Labeling DNA Replication Foci to Visualize Chromosome Territories In Vivo

Apolinar Maya‐Mendoza1, Dean A. Jackson2

1 Genome Integrity Unit, Danish Cancer Society Research Centre, Copenhagen, 2 Systems Microscopy Centre, Faculty of Life Sciences, The University of Manchester, Manchester
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.21
DOI:  10.1002/cpcb.19
Online Posting Date:  June, 2017
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Abstract

While a detailed understanding of chromatin dynamics is needed to explain how higher‐order chromatin organization influences nuclear function, the molecular principles that regulate chromatin mobility in mammalian nuclei remain largely unknown. Here we describe experimental tools to follow chromatin dynamics by labeling DNA during S phase. Using these methods, we have found that foci labeled during early and mid/late S phase have significantly different dynamic behavior. Spatially constrained heterochromatic foci restrict long‐range transformations of the chromosome territory (CT) structure while providing a structural framework on which highly mobile euchromatic foci undergo positional oscillations that drive local changes in the chromosome shape. Despite often dramatic mobility, we have demonstrated a preservation of structural integrity which ensures that DNA from neighboring CTs is not able to mix freely within the same nuclear space. Finally, other potential applications of the presented protocols are discussed. © 2017 by John Wiley & Sons, Inc.

Keywords: cell fate; chromatin; chromosome territories; developmental biology; DNA replication foci; dynamics; fluorescence; fluorescent‐tagged dNTPs; imaging; stem cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Visualization of Full Chromosome Territories with Bromodeoxyuridine (BrdU)
  • Basic Protocol 2: Visualization of the Chromosome Territories In Vivo
  • Basic Protocol 3: Tracking Cell Fate in 3‐D Cultures by Labeling Replication Foci
  • Basic Protocol 4: Microscopy and Image Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Visualization of Full Chromosome Territories with Bromodeoxyuridine (BrdU)

  Materials
  • Adherent cells of interest
  • Appropriate culture medium
  • 10 mM 5‐bromo‐2′‐deoxyuridine (BrdU; Sigma‐Aldrich, cat. no. B5002) stock solution in sterile cell culture grade water or PBS (store in small aliquots at −20°C)
  • Phosphate‐buffered saline (PBS) without Ca++ and Mg++ (Gibco, cat. no. 14190094)
  • 4% (w/v) formaldehyde solution (Prolabo/VWR, cat. no. 9713.1000)
  • 2 M HCl (Sigma‐Aldrich, cat. no. H1785)
  • DNase I (RNase‐Free DNase, Promega, cat. no. M6101; optional)
  • DNase buffer (optional): 40 mM Tris·Cl (pH 7.9), 10 mM NaCl, 6 mM MgCl 2, and 10 mM CaCl 2 in PBS
  • 0.5% (v/v) Triton X‐100 (Sigma‐Aldrich, cat. no. 93443) in PBS
  • PBS+: PBS (Gibco, cat. no. 14190094) containing 1% (w/v) bovine serum albumin (BSA, Sigma‐Aldrich, cat. no. A2153) and 0.1% (v/v) Tween 20 (Sigma‐Aldrich, cat. no. P1379); to avoid bacterial contamination, filter the solution and add 0.002% sodium azide (NaN 3; Sigma‐Aldrich, cat. no. S2002) final concentration
  • Primary antibody: mouse anti‐BrdU B44 (BD Biosciences, cat. no. 347580)
  • Secondary antibody: Alexa Fluor 568–conjugated goat anti‐mouse (Molecular Probes, cat. no. A‐11004)
  • 2 ng/ml Hoechst 33342 (Molecular Probes, cat. no. H3570) in PBS
  • Vectashield (Vector Laboratories, cat. no. H1000)
  • ProLong Gold Antifade Mountant (Molecular Probes, cat. no. P36930)
  • Glass microscope slides (ThermoFisher Scientific, cat. no. 10143560W90)
  • Transparent nail polish
  • 35‐mm (or appropriately sized) culture dishes
  • Nunc glass bottom dishes (12‐mm inside bottom diameter; ThermoFisher Scientific, cat. no. 150680)
  • Optional: microscope slide coverslips (12 mm; VWR, cat. no. 631‐0713) and 24‐well plates (Thermo Fisher Scientific, cat. no. 142475)
  • Additional reagents and equipment for cell culture (unit 1.1; Phelan & May, )

