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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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 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?link 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. This unit describes the ChIP protocol for Saccharomyces cerevisiae ; however, it is also applicable to other organisms.

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Basic Protocol 1: Chromatin Immunoprecipitation
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
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Chromatin Immunoprecipitation

Materials

Saccharomyces cerevisae cells to be studied
37% formaldehyde: store up to 1 year at room temperature
2.5 M glycine, heat sterilized
TBS ( appendix 2A ), 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 2A )
20 mg/ml DNase‐free RNase A (see recipe )
10× loading buffer (see recipe )
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 (see recipe ), room temperature
FA lysis buffer (see recipe )/0.5 M NaCl
ChIP wash buffer (see recipe )
Primers (see )
3000 Ci/mmol [³² P]dATP (optional; see annotation to step )

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 ( appendix 3A ), phenol/chloroform extraction and ethanol precipitation ( appendix 3A ), PCR ( appendix 3F ), agarose gel electrophoresis ( appendix 3A ), and nondenaturing acrylamide gel electrophoresis (unit 6.5 )

CAUTION: When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see appendix 1D ).

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 2A for buffer)

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 )
Immunoprecipitated fragments (“IP” sample; see protocol 1 , step )
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 17.7.1 Scheme for chromatin immunoprecipitation from yeast whole‐cell extracts.

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Figure 17.7.2 Anticipated results from chromatin immunoprecipitation analysis of origin recognition complex (ORC) with replication origin and nonorigin DNA sequences.

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Videos

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.
	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.
	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 crosslinking: 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 References
	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 et al., 1988. See above.
	Describes original formaldehyde cross‐linking and immunoprecipitation technique for mapping protein‐DNA interactions.
	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.

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

Abstract

Table of Contents

Materials

Basic Protocol 1: Chromatin Immunoprecipitation

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

Figures

Videos

Literature Cited