Chromatin Interaction Analysis Using Paired‐End Tag Sequencing

互联网2013-12-31

2378

Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Chromatin Interaction Analysis using Paired?End Tag sequencing (ChIA?PET) is a technique developed for large?scale, de novo analysis of higher?order chromatin structures. Cells are treated with formaldehyde to cross?link chromatin interactions, DNA segments bound by protein factors are enriched by chromatin immunoprecipitation, and interacting DNA fragments are then captured by proximity ligation. The Paired?End Tag (PET) strategy is applied to the construction of ChIA?PET libraries, which are sequenced by high?throughput next?generation sequencing technologies. Finally, raw PET sequences are subjected to bioinformatics analysis, resulting in a genome?wide map of binding sites and chromatin interactions mediated by the protein factor under study. This unit describes ChIA?PET for genome?wide analysis of chromatin interactions in mammalian cells, with the application of Roche/454 and Illumina sequencing technologies. Curr. Protoc. Mol. Biol. 89:21.15.1?21.15.25. © 2010 by John Wiley & Sons, Inc.

Keywords: PET; paired?end; mate?pair; SAGE; DNA sequencing; ChIA?PET; 454 sequencing; Illumina sequencing; transcription factor binding sites; chromatin interactions; chromosome conformation capture; chromatin immunoprecipitation

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Introduction
Basic Protocol 1: Construction of a ChIA‐PET Library
Support Protocol 1: Preparation of Linkers and Adapters for ChIA‐PET
Support Protocol 2: Validation of Linkers for ChIA‐PET
Reagents and Solutions
Commentary
Literature Cited
Figures

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Construction of a ChIA‐PET Library

Materials

ChIP DNA bound to Sepharose beads (unit 21.3 )
TE buffer, pH 8.0 ( appendix 22 )
Elution buffer (see recipe )
20 mg/ml proteinase K (Invitrogen)
Buffer EB (Qiagen)
10× T4 polymerase buffer (Promega)
10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, and dTTP)
Nuclease‐free H₂ O (see unit 4.1 )
9.7 U/µl T4 DNA polymerase (Promega)
Wash buffer (see recipe )
200 ng/µl biotinylated half‐linkers A and B (Fig. ; see Support Protocols protocol 31 and protocol 42 )
5× T4 DNA ligase buffer with PEG (Invitrogen)
30 U/µl T4 DNA ligase (Fermentas)
10× T4 DNA ligase buffer (without PEG; New England Biolabs)
10 U/µl T4 DNA polynucleotide kinase (New England Biolabs)
Elution buffer (see recipe )
20% (v/v) Triton X‐100
25:24:1 phenol/chloroform/isoamyl alcohol, pH 7.9 (Ambion)
3 M sodium acetate, pH 5.2 ( appendix 22 )
15 mg/ml GlycoBlue (Ambion)
Isopropanol
75% ethanol
10× NEBuffer 4 (New England Biolabs)
10× (500 µM) S‐adenosylmethionine (SAM; New England Biolabs), freshly prepared, by adding 1 µl 640× SAM from the manufacturer to 63 µl H₂ O
200 ng/µl nonbiotinylated linker (Fig. ; Support Protocols protocol 31 and protocol 42 )
2 U/µl Mme I restriction endonuclease (New England Biolabs)
Dynabeads M‐280 Streptavidin (Invitrogen)
1× and 2× B&W buffer (see recipe )
200 ng/µl 454 GS20 Adapters A and B (Fig. ; see Support Protocols protocol 31 and protocol 42 )
10× NEBuffer 2 (New England Biolabs)
10 U/µl E. coli DNA polymerase I (New England Biolabs)
10 µM 454 PCR Primer A and biotinylated Primer B (Fig. )
HotStarTaq Master Mix (Qiagen)
6% TBE PAGE gel (5 wells; Invitrogen, by special request only; also see unit 2.7 )
10× TBE buffer ( appendix 22 )
SYBR Green I (Invitrogen Molecular Probes)
QIAquick PCR Purification Kit (Qiagen)
25 µM Illumina 1‐454 primer (Fig. )
25µM Illumina 2‐454 primer (Fig. )
Phusion High‐Fidelity PCR Master Mix with HF buffer (Finnzymes; http://www.finnzymes.us/)

