Combined 3C-ChIP-Cloning (6C) Assay: A Tool to Unravel Protein-Mediated Genome Architecture

INTRODUCTION

Progress in technologies to address long-range chromosomal interactions in vivo has extensively revised concepts about different aspects of transcriptional regulation. These methods allow probing physical proximities between chromatin elements without specifically identifying the protein components that mediate such interactions. Here we describe a detailed protocol for Combined 3C-ChIP-Cloning (6C) technology, which combines multiple techniques to identify the proteins that bridge distant genomic regions, while simultaneously identifying such physical proximities. This method is also useful for determining if a candidate protein might mediate long-range interactions, both in cis and in trans in the nucleus. We discuss how the 6C technique can be incorporated with other techniques to discover all the chromatin regions in the nucleus that interact with a given gene or chromatin region of interest in a specific protein-dependent manner. Such information allows complete, cell-type-specific mapping of all the chromatin interactions mediated by specific proteins. The 6C assay advances our understanding of the three-dimensional aspects of the higher-order folding of chromatin and provides an important tool to examine the role of specific proteins in nuclear organization. In addition to providing a detailed protocol of the 6C technique, we discuss how this technology can be used by investigators working in the area of chromatin biology, with special interest in chromatin long-range interactions.