Mapping Networks of Protein‐Mediated Physical Interactions Between Chromatin Elements

Understanding of transcriptional regulation has advanced in recent years in part due to development of technologies which allow determination of physical proximities between interacting chromatin regions at a resolution beyond that offered by conventional microscopy techniques. However, these methods do not specifically identify the protein component(s) that might mediate such interactions. This unit provides a detailed protocol for Combined 3C‐ChIP‐Cloning (6C) technology, which combines multiple techniques to simultaneously identify physical proximities between chromatin elements as well as the proteins that mediate these interactions. The unit further explores how the 6C assay can be combined with other techniques for a complete, cell‐type‐specific mapping of all inter‐ and intrachromosomal interactions mediated by specific proteins. Thus, the 6C assay provides a useful tool to address the role of specific proteins in nuclear organization and to advance our understanding about the relation of chromatin higher‐order organization and transcriptional regulation. Curr. Protoc. Mol. Biol . 89:21.16.1‐21.16.13. © 2010 by John Wiley & Sons, Inc.