Modulation of Higher Order Chromatin Conformation in Mammalian Cell Nuclei Can Be Mediated by Polyamines and Divalent Cations

Visvanathan, Ashwat; Ahmed, Kashif; Even-Faitelson, Liron; Llères, David; Bazett-Jones, David P.; Lamond, Angus I.

doi:10.1371/journal.pone.0067689

Cited by 69 publications

(67 citation statements)

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“…Therefore, understanding the interplay between the structural organization of chromatin and mitochondrial function is pivotal to understanding numerous diseases. Recent fluorescence microscopy studies have suggested that impairment of cellular metabolism induces rapid (<15-min) transformation of chromatin (42,43). However, these studies often require the production of specialized transfection models (H2B-GFP) or the use of DNA-binding dyes such as Hoechst 33342 and, as such, are limited in their ability to study multiple cell lines and/or over significant periods of time without perturbing the natural cell behavior (42,43).…”

Section: Resultsmentioning

confidence: 99%

Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

Almassalha

Bauer

Chandler

et al. 2016

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

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The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a livecell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higherorder chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure-function relationship in live cells.E very cellular and extracellular structure has a complex nanoscale organization ranging from individual macromolecules that are a few nanometers in size (e.g., protein and DNA) to macromolecular assemblies that are tens to hundreds of nanometers in size (e.g., cell membranes, higher-order chromatin structure, cytoskeleton, and extracellular matrix fibers). A major scientific challenge is to understand these macromolecular structures, specifically their function and interactions in structurally and dynamically complex living cellular systems. To meet these goals, the ideal live-cell imaging technology would satisfy six key requirements: being (i) nanoscale sensitive (<200 nm), (ii) label free, (iii) nonperturbing, (iv) quantitative, (v) high throughput, and (vi) molecularly informative.Current approaches are unable to meet all of these criteria alone. The breakthroughs in superresolution fluorescence microscopy (SRM) have enabled new imaging technologies capable of providing unprecedented molecular identification at the highest resolutions currently available in live cells, but require the use of exogenous fluorophores to visualize macromolecular structures (1-3). For some applications, these labels are indispensable to achieve molecular specificity. However, there are significant drawbacks to the exclusive use of molec...

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Section: Resultsmentioning

confidence: 99%

Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

Almassalha

Bauer

Chandler

et al. 2016

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

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“…In E. coli, the nucleoid body in late stationary phase is very compact, even though the average superhelical density is low (Frenkiel-Krispin et al 2001). Similarly, when HeLa cells are deprived of energy, the chromatin compacts, yet, in this case, there is no evidence for a structurally distinct chromatin fibre (Visvanathan et al 2013). …”

Section: Nucleoprotein Complexesmentioning

confidence: 99%

The regulatory role of DNA supercoiling in nucleoprotein complex assembly and genetic activity

