Abstract
The Polycomb group (PcG) proteins form regulatory complexes that modify the chromatin structure and silence their target genes. Recent works have found that the composition of Polycomb complexes is highly dynamic. Defining the different protein components of each complex is fundamental for better understanding their biological functions. Fluorescent resonance energy transfer (FRET) is a powerful tool to measure protein–protein interactions, in nanometer order and in their native cellular environment. Here we describe the preparation and execution of a typical FRET experiment using CFP-tagged protein as donor and YFP-tagged protein as acceptor. We further show that FRET can be used in a competition assay to measure binding affinities of different components of the same chromatin complex.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli D (2007) Genome regulation by polycomb and trithorax proteins. Cell 128(4):735–745
Levine SS, Weiss A, Erdjument-Bromage H, Shao Z, Tempst P, Kingston RE (2002) The core of the polycomb repressive complex is compositionally and functionally conserved in flies and humans. Mol Cell Biol 22(17):6070–6078
Czermin B, Melfi R, McCabe D, Seitz V, Imhof A, Pirrotta V (2002) Drosophila enhancer of Zeste (ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites. Cell 111(2):185–196
Ciferri C, Lander GC, Maiolica A, Herzog F, Aebersold R, Nogales E (2012) Molecular architecture of human polycomb repressive complex 2. Elife 1:e00005
Farcas AM, Blackledge NP, Sudbery I, Long HK, McGouran JF, Rose NR, Lee S, Sims D, Cerase A, Sheahan TW, Koseki H, Brockdorff N, Ponting CP, Kessler BM, Klose RJ (2012) KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands. Elife 1:e00205
Gao Z, Zhang J, Bonasio R, Strino F, Sawai A, Parisi F, Kluger Y, Reinberg D (2012) PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes. Mol Cell 45(3):344–356
Sun Y, Wallrabe H, Seo SA, Periasamy A (2011) FRET microscopy in 2010: the legacy of Theodor Förster on the 100th anniversary of his birth. ChemPhysChem 12(3):462–474
Bastiaens PI, Pepperkok R (2000) Observing proteins in their natural habitat: the living cell. Trends Biochem Sci 25(12):631–637
Selvin PR (2000) The renaissance of fluorescence resonance energy transfer. Nat Struct Biol 7(9):730–734
Chen Y, Mills JD, Periasamy A (2003) Protein localization in living cells and tissues using FRET and FLIM. Differentiation 71(9–10):528–541
Zhang WX, Wang R, Wisniewski D, Marcy AI, LoGrasso P, Lisnock JM, Cummings RT, Thompson JE (2005) Time-resolved Forster resonance energy transfer assays for the binding of nucleotide and protein substrates to p38alpha protein kinase. Anal Biochem 343(1):76–83
Tirat A, Freuler F, Stettler T, Mayr LM, Leder L (2006) Evaluation of two novel tag-based labelling technologies for site-specific modification of proteins. Int J Biol Macromol 39(1–3):66–76
Sharma B, Deo SK, Bachas LG, Daunert S (2005) Competitive binding assay using fluorescence resonance energy transfer for the identification of calmodulin antagonists. Bioconjug Chem 16(5):1257–1263
Tian H, Ip L, Luo H, Chang DC, Luo KQ (2007) A high throughput drug screen based on fluorescence resonance energy transfer (FRET) for anticancer activity of compounds from herbal medicine. Br J Pharmacol 150(3):321–334
Rogers MS, Cryan LM, Habeshian KA, Bazinet L, Caldwell TP, Ackroyd PC, Christensen KA (2012) A FRET-based high throughput screening assay to identify inhibitors of anthrax protective antigen binding to capillary morphogenesis gene 2 protein. PLoS One 7(6):e39911
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Cherubini, A., Zippo, A. (2016). Fluorescence Resonance Energy Transfer Microscopy for Measuring Chromatin Complex Structure and Dynamics. In: Lanzuolo, C., Bodega, B. (eds) Polycomb Group Proteins. Methods in Molecular Biology, vol 1480. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6380-5_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6380-5_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6378-2
Online ISBN: 978-1-4939-6380-5
eBook Packages: Springer Protocols