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1
Zofall, Martin, Jim Persinger, and Blaine Bartholomew. "Functional Role of Extranucleosomal DNA and the Entry Site of the Nucleosome in Chromatin Remodeling by ISW2." Molecular and Cellular Biology 24, no. 22 (November 15, 2004): 10047–57. http://dx.doi.org/10.1128/mcb.24.22.10047-10057.2004.
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ABSTRACT A minimal amount of extranucleosomal DNA was required for nucleosome mobilization by ISW2 as shown by using a photochemical histone mapping approach to analyze nucleosome movement on a set of nucleosomes with varied lengths of extranucleosomal DNA. ISW2 was ineffective in repositioning or mobilizing nucleosomes with ≤20 bp of extranucleosomal DNA. In addition, ISW2 was able to slide nucleosomes to within only 10 to 13 bp of the edge of DNA fragments. The nucleosome mobilization was promoted by extranucleosomal single-stranded DNA with modest strand preference. Gaps (10 bp) just inside the nucleosome and in the extranucleosomal DNA showed that the transfer of torsional strain (twist) into the nucleosomal DNA region was not required for mobilizing nucleosomes. However, indications are that the extranucleosomal DNA immediately adjacent to the nucleosome has an important role in the initial stage of nucleosome movement by ISW2.
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Anderson, J. D., A. Thåström, and J. Widom. "Spontaneous Access of Proteins to Buried Nucleosomal DNA Target Sites Occurs via a Mechanism That Is Distinct from Nucleosome Translocation." Molecular and Cellular Biology 22, no. 20 (October 15, 2002): 7147–57. http://dx.doi.org/10.1128/mcb.22.20.7147-7157.2002.
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ABSTRACT 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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Feng, Jianxun, Haiyun Gan, Matthew L. Eaton, Hui Zhou, Shuqi Li, Jason A. Belsky, David M. MacAlpine, Zhiguo Zhang, and Qing Li. "Noncoding Transcription Is a Driving Force for Nucleosome Instability inspt16Mutant Cells." Molecular and Cellular Biology 36, no. 13 (May 2, 2016): 1856–67. http://dx.doi.org/10.1128/mcb.00152-16.
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FACT (facilitateschromatintranscription) consists of two essential subunits, Spt16 and Pob3, and functions as a histone chaperone. Mutation ofspt16results in a global loss of nucleosomes as well as aberrant transcription. Here, we show that the majority of nucleosome changes upon Spt16 depletion are alterations in nucleosome fuzziness and position shift. Most nucleosomal changes are suppressed by the inhibition of RNA polymerase II (Pol II) activity. Surprisingly, a small subgroup of nucleosome changes is resistant to transcriptional inhibition. Notably, Spt16 and distinct histone modifications are enriched at this subgroup of nucleosomes. We also report 1,037Spt16-suppressednoncodingtranscripts (SNTs) and found that the SNT start sites are enriched with the subgroup of nucleosomes resistant to Pol II inhibition. Finally, the nucleosomes at genes overlapping SNTs are more susceptible to changes upon Spt16 depletion than those without SNTs. Taken together, our results support a model in which Spt16 has a role in maintaining local nucleosome stability to inhibit initiation of SNT transcription, which once initiated drives additional nucleosome loss upon Spt16 depletion.
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Venturi, Christina Bourgeois, Alexander M. Erkine, and David S. Gross. "Cell Cycle-Dependent Binding of Yeast Heat Shock Factor to Nucleosomes." Molecular and Cellular Biology 20, no. 17 (September 1, 2000): 6435–48. http://dx.doi.org/10.1128/mcb.20.17.6435-6448.2000.
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ABSTRACT In the nucleus, transcription factors must contend with the presence of chromatin in order to gain access to their cognate regulatory sequences. As most nuclear DNA is assembled into nucleosomes, activators must either invade a stable, preassembled nucleosome or preempt the formation of nucleosomes on newly replicated DNA, which is transiently free of histones. We have investigated the mechanism by which heat shock factor (HSF) binds to target nucleosomal heat shock elements (HSEs), using as our model a dinucleosomal heat shock promoter (hsp82-ΔHSE1). We find that activated HSF cannot bind a stable, sequence-positioned nucleosome in G1-arrested cells. It can do so readily, however, following release from G1 arrest or after the imposition of either an early S- or late G2-phase arrest. Surprisingly, despite the S-phase requirement, HSF nucleosomal binding activity is restored in the absence of hsp82 replication. These results contrast with the prevailing paradigm for activator-nucleosome interactions and implicate a nonreplicative, S-phase-specific event as a prerequisite for HSF binding to nucleosomal sites in vivo.
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Kelbauskas, L., N. Woodbury, and D. Lohr. "DNA sequence-dependent variation in nucleosome structure, stability, and dynamics detected by a FRET-based analysisThis paper is one of a selection of papers published in this Special Issue, entitled 29th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process." Biochemistry and Cell Biology 87, no. 1 (February 2009): 323–35. http://dx.doi.org/10.1139/o08-126.
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Förster resonance energy transfer (FRET) techniques provide powerful and sensitive methods for the study of conformational features in biomolecules. Here, we review FRET-based studies of nucleosomes, focusing particularly on our work comparing the widely used nucleosome standard, 5S rDNA, and 2 promoter-derived regulatory element-containing nucleosomes, mouse mammary tumor virus (MMTV)-B and GAL10. Using several FRET approaches, we detected significant DNA sequence-dependent structure, stability, and dynamics differences among the three. In particular, 5S nucleosomes and 5S H2A/H2B-depleted nucleosomal particles have enhanced stability and diminished DNA dynamics, compared with MMTV-B and GAL10 nucleosomes and particles. H2A/H2B-depleted nucleosomes are of interest because they are produced by the activities of many transcription-associated complexes. Significant location-dependent (intranucleosomal) stability and dynamics variations were also observed. These also vary among nucleosome types. Nucleosomes restrict regulatory factor access to DNA, thereby impeding genetic processes. Eukaryotic cells possess mechanisms to alter nucleosome structure, to generate DNA access, but alterations often must be targeted to specific nucleosomes on critical regulatory DNA elements. By endowing specific nucleosomes with intrinsically higher DNA accessibility and (or) enhanced facility for conformational transitions, DNA sequence-dependent nucleosome dynamics and stability variations have the potential to facilitate nucleosome recognition and, thus, aid in the crucial targeting process. This and other nucleosome structure and function conclusions from FRET analyses are discussed.
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Szerlong, Heather J., and Jeffrey C. Hansen. "Nucleosome distribution and linker DNA: connecting nuclear function to dynamic chromatin structureThis paper is one of a selection of papers published in a Special Issue entitled 31st Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process." Biochemistry and Cell Biology 89, no. 1 (February 2011): 24–34. http://dx.doi.org/10.1139/o10-139.
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Genetic information in eukaryotes is managed by strategic hierarchical organization of chromatin structure. Primary chromatin structure describes an unfolded nucleosomal array, often referred to as “beads on a string”. Chromatin is compacted by the nonlinear rearrangement of nucleosomes to form stable secondary chromatin structures. Chromatin conformational transitions between primary and secondary structures are mediated by both nucleosome-stacking interactions and the intervening linker DNA. Chromatin model system studies find that the topography of secondary structures is sensitive to the spacing of nucleosomes within an array. Understanding the relationship between nucleosome spacing and higher order chromatin structure will likely yield important insights into the dynamic nature of secondary chromatin structure as it occurs in vivo. Genome-wide nucleosome mapping studies find the distance between nucleosomes varies, and regions of uniformly spaced nucleosomes are often interrupted by regions of nonuniform spacing. This type of organization is found at a subset of actively transcribed genes in which a nucleosome-depleted region near the transcription start site is directly adjacent to uniformly spaced nucleosomes in the coding region. Here, we evaluate secondary chromatin structure and discuss the structural and functional implications of variable nucleosome distributions in different organisms and at gene regulatory junctions.
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Clapier, Cedric R., Gernot Längst, Davide F. V. Corona, Peter B. Becker, and Karl P. Nightingale. "Critical Role for the Histone H4 N Terminus in Nucleosome Remodeling by ISWI." Molecular and Cellular Biology 21, no. 3 (February 1, 2001): 875–83. http://dx.doi.org/10.1128/mcb.21.3.875-883.2001.
