Freshly brewed coffee, a lightly toasted bagel, the sweetness of orange juice; what makes you feel “chirpy” first thing in the morning? The good people from the labs of William J. Greenleaf and Howard Y. Chang (Stanford University, CA, USA) have been waking up, working hard, and feeling good, all thanks to the development of a new means to uncover how long non-coding RNAs (lncRNAs) act as molecular scaffolds through the identification of genome-wide RNA-associated chromatin contacts: a protocol they call HiChIRP!
HiChIRP swaps the chromatin immunoprecipitation in the HiChIP protocol for chromatin isolation by RNA purification (ChIRP) enrichment. As ChIRP requires RNA capture by biotinylated probes, the protocol incorporates an azido-modified nucleotide into chromatin contacts during chromosome conformation capture (3C). Subsequently, CLICK chemistry covalently conjugates azido-containing chromatin contacts to biotin for subsequent contact enrichment following RNA enrichment with biotinylated probes. Additionally, the exhaustive optimization of the 3C protocol while generating chromatin contact libraries preserves RNA integrity and maintains long-range ligation efficiency.
HiChIRP enables the interrogation of the scaffolding function of lncRNAs present down to around ten copies per cell, and so the team next applied HiChIRP to various lncRNAs and compared their results to data generated through complementary methodologies
- The 7SK small nuclear RNA, a high copy number
lncRNA, promotes transcriptional pause release by regulating chromatin
- Analyses in mouse embryonic stem cells revealed that 7SK-enriched chromatin loops span short distances and associate with active regulatory elements
- These findings confirm known functions of 7SK at promoters and the concept of shorter gene regulatory loops within topological domains
- The TERC telomerase
RNA component, a medium copy-number lncRNA, represents one of the critical
components of the human telomerase holoenzyme
- Most TERC reads occur at telomeric and subtelomeric regions, as expected
- However, analyses also implicate TERC in translocations at the IGH immunoglobulin heavy locus in a neoplastic B cell line, agreeing with reports that chromosome conformation can predict certain classes of translocations, while also hinting at a role for TERC in the regulation of oncogene expression
- lincRNA-EPS, an extremely low copy number
lncRNA (specifically, a long intergenic ncRNA), regulates inflammatory gene
- Analysis of bone marrow-derived macrophages suggests that lincRNA-EPS encourages chromatin interactions between topologically associated domain boundaries and the promoters of immune response genes to mediate immune-related gene repression
Overall, the authors highlight their upbeat and ever-ready new protocol as an accurate and efficient means to study the role of lncRNAs in the structural organization of the genome.
Start that day off feeling a little more “chirpy” by reading all the details of this tasty new protocol over at Nature Methods, May 2019.