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Jonathan Widom - One of the best experts on this subject based on the ideXlab platform.
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chemical map of schizosaccharomyces pombe reveals species specific features in Nucleosome Positioning
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Georgette Moyleheyrman, Jonathan Widom, Tetiana Zaichuk, Quanwei Zhang, Olke C Uhlenbeck, Robert A Holmgren, Jiping WangAbstract:Using a recently developed chemical approach, we have generated a genome-wide map of Nucleosomes in vivo in Schizosaccharomyces pombe (S. pombe) at base pair resolution. The shorter linker length previously identified in S. pombe is due to a preponderance of Nucleosomes separated by ∼4/5 bp, placing Nucleosomes on opposite faces of the DNA. The periodic dinucleotide feature thought to position Nucleosomes is equally strong in exons as in introns, demonstrating that Nucleosome Positioning information can be superimposed on coding information. Unlike the case in Saccharomyces cerevisiae, A/T-rich sequences are enriched in S. pombe Nucleosomes, particularly at ±20 bp around the dyad. This difference in Nucleosome binding preference gives rise to a major distinction downstream of the transcription start site, where Nucleosome phasing is highly predictable by A/T frequency in S. pombe but not in S. cerevisiae, suggesting that the genomes and DNA binding preferences of Nucleosomes have coevolved in different species. The poly (dA-dT) tracts affect but do not deplete Nucleosomes in S. pombe, and they prefer special rotational positions within the Nucleosome, with longer tracts enriched in the 10- to 30-bp region from the dyad. S. pombe does not have a well-defined Nucleosome-depleted region immediately upstream of most transcription start sites; instead, the −1 Nucleosome is positioned with the expected spacing relative to the +1 Nucleosome, and its occupancy is negatively correlated with gene expression. Although there is generally very good agreement between Nucleosome maps generated by chemical cleavage and micrococcal nuclease digestion, the chemical map shows consistently higher Nucleosome occupancy on DNA with high A/T content.
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what controls Nucleosome positions
Trends in Genetics, 2009Co-Authors: Eran Segal, Jonathan WidomAbstract: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.
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distinct modes of regulation by chromatin encoded through Nucleosome Positioning signals
PLOS Computational Biology, 2008Co-Authors: Yair Field, Irene K. Moore, Jonathan Widom, N Kaplan, Yvonne N Fondufemittendorf, Eilon Sharon, Yaniv Lubling, Eran SegalAbstract:The detailed positions of Nucleosomes profoundly impact gene regulation and are partly encoded by the genomic DNA sequence. However, less is known about the functional consequences of this encoding. Here, we address this question using a genome-wide map of ∼380,000 yeast Nucleosomes that we sequenced in their entirety. Utilizing the high resolution of our map, we refine our understanding of how Nucleosome organizations are encoded by the DNA sequence and demonstrate that the genomic sequence is highly predictive of the in vivo Nucleosome organization, even across new Nucleosome-bound sequences that we isolated from fly and human. We find that Poly(dA:dT) tracts are an important component of these Nucleosome Positioning signals and that their Nucleosome-disfavoring action results in large Nucleosome depletion over them and over their flanking regions and enhances the accessibility of transcription factors to their cognate sites. Our results suggest that the yeast genome may utilize these Nucleosome Positioning signals to regulate gene expression with different transcriptional noise and activation kinetics and DNA replication with different origin efficiency. These distinct functions may be achieved by encoding both relatively closed (Nucleosome-covered) chromatin organizations over some factor binding sites, where factors must compete with Nucleosomes for DNA access, and relatively open (Nucleosome-depleted) organizations over other factor sites, where factors bind without competition.
