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Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific Genomic Sequences In Vivo

Oscar Aparicio¹, Joseph V. Geisberg², Edward Sekinger², Annie Yang², Zarmik Moqtaderi², Kevin Struhl²

¹University of Southern California, Los Angeles, California
²Harvard Medical School, Boston, Massachusetts

Publication Name:

Current Protocols in Molecular Biology

Unit Number:

Unit 21.3

DOI:

10.1002/0471142727.mb2103s69

Online Posting Date:

February, 2005

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Abstract

Chromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting the association of individual proteins with specific genomic regions in vivo. Live cells are treated with formaldehyde to generate protein-protein and protein-DNA cross-links between molecules that are in close proximity on the chromatin template in vivo. DNA sequences that cross-link with a given protein are selectively enriched, and reversal of the formaldehyde cross-linking permits recovery and quantitative analysis of the immunoprecipitated DNA. As formaldehyde inactivates cellular enzymes essentially immediately upon addition to cells, ChIP provides snapshots of protein-protein and protein-DNA interactions at a particular time point, and hence is useful for kinetic analysis of events occurring on chromosomal sequences in vivo. In addition, ChIP can be combined with microarray technology to identify the location of specific proteins on a genome-wide basis. Basic Protocol 1 in this unit describes the ChIP procedure for Saccharomyces cerevisiae; Basic Protocol 2 describes the corresponding steps for mammalian cells.

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Unit Introduction
Basic Protocol 1: Chromatin Immunoprecipitation in Yeast Cells
Basic Protocol 2: Chromatin Immunoprecipitation in Mammalian Cells
Alternate Protocol 1: Specific Peptide Elution of Protein-DNA Complexes Immunoprecipitated from Cross-Linked Chromatin
Alternate Protocol 2: Analysis of Chromatin Immunoprecipitation Experiments by Real-Time Quantitative PCR with SYBR Green
Alternate Protocol 3: Mapping Protein Binding Sites In Vivo using Chromatin Immunoprecipitation Followed by Quantitative PCR Scanning
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Chromatin Immunoprecipitation in Yeast Cells

Materials

Saccharomyces cerevisiae cells (Chapter 13) to be studied
37% formaldehyde: store up to 1 year at room temperature
2.5 M glycine, heat sterilized
TBS (APPENDIX 2), ice cold
FA lysis buffer with and without 2 mM PMSF (see recipe), ice cold
ChIP elution buffer (see recipe)
20 mg/ml Pronase (Roche) in TBS; store up to 1 year at –20°C
TE buffer, pH 7.5 (APPENDIX 2)
20 mg/ml DNase-free RNase A (UNIT 3.13)
10× loading buffer (UNIT 2.5A)
Primary antibody against protein or epitope of interest
50% (v/v) protein A–Sepharose beads (Amersham Pharmacia Biotech) or equivalent in TBS
FA lysis buffer, room temperature
FA lysis buffer/0.5 M NaCl
ChIP wash buffer (see recipe)
Primers (see Critical Parameters and Troubleshooting)
3000 Ci/mmol [³²P]dATP

2-ml screw-cap microcentrifuge tubes with (relatively) flat bottoms
~0.5-mm-diameter silica-zirconia (BioSpec; preferred) or glass beads
Mini bead beater (BioSpec; preferred) or individual or multivortexer
5-ml syringe
15-ml conical tubes, disposable
25-G needles
Sonicator with microtip probe (e.g., Branson Sonifier 250)
End-over-end rotator
0.5-ml PCR tube
Spin-X centrifuge-tube filter (e.g., Corning)
65°C water bath
PCR-purification spin column (Qiagen)
Software for analyzing PCR primers and products

Additional reagents and equipment for growth of Saccharomyces cerevisiae cultures (UNITS 13.1 & 13.2), phenol/chloroform extraction and ethanol precipitation (UNIT 2.1A), PCR (UNITS 15.1 & 15.7), agarose gel electrophoresis (UNIT 2.5A), and nondenaturing acrylamide gel electrophoresis (UNIT 2.7)

