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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Abstract

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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Materials

Basic Protocol 1: FRAP‐Based Quantitative Measurement of MTase Activity in Living Cells

  Materials
  • 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

  Materials
  • 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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Figures

  •   FigureFigure 22.12.1 Principle of the trapping assay (shown here for the maintenance MTase Dnmt1 with hemimethylated CpG substrate sites). (A) Methylated and non‐methylated cytosines are depicted as black and white hexagons, respectively. MTase binds to (hemimethylated) CpG sites produced during DNA replication and forms a transient covalent complex with the cytosine residue (gray line). After methyl group transfer, the enzyme is released and becomes available for another round of methylation (dotted arrow). (B) Mechanism‐based inhibitors such as 5‐aza‐dC (hexagons with red N at position 5) are incorporated into DNA during S‐phase. The MTase forms a stable covalent complex with 5‐aza‐dC (black line) and becomes trapped. (C) Dynamic exchange of MTases is visualized as fluorescence recovery after photobleaching of GFP‐MTase in the outlined region (top row). Immobilization of GFP‐MTase is detected as reduced recovery after photobleaching and decrease of mobile fraction ( Mf) and increase of the immobile fraction, respectively (bottom row). Dots indicate MTase accumulation at replication foci (RF). Covalent trapping leads to an increase of the RF‐associated fraction and a decrease of diffuse fraction. (D) To compare, e.g., specific mutant MTases with wild‐type MTase, the immobile fractions (1 – Mf) are plotted as a function of incubation time. Such a comparison may also be performed simultaneously in single living cells when wild‐type and mutant MTase tagged with spectrally distinct fluorescent proteins are co‐expressed. (E) Example of ROI selection for quantitative FRAP evaluation. The left panel shows a high S/N reference image of an early S‐phase mouse myoblast cell expressing GFP‐Dnmt1 before bleaching (n, nucleoli). The right panel shows the corresponding first postbleach frame of the FRAP series. Bleach ROI (a), total ROI (b), and background ROI (c) are indicated. Bar = 5 µm.
  •   FigureFigure 22.12.2 Fixed cell assay to visualize Dnmt1 immobilization after 5‐aza‐dC treatment. Typical early S‐phase (A) and late S‐phase nuclei (B) of mouse myoblast cells are shown. Cells were BrdU labeled and fixed without or after a 60‐ and 120‐min treatment with 5‐aza‐dC. Replication foci were marked by BrdU immunostaining of nascent DNA. Note the stronger association of Dnmt1 at or near replication sites after 5‐aza‐dC treatment. At 120 min, a clear loss of co‐localization is observed as the pool of initially mobile Dnmt1 is fully trapped at earlier replicated genomic DNA. The effect seems more pronounced in late S‐phase, likely reflecting the higher density of methylated CpG sites in late replicating constitutive heterochromatin. Bar = 5 µm.
  •   FigureFigure 22.12.3 FRAP analysis of the Dnmt1 mobility before and during 5‐aza‐dC treatment in a single living cell. (A) Reference image (confocal mid section) of a mouse myoblast cell in late S‐phase co‐transfected with RFP‐PCNA and GFP‐Dnmt1 (wild‐type). PCNA and Dnmt1 largely co‐localize at replication foci. Bar = 5 µm. (B) FRAP of GFP‐Dnmt1 (shown in false color) localized at a late replication focus is shown before treatment (control), as well as after a 15‐ and 40‐min incubation of the same nucleus with 30 µM 5‐aza‐dC. The last prebleach, the postbleach and the final frame of each series is shown. Boxes indicate the bleach ROIs. FRAP series were recorded at different z‐levels. Note the continuous loss of the diffuse fraction with longer incubation time. (C) Quantitative evaluation of the FRAP series shown in B demonstrating the decrease of the mobile fraction with 5‐aza‐dC incubation.
  •   FigureFigure 22.12.4 Summary of factors affecting the trapping rate of GFP‐MTase fusion proteins. For comparability of results, experimental conditions that potentially affect the trap production rate should be kept constant. Two enzymes may also be directly compared side‐by‐side in single living cells using, e.g., red and green fluorescent fusions (see text for details).

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Literature Cited

Literature Cited
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   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.
   Sporbert, A., Domaing, P., Leonhardt, H., and Cardoso, M.C. 2005. PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins. Nucleic Acids Res. 33:3521‐3528.
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