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archaeal histone selection of Nucleosome Positioning sequences and the procaryotic origin of histone dependent genome evolution
Journal of Molecular Biology, 2000Co-Authors: Kathryn A Bailey, Jonathan Widom, Suzette L Pereira, John N ReeveAbstract:Archaeal histones and the eucaryal (eucaryotic) Nucleosome core histones have almost identical histone folds. Here, we show that DNA molecules selectively incorporated by rHMfB (recombinant archaeal histone B from Methanothermus fervidus) into archaeal Nucleosomes from a mixture of approximately 10(14) random sequence molecules contain sequence motifs shown previously to direct eucaryal Nucleosome Positioning. The dinucleotides GC, AA (=TT) and TA are repeated at approximately 10 bp intervals, with the GC harmonic displaced approximately 5 bp from the AA and TA harmonics [(GCN(3)AA or TA)(n)]. AT and CG were not strongly selected, indicating that TA not equalAT and GC not equalCG in terms of facilitating archaeal Nucleosome assembly. The selected molecules have affinities for rHMfB ranging from approximately 9 to 18-fold higher than the level of affinity of the starting population, and direct the positioned assembly of archaeal Nucleosomes. Fourier-transform analyses have revealed that AA dinucleotides are much enriched at approximately 10. 1 bp intervals, the helical repeat of DNA wrapped around a Nucleosome, in the genomes of Eucarya and the histone-containing Euryarchaeota, but not in the genomes of Bacteria and Crenarchaeota, procaryotes that do not have histones. Facilitating histone packaging of genomic DNA has apparently therefore imposed constraints on genome sequence evolution, and since archaeal histones have no structure in addition to the histone fold, these constraints must result predominantly from histone fold-DNA contacts. Based on the three-domain universal phylogeny, histones and histone-dependent genome sequence evolution most likely evolved after the bacterial-archaeal divergence but before the archaeal-eucaryal divergence, and were subsequently lost in the Crenarchaeota. However, with lateral gene transfer, the first histone fold could alternatively have evolved after the archaeal-eucaryal divergence, early in either the euryarchaeal or eucaryal lineages.
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Nucleosome PACKAGING AND Nucleosome Positioning OF GENOMIC DNA
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: P. T. Lowary, Jonathan WidomAbstract:The goals of this study were to assess the extent to which bulk genomic DNA sequences contribute to their own packaging in Nucleosomes and to reveal the relationship between Nucleosome packaging and Positioning. Using a competitive Nucleosome reconstitution assay, we found that at least 95% of bulk DNA sequences have an affinity for histone octamer in Nucleosomes that is similar to that of randomly synthesized DNA; they contribute little to their own packaging at the level of individual Nucleosomes. An equation was developed that relates the measured free energy to the fractional occupancy of specific Nucleosome positions. Evidently, the bulk of eukaryotic genomic DNA is also not evolved or constrained for significant sequence-directed Nucleosome Positioning at the level of individual Nucleosomes. Implications for gene regulation in vivo are discussed.
Philipp Korber - One of the best experts on this subject based on the ideXlab platform.
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The nuclear actin-containing Arp8 module is a linker DNA sensor driving INO80 chromatin remodeling
Nature Structural & Molecular Biology, 2018Co-Authors: Kilian R. Knoll, Sebastian Eustermann, Vanessa Niebauer, Elisa Oberbeckmann, Gabriele Stoehr, Kevin Schall, Alessandro Tosi, Marianne Schwarz, Andrea Buchfellner, Philipp KorberAbstract:X-ray crystal structures and mutational analysis of the Arp8 module of the yeast chromatin remodeler INO80 reveal its function as a linker DNA sensor required for Nucleosome Positioning. Nuclear actin (N-actin) and actin-related proteins (Arps) are critical components of several chromatin modulating complexes, including the chromatin remodeler INO80, but their function is largely elusive. Here, we report the crystal structure of the 180-kDa Arp8 module of Saccharomyces cerevisiae INO80 and establish its role in recognition of extranucleosomal linker DNA. Arp8 engages N-actin in a manner distinct from that of other actin-fold proteins and thereby specifies recruitment of the Arp4–N-actin heterodimer to a segmented scaffold of the helicase-SANT-associated (HSA) domain of Ino80. The helical HSA domain spans over 120 Å and provides an extended binding platform for extranucleosomal entry DNA that is required for Nucleosome sliding and genome-wide Nucleosome Positioning. Together with the recent cryo-electron microscopy structure of INO80^Core–Nucleosome complex, our findings suggest an allosteric mechanism by which INO80 senses 40-bp linker DNA to conduct highly processive chromatin remodeling.
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chd1 remodelers regulate Nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in s pombe
The EMBO Journal, 2012Co-Authors: Julia Pointner, Annelie Stralfors, Karl Ekwall, Jenna Persson, Punit Prasad, Ulrika Normanaxelsson, Olga Khorosjutina, Nils Krietenstein, Peter J Svensson, Philipp KorberAbstract:Nucleosome Positioning governs access to eukaryotic genomes. Many genes show a stereotypic organisation at their 5′end: a Nucleosome free region just upstream of the transcription start site (TSS) followed by a regular nucleosomal array over the coding region. The determinants for this pattern are unclear, but Nucleosome remodelers are likely critical. Here we study the role of remodelers in global Nucleosome Positioning in S. pombe and the corresponding changes in expression. We find a striking evolutionary shift in remodeler usage between budding and fission yeast. The S. pombe RSC complex does not seem to be involved in Nucleosome Positioning, despite its prominent role in S. cerevisiae. While S. pombe lacks ISWI-type remodelers, it has two CHD1-type ATPases, Hrp1 and Hrp3. We demonstrate Nucleosome spacing activity for Hrp1 and Hrp3 in vitro, and that together they are essential for linking regular genic arrays to most TSSs in vivo. Impaired arrays in the absence of either or both remodelers may lead to increased cryptic antisense transcription, but overall gene expression levels are only mildly affected.
