Mapping Networks of Protein‐Mediated Physical Interactions Between Chromatin Elements

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Abstract
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Abstract

Understanding of transcriptional regulation has advanced in recent years in part due to development of technologies which allow determination of physical proximities between interacting chromatin regions at a resolution beyond that offered by conventional microscopy techniques. However, these methods do not specifically identify the protein component(s) that might mediate such interactions. This unit provides a detailed protocol for Combined 3C?ChIP?Cloning (6C) technology, which combines multiple techniques to simultaneously identify physical proximities between chromatin elements as well as the proteins that mediate these interactions. The unit further explores how the 6C assay can be combined with other techniques for a complete, cell?type?specific mapping of all inter? and intrachromosomal interactions mediated by specific proteins. Thus, the 6C assay provides a useful tool to address the role of specific proteins in nuclear organization and to advance our understanding about the relation of chromatin higher?order organization and transcriptional regulation. Curr. Protoc. Mol. Biol. 89:21.16.1?21.16.13. © 2010 by John Wiley & Sons, Inc.

Keywords: transcriptional regulation; chromatin folding; nuclear organization

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Materials

Basic Protocol 1:

Materials

Cell type of interest, grown under appropriate cell culture conditions in appropriate medium
≥36.5% formaldehyde solution (Sigma‐Aldrich, cat. no. 33220)
2 M glycine (Fisher, cat. no. BP381)
Phosphate‐buffered saline (PBS), pH 7.2 (Invitrogen, cat. no. 20012)
Protease inhibitor cocktail (Sigma, P8340)
1× trypsin‐EDTA (0.05% trypsin in tetrasodium EDTA), liquid (Invitrogen, cat. no. 25300120)
Fetal bovine serum (FBS)
Cell lysis solution (see recipe )
10× restriction enzyme buffer
20% (w/v) sodium dodecyl sulfate (SDS; Fisher, cat. no. BP166)
20% (v/v) Triton X‐100 (VWR Scientific)
Restriction enzyme (see Critical Parameters)
1.15× ligation buffer (see recipe for 10×)
400 U/µl T4 DNA ligase (New England Biolabs)
ChIP dilution buffer (see recipe )
Antibody for immunoprecipitation, specific for the protein of interest
Magnetic beads, protein A‐conjugated (Invitrogen)
Magnetic beads, protein G‐conjugated (Invitrogen)
0.5% bovine serum albumin (BSA; New England Biolabs) in PBS
Low‐salt wash buffer (see recipe )
High‐salt wash buffer (see recipe )
TE buffer ( appendix 22 )
Elution buffer (see recipe )
10 mg/ml RNase A, DNase‐free (Sigma, cat. no. R‐6513)
10 mg/ml proteinase K (Invitrogen) in TE buffer, pH 8.0 (see appendix 22 for TE buffer)
Phenol:chloroform:isoamyl alcohol, UltraPure (25:24:1 v/v/v; Invitrogen)
3 M sodium acetate, pH 5.2 (Fisher, cat. no. BP333)
20 mg/ml glycogen (Roche)
Absolute ethanol, cold
70% ethanol
REDTaq DNA polymerase (Sigma, cat. no. D‐4309)
100 mM dNTP set (Invitrogen)
PCR primers for sequencing the plasmid as well as for validating physical interactions between captured chromatin elements by 3C assay and protein occupancy by ChIP assay
E.coli cells , competent, high‐efficiency (≥5 × 10⁹ cfu/µg DNA; e.g., XL10‐Gold ultracompetent cells from Stratagene; also see unit 1.8 )
LB plates with antibiotic of choice (e.g., ampicillin or kanamycin) and Xgal and IPTG (unit 1.1 )
LB plates with antibiotic or choice (e.g., ampicillin or kanamycin; unit 1.1 )
PureLink HQ 96 Mini Plasmid DNA Purification Kit (Invitrogen)
1% agarose gel (UltraPure; Invitrogen; also see unit 2.5 )
DNA gel stain (SYBR Safe, 10,000× concentrate in DMSO; Invitrogen)
DNA ladder, 100‐bp and/or 1‐kb (New England Biolabs)

15‐cm tissue culture dishes
15‐ and 50‐ml conical centrifuge tubes
Centrifuge, clinical
Centrifuge, high‐speed, refrigerated, equipped with swinging‐bucket rotor for 15‐ml tubes
65°C and 50°C water baths or heat block
16° water bath
End‐over‐end rotator
Magnetic stand for 1.5‐ml microcentrifuge tubes (Invitrogen)
Gel imaging system for quantifying PCR products
DNA analysis software (Vector NTI)
DNA sequence analysis software (Finch TV)

Additional reagents and equipment for restriction digestion (unit 3.1 ), PCR (unit 15.1 ), cloning of DNA (unit 3.16 ), transformation of competent E. coli (unit 1.8 ), plasmid miniprep (unit 1.6 ), agarose gel electrophoresis (unit 2.5 ), and DNA sequencing (Chapter 7)

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Figures

Figure 21.16.1 Summary of the Combined 3C‐ChIP‐Cloning (6C) method. Briefly, the conventional 3C assay (Tolhuis et al., ) is performed up to the ligation step, following which the chromatin is subjected to chromatin immunoprecipitation (ChIP) using an antibody against the protein of interest. Then, the purified DNA is ligated into a cloning vector bearing the sequence overhangs generated in the enzyme digestion used in the 3C assay to facilitate insert cloning and further screening. Clones are then digested with the restriction enzyme used for the 3C assay, and the ones showing multiple inserts are subjected to sequencing from both directions in the vector to reveal the identity of the partners.

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Figure 21.16.2 Further applications and the future of Combined 3C‐ChIP‐Cloning (6C) methodology. The 6C method can be combined with other techniques to identify the entire “interactome” in the nucleus for a gene or chromatin region of choice that is mediated by a specific protein of interest. Following chromatin immunoprecipitation (ChIP), the samples may be processed for 4C analysis (Zhao et al., ) or reverse cross‐linked, purified, digested with a four‐base cutter, and further subjected to either 3C‐chip (Simonis et al., ) or ACT assay (Ling et al., ). This technique could also be used to investigate whether two known chromatin regions are brought in close physical proximity by a protein of interest. For this purpose, subsequent to the reversal of cross‐linking and purification, the amplification criteria used in the original 3C assay should be followed (Dekker et al., ).

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Literature Cited

Literature Cited
	Dekker, J., Rippe, K., Dekker, M., and Kleckner, N. 2002. Capturing chromosome conformation. Science 295:1306‐1311.
	Dostie, J., Richmond, T.A., Arnaout, R.A., Selzer, R.R., Lee, W.L., Honan, T.A., Rubio, E.D., 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.
	Kurukuti, S., Tiwari, V.K., Tavoosidana, G., Pugacheva, E., Murrell, A., Zhao, Z., Lobanenkov, V., Reik, W., and Ohlsson, R. 2006. CTCF binding at the H19 imprinting control region mediates maternally inherited higher‐order chromatin conformation to restrict enhancer access to Igf2. Proc. Natl. Acad. Sci. U.S.A. 103:10684‐10689.
	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.
	Simonis, M., Klous, P., Splinter, E., Moshkin, Y., Willemsen, R., de Wit, E., van Steensel, B., and de Laat, W. 2006. Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture‐on‐chip (4C). Nat. Genet. 38:1348‐1354.
	Simonis, M., Kooren, J., and de Laat, W. 2007. An evaluation of 3C‐based methods to capture DNA interactions. Nat. Methods 4:895‐901.
	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.
	Tiwari, V.K., Cope, L., McGarvey, K.M., Ohm, J.E., and Baylin, S.B. 2008. A novel 6C assay uncovers Polycomb‐mediated higher order chromatin conformations. Genome Res. 18:1171‐1179.
	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.
	Zhao, Z., Tavoosidana, G., Sjolinder, M., Gondor, A., Mariano, P., Wang, S., Kanduri, C., Lezcano, M., Sandhu, K.S., Singh, U., Pant, V., Tiwari, V., Kurukuti, S., and Ohlsson, R. 2006. Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra‐ and interchromosomal interactions. Nat. Genet. 38:1341‐1347.

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Mapping Networks of Protein‐Mediated Physical Interactions Between Chromatin Elements

Abstract

Table of Contents

Materials

Basic Protocol 1:

Figures

Videos

Literature Cited