Once in a while, an epigenetic technique comes along to rock your world more than your favorite Led Zeppelin or Foo Fighters album! Now, a band of researchers fronted by the dynamic duo of John L. Rinn and Thomas R. Cech (University of Colorado, Boulder, CO, USA) has just released a chart-topping masterpiece with their RNA-dependent ChIP-deep sequencing (rChIP-seq) protocol. For an encore, the team employed rChIP-seq to establish that Polycomb repressive complex 2 (PRC2), which lays down the H3K27me3 mark of facultative heterochromatin and controls stem cell differentiation, fundamentally requires RNA binding for chromatin localization in human pluripotent stem cells (hPSCs) and the definition of cellular state.
Although sonically-charged studies have highlighted the relative importance of histone modifications, DNA sequences, and non-histone DNA-binding proteins in the proper localization of PRC2 and the epigenetic repression of gene expression, the role of RNA in this relationship had remained controversial. However, PRC2 does interact with high abundance RNA motifs (G-quadruplexes and G-tracts), and a recent study also revealed that G-quadruplexes evict PRC2 from chromatin during gene activation.
Drum roll, please! Let’s hear the full details (all killer, no filler!) of how Long, Hwang, and Colleagues took advantage of rChIP-seq to highlight the critical contribution of RNA to PRC2 function in hPSCs:
- rChIP-seq couples deep sequencing with an adaptation of a previously developed RNA-dependent ChIP protocol to discriminate between RNA-dependent and RNA-independent chromatin localization
- rChIP-seq of hPSCs demonstrated that the genome-wide localization of the PRC2 components EZH2 and SUZ12 to chromatin significantly decreases following the addition of RNase A (to remove steady-state RNA) or triptolide (to deplete nascent RNA) at the immunoprecipitation step of the ChIP procedure
- Additional experiments with an EZH2 mutant that cannot bind RNA (but retains all other known functions) provided more evidence that the disruption of the PRC2–RNA interaction causes a loss of PRC2 occupancy and histone methyltransferase activity (for H3K27me3) on a subset of genes in hPSCs
- RNA-seq analysis of EZH2 mutant hPSCs suggests that the loss of the PRC2-RNA interaction prompts the premature expression of cardiac genes before the initiation of differentiation, which may cause defects in cardiac differentiation
- Directed differentiation of hPSCs confirmed that the altered epigenetic silencing of several hundred genes due to the failure of PRC2 to bind to RNA leads to a loss of normal cardiomyocyte differentiation
- This finding suggests an essential role for RNA in cell fate determination in hPSCs
The authors suggest that RNA forms a direct or indirect bridge (not a musical one!) between PRC2 and chromatin, perhaps keeping PRC2 in a temporarily inhibited state, but positioned in proximity to chromatin. The loss of the bridge leads to the loss of PRC2 occupancy at canonical targets and a possible gain at de novo targets – a mechanism that the authors claim can unite previous seemingly contradictory observations (See the original study for an exciting discussion!).
“We were able to use data science approaches and high-powered computing to understand molecular patterns and evaluate RNA’s role in a novel, quantitative way,” said co-first-author Hwang. “RNA seemed to be playing the role of air traffic controller, guiding the plane – or protein complex – to the right spot on the DNA to land and silence genes,” said co-first-author Long, who continues “We can now say, unequivocally, that RNA is critical in this process of cell differentiation.” “These findings will set a new scientific stage showing an inextricable link between epigenetics and RNA biology,” said co-senior-author Rinn. “They could have broad implications for understanding, and addressing, human disease going forward.”
For more on rCHiP-seq and how RNA plays a central role in the rock and roll lifestyle of PRC2, guitar lick and drum roll your way over to Nature Genetics, July 2020.