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ABSTRACT The ATPase ISWI can be considered the catalytic core of several multiprotein nucleosome remodeling machines. Alone or in the context of nucleosome remodeling factor, the chromatin accessibility complex (CHRAC), or ACF, ISWI catalyzes a number of ATP-dependent transitions of chromatin structure that are currently best explained by its ability to induce nucleosome sliding. In addition, ISWI can function as a nucleosome spacing factor during chromatin assembly, where it will trigger the ordering of newly assembled nucleosomes into regular arrays. Both nucleosome remodeling and nucleosome spacing reactions are mechanistically unexplained. As a step toward defining the interaction of ISWI with its substrate during nucleosome remodeling and chromatin assembly we generated a set of nucleosomes lacking individual histone N termini from recombinant histones. We found the conserved N termini (the N-terminal tails) of histone H4 essential to stimulate ISWI ATPase activity, in contrast to other histone tails. Remarkably, the H4 N terminus, but none of the other tails, was critical for CHRAC-induced nucleosome sliding and for the generation of regularity in nucleosomal arrays by ISWI. Direct nucleosome binding studies did not reflect a dependence on the H4 tail for ISWI-nucleosome interactions. We conclude that the H4 tail is critically required for nucleosome remodeling and spacing at a step subsequent to interaction with the substrate.
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Knezetic, J. A., G. A. Jacob, and D. S. Luse. "Assembly of RNA polymerase II preinitiation complexes before assembly of nucleosomes allows efficient initiation of transcription on nucleosomal templates." Molecular and Cellular Biology 8, no. 8 (August 1988): 3114–21. http://dx.doi.org/10.1128/mcb.8.8.3114-3121.1988.
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We have previously shown that assembly of nucleosomes on the DNA template blocks transcription initiation by RNA polymerase II in vitro. In the studies reported here, we demonstrate that assembly of a complete RNA polymerase II preinitiation complex before nucleosome assembly results in nucleosomal templates which support initiation in vitro as efficiently as naked DNA. Control experiments prove that our observations are not the result of slow displacement of nucleosomes by the transcription machinery during chromatin assembly, nor are they an artifact of inefficient nucleosome deposition on templates already bearing an RNA polymerase. Thus, the RNA polymerase II preinitiation complex appears to be resistant to disruption by subsequent nucleosome assembly.
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Anderson, J. D., and J. Widom. "Poly(dA-dT) Promoter Elements Increase the Equilibrium Accessibility of Nucleosomal DNA Target Sites." Molecular and Cellular Biology 21, no. 11 (June 1, 2001): 3830–39. http://dx.doi.org/10.1128/mcb.21.11.3830-3839.2001.
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ABSTRACT Polypurine tracts are important elements of eukaryotic promoters. They are believed to somehow destabilize chromatin, but the mechanism of their action is not known. We show that incorporating an A16 element at an end of the nucleosomal DNA and further inward destabilizes histone-DNA interactions by 0.1 ± 0.03 and 0.35 ± 0.04 kcal mol−1, respectively, and is accompanied by 1.5- ± 0.1-fold and 1.7- ± 0.1-fold increases in position-averaged equilibrium accessibility of nucleosomal DNA target sites. These effects are comparable in magnitude to effects of A16 elements that correlate with transcription in vivo, suggesting that our system may capture most of their physiological role. These results point to two distinct but interrelated models for the mechanism of action of polypurine tract promoter elements in vivo. Given a nucleosome positioned over a promoter region, the presence of a polypurine tract in that nucleosome's DNA decreases the stability of the DNA wrapping, increasing the equilibrium accessibility of other DNA target sites buried inside that nucleosome. Alternatively (if nucleosomes are freely mobile), the presence of a polypurine tract provides a free energy bias for the nucleosome to move to alternative locations, thereby changing the equilibrium accessibilities of other nearby DNA target sites.
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Gangaraju, Vamsi K., and Blaine Bartholomew. "Dependency of ISW1a Chromatin Remodeling on Extranucleosomal DNA." Molecular and Cellular Biology 27, no. 8 (February 5, 2007): 3217–25. http://dx.doi.org/10.1128/mcb.01731-06.
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ABSTRACT The nucleosome remodeling activity of ISW1a was dependent on whether ISW1a was bound to one or both extranucleosomal DNAs. ISW1a preferentially bound nucleosomes with an optimal length of ∼33 to 35 bp of extranucleosomal DNA at both the entry and exit sites over nucleosomes with extranucleosomal DNA at only one entry or exit site. Nucleosomes with extranucleosomal DNA at one of the entry/exit sites were readily remodeled by ISW1a and stimulated the ATPase activity of ISW1a, while conversely, nucleosomes with extranucleosomal DNA at both entry/exit sites were unable either to stimulate the ATPase activity of ISW1a or to be mobilized. DNA footprinting revealed that a major conformational difference between the nucleosomes was the lack of ISW1a binding to nucleosomal DNA two helical turns from the dyad axis in nucleosomes with extranucleosomal DNA at both entry/exit sites. The Ioc3 subunit of ISW1a was found to be the predominant subunit associated with extranucleosomal DNA when ISW1a is bound either to one or to both extranucleosomal DNAs. These two conformations of the ISW1a-nucleosome complex are suggested to be the molecular basis for the nucleosome spacing activity of ISW1a on nucleosomal arrays. ISW1b, the other isoform of ISW1, does not have the same dependency for extranucleosomal DNA as ISW1a and, likewise, is not able to space nucleosomes.
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Bazett-Jones, David P., Jacques Côté, Carolyn C. Landel, Craig L. Peterson, and Jerry L. Workman. "The SWI/SNF Complex Creates Loop Domains in DNA and Polynucleosome Arrays and Can Disrupt DNA-Histone Contacts within These Domains." Molecular and Cellular Biology 19, no. 2 (February 1, 1999): 1470–78. http://dx.doi.org/10.1128/mcb.19.2.1470.
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ABSTRACT To understand the mechanisms by which the chromatin-remodeling SWI/SNF complex interacts with DNA and alters nucleosome organization, we have imaged the SWI/SNF complex with both naked DNA and nucleosomal arrays by using energy-filtered microscopy. By making ATP-independent contacts with DNA at multiple sites on its surface, SWI/SNF creates loops, bringing otherwise-distant sites into close proximity. In the presence of ATP, SWI/SNF action leads to the disruption of nucleosomes within domains that appear to be topologically constrained by the complex. The data indicate that the action of one SWI/SNF complex on an array of nucleosomes can lead to the formation of a region where multiple nucleosomes are disrupted. Importantly, nucleosome disruption by SWI/SNF results in a loss of DNA content from the nucleosomes. This indicates a mechanism by which SWI/SNF unwraps part of the nucleosomal DNA.
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Pederson, D. S., and T. Fidrych. "Heat shock factor can activate transcription while bound to nucleosomal DNA in Saccharomyces cerevisiae." Molecular and Cellular Biology 14, no. 1 (January 1994): 189–99. http://dx.doi.org/10.1128/mcb.14.1.189-199.1994.
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After each round of replication, new transcription initiation complexes must assemble on promoter DNA. This process may compete with packaging of the same promoter sequences into nucleosomes. To elucidate interactions between regulatory transcription factors and nucleosomes on newly replicated DNA, we asked whether heat shock factor (HSF) could be made to bind to nucleosomal DNA in vivo. A heat shock element (HSE) was embedded at either of two different sites within a DNA segment that directs the formation of a stable, positioned nucleosome. The resulting DNA segments were coupled to a reporter gene and transfected into the yeast Saccharomyces cerevisiae. Transcription from these two plasmid constructions after induction by heat shock was similar in amount to that from a control plasmid in which HSF binds to nucleosome-free DNA. High-resolution genomic footprint mapping of DNase I and micrococcal nuclease cleavage sites indicated that the HSE in these two plasmids was, nevertheless, packaged in a nucleosome. The inclusion of HSE sequences within (but relatively close to the edge of) the nucleosome did not alter the position of the nucleosome which formed with the parental DNA fragment. Genomic footprint analyses also suggested that the HSE-containing nucleosome was unchanged by the induction of transcription. Quantitative comparisons with control plasmids ruled out the possibility that HSF was bound only to a small fraction of molecules that might have escaped nucleosome assembly. Analysis of the helical orientation of HSE DNA in the nucleosome indicated that HSF contacted DNA residues that faced outward from the histone octamer. We discuss the significance of these results with regard to the role of nucleosomes in inhibiting transcription and the normal occurrence of nucleosome-free regions in promoters.
