselves, without changes in the underlying DNA sequence or nucleosome positioning. We suggest that meiosis-specific changes in chromatin at a DSB site are a signal reflecting a pivotal step in DSB formation.
PMCID: PMC148437  PMID: 10219090
19.  Long-Range Nucleosome Ordering Is Associated with Gene Silencing in Drosophila melanogaster Pericentric Heterochromatin 
Molecular and Cellular Biology  2001;21(8):2867-2879.
We have used line HS-2 of Drosophila melanogaster, carrying a silenced transgene in the pericentric heterochromatin, to investigate in detail the chromatin structure imposed by this environment. Digestion of the chromatin with micrococcal nuclease (MNase) shows a nucleosome array with extensive long-range order, indicating regular spacing, and with well-defined MNase cleavage fragments, indicating a smaller MNase target in the linker region. The repeating unit is ca. 10 bp larger than that observed for bulk Drosophila chromatin. The silenced transgene shows both a loss of DNase I-hypersensitive sites and decreased sensitivity to DNase I digestion within an array of nucleosomes lacking such sites; within such an array, sensitivity to digestion by MNase is unchanged. The ordered nucleosome array extends across the regulatory region of the transgene, a shift that could explain the loss of transgene expression in heterochromatin. Highly regular nucleosome arrays are observed over several endogenous heterochromatic sequences, indicating that this is a general feature of heterochromatin. However, genes normally active within heterochromatin (rolled and light) do not show this pattern, suggesting that the altered chromatin structure observed is associated with regions that are silent, rather than being a property of the domain as a whole. The results indicate that long-range nucleosomal ordering is linked with the heterochromatic packaging that imposes gene silencing.
doi:10.1128/MCB.21.8.2867-2879.2001
PMCID: PMC86916  PMID: 11283265
20.  Accurate Prediction of Inducible Transcription Factor Binding Intensities In Vivo 
PLoS Genetics  2012;8(3):e1002610.
DNA sequence and local chromatin landscape act jointly to determine transcription factor (TF) binding intensity profiles. To disentangle these influences, we developed an experimental approach, called protein/DNA binding followed by high-throughput sequencing (PB–seq), that allows the binding energy landscape to be characterized genome-wide in the absence of chromatin. We applied our methods to the Drosophila Heat Shock Factor (HSF), which inducibly binds a target DNA sequence element (HSE) following heat shock stress. PB–seq involves incubating sheared naked genomic DNA with recombinant HSF, partitioning the HSF–bound and HSF–free DNA, and then detecting HSF–bound DNA by high-throughput sequencing. We compared PB–seq binding profiles with ones observed in vivo by ChIP–seq and developed statistical models to predict the observed departures from idealized binding patterns based on covariates describing the local chromatin environment. We found that DNase I hypersensitivity and tetra-acetylation of H4 were the most influential covariates in predicting changes in HSF binding affinity. We also investigated the extent to which DNA accessibility, as measured by digital DNase I footprinting data, could be predicted from MNase–seq data and the ChIP–chip profiles for many histone modifications and TFs, and found GAGA element associated factor (GAF), tetra-acetylation of H4, and H4K16 acetylation to be the most predictive covariates. Lastly, we generated an unbiased model of HSF binding sequences, which revealed distinct biophysical properties of the HSF/HSE interaction and a previously unrecognized substructure within the HSE. These findings provide new insights into the interplay between the genomic sequence and the chromatin landscape in determining transcription factor binding intensity.
Author Summary
Transcription factors (TFs) bind DNA to modulate levels of gene expression. TF binding sites change throughout development, in response to environmental stimuli, and different tissues have distinct TF binding profiles. The mechanism by which TFs discriminate between binding sites in a context dependent manner is an area of active research, but it is clear that the chromatin environment in which potential binding sites reside strongly influences binding. This study used the Heat Shock TF (HSF) to study the effect chromatin has upon induced HSF binding. We implemented an experimental technique to quantify all potential HSF binding sites in the genome. These data were incorporated into a modeling framework along with chromatin landscape information prior to HSF binding to accurately predict the intensities of inducible HSF binding sites. DNase I hypersensitivity and tetra-acetylation of H4 were the most influential covariates in the model. The binding data enabled the development of a more complete HSF/DNA interaction model, providing insight into the biophysical interaction of HSF trimer subunits and target DNA pentamers.
doi:10.1371/journal.pgen.1002610
PMCID: PMC3315474  PMID: 22479205
21.  Multiple protein-DNA interactions over the yeast HSC82 heat shock gene promoter. 
Nucleic Acids Research  1995;23(10):1822-1829.