1.5‐ml screw‐cap microcentrifuge tubes
Refrigerated microcentrifuge
Intelli‐Mixer RM‐2L (Palico Biotech, http://www.palicobio.com/)
2‐ml Phase‐Lock Gel tubes (Eppendorf)
Microspin plastic centrifuge tube filter units (0.45‐µm, e.g., Corning Costar Spin‐X)
50‐ml MaXtract High Density tubes (Qiagen)
50‐ml polypropylene copolymer (PPCO) centrifuge tubes (Nalgene)
Magnetic Particle Collector (Invitrogen)
1.5‐ml DNA LoBind Tubes (Eppendorf)
Thermal cycler
0.2‐ml PCR tubes
Dark Reader Transilluminator (Clare Chemical Research; http://www.clarechemical.com/)
0.6‐ml microcentrifuge tubes pierced at bottom with 21‐G needle
Agilent 2100 Bioanalyzer (Agilent Technologies)
Agilent DNA 1000 Kit (Agilent Technologies)

Additional reagents and equipment for phenol/chloroform extraction and isopropanol precipitation of DNA (unit 2.1 ), PicoGreen dsDNA quantitation ( appendix 3D and unit 21.12 ), PCR (unit 15.1 ), and analysis of PCR products by electrophoresis (unit 2.7 )

Support Protocol 1: Preparation of Linkers and Adapters for ChIA‐PET

Materials

Linker and adapter oligonucleotides (Fig. )
1× TNE buffer (see recipe )
4% to 20% TBE PAGE gel (10 wells; Invitrogen)
25‐bp DNA ladder

Additional reagents and equipment for annealing of oligonucleotides (unit 21.12 , Support Protocol 2) and electrophoresis of DNA (unit 2.7 )

Support Protocol 2: Validation of Linkers for ChIA‐PET

Materials

DNA fragment X of known size (blunt‐ended, 5′‐phosphorylated, ∼1 to 3 kb)
200 ng/µl annealed biotinylated half‐linkers A and B (Fig. )
5× T4 DNA ligase buffer with PEG (Invitrogen)
30 U/µl T4 DNA ligase (Fermentas)
Nuclease‐free H₂ O (see unit 4.1 )
QIAquick PCR Purification Kit (Qiagen)
Buffer EB (Qiagen)
10× T4 DNA ligase buffer (without PEG; New England Biolabs)
10 U/µl T4 DNA polynucleotide kinase (New England Biolabs)
3 M sodium acetate, pH 5.2 ( appendix 22 )
15 mg/ml GlycoBlue (Ambion)
Isopropanol
75% ethanol
10× NEBuffer 4 (New England Biolabs)
10× (500 µM) S‐adenosylmethionine (SAM; New England Biolabs), freshly prepared by adding 1 µl 640× buffer from the manufacturer to 63 µl H₂ O
200 ng/µl nonbiotinylated linker (Fig. ; Support Protocols protocol 31 and protocol 42 )
2 U/µl Mme I restriction endonuclease (New England Biolabs)
Dynabeads M‐280 Streptavidin (Invitrogen)
1× and 2× B&W buffer (see recipe )
200 ng/µl 454 GS20 Adapters A and B (Fig. ; see Support Protocols protocol 31 and protocol 42 )
10× NEBuffer 2 (New England Biolabs)
10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, and dTTP)
10 U/µl E. coli DNA polymerase I (New England Biolabs)
10 µM 454 PCR Primer A and biotinylated Primer B (Fig. )
HotStarTaq Master Mix (Qiagen)
4% to 20% TBE PAGE gel (10 wells; Invitrogen)