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Dang, Weiwei, and Blaine Bartholomew. "Domain Architecture of the Catalytic Subunit in the ISW2-Nucleosome Complex." Molecular and Cellular Biology 27, no. 23 (October 1, 2007): 8306–17. http://dx.doi.org/10.1128/mcb.01351-07.
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ABSTRACT ATP-dependent chromatin remodeling has an important role in the regulation of cellular differentiation and development. For the first time, a topological view of one of these complexes has been revealed, by mapping the interactions of the catalytic subunit Isw2 with nucleosomal and extranucleosomal DNA in the complex with all four subunits of ISW2 bound to nucleosomes. Different domains of Isw2 were shown to interact with the nucleosome near the dyad axis, another near the entry site of the nucleosome, and another with extranucleosomal DNA. The conserved DEXD or ATPase domain was found to contact the superhelical location 2 (SHL2) of the nucleosome, providing a direct physical connection of ATP hydrolysis with this region of nucleosomes. The C terminus of Isw2, comprising the SLIDE (SANT-like domain) and HAND domains, was found to be associated with extranucleosomal DNA and the entry site of nucleosomes. It is thus proposed that the C-terminal domains of Isw2 are involved in anchoring the complex to nucleosomes through their interactions with linker DNA and that they facilitate the movement of DNA along the surface of nucleosomes.
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Guyon, Jeffrey R., Geeta J. Narlikar, Saïd Sif, and Robert E. Kingston. "Stable Remodeling of Tailless Nucleosomes by the Human SWI-SNF Complex." Molecular and Cellular Biology 19, no. 3 (March 1, 1999): 2088–97. http://dx.doi.org/10.1128/mcb.19.3.2088.
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ABSTRACT The histone N-terminal tails have been shown previously to be important for chromatin assembly, remodeling, and stability. We have tested the ability of human SWI-SNF (hSWI-SNF) to remodel nucleosomes whose tails have been cleaved through a limited trypsin digestion. We show that hSWI-SNF is able to remodel tailless mononucleosomes and nucleosomal arrays, although hSWI-SNF remodeling of tailless nucleosomes is less effective than remodeling of nucleosomes with tails. Analogous to previous observations with tailed nucleosomal templates, we show both (i) that hSWI-SNF-remodeled trypsinized mononucleosomes and arrays are stable for 30 min in the remodeled conformation after removal of ATP and (ii) that the remodeled tailless mononucleosome can be isolated on a nondenaturing acrylamide gel as a novel species. Thus, nucleosome remodeling by hSWI-SNF can occur via interactions with a tailless nucleosome core.
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Morrison, Ashby J., Claude Sardet, and Rafael E. Herrera. "Retinoblastoma Protein Transcriptional Repression through Histone Deacetylation of a Single Nucleosome." Molecular and Cellular Biology 22, no. 3 (February 1, 2002): 856–65. http://dx.doi.org/10.1128/mcb.22.3.856-865.2002.
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ABSTRACT The retinoblastoma protein, pRb, controls transcription through recruitment of histone deacetylase to particular E2F-responsive genes. We determined the acetylation level of individual nucleosomes present in the cyclin E promoter of RB +/+ and RB −/− mouse embryo fibroblasts. We also determined the effects of pRb on nucleosomal conformation by examining the thiol reactivity of histone H3 of individual nucleosomes. We found that pRb represses the cyclin E promoter through histone deacetylation of a single nucleosome, to which it and histone deacetylase 1 bind. In addition, the conformation of this nucleosome is modulated by pRb-directed histone deacetylase activity. Thus, the repressive role of pRb in cyclin E transcription and therefore cell cycle progression can be mapped to its control of the acetylation status and conformation of a single nucleosome.
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Holde, K. van, and T. Yager. "Models for chromatin remodeling: a critical comparison." Biochemistry and Cell Biology 81, no. 3 (June 1, 2003): 169–72. http://dx.doi.org/10.1139/o03-038.
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Nucleosome remodeling has been shown, in many cases, to involve cis displacement of nucleosomes on the DNA. This process seems similar to the long-recognized random diffusion of nucleosomes along DNA, but the remodeling process is unidirectional and ATP dependent. Several years ago, we developed a model for nucleosome migration, based on the diffusion of "twist-defects" within the nucleosomal DNA. This has been modified into a model that incorporates ATP-dependent defect generation, and can account for many observations concerning remodeling. However, certain experimental studies in recent years have cast doubt on the applicability of the twist-diffusion model for remodeling, and seem to favor instead a "reptation" model. We discuss herein these problems and propose a resolution.Key words: nucleosome, remodeling, chromatin.
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Barbier, Jérémy, Cédric Vaillant, Jean-Nicolas Volff, Frédéric G. Brunet, and Benjamin Audit. "Coupling between Sequence-Mediated Nucleosome Organization and Genome Evolution." Genes 12, no. 6 (June 1, 2021): 851. http://dx.doi.org/10.3390/genes12060851.
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The nucleosome is a major modulator of DNA accessibility to other cellular factors. Nucleosome positioning has a critical importance in regulating cell processes such as transcription, replication, recombination or DNA repair. The DNA sequence has an influence on the position of nucleosomes on genomes, although other factors are also implicated, such as ATP-dependent remodelers or competition of the nucleosome with DNA binding proteins. Different sequence motifs can promote or inhibit the nucleosome formation, thus influencing the accessibility to the DNA. Sequence-encoded nucleosome positioning having functional consequences on cell processes can then be selected or counter-selected during evolution. We review the interplay between sequence evolution and nucleosome positioning evolution. We first focus on the different ways to encode nucleosome positions in the DNA sequence, and to which extent these mechanisms are responsible of genome-wide nucleosome positioning in vivo. Then, we discuss the findings about selection of sequences for their nucleosomal properties. Finally, we illustrate how the nucleosome can directly influence sequence evolution through its interactions with DNA damage and repair mechanisms. This review aims to provide an overview of the mutual influence of sequence evolution and nucleosome positioning evolution, possibly leading to complex evolutionary dynamics.
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Lakadamyali, Melike. "SUPERRESOLUTION IMAGING OF CHROMATIN IN HEALTH AND DISEASE." Innovation in Aging 6, Supplement_1 (November 1, 2022): 28–29. http://dx.doi.org/10.1093/geroni/igac059.105.
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Abstract Super-resolution microscopy has been playing an instrumental role in providing new insights into how the genome is folded and packaged inside intact nuclei in single cells. I will present our work on using super-resolution microscopy to visualize and quantify the spatial organization of chromatin with nanoscale spatial resolution in single cells. Our work has revealed that at the nucleosomal level chromatin is a disordered fiber composed of groups of nucleosomes packaged at varying densities, which we named nucleosome clutches. Despite the heterogeneity of nucleosome clutch organization, the size and packing density of nucleosome clutches is cell-type specific and correlates with cell fate. Our recent results also show that nucleosome clutches and chromatin nano-structure can be remodeled via chemo-mechanical cues. In particular degenerative chemo-mechanical cues during disease lead to aberrant chromatin nano-structure and loss of mechano-epigenetic memory, potentially leading to alterations in cell phenotype.
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Kim, Yeonjung, Neil McLaughlin, Kim Lindstrom, Toshio Tsukiyama, and David J. Clark. "Activation of Saccharomyces cerevisiae HIS3 Results in Gcn4p-Dependent, SWI/SNF-Dependent Mobilization of Nucleosomes over the EntireGene." Molecular and Cellular Biology 26, no. 22 (September 18, 2006): 8607–22. http://dx.doi.org/10.1128/mcb.00678-06.
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ABSTRACT The effects of transcriptional activation on the chromatin structure of the Saccharomyces cerevisiae HIS3 gene were addressed by mapping the precise positions of nucleosomes in uninduced and induced chromatin. In the absence of the Gcn4p activator, the HIS3 gene is organized into a predominant nucleosomal array. In wild-type chromatin, this array is disrupted, and several alternative overlapping nucleosomal arrays are formed. The disruption of the predominant array also requires the SWI/SNF remodeling machine, indicating that the SWI/SNF complex plays an important role in nucleosome mobilization over the entire HIS3 gene. The Isw1 remodeling complex plays a more subtle role in determining nucleosome positions on HIS3, favoring positions different from those preferred by the SWI/SNF complex. Both the SWI/SNF and Isw1 complexes are constitutively present in HIS3 chromatin, although Isw1 tends to be excluded from the HIS3 promoter. Despite the apparent disorder of HIS3 chromatin generated by the formation of multiple nucleosomal arrays, nucleosome density profiles indicate that some long-range order is always present. We propose that Gcn4p stimulates nucleosome mobilization over the entire HIS3 gene by the SWI/SNF complex. We suggest that the net effect of interplay among remodeling machines at HIS3 is to create a highly dynamic chromatin structure.