We have utilized DNase I and micrococcal nuclease (MNase) to map the chromatin structure of the HSC82 heat shock gene of Saccharomyces cerevisiae. The gene is expressed at a high basal level which is enhanced 2-3-fold by thermal stress. A single, heat-shock invariant DNase I hypersensitive domain is found within the HSC82 chromosomal locus; it maps to the gene's 5' end and spans 250 bp of promoter sequence. DNase I genomic footprinting reveals that within this hypersensitive region are four constitutive protein-DNA interactions. These map to the transcription initiation site, the TATA box, the promoter-distal heat shock element (HSE1) and a consensus GRF2 (REB1/Factor Y) sequence. However, two other potential regulatory sites, the promoter-proximal heat shock element (HSE0) and a consensus upstream repressor sequence (URS1), are not detectably occupied under either transcriptional state. In contrast to its sensitivity to DNAase I, the nucleosome-free promoter region is relatively protected from MNase; the enzyme excises a stable nucleoprotein fragment of approximately 210 bp. As detected by MNase, there are at least two sequence-positioned nucleosomes arrayed 5' of the promoter; regularly spaced nucleosomes exhibiting an average repeat length of 160-170 bp span several kilobases of both upstream and downstream regions. Similarly, the body of the gene, which exhibits heightened sensitivity to DNase I, displays a nucleosomal organization under both basal and induced states, but these nucleosomes are not detectably positioned with respect to the underlying DNA sequence and may be irregularly spaced and/or structurally altered. We present a model of the chromatin structure of HSC82 and compare it to one previously derived for the closely related, but differentially regulated, HSP82 heat shock gene.
Images
PMCID: PMC306942  PMID: 7784189
22.  Butyrate-induced changes in nuclease sensitivity of chromatin cannot be correlated with transcriptional activation. 
Molecular and Cellular Biology  1987;7(11):3863-3870.
We examined in the H4IIE rat hepatoma cell line the relationship between butyrate-induced changes in the nuclease sensitivity of chromatin and changes in transcriptional activity of specific genes. The butyrate-inducible metallothionein I (MT-I) gene underwent a dramatic increase in DNase I sensitivity after 3 h of butyrate treatment. However, genes not transcribed in H4IIE cells underwent the same changes in DNase I sensitivity. Thus, butyrate-induced increases in DNase I sensitivity are not sufficient for the transcriptional activation of a gene. Butyrate treatment has also been reported to alter the sensitivity of sequences to micrococcal nuclease (MNase) in a manner reflecting their tissue-specific expression. Butyrate exposure caused increased digestion of the MT-I gene by MNase. However, butyrate-induced MNase sensitivity also occurred for genes which are neither transcribed in untreated cells nor butyrate inducible. Moreover, cadmium, a potent transcriptional activator of the MT-I gene, does not alter the sensitivity of the MT-I gene to MNase. Thus, the butyrate-induced alterations in MNase sensitivity are neither sufficient for, necessary for, nor indicative of transcriptional activation.
Images
PMCID: PMC368053  PMID: 3431545
23.  Chromatin particle spectrum analysis: a method for comparative chromatin structure analysis using paired-end mode next-generation DNA sequencing 
Nucleic Acids Research  2010;39(5):e26.
Microarray and next-generation sequencing techniques which allow whole genome analysis of chromatin structure and sequence-specific protein binding are revolutionizing our view of chromosome architecture and function. However, many current methods in this field rely on biochemical purification of highly specific fractions of DNA prepared from chromatin digested with either micrococcal nuclease or DNaseI and are restricted in the parameters they can measure. Here, we show that a broad size-range of genomic DNA species, produced by partial micrococcal nuclease digestion of chromatin, can be sequenced using paired-end mode next-generation technology. The paired sequence reads, rather than DNA molecules, can then be size-selected and mapped as particle classes to the target genome. Using budding yeast as a model, we show that this approach reveals position and structural information for a spectrum of nuclease resistant complexes ranging from transcription factor-bound DNA elements up to mono- and poly-nucleosomes. We illustrate the utility of this approach in visualizing the MNase digestion landscape of protein-coding gene transcriptional start sites, and demonstrate a comparative analysis which probes the function of the chromatin-remodelling transcription factor Cbf1p.
doi:10.1093/nar/gkq1183
PMCID: PMC3061068  PMID: 21131275
24.  TRF2 Controls Telomeric Nucleosome Organization in a Cell Cycle Phase-Dependent Manner 
PLoS ONE  2012;7(4):e34386.