Intelli‐Mixer RM‐2L (Palico Biotech, http://www.palicobio.com/)
Magnetic Particle Collector (Invitrogen)
1.5‐ml DNA LoBind tubes (Eppendorf)
0.2‐ml PCR tubes
Thermal cycler

Additional reagents and equipment for phenol/chloroform extraction of DNA (unit 2.1 ), PCR (unit 15.1 ), and analysis of PCR products by electrophoresis (unit 2.7 )

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 21.15.1 Schematic overview of ChIA‐PET method. (A ) Outline of ChIA‐PET library construction procedure. Chromatin samples from cell cultures are cross‐linked, sonicated, and immunoprecipitated. Separate aliquots of ChIP DNA are ligated to barcoded half‐linkers, and proximity ligation is carried out. PETs are released by restriction digest, purified on streptavidin‐coated magnetic beads, and ligated to adapters for next‐generation sequencing. (B ) Examples of experimental and chimeric ChIA‐PETs. Note the A/B linker composition of chimeric PETs. Tags flanking the central linker sequence are read out and mapped to the genome. (C ) Binding sites and interactions are identified by clusters of overlapping PETs; singleton PETs indicate nonspecific ligations that do not represent true binding sites or interactions.

View Image

Figure 21.15.2 Custom oligonucleotide sequences for ChIA‐PET.

View Image

Figure 21.15.3 Gel analysis of ChIA‐PETs after PCR amplification (, step 32). A 25‐bp DNA ladder is shown in lane 1 for size reference. Lanes 2 and 3 are the PCR products generated after 20 cycles of PCR amplification from 1 µl and 2 µl of bead‐immobilized template, respectively. 25 cycles were used to generate the PCR products in lanes 4 and 5, from 1 µl and 2 µl of beads, respectively. (A ) This is a successful library, as indicated by the bright, well‐defined bands at the expected size of 166 bp. (B ) On this gel, PCR amplification has failed to yield sufficient ChIA‐PET DNA, as seen from the very weak band present in lane 5. This could indicate that PCR conditions need to be optimized, or that library construction has failed.

View Image

Figure 21.15.4 Agilent 2100 Bioanalyzer analysis of purified 454 adapter‐ligated ChIA‐PETs (, step 40). (A ) Screen capture of Agilent 2100 Bioanalyzer electropherograms profiling a successful library, with a single intense peak at the expected size of 166 bp, and (B ) an unsuccessful library, with a faint signal indicating insufficient ChIA‐PET DNA. Peaks of interest are indicated by arrows. Note that the Agilent Bioanalyzer assay usually reports a slightly higher‐than‐expected size; in this case, the desired peak is displayed at 180 bp instead of 166 bp. This is within the 10% error range of the Agilent assay.

View Image

Figure 21.15.5 Agilent 2100 Bioanalyzer analysis of purified ChIA‐PETs after conversion for Illumina sequencing (step 44). A successful library is characterized by a single intense peak (indicated by an arrow) with an expected size of 223 bp.

View Image

Figure 21.15.6 Screen captures of ChIA‐PET whole‐genome interaction views. Chromatin interactions are displayed with purple loop structures where the interactions are located. Interactions are colored according to cluster size; darker purple loops represent more interaction PETs in an interaction unit, and hence, higher‐confidence interactions. Regions with multiple unique interactions appear “onion‐shaped.” An example of a high‐quality ChIA‐PET library containing many high‐confidence interactions is shown in (A ). In contrast, an unsuccessful library (B ) has few interactions, and these interactions are mostly low‐confidence.

View Image

Figure 21.15.7 Example of PET genomic‐span histograms. Genomic span is defined as the genomic distance between the two mapped tags within each PET sequence. The histogram in (A ) represents the distribution of genomic spans for a successful ChIA‐PET library: it shows a high number of self‐ligation and inter‐ligation PETs with an exponentially decreasing distribution of genomic spans. An example of a low‐quality library, shown in (B ), has an abnormally low PET count and few intrachromosomal inter‐ligation PETs with genomic spans exceeding 3 kb. Panel (C ) is an example of a library with a broad distribution of genomic spans, most likely due to poor sonication quality of ChIP DNA.

