Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation.
The standard chromatin immunoprecipitation (ChIP) assay is used to examine the specific association of transcription factors with DNA in the context of living cells. Here we review two modifications to this protocol which are designed to identify novel target genes of transcription factors in mammalian cells. The main advantage to both of these approaches is that only DNA sequences directly bound by a factor within the context of a living cell will be identified. Therefore, artifacts associated with overexpression and/or alterations in signaling pathways are avoided. The first modification we describe, a ChIP cloning strategy, can be used to isolate any genomic fragment specifically associated with a particular factor. It requires no special equipment or reagents other than a high-affinity antibody to be used for immunoprecipitation of the factor of interest. However, it is most useful for the isolation of a small number of genomic targets. In contrast, the second modification, which combines ChIP with specialized CpG microarrays, is ideal for a more global analysis of target genes. Advantages, common problems, and detailed protocols for these two ChIP techniques are discussed.
Weinmann
Amy S
AS
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, 1400 University Avenue, Madison 53706, USA.
Farnham
Peggy J
PJ
D000818:Animals; D002843:Chromatin / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D018899:CpG Islands; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D011233:Precipitin Tests / Q000379:methods*; D014157:Transcription Factors / Q000378:metabolism*
D002843:Chromatin; D014157:Transcription Factors