Active RNA Polymerases: Mobile or Immobile Molecular Machines?

Papantonis, Argyris; Larkin, Joshua D.; Wada, Youichiro; Ohta, Yoshihiro; Ihara, Sigeo; Kodama, Tatsuhiko; Cook, Peter R.

doi:10.1371/journal.pbio.1000419

Cited by 88 publications

(152 citation statements)

References 30 publications

(43 reference statements)

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“…However, 30 min after adding the cytokine (the situation modeled), NFκB binds to the promoter, pioneering polymerases now transcribe the first half of this 221-kbp gene (Fig. 5A) (27), and ChIA-PET reveals that this transcribed half contacts many other segments on the same and other chromosomes; these also tend to bind the polymerase and/or NFκB (16,28). The contacts seen in the simulation mirror those detected by ChIA-PET (Fig.…”

Section: Rna Polymerase II and Nfκb Cluster On Binding To Whole Humanmentioning

confidence: 70%

“…NFκB is normally cytoplasmic, but addition of TNFα to diploid (G0) human umbilical vein endothelial cells (HUVECs) induces phosphorylation of the p65 subunit of NFκB, nuclear import, and binding to thousands of sites around the genome; then, several hundred genes are up-/down-regulated as new intra/ interchromosomal contacts appear. Here, we model the situation 30 min after adding TNFα, a time when we have detailed information on protein binding and how binding influences transcriptional activity and inter/intrachromosomal contacts (16,27,28). To make our model more realistic, the number of monomers in each polymer reflects chromosome length.…”

Section: Rna Polymerase II and Nfκb Cluster On Binding To Whole Humanmentioning

confidence: 99%

See 1 more Smart Citation

Nonspecific bridging-induced attraction drives clustering of DNA-binding proteins and genome organization

Brackley

Taylor²,

Papantonis

et al. 2013

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

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240

344

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Molecular dynamics simulations are used to model proteins that diffuse to DNA, bind, and dissociate; in the absence of any explicit interaction between proteins, or between templates, binding spontaneously induces local DNA compaction and protein aggregation. Small bivalent proteins form into rows [as on binding of the bacterial histone-like nucleoid-structuring protein (H-NS)], large proteins into quasi-spherical aggregates (as on nanoparticle binding), and cylinders with eight binding sites (representing octameric nucleosomal cores) into irregularly folded clusters (like those seen in nucleosomal strings). Binding of RNA polymerase II and a transcription factor (NFκB) to the appropriate sites on four human chromosomes generates protein clusters analogous to transcription factories, multiscale loops, and intrachromosomal contacts that mimic those found in vivo. We suggest that this emergent behavior of clustering is driven by an entropic bridging-induced attraction that minimizes bending and looping penalties in the template.polymer physics | Brownian dynamics | chromatin looping | nucleosome D NA in living cells associates with proteins that continuously bind and dissociate. Some proteins affect local structure (such as histones and histone-like proteins), whereas others act globally to compact whole chromosomal segments [such as CCCTC-binding factor (CTCF)] (1-3). Bound proteins also cluster into supramolecular structures; for example, different transcription factors often bind to the same hot spots in the fly genome (4), and active molecules of RNA polymerase II coassociate in transcription factories (5, 6). In the latter case, clustering generates high local concentrations that facilitate production of the appropriate transcripts, as well as organizing the genome in 3D space.Against this background, biophysicists have begun to model DNA folding driven by DNA-binding proteins (3,(7)(8)(9)(10)(11)(12). Usually, the effects of DNA binding are incorporated into an effective potential that influences DNA dynamics; for instance, by stipulating that selected protein-binding regions in the polymer attract each other (11,12). Here, we use molecular dynamics (MD) to model proteins that diffuse to DNA, bind, and dissociate. In the absence of any explicit mutual attraction between proteins or between monomers in the polymer, we uncover an emergent property of the system: binding spontaneously induces protein clustering and genome compaction. For example, simulations yield structures seen experimentally when proteins representing bacterial histone-like nucleoid-structuring protein (H-NS) (1, 2, 13), gold nanoparticles (14, 15), and nucleosome cores bind to DNA. Using data derived from ChIP coupled to high-throughput sequencing (ChIP-seq) (16), we also model binding of RNA polymerase II and its transcription factor, NFκB, to the appropriate (cognate) sites on four human chromosomes; the two proteins spontaneously cluster into factories that are surrounded by loops that reflect those detected in cells using chromatin inte...

