Abstract
Chromatin-protein interactions are important in determining chromosome structure and function, thereby regulating gene expression patterns. Most chromatin associated proteins bind chromatin in a transient manner, with residence times on the order of a few seconds to minutes. This is especially pertinent in mouse embryonic stem cells (ESCs), where hyperdynamic binding of chromatin associated proteins to chromatin is thought to regulate genome plasticity. In order to quantitatively measure binding dynamics of such chromatin proteins in living cells, a combination of GFP-fusion proteins and photobleaching-based assays such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are advantageous over other existing biochemical assays, because they are applied in living cells at a single cell level. In this chapter we describe a detailed protocol for performing FRAP and FLIP assays for measuring structural chromatin protein dynamics such as Heterochromatin Protein 1 (HP1) and linker histone H1 in mouse ESCs and during ESC differentiation.
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Acknowledgements
We thank the Abisch Frenkel Foundation, the Israel Cancer Research Foundation (ICRF), the Human Frontiers Science Foundation (HFSP), the Israel Science Foundation (ISF 657/12; 1252/12), the Israel Ministry of Science, and the European Research Council (ERC-281781) for financial support. AH is Marie Curie Nucleosome4D fellow.
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Harikumar, A., Meshorer, E. (2013). Measuring the Dynamics of Chromatin Proteins During Differentiation. In: Shav-Tal, Y. (eds) Imaging Gene Expression. Methods in Molecular Biology, vol 1042. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-526-2_12
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DOI: https://doi.org/10.1007/978-1-62703-526-2_12
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-525-5
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