Chromatin Immunoprecipitation (ChIP) provides data on the distribution of protein factors on DNA with high temporal and spatial resolution.
The crosslinking of proteins to DNA freezes interactions in time and stabilizes weak interactions that might not persist through the immunoprecipitation protocol. We use two methods for crosslinking, chemical fixation via formaldehyde and photophysical fixation via UV light. Formaldehyde crosslinks proteins to other proteins as well as to DNA, whereas UV light predominantly forms DNA-protein crosslinks. The two methods can provide complementary information about the structure of a protein-DNA complex.
The crosslinked DNA is fragmented, and an antibody is used to purify the DNA fragments bound to a specific protein. Quantitative real-time PCR measures the relative amounts of the protein that are found at various DNA sequences. The genome-wide distribution of the protein can now be conveniently analyzed by ChIP-seq and analysis pipelines developed in our lab.
- Guertin MJ, Martins AL, Siepel A, Lis JT. (2012) “Accurate prediction of inducible transcription factor binding intensities in vivo.” PLoS Genet. 8(3):e1002610. (PubMed)
- Zhang X, Bolt M, Guertin MJ, Chen W, Zhang S, Cherrington BD, Slade DJ, Dreyton CJ, Subramanian V, Bicker KL, Thompson PR, Mancini MA, Lis JT, Coonrod SA. (2012) “Peptidylarginine deiminase 2-catalyzed histone H3 arginine 26 citrullination facilitates estrogen receptor α target gene activation.” Proc Natl Acad Sci USA. 109(33):13331-6. (PubMed)
- Fuda NJ, Buckley MS, Wei W, Core LJ, Waters CT, Reinberg D, Lis JT. (2012) “Fcp1 dephosphorylation of the RNA polymerase II C-terminal domain is required for efficient transcription of heat shock genes.” Mol Cell Biol. 32(17):3428-37. (PubMed)
- Guertin MJ, Lis JT. (2010) “Chromatin landscape dictates HSF binding to target DNA elements.”PLoS Genet. 9;6(9). (PubMed)
- Ni Z et al. (2008) “P-TEFb is critical for the maturation of RNA polymerase II into productive elongation in vivo.” Mol Cell Biol. 28(3):1161-70. (PubMed)
- Adelman K et al. (2006) “Drosophila Paf1 modulates chromatin structure at actively transcribed genes.” Mol Cell Biol. 26(1):250-60. (PubMed)
- Adelman K et al. (2005) “Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS.” Mol Cell. 17(1):103-12. (PubMed)
- Boehm AK et al. (2003) “Transcription factor and polymerase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock.” Mol Cell Biol. 23(21):7628-37. (PubMed)
- Gilmour DS & Lis JT. (1986) “RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells.” Mol. Cell. Biol. 6, 3984–3989 (PubMed)
- First use of UV crosslinking and ChIP showing the presence of Pol II on the uninduced Hsp70 gene.
- Gilmour DS & Lis JT (1985) “In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster.” Mol Cell Biol. 5(8):2009-18. (PubMed)
- First protein-DNA crosslinking to map a protein’s density on specific genes in vivo in eukaryotes.
Footprinting involves treating live cells with a chemical agent that causes DNA damage. If a protein is bound to the DNA, it can protect the strands it covers from this damage, and its binding site can be deduced.
- Giardina C & Lis JT. (1995) “Dynamic protein-DNA architecture of a yeast heat shock promoter.” Mol Cell Biol. 15(5):2737-44. (PubMed)