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9.8: Chromatin Immunoprecipitation

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  • Many DNA-binding proteins, such as transcription factors, bind to specific sequences of nucleotides in, for example, promoters and enhancers of genes. Some examples:

    • a generalized eukaryotic promoter
    • multiple transcription factors bound to the Drosophila eve promoter
    • the glucocorticoid receptor (protein) bound to its response element (DNA)
    • the tryptophan repressor bound to its operator

    The binding of protein to DNA is done by noncovalent forces and is easily reversible. In fact, as conditions in a cell change, there is a dynamic coming and going of DNA-binding proteins throughout the genome. The identification of a specific site in DNA bound by a particular protein at a particular time can be discovered by the technique of chromatin immunoprecipitation (ChIP).

    The Procedure

    1. Harvest your cell population at the desired time. Some examples:
      • yeast cells growing on a particular nutrient;
      • HeLa cells exposed to a particular cytokine;
      • salivary gland cells of Drosophila at the time of pupation.
    2. Treat the cells with formaldehyde (HCHO) which creates covalent bonds between the proteins and nucleotides to which they have been bound noncovalently.
    3. Break open the cells releasing their contents.
    4. Use ultrasound to break the DNA into fragments averaging about 500 bp long.
    5. Add an antibody that specifically binds to the protein you are interested in.
    6. Add beads coated with Protein A or Protein S — both proteins that bind to any antibody.
    7. Centrifuge down the complexes of bead—antibody—target protein—DNA.
    8. Heat the complexes to break the covalent crosslinks between the target protein and the DNA.
    9. Digest the protein with a protease leaving purified DNA fragments.
    10. Perform PCR on the fragments.
    11. Use any of several methods to identify the amplified DNA, for example,
      • Southern blotting
      • DNA chip analysis ("ChIP on chip")
      • clone and sequence ("ChIP-Seq")