TY - CHAP
T1 - Double chromatin immunoprecipitation
T2 - Analysis of target co-occupancy of retinal transcription factors
AU - Peng, Guang Hua
AU - Chen, Shiming
PY - 2013
Y1 - 2013
N2 - 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.
AB - 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.
KW - Double chromatin immunoprecipitation
KW - Retinal photoreceptors
KW - Target co-occupancy
KW - Transcription factor interactions
UR - http://www.scopus.com/inward/record.url?scp=84871995786&partnerID=8YFLogxK
U2 - 10.1007/978-1-62703-080-9-22
DO - 10.1007/978-1-62703-080-9-22
M3 - Chapter
C2 - 23150378
AN - SCOPUS:84871995786
SN - 9781627030793
T3 - Methods in Molecular Biology
SP - 311
EP - 328
BT - Retinal Degeneration
A2 - Weber, Bernhard H.F.
A2 - Langmann, Thomas
ER -