Basic Protocol 2: Visualization of the Chromosome Territories In Vivo

  Materials
  • Adherent cells of interest
  • CO 2‐independent cell culture medium
  • Phosphate‐buffered saline (PBS) without Ca++ or Mg++ (Gibco, cat. no. 14190094)
  • FuGENE 6 (preferred; Roche, cat. no. 11 815 091 001); optionally use FuGENE 6 (Promega, cat. no. E2693), or as a last resort, FuGENE HD (Promega, cat. no. E2311)
  • Alexa Fluor 488–5‐dUTP (Molecular Probes, Cat. C11397)
  • Cy3‐dUTP (GE Healthcare, cat. no. PA53022; Sigma‐Aldrich, cat. no. GEPA3022)
  • Aphidicolin (Sigma‐Aldrich, Cat. A0781) in DMSO
  • Nunc glass bottom dishes (12‐mm inside bottom diameter; Thermo Fisher Scientific, cat. no. 150680), 24‐well plates (Thermo Fisher Scientific, cat. no. 142475), or multi‐well glass‐bottom microscope slides (Millicell EZ slide 4‐well glass; Merck Millipore, cat. no. PEZG S0416)
  • Additional reagents and equipment for cell culture (unit 1.1; Phelan & May, )
NOTE: CO 2‐independent medium is desirable, in addition to a good CO 2 controller installed in the microscope. Unfortunately, Gibco has discontinued production of the CO 2‐independent phenol red–free medium, which worked very well. Currently available options are FluoroBrite DMEM (Gibco, cat. no. A1896702) or CO 2‐independent medium with phenol red (Gibco, cat. no. 18045054). Because different models are available for the live cell imaging chambers and CO 2 controllers, pilot experiments and medium optimization must be carried out for any particular microscope configuration.

Basic Protocol 3: Tracking Cell Fate in 3‐D Cultures by Labeling Replication Foci

  Materials
  • Adherent cells: human mammary gland MCF10A cells (ATCC, Cat. CRL‐10317), which can be grown as a monolayer (2‐D) in uncoated cell culture dishes or as acini (3‐D) in cell culture dishes coated with laminin‐rich extracellular matrix (lrECM, Matrigel)
  • Nonadherent cells:
    • DT40 chicken cells (ATCC #CRL‐2111)
    • HeLa‐S3 cells (ATCC #CCL‐2.2)
  • MCF10A cell culture medium (see recipe)
  • Phosphate‐buffered saline (PBS) without Ca++ or Mg++ (Gibco, cat. no. 14190094)
  • FuGENE 6 (preferred; Roche, cat. no. 11 815 091 001); optionally use FuGENE 6 (Promega, cat. no. E2693), or as a last resort, FuGENE HD (Promega, cat. no. E2311)
  • Fluorescent dUTP:
    • Alexa Fluor 488–5‐dUTP (Molecular Probes, Cat. C11397)
    • Cy3‐dUTP (GE Healthcare, cat. no. PA53022; Sigma‐Aldrich, cat. no. GEPA3022)
  • Trypsin‐EDTA solution (Gibco, cat. no. 15400‐054)
  • 3‐D medium: MCF10A cell culture medium (see recipe) supplemented with 2% (w/v) lrECM (Matrigel; Corning, cat. no. 356231)
  • DT40 cell culture medium (see recipe)
  • HeLa‐S3 cell culture medium (see recipe)
  • 24‐well culture plates
  • Tissue culture microscope
  • Centrifuge
  • 100‐mm cell culture dishes
  • Additional reagents and equipment for cell culture, including trypsinization and counting cells (unit 1.1; Phelan & May, )

Basic Protocol 4: Microscopy and Image Analysis

  Materials
  • Multi‐colored TetraSpeck fluorescent beads (Molecular Probes, cat. no. T7284)
  • Images of the labeled DNA replication foci and CTs in vivo taken using a microscope with an appropriate CO 2‐control chamber and a 100× magnification objective (in our experience, the microscope objectives from Carl Zeiss are the best on the market)
  • Laser scanning microscope (LSM; Zeiss, 510, 700, or 800 series (we recommend alpha Plan‐Apochromat 100×/1.46 oil iris M27 objective; Zeiss, Cat. 420796‐9800‐000)
  • For long‐term time‐lapse experiments, the DeltaVision system is a very good option with a good detection system when a CoolSNAP‐HQ2 camera is available (a few years ago, when the image acquisition speed was the bottleneck, this system was an excellent choice)
  • 100 × /1.4 oil Super‐Plan APO objective (Olympus)
  • Imaris software (Bitplane)
  • Velocity software (Perkin Elmer)
  • ImageJ and Fiji free software
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Figures

Videos

Literature Cited

Literature Cited
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