Basic Protocol 2: Chromatin Immunoprecipitation in Mammalian Cells

Materials

Mammalian cells growing in culture
Culture medium (see APPENDIX 3F)
37% (v/v) formaldehyde
2.5 M glycine
Tris-buffered saline (TBS; APPENDIX 2), ice cold
MC lysis buffer (see recipe), ice cold and room temperature
Liquid nitrogen
MNase buffer (see recipe), room temperature
100 mM (50×) phenylmethylsulfonyl fluoride (PMSF) in ethanol (prepare fresh)
20× protease inhibitors: 1 mini complete-EDTA free tablet (Roche) in 500 µl MNase buffer (prepare fresh)
Micrococcal nuclease (MNase; USB; optional)
0.2 M EGTA
20% (w/v) sodium dodecyl sulfate (SDS)
Protein A/G–Sepharose beads (Amersham Biosciences)
FA lysis buffer (with 150 mM NaCl; see recipe) with 2 mM (1×) PMSF and 1× protease inhibitors (added from 20× stock; see above)

Platform shaker
Refrigerated centrifuge
2-ml screw-cap microcentrifuge tubes
Probe sonicator (e.g., Branson)
15-ml conical tubes
End-over-end rotator

Additional reagents and equipment for mammalian cell culture (APPENDIX 3F) and checking DNA fragment size, immunoprecipitation, reversing cross-links, purification of immunoprecipitated DNA, and quantitative PCR (as for ChIP with yeast; see Basic Protocol 1)

Alternate Protocol 1: Specific Peptide Elution of Protein-DNA Complexes Immunoprecipitated from Cross-Linked Chromatin

Additional Materials (also see Basic Protocol 1)

1 mg/ml peptide (e.g., myc, HA) in TBS (see APPENDIX 2 for TBS)

For this protocol, follow steps to of the main method (see Basic Protocol 1), replace steps to with the following, and continue with step onwards.

Alternate Protocol 2: Analysis of Chromatin Immunoprecipitation Experiments by Real-Time Quantitative PCR with SYBR Green

Materials

Input DNA (see Basic Protocol 1, step , and Basic Protocol 2, step )
Immunoprecipitated fragments (“IP” sample; see Basic Protocol 1, step , and Basic Protocol 2, step )
TE buffer, pH 7.5 (APPENDIX 2A)
Primers (see Critical Parameters and Troubleshooting)
2× SYBR Green Taq mix (see recipe)
Real-time PCR machine and corresponding software (e.g., ABI)
96-well PCR plates (ABI, cat. no. 4306737) and optical adhesive covers
Centrifuge with swinging-bucket rotor and microtiter plate adapter
Spreadsheet program (e.g., Microsoft Excel)

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Figures

Figure 21.3.1

Scheme for chromatin immunoprecipitation from yeast whole-cell extracts.

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Figure 21.3.2

Effect of immunoprecipitated DNA fragment size on the distance from a binding site from which PCR signal will be observed. As DNA fragment size increases, PCR signal is observed upon amplification with primer pairs further from the binding site.

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Figure 21.3.3

Distribution of PCR signal observed after ChIP in two idealized DNA populations of different sonicated fragment size. (A) When fragment size is short, the distribution of signal around a binding site is fairly narrow. (B) With a population of larger DNA fragments, the distribution of PCR signal is broader, though it will be more intense at its peak, since more immunoprecipitated fragments can be amplified by the central primer pair. Peak signal occurs in the same position regardless of fragment size.

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Figure 21.3.4

With fewer and larger PCR products, information about the position of the binding site within the peak PCR product is limited. Here, all that can be said is that the binding site is contained somewhere within the section of DNA amplified by the central PCR primer pair.

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Figure 21.3.5

When the midpoint-to-midpoint distances of the PCR products are more closely spaced, the density of information and the symmetry of the signal plot make it possible to infer that the binding site is in the middle, rather than close to either end, of the DNA amplified by the central primer pair.

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Figure 21.3.6

When the binding site is much closer to one or the other end of the fragment amplified by the central primer pair, the signal distribution will be skewed toward the neighboring PCR product. In (A), peak binding occurs at the very end of the region amplified by primer pair 3 and slightly off-center with respect to that amplified by primer pair 2. In (B) peak binding occurs at the opposite end of the region amplified by primer pair 3 and is slightly off-center with respect to the region amplified by primer pair 4.

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Figure 21.3.7

Anticipated results from chromatin immunoprecipitation analysis of origin recognition complex (ORC) with replication origin and nonorigin DNA sequences.

