Chromosome conformation capture (3C) of tandem arrays in yeast.

Abstract

Studying interphase chromosome arrangements at the molecular level can provide important details on the function and coordination of many metabolic processes that take place on DNA, such as transcription or DNA repair. The chromosome conformation capture (3C) methodology was originally developed in yeast to study the interphase organization of a single-yeast chromosome. 3C assays allow the identification of physical interactions between distant DNA segments and thus provide detailed information on the folding of chromatin in the native cellular state. Since its initial development, the technique has been used to advance our understanding of the folding of gene loci and has yielded important discoveries, like the demonstration that distant transcriptional regulatory elements interact with their target promoters through chromatin loops. The 3C method uses formaldehyde cross-linking to covalently link interacting chromatin segments in intact cells. After chromatin digestion and ligation of cross-linked fragments, the frequency of interaction between distant DNA elements can be quantified. However, the design, analysis, and controls used are critical when using 3C. In this chapter, we describe the general protocol for 3C analysis and demonstrate it through analysis of interactions in repetitive sequences of the ribosomal gene array of Saccharomyces cerevisiae.