4C is the sequel to 3C, and like Terminator 2 it’s even better than the original. 4C offers several innovations to the basic 3C protocol. Most importantly it allows for the detection of unknown DNA regions interaction with the region of interest.
The 4C procedure is the same as 3C until the crosslinks are reversed. Then the DNA is treated with a second, frequently cutting restriction enzyme with a different recognition sequence than the first. This creates fragments with sticky ends that can circularize, containing the known DNA of interest and its interacting DNA. Primers designed to bind the known DNA are then used to amplify the unknown DNA outward around the circle. This library can then be characterized by microarray, or DNA sequencing. 4C’s compatibly with whole-genome technologies and its ability to amplify unknown interacters drastically increases its scale over 3C. 4C, like 3C has a large amount of background noise (Dekker, 2006).
4C was first introduced in two independent articles in the same journal in 2006. These papers examined the structure of the H19 imprinting locus (Zhao et al., 2006) and the organization of active and inactive chromatin (Simonis et al., 2006). Since then it has been applied to diverse studies such as tRNA function (Raab et al., 2012) and breast cancer (Zeitz et al., 2013).
For additional reading about chromosome conformation capture methods, check out this article on Hi-C and related methods from our friends at Active Motif.
4C Additional Reading
This book chapter provides detailed information on all aspects of 4C. It gives a good background on the technology as well as a detailed 4C protocol and complete data analysis and example results.
Splinter, E., de Wit, E., van de Werken, H.J., Klous, P., and de Laat, W. (2012). Determining long-range chromatin interactions for selected genomic sites using 4C-seq technology: from fixation to computation. Methods 58, 221-230.
This paper provides a detailed explanation and protocol for 4C. It also goes into depth on the data analysis methods and their justification.
- Dekker, J. (2006). The three ‘C’ s of chromosome conformation capture: controls, controls, controls. Nat. Methods 3, 17-21.
- Raab, J.R., Chiu, J., Zhu, J., Katzman, S., Kurukuti, S., Wade, P.A., Haussler, D., and Kamakaka, R.T. (2012). Human tRNA genes function as chromatin insulators. EMBO J. 31, 330-350.
- Simonis, M., Klous, P., Splinter, E., Moshkin, Y., Willemsen, R., de Wit, E., van Steensel, B., and de Laat, W. (2006). Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C). Nat. Genet. 38, 1348-1354.
- Zeitz, M.J., Ay, F., Heidmann, J.D., Lerner, P.L., Noble, W.S., Steelman, B.N., and Hoffman, A.R. (2013). Genomic interaction profiles in breast cancer reveal altered chromatin architecture. PLoS One 8, e73974.
- Zhao, Z., Tavoosidana, G., Sjolinder, M., Gondor, A., Mariano, P., Wang, S., Kanduri, C., Lezcano, M., Sandhu, K.S., Singh, U., et al. (2006). Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. Nat. Genet. 38, 1341-1347.