The publication of a new technique is like waving a red rag to a bull for many in the epigenetics arena. Now, “RedChIP” has us stampeding in anticipation towards the exploration of the complex interactions taking place between RNA, DNA, and proteins in the nucleus. Olé!
Non-coding RNAs (ncRNAs) interact with proteins and DNA to regulate gene expression and influence genome three-dimensional organization by, for example, targeting Polycomb repressive complexes or constructing CTCF-dependent chromatin loops; however, we don’t fully understand which RNA species take part. While RNA-DNA proximity ligation techniques map genome-wide ncRNA interactions, they don´t describe the proteins involved.
Bullish researchers led by Sergey V. Razin (Russian Academy of Sciences, Moscow, Russia) tried not to see red when faced with this perplexing situation; instead, they combined RNA-DNA proximity ligation (Red-C) with chromatin immunoprecipitation (ChIP) to create RedChIP, which identifies RNAs associated with a DNA-bound protein of interest.
As a parallel to HiChIP, which takes a protein-centric view of mapping DNA-DNA interactions, RedChIP evaluates RNA-DNA interactions occurring with a given protein. After crosslinking RNA-protein-DNA complexes in living cells, RNA and DNA fragments undergo in situ ligation using a bridge adapter; then, a specific antibody immunoprecipitates protein-associated RNA-DNA complexes. Finally, RNA-DNA sequencing reports on RNA-DNA interactions mediated by the protein and protein-DNA interactions mediated by RNAs.
Take the epigenetic bull by the horns and learn more about RedChIP from Gavrilov and colleagues:
- RedChIP identified cis– and trans-acting ncRNAs enriched at binding sites for EZH2 (catalytic subunit of the PRC2 complex) in human embryonic stem cells and CTCF in human myelogenous leukemia (K562) cells
- Cis-acting ncRNAs at EZH2 binding sites include Kcnq1ot1 (a long ncRNA [lncRNA] involved in Polycomb-mediated silencing) and antisense lncRNAs from the HOXC locus
- Trans-acting RNAs at EZH2 binding sites include the lncRNA KIF5C-AS1, the small nuclear (sn)RNA RNU5B-1, and the small nucleolar RNA SNORD3a, which represent candidate mediators of global Polycomb activity
- Cis-acting ncRNAs at CTCF binding sites include seven lncRNAs that may aid CTCF loading or the organization of chromatin loops (such as promoter-enhancer loops) within lncRNA-associated genomic loci
- Trans-acting ncRNAs at CTCF binding sites include the snRNA RNU12, which represents over 1% of all RNA contacts in K562 cells (the second most of all RNAs)
- RedChIP-enriched RNAs significantly intersect with RNAs identified from more traditional formaldehyde RNA immunoprecipitation-sequencing experiments (18/22), providing evidence for the robustness of RedChIP
Overall, the authors see RedChIP as a straightforward and accurate means of identifying RNAs associated with genomic regions occupied by a protein of interest. Indeed, the identification of Kcnq1ot1, which targets Polycomb complexes to repressed genomic domains, and the significant overlap of ncRNAs isolated via a conventional technique suggest that RedChIP represents the way forward for studies of RNA-protein-DNA interactions in live cells.
Charge on over to PNAS, January 2022 for more!