Z-DNA occurs when the DNA molecule forms a left-handed double helix rather than the right-handed structure that we’re used to. This inside-out conformation is enriched at actively transcribed genes and enhancers, where it allows chromatin states to be epigenetically altered in a way that extends beyond the information contained in the DNA sequence. For example, the RNA editing enzyme, ADAR1, is known to bind to Z-DNA and a new study from the lab of Timothy Bredy (University of Queensland, Australia) suggests that they both add to the mix of molecules needed for fear memory and extinction.
The talented team used mice that went through fear extinction training, which involves learning that a stimulus is no longer associated with a foot shock, even though it used to be, to find that:
- Adar1 mRNA is transiently increased in the prefrontal cortex (PFC) after training, especially in neurons that are separated by fluorescence activated cell sorting (FACS) based on Arc, a marker of recent activity
- The ADAR1 protein isoform that localizes to the nucleus is particularly increased after fear extinction training, as measured by Western Blot
- Extinction training increases ADAR1 binding to 122 genomic loci in active neurons, when compared to inactive neurons, as measured by ChIP-seq
- Knocking down ADAR1 with a short hairpin RNA (shRNA) prevents mice from extinguishing fear memories, but has no impact on their ability to consolidate the original memory, or simply forget it over time
Next, the savvy scientists used doxorubicin to block the formation of Z-DNA and found that:
- Blocking Z-DNA reduces Adar1 binding in primary cortical neurons
- Normally, Z-DNA levels increase in the PFC immediately after fear conditioning, as measured by anti-Z-DNA ChIP-qPCR, and then decrease before Adar1 is induced by fear extinction
- Blocking Z-DNA formation in the PFC before training has no effect on acquiring or consolidating fear memories, but it prevents fear extinction
- At target sites, Z-DNA levels increase during fear learning, when ADAR1 binding is low, and decrease during extinction, when ADAR1 binding is high
Since ADAR1 is an RNA editing enzyme, the intelligent investigators looked for instances of RNA editing in their previous data, that overlapped with ADAR1 binding sites and found that:
- Transcripts expressed by active neurons are enriched for ADAR1 binding and RNA editing
- Edited transcripts are enriched for repetitive elements, like LINES and SINES
- Transcripts that are edited during fear learning are largely distinct from those that are edited during extinction
- Adar1 knockdown prevents RNA editing at sites that normally gain ADAR1 during fear extinction
Importantly, there are no substitutions in this recipe that can replace a fully functioning ADAR1 protein; both the RNA editing, and Z-DNA binding domains are necessary for proper fear extinction.
First author Paul Marshall shares, “It seems that the more easily you can switch between DNA structures, the more plastic your memory is. Flexibility of DNA structure, flexibility of memory. This enables an agile response to our environment. Fear memories need to be plastic. They can be very useful for survival, but they can also get in the way of normal functioning.”
Senior author Timothy Bredy concludes, “The balance between fear and fear-extinction is critical to cognitive flexibility. Indeed, the impairment of fear extinction is a key feature of PTSD and phobias. The more we understand about how fear extinction works, the more chance we have of finding better treatments for those conditions.”
Check out the structural complexities of fear extinction from A to Z in Nature Neuroscience, May 2020.