Abstract
Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNA–DNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNA–chromatin interactions. Here, we introduce RedChIP, a technique combining RNA–DNA proximity ligation and chromatin immunoprecipitation for identifying RNA–chromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis- and trans-acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNA–DNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs.
Highlights
Nuclear noncoding RNAs are key regulators of gene expression and chromatin organization
To identify RNAs that could be involved in the functioning of DNA-bound proteins, we developed a hybrid approach— RedChIP—combining an RNA–DNA proximity ligation technique [Red-C [5]] with chromatin immunoprecipitation (ChIP)
The RedChIP experimental procedure is analogous to HiChIP used for protein-centric mapping of DNA–DNA interactions [6], with the difference being that RNA–DNA interactions are analyzed instead of DNA–DNA interactions
Summary
Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. Noncoding RNA j cell nucleus j RNA–DNA interactome j CTCF j Polycomb Recent studies indicate that RNA is essential for the chromatin targeting of Polycomb repressive complexes [3] and the organization of CTCF-dependent chromatin loops [4].
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