Nuclear myosin VI regulates the spatial organization of mammalian transcription initiation

Hari-Gupta, Yukti; Fili, Natalia; Santos, Ália dos; Aw, Cook; Gough, R. E.; Hcw, Reed; Wang, Lin; Aaron, Jesse; Venit, Tomáš; Wait, Eric; Große-Berkenbusch, Andreas; Gebhardt, Christof; Percipalle, Piergiorgio; Chew, Teng‐Leong; Martin-Fernandez, Marisa L.; Cp, Toseland

doi:10.1101/2020.04.21.053124

Cited by 11 publications

(13 citation statements)

References 69 publications

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“…To further address this point, we performed single molecule localisation microscopy (STORM) analysis with the 50A guide 2 h measurement ( Figure 4 G). This approach is equivalent to the widefield imaging performed in Figure 2 and Figure 3 , but with approximately five-fold increased resolution and the ability to count single molecules and quantify clusters [ 21 , 22 ]. We detected γH2AX foci and then quantified the foci using cluster analysis ( Figure 4 H).…”

Section: Resultsmentioning

confidence: 99%

A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9

et al. 2021

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Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-strand breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease enzyme is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex, which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.

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Section: Resultsmentioning

confidence: 99%

A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9

et al. 2021

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“…It would therefore be interesting to assess how Pol II clusters are reestablished following cell division. The reshaping and reestablishment of Pol II foci have recently also been connected to ATP-dependent catalytic processes associated with nuclear actin and myosin 53,54 . Taken together, there is indication of a number of catalytic and mechanochemical processes that might contribute to the establishment and changes of Pol II cluster morphology and await further investigation.…”

Section: Discussionmentioning

confidence: 99%

RNA polymerase II clusters form in line with liquid phase wetting of chromatin

Pancholi

Klingberg

Zhang

et al. 2021

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It is essential for cells to control which genes are transcribed into RNA. In eukaryotes, two major control points are recruitment of RNA polymerase II (Pol II) into a paused state and subsequent pause release to begin transcript elongation. Pol II associates with macromolecular clusters during recruitment, but it remains unclear how Pol II recruitment and pause release might affect these clusters. Here, we show that clusters exhibit morphologies that are in line with wetting of chromatin by a liquid phase enriched in recruited Pol II. Applying instantaneous structured illumination microscopy and stimulated emission double depletion microscopy to pluripotent zebrafish embryos, we find recruited Pol II associated with large clusters, and elongating Pol II with dispersed clusters. A lattice kinetic Monte Carlo model representing recruited Pol II as a liquid phase reproduced the observed cluster morphologies. In this model, chromatin is a copolymer chain containing regions that attract or repel recruited Pol II, supporting droplet formation by wetting or droplet dispersal, respectively.

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“…Many studies use drugs to disrupt cytoskeletal forces, and it is unclear how these may directly affect nuclear events, such as transcription or chromatin organisation. Latrunculin B, for example, disrupts cytoskeletal formation through inhibition of actin polymerisation, but it also disrupts nuclear actin and myosin functions, directly affecting transcription levels and RNA Polymerase II (RNAPII) spatial organisation [ 34 ]. Methods such as micropatterning [ 35 , 36 ] or the use of gels as substrates for cells [ 37 , 38 , 39 ] are becoming increasingly common to change the stresses exerted upon the cytoskeleton.…”

Section: Contributing Factors To Nuclear Mechanicsmentioning

confidence: 99%

Regulation of Nuclear Mechanics and the Impact on DNA Damage

Santos

Toseland

2021

IJMS

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In eukaryotic cells, the nucleus houses the genomic material of the cell. The physical properties of the nucleus and its ability to sense external mechanical cues are tightly linked to the regulation of cellular events, such as gene expression. Nuclear mechanics and morphology are altered in many diseases such as cancer and premature ageing syndromes. Therefore, it is important to understand how different components contribute to nuclear processes, organisation and mechanics, and how they are misregulated in disease. Although, over the years, studies have focused on the nuclear lamina—a mesh of intermediate filament proteins residing between the chromatin and the nuclear membrane—there is growing evidence that chromatin structure and factors that regulate chromatin organisation are essential contributors to the physical properties of the nucleus. Here, we review the main structural components that contribute to the mechanical properties of the nucleus, with particular emphasis on chromatin structure. We also provide an example of how nuclear stiffness can both impact and be affected by cellular processes such as DNA damage and repair.

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Nuclear myosin VI regulates the spatial organization of mammalian transcription initiation

Cited by 11 publications

References 69 publications

A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9

A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9

RNA polymerase II clusters form in line with liquid phase wetting of chromatin

Regulation of Nuclear Mechanics and the Impact on DNA Damage

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