Document Detail


Double chromatin immunoprecipitation: analysis of target co-occupancy of retinal transcription factors.
MedLine Citation:
PMID:  23150378     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Guang-Hua Peng; Shiming Chen
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Methods in molecular biology (Clifton, N.J.)     Volume:  935     ISSN:  1940-6029     ISO Abbreviation:  Methods Mol. Biol.     Publication Date:  2013  
Date Detail:
Created Date:  2012-11-15     Completed Date:  2013-04-17     Revised Date:  2013-08-01    
Medline Journal Info:
Nlm Unique ID:  9214969     Medline TA:  Methods Mol Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  311-28     Citation Subset:  IM    
Affiliation:
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Chromatin Immunoprecipitation / methods*
Homeodomain Proteins / analysis
Mice
Orphan Nuclear Receptors / analysis
Promoter Regions, Genetic
Retina / cytology*,  metabolism*
Retinal Cone Photoreceptor Cells / metabolism
Retinal Rod Photoreceptor Cells / metabolism
Trans-Activators / analysis
Transcription Factors / analysis*
Grant Support
ID/Acronym/Agency:
EY012543/EY/NEI NIH HHS; EY02687/EY/NEI NIH HHS; R01 EY012543/EY/NEI NIH HHS
Chemical
Reg. No./Substance:
0/Homeodomain Proteins; 0/Nr2e3 protein, mouse; 0/Orphan Nuclear Receptors; 0/Trans-Activators; 0/Transcription Factors; 0/cone rod homeobox protein

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