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a packing mechanism for Nucleosome organization reconstituted across a eukaryotic genome
Science, 2011Co-Authors: Zhenhai Zhang, Philipp Korber, Christian J Wippo, Megha Wal, Elissa Ward, Franklin B PughAbstract:Near the 5′ end of most eukaryotic genes, Nucleosomes form highly regular arrays that begin at canonical distances from the transcriptional start site. Determinants of this and other aspects of genomic Nucleosome organization have been ascribed to statistical Positioning, intrinsically DNA-encoded Positioning, or some aspect of transcription initiation. Here, we provide evidence for a different explanation. Biochemical reconstitution of proper Nucleosome Positioning, spacing, and occupancy levels was achieved across the 5′ ends of most yeast genes by adenosine triphosphate–dependent trans-acting factors. These transcription-independent activities override DNA-intrinsic Positioning and maintain uniform spacing at the 5′ ends of genes even at low Nucleosome densities. Thus, an active, nonstatistical Nucleosome packing mechanism creates chromatin organizing centers at the 5′ ends of genes where important regulatory elements reside.
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schizosaccharomyces pombe genome wide Nucleosome mapping reveals Positioning mechanisms distinct from those of saccharomyces cerevisiae
Nature Structural & Molecular Biology, 2010Co-Authors: Alexandra Lantermann, Guocheng Yuan, Tobias Straub, Annelie Stralfors, Karl Ekwall, Philipp KorberAbstract:Nucleosome occupancy can affect the accessibility of DNA to other factors. A genome-wide map of Nucleosomes in Schizosaccharomyces pombe is now presented. Comparisons to published Saccharomyces cerevisiae maps reveal species-specific differences arguing for evolutionary plasticity of Nucleosome Positioning mechanisms. Positioned Nucleosomes limit the access of proteins to DNA and implement regulatory features encoded in eukaryotic genomes. Here we have generated the first genome-wide Nucleosome Positioning map for Schizosaccharomyces pombe and annotated transcription start and termination sites genome wide. Using this resource, we found surprising differences from the previously published Nucleosome organization of the distantly related yeast Saccharomyces cerevisiae. DNA sequence guides Nucleosome Positioning differently: for example, poly(dA-dT) elements are not enriched in S. pombe Nucleosome-depleted regions. Regular nucleosomal arrays emanate more asymmetrically—mainly codirectionally with transcription—from promoter Nucleosome-depleted regions, but promoters harboring the histone variant H2A.Z also show regular arrays upstream of these regions. Regular Nucleosome phasing in S. pombe has a very short repeat length of 154 base pairs and requires a remodeler, Mit1, that is conserved in humans but is not found in S. cerevisiae. Nucleosome Positioning mechanisms are evidently not universal but evolutionarily plastic.
Naama Barkai - One of the best experts on this subject based on the ideXlab platform.
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divergence of Nucleosome Positioning between two closely related yeast species genetic basis and functional consequences
Molecular Systems Biology, 2010Co-Authors: Itay Tirosh, Nadejda Sigal, Naama BarkaiAbstract:Gene regulation differs greatly between related species, constituting a major source of phenotypic diversity. Recent studies characterized extensive differences in the gene expression programs of closely related species. In contrast, virtually nothing is known about the evolution of chromatin structure and how it influences the divergence of gene expression. Here, we compare the genome-wide Nucleosome Positioning of two closely related yeast species and, by profiling their inter-specific hybrid, trace the genetic basis of the observed differences into mutations affecting the local DNA sequences (cis effects) or the upstream regulators (trans effects). The majority (approximately 70%) of inter-species differences is due to cis effects, leaving a significant contribution (30%) for trans factors. We show that cis effects are well explained by mutations in Nucleosome-disfavoring AT-rich sequences, but are not associated with divergence of Nucleosome-favoring sequences. Differences in Nucleosome Positioning propagate to multiple adjacent Nucleosomes, supporting the statistical Positioning hypothesis, and we provide evidence that Nucleosome-free regions, but not the +1 Nucleosome, serve as stable border elements. Surprisingly, although we find that differential Nucleosome Positioning among cell types is strongly correlated with differential expression, this does not seem to be the case for evolutionary changes: divergence of Nucleosome Positioning is excluded from regulatory elements and is not correlated with gene expression divergence, suggesting a primarily neutral mode of evolution. Our results provide evolutionary insights to the genetic determinants and regulatory function of Nucleosome Positioning.
