Chromatin Conformation Capture (3C) is an important technique used to study chromatin structure, as well as the basis for several other derivative techniques. 3C provides information on 3D chromatin structures that occur in living cells. The first study using 3C was published in 2002, in which the authors used it to discover that the yeast third chromosome forms a 3D contorted ring structure (Dekker et al., 2002).
The protocol involves formaldehyde cross-linking of cells followed by chromatin isolation and digestion with a restriction enzyme. The specific enzymes used are chosen in order to free a known or predicted DNA-DNA interaction mediated by a protein complex (Dekker, 2006). These freed fragments are then ligated into rings and the crosslinks are reversed. Real-time PCR is then used to determine the ligation product abundance in the purified DNA. The abundance of these recombinant fragments directly correlates to the interaction frequency of the two ligated regions. This basic principle can be combined with other technologies to increase scale or specificity of the DNA loops being interrogated.
3C has many derivative techniques, each with a more eccentric name than the last. The Circularized Chromosome Conformation Capture (4C) protocol detects unknown DNA regions interacting with a locus of interest. Carbon Copy Chromosome Conformation Capture (5C) uses unique primers to generate a library of all ligation products created, which are then detected with next-gen sequencing. ChIP-Loop combines ChIP and 3C by using an antibody against a protein known to bind the DNA locus of interest. Hi-C is a dramatic increase in scale over other 3C techniques: successful ligations products only are enriched, then DNA sequenced, quantifying frequencies across the enter genome. Finally, Capture-C is a recent innovation that offers high resolution and high throughput; it combines oligonucleotide capture technology (OCT), 3C and high-throughput sequencing to examine hundreds of loci simultaneously.
3C Based Techniques
Circularized Chromosome Conformation Capture (4C): The sequel to 3C offers several innovations to the basic protocol, and reveals how unknown DNA regions interact with a region of interest.
Carbon Copy Chromosome Conformation Capture (5C): Useful for examining interactions with particular loci of interest in detail.
ChIP-Loop: If 3C is chocolate and ChIP is peanut butter then ChIP-Loop is a peanut butter cup: the combination is better than either on its own. The major advantage of ChIP-Loop is it reduces the background noise in 3C experiments and increases the specificity by selecting for a known protein mediating the DNA-DNA interaction.
Hi-C: Tthe first of the 3C derivate technologies to be truly genome-wide. Biotin pull-down isolates specific fragments and the resulting library is then sequenced and mapped to the genome allowing fragment identity and abundance to be detected.
Capture-C: The newest, and in many ways most advanced 3C technology to be developed, is both high resolution and high throughput. It combines 3C and next-gen sequencing with oligonucleotide capture technology (OCT).
3C Techniques Additional reading
This review is a great resource for understanding many aspects of 3C technologies, particularly data analysis. The authors give examples of what the data output for each technologies look like and how they compare to each other.
This review compares and contrasts the most common 3C techniques. It also gives many references for how different studies have used each technologies to address specific biological questions.
- Dekker, J. (2006). The three ‘C’ s of chromosome conformation capture: controls, controls, controls. Nat. Methods 3, 17-21.
- Dekker, J., Rippe, K., Dekker, M., and Kleckner, N. (2002). Capturing chromosome conformation. Science 295, 1306-1311.