Visualization and Measurement of DNA Methyltransferase Activity in Living Cells

Lothar Schermelleh1, Fabio Spada1, Heinrich Leonhardt1

1 Ludwig Maximilians University Munich (LMU), Department of Biology II, Martinsried, Germany
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.12
DOI:  10.1002/0471143030.cb2212s39
Online Posting Date:  June, 2008
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In this unit, a live‐cell assay to measure DNA (cytosine‐5) methyltransferase (MTase) activity at the single‐cell level is described. This method takes advantage of the irreversible binding of enzymatically active MTases to genomic DNA substituted with the mechanism‐based inhibitor 5‐aza‐2′‐deoxycytidine (5‐aza‐dC). The procedure comprises incorporation of this nucleoside analog into DNA during replication and quantification of the time‐dependent MTase immobilization by fluorescence recovery after photobleaching (FRAP). This trapping assay monitors kinetic properties and activity‐dependent immobilization of MTases in their native environment and enables direct comparison of mutations and inhibitors that affect MTase regulation and catalytic activity in single living cells. In addition, a simplified protocol to obtain qualitative information on the activity of either endogenously or exogenously expressed MTases is provided. Curr. Protoc. Cell Biol. 39:22.12.1‐22.12.16. © 2008 by John Wiley & Sons, Inc.

Keywords: DNA methylation; DNA (cytosine‐5) methyltransferase; Dnmt1; trapping assay; fluorescence recovery after photobleaching; 5‐aza‐2′‐deoxycytidine; mechanism‐based inhibitor

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: FRAP‐Based Quantitative Measurement of MTase Activity in Living Cells
  • Alternate Protocol 1: Qualitative MTase Activity Assay on Fixed Cells Using BrdU Replication Labeling
  • Alternate Protocol 2: Immunodetect PCNA
  • Alternate Protocol 3: Immunodetect Endogenous MTases
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: FRAP‐Based Quantitative Measurement of MTase Activity in Living Cells

  • Cultured cells
  • Expression vectors encoding MTase fusions with fluorescent proteins suitable for FRAP analysis (e.g., eGFP and RFP)
  • Transfection reagent (e.g., Transfectin, BioRad) or stably expressing cell line
  • 5‐Aza‐2′‐deoxycytidine (5‐aza‐dC; see recipe)
  • Live‐cell chamber slides (e.g., Lab‐Tek, Nunc or µ‐Slides, Ibidi)
  • Confocal laser scanning microscope (CLSM) system (e.g., Leica TCS SP2/5 AOBS) suited for live‐cell microscopy (inverted set up) equipped with the following:
    • High numerical aperture (1.2 to 1.4 NA) oil, glycerol, or water objectives (60×, 63×, or 100×)
    • High‐power blue laser to provide a 488‐nm line for bleaching and GFP excitation
    • Green laser (543‐nm/561‐nm) to excite red fluorescent protein
    • Temperature‐controlled incubator box (recommended)
    • Motorized xy‐stage (optional)

Alternate Protocol 1: Qualitative MTase Activity Assay on Fixed Cells Using BrdU Replication Labeling

  • Cultured cells grown on coverslips of appropriate size (e.g., 15 × 15–mm) and thickness (e.g., 0.17 ± 0.01 mm) in small plastic petri dishes
  • 5‐Aza‐2′‐deoxycytidine (5‐aza‐dC; see recipe)
  • 5‐Bromo‐2′‐deoxyuridine (BrdU; Sigma‐Aldrich)
  • Phosphate‐buffered saline (PBS, appendix 2A)
  • Fixation solution (see recipe)
  • PBST (see recipe)
  • 10% (w/v) Triton X‐100 ( appendix 2A)
  • Blocking solution (see recipe)
  • 1 M MgCl 2 ( appendix 2A)
  • 1:1000 mouse anti‐BrdU antibodies (e.g., MAb IU‐4, CALTAG Labs)
  • DNase I (see recipe)
  • PBSTE (see recipe)
  • Green and/or red fluorescent secondary antibodies (e.g., Alexa Fluor 488 and 555 conjugates, Invitrogen)
  • 4′,6‐diamidino‐2‐phenylindole (DAPI, see recipe)
  • Anti‐fade mounting medium (e.g., Vectashield, Vector Laboratories)
  • Transparent nail polish (base coat)
  • 6‐well plates or 35‐mm culture dishes
  • Fine‐tip forceps
  • 37°C incubator
  • Glass microscope slides
  • CLSM or epifluorescence microscope system appropriately equipped with CCD camera, fluorescence filter sets, and high‐NA objectives
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Literature Cited

Literature Cited
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   Juttermann, R., Li, E., and Jaenisch, R. 1994. Toxicity of 5‐aza‐2′‐deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proc. Natl. Acad. Sci. U.S.A. 91:11797‐11801.
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   Okano, M., Xie, S., and Li, E. 1998. Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells. Nucleic Acids Res. 26:2536‐2540.
   Okano, M., Bell, D.W., Haber, D.A., and Li, E. 1999. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell. 99:247‐257.
   Schermelleh, L., Spada, F., Easwaran, H.P., Zolghadr, K., Margot, J.B., Cardoso, M.C., and Leonhardt, H. 2005. Trapped in action: Direct visualization of DNA methyltransferase activity in living cells. Nat. Methods 2:751‐756.
   Schermelleh, L., Haemmer, A., Spada, F., Rösing, N., Meilinger, D., Rothbauer, U., Cardoso, M.C., and Leonhardt, H. 2007. Dynamics of Dnmt1 interaction with the replication machinery and its role in postreplicative maintenance of DNA methylation. Nucleic Acids Res. 35:4301‐4312.
   Spada, F., Haemmer, A., Kuch, D., Rothbauer, U., Schermelleh, L., Kremmer, E., Carell, T., Langst, G., and Leonhardt, H. 2007. Dnmt1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells. J. Cell Biol. 176:565‐571.
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