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divergence of Nucleosome Positioning between two closely related yeast species genetic basis and functional consequences
Molecular Systems Biology, 2010Co-Authors: Itay Tirosh, Nadejda Sigal, Naama BarkaiAbstract:Gene regulation differs greatly between related species, constituting a major source of phenotypic diversity. Recent studies characterized extensive differences in the gene expression programs of closely related species. In contrast, virtually nothing is known about the evolution of chromatin structure and how it influences the divergence of gene expression. Here, we compare the genomewide Nucleosome Positioning of two closely related yeast species and, by profiling their interspecific hybrid, trace the genetic basis of the observed differences into mutations affecting the local DNA sequences (cis effects) or the upstream regulators (trans effects). The majority (B70%) of inter-species differences is due to cis effects, leaving a significant contribution (30%) for trans factors. We show that cis effects are well explained by mutations in Nucleosome-disfavoring AT-rich sequences,but arenot associated with divergence of Nucleosome-favoring sequences.Differences in Nucleosome Positioning propagate to multiple adjacent Nucleosomes, supporting the statistical Positioning hypothesis, and we provide evidence that Nucleosome-free regions, but not the þ1 Nucleosome, serve as stable border elements. Surprisingly, although we find that differential Nucleosome Positioning among cell types is strongly correlated with differential expression, this does not seem to be the case for evolutionary changes: divergence of Nucleosome Positioning is excluded from regulatory elements and is not correlated with gene expression divergence, suggesting a primarily neutral mode of evolution. Our results provide evolutionary insights to the genetic determinants and regulatory function of Nucleosome Positioning.
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two strategies for gene regulation by promoter Nucleosomes
Genome Research, 2008Co-Authors: Itay Tirosh, Naama BarkaiAbstract:Chromatin structure is central for the regulation of gene expression, but its genome-wide organization is only beginning to be understood. Here, we examine the connection between patterns of Nucleosome occupancy and the capacity to modulate gene expression upon changing conditions, i.e., transcriptional plasticity. By analyzing genome-wide data of Nucleosome Positioning in yeast, we find that the presence of Nucleosomes close to the transcription start site is associated with high transcriptional plasticity, while Nucleosomes at more distant upstream positions are negatively correlated with transcriptional plasticity. Based on this, we identify two typical promoter structures associated with low or high plasticity, respectively. The first class is characterized by a relatively large Nucleosome-free region close to the start site coupled with well-positioned Nucleosomes further upstream, whereas the second class displays a more evenly distributed and dynamic Nucleosome Positioning, with high occupancy close to the start site. The two classes are further distinguished by multiple promoter features, including histone turnover, binding site locations, H2A.Z occupancy, expression noise, and expression diversity. Analysis of Nucleosome Positioning in human promoters reproduces the main observations. Our results suggest two distinct strategies for gene regulation by chromatin, which are selectively employed by different genes.
Oliver J Rando - One of the best experts on this subject based on the ideXlab platform.
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a functional evolutionary approach to identify determinants of Nucleosome Positioning a unifying model for establishing the genome wide pattern
Molecular Cell, 2012Co-Authors: Amanda L Hughes, Oliver J Rando, Yi Jin, Kevin StruhlAbstract:Although the genomic pattern of Nucleosome Positioning is broadly conserved, quantitative aspects vary over evolutionary timescales. We identify the cis and trans determinants of Nucleosome Positioning using a functional evolutionary approach involving S. cerevisiae strains containing large genomic regions from other yeast species. In a foreign species, Nucleosome depletion at promoters is maintained over poly(dA:dT) tracts, whereas interNucleosome spacing and all other aspects of Nucleosome Positioning tested are not. Interestingly, the locations of the +1 Nucleosome and RNA start sites shift in concert. Strikingly, in a foreign species, Nucleosome-depleted regions occur fortuitously in coding regions, and they often act as promoters that are associated with a positioned Nucleosome array linked to the length of the transcription unit. We suggest a three-step model in which Nucleosome remodelers, general transcription factors, and the transcriptional elongation machinery are primarily involved in generating the Nucleosome Positioning pattern in vivo.