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Literature Cited

Literature Cited
	Aparicio, O.M., Weinstein, D.M., and Bell, S.P. 1997. Components and dynamics of DNA replication complexes in S. cerevisiae: Redistribution of MCM proteins and Cdc45p during S phase. Cell 91:59-69.
	Braunstein, M., Rose, A.B., Holmes, S.G., Allis, C.D., and Broach, J.R. 1993. Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev. 7:592-604.
	Cosma, M.P., Tanaka, T., and Nasmyth, K. 1999. Ordered recruitment of transcription and chromatin remodeling factors to a cell cycle-and developmentally regulated promoter. Cell 97:299-311.
	Dedon, P.C., Soults, J.A., Allis, C.D., and Gorovsky, M.A. 1991. A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions. Anal. Biochem. 197:83-90.
	Gilmour, D.S. and Lis, J.T. 1984. Detecting protein-DNA interactions in vivo: Distribution of RNA polymerase on specific bacterial genes. Proc. Natl. Acad. Sci. U.S.A. 81:4275-4279.
	Gilmour, D.S., Rougvie, A.E., and Lis, J.T. 1991. Protein-DNA cross-linking as a means to determine the distribution of proteins on DNA in vivo. Meth. Cell Biol. 35:369-381.
	Harlow, E. and Lane, D. 1998. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
	Hecht, A., Strahl-Bolsinger, S., and Grunstein, M. 1996. Spreading of transcriptional repressor SIR3 from telomeric heterochromatin. Nature 383:92-96.
	Iyer, V.R., Horak, C.E., Scafe, C.S., Botstein, D., Snyder, M., and Brown, P.O. 2001. Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Nature 409:533-538.
	Kadosh, D. and Struhl, K. 1998. Targeted recruitment of the Sin3-Rpd3 histone deacetylase complex generates a highly localized domain of repressed chromatin in vivo. Mol. Cell. Biol. 18:5121-5127.
	Kuras, L. and Struhl, K. 1999. Binding of TBP to promoters in vivo is stimulated by activators and requires Pol II holoenzyme. Nature 399:609-612.
	Kuras, L., Kosa, P., Mencia, M., and Struhl, K. 2000. TAF-containing and TAF-independent forms of transcriptionally active TBP in vivo. Science 288:1244-1248.
	Li, X.-Y., Virbasius, A., Zhu, X., and Green, M.R. 1999. Enhancement of TBP binding by activators and general transcription factors. Nature 399:605-609.
	Meluh, P.B. and Koshland, D. 1997. Budding yeast centromere composition and assembly as revealed by in vivo cross-linking. Genes Dev. 11:3401-3412.
	Mencia, M. and Struhl, K. 2001. A region of TAF130 required for the TFIID complex to associate with promoters. Mol. Cell. Biol. 21:1145-1154.
	Moqtaderi, Z. and Struhl, K. 2004. Genome-wide occupancy profile of the RNA polymerase III machinery in Saccharomyces cerevisiae reveals loci with incomplete transcription complexes. Mol. Cell. Biol. 24:4118-4127.
	Orlando, V., Strutt, H., and Paro, R. 1997. Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods 11:205-214.
	Ren, B. et al. 2000. Genome-wide location and function of DNA binding proteins. Science 290:2306-2309.
	Solomon, M.J. and Varshavsky, A. 1985. Formaldehyde-mediated DNA-protein cross-linking: A probe for in vivo chromatin structures. Proc. Natl. Acad. Sci. U.S.A. 82:6470-6474.
	Solomon, M.J., Larsen, P.L., and Varshavsky, A. 1988. Mapping protein-DNA interactions in vivo with formaldehyde: Evidence that histone H4 is retained on a highly transcribed gene. Cell 53:937-947.
	Strahl-Bolsinger, S., Hecht, A., Luo, K., and Grunstein, M. 1997. SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev. 11:83-93.
	Tanaka, T., Knapp, D., and Nasmyth, K. 1997. Loading of an MCM protein onto DNA replication origins is regulated by cdc6p and CDKs. Cell 90:649-660.
Key Reference
	Hecht et al., 1996. See above.
Describes the technique from which the Basic Protocol 1 was adapted.
	Orlando et al., 1997. See above.
Describes formaldehyde cross-linking and immunoprecipitation for chromatin analysis in Drosophila, and discusses various parameters of the technique.
	Solomon and Varshavsky, 1985. See above.
Characterizes formaldehyde cross-linking, cross-link reversal, and sensitivity of cross-linked protein-DNA complexes to proteases and endonucleases.
	Solomon et al., 1988. See above.
Describes original formaldehyde cross-linking and immunoprecipitation technique for mapping protein-DNA interactions.