Dynamics of Nucleosomes Revealed by Time-Lapse Atomic Force Microscopy

Shlyakhtenko, Luda S.; Lushnikov, Alexander Y.; Lyubchenko, Yuri L.

doi:10.1021/bi900977t

Cited by 77 publications

(132 citation statements)

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“…S1 of the SI Appendix), which was recently shown to prevent the nucleosome from sliding (32). This first series of experiments demonstrates the excluding role of high-energy barriers that are encoded in the sequence and that preclude nucleosome formation.…”

Section: Resultsmentioning

confidence: 60%

Nucleosome positioning by genomic excluding-energy barriers

Milani

Chevereau

Vaillant

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Recent genome-wide nucleosome mappings along with bioinformatics studies have confirmed that the DNA sequence plays a more important role in the collective organization of nucleosomes in vivo than previously thought. Yet in living cells, this organization also results from the action of various external factors like DNAbinding proteins and chromatin remodelers. To decipher the code for intrinsic chromatin organization, there is thus a need for in vitro experiments to bridge the gap between computational models of nucleosome sequence preferences and in vivo nucleosome occupancy data. Here we combine atomic force microscopy in liquid and theoretical modeling to demonstrate that a major sequence signaling in vivo are high-energy barriers that locally inhibit nucleosome formation rather than favorable positioning motifs. We show that these genomic excluding-energy barriers condition the collective assembly of neighboring nucleosomes consistently with equilibrium statistical ordering principles. The analysis of two gene promoter regions in Saccharomyces cerevisiae and the human genome indicates that these genomic barriers direct the intrinsic nucleosome occupancy of regulatory sites, thereby contributing to gene expression regulation.nucleosome statistical ordering | chromatin-mediated gene regulation | physical modeling | atomic force microscopy T he recent flowering of tiled micro-arrays (1, 2) and chipsequencing (3) approaches has provided an unprecedented opportunity to elucidate the extent to which the DNA sequence participates in the positioning of nucleosomes observed in vivo along eukaryotic chromosomes (4, 5). Among the results indicating that nucleosome formation is facilitated by the DNA sequence, it is known that some genomic sequences presenting a ≈10 bp periodicity of some di-or trinucleotides (e.g., AA/TT) show higher affinity for nucleosomes (6-9). The periodic positioning of these motifs over a few helical pitches would contribute to a global spontaneous curvature of DNA that would favor its wrapping on the histone surface (10, 11). However, the statistical significance of this 10-bp periodicity nucleosomal positioning signal remains a subject of great debate (4,5,12,13). According to previous reports (12, 13), in Saccharomyces cerevisiae (1, 2), no more than 20% of the in vivo nucleosome positioning, above what is expected by chance, is determined by intrinsic signals in the genomic DNA. An alternative antipositioning signaling picture has recently emerged from bioinformatic studies (13-16) that bring to light the fact that the sequence is actually highly predictive of the nucleosome-free regions (NFRs) observed in vivo at gene promoters and terminations (1, 2). Excluding-energy barriers coded in the sequence would locally impair nucleosome formation and nonlocally influence the overall nucleosomal chromatin organization according to equilibrium statistical ordering principles (14, 17). Furthermore, by conditioning an activatory or inhibitory nucleosomal chromatin environment, these genomic energy b...

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Section: Resultsmentioning

confidence: 60%

Nucleosome positioning by genomic excluding-energy barriers

Milani

Chevereau

Vaillant

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…1a to c). For AFM images, it is possible to estimate the volume of a particle and derive the approximate mass (41,40,42,58,59). The volumes of these foci show a bimodal distribution (Fig.…”

Section: Resultsmentioning

confidence: 99%

MeCP2 Binds Cooperatively to Its Substrate and Competes with Histone H1 for Chromatin Binding Sites