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the role of Nucleosome Positioning in the evolution of gene regulation
PLoS, 2010Co-Authors: Alexander M Tsankov, Dawn A Thompson, Amanda Socha, Aviv Regev, Oliver J RandoAbstract:Chromatin organization plays a major role in gene regulation and can affect the function and evolution of new transcriptional programs. However, it can be difficult to decipher the basis of changes in chromatin organization and their functional effect on gene expression. Here, we present a large-scale comparative genomic analysis of the relationship between chromatin organization and gene expression, by measuring mRNA abundance and Nucleosome positions genome-wide in 12 Hemiascomycota yeast species. We found substantial conservation of global and functional chromatin organization in all species, including prominent Nucleosome-free regions (NFRs) at gene promoters, and distinct chromatin architecture in growth and stress genes. Chromatin organization has also substantially diverged in both global quantitative features, such as spacing between adjacent Nucleosomes, and in functional groups of genes. Expression levels, intrinsic antinucleosomal sequences, and trans-acting chromatin modifiers all play important, complementary, and evolvable roles in determining NFRs. We identify five mechanisms that couple chromatin organization to evolution of gene regulation and have contributed to the evolution of respiro-fermentation and other key systems, including (1) compensatory evolution of alternative modifiers associated with conserved chromatin organization, (2) a gradual transition from constitutive to transregulated NFRs, (3) a loss of intrinsic anti-nucleosomal sequences accompanying changes in chromatin organization and gene expression, (4) re-Positioning of motifs from NFRs to Nucleosome-occluded regions, and (5) the expanded use of NFRs by paralogous activator-repressor pairs. Our study sheds light on the molecular basis of chromatin organization, and on the role of chromatin organization in the evolution of gene regulation.
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Nucleosome Positioning how is it established and why does it matter
Developmental Biology, 2010Co-Authors: Marta Radmanlivaja, Oliver J RandoAbstract:Packaging of eukaryotic genomes into chromatin affects every process that occurs on DNA. The Positioning of Nucleosomes on underlying DNA plays a key role in the regulation of these processes, as the Nucleosome occludes underlying DNA sequences. Here, we review the literature on mapping Nucleosome positions in various organisms, and discuss how Nucleosome positions are established, what effect Nucleosome Positioning has on control of gene expression, and touch on the correlations between chromatin packaging, sequence evolution, and the evolution of gene expression programs.
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genome scale identification of Nucleosome positions in s cerevisiae
Science, 2005Co-Authors: Guocheng Yuan, Yuenjong Liu, Michael F Dion, Michael Slack, Steven J Altschuler, Oliver J RandoAbstract:The Positioning of Nucleosomes along chromatin has been implicated in the regulation of gene expression in eukaryotic cells, because packaging DNA into Nucleosomes affects sequence accessibility. We developed a tiled microarray approach to identify at high resolution the translational positions of 2278 Nucleosomes over 482 kilobases of Saccharomyces cerevisiae DNA, including almost all of chromosome III and 223 additional regulatory regions. The majority of the Nucleosomes identified were well-positioned. We found a stereotyped chromatin organization at Pol II promoters consisting of a Nucleosome-free region ∼200 base pairs upstream of the start codon flanked on both sides by positioned Nucleosomes. The Nucleosome-free sequences were evolutionarily conserved and were enriched in poly-deoxyadenosine or poly-deoxythymidine sequences. Most occupied transcription factor binding motifs were devoid of Nucleosomes, strongly suggesting that Nucleosome Positioning is a global determinant of transcription factor access.
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genome scale identification of Nucleosome positions in s cerevisiae
Science, 2005Co-Authors: Guocheng Yuan, Yuenjong Liu, Michael F Dion, Michael Slack, Steven J Altschuler, Oliver J RandoAbstract:The Positioning of Nucleosomes along chromatin has been implicated in the regulation of gene expression in eukaryotic cells, because packaging DNA into Nucleosomes affects sequence accessibility. We developed a tiled microarray approach to identify at high resolution the translational positions of 2278 Nucleosomes over 482 kilobases of Saccharomyces cerevisiae DNA, including almost all of chromosome III and 223 additional regulatory regions. The majority of the Nucleosomes identified were well-positioned. We found a stereotyped chromatin organization at Pol II promoters consisting of a Nucleosome-free region approximately 200 base pairs upstream of the start codon flanked on both sides by positioned Nucleosomes. The Nucleosome-free sequences were evolutionarily conserved and were enriched in poly-deoxyadenosine or poly-deoxythymidine sequences. Most occupied transcription factor binding motifs were devoid of Nucleosomes, strongly suggesting that Nucleosome Positioning is a global determinant of transcription factor access.
Julia R. Widom - One of the best experts on this subject based on the ideXlab platform.
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measurement of histone dna interaction free energy in Nucleosomes
Methods, 2004Co-Authors: A Thastrom, P. T. Lowary, Julia R. WidomAbstract:Nucleosome Positioning DNA sequences are of increasing interest because of their proposed roles in gene regulation and other chromosome functions in vivo, and because they have revealed new insights into the sequence-dependent structures and mechanics of DNA itself. Here, we describe methods to quantify the relative affinities of histone-DNA interactions in Nucleosomes, i.e., the Nucleosome Positioning power of differing DNA sequences. We review methods developed by others and then discuss in detail our own approach to measurement of histone-DNA interaction free energies. Compared to earlier methods, our dialysis-based approach reduces the possibility that non-equilibrium or irreproducible results could be obtained. It facilitates a direct comparison of free energies for many sequences at the same time and it allows analysis of DNAs having a wide range of relative affinities.
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spontaneous access of proteins to buried nucleosomal dna target sites occurs via a mechanism that is distinct from Nucleosome translocation
Molecular and Cellular Biology, 2002Co-Authors: J D Anderson, A Thastrom, Julia R. WidomAbstract:Intrinsic Nucleosome dynamics termed "site exposure" provides spontaneous and cooperative access to buried regions of nucleosomal DNA in vitro. Two different mechanisms for site exposure have been proposed, one based on Nucleosome translocation, the other on dynamic Nucleosome conformational changes in which a stretch of the nucleosomal DNA is transiently released off the histone surface. Here we report on three experiments that distinguish between these mechanisms. One experiment investigates the effects on the accessibilities of restriction enzyme target sites inside Nucleosomes when extra DNA (onto which the Nucleosome may move at low energetic cost) is appended onto one end. The other two experiments test directly for Nucleosome mobility under the conditions used to probe accessibility to restriction enzymes: one on a selected nonnatural Nucleosome Positioning sequence, the other on the well-studied 5S rRNA gene Nucleosome Positioning sequence. We find from all three assays that restriction enzymes gain access to sites throughout the entire length of the nucleosomal DNA without contribution from Nucleosome translocation. We conclude that site exposure in Nucleosomes in vitro occurs via a Nucleosome conformational change that leads to transient release of a stretch of DNA from the histone surface, most likely involving progressive uncoiling from an end. Recapture at a distal site along DNA that has partially uncoiled would result in looped structures which are believed to contribute to RNA polymerase elongation and may contribute to spontaneous or ATP-driven Nucleosome mobility. Transient open states may facilitate the initial entry of transcription factors and enzymes in vivo.
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New DNA sequence rules for high affinity binding to histone octamer and sequence-directed Nucleosome Positioning.
Journal of molecular biology, 1998Co-Authors: P. T. Lowary, Julia R. WidomAbstract:DNA sequences that position Nucleosomes are of increasing interest because of their relationship to gene regulation in vivo and because of their utility in studies of Nucleosome structure and function in vitro. However, at present our understanding of the rules for DNA sequence-directed Nucleosome Positioning is fragmentary, and existing Positioning sequences have many limitations. We carried out a SELEX experiment starting with a large pool of chemically synthetic random. DNA molecules to identify those individuals having the highest affinity for histone octamer. A set of highest-affinity molecules were selected, cloned, and sequenced, their affinities (free energies) for histone octamer in Nucleosome reconstitution measured, and their ability to position Nucleosomes in vitro assessed by native gel electrophoresis. The selected sequences have higher affinity than previously known natural or non-natural sequences, and have a correspondingly strong Nucleosome Positioning ability. A variety of analyses including Fourier transform, real-space correlation, and direct counting computations were carried out to assess non-random features in the selected sequences. The results reveal sequence rules that were already identified in earlier studies of natural nucleosomal DNA, together with a large set of new rules having even stronger statistical significance. Possible physical origins of the selected molecules' high affinities are discussed. The sequences isolated in this study should prove valuable for studies of chromatin structure and function in vitro and, potentially, for studies in vivo.
