Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Dynamic regulation of Z-DNA in the mouse prefrontal cortex by the RNA-editing enzyme Adar1 is required for fear extinction

Nature Neuroscience volume 23, pages 718–729 (2020)Cite this article

Subjects

A Publisher Correction to this article was published on 26 June 2020

This article has been updated

Abstract

DNA forms conformational states beyond the right-handed double helix; however, the functional relevance of these noncanonical structures in the brain remains unknown. Here we show that, in the prefrontal cortex of mice, the formation of one such structure, Z-DNA, is involved in the regulation of extinction memory. Z-DNA is formed during fear learning and reduced during extinction learning, which is mediated, in part, by a direct interaction between Z-DNA and the RNA-editing enzyme Adar1. Adar1 binds to Z-DNA during fear extinction learning, which leads to a reduction in Z-DNA at sites where Adar1 is recruited. Knockdown of Adar1 leads to an inability to modify a previously acquired fear memory and blocks activity-dependent changes in DNA structure and RNA state—effects that are fully rescued by the introduction of full-length Adar1. These findings suggest a new mechanism of learning-induced gene regulation that is dependent on proteins that recognize alternate DNA structure states, which are required for memory flexibility.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Adar1 binds to DNA and targets DNA repetitive elements in response to behavioral training.
Fig. 2: Adar1 knockdown impairs memory updating after extinction training.
Fig. 3: Z-DNA is a critical modulator of activity-dependent transcription.
Fig. 4: Adar1–Z-DNA interaction at the Nrxn3 locus modulates the expression of transcripts derived from a noncoding region enriched with SINEs.
Fig. 5: RNA editing enriched at SINEs and LINEs in activated neurons.
Fig. 6: Editing of RNA derived from SINEs and LINEs requires Adar1 binding to DNA.
Fig. 7: Fear extinction requires both Adar1 Z-DNA-binding and RNA-editing domains.

Similar content being viewed by others

Data availability

All data and code used to generate the data are available upon reasonable request to the authors.

Change history

References

  1. Schwartz, T., Rould, M. A., Lowenhaupt, K., Herbert, A. & Rich, A. Crystal structure of the Zα domain of the human editing enzyme ADAR1 bound to left-handed Z-DNA. Science 284, 1841–1845 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Herbert, A. et al. The Zα domain from human ADAR1 binds to the Z-DNA conformer of many different sequences. Nucleic Acids Res. 26, 3486–3493 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Crick, F. H. C. & Watson, J. D. The complementary structure of deoxyribonucleic acid. Proc. R. Soc. A Math. Phys. Eng. Sci. 223, 80–96 (1954).

    CAS  Google Scholar 

  4. Franklin, R. E. & Gosling, R. G. The structure of sodium thymonucleate fibres. I. The influence of water content. Acta Crystallogr. 6, 673–677 (1953).

    Article  CAS  Google Scholar 

  5. Wang, A. H. J. et al. Molecular structure of a left-handed double helical DNA fragment at atomic resolution. Nature 282, 680–686 (1979).

    Article  CAS  PubMed  Google Scholar 

  6. Pohl, F. M. Hysteretic behaviour of a Z-DNA-antibody complex. Biophys. Chem. 26, 385–390 (1987).

    Article  CAS  PubMed  Google Scholar 

  7. Herbert, A. A genetic instruction code based on DNA conformation. Trends Genet. 35, 887–890 (2019).

    Article  CAS  PubMed  Google Scholar 

  8. Haniford, D. B. & Pulleyblank, D. E. Facile transition of poly[d(TG)·d(CA)] into a left-handed helix in physiological conditions. Nature 302, 632–634 (1983).

    Article  CAS  PubMed  Google Scholar 

  9. Dobi, A. & Agoston, D. V. Submillimolar levels of calcium regulates DNA structure at the dinucleotide repeat (TG/AC)n. Chem. Biochem. 95, 5981–5986 (1998).

    CAS  Google Scholar 

  10. Behe, M. & Felsenfeld, G. Effects of methylation on a synthetic polynucleotide: the B–Z transition in poly(dG-m5dC).poly(dG-m5dC). Proc. Natl Acad. Sci. USA 78, 1619–1623 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Marshall, P. & Bredy, T. W. Cognitive neuroepigenetics: the next evolution in our understanding of the molecular mechanisms underlying learning and memory? NPJ Sci. Learn. 1, 16014 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  12. McGaugh, J. L. Memory–a century of consolidation. Science 287, 248–251 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Schade, M. et al. The solution structure of the Zα domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA. Proc. Natl Acad. Sci. USA 96, 12465–12470 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lee, Y. M. et al. NMR investigation on the DNA binding and B–Z transition pathway of the Zα domain of human ADAR1. Biophys. Chem. 172, 18–25 (2013).

    Article  CAS  PubMed  Google Scholar 

  15. Lee, A.-R. et al. NMR dynamics study reveals the Zα domain of human ADAR1 associates with and dissociates from Z-RNA more slowly than Z-DNA. ACS Chem. Biol. 14, 245–255 (2019).

    Article  CAS  PubMed  Google Scholar 

  16. Rudenko, A. et al. Tet1 is critical for neuronal activity-regulated gene expression and memory extinction. Neuron 79, 1109–1122 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Marshall, P. R. & Bredy, T. W. Neuroepigenetic mechanisms underlying fear extinction: emerging concepts. Psychopharmacology 236, 133–142 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Li, X. et al. The DNA modification N6-methyl-2′-deoxyadenosine (m6dA) drives activity-induced gene expression and is required for fear extinction. Nat. Neurosci. 22, 534–544 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nie, Y., Zhao, Q., Su, Y. & Yang, J. H. Subcellular distribution of AdAR1 isoforms is synergistically determined by three nuclear discrimination signals and a regulatory motif. J. Biol. Chem. 279, 13249–13255 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Keum, S. et al. A missense variant at the Nrxn3 locus enhances empathy fear in the mouse. Neuron 98, 588–601 (2018).

    Article  CAS  PubMed  Google Scholar 

  21. Thomