Abstract
We describe a modified 3C (“chromosome conformation capture”) protocol for detection of transient, short-range chromatin interactions in the yeast Saccharomyces cerevisiae. 3C was initially described by Job Dekker and involves formaldehyde cross-linking to stabilize transient chromatin interactions, followed by restriction digestion, ligation, and locus-specific PCR. As such, 3C reveals complex three-dimensional interactions between distal genetic elements within intact cells at high resolution. Using a modified version of Dekker’s protocol, we are able to detect gene loops that juxtapose promoter and terminator regions of yeast genes with ORFs as short as 1 kb. We are using this technique to define the cis- and trans-acting requirements for the formation and maintenance of gene loops, and to elucidate their physiological consequences. We anticipate that this method will be generally applicable to detect dynamic, short-range chromatin interactions, not limited to gene loops.
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Acknowledgments
We are grateful to Athar Ansari (Wayne State University) for initially developing the 3C protocol in our laboratory and for his insight into the application of 3C to study gene loops. The work in our laboratory is supported by NIH RO1 grants GM39484 (to M.H.) and GM068887 (to Claire Moore (Tufts Medical School) and M. H.).
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Singh, B.N., Hampsey, M. (2014). Detection of Short-Range Chromatin Interactions by Chromosome Conformation Capture (3C) in Yeast. In: Smith, J., Burke, D. (eds) Yeast Genetics. Methods in Molecular Biology, vol 1205. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1363-3_13
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DOI: https://doi.org/10.1007/978-1-4939-1363-3_13
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