View Image

Figure 21.15.8 Screenshots for ChIA‐PET binding site and interaction data. (A ) High‐confidence interaction cluster with good binding site peaks. (B ) Weak interaction cluster with good binding site peaks. (C ) Region with poor binding site peaks and no interactions. Self‐ligation PETs show transcription factor binding sites, and interaction clusters comprise multiple overlapping inter‐ligation PETs. A successful ChIA‐PET library should have an abundance of strong binding sites and a high number of interaction clusters, as shown in examples (A) and (B). An unsuccessful library primarily contains weak binding sites and few, if any, interactions, as in (C). This problem is possibly due to poor ChIP enrichment, and may be resolved by optimizing ChIP procedures.

View Image

Videos

Literature Cited

Literature Cited
	Branco, M.R. and Pombo, A. 2006. Intermingling of chromosome territories in interphase suggests role in translocations and transcription‐dependent associations. PLoS Biol. 4:e138.
	Cai, S., Lee, C.C., and Kohwi‐Shigematsu, T. 2006. SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes. Nat. Genet. 38:1278‐1288.
	Carter, D., Chakalova, L., Osborne, C.S., Dai, Y.F., and Fraser, P. 2002. Long‐range chromatin regulatory interactions in vivo. Nat. Genet. 32:623‐626.
	Chen, X., Xu, H., Yuan, P., Fang, F., Huss, M., Vega, V.B., Wong, E., Orlov, Y.L., Zhang, W., Jiang, J., Loh, Y.H., Yeo, H.C., Yeo, Z.X., Narang, V., Govindarajan, K.R., Leong, B., Shahab, A., Ruan, Y., Bourque, G., Sung, W.K., Clarke, N.D., Wei, C.L., and Ng, H.H. 2008. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133:1106‐1117.
	Chiu, K.P., Wong, C.H., Chen, Q., Ariyaratne, P., Ooi, H.S., Wei, C.L., Sung, W.K., and Ruan, Y. 2006. PET‐Tool: A software suite for comprehensive processing and managing of Paired‐End diTag (PET) sequence data. BMC Bioinformatics 7:390.
	Cremer, T. and Cremer, C. 2001. Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat. Rev. Genet. 2:292‐301.
	Dekker, J. 2006. The three ‘C’ s of chromosome conformation capture: controls, controls, controls. Nat. Methods 3:17‐21.
	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.
	Fullwood, M.J. and Ruan, Y. 2009. ChIP‐based methods for the identification of long‐range chromatin interactions. J. Cell Biochem. 107:30‐39.
	Fullwood, M.J., Wei, C.L., Liu, E.T., and Ruan, Y. 2009. Next‐generation DNA sequencing of paired‐end tags (PET) for transcriptome and genome analyses. Genome Res. 19:521‐532.
	Gondor, A., Rougier, C., and Ohlsson, R. 2008. High‐resolution circular chromosome conformation capture assay. Nat. Protoc. 3:303‐313.
	Harris, T.D., Buzby, P.R., Babcock, H., Beer, E., Bowers, J., Braslavsky, I., Causey, M., Colonell, J., Dimeo, J., Efcavitch, J.W., Giladi, E., Gill, J., Healy, J., Jarosz, M., Lapen, D., Moulton, K., Quake, S.R., Steinmann, K., Thayer, E., Tyurina, A., Ward, R., Weiss, H., Xie, Z. 2008. Single‐molecule DNA sequencing of a viral genome. Science 320:106‐109.
	Holt, R.A. and Jones, S.J. 2008. The new paradigm of flow cell sequencing. Genome Res. 18:839‐846.
