Mapping Chromatin Interactions by Chromosome Conformation Capture

Adriana Miele1, Nele Gheldof1, Tomoko M. Tabuchi1, Josée Dostie1, Job Dekker1

1 University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.11
DOI:  10.1002/0471142727.mb2111s74
Online Posting Date:  May, 2006
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Abstract

Chromosome conformation capture (3C) is one of the only techniques that allows for analysis of an intermediate level of chromosome structure ranging from a few to hundreds of kilobases, a level most relevant for gene regulation. The 3C technique is used to detect physical interactions between sequence elements that are located on the same or on different chromosomes. For instance, physical interactions between distant enhancers and target genes can be measured. The 3C assay uses formaldehyde cross‐linking to trap connections between chromatin segments that can, after a number of manipulations, be detected by PCR. This unit describes detailed protocols for performing 3C with yeast Saccharomyces cerevisiae and mammalian cells.

Keywords: DNA; chromatin; Saccharomyces cerevisiae; mammalian cells; interaction

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Generation of 3C Template from Intact Yeast Cells
  • Basic Protocol 2: Generation of Control Template from Yeast Genomic DNA
  • Basic Protocol 3: Generation of 3C Template from Mammalian Cells
  • Basic Protocol 4: Generation of Control Template from Mammalian DNA
  • Basic Protocol 5: Analysis of Interaction Frequencies Using 3C and Control Templates by Quantitative PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generation of 3C Template from Intact Yeast Cells

  Materials
  • Saccharomyces cerevisiae cells of interest (unit 13.2)
  • Spheroplasting buffer I (see recipe)
  • 20 mg/ml zymolyase 100‐T solution (see recipe)
  • MES wash buffer (see recipe)
  • 37% (v/v) formaldehyde
  • 2.5 M glycine
  • Restriction enzyme and corresponding 10× restriction enzyme buffer
  • 1% and 10% (w/v) sodium dodecyl sulfate (SDS; appendix 22)
  • 10% (v/v) Triton X‐100
  • 10× ligation buffer (see recipe)
  • 10 mg/ml bovine serum albumin (BSA)
  • 100 mM adenosine triphosphate (ATP)
  • T4 DNA ligase
  • 10 mg/ml proteinase K in TE buffer, pH 8.0
  • 1:1 (v/v) phenol/chloroform (unit 2.1)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 100% ethanol
  • TE buffer, pH 8.0 ( appendix 22)
  • 10 mg/ml DNase‐free RNase A (unit 3.13)
  • 15‐, 50‐, and 250‐ml disposable conical tubes
  • Roller drum in 30°C incubator
  • Refrigerated tabletop centrifuge
  • 1.7‐ml microcentrifuge tubes
  • 16°, 37°, 42°, and 65°C water baths
  • 30‐ml screw‐cap centrifuge tubes

Basic Protocol 2: Generation of Control Template from Yeast Genomic DNA

  Materials
  • Saccharomyces cerevisiae cells of interest (unit 13.2)
  • Spheroplasting buffer II (see recipe)
  • Lysing buffer I (see recipe)
  • 20 mg/ml proteinase K in TE buffer, pH 8.0
  • 5 M potassium acetate
  • 80% and 100% ethanol, ice cold
  • TE buffer, pH 8.0 ( appendix 22), containing 10 µg/ml DNase‐free RNase A
  • 1:1 (v/v) phenol/chloroform (unit 2.1)
  • 100% isopropanol
  • Restriction enzyme and corresponding 10× restriction enzyme buffer
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 10× ligation buffer (see recipe)
  • 1 mg/ml bovine serum albumin (BSA)
  • 10 mM adenosine triphosphate (ATP)
  • T4 DNA ligase
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • 250‐ml disposable conical tubes
  • 16°, 37°, and 65°C water baths
  • 1.7‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • Additional reagents and equipment for quantifying DNA by absorption spectroscopy ( appendix 3D)