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Vitolo, Joseph M., Zungyoon Yang, Ravi Basavappa, and Jeffrey J. Hayes. "Structural Features of Transcription Factor IIIA Bound to a Nucleosome in Solution." Molecular and Cellular Biology 24, no. 2 (January 15, 2004): 697–707. http://dx.doi.org/10.1128/mcb.24.2.697-707.2004.
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ABSTRACT Assembly of a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene into a nucleosome greatly restricts binding of the 5S gene-specific transcription factor IIIA (TFIIIA) to the 5S internal promoter. However, TFIIIA binds with high affinity to 5S nucleosomes lacking the N-terminal tail domains of the core histones or to nucleosomes in which these domains are hyperacetylated. The degree to which tail acetylation or removal improves TFIIIA binding cannot be simply explained by a commensurate change in the general accessibility of nucleosomal DNA. In order to investigate the molecular basis of how TFIIIA binds to the nucleosome and to ascertain if binding involves all nine zinc fingers and/or displacement of histone-DNA interactions, we examined the TFIIIA-nucleosome complex by hydroxyl radical footprinting and site-directed protein-DNA cross-linking. Our data reveal that the first six fingers of TFIIIA bind and displace approximately 20 bp of histone-DNA interactions at the periphery of the nucleosome, while binding of fingers 7 to 9 appears to overlap with histone-DNA interactions. Molecular modeling based on these results and the crystal structures of a nucleosome core and a TFIIIA-DNA cocomplex yields a precise picture of the ternary complex and a potentially important intermediate in the transition from naïve chromatin structure to productive polymerase III transcription complex.
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Georgel, Philippe T. "Chromatin potentiation of the hsp70 promoter is linked to GAGA-factor recruitment." Biochemistry and Cell Biology 83, no. 4 (August 1, 2005): 555–65. http://dx.doi.org/10.1139/o05-060.
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The events leading to transcription initiation of the Drosophila melanogaster heat-shock protein (hsp)70 gene have been demonstrated to be directly connected with nucleosome remodeling factor and GAGA-dependent chromatin remodeling on its promoter region. To investigate the relative importance of the multiple GAGA-factor binding sites in the process of chromatin remodeling and their effect on DNA conformation, the position of nucleosomes over the proximal region of the promoter was mapped. No real-positioned nucleosome was detected. By matching the relative position of the GAGA-factor binding sites with the distribution of nucleosomes over the hsp70 promoter, the GAGA site 2 appeared to be the most accessible, i.e., located close to a nucleosomal edge or within the linker DNA. This result, combined with previous observations, suggest a link between increased GAGA-factor accessibility and efficiency of transcription initiation. The effect of GAGA-binding-site mutations, both individually and in combination, on DNA structure and nucleosome remodeling was assessed using free DNA and fly embryo extract chromatin templates assembled in vitro. Results indicated that both the number of functional sites and their positions within the chromatin were important determinants for nucleosome-remodeling efficiency. Ultimately, the degree of accessibility of the GAGA factor to its cognate binding site(s) appears to be proportional to chromatin-remodeling competency of the hsp70 promoter.Key words: chromatin, remodeling, nucleosome, hsp70, GAGA, Drosophila.
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Xu, Mai, Robert T. Simpson, and Michael P. Kladde. "Gal4p-Mediated Chromatin Remodeling Depends on Binding Site Position in Nucleosomes but Does Not Require DNA Replication." Molecular and Cellular Biology 18, no. 3 (March 1, 1998): 1201–12. http://dx.doi.org/10.1128/mcb.18.3.1201.
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ABSTRACT Biochemical studies have demonstrated decreased binding of various proteins to DNA in nucleosome cores as their cognate sites are moved from the edge of the nucleosome to the pseudodyad (center). However, to date no study has addressed whether this structural characteristic of nucleosomes modulates the function of a transcription factor in living cells, where processes of DNA replication and chromatin modification or remodeling could significantly affect factor binding. Using a sensitive, high-resolution methyltransferase assay, we have monitored the ability of Gal4p in vivo to interact with a nucleosome at positions that are known to be inaccessible in nucleosome cores in vitro. Gal4p efficiently bound a single cognate site (UASG) centered at 41 bp from the edge of a positioned nucleosome, perturbing chromatin structure and inducing transcription. DNA binding and chromatin perturbation accompanying this interaction also occurred in the presence of hydroxyurea, indicating that DNA replication is not necessary for Gal4p-mediated nucleosome disruption. These data extend previous studies, which demonstrated DNA replication-independent chromatin remodeling, by showing that a single dimer of Gal4p, without the benefit of cooperative interactions that occur at complex wild-type promoters, is competent for invasion of a preestablished nucleosome. When the UASG was localized at the nucleosomal pseudodyad, relative occupancy by Gal4p, nucleosome disruption, and transcriptional activation were substantially compromised. Therefore, despite the increased nucleosome binding capability of Gal4p in cells, the precise translational position of a factor binding site in one nucleosome in an array can affect the ability of a transcriptional regulator to overcome the repressive influence of chromatin.
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Maluchenko, Natalya, Darya Koshkina, Anna Korovina, Vasily Studitsky, and Alexey Feofanov. "Interactions of PARP1 Inhibitors with PARP1-Nucleosome Complexes." Cells 11, no. 21 (October 23, 2022): 3343. http://dx.doi.org/10.3390/cells11213343.
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Inhibitors (PARPi) of poly(ADP-ribose-)polymerase-1 (PARP1) are used in antitumor therapy; their cytotoxicity correlates with the efficiency of PARP1 trapping in cell chromatin. Previous studies have demonstrated the PARPi-induced trapping of PARP1 on DNA, although details of the mechanism remain controversial. Here, the interactions of PARP1-nucleosome complexes with PARPi, olaparib (Ola), talazoparib (Tala), and veliparib (Veli) were studied. PARPi trap PARP1 on nucleosomes without affecting the structure of PARP1-nucleosome complexes. The efficiency of PARP1 trapping on nucleosomes increases in the order of Tala>Ola>>Veli, recapitulating the relative trapping efficiencies of PARPi in cells, but different from the relative potency of PARPi to inhibit the catalytic activity of PARP1. The efficiency of PARP1 trapping on nucleosomes correlates with the level of inhibition of auto-PARylation, which otherwise promotes the dissociation of PARP1-nucleosome complexes. The trapping efficiencies of Tala and Ola (but not Veli) are additionally modulated by the enhanced PARP1 binding to nucleosomes. The dissociation of PARP1-nucleosome complexes occurs without a loss of histones and leads to the restoration of the intact structure of nucleosomal DNA. The data suggest that the chromatin structure can considerably affect the efficiency of the PARPi action.
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Merkl, Philipp E., Michael Pilsl, Tobias Fremter, Katrin Schwank, Christoph Engel, Gernot Längst, Philipp Milkereit, Joachim Griesenbeck, and Herbert Tschochner. "RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure." Journal of Biological Chemistry 295, no. 15 (February 14, 2020): 4782–95. http://dx.doi.org/10.1074/jbc.ra119.011827.
Full textAbstract:
RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I–associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I–specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity.
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Hedberg, Annica, Premasany Kanapathippillai, Ole Petter Rekvig, and Kristin Andreassen Fenton. "LMW Heparin Prevents Increased Kidney Expression of Proinflammatory Mediators in (NZBxNZW)F1 Mice." Clinical and Developmental Immunology 2013 (2013): 1–11. http://dx.doi.org/10.1155/2013/791262.
Full textAbstract:
We have previously demonstrated that continuous infusion of low molecular weight (LMW) heparin delays autoantibody production and development of lupus nephritis in (NZBxNZW)F1 (B/W) mice. In this study we investigated the effect of LMW heparin on renal cytokine and chemokine expression and on nucleosome-mediated activation of nucleosome-specific splenocytes. Total mRNA extracted from kidneys of heparin-treated or -untreated B/W mice was analysed by qPCR for the expression of several cytokines, chemokines, and Toll-like receptors. Splenocytes taken from B/W mice were stimulated with nucleosomes with or without the presence of heparin. Splenocyte cell proliferation as thymidine incorporation and the expression of costimulatory molecules and cell activation markers were measured. Heparin treatment of B/W mice reduced thein vivoexpression of CCR2, IL1β, and TLR7 compared to untreated B/W mice. Nucleosome-induced cell proliferation of splenocytes was not influenced by heparin. The expression of CD80, CD86, CD69, CD25, CTLA-4, and TLR 2, 7, 8, and 9 was upregulated upon stimulation by nucleosomes, irrespective of whether heparin was added to the cell culture or not. In conclusion, treatment with heparin lowers the kidney expression of proinflammatory mediators in B/W mice but does not affect nucleosomal activation of splenocytes.
