Double Chromatin Immunoprecipitation: Analysis of Target Co-occupancy of Retinal Transcription Factors

Abstract

Combinatorial binding of transcription factors (TFs) and cofactors to speci fi c 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 speci fi c 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-speci fi c 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.