A perspective of the dynamic structure of the nucleus explored at the single-molecule level

Dange, Thomas; Joseph, Aviva; Grünwald, David

doi:10.1007/s10577-010-9156-5

Cited by 6 publications

(10 citation statements)

References 94 publications

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“…Therefore, the ultimate challenge is the development of strategies that analyze structures in single cells. Live-cell imaging methods that can track a single locus must be employed (Robinett et al 1996;Janicki et al 2004), possibly in combination with single-molecule detection methods, in order to provide further insight into the dynamics of the nuclear structure (Dange et al 2011). Integration of 3C/4C/5C/HiC/ChIA-PET data with highresolution live-cell imaging of tagged loci and singlemolecule tracking data will lead to a better understanding of the dynamics underlying chromosomal interaction data and the cell-to-cell variation of these interactions.…”

Section: Future Directionsmentioning

confidence: 99%

A decade of 3C technologies: insights into nuclear organization

Wit

Laat²

2012

Genes Dev.

657

487

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Over the past 10 years, the development of chromosome conformation capture (3C) technology and the subsequent genomic variants thereof have enabled the analysis of nuclear organization at an unprecedented resolution and throughput. The technology relies on the original and, in hindsight, remarkably simple idea that digestion and religation of fixed chromatin in cells, followed by the quantification of ligation junctions, allows for the determination of DNA contact frequencies and insight into chromosome topology. Here we evaluate and compare the current 3C-based methods (including 4C [chromosome conformation capture-on-chip], 5C [chromosome conformation capture carbon copy], HiC, and ChIA-PET), summarize their contribution to our current understanding of genome structure, and discuss how shape influences genome function.For more than a century, researchers have been making inquiries into the organization of the nucleus, the largest and most easily discernable organelle in the eukaryotic cell. Early in the 20th Century, Cajal (1903) identified subnuclear structures that were later named Cajal bodies. Twenty-five years later, Heitz (1928) observed differentially staining chromatin in interphase nuclei of mosses and described it as heterochromatin and euchromatin. Interest in nuclear structure was further fueled by the realization that the nucleus contains genetic material in the form of DNA fibers. In humans, when unwound, the DNA measures ;2 m, ;200,000 times the diameter of an average mammalian cell nucleus. Packing DNA inside the nucleus therefore imposes tremendous organizational challenges. While already conceptually interesting, the shape of the genome becomes even more fascinating when one realizes that it also relates to genome functioning. Although we are still far from understanding this exact relationship, breakthrough technologies are now available for the systematic and detailed analysis of nuclear organization.Traditionally, nuclear organization is studied by microscopy, and thus it is appropriate to start by highlighting some important observations made under the microscope.However, the emphasis of this review is on novel genomics strategies that are based on chromosome conformation capture (3C) technology. Ten years ago, Dekker et al. (2002) developed 3C technology, a biochemical strategy to analyze contact frequencies between selected genomic sites in cell populations. Since then, various 3C-derived genomics methods have been developed. In comparison with microscopy, 3C-based methods enable more systematic DNA topology studies at a higher resolution: These technologies can put observations made on single genes in selected cells in the context of genomic behavior in cell populations. The generated DNA contact maps start teaching us the rules that dictate genome structure and functioning inside the cell. As we explain here, these rules are probabilistic, not deterministic, implying that they cannot predict the shape and functioning of the genome in individual cells. To investigate this, microscopy i...

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Section: Future Directionsmentioning

confidence: 99%

A decade of 3C technologies: insights into nuclear organization

Wit

Laat²

2012

Genes Dev.

657

487

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“…The integration of complementary techniques like single-molecule tracking (SMT) and fluorescence correlation spectroscopy will be of great value in unravelling the full picture of the dynamic behaviour of proteins. These approaches will be discussed elsewhere in this issue (Erdel et al 2011;Dange et al 2011). …”

Section: Frap Analysismentioning

confidence: 99%

Nuclear proteins: finding and binding target sites in chromatin

et al. 2010

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Fluorescent protein labelling, as well as impressive progress in live cell imaging have revolutionised the view on how essential nuclear functions like gene transcription regulation and DNA repair are organised. Here, we address questions like how DNAinteracting molecules find and bind their target sequences in the vast amount of DNA. In addition, we discuss methods that have been developed for quantitative analysis of data from fluorescence recovery after photobleaching experiments (FRAP).

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“…In theory, anomalous diffusion can be derived from generalized Langevin equations (8), fractional Brownian motion (FBM) (16),percolation (17), diffusion in fractals (18), diffusion in a heterogeneous landscape (19,20), or using power law jump size or waiting time distributions in continuous time random walk (CTRW) models (21).…”

Section: Introductionmentioning

confidence: 99%

Inferring Diffusion Dynamics from FCS in Heterogeneous Nuclear Environments

Tsekouras

Siegel

Day

et al. 2015

Biophysical Journal

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Fluorescence correlation spectroscopy (FCS) is a noninvasive technique that probes the diffusion dynamics of proteins down to single-molecule sensitivity in living cells. Critical mechanistic insight is often drawn from FCS experiments by fitting the resulting time-intensity correlation function, G(t), to known diffusion models. When simple models fail, the complex diffusion dynamics of proteins within heterogeneous cellular environments can be fit to anomalous diffusion models with adjustable anomalous exponents. Here, we take a different approach. We use the maximum entropy method to show-first using synthetic data-that a model for proteins diffusing while stochastically binding/unbinding to various affinity sites in living cells gives rise to a G(t) that could otherwise be equally well fit using anomalous diffusion models. We explain the mechanistic insight derived from our method. In particular, using real FCS data, we describe how the effects of cell crowding and binding to affinity sites manifest themselves in the behavior of G(t). Our focus is on the diffusive behavior of an engineered protein in 1) the heterochromatin region of the cell's nucleus as well as 2) in the cell's cytoplasm and 3) in solution. The protein consists of the basic region-leucine zipper (BZip) domain of the CCAAT/enhancer-binding protein (C/EBP) fused to fluorescent proteins.

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A perspective of the dynamic structure of the nucleus explored at the single-molecule level

Cited by 6 publications

References 94 publications

A decade of 3C technologies: insights into nuclear organization

A decade of 3C technologies: insights into nuclear organization

Nuclear proteins: finding and binding target sites in chromatin

Inferring Diffusion Dynamics from FCS in Heterogeneous Nuclear Environments

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