Defining In Vivo Targets of Nuclear Proteins by Chromatin Immunoprecipitation and Microarray Analysis

Zarmik Moqtaderi1, Kevin Struhl1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.9
DOI:  10.1002/0471142727.mb2109s68
Online Posting Date:  November, 2004
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This unit describes the combination of chromatin immunoprecipitation (ChIP) with microarray hybridization to determine the genome‐wide occupancy profile of a DNA‐associated protein. After conventional ChIP, the immunoprecipitated material is amplified by a two‐step process involving primer extension followed by PCR in the presence of a modified nucleotide. The amplified DNA is fluorescently labeled in a reaction that couples dye to the modified nucleotide, and the labeled sample is hybridized to a microarray representing a complete genome. This method allows the study of a protein's pattern of DNA association across an entire genome with no need for prior knowledge of potential DNA targets.

Keywords: Chromatin immunoprecipitation; ChIP; microarray; amplification; PCR; dye coupling; protein‐DNA interactions; ChIP‐chip; ChIP‐on‐chip; genome‐wide location; hybridization; whole‐genome analysis

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1:

  • De‐cross‐linked DNA samples: typically yeast DNA from chromatin immunoprecipitation and input DNA control; unit 21.3)
  • 5× Sequenase buffer (USB)
  • 80 µM primer A: GTTTCCCAGTCACGATCNNNNNNNNN (see unit 2.11 for oligonucleotide synthesis)
  • 10 mg/ml BSA
  • 0.1 M DTT
  • 10 mM dNTP mix: 10 mM each dATP, dCTP, dGPT, and dTTP (unit 3.4)
  • 13 U/µl Sequenase (USB) stock: dilute 1/10 immediately before use
  • 10× PCR buffer (see recipe)
  • Amino‐allyl dNTP mix (see recipe)
  • 100 µM primer B: GTTTCCCAGTCACGATC (see unit 2.11 for oligonucleotide synthesis)
  • 5 U/µl Taq DNA polymerase
  • 0.1 M sodium bicarbonate, pH 9.0
  • Cy3 or Cy5 monoreactive dye (Amersham): resuspend entire tube of Cy5 or Cy3 dye in 45 µl DMSO
  • 3 M sodium acetate ( appendix 222)
  • 10 mg/ml sheared salmon sperm DNA (unit 20.1)
  • 2.5× array hybridization buffer (see recipe)
  • Array prehybridization buffer (see recipe)
  • 95% ethanol
  • Array wash buffer (see recipe)
  • 0.2× SSC ( appendix 222)
  • QIAquick PCR purification kit (Qiagen)
  • Thermal cycler
  • MinElute PCR purification kit (Qiagen)
  • Microarrays (unit 22.1)
  • Coplin jar or petri dish
  • Plastic racks for holding microscope slides
  • Plastic containers for washing microscope slides in holders
  • Tabletop centrifuge with adaptors suitable for holding plastic microscope slide racks (typically the same as the adaptors meant for holding 96‐well plates)
  • 95°C heating block or water bath
  • LifterSlip coverslips for microarrays (Erie Scientific Company)
  • Rubber cement
  • Humidified chamber (see recipe)
  • 45°C oven
  • Platform shaker
  • Additional reagents and equipment for PCR amplification (unit 15.1) and separation of small DNA fragments by agarose gel electrophoresis (unit 2.5)
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Literature Cited

Literature Cited
   Bohlander, S.K., Espinosa, R. III, Le Beau, M.M., Rowley, J.D., and Diaz, M.O. 1992. A method for the rapid sequence‐independent amplification of microdissected chromosomal material. Genomics 13:1322‐1324.
   DeRisi, J., Penland, L., Brown, P.O., Bittner, M.L., Meltzer, P.S., Ray, M., Chen, Y., Su, Y.A., and Trent, J.M. 1996. Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nature Genet. 14:457‐460.
   DeRisi, J.L., Iyer, V.R., and Brown, P.O. 1997. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278:680‐686.
   Eberwine, J. 1996. Amplification of mRNA populations using aRNA generated from immobilized oligo(dT)‐T7 primed cDNA. BioTechniques 20:584‐591.
   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.
   Kuras, L. and Struhl, K. 1999. Binding of TBP to promoters in vivo is stimulated by activators and requires Pol II holoenzyme. Nature 399:609‐613.
   Lee, T.I., Rinaldi, N.J., Robert, F., Odom, D.T., Bar‐Joseph, Z., Gerber, G.K., Hannett, N.M., Harbison, C.T., Thompson, C.M., Simon, I., Zeitlinger, J., Jennings, E.G., Murray, H.L., Gordon, D.B., Ren, B., Wyrick, J.J., Tagne, J.B., Volkert, T.L., Fraenkel, E., Gifford, D.K., and Young, R.A. 2002. Transcriptional regulatory networks in Saccharomyces cerevisiae. Science 298:799‐804.
   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.
   Lockhart, D.J., Dong, H., Byrne, M.C., Follettie, M.T., Gallo, M.V., Chee, M.S., Mittmann, M., Wang, C., Kobayashi, M., Horton, H., and Brown, E.L. 1996. Expression monitoring by hybridization to high‐density oligonucleotide arrays. Nat. Biotechnol. 14:1675‐1680.
   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.
   Reid, J.L., Iyer, V.R., Brown, P.O., and Struhl, K. 2000. Coordinate regulation of yeast ribosomal protein genes is associated with targeted recruitment of Esa1 histone acetylase. Mol. Cell. 6:1297‐1307.
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