Measurement of protein-DNA interactions in vivo by chromatin immunoprecipitation.
Elucidating mechanisms controlling nuclear processes requires an understanding of the nucleoprotein structure of genes at endogenous chromosomal loci. Traditional approaches to measuring protein-DNA interactions in vitro have often failed to provide insights into physiological mechanisms. Given that most transcription factors interact with simple DNA sequence motifs, which are abundantly distributed throughout a genome, it is essential to pinpoint the small subset of sites bound by factors in vivo. Signaling mechanisms induce the assembly and modulation of complex patterns of histone acetylation, methylation, phosphorylation, and ubiquitination, which are crucial determinants of chromatin accessibility. These seemingly complex issues can be directly addressed by a powerful methodology termed the chromatin immunoprecipitation (ChIP) assay. ChIP analysis involves covalently trapping endogenous proteins at chromatin sites, thereby yielding snapshots of protein-DNA interactions and histone modifications within living cells. The chromatin is sonicated to generate small fragments, and an immunoprecipitation is conducted with an antibody against the desired factor or histone modification. Crosslinks are reversed, and polymerase chain reaction (PCR) is used to assess whether DNA sequences are recovered immune-specifically. Chromatin-domain scanning coupled with quantitative analysis is a powerful means of dissecting mechanisms by which signaling pathways target genes within a complex genome.
Im
Hogune
H
Department of Pharmacology, Molecular and Cellular Pharmacology Program, University of Wisconsin Medical School, Madison, USA.
Grass
Jeffrey A
JA
Johnson
Kirby D
KD
Boyer
Meghan E
ME
Wu
Jing
J
Bresnick
Emery H
EH
D000107:Acetylation; D000818:Animals; D002467:Cell Nucleus / Q000235:genetics; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008745:Methylation; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests / Q000379:methods*; D011499:Protein Processing, Post-Translational; D015203:Reproducibility of Results; D015398:Signal Transduction; D014157:Transcription Factors / Q000032:analysis* / Q000276:immunology / Q000378:metabolism
D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; D014157:Transcription Factors; D004247:DNA