Ghosh

Horowitz-Scherer

Nikitina

et al. 2010

Molecular and Cellular Biology

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Sporadic mutations in the hMeCP2 gene, coding for a protein that preferentially binds symmetrically methylated CpGs, result in the severe neurological disorder Rett syndrome (RTT). In the present work, employing a wide range of experimental approaches, we shed new light on the many levels of MeCP2 interaction with DNA and chromatin. We show that strong methylation-independent as well as methylation-dependent binding by MeCP2 is influenced by DNA length. Although MeCP2 is strictly monomeric in solution, its binding to DNA is cooperative, with dimeric binding strongly correlated with methylation density, and strengthened by nearby A/T repeats. Dimeric binding is abolished in the F155S and R294X severe RTT mutants. MeCP2 also binds chromatin in vitro, resulting in compaction-related changes in nucleosome architecture that resemble the classical zigzag motif induced by histone H1 and considered important for 30-nm-fiber formation. In vivo chromatin binding kinetics and in vitro steady-state nucleosome binding of both MeCP2 and H1 provide strong evidence for competition between MeCP2 and H1 for common binding sites. This suggests that chromatin binding by MeCP2 and H1 in vivo should be viewed in the context of competitive multifactorial regulation.DNA methylation constitutes an important epigenetic component in transcriptional regulation, with methylation generally leading to repression of nearby genes (6). However, the mechanism by which the epigenetic signal is passed to the regulatory machinery is not well understood. Research in this area has been focused on a small family of methyl-CpG binding proteins, best characterized by MeCP2 (19), mutations in which result in Rett syndrome (RTT), a debilitating neurodevelopmental disease in humans (2).A mechanism of MeCP2-mediated gene silencing may involve recruitment of histone deacetylases upon methyl-specific binding (57). However, other mechanisms, which are not necessarily mutually exclusive, such as stabilization of large chromatin loops (29) and promotion of chromatin compaction (51), have also been suggested (14). Studies on in vivo distribution of MeCP2 in nuclei have revealed that, in addition to the expected occupancy of sites of CpG methylation, MeCP2 shows significant binding to unmethylated DNA (71). However, a recent analysis of MeCP2 occupancy has revealed that the genomic distribution of MeCP2 in mammalian neurons closely tracks methyl-CpG density (60). These results highlight our current lack of understanding of key questions pertinent to the binding of MeCP2 to DNA and chromatin. It is especially important, for example, to quantitate the modulation of binding by factors such as methylation density (8,37,48,60) and the presence of adjacent A/T-rich sequences (31) that are reported to influence binding. In the present work, we have used a variety of quantitative approaches to show that, when bound to DNA, MeCP2 exhibits a cooperative monomerdimer equilibrium, which is influenced by DNA length, methylation density, and the presence of nearby A/T repeats.Th...

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“…DNA used for the nucleosome assembly is similar to that used in previous studies. 26 DNA substrate was generated by polymerase chain reaction using plasmid pGEM3Z-601 as a template, which encodes a high-affinity nucleosome positioning sequence 601. 32 The 601 sequence (147 bp) was located inside the 353 bp DNA fragment with 127 and 79 bp arm DNA, so that the mononucleosome reconstituted with the DNA substrate has two DNA arms that differ in length by 1.6-fold.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…24 Note in this regard, recent studies using time-lapse observations allowed direct observation of the dynamics and unwrapping of nucleosomes. 25,26 The unwrapping of nucleosomes proceeds from the ends of the particle, resulting in unwrapping of DNA regions as large as dozens of base pairs. This process may lead to a complete unfolding of nucleosomes and dissociation of the histone core from the complex.…”

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confidence: 99%

Dynamics of Nucleosomes Assessed with Time-Lapse High-Speed Atomic Force Microscopy

2011

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A fundamental challenge of gene regulation is the accessibility of DNA within nucleosomes. Recent studies performed by various techniques, including single-molecule approaches, led to the realization that nucleosomes are quite dynamic rather than static systems, as they were once considered. Direct data are needed to characterize the dynamics of nucleosomes. Specifically, if nucleosomes are dynamic, the following questions need to be answered. What is the range of nucleosome dynamics? Is a non-ATP-dependent unwrapping of nucleosomes possible? What are the factors facilitating the large-scale opening and unwrapping of nucleosomes? In previous studies using time-lapse atomic force microscopy (AFM) imaging, we were able, for the first time, to observe spontaneous, ATP-independent unwrapping of nucleosomes. However, low temporal resolution did not allow visualization of various pathways of nucleosome dynamics. In the studies described here, we applied high-speed time-lapse AFM (HS-AFM) capable of visualizing molecular dynamics on the millisecond time scale to study the nucleosome dynamics. The mononucleosomes were assembled on a 353 bp DNA substrate containing nucleosome-specific 601 sequence. With HS-AFM, we were able to observe the dynamics of nucleosome on a subsecond time scale and visualize various pathways of nucleosome dynamics, such as sliding and unwrapping to various extents, including complete dissociation. These studies highlight an important role of electrostatic interactions in chromatin dynamics. Overall, our findings shed new light on nucleosome dynamics and provide a novel hypothesis for the mechanisms controlling the spontaneous dynamics of chromatin.

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Dynamics of Nucleosomes Revealed by Time-Lapse Atomic Force Microscopy

Cited by 77 publications

References 47 publications

Nucleosome positioning by genomic excluding-energy barriers

Nucleosome positioning by genomic excluding-energy barriers

MeCP2 Binds Cooperatively to Its Substrate and Competes with Histone H1 for Chromatin Binding Sites

Dynamics of Nucleosomes Assessed with Time-Lapse High-Speed Atomic Force Microscopy

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