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Ramachandran, Srinivas, and Steven Henikoff. "Replicating nucleosomes." Science Advances 1, no. 7 (August 2015): e1500587. http://dx.doi.org/10.1126/sciadv.1500587.
Full textAbstract:
Eukaryotic replication disrupts each nucleosome as the fork passes, followed by reassembly of disrupted nucleosomes and incorporation of newly synthesized histones into nucleosomes in the daughter genomes. In this review, we examine this process of replication-coupled nucleosome assembly to understand how characteristic steady-state nucleosome landscapes are attained. Recent studies have begun to elucidate mechanisms involved in histone transfer during replication and maturation of the nucleosome landscape after disruption by replication. A fuller understanding of replication-coupled nucleosome assembly will be needed to explain how epigenetic information is replicated at every cell division.
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Lieleg, Corinna, Philip Ketterer, Johannes Nuebler, Johanna Ludwigsen, Ulrich Gerland, Hendrik Dietz, Felix Mueller-Planitz, and Philipp Korber. "Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density." Molecular and Cellular Biology 35, no. 9 (March 2, 2015): 1588–605. http://dx.doi.org/10.1128/mcb.01070-14.
Full textAbstract:
Arrays of regularly spaced nucleosomes are a hallmark of chromatin, but it remains unclear how they are generated. Recent genome-wide studies,in vitroandin vivo, showed constant nucleosome spacing even if the histone concentration was experimentally reduced. This counters the long-held assumption that nucleosome density determines spacing and calls for factors keeping spacing constant regardless of nucleosome density. We call this a clamping activity. Here, we show in a purified system that ISWI- and CHD1-type nucleosome remodelers have a clamping activity such that they not only generate regularly spaced nucleosome arrays but also generate constant spacing regardless of nucleosome density. This points to a functionally attractive nucleosome interaction that could be mediated either directly by nucleosome-nucleosome contacts or indirectly through the remodelers. MutantDrosophila melanogasterISWI without the HAND-SANT-SLIDE (HSS) domain had no detectable spacing activity even though it is known to remodel and slide nucleosomes. This suggests that the role of ISWI remodelers in generating constant spacing is not just to mediate nucleosome sliding; they actively contribute to the attractive interaction. Additional factors are necessary to set physiological spacing in absolute terms.
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Neumann, Heinz, and Bryan J. Wilkins. "Spanning the gap: unraveling RSC dynamics in vivo." Current Genetics 67, no. 3 (January 23, 2021): 399–406. http://dx.doi.org/10.1007/s00294-020-01144-1.
Full textAbstract:
AbstractMultiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.
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Vasilev, V. A., D. M. Ryabov, A. K. Shaytan, and G. A. Armeev. "Updating nucleosome positions within individual genes using molecular modeling methods and mnase sequencing data." Биофизика 68, no. 5 (October 15, 2023): 911–19. http://dx.doi.org/10.31857/s0006302923050101.
Full textAbstract:
Organization of chromatin plays an important role in regulating the genetic machinery of the cell. The basic unit of chromatin packaging is a nucleosome, which harbors DNA of about 145 base pairs in length. The packaging of genetic material and its accessibility to transcription enzymes and other regulatory chromatin proteins depends on the positions of nucleosomes. MNase sequencing is used to examine nucleosome positions in a genome. MNase sequencing data are sufficient for detecting the presence of nucleosomes on the sequence, but a determination of the precise locations of nucleosomes can be problematic. Accurate determination of nucleosome positions requires additional data filtering and processing. In this study, using MNase sequencing data, a combined method based on geometric analysis of nucleosome chain molecular models is proposed for selecting possible nucleosome positions. The developed algorithm efficiently eliminates inaccessible nucleosome chain combinations and conformationally prohibited nucleosome positions.
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Horowitz, R. A., D. A. Agard, J. W. Sedat, and C. L. Woodcock. "The three-dimensional architecture of chromatin in situ: electron tomography reveals fibers composed of a continuously variable zig-zag nucleosomal ribbon." Journal of Cell Biology 125, no. 1 (April 1, 1994): 1–10. http://dx.doi.org/10.1083/jcb.125.1.1.
Full textAbstract:
The three dimensional (3D) structure of chromatin fibers in sections of nuclei has been determined using electron tomography. Low temperature embedding and nucleic acid-specific staining allowed individual nucleosomes to be clearly seen, and the tomographic data collection parameters provided a reconstruction resolution of 2.5 nm. Chromatin fibers have complex 3D trajectories, with smoothly bending regions interspersed with abrupt changes in direction, and U turns. Nucleosomes are located predominantly at the fiber periphery, and linker DNA tends to project toward the fiber interior. Within the fibers, a unifying structural motif is a two nucleosome-wide ribbon that is variably bent and twisted, and in which there is little face-to-face contact between nucleosomes. It is suggested that this asymmetric 3D zig-zag of nucleosomes and linker DNA represents a basic principle of chromatin folding that is determined by the properties of the nucleosome-linker unit. This concept of chromatin fiber architecture is contrasted with helical models in which specific nucleosome-nucleosome contacts play a major role in generating a symmetrical higher order structure. The transcriptional control implications of a more open and irregular chromatin structure are discussed.
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Vettese-Dadey, M., P. Walter, H. Chen, L. J. Juan, and J. L. Workman. "Role of the histone amino termini in facilitated binding of a transcription factor, GAL4-AH, to nucleosome cores." Molecular and Cellular Biology 14, no. 2 (February 1994): 970–81. http://dx.doi.org/10.1128/mcb.14.2.970-981.1994.
Full textAbstract:
Facilitated, "cooperative" binding of GAL4-AH to nucleosomal DNA occurred in response to inhibition from the core histone amino termini. The binding of GAL4-AH (which contains the DNA-binding and dimerization domains of GAL4) to nucleosome cores containing multiple binding sites initiated at the end of a nucleosome core and proceeded in a cooperative manner until all sites were occupied. However, following tryptic removal of the core histone amino termini, GAL4-AH binding appeared to be noncooperative, similar to binding naked DNA. Binding of GAL4-AH to nucleosomes bearing a single GAL4 site at different positions indicated that inhibition of GAL4 binding was largely mediated by the histone amino termini and primarily occurred at sites well within the core and not near the end. When the histone amino termini were intact, binding of GAL4-AH to sites near the center of a nucleosome core was greatly enhanced by the presence of additional GAL4 dimers bound to more-accessible positions. These data illustrate that the binding of a factor to more-accessible sites, near the end of a nucleosome, allows facilitated binding of additional factors to the center of the nucleosome, thereby overcoming repression from the core histone amino termini. This mechanism may contribute to the binding of multiple factors to complex promoter and enhancer elements in cellular chromatin.
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Baldi, Sandro. "Nucleosome positioning and spacing: from genome-wide maps to single arrays." Essays in Biochemistry 63, no. 1 (February 21, 2019): 5–14. http://dx.doi.org/10.1042/ebc20180058.
Full textAbstract:
Abstract The positioning of nucleosomes relative to DNA and their neighboring nucleosomes represents a fundamental layer of chromatin organization. Changes in nucleosome positioning and spacing affect the accessibility of DNA to regulatory factors and the formation of higher order chromatin structures. Sequencing of mononucleosomal fragments allowed mapping nucleosome positions on a genome-wide level in many organisms. This revealed that successions of evenly spaced and well-positioned nucleosomes—so called phased nucleosome arrays—occur at the 5′ end of many active genes and in the vicinity of transcription factor and other protein binding sites. Phased arrays arise from the interplay of barrier elements on the DNA, which position adjacent nucleosomes, and the nucleosome spacing activity of ATP-dependent chromatin remodelers. A shortcoming of classic mononucleosomal mapping experiments is that they only reveal nucleosome spacing and array regularity at select sites in the genome with well-positioned nucleosomes. However, new technological approaches elucidate nucleosome array structure throughout the genome and with single-cell resolution. In the future, it will be interesting to see whether changes in nucleosome array regularity and spacing contribute to the formation of higher order chromatin structures and the spatial organization of the genome in vivo.
