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

1 University of Southern California, Los Angeles, California, 2 Harvard 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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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. in this unit describes the ChIP procedure for Saccharomyces cerevisiae; describes the corresponding steps for mammalian cells.

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Table of Contents

  • 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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Basic Protocol 1: Chromatin Immunoprecipitation in Yeast Cells

  • 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 22), 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 22)
  • 20 mg/ml DNase‐free RNase A (unit 3.13)
  • 10× loading buffer (unit 2.5)
  • 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 )
  • 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 2.1), PCR (units 15.1& 15.7), agarose gel electrophoresis (unit 2.5), and nondenaturing acrylamide gel electrophoresis (unit 2.7)

Basic Protocol 2: Chromatin Immunoprecipitation in Mammalian Cells

  • Mammalian cells growing in culture
  • Culture medium (see appendix 3F)
  • 37% (v/v) formaldehyde
  • 2.5 M glycine
  • Tris‐buffered saline (TBS; appendix 22), 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 protocol 1)

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

  • 1 mg/ml peptide (e.g., myc, HA) in TBS (see appendix 22 for TBS)
For this protocol, follow steps to of the main method (see 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

  • Input DNA (see protocol 1, step , and protocol 2, step )
  • Immunoprecipitated fragments (“IP” sample; see protocol 1, step , and protocol 2, step )
  • TE buffer, pH 7.5 ( appendix 2A)
  • Primers (see )
  • 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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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 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.
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