Real-time imaging of specific genomic loci in eukaryotic cells using the ANCHOR DNA labelling system

Germier, Thomas; Audibert, Sylvain; Kočanová, Silvia; Lane, David; Bystricky, Kerstin

doi:10.1016/j.ymeth.2018.04.008

Cited by 33 publications

(30 citation statements)

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“…Our ability to study these chromatin dynamics have been revolutionised by technologies based on genome editing that allow specific genomic loci to be targeted in live cells. Early iterations of this approach were rather laborious; cell lines needed to be created in which the target locus was tagged with DNA binding site arrays that recruit a fluorescently-tagged cognate DNA-binding protein (such as the Lac operatorrepressor (Belmont and Straight, 1998;Robinett et al, 1996), Tet operator-repressor (Lucas et al, 2014) and ANCHOR (Germier et al, 2018) systems). Now, loci can be targeted in live cells with a version of the CRISPR (clustered regularly interspaced short palindromic repeats) system that uses an endonuclease-deficient form of Cas9 (dead-Cas9 (dCas9)) fused with a fluorescent protein .…”

Section: Live-cell Imaging Of Nuclear Structuresmentioning

confidence: 99%

Methods for mapping 3D chromosome architecture

2019

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Several people have supported and helped me to during my PhD with guidance and advice, with contributions to the here presented work, but also with their friendship. First, I would like to thank my supervisor Ana for guiding me though my PhD and helping me manage three interesting but also challenging projects with ups and downs along the way. Thank you for lots of great advice and for helping me stay positive in difficult times.Many thanks to Uwe and Robert for our annual committee meetings, your time and advice, and my PhD committee for taking the time to review this thesis.I would like to thank Sasha (Alexander) for working with me on what sometimes seemed to be endless GAM optimisations, and for advice in all possible and impossible challenges that the wet lab provides. Many thanks go to all bioinformaticians that worked with me during my PhD; Ibai, Sasha (again), Ehsan, Christoph, Dominik, Tom, Rob, Mariano, and Markus. All of you are indispensable to making these projects a success, and you did not only do an amazing job but also managed to teach me a basic understanding of data analysis. Many thanks to Rob for all his prior work on GAM and for teaching me GAM when I joined the lab. Thank you, Enric, for joining our lab for the task of setting up an in-house WGA, your initiative and your work made this possible. Special thanks go to Gesa for helping me out when collecting nuclear profiles got too much for just one person, and for being a great friend. Without you the lab would have been only half the fun. I would also like to thank Izabela for her contribution to our final optimisations, which gave me a lot of positive energy when I really needed it, and everyone else in the Pombo lab for their help and/or friendship;

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Section: Live-cell Imaging Of Nuclear Structuresmentioning

confidence: 99%

Methods for mapping 3D chromosome architecture

2019

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“…However, while a useful tool, this system has important drawbacks, specifically the high repeats present in the LacO sequence can disrupt chromatin organization/function and importantly this system is often inserted in the genome in multiple copies at random locations and is not suitable for visualizing endogenous genomic sites. Alternatively, the Anchor (ParB/parS) system takes advantage of protein oligomerization at a site specific locus, rather than a repetitive gene array to achieve the signal amplification needed to visualize this locus (Figure 3C) 59 . Despite not using a large repetitive gene array, the parS sequence still needs to be incorporated into the genome and doing so at a specific locus without perturbing the locus dynamics, organization and function may be challenging.…”

Section: Live Cell Imaging Of Genome Dynamicsmentioning

confidence: 99%

Visualizing the genome in high resolution challenges our textbook understanding

Lakadamyali

Cosma

2020

Nat Methods

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In the recent decade, new methods have been developed that probe the 4D folding of the genome in space and time with unprecedented resolution. These methods, including chromosome conformation capture and high resolution light and electron microscopy, are shedding new light onto genome architecture and function. We review the emerging picture on genome organization revealed by super-resolution and live-cell imaging. We compare and contrast population based chromosome conformation capture approaches and imaging based approaches and highlight the future challenges ahead.

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“…More recently, it has been used to monitor the mobility of a genomic locus upon activation of transcription [25,26] and to visualize viral replication [27] in live mammalian cells. We made the system inducible by fusing ParB to a domain of the A. thaliana protein ABSCISIC ACID INSENSITIVE 1 (ABI), which dimerizes with a domain of PYRABACTIN RESISTANCE1-LIKE 1 (PYL) upon addition of abscisic acid (ABA) to the culture medium [28].…”

Section: Resultsmentioning

confidence: 99%

Expanded CAG/CTG Repeats Resist Gene Silencing Mediated by Targeted Epigenome Editing

Yang

Borgeaud

Aeschbach

et al. 2018

Preprint

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Epigenome editing is an attractive way to manipulate gene expression. However, editing efficiencies depend on the DNA sequence context in a manner that remains poorly understood.Here we developed a novel system in which any protein can be recruited at will to a GFP reporter.We named it ParB/ANCHOR-mediated Inducible Targeting (PInT). Using PInT, we tested how CAG/CTG repeat size affects the ability of histone deacetylases to modulate gene expression. We found that repeat expansion reduces the effectiveness of silencing brought about by HDAC5targeting. This repeat-length specificity was abolished when we inhibited HDAC3 activity. Our data guide the use of these histone deacetylases in manipulating chromatin. PInT can be adapted to study the effect of virtually any sequence on epigenome editing.

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Real-time imaging of specific genomic loci in eukaryotic cells using the ANCHOR DNA labelling system

Cited by 33 publications

References 50 publications

Methods for mapping 3D chromosome architecture

Methods for mapping 3D chromosome architecture

Visualizing the genome in high resolution challenges our textbook understanding

Expanded CAG/CTG Repeats Resist Gene Silencing Mediated by Targeted Epigenome Editing

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