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Monitoring Dynamic Binding of Chromatin Proteins In Vivo by Fluorescence Recovery After Photobleaching

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Chromatin Remodeling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 833))

Abstract

Fluorescence recovery after photobleaching (FRAP) has now become widely used to investigate nuclear protein binding to chromatin in live cells. FRAP can be applied qualitatively to assess if chromatin binding interactions are altered by various biological perturbations. It can also be applied semi-quantitatively to allow numerical comparisons between FRAP curves, and even fully quantitatively to yield estimates of in vivo diffusion constants and nuclear protein binding rates to chromatin. Here we describe how FRAP data should be collected and processed for these qualitative, semi-quantitative, and quantitative analyses.

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References

  1. Houtsmuller AB (2005) Fluorescence recovery after photobleaching: application to nuclear proteins. Adv Biochem Eng Biotechnol 95:177–199

    PubMed  CAS  Google Scholar 

  2. Sprague BL, McNally JG (2005) FRAP analysis of binding: proper and fitting. Trends Cell Biol 15:84–91

    Article  PubMed  CAS  Google Scholar 

  3. Hager GL, McNally JG, Misteli T (2009) Transcription dynamics. Mol Cell 35:741–753

    Article  PubMed  CAS  Google Scholar 

  4. Sprouse RO, Karpova TS, Mueller F, Dasgupta A, McNally JG, Auble DT (2008) Regulation of TATA-binding protein dynamics in living yeast cells. Proc Natl Acad Sci USA 105:13304–13308

    Article  PubMed  CAS  Google Scholar 

  5. Nishiyama A, Mochizuki K, Mueller F, Karpova T, McNally JG, Ozato K (2008) Intracellular delivery of acetyl-histone peptides inhibits native bromodomain-chromatin interactions and impairs mitotic progression. FEBS Lett 582:1501–1507

    Article  PubMed  CAS  Google Scholar 

  6. Phair RD, Misteli T (2001) Kinetic modelling approaches to in vivo imaging. Nat Rev Mol Cell Biol 2:898–907

    Article  PubMed  CAS  Google Scholar 

  7. Karpova TS, Chen TY, Sprague BL, McNally JG (2004) Dynamic interactions of a transcription factor with DNA are accelerated by a chromatin remodeller. EMBO Rep 5:1064–1070

    Article  PubMed  CAS  Google Scholar 

  8. Stavreva DA, Muller WG, Hager GL, Smith CL, McNally JG (2004) Rapid glucocorticoid receptor exchange at a promoter is coupled to transcription and regulated by chaperones and proteasomes. Mol Cell Biol 24:2682–2697

    Article  PubMed  CAS  Google Scholar 

  9. Mueller F, Mazza D, Stasevich TJ, McNally JG (2010) FRAP and kinetic modeling in the analysis of nuclear protein dynamics: what do we really know? Curr Opin Cell Biol 22:403–411

    Article  PubMed  CAS  Google Scholar 

  10. Mueller F, Wach P, McNally JG (2008) Evidence for a common mode of transcription factor interaction with chromatin as revealed by improved quantitative fluorescence recovery after photobleaching. Biophys J 94:3323–3339

    Article  PubMed  CAS  Google Scholar 

  11. Waharte F, Brown CM, Coscoy S, Coudrier E, Amblard F (2005) A two-photon FRAP analysis of the cytoskeleton dynamics in the microvilli of intestinal cells. Biophys J 88:1467–1478

    Article  PubMed  CAS  Google Scholar 

  12. Sprague BL, Pego RL, Stavreva DA, McNally JG (2004) Analysis of binding reactions by fluorescence recovery after photobleaching. Biophys J 86:3473–3495

    Article  PubMed  CAS  Google Scholar 

  13. Beaudouin J, Mora-Bermudez F, Klee T, Daigle N, Ellenberg J (2006) Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins. Biophys J 90:1878–1894

    Article  PubMed  CAS  Google Scholar 

  14. Hinow P, Rogers CE, Barbieri CE, Pietenpol JA, Kenworthy AK, DiBenedetto E (2006) The DNA binding activity of p53 displays reaction-diffusion kinetics. Biophys J 91:330–342

    Article  PubMed  CAS  Google Scholar 

  15. Michelman-Ribeiro A, Mazza D, Rosales T, Stasevich TJ, Boukari H, Rishi V, Vinson C, Knutson JR, McNally JG (2009) Direct measurement of association and dissociation rates of DNA binding in live cells by fluorescence correlation spectroscopy. Biophys J 97:337–346

    Article  PubMed  CAS  Google Scholar 

  16. Stasevich TJ, Mueller F, Michelman-Ribeiro A, Rosales T, Knutson JR, McNally JG (2010) Cross-validating FRAP and FCS to quantify the impact of photobleaching on in vivo binding estimates. Biophys J 99:3093–3101

    Article  PubMed  CAS  Google Scholar 

  17. Mazza D, Cella F, Vicidomini G, Krol S, Diaspro A (2007) Role of three-dimensional bleach distribution in confocal and two-photon fluorescence recovery after photobleaching experiments. Appl Opt 46:7401–7411

    Article  PubMed  CAS  Google Scholar 

  18. Sinnecker D, Voigt P, Hellwig N, Schaefer M (2005) Reversible photobleaching of enhanced green fluorescent proteins. Biochemistry 44:7085–7094

    Article  PubMed  CAS  Google Scholar 

  19. Weiss M (2004) Challenges and artifacts in quantitative photobleaching experiments. Traffic 5:662–671

    Article  PubMed  CAS  Google Scholar 

  20. Kang M, Kenworthy AK (2008) A closed-form analytic expression for FRAP formula for the binding diffusion model. Biophys J 95:L13–L15

    Article  PubMed  CAS  Google Scholar 

  21. Carrero G, McDonald D, Crawford E, de Vries G, Hendzel MJ (2003) Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins. Methods 29:14–28

    Article  PubMed  CAS  Google Scholar 

  22. Phair RD, Scaffidi P, Elbi C, Vecerova J, Dey A, Ozato K, Brown DT, Hager G, Bustin M, Misteli T (2004) Global nature of dynamic protein-chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins. Mol Cell Biol 24:6393–6402

    Article  PubMed  CAS  Google Scholar 

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Correspondence to James G. McNally .

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Mueller, F., Karpova, T.S., Mazza, D., McNally, J.G. (2012). Monitoring Dynamic Binding of Chromatin Proteins In Vivo by Fluorescence Recovery After Photobleaching. In: Morse, R. (eds) Chromatin Remodeling. Methods in Molecular Biology, vol 833. Humana Press. https://doi.org/10.1007/978-1-61779-477-3_11

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  • DOI: https://doi.org/10.1007/978-1-61779-477-3_11

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-476-6

  • Online ISBN: 978-1-61779-477-3

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