Dissecting long-range transcriptional mechanisms by chromatin immunoprecipitation.
Analysis of physiological mechanisms that control transcription often requires extrapolation of in vitro measurements into in vivo mechanisms. This extrapolation is complex, as mammalian genes are commonly organized into broad chromosomal domains, and such domains cannot be readily reconstituted in vitro. Thus, the nucleoprotein structure of chromosomes constitutes a considerable impediment to elucidating transcriptional mechanisms. The development of assays to measure protein-DNA interactions and chromatin structure in living cells has greatly facilitated progress in understanding physiological transcriptional mechanisms. Chromatin immunoprecipitation (ChIP) is a powerful approach that allows one to define the interaction of factors with specific chromosomal sites in living cells, thereby providing a snapshot of the native chromatin structure and factors bound to genes in different functional states. ChIP involves treating cells or tissue briefly with formaldehyde to crosslink proteins to DNA. An antibody against a protein suspected of binding a given cis-element is then used to immunoprecipitate chromatin fragments. Polymerase chain reaction analysis of the immunoprecipitate with primers flanking the cis-element reveals whether a specific DNA sequence is recovered in an immune-specific manner and therefore whether the protein contacted the site in living cells. The central focus of this review is the use of ChIP to study transcriptional activation over long distances on chromosomes.
Johnson
Kirby D
KD
Molecular and Cellular Pharmacology Program, Department of Pharmacology, University of Wisconsin Medical School, 387 Medical Sciences Center,1300 University Avenue, Madison 53706, USA.
Bresnick
Emery H
EH
D000107:Acetylation; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D005914:Globins / Q000235:genetics; D006657:Histones / Q000378:metabolism; D006801:Humans; D011233:Precipitin Tests / Q000379:methods*; D014158:Transcription, Genetic / Q000502:physiology*
D002843:Chromatin; D006657:Histones; D005914:Globins; D004247:DNA