Analysis of Protein Co‐Occupancy by Quantitative Sequential Chromatin Immunoprecipitation

Joseph V. Geisberg1, Kevin Struhl1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.8
DOI:  10.1002/0471142727.mb2108s70
Online Posting Date:  May, 2005
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Sequential Chromatin Immunoprecipitation (SeqChIP) is a powerful technique for analyzing the simultaneous association of two different proteins with genomic DNA sequences in vivo. Cellular Protein‐DNA complexes are cross‐linked with formaldehyde (UNIT), and are purified via two successive immunoprecipitations, with each immunoprecipitation targeting a different protein. Protein‐DNA cross‐links are then reversed and DNA sequences of interest are analyzed by quantitative PCR. At each genomic region, calculated SeqChIP co‐occupancy values are compared to occupancy values of singly immunoprecipitated samples. The extent of enrichment brought about by the second immunoprecipitation relative to the singly immunoprecipitated sample is directly correlated with the degree of co‐occupancy between the two proteins at the genomic location assayed. In principle, the technique is not limited to Saccharomyces cerevisiae. Cells from a wide variety of organisms can be used.

Keywords: chromatin; immunoprecipitation; protein-DNA interactions; in vivo crosslinking

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • 100 mg/ml bovine serum albumin (BSA, Fraction V; Sigma) in water (store at −20°C)
  • 500 µg/ml λ phage DNA (not sheared; New England Biolabs)
  • 10 mg/ml E. coli tRNA in water (store at −20°C)
  • 20 mg/ml glycogen, or Pellet Paint (Novagen) as DNA carrier
  • Additional reagents and equipment for ChIP (unit 21.3, Basic Protocol), growth of Saccharomyces cerevisiae (units 13.1& 13.2), extraction and purification of DNA (unit 2.1), and real‐time quantitative PCR (unit 21.3, Alternate Protocol 2)
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Literature Cited

Literature Cited
   Geisberg, J.V. and Struhl, K. 2004. Quantitative sequential chromatin immunoprecipitation, a method for analyzing co‐occupancy of proteins at genomic regions in vivo. Submitted.
   Ijpenberg, A., Tan, N.S., Gelman, L., Kersten, S., Seydoux, J., Xu, J., Metzger, D., Canaple, L., Chambon, P., Wahli, W., and Desvergne, B. 2004. In vivo activation of PPAR target genes by RXR homodimers. EMBO J. 23:2083‐2091.
   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., Bhaumik, S.R., and Green, M.R. 2000. Distinct classes of yeast promoters revealed by differential TAF recruitment. Science 288:1242‐1244.
   Metivier, R., Penot, G., Hubner, M.R., Reid, G., Brand, H., Kos, M., and Gannon, F. 2003. Estrogen receptor‐alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115:751‐763.
   Proft, M. and Struhl, K. 2002. Hog1 kinase converts the Sko1‐Cyc8‐Tup1 repressor complex into an activator that recruits SAGA and SWI/SNF in response to osmotic stress. Mol. Cell 9:1307‐1317.
   Soutoglou, E. and Talianidis, I. 2002. Coordination of PIC assembly and chromatin remodeling during differentiation‐induced gene activation. Science 295:1901‐1904.
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