Chromosome Conformation Capture Carbon Copy Technology

Josée Dostie1, Ye Zhan2, Job Dekker2

1 McGill University, Montréal, Québec, Canada, 2 University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.14
DOI:  10.1002/0471142727.mb2114s80
Online Posting Date:  October, 2007
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Abstract

Chromosome conformation capture (3C) is used to quantify physical DNA contacts in vivo at high resolution. 3C was first used in yeast to map the spatial chromatin organization of chromosome III, and in higher eukaryotes to demonstrate that genomic DNA elements regulate target genes by physically interacting with them. 3C has been widely adopted for small‐scale analysis of functional chromatin interactions along (cis) or between (trans) chromosomes. For larger‐scale applications, chromosome conformation capture carbon copy (5C) combines 3C with ligation‐mediated amplification (LMA) to simultaneously quantify hundreds of thousands of physical DNA contacts by microarray or ultra‐high‐throughput DNA sequencing. 5C allows the mapping of extensive networks of physical interactions among large sets of genomic elements throughout the genome. Such networks can provide important biological insights, e.g., by identifying relationships between regulatory elements and their target genes. This unit describes 5C for large‐scale analysis of cis‐ and trans‐chromatin interactions in mammalian cells. Curr. Protoc. Mol. Biol. 80:21.14.1‐21.14.13. © 2007 by John Wiley & Sons, Inc.

Keywords: chromatin structure; epigenetic regulation; genomics; systems/network biology; DNA looping; transcription; high throughput

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Generation of 5C Libraries from Cells and Control BAC DNA
  • Basic Protocol 2: Quality Control of 5C Libraries by Nested PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generation of 5C Libraries from Cells and Control BAC DNA

  Materials
  • ∼125 ng/µl cellular 3C template library in TE buffer (see unit 21.11)
  • ∼50 ng/µl control template library in TE buffer (see unit 21.11)
  • 9.9 mg/ml salmon testis DNA (Sigma)
  • Deionized water, autoclaved
  • 20 µM 5C primers (see ) in 1× TE buffer ( appendix 22)
  • 10× 5C annealing buffer (see recipe)
  • 10× 5C ligation buffer (see recipe)
  • 40 U/µl Taq DNA ligase (NEB)
  • 10× PCR buffer (see recipe)
  • 50 mM MgSO 4
  • 25 mM dNTP mix
  • 80 µM forward T7 and reverse T3 universal PCR primer stock solutions in 1× TE buffer ( appendix 22)
  • 5 U/µl Taq DNA polymerase (NEB)
  • 2% agarose gels containing 0.5× TBE and 0.5 µg/ml ethidium bromide (see unit 2.5)
  • DNA molecular weight marker of known concentration (e.g., 100‐bp ladder; NEB)
  • 0.5‐ml PCR tubes or 96‐well plates
  • Refrigerated tabletop centrifuge
  • Automated thermal cycler
  • MinElute PCR purification kit (Qiagen)
  • Gel documentation system with appropriate software for quantifying PCR products
  • Additional reagents and equipment for performing PCR (units 15.1& 15.7) and agarose gel electrophoresis (unit 2.5)
NOTE: It is important to read all protocol steps before starting the experiment.

Basic Protocol 2: Quality Control of 5C Libraries by Nested PCR

  Materials
  • 5C library ( protocol 1)
  • 10× PCR buffer (see recipe)
  • 50 mM MgSO 4
  • 25 mM dNTP mix
  • 80 µM nested 5C primers (see ) in 1× TE buffer ( appendix 22)
  • 5 U/µl Taq DNA polymerase (NEB)
  • 2% agarose gels containing 0.5× TBE and 0.5 µg/ml ethidium bromide (see unit 2.5)
  • DNA molecular weight markers of known concentration (e.g., 100‐bp ladder; NEB)
  • 96‐well plates
  • Automated thermal cycler
  • Gel documentation system with appropriate software for quantifying PCR products
  • Additional reagents and equipment for performing PCR (units 15.1& 15.7) and agarose gel electrophoresis (unit 2.5)
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Figures

Videos

Literature Cited

Literature Cited
   Dekker, J. 2003. A closer look at long‐range chromosomal interactions. Trends Biochem. Sci. 28:277‐280.
   Dekker, J. 2006. The 3 C's of chromosome conformation capture: Controls, controls, controls. Nat. Methods 3:17‐21.
   Dekker, J. 2007. GC‐ and AT‐rich chromatin domains differ in conformation and histone modification status and are differentially modulated by Rpd3p. Genome Biol. 8:R116.
   Dekker, J., Rippe, K., Dekker, M., and Kleckner, N. 2002. Capturing chromosome conformation. Science 295:1306‐1311.
   Dostie, J. and Dekker, J. 2007. Mapping networks of physical interactions between genomic elements using 5C technology. Nat. Protoc. 2:988‐1002.
   Dostie, J., Richmond, T.A., Arnaout, R.A., Selzer, R.R., Lee, W.L., Honan, T.A., Rubio, E.H., Krumm, A., Lamb, J., Nusbaum, C., Green, R.D., and Dekker, J. 2006. Chromosome conformation capture carbon copy (5C): A massively parallel solution for mapping interactions between genomic elements. Genome Res. 16:1299‐1309.
   Gheldof, N., Tabuchi, T.M., and Dekker, J. 2006. The active FMR1 promoter is associated with a large domain of altered chromatin conformation with embedded local histone modifications. Proc. Natl. Acad. Sci. U.S.A. 103:12463‐12468.
   Landegren, U., Kaiser, R., Sanders, J., and Hood, L. 1988. A ligase‐mediated gene detection technique. Science 241:1077‐1080.
   Ling, J.Q., Li, T., Hu, J.F., Vu, T.H., Chen, H.L., Qiu, X.W., Cherry, A.M., and Hoffman, A.R. 2006. CTCF mediates interchromosomal colocalization between Igf2/H19 and Wsb1/Nf1. Science 312:269‐272.
   Liu, Z. and Garrard, W.T. 2005. Long‐range interactions between three transcriptional enhancers, active Vkappa gene promoters, and a 3′ boundary sequence spanning 46 kilobases. Mol. Cell. Biol. 25:3220‐3231.
   Lomvardas, S., Barnea, G., Pisapia, D.J., Mendelsohn, M., Kirkland, J., and Axel, R. 2006. Interchromosomal interactions and olfactory receptor choice. Cell 126:403‐413.
   Murrell, A., Heeson, S., and Reik, W. 2004. Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent‐specific chromatin loops. Nat. Genet. 36:889‐893.
   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.
   Splinter, E., Grosveld, F., and de Laat, W. 2004. 3C technology: Analyzing the spatial organization of genomic loci in vivo. Methods Enzymol. 375:493‐507.
   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.
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