	Horike, S., Cai, S., Miyano, M., Cheng, J.F., and Kohwi‐Shigematsu, T. 2005. Loss of silent‐chromatin looping and impaired imprinting of DLX5 in Rett syndrome. Nat Genet. 37:31‐40.
	Margulies, M., Egholm, M., Altman, W.E., Attiya, S., Bader, J.S., Bemben, L.A., Berka, J., Braverman, M.S., Chen, Y.J., Chen, Z., Dewell, S.B., Du, L., Fierro, J.M., Gomes, X.V., Godwin, B.C., He, W., Helgesen, S., Ho, C.H., Irzyk, G.P., Jando, S.C., Alenquer, M.L., Jarvie, T.P., Jirage, K.B., Kim, J.B., Knight, J.R., Lanza, J.R., Leamon, J.H., Lefkowitz, S.M., Lei, M., Li, J., Lohman, K.L., Lu, H., Makhijani, V.B., McDade, K.E., McKenna, M.P., Myers, E.W., Nickerson, E., Nobile, J.R., Plant, R., Puc, B.P., Ronan, M.T., Roth, G.T., Sarkis, G.J., Simons, J.F., Simpson, J.W., Srinivasan, M., Tartaro, K.R., Tomasz, A., Vogt, K.A., Volkmer, G.A., Wang, S.H., Wang, Y., Weiner, M.P., Yu, P., Begley, R.F., and Rothberg, J.M. 2005. Genome sequencing in microfabricated high‐density picolitre reactors. Nature 437:376‐380.
	Ng, P., Wei, C.L., Sung, W.K., Chiu, K.P., Lipovich, L., Ang, C.C., Gupta, S., Shahab, A., Ridwan, A., Wong, C.H., Liu, E.T., Ruan, Y. 2005. Gene identification signature (GIS) analysis for transcriptome characterization and genome annotation. Nat. Methods 2:105‐111.
	Osborne, C.S., Chakalova, L., Brown, K.E., Carter, D., Horton, A., Debrand, E., Goyenechea, B., Mitchell, J.A., Lopes, S., Reik, W., and Fraser, P. 2004. Active genes dynamically colocalize to shared sites of ongoing transcription. Nat. Genet. 36:1065‐1071.
	Shendure, J., Porreca, G.J., Reppas, N.B., Lin, X., McCutcheon, J.P., Rosenbaum, A.M., Wang, M.D., Zhang, K., Mitra, R.D., and Church, G.M. 2005. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309:1728‐1732.
	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.
	Stein, L.D., Mungall, C., Shu, S., Caudy, M., Mangone, M., Day, A., Nickerson, E., Stajich, J.E., Harris, T.W., Arva, A., and Lewis, S. 2002. The generic genome browser: A building block for a model organism system database. Genome Res. 12:1599‐1610.
	Su, W., Porter, S., Kustu, S., and Echols, H. 1990. DNA‐looping and enhancer activity: Association between DNA‐bound NtrC activator and RNA polymerase at the bacterial glnA promoter. Proc. Natl. Acad. Sci. U.S.A. 87:5504‐5508.
	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.
	Wei, C.L., Wu, Q., Vega, V.B., Chiu, K.P., Ng, P., Zhang, T., Shahab, A., Yong, H.C., Fu, Y., Weng, Z., Liu, J., Zhao, X.D., Chew, J.L., Lee, Y.L., Kuznetsov, V.A., Sung, W.K., Miller, L.D., Lim, B., Liu, E.T., Yu, Q., Ng, H.H., and Ruan, Y. 2006. A global map of p53 transcription‐factor binding sites in the human genome. Cell 124:207‐219.
	Yoshimura, S.H., Maruyama, H., Ishikawa, F., Ohki, R., and Takeyasu, K. 2004. Molecular mechanisms of DNA end‐loop formation by TRF2. Genes Cells 9:205‐218.
	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.

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Chromatin Interaction Analysis Using Paired‐End Tag Sequencing

Abstract

Table of Contents

Materials

Basic Protocol 1: Construction of a ChIA‐PET Library

Support Protocol 1: Preparation of Linkers and Adapters for ChIA‐PET

Support Protocol 2: Validation of Linkers for ChIA‐PET

Figures

Videos

Literature Cited