3D FISH to analyse gene domain-specific chromatin re-modeling in human cancer cell lines

Abstract Fluorescence in situ hybridization (FISH) is a common technique used to label DNA and/or RNA for detection of a genomic region of interest. However, the technique can be challenging, in particular when applied to single genes in human cancer cells. Here, we provide a step-by-step protocol for analysis of short (35 kb–300 kb) genomic regions in three dimensions (3D). We discuss the experimental design and provide practical considerations for 3D imaging and data analysis to determine chromatin folding. We demonstrate that 3D FISH using BACs (Bacterial Artificial Chromosomes) or fosmids can provide detailed information of the architecture of gene domains. More specifically, we show that mapping of specific chromatin landscapes informs on changes associated with estrogen stimulated gene activity in human breast cancer cell lines.