title: Double chromatin immunoprecipitation: analysis of target co-occupancy of retinal transcription factors.
abstract: Combinatorial binding of transcription factors (TFs) and cofactors to specific regulatory regions of target genes in vivo is an important mechanism of transcriptional regulation. Chromatin immunoprecipitation (ChIP) is a powerful technique to detect protein binding to specific regions of target genes in vivo. However, conventional ChIP analysis for individual factors (single ChIP) does not provide information on co-occupancy of two interacting TFs on target genes, even if both bind to the same chromatin regions. Double ChIP analysis involves sequential (double) immunoprecipitation of two chromatin-binding proteins and can be used to study co-occupancy of two or more factors on specific regions of the same DNA allele. Furthermore, by including a cell type-specific protein in double-ChIP, target co-occupancy in a specific cell type can be studied even if the other partner is more widely expressed. In this chapter, we describe a detailed protocol for double ChIP analysis in mouse retinas. Using the rod-specific transcription factor NR2E3 and the cone/rod homeobox protein CRX as examples, we show that NR2E3 and CRX are co-enriched on the promoter of active Rho and Rbp3 genes in rods, but are present to a much lesser degree on the promoters of silent cone opsin genes. These results suggest a new mechanism by which rod and cone genes are differentially regulated by these transcription factors in rod photoreceptors.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA.
authors.forename: Guang-Hua
authors.initials: GH
authors.lastname: Peng
authors.forename: Shiming
authors.initials: S
authors.lastname: Chen
mesh_terms: D000818:Animals; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D018398:Homeodomain Proteins / Q000032:analysis; D051379:Mice; D057093:Orphan Nuclear Receptors / Q000032:analysis; D011401:Promoter Regions, Genetic; D012160:Retina / Q000166:cytology* / Q000378:metabolism*; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism; D015534:Trans-Activators / Q000032:analysis; D014157:Transcription Factors / Q000032:analysis*
chemical_list: D018398:Homeodomain Proteins; C485736:Nr2e3 protein, mouse; D057093:Orphan Nuclear Receptors; D015534:Trans-Activators; D014157:Transcription Factors; C109288:cone rod homeobox protein