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Morse, Randall H. "Getting into chromatin: how do transcription factors get past the histones?" Biochemistry and Cell Biology 81, no. 3 (June 1, 2003): 101–12. http://dx.doi.org/10.1139/o03-039.
Full textAbstract:
Transcriptional activators and the general transcription machinery must gain access to DNA that in eukaryotes may be packaged into nucleosomes. In this review, I discuss this problem from the standpoint of the types of chromatin structures that these DNA-binding proteins may encounter, and the mechanisms by which they may contend with various chromatin structures. The discussion includes consideration of experiments in which chromatin structure is manipulated in vivo to confront activators with nucleosomal binding sites, and the roles of nucleosome dynamics and activation domains in facilitating access to such sites. Finally, the role of activators in facilitating access of the general transcriptional machinery to sites in chromatin is discussed. Key words: nucleosome, chromatin, transcriptional activation, Saccharomyces cerevisiae.
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Maluchenko, N. V., T. V. Andreeva, O. V. Geraskina, N. S. Gerasimova, A. V. Lubitelev, A. V. Feofanov, and V. M. Studitsky. "On the interaction of resveratrol with nucleosomes." Биофизика 68, no. 3 (June 15, 2023): 466–73. http://dx.doi.org/10.31857/s0006302923030067.
Full textAbstract:
The natural polyphenol resveratrol has anti-inflammatory, antioxidant, antitumor and geroprotective properties. The wide range of resveratrol activities is determined by its ability to modulate a variety of signaling pathways in the cell and interact with various target molecules. It is known that resveratrol interacts with DNA, but the effect of this interaction on the structure of chromatin has not been studied. In this work, we studied the effect of resveratrol on the structure of the nucleosome, the functional and structural unit of chromatin. Fluorescent microscopy of single nucleosomes based on Forster resonance energy transfer and analysis of changes in the electrophoretic mobility of nucleosomes in polyacrylamide gel showed that, at a concentration of ~100 μM, resveratrol affects the conformation of DNA linker regions, limits the conformational dynamics of DNA near the nucleosome boundary, but does not cause significant changes in the folding of nucleosomal DNA on the histone octamer. A small effect of resveratrol on the structure of the nucleosome compared to quercetin is presumably determined by the binding mode of resveratrol in a minor groove of DNA.
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Rube, H. Tomas, and Jun S. Song. "Quantifying the role of steric constraints in nucleosome positioning." Nucleic Acids Research 42, no. 4 (November 27, 2013): 2147–58. http://dx.doi.org/10.1093/nar/gkt1239.
Full textAbstract:
Abstract Statistical positioning, the localization of nucleosomes packed against a fixed barrier, is conjectured to explain the array of well-positioned nucleosomes at the 5′ end of genes, but the extent and precise implications of statistical positioning in vivo are unclear. We examine this hypothesis quantitatively and generalize the idea to include moving barriers as well as nucleosomes actively packed against a barrier. Early experiments noted a similarity between the nucleosome profile aligned and averaged across genes and that predicted by statistical positioning; however, we demonstrate that aligning random nucleosomes also generates the same profile, calling the previous interpretation into question. New rigorous results reformulate statistical positioning as predictions on the variance structure of nucleosome locations in individual genes. In particular, a quantity termed the variance gradient, describing the change in variance between adjacent nucleosomes, is tested against recent high-throughput nucleosome sequencing data. Constant variance gradients provide support for generalized statistical positioning in ∼50% of long genes. Genes that deviate from predictions have high nucleosome turnover and cell-to-cell gene expression variability. The observed variance gradient suggests an effective nucleosome size of 158 bp, instead of the commonly perceived 147 bp. Our analyses thus clarify the role of statistical positioning in vivo.
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Bednar, J., R. A. Horowitz, J. Dubochet, and C. L. Woodcock. "Chromatin conformation and salt-induced compaction: three-dimensional structural information from cryoelectron microscopy." Journal of Cell Biology 131, no. 6 (December 15, 1995): 1365–76. http://dx.doi.org/10.1083/jcb.131.6.1365.
Full textAbstract:
Cryoelectron microscopy has been used to examine the three-dimensional (3-D) conformation of small oligonucleosomes from chicken erythrocyte nuclei after vitrification in solutions of differing ionic strength. From tilt pairs of micrographs, the 3-D location and orientation of the nucleosomal disks, and the paths of segments of exposed linker can be obtained. In "low-salt" conditions (5 mM NaCl, 1 mM EDTA, pH 7.5), the average trinucleosome assumes the shape of an equilateral triangle, with nucleosomes at the vertices, and a length of exposed linker DNA between consecutive nucleosomes equivalent to approximately 46 bp. The two linker DNA segments converge at the central nucleosome. Removal of histones H1 and H5 results in a much more variable trinucleosome morphology, and the two linker DNA segments usually join the central nucleosome at different locations. Trinucleosomes vitrified in 20 mM NaCl, 1 mM EDTA, (the salt concentration producing the maximal increase in sedimentation), reveal that compaction occurs by a reduction in the included angle made by the linker DNA segments at the central nucleosome, and does not involve a reduction in the distance between consecutive nucleosomes. Frequently, there is also a change in morphology at the linker entry-exit site. At 40 mM NaCl, there is no further change in trinucleosome morphology, but polynucleosomes are appreciably more compact. Nevertheless, the 3-D zig-zag conformation observed in polynucleosomes at low salt is retained at 40 mM NaCl, and individual nucleosome disks remain separated from each other. There is no evidence for the formation of solenoidal arrangements within polynucleosomes. Comparison of the solution conformation of individual oligonucleosomes with data from physical measurements on bulk chromatin samples suggests that the latter should be reinterpreted. The new data support the concept of an irregular zig-zag chromatin conformation in solution over a range of ionic strengths, in agreement with other in situ (McDowall, A.W., J.M. Smith, and J. Dubochet. 1986, EMBO (Eur. Mol. Biol. Organ.) J.5: 1395-1402; Horowitz, R.A., D.A. Agard, J.W. Sedat, and C.L. Woodcock, 1994. J. Cell Biol. 125:1-10), and in vitro conclusions (van Holde, K., and J. Zlatanova. 1995. J. Biol. Chem. 270:8373-8376). Cryoelectron microscopy also provides a way to determine the 3-D conformation of naturally occurring chromatins in which precise nucleosome positioning plays a role in transcriptional regulation.
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Morse, R. H., S. Y. Roth, and R. T. Simpson. "A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo." Molecular and Cellular Biology 12, no. 9 (September 1992): 4015–25. http://dx.doi.org/10.1128/mcb.12.9.4015-4025.1992.
Full textAbstract:
Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.
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Zhou, Keda, Magdalena Gebala, Dustin Woods, Kousik Sundararajan, Garrett Edwards, Dan Krzizike, Jeff Wereszczynski, Aaron F. Straight, and Karolin Luger. "CENP-N promotes the compaction of centromeric chromatin." Nature Structural & Molecular Biology 29, no. 4 (April 2022): 403–13. http://dx.doi.org/10.1038/s41594-022-00758-y.
Full textAbstract:
AbstractThe histone variant CENP-A is the epigenetic determinant for the centromere, where it is interspersed with canonical H3 to form a specialized chromatin structure that nucleates the kinetochore. How nucleosomes at the centromere arrange into higher order structures is unknown. Here we demonstrate that the human CENP-A-interacting protein CENP-N promotes the stacking of CENP-A-containing mononucleosomes and nucleosomal arrays through a previously undefined interaction between the α6 helix of CENP-N with the DNA of a neighboring nucleosome. We describe the cryo-EM structures and biophysical characterization of such CENP-N-mediated nucleosome stacks and nucleosomal arrays and demonstrate that this interaction is responsible for the formation of densely packed chromatin at the centromere in the cell. Our results provide first evidence that CENP-A, together with CENP-N, promotes specific chromatin higher order structure at the centromere.
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Brambilla, Francesca, Jose Manuel Garcia-Manteiga, Emanuele Monteleone, Lena Hoelzen, Angelica Zocchi, Alessandra Agresti, and Marco E. Bianchi. "Nucleosomes effectively shield DNA from radiation damage in living cells." Nucleic Acids Research 48, no. 16 (July 25, 2020): 8993–9006. http://dx.doi.org/10.1093/nar/gkaa613.
