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. Just as in standard 3C, chromatin is formaldehyde cross-linked then restriction digested. The cross-linked chromatin is then immunoprecipitated using an antibody against a protein of interest. The ligation step occurs while the chromatin is still coupled to the beads. qPCR is then used to identify interactions of interest.
ChIP-Loop was first described in a 2005 paper in which the authors combined ChIP and 3C for the first time to better understand Rhett Syndrome (Horike et al., 2005). They found that the formation of a silent-chromatin loop is and important function of the MECP2 gene that is lost in Rhett Syndrome. 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.
ChIP-Loop may not be fully informative on its own; some argue that ChIP-Loop data must be clarified using ChIP data. For example, if two ChIP-Loop interacting segments are not both enriched in a ChIP sample, then the interaction should not be considered real (Simonis et al., 2007). In a noteworthy study, Ren et al., (2012) combined ChIP-loop with similar assays to show that the CTCF transcription factor mediates many chromatin loop interactions at individual gene loci.
ChIP-Loop Additional Reading
Many 3C technologies are described in this review, but it goes into detail on ChIP-Loop and give a good explanation of the technology and how it compares to similar approaches.
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 is a text book chapter that describes many 3C technologies and includes a section on ChIP-Loop.
- Horike, S., Cai, S., Miyano, M., Cheng, J.F., and Kohwi-Shigematsu, T. (2005). Loss of silent-chromatin looping and impaired imprinting of DLX5 in Rett syndrome. Nat. Genet. 37, 31-40.
- Ren, L., Wang, Y., Shi, M., Wang, X., Yang, Z., and Zhao, Z. (2012). CTCF mediates the cell-type specific spatial organization of the Kcnq5 locus and the local gene regulation. PLoS One 7, e31416.
- Simonis, M., Kooren, J., and de Laat, W. (2007). An evaluation of 3C-based methods to capture DNA interactions. Nat. Methods 4, 895-901.