Epigenetic regulatory cascades can be complex affairs…to say the least. Thankfully, a recent chromatin effector coregulator-based study now keeps us firmly in the loop by revealing how a histone demethylase induces gene repression by triggering a regulatory cascade that removes RNA:DNA hybrid structures known as R-loops!
A talented team led by Philipp Rathert (University of Stuttgart, Germany) sought to explore the co-regulatory proteins that modulate lysine-specific demethylase 1 (LSD1, or KDM1A) function in order to decipher its complex role in gene transcription and cancer development. The authors combined a chromatin in vivo assay system, which profiles the consequences of epigenetic effector recruitment using a time-resolved fluorescent reporter system, with a microRNA-embedded short hairpin RNA library focused on epigenetic effectors to perform a chromatin effector co-regulator screen (or ChECS) in living cells.
Let’s get up-to-date with the findings of Pinter and colleagues and read up on a novel regulatory cascade involving LSD1 and the removal of R-loops!
- Three ATP-dependent RNA helicases lay among the top ten co-regulators of LSD1 found in mouse cells
- Only DEAD-box helicase 19A (DDX19A) positively regulates LSD1-mediated silencing and represents the screen’s top-hit
- While DDX19A and LSD1 seem not to interact directly, Ddx19a downregulation induces R-loop formation and inhibits LSD1-mediated gene silencing
- Subsequent detailed analysis using immunofluorescence microscopy, DNA-RNA immunoprecipitation, ChIP-sequencing, R-loop immunoprecipitation, histone modification analysis, and epigenetic inhibitor treatment (among others) reveals a novel transcriptional regulatory cascade
- LSD1-mediated demethylation of H3K4me2 allows the polycomb repressive complex 2 (PRC2) to methylate H3K27me3
- DDX19A binds to H3K27me3 through a currently unknown motif and removes R-loops at the active gene promoters
- The subsequent derepression of LSD1 and PRC2 establishes a positive feedback loop that supports gene repression
Overall, this exciting new LSD1 study gets us firmly back in the loop about the regulatory roles of R-loops. Senior author Philipp Rathert concludes, “We are very excited about the publication due to several reasons. First, we were able to probe the regulatory network of LSD1 using a hypothesis-free approach for the first time. Second, using this approach, we could identify a novel transcriptional regulatory cascade where the removal of R-loops is required to induce a robust transcriptional shutdown downstream of LSD1 activity, which does show that R-loops have a direct regulatory effect on transcription. Finally, the finding that DDX19A has a so-far undisclosed H3K27me3 binding site took us by surprise and could open up a completely new line of research in the helicase field since it suggests that many other chromatin-associated DEAD/DEAH-box helicases might be recruited or regulated in a similar way.”
Stay in the loop by finding out how removing R-loops regulates repression at Nucleic Acids Research, April 2021.