Full textAbstract:
Abstract Eukaryotic DNA is organized in nucleosomes, which package DNA and regulate its accessibility to transcription, replication, recombination and repair. Here, we show that in living cells nucleosomes protect DNA from high-energy radiation and reactive oxygen species. We combined sequence-based methods (ATAC-seq and BLISS) to determine the position of both nucleosomes and double strand breaks (DSBs) in the genome of nucleosome-rich malignant mesothelioma cells, and of the same cells partially depleted of nucleosomes. The results were replicated in the human MCF-7 breast carcinoma cell line. We found that, for each genomic sequence, the probability of DSB formation is directly proportional to the fraction of time it is nucleosome-free; DSBs accumulate distal from the nucleosome dyad axis. Nucleosome free regions and promoters of actively transcribed genes are more sensitive to DSB formation, and consequently to mutation. We argue that this may be true for a variety of chemical and physical DNA damaging agents.
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Cutter, Amber R., and Jeffrey J. Hayes. "Linker histones: novel insights into structure-specific recognition of the nucleosome." Biochemistry and Cell Biology 95, no. 2 (April 2017): 171–78. http://dx.doi.org/10.1139/bcb-2016-0097.
Full textAbstract:
Linker histones (H1s) are a primary component of metazoan chromatin, fulfilling numerous functions, both in vitro and in vivo, including stabilizing the wrapping of DNA around the nucleosome, promoting folding and assembly of higher order chromatin structures, influencing nucleosome spacing on DNA, and regulating specific gene expression. However, many molecular details of how H1 binds to nucleosomes and recognizes unique structural features on the nucleosome surface remain undefined. Numerous, confounding studies are complicated not only by experimental limitations, but the use of different linker histone isoforms and nucleosome constructions. This review summarizes the decades of research that has resulted in several models of H1 association with nucleosomes, with a focus on recent advances that suggest multiple modes of H1 interaction in chromatin, while highlighting the remaining questions.
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Nikitina, Tatiana, Rajarshi P. Ghosh, Rachel A. Horowitz-Scherer, Jeffrey C. Hansen, Sergei A. Grigoryev, and Christopher L. Woodcock. "MeCP2-Chromatin Interactions Include the Formation of Chromatosome-like Structures and Are Altered in Mutations Causing Rett Syndrome." Journal of Biological Chemistry 282, no. 38 (July 27, 2007): 28237–45. http://dx.doi.org/10.1074/jbc.m704304200.
Full textAbstract:
hMeCP2 (human methylated DNA-binding protein 2), mutations of which cause most cases of Rett syndrome (RTT), is involved in the transmission of repressive epigenetic signals encoded by DNA methylation. The present work focuses on the modifications of chromatin architecture induced by MeCP2 and the effects of RTT-causing mutants. hMeCP2 binds to nucleosomes close to the linker DNA entry-exit site and protects ∼11 bp of linker DNA from micrococcal nuclease. MeCP2 mutants differ in this property; the R106W mutant gives very little extra protection beyond the ∼146-bp nucleosome core, whereas the large C-terminal truncation R294X reveals wild type behavior. Gel mobility assays show that linker DNA is essential for proper MeCP2 binding to nucleosomes, and electron microscopy visualization shows that the protein induces distinct conformational changes in the linker DNA. When bound to nucleosomes, MeCP2 is in close proximity to histone H3, which exits the nucleosome core close to the proposed MeCP2-binding site. These findings firmly establish nucleosomal linker DNA as a crucial binding partner of MeCP2 and show that different RTT-causing mutations of MeCP2 are correspondingly defective in different aspects of the interactions that alter chromatin architecture.
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Skrajna, Aleksandra, Dennis Goldfarb, Katarzyna M. Kedziora, Emily M. Cousins, Gavin D. Grant, Cathy J. Spangler, Emily H. Barbour, et al. "Comprehensive nucleosome interactome screen establishes fundamental principles of nucleosome binding." Nucleic Acids Research 48, no. 17 (July 7, 2020): 9415–32. http://dx.doi.org/10.1093/nar/gkaa544.
Full textAbstract:
Abstract Nuclear proteins bind chromatin to execute and regulate genome-templated processes. While studies of individual nucleosome interactions have suggested that an acidic patch on the nucleosome disk may be a common site for recruitment to chromatin, the pervasiveness of acidic patch binding and whether other nucleosome binding hot-spots exist remain unclear. Here, we use nucleosome affinity proteomics with a library of nucleosomes that disrupts all exposed histone surfaces to comprehensively assess how proteins recognize nucleosomes. We find that the acidic patch and two adjacent surfaces are the primary hot-spots for nucleosome disk interactions, whereas nearly half of the nucleosome disk participates only minimally in protein binding. Our screen defines nucleosome surface requirements of nearly 300 nucleosome interacting proteins implicated in diverse nuclear processes including transcription, DNA damage repair, cell cycle regulation and nuclear architecture. Building from our screen, we demonstrate that the Anaphase-Promoting Complex/Cyclosome directly engages the acidic patch, and we elucidate a redundant mechanism of acidic patch binding by nuclear pore protein ELYS. Overall, our interactome screen illuminates a highly competitive nucleosome binding hub and establishes universal principles of nucleosome recognition.
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Whitehouse, Iestyn, Chris Stockdale, Andrew Flaus, Mark D. Szczelkun, and Tom Owen-Hughes. "Evidence for DNA Translocation by the ISWI Chromatin-Remodeling Enzyme." Molecular and Cellular Biology 23, no. 6 (March 15, 2003): 1935–45. http://dx.doi.org/10.1128/mcb.23.6.1935-1945.2003.
Full textAbstract:
ABSTRACT The ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin-remodeling activities. Here, we studied the interaction of the ISWI protein with nucleosomal substrates. We found that the ability of nucleic acids to bind and stimulate the ATPase activity of ISWI depends on length. We also found that ISWI is able to displace triplex-forming oligonucleotides efficiently when they are introduced at sites close to a nucleosome but successively less efficiently 30 to 60 bp from its edge. The ability of ISWI to direct triplex displacement was specifically impeded by the introduction of 5- or 10-bp gaps in the 3′-5′ strand between the triplex and the nucleosome. In combination, these observations suggest that ISWI is a 3′-5′-strand-specific, ATP-dependent DNA translocase that may be capable of forcing DNA over the surface of nucleosomes.
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Mohan, C., S. Adams, V. Stanik, and S. K. Datta. "Nucleosome: a major immunogen for pathogenic autoantibody-inducing T cells of lupus." Journal of Experimental Medicine 177, no. 5 (May 1, 1993): 1367–81. http://dx.doi.org/10.1084/jem.177.5.1367.
Full textAbstract:
Only a fraction (12%) of 268 "autoreactive" T cell clones derived from lupus-prone mice can selectively induce the production of pathogenic anti-DNA autoantibodies in vitro and accelerate the development of lupus nephritis when transferred in vivo. The CDR3 loops of T cell receptor beta chains expressed by these pathogenic T helper (Th) clones contain a recurrent motif of anionic residues suggesting that they are selected by autoantigens with cationic residues. Herein, we found that approximately 50% of these pathogenic Th clones were specific for nucleosomal antigens, but none of them responded to cationic idiopeptides shared by variable regions of pathogenic anti-DNA autoantibodies. Nucleosomes did not stimulate the T cells as a nonspecific mitogen or superantigen. Only the pathogenic Th cells of lupus responded to nucleosomal antigens that were processed and presented via the major histocompatibility class II pathway. Although the presentation of purified mononucleosomes to the Th clones could be blocked by inhibitors of endosomal proteases, neither of the two components of the nucleosomes--free DNA or histones by themselves--could stimulate the Th clones. Thus critical peptide epitopes for the Th cells were probably protected during uptake and processing of the nucleosome particle as a whole. The nucleosome-specific Th clones preferentially augmented the production of IgG autoantibodies to histone-DNA complex in vitro. In vivo, nucleosome-specific, CD4+ T cells were not detectable in normal mice, but they were found in the spleens of lupus-prone mice as early as 1 mo of age, long before other autoimmune manifestations. Immunization of young, preautoimmune lupus mice with nucleosomes augmented the production of autoantibodies and markedly accelerated the development of severe glomerulonephritis. Previously, crude preparations containing nucleosomes were shown by others to have polyclonal mitogenic activity for B cells from normal as well as lupus mice. Identification here of pure mononucleosome as a lupus-specific immunogen for the Th cells that selectively help the pathogenic anti-DNA autoantibody producing B cells of lupus could lead to the design of specific therapy against this pathogenic autoimmune response.
