RedChIP identifies noncoding RNAs associated with genomic sites occupied by Polycomb and CTCF proteins

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.

Keywords: CTCF; Polycomb; RNA–DNA interactome; cell nucleus; noncoding RNA.

Fig. 1.

RedChIP technique. (A) Outline of the experimental procedure. (B) A region of Chr17 encompassing Hoxb genes showing distribution of DNA and RNA portions in IP (RedChIP) and input (RedC) fractions from experiments with EZH2 and CTCF antibodies. Shown alongside are ChIP-seq peaks of EZH2 in H1-hESCs (human embryonic stem cells) and ChIP-seq peaks of CTCF in K562 cells as well as total RNA-seq profiles for H1-hESCs and K562 cells (from ENCODE). (C) Distribution of DNA portions around EZH2 peaks in hESCs and CTCF peaks in K562. (D) Ratio of the number of RNA contacts detected in different chromatin types in IP fraction to the number of RNA contacts detected in the same chromatin types in input fraction.

Fig. 2.

Identification of ncRNAs associated with genomic regions occupied by EZH2 in hESCs and by CTCF in K562 cells. (A) Ratio of the number of cis contacts of individual RNAs between EZH2-precipitated and input fractions (x axis) vs. the percentage of cis contacts detected in different chromatin types in EZH2-precipitated fraction (y axis). (B) The same as A for CTCF-precipitated fraction. (C and D) Ratio of the number of cis (C) or trans (D) contacts of individual RNAs between EZH2-precipitated and input fractions for rep1 and rep2. (E and F) Ratio of the number of cis (E) or trans (F) contacts of individual RNAs between CTCF-precipitated and input fractions for rep1 and rep2. (G) Distribution of fold changes from C for different RNA biotypes (antisense, n = 44; linc, n = 162; protein coding, n = 4,184). *P < 0.05, ***P < 0.001. (H) Intersection of RNAs enriched in RedChIP and fRIP-seq.