CHAPTER

Chapter Six: Chromatin immunoprecipitation (ChIP)

Contents Page

Chromatin immunoprecipitation overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 Chromatin immunoprecipitation formats Antibody format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21 Antibody-free format; Use of HaloTag fusion proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22 When to use chromatin immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23 Primary reagent requirements for chromatin immunoprecipitation assays . . . . . . . . . . . . . . . . . . . . .23

CHROMATIN IMMUNOPRECIPITATION

Chromatin immunoprecipitation overview

Chromatin immunoprecipitation (ChIP) is an experimental method used to determine whether proteins, such as certain transcription factors, are associated with a specic genomic region in living cells or tissues. This cell-based technique is often used together with non-cell-based assays to characterize protein:DNA interactions. The method is based on the principle that formaldehyde reacts with primary amines located on amino acids and the bases on DNA or RNA molecules, forming a covalent crosslink between the specic protein to the DNA on which they are situated. Following crosslinking, the cells are lysed and the crude cell extracts are sonicated to shear the DNA to a smaller size. The protein:DNA complex is immunoprecipitated using an antibody against the protein of interest. The DNA protein cross-links are reversed by heating and the proteins removed by treatment with proteinase K. The DNA portion of the complex is then puried and identied by PCR using specic primers to the suspected binding region.

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Chromatin immunoprecipitation (ChIP) formats

Antibody format
The classic format of the ChIP assay follows the basic procedure noted in Figure 10 and requires four days for completion. The procedure requires highly specic antibodies

to the protein of interest. If antibodies are not available the proteins can be fused to tags such as HA or c-myc, which are recognized by commercially available antibodies. The success of the procedure relies on the ability of the antibody to bind to the target protein after crosslinking (crosslinking changes epitope recognition of the antibody).

CHAPTER

CHROMATIN IMMUNOPRECIPITATION

Transcription Factor
TF Crosslink with Formaldehyde TF TF Lysis of Cytoplasm Nuclei Lysis in 1% SDS

Experimental
TF Antibody TF

Control
TF Sonicate Split Sample Incubate +/- Antibody O/N at 4C Incubate with Protein A/G Agarose 2 hours at 4C A/G Wash Elute with TE +1% SDS Proteinase K, Reverse CrosslinksO/N at 65C

TF

REFERENCES

Key original reference for chromatin immunoprecipitation

1. Solomon, M. et al. (1985) Proc. Natl. Acad. Sci. 82, 647074.

A/G

TF

TF

Antibody format
1. Benson, L. et al. (2006) J. Biol. Chem. 281, 928796. 2. Ghosh, M. et al. (2006) Mol. Cell. Biol. 26, 527083. 3. White, D. et al. (2006) Cancer Res. 66, 346370. 4. Lei, H. et al. (2006) Proc. Natl. Acad. Sci. 103, 1030509. 5. Shur, I. et al. (2006) Stem Cells 24, 128893. 6. Natesampillai, S. et al. (2006) J. Biol. Chem. 281, 304047.

TF

TF
6641MA

Purify DNA

Figure 10. Overview of chromatin immunoprecipitation using antibodies. Mammalian cells are grown using the appropriate conditions to modulate the formation of protein:DNA interactions. To conserve the DNA:protein structure during cell lysis formaldehyde is added resulting in the formation of cross-links between the DNA and the bound protein. A whole-cell extract is prepared, and the cross-linked chromatin is sheared by sonication to reduce average DNA fragment size. Either a polyclonal or monoclonal antibody to the target protein is added. The success of the procedure relies on the use of an antibody that will specically and tightly bind its target protein under the buffer and wash conditions used. Protein A/G agarose beads are added to capture the complex and incubated overnight. Reversal of the formaldehyde cross-linking by heating permits the recovery and quantitative analysis of the immunoprecipitated DNA.

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CHROMATIN IMMUNOPRECIPITATION

Antibody-free format; use of HaloTag fusion proteins (Figure 11)

An assay format that does not require the use of antibodies is based on the HaloTag Technology. A HaloTag vector containing the protein-coding sequence of interest is transfected into the appropriate mammalian cell line. The cells are then xed with formaldehyde, allowing the HaloTag fusion protein to be crosslinked to the specic region

of chromatin, lysed and then sonicated. Then, instead of using an antibody to capture the DNA:protein complex, the complex is captured directly onto the HaloLink Resin. The HaloLink Resin provides a method for covalent, oriented attachment of HaloTag fusion proteins onto a solid surface. The DNA:protein crosslinks are reversed by heating, and the DNA can then be puried using commercially available columns.

Covalent Capture of Chromatin Complexes Using HaloTag Technology

Transfection

HT

HaloTag Vector

TF
TF HT

Expression of HaloTag Fusion Protein

HT

TF
HaloCHIP Blocking Ligand

Formaldehyde Cross-linking

Experimental Sample
HaloLink

Control Sample
HaloLink

Lysis, Sonication

11.5 days

HT

HT

Split Sample
Add HaloCHIP Blocking Ligand to the control sample to prevent binding to HaloLink Resin

TF

TF

Capture on HaloLink Resin

HaloLink

HT

HaloLink

TF
REFERENCES (CONTINUED)

Wash HaloLink Resin

Covalent capture allows for highly stringent washes to remove non-specific proteins and DNA

Genome-wide chromatin immunoprecipitation assays

1. Krieg, A. et al. (2006) Mol. Cell. Biol. 26, 703045. 2. Mayanil, C. et al. (2006) J. Biol. Chem. 281, 2454452. 3. Kajiyama, Y. et al. (2006) J. Biol. Chem. 281, 3012231.
Analyze Analyze Sample DNA Background DNA

Release of DNA by reversal of cross-links

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Figure 11. Capture of protein:chromatin interactions using HaloTag technology. The protein-coding sequence of a transcription factor (TF) is cloned into a HaloTag (HT) vector containing the necessary elements for protein expression in mammalian cells. This recombinant vector is transfected into mammalian cells. Mammalian cells are then grown under the appropriate conditions to modulate the formation of protein:DNA interactions. In order to conserve the DNA:protein structure formaldehyde is added resulting in the formation of cross-links between the DNA and the bound protein. A whole-cell extract is prepared, and the cross-linked chromatin is sheared by sonication to reduce average DNA fragment size. The complex is then immobilized by the addition of the HaloLink Resin followed by a short incubation. Reversal of the formaldehyde cross-linking by heating permits the recovery and quantitative analysis of the immunoprecipitated DNA.

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When to use chromatin immunoprecipitation

Chromatin immunoprecipitation can be used to conrm the location of individual or multiple transcription factors during cell growth or upon exposure to abnormal conditions such as UV radiation. The technique can also be utilized in conjunction with microarrays to discover the location of various transcription factors on a genome-wide basis.

Primary reagent requirements for chromatin immunoprecipitation assays

Mammalian cell line of choice Cell culture media Polyclonal or monoclonal antibody Cell lysis reagent Formaldehyde Protein A/G agarose Proteinase K DNA purication reagents PCR reagents (including primers specic for the DNA region of interest) HaloTag fusion protein (specic for HaloTag procedure) HaloLink Resin (specic for HaloTag procedure)

CHAPTER

CHROMATIN IMMUNOPRECIPITATION

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