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Section: Rna Polymerase II and Nfκb Cluster On Binding To Whole Humanmentioning

confidence: 70%

Section: Rna Polymerase II and Nfκb Cluster On Binding To Whole Humanmentioning

confidence: 99%

Nonspecific bridging-induced attraction drives clustering of DNA-binding proteins and genome organization

Brackley

Taylor²,

Papantonis

et al. 2013

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

Self Cite

240

344

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“…Live-cell PWS is a natural supplement to superresolution fluorescence techniques, providing quantifiable information about unstained cellular organization to examine the role of the nanoarchitecture on molecular interactions in live cells. In the future, we envision that live-cell PWS can be applied to a broad range of critical studies of structure-function in live cells, leveraging the multimodal potential in conjunction with existing SRM to study: (i) the interaction between chromatin structure and mRNA transport; (ii) the accessibility of euchromatin and heterochromatin to transcription factors (45)(46)(47); (iii) the relationship between chromatin looping, as measured by techniques such as Hi-C, to the physical chromatin structure (6,16,48); (iv) why and how higher-order chromatin structure changes in cancer (14); (v) the role of nuclear architecture as an epigenetic regulator of gene expression (6,16,48); (vi) the effect of metabolism on chromatin structure (40,49); and (vii) the role of chromatin dynamics in stem cell development (50,51).…”

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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“…This microscopybased assay, which can target RNA or DNA (Table 1), reveals proximal FISH foci in a subset of the population. 3,4,6 Collectively, 3C and FISH data suggest that a special pool of cells in the population possesses the correct chromatin organization to permit specific chromosomal interactions.…”

Section: Introductionmentioning

confidence: 97%

“…1 Chromosomal contact is strongly correlated with the transcriptional activity of interacting DNA elements. [2][3][4][5][6][7] Therefore, the physical contact between looped chromatin appears to be fundamental to gene regulation. However, whether the "kissing" of chromatin is a direct cause, or consequence, of transcription is a longstanding question in molecular biology and the subject of intense scrutiny.…”

Section: Introductionmentioning

confidence: 99%

Are genes switched on when they kiss?

Fanucchi

Shibayama

Mhlanga

2014

Nucleus

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Chromatin loops are pervasive and permit the tight compaction of DNA within the confined space of the nucleus. Looping enables distal genes and DNA elements to engage in chromosomal contact, to form multigene complexes. Advances in biochemical and imaging techniques reveal that loopmediated contact is strongly correlated with transcription of interacting DNA. However, these approaches only provide a snapshot of events and therefore are unable to reveal the dynamics of multigene complex assembly. This highlights the necessity to develop single cell-based assays that provide single molecule resolution, and are able to functionally interrogate the role of chromosomal contact on gene regulation. To this end, high-resolution single cell imaging regimes, combined with genome editing approaches, are proving to be pivotal to advancing our understanding of loop-mediated dynamics.

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Active RNA Polymerases: Mobile or Immobile Molecular Machines?

Abstract: Although it is widely assumed that active RNA polymerase tracks along its template, we find that DNA, not the polymerase, moves, suggesting that polymerase works by reeling in the template.

Cited by 88 publications

References 30 publications

Nonspecific bridging-induced attraction drives clustering of DNA-binding proteins and genome organization

Nonspecific bridging-induced attraction drives clustering of DNA-binding proteins and genome organization

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

Are genes switched on when they kiss?

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