Muskhelishvili

Travers

2016

Biophys Rev

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We argue that dynamic changes in DNA supercoiling in vivo determine both how DNA is packaged and how it is accessed for transcription and for other manipulations such as recombination. In both bacteria and eukaryotes, the principal generators of DNA superhelicity are DNA translocases, supplemented in bacteria by DNA gyrase. By generating gradients of superhelicity upstream and downstream of their site of activity, translocases enable the differential binding of proteins which preferentially interact with respectively more untwisted or more writhed DNA. Such preferences enable, in principle, the sequential binding of different classes of protein and so constitute an essential driver of chromatin organization.Keywords DNA supercoiling . DNA translocases . Chromatin organization . Superhelicity gradients . DNA untwisting . DNAwrithing Dynamic changes of DNA supercoiling act as a driving force behind the alterations of genetic activity and DNA compaction both in eukaryotes and prokaryotes. Both these processes involve formation of spatially organized nucleoprotein structures by DNA architectural proteins. Whereas in eukaryotes the paradigmal example is the histone octamer, in prokaryotes a similar function is attributed to a small class of highly abundant nucleoid-associated proteins. We argue that, depending on the primary sequence organization, the changes of superhelicity elicit various alterations of local DNA geometry, while these, in turn, facilitate the assembly of distinct nucleoprotein structures pertinent to genetic function. Genome-wide conversion of the superhelical energy into various three-dimensional DNA structures thus acts as an analogue code coordinating the energy supply with the genetic expression of the chromosome.Recent studies make it increasingly clear that the DNA double helix carries at least two types of encoded information and that these include the well-known genetic code and the structural information determining the form and physical properties of the double helix itself. Since both these information types are inscribed in the same DNA sequence, and yet one is discrete whereas the other is continuous, they have been respectively dubbed the digital and analog DNA codes (Marr et al. 2008;Travers et al. 2012;Muskhelishvili and Travers 2013). The potential of the DNA to adopt distinct configurations is strongly modulated by DNA supercoiling, which is increasingly recognized as one of the major forces coordinating the cellular DNA transactions in both prokaryotes (including Archaea) and eukaryotes.DNA supercoiling can be generated by the simple application of torque to the double helix (Strick et al. 1998) but, importantly, because the DNA molecule itself possesses chirality-it is, after all, a right-handed double helix-the local structures stabilized by changes in superhelical density depend on both the sign and strength of the applied torque. For example, both positive and negative torque (respectively overand underwinding of the double helix) can change the intrinsic coiling of ...

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“…Polyamines caused chromatin compaction in an array made of positioning nucleosomes with 177 bp in length (Korolev et al 2010). Further, increasing concentrations of SPM and SPD caused an increase in chromatin compaction in permeabilized HeLa cells with maximum compaction seen at 1.5 mM of SPD and at 0.4 mM of SPM (Visvanathan et al 2013). Therefore, conjugation of polyamines in histone proteins catalyzed by TG2 may cause chromatin compaction nearby and possibly modulate chromatin remodeling.…”

Section: Discussionmentioning

confidence: 97%

Uncovering protein polyamination by the spermine-specific antiserum and mass spectrometric analysis

Chou

Lee

et al. 2014

Amino Acids

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The polyamines spermidine and spermine, and their precursor putrescine, have been shown to play an important role in cell migration, proliferation, and differentiation. Because of their polycationic property, polyamines are traditionally thought to be involved in DNA replication, gene expression, and protein translation. However, polyamines can also be covalently conjugated to proteins by transglutaminase 2 (TG2). This modification leads to an increase in positive charge in the polyamine-incorporated region which significantly alters the structure of proteins. It is anticipated that protein polyamine conjugation may affect the protein-protein interaction, protein localization, and protein function of the TG2 substrates. In order to investigate the roles of polyamine modification, we synthesized a spermine-conjugated antigen and generated an antiserum against spermine. In vitro TG2-catalyzed spermine incorporation assays were carried out to show that actin, tubulins, heat shock protein 70 and five types of histone proteins were modified with spermine, and modification sites were also identified by liquid chromatography and linear ion trap-orbitrap hybrid mass spectrometry. Subsequent mass spectrometry-based shotgun proteomic analysis also identified 254 polyaminated sites in 233 proteins from the HeLa cell lysate catalyzed by human TG2 with spermine, thus allowing, for the first time, a global appraisal of site-specific protein polyamination. Global analysis of mouse tissues showed that this modification really exists in vivo. Importantly, we have demonstrated that there is a new histone modification, polyamination, in cells. However, the functional significance of histone polyamination demands further investigations.

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Modulation of Higher Order Chromatin Conformation in Mammalian Cell Nuclei Can Be Mediated by Polyamines and Divalent Cations

Cited by 69 publications

References 50 publications

Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

The regulatory role of DNA supercoiling in nucleoprotein complex assembly and genetic activity

Uncovering protein polyamination by the spermine-specific antiserum and mass spectrometric analysis

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