Basic Protocol 3: Generation of 3C Template from Mammalian Cells

  Materials
  • Mammalian cells growing in appropriate culture medium ( appendix 3F)
  • 37% (v/v) formaldehyde
  • 2.5 M glycine
  • Lysing buffer II (see recipe), ice cold
  • Protease inhibitor cocktail for use with mammalian cells
  • Restriction enzyme and corresponding 10× restriction enzyme buffer
  • 1% and 10% (w/v) SDS
  • 10% (v/v) Triton X‐100
  • 10× ligation buffer (see recipe)
  • 10 mg/ml BSA
  • 100 mM ATP
  • T4 DNA ligase
  • 10 mg/ml proteinase K in TE buffer, pH 8.0
  • Phenol (unit 2.1)
  • 1:1 (v/v) phenol/chloroform (unit 2.1)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 70% and 100% (v/v) ethanol
  • TE buffer, pH 8.0 ( appendix 22)
  • Chloroform
  • 10 mg/ml DNase‐free RNase A
  • Dounce homogenizer with pestle B
  • 1.7‐ml microcentrifuge tubes
  • 16°, 37°, 42°, and 65°C water baths
  • 15‐ and 50‐ml disposable conical tubes
  • 250‐ml screw‐cap centrifuge bottles

Basic Protocol 4: Generation of Control Template from Mammalian DNA

  Materials
  • BAC clones (e.g., Invitrogen and CHORI; http://bacpac.chori.org)
  • TE buffer, pH 8.0 ( appendix 22)
  • 0.8% agarose/0.5× TBE gel (unit 2.5)
  • Molecular weight standard of known concentration
  • Restriction enzyme and corresponding 10× restriction enzyme buffer
  • 10 mg/ml BSA
  • 1:1 (v/v) phenol/chloroform (unit 2.1)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 70% and 100% (v/v) ethanol, ice cold
  • 10× ligation buffer (see recipe)
  • 100 mM ATP
  • T4 DNA ligase
  • Chloroform
  • 1.7‐ and 2‐ml microcentrifuge tubes
  • 16°, 37°, and 65°C water baths
  • Additional reagents and equipment for recovery of DNA from PAC/BAC clones (unit 5.9), for real‐time PCR if applicable (unit 21.3), and for agarose gel electrophoresis (unit 2.5)

Basic Protocol 5: Analysis of Interaction Frequencies Using 3C and Control Templates by Quantitative PCR

  Materials
  • DNA templates (see Basic Protocols protocol 11 through protocol 44)
  • Molecular weight standard of known concentration
  • 10× PCR buffer for yeast or mammalian templates (see recipes)
  • 100 mM dNTPs
  • Primers (see )
  • Taq DNA polymerase
  • 50 mM MgSO 4
  • Automated thermal cycler
  • Gel documentation setup with appropriate software for quantifying PCR products
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5) and PCR (units 15.1& 15.7)
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Figures

Videos

Literature Cited

   Dekker, J. 2003. A closer look at long‐range chromosomal interactions. Trends Biochem. Sci. 28:277‐280.
   Dekker, J., Rippe, K., Dekker, M., and Kleckner, N. 2002. Capturing chromosome conformation. Science 295:1306‐1311.
   Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J. 1997. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389:251‐260.
   Spilianakis, C.G. and Flavell, R.A. 2004. Long‐range intrachromosomal interactions in the T helper type 2 cytokine locus. Nat. Immunol. 5:1017‐1027.
   Spilianakis, C.G., Lalioti, M.D., Town, T., Lee, G.R., and Flavell, R.A. 2005. Interchromosomal associations between alternatively expressed loci. Nature 435:637‐645.
   Tolhuis, B., Palstra, R.J., Splinter, E., Grosveld, F., and de Laat, W. 2002. Looping and interaction between hypersensitive sites in the active beta‐globin locus. Mol. Cell 10:1453‐1465.
   Vakoc, C., Letting, D.L., Gheldof, N., Sawado, T., Bender, M.A., Groudine, M., Weiss, M.J., Dekker, J., and Blobel, G.A. 2005. Proximity among distant regulatory elements at the beta‐globin locus requires GATA‐1 and FOG‐1. Mol. Cell 17:453‐462.
Internet Resources
   http://bacpac.chori.org
  Website for information and to purchase BAC clones from various sources.
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