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Wippo, Christian J., Bojana Silic Krstulovic, Franziska Ertel, Sanja Musladin, Dorothea Blaschke, Sabrina Stürzl, Guo-Cheng Yuan, Wolfram Hörz, Philipp Korber, and Slobodan Barbaric. "Differential Cofactor Requirements for Histone Eviction from Two Nucleosomes at the Yeast PHO84 Promoter Are Determined by Intrinsic Nucleosome Stability." Molecular and Cellular Biology 29, no. 11 (March 23, 2009): 2960–81. http://dx.doi.org/10.1128/mcb.01054-08.
Full textAbstract:
ABSTRACT We showed previously that the strong PHO5 promoter is less dependent on chromatin cofactors than the weaker coregulated PHO8 promoter. In this study we asked if chromatin remodeling at the even stronger PHO84 promoter was correspondingly less cofactor dependent. The repressed PHO84 promoter showed a short hypersensitive region that was flanked upstream and downstream by a positioned nucleosome and contained two transactivator Pho4 sites. Promoter induction generated an extensive hypersensitive and histone-depleted region, yielding two more Pho4 sites accessible. This remodeling was strictly Pho4 dependent, strongly dependent on the remodelers Snf2 and Ino80 and on the histone acetyltransferase Gcn5, and more weakly on the acetyltransferase Rtt109. Importantly, remodeling of each of the two positioned nucleosomes required Snf2 and Ino80 to different degrees. Only remodeling of the upstream nucleosome was strictly dependent on Snf2. Further, remodeling of the upstream nucleosome was more dependent on Ino80 than remodeling of the downstream nucleosome. Both nucleosomes differed in their intrinsic stabilities as predicted in silico and measured in vitro. The causal relationship between the different nucleosome stabilities and the different cofactor requirements was shown by introducing destabilizing mutations in vivo. Therefore, chromatin cofactor requirements were determined by intrinsic nucleosome stabilities rather than correlated to promoter strength.
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Hara, Ryujiro, Jinyao Mo, and Aziz Sancar. "DNA Damage in the Nucleosome Core Is Refractory to Repair by Human Excision Nuclease." Molecular and Cellular Biology 20, no. 24 (December 15, 2000): 9173–81. http://dx.doi.org/10.1128/mcb.20.24.9173-9181.2000.
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ABSTRACT To investigate the effect of nucleosomes on nucleotide excision repair in humans, we prepared a mononucleosome containing a (6-4) photoproduct in the nucleosome core and examined its repair with the reconstituted human excision nuclease system and with cell extracts. Nucleosomal DNA is repaired at a rate of about 10% of that for naked DNA in both systems. These results are in agreement with in vivo data showing a considerably slower rate of repair of overall genomic DNA relative to that for transcriptionally active DNA. Furthermore, our results indicate that the first-order packing of DNA in nucleosomes is a primary determinant of slow repair of DNA in chromatin.
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Cole, Lauren, and Jonathan Dennis. "MNase Profiling of Promoter Chromatin in Salmonella typhimurium-Stimulated GM12878 Cells Reveals Dynamic and Response-Specific Nucleosome Architecture." G3: Genes|Genomes|Genetics 10, no. 7 (May 13, 2020): 2171–78. http://dx.doi.org/10.1534/g3.120.401266.
Full textAbstract:
The nucleosome is the primary unit of chromatin structure and commonly imputed as a regulator of nuclear events, although the exact mechanisms remain unclear. Recent studies have shown that certain nucleosomes can have different sensitivities to micrococcal nuclease (MNase) digestion, resulting in the release of populations of nucleosomes dependent on the concentration of MNase. Mapping MNase sensitivity of nucleosomes at transcription start sites genome-wide reveals an important functional nucleosome organization that correlates with gene expression levels and transcription factor binding. In order to understand nucleosome distribution and sensitivity dynamics during a robust genome response, we mapped nucleosome position and sensitivity using multiple concentrations of MNase. We used the innate immune response as a model system to understand chromatin-mediated regulation. Herein we demonstrate that stimulation of a human lymphoblastoid cell line (GM12878) with heat-killed Salmonella typhimurium (HKST) results in changes in nucleosome sensitivity to MNase. We show that the HKST response alters the sensitivity of -1 nucleosomes at highly expressed promoters. Finally, we correlate the increased sensitivity with response-specific transcription factor binding. These results indicate that nucleosome sensitivity dynamics reflect the cellular response to HKST and pave the way for further studies that will deepen our understanding of the specificity of genome response.
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Eckey, Maren, Wei Hong, Maria Papaioannou, and Aria Baniahmad. "The Nucleosome Assembly Activity of NAP1 Is Enhanced by Alien." Molecular and Cellular Biology 27, no. 10 (March 5, 2007): 3557–68. http://dx.doi.org/10.1128/mcb.01106-06.
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ABSTRACT The assembly of nucleosomes into chromatin is essential for the compaction of DNA and inactivation of the DNA template to modulate and repress gene expression. The nucleosome assembly protein 1, NAP1, assembles nucleosomes independent of DNA synthesis and was shown to enhance coactivator-mediated gene expression, suggesting a role for NAP1 in transcriptional regulation. Here, we show that Alien, known to harbor characteristics of a corepressor of nuclear hormone receptors such as of the vitamin D receptor (VDR), binds in vivo and in vitro to NAP1 and modulates its activity by enhancing NAP1-mediated nucleosome assembly on DNA. Furthermore, Alien reduces the accessibility of the histones H3 and H4 for NAP1-promoted assembly reaction. This indicates that Alien sustains and reinforces the formation of nucleosomes. Employing deletion mutants of Alien suggests that different regions of Alien are involved in enhancement of NAP1-mediated nucleosome assembly and in inhibiting the accessibility of the histones H3 and H4. In addition, we provide evidence that Alien is associated with chromatin and with micrococcus nuclease-prepared nucleosome fractions and interacts with the histones H3 and H4. Furthermore, chromatin immunoprecipitation and reimmunoprecipitation experiments suggest that NAP1 and Alien localize to the endogenous CYP24 promoter in vivo, a VDR target gene. Based on these findings, we present here a novel pathway linking corepressor function with nucleosome assembly activity.
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Saunders, M. J., E. Yeh, M. Grunstein, and K. Bloom. "Nucleosome depletion alters the chromatin structure of Saccharomyces cerevisiae centromeres." Molecular and Cellular Biology 10, no. 11 (November 1990): 5721–27. http://dx.doi.org/10.1128/mcb.10.11.5721-5727.1990.
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Saccharomyces cerevisiae centromeric DNA is packaged into a highly nuclease-resistant chromatin core of approximately 200 base pairs of DNA. The structure of the centromere in chromosome III is somewhat larger than a 160-base-pair nucleosomal core and encompasses the conserved centromere DNA elements (CDE I, II, and III). Extensive mutational analysis has revealed the sequence requirements for centromere function. Mutations affecting the segregation properties of centromeres also exhibit altered chromatin structures in vivo. Thus the structure, as delineated by nuclease digestion, correlated with functional centromeres. We have determined the contribution of histone proteins to this unique structural organization. Nucleosome depletion by repression of either histone H2B or H4 rendered the cell incapable of chromosome segregation. Histone repression resulted in increased nuclease sensitivity of centromere DNA, with up to 40% of CEN3 DNA molecules becoming accessible to nucleolytic attack. Nucleosome depletion also resulted in an alteration in the distribution of nuclease cutting sites in the DNA surrounding CEN3. These data provide the first indication that authentic nucleosomal subunits flank the centromere and suggest that nucleosomes may be the central core of the centromere itself.
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Morillon, Antonin. "Is histone loss a common feature of DNA metabolism regulation?This paper is one of a selection of papers published in this Special Issue, entitled 27th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process." Biochemistry and Cell Biology 84, no. 4 (August 2006): 450–52. http://dx.doi.org/10.1139/o06-070.
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Chromatin modifications play a crucial role in regulating DNA metabolism. Chromatin structures can be remodeled by covalently modifying histones, by shifting nucleosomes along the DNA, and by changing the histone composition of nucleosomes. Lately, nucleosome displacement has been extensively described within transcribed genes and DNA breaks. This review focuses on recently published work that describes the relationships between histone modification/exchange and nucleosome displacement.