Mapping Chromatin Interactions by Chromosome Conformation Capture

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 recipe s)
100 mM dNTPs
Primers (see )
Taq DNA polymerase
50 mM MgSO₄

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

Figure 21.11.1 Schematic representation of the chromosome conformation capture (3C) technology. Cells are treated with formaldehyde to induce a cross‐link resulting in covalent linkage of interacting chromatin segments (indicated by the solid center oval). Cross‐linked chromatin is digested and ligated under dilute DNA concentrations. After reversal of cross‐links, ligation products can be detected by semi‐quantitative PCR (arrows indicate PCR primers).

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Figure 21.11.2 Gel analysis of BAC DNA during generation of mammalian control template. A 1‐kb ladder is shown in lane 1 for size reference. Lane 2 is undigested BAC DNA, which runs on an agarose gel at slightly more than 10 kb. Lane 3 is digested BAC DNA, which is smear‐like in appearance. Lane 4 is ligated BAC DNA, which shows the disappearance of most of the smear‐like trail. Similar results are obtained for a Saccharomyces cerevisiae control template at the corresponding stages.

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Figure 21.11.3 Titration and quantification of Saccharomyces cerevisiae 3C and control templates. The panels depict titration and analysis of a control template (left) and 3C template (right) using the same two primer pairs. The PCR products were run on agarose gels, PCR products were quantified, and the amount of PCR product was plotted versus DNA concentration in micrograms for the two primers pairs, one 11 kb apart in genomic distance (closed circles) and one 85 kb apart (open circles). For both primer pairs, the control template yields similar amounts of PCR product. The 3C template yields lower PCR products for the 85‐kb primer pair than for the 11‐kb primer pair. The linear range for PCR amplification for 3C and control templates is to the left of the dashed line. The DNA concentration for use in subsequent reactions should be in this range, but should also be high enough so that PCR products can be accurately quantified on an agarose gel. Similar results are obtained for a mammalian control template and 3C template.

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Figure 21.11.4 Hypothetical results for a 3C experiment. (A ) A 3C dataset with no looping interaction between sequence elements x and y (black boxes). A number of interactions have been tested between sequence element x and sites located further away from it including sequence element y. Neighboring sites of x, those separated by a small genomic distance, exhibit high interaction frequencies. As genomic distance between sites increases, the interaction frequencies decrease progressively. A low interaction frequency is detected between sequence elements x and y. The exact shape of the curve is dependent on flexibility and the level of compaction of the chromatin fiber. (B ) In a 3C dataset with a looping interaction between sequence elements x and y, a local peak of interaction frequencies is observed. This peak (at ∼80 kb) shows that sequence elements x and y interact more frequently than expected, which is indicative of a looping interaction.

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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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Mapping Chromatin Interactions by Chromosome Conformation Capture

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited