ChIP: How to Find Which DNA Regions Interact with Protein
Chromatin immunoprecipitation sequencing (ChIP-seq) is a technique used to identify sequences of DNA where proteins (like transcription factors) bind. The process is as follows.
1. Cellular fixation
Fix cells with formaldehyde to link any DNA-binding proteins to the DNA. This will allow us to reliably see where proteins bound DNA at the time of fixation.
2. Chromatin digestion
Digest DNA-protein complexes with micrococcal nuclease, which is an endo-exonuclease because it chops up all DNA, regardless of if the DNA is on the ends or in the middle of the strand. DNase can also be used in this procedure; however, DNase is more selective in what it can cut; thus, DNase digestion is more useful to determine which sites on the DNA are more sensitive to DNase cutting or more similar to DNase cutting site sequences, relative to the locations of proteins. DNase-seq's goal is thus to determine how accessible the chromatin (proteins+DNA) is to DNase enzymes, rather than determining which DNA sequences are bound to proteins. Anyways, for ChIP-seq, digestion with micrococcal nuclease results in all the DNA going away except the DNA cross-linked to the proteins.
3. Immunoprecipitation
"Immunoprecipitation" means precipitating (making a solid lump) via an immune method (using antibodies). Antibodies against DNA-binding proteins of interest are constructed and released into the solution. Immunoaffinity beads (heavy balls that like to bind to antibodies) like those coated with Protein A or Protein G are used to precipitate the Ig-tagged proteins of interest out of solution. This precipitate is then collected and washed to remove any excess materials.
4. Crosslink reversal
This step can be done with various agents. Heat can denature the proteins, disrupting the protein-DNA interaction. Sonication and glycine addition are alternative methods to achieve the same result of disrupting the interaction. Crosslink reversal means that in our protein-DNA complexes, the proteins and DNA are separated.
5. Purification
Depending on the researcher's interest, either the DNA, protein, or both, can be purified and analyzed separately. This analysis can give valuable information about which transcription factors/other DNA-binding proteins associate with the DNA at a given time in a specific cell type.
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DNase vs. micrococcal nuclease: https://www-sciencedirect-com.ucsf.idm.oclc.org/topics/biochemistry-genetics-and-molecular-biology/deoxyribonuclease-i; https://www.neb.com/en-us/products/m0247-micrococcal-nuclease.
ChIP vs DNase-seq: https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-016-1274-4.
ChIP Protocol: https://star-protocols.cell.com/protocols/71.
