Analysis of Interactions Between Genomic Loci Through Chromosome Conformation Capture (3C)

Belal El Kaderi1, Scott Medler1, Athar Ansari1

1 Department of Biological Sciences, Wayne State University, Detroit, Michigan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.15
DOI:  10.1002/0471143030.cb2215s56
Online Posting Date:  September, 2012
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Abstract

Genome architecture plays a significant role in the regulation of DNA‐based cellular processes such as transcription and recombination. The successful accomplishment of these processes involves coordinated interaction of DNA elements located at a distance from each other. The ‘Chromosome Conformation Capture’ (3C) assay is a convenient tool for identification of physical association between spatially separated DNA elements in a cell under physiological conditions. The principle of 3C is to convert physical chromosomal interactions into specific DNA ligation products, which are then detected by PCR. The 3C protocol was originally used to identify long‐range, stable chromosomal interactions in yeast. Here we describe a modified 3C procedure that can detect transient, short‐range interactions of DNA elements separated by a distance of less than 700 bp. This method has been successfully used to detect dynamic interaction of transcription regulatory elements in yeast and can be used for detecting similar interactions of other genomic regions. Curr. Protoc. Cell Biol. 56:22.15.1‐22.15.21. © 2012 by John Wiley & Sons, Inc.

Keywords: chromosome conformation; DNA element interaction; yeast

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Chromosome Conformation Capture (3C) Assay
  • Alternate Protocol 1: Two‐Step Digestion of Cross‐Linked Chromatin
  • Alternate Protocol 2: DNA Purification after Reversing the Cross‐Links
  • Support Protocol 1: Gel Band Quantification by Densitometry
  • Support Protocol 2: Checking Restriction Digestion of Chromatin
  • Support Protocol 3: Checking Amplification Efficiency of PCR Primers Used in 3C
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Chromosome Conformation Capture (3C) Assay

  Materials
  • Saccharomyces cerevisiae cells growing on freshly streaked plate (unit 1.6)
  • Appropriate liquid medium for growing yeast cells (unit 1.6)
  • 37% formaldehyde solution (store up to 1 year at room temperature)
  • 2.5 M glycine, sterilized by autoclaving
  • Tris‐buffered saline (TBS; appendix 2A) containing 1% (v/v) Triton X‐100, ice cold
  • Tris‐buffered saline (TBS; appendix 2A), ice cold
  • FA lysis buffer (see recipe), ice cold
  • 10% (w/v) SDS (see recipe for 20% in appendix 2A)
  • 100 mM PMSF ( appendix 2A; store at 4°C)
  • TM buffer (see recipe)
  • Liquid nitrogen
  • 1% (w/v) SDS (see recipe for 20% in appendix 2A)
  • 10% Triton X‐100 ( appendix 2A)
  • Restriction enzyme buffer and 10 U/µl enzyme
  • Quick Ligation Kit (New England Biolabs)
  • 5 M NaCl
  • 20 mg/ml proteinase K (see recipe)
  • 20 mg/ml DNase‐free RNase A (see recipe)
  • 25:24:1 phenol/chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 100% ethanol
  • 20 mg/ml glycogen
  • TE buffer, pH 7.5 ( appendix 2A)
  • Primer pairs (see Critical Parameters and Troubleshooting)
  • Taq polymerase and 10× PCR buffer (available from various suppliers)
  • 10 mM deoxyribonucleotide triphosphate (dNTP) mix (available from various suppliers)
  • 50‐ and 15‐ml sterile, disposable, conical tubes (e.g., BD Falcon)
  • Temperature regulated shakers and incubators for growing yeast cells
  • 250‐ml baffled Erlenmeyer flasks
  • Platform shaker
  • Refrigerated Sorvall centrifuge with rotor for 50‐ml conical tubes
  • 1.5‐ml LockTop microcentrifuge tubes
  • Acid‐washed glass beads (∼0.5‐mm diameter; Sigma, cat. no. G8772)
  • Multihead vortexer
  • 22‐G needles
  • Refrigerated centrifuge
  • 1.5‐ml standard microcentrifuge tubes
  • Refrigerated microcentrifuge
  • Nutator
  • 2‐ml flat‐bottom microcentrifuge tubes
  • 65°C water bath
  • 42°C water bath
  • 2‐ml conical‐bottom microcentrifuge tubes
  • Spectrophotometer
  • 0.2‐ml PCR tubes
  • Thermal cycler
  • Additional reagents and equipment for growing Saccharomyces cerevisiae (unit 1.6), quantification of DNA by absorbance spectrometry ( appendix 3D), and quantification of PCR products ( protocol 4)

Alternate Protocol 1: Two‐Step Digestion of Cross‐Linked Chromatin

  Materials
  • SDS‐solubilized chromatin from step 17 of the protocol 1
  • Additional reagents and equipment for 3C assay ( protocol 1)

Alternate Protocol 2: DNA Purification after Reversing the Cross‐Links

  • 7.5 M ammonium acetate

Support Protocol 1: Gel Band Quantification by Densitometry

  Materials
  • 1.5% agarose solution
  • 1× TAE buffer ( appendix 2A)
  • 10× loading buffer (see recipe)
  • PCR product samples ( protocol 1)
  • DNA marker (100 bp Quick Load, New England Biolabs)
  • 10 mg/ml ethidium bromide
  • 75 × 50 × 1–mm glass slides (Fisherbrand, cat. no. 12‐550C)
  • Well combs
  • Horizontal gel electrophoresis system
  • KODAK Gel Logic 200 Imaging System Windows computer running Kodak 1D software and Microsoft Excel
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )
CAUTION: When working with ethidium bromide, take appropriate precautions to avoid contamination. Proper management of ethidium bromide and phenol/chloroform/isoamyl alcohol is very important.

Support Protocol 2: Checking Restriction Digestion of Chromatin

  Materials
  • Restriction digested cross‐linked chromatin ( protocol 1)
  • 10% (w/v) SDS (see recipe for 20% in appendix 2A)
  • 20 mg/ml proteinase K (see recipe)
  • 25:24:1 phenol/chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 100% ethanol
  • 20 mg/ml glycogen
  • TE buffer, pH 8.0 ( appendix 2A)
  • Primer pairs (see Critical Parameters and Troubleshooting)
  • Taq polymerase and 10× PCR buffer (available from various suppliers)
  • 10 mM deoxyribonucleotide triphosphate (dNTP) mix (available from various suppliers)
  • 65°C water bath
  • 0.2‐ml PCR tubes
  • Thermal cycler
  • Additional reagents and equipment for quantification of DNA by absorbance spectrometry ( appendix 3D)

Support Protocol 3: Checking Amplification Efficiency of PCR Primers Used in 3C

  Materials
  • Purified genomic DNA from the strain used in 3C analysis
  • Restriction enzyme (5 U/µl) used in 3C analysis
  • 25:24:1 phenol/chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 100% ethanol
  • Quick Ligation Kit (New England Biolabs)
  • TE buffer, pH 8.0 ( appendix 2A)
  • 65°C water bath (and water bath at temperature optimal for restriction enzyme)
  • Additional reagents and equipment for 3C assay (Basic Protocol 1) and gel band quantitation ( protocol 4)
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Figures

Videos

Literature Cited

Literature Cited
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