Karen Ng

Research Institute of Molecular Pathology

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Anton Wutz

ETH Zurich

Martin Leeb

Max Perutz Labs

Rachael Walker

Babraham Institute

Aurélien Bancaud

Laboratoire d'Analyse et d'Architecture des Systèmes

Jan Ellenberg

European Molecular Biology Laboratory

Peter Humphreys

Shanghai Institute for Science of Science

Tatsuya Ohhata

Hamamatsu University School of Medicine

Nathalie Daigle

National Institute of Arthritis and Musculoskeletal and Skin Diseases

Dieter Pullirsch

Austrian Agency for Health and Food Safety

Cooperative Institutions

Wellcome/MRC Cambridge Stem Cell Institute

University of Cambridge

Medical Research Council

Vienna Biocenter

Research Institute of Molecular Pathology

Max Perutz Labs

University of Vienna

Centre National de la Recherche Scientifique

European Molecular Biology Laboratory

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RNA FISH on Cultured Cells in Interphase: Figure 1.

Cold Spring Harbor Protocols (2007)

Karen Ng Anton Wutz

INTRODUCTIONFluorescence in situ hybridization (FISH) has become a widely used method in genome and molecular genetic studies. The technique is highly versatile and has been adapted to carry out genome-wide screenings, microarray quantifications, cancer cytogenetics analysis, and RNA expression and localization studies. The study of intracellular RNA localization using RNA FISH provides insights into the in situ physical characteristics of transcription and intracellular RNA transport in individual cells. In our lab, we use RNA FISH to detect the localization of Xist RNA, a nuclear noncoding transcript that coats the entire chromosome from which it is transcribed. The advantage of using RNA FISH in our case is to extract precise molecular information directly in the context of cellular structure. The RNA FISH technique requires the generation of a labeled probe, hybridization of the probe to a fixed sample, and detection of the labeled probe using microscopy.

XIST

Transcription

Small nuclear RNA

10.1101/pdb.prot4763

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Citations (3)

Xist and the order of silencing

EMBO Reports (2007)

Karen Ng Dieter Pullirsch Martin Leeb Anton Wutz

XIST

Dosage compensation

Gene dosage

10.1038/sj.embor.7400871

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Citations (156)

A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells

Molecular Biology of the Cell (2011)

Karen Ng Nathalie Daigle Aurélien Bancaud Tatsuya Ohhata Peter Humphreys

In mammals, silencing of one of the two X chromosomes in female cells compensates for the different number of X chromosomes between the sexes. The noncoding Xist RNA initiates X chromosome inactivation. Xist spreads from its transcription site over the X chromosome territory and triggers the formation of a repressive chromatin domain. To understand localization of Xist over one X chromosome we aimed to develop a system for investigating Xist in living cells. Here we report successful visualization of transgenically expressed MS2-tagged Xist in mouse embryonic stem cells. Imaging of Xist during an entire cell cycle shows that Xist spreads from a single point to a steady state when the chromosome is covered with a constant amount of Xist. Photobleaching experiments of the established Xist cluster indicate that chromosome-bound Xist is dynamic and turns over on the fully Xist covered chromosome. It appears that in interphase the loss of bound Xist and newly produced Xist are in equilibrium. We also show that the turnover of bound Xist requires transcription, and Xist binding becomes stable when transcription is inhibited. Our data reveal a strategy for visualizing Xist and indicate that spreading over the chromosome might involve dynamic binding and displacement.

XIST

Transcription

10.1091/mbc.e11-02-0146

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Citations (47)