Mammalian telomeres stabilize chromosome ends as a result of their assembly into a peculiar form of chromatin comprising a complex of non-histone proteins named shelterin. TRF2, one of the shelterin components, binds to the duplex part of telomeric DNA and is essential to fold the telomeric chromatin into a protective cap. Although most of the human telomeric DNA is organized into tightly spaced nucleosomes, their role in telomere protection and how they interplay with telomere-specific factors in telomere organization is still unclear. In this study we investigated whether TRF2 can regulate nucleosome assembly at telomeres.
By means of chromatin immunoprecipitation (ChIP) and Micrococcal Nuclease (MNase) mapping assay, we found that the density of telomeric nucleosomes in human cells was inversely proportional to the dosage of TRF2 at telomeres. This effect was not observed in the G1 phase of the cell cycle but appeared coincident of late or post-replicative events. Moreover, we showed that TRF2 overexpression altered nucleosome spacing at telomeres increasing internucleosomal distance. By means of an in vitro nucleosome assembly system containing purified histones and remodeling factors, we reproduced the short nucleosome spacing found in telomeric chromatin. Importantly, when in vitro assembly was performed in the presence of purified TRF2, nucleosome spacing on a telomeric DNA template increased, in agreement with in vivo MNase mapping.
Our results demonstrate that TRF2 negatively regulates the number of nucleosomes at human telomeres by a cell cycle-dependent mechanism that alters internucleosomal distance. These findings raise the intriguing possibility that telomere protection is mediated, at least in part, by the TRF2-dependent regulation of nucleosome organization.
doi:10.1371/journal.pone.0034386
PMCID: PMC3335031  PMID: 22536324
25.  DNA-encoded nucleosome occupancy is associated with transcription levels in the human malaria parasite Plasmodium falciparum 
BMC Genomics  2014;15(1):347.
Background
In eukaryotic organisms, packaging of DNA into nucleosomes controls gene expression by regulating access of the promoter to transcription factors. The human malaria parasite Plasmodium falciparum encodes relatively few transcription factors, while extensive nucleosome remodeling occurs during its replicative cycle in red blood cells. These observations point towards an important role of the nucleosome landscape in regulating gene expression. However, the relation between nucleosome positioning and transcriptional activity has thus far not been explored in detail in the parasite.
Results
Here, we analyzed nucleosome positioning in the asexual and sexual stages of the parasite’s erythrocytic cycle using chromatin immunoprecipitation of MNase-digested chromatin, followed by next-generation sequencing. We observed a relatively open chromatin structure at the trophozoite and gametocyte stages, consistent with high levels of transcriptional activity in these stages. Nucleosome occupancy of genes and promoter regions were subsequently compared to steady-state mRNA expression levels. Transcript abundance showed a strong inverse correlation with nucleosome occupancy levels in promoter regions. In addition, AT-repeat sequences were strongly unfavorable for nucleosome binding in P. falciparum, and were overrepresented in promoters of highly expressed genes.
Conclusions
The connection between chromatin structure and gene expression in P. falciparum shares similarities with other eukaryotes. However, the remarkable nucleosome dynamics during the erythrocytic stages and the absence of a large variety of transcription factors may indicate that nucleosome binding and remodeling are critical regulators of transcript levels. Moreover, the strong dependency between chromatin structure and DNA sequence suggests that the P. falciparum genome may have been shaped by nucleosome binding preferences. Nucleosome remodeling mechanisms in this deadly parasite could thus provide potent novel anti-malarial targets.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-347) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-347
PMCID: PMC4035074  PMID: 24885191
Malaria; Cell cycle; Nucleosome; Transcription; Sequence

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