As the saying goes, the shortest path from A to B is a straight line and if you’re studying DNA demethylation, you know just how complicated things can get along the road from methylated to unmethylated cytosines. Mammals use Ten-Eleven Translocation proteins (TET) to oxidize 5mC, but the process creates pit stops that may be independent epigenetic states of their own: 5-hydroxy-, -formyl- and –carboxycytosine (5hmC, 5fC, 5caC). Plants, on the other hand, have evolved a more direct route to demethylation with the DNA glycosylase REPRESSOR OF SILENCING 1 (ROS1), which directly targets 5mC for base excision repair (BER). New research is taking advantage of this epigenetic beeline, and the result is a more direct targeted demethylation tool.
The talented team from the lab of Teresa Roldán-Arjona (University of Cόrdoba, Spain) fused a deactivated Cas9 (dCAs9) protein to the active domain of ROS1 (dCas9-ROS1) and transfected it, along with a methylated reported construct, into HEK293 cells and found that:
- dCas9-ROS1 is able to reactivate the expression
of a methylation-silenced luciferase gene in a density dependent manner
- However, it is unable to reactivate a plasmid that’s 100% methylated
- In contrast, dCas9 fused to the active domain of TET1 (dCas9-TET1) fails to reactivate luciferase under any level of methylation
- dCas9-ROS1 also reactivates a
methylation-silenced GFP construct, but is less efficient than traditional
dCas9-p300 or dCas9-VP160 activators
- All three constructs are unable to re-activate GFP constructs that are 90% methylated
- Co-transfecting dCas9-ROS1 with other constructs actually decreases luciferase activity compared to transfecting each construct individually
- Co-transfecting dCas9-ROS1 with additional, targeted downstream BER factors does not improve its ability to re-activate a methylated luciferase construct
- Active dCas9-ROS1 transfection decreases methylation across multiple CpGs in and near the promoter of the luciferase expression construct
Interestingly, dCas9-ROS1 also increases the expression of an unmethylated luciferase construct, which means that even newly transfected constructs are subject to de novo methylation, or that dCas9-ROS1 may have activator activities outside its role as a demethylase. Either way, you’ll be happy to have this technique ride shotgun on your next mammalian demethylation experiment!
Head directly to the original article in the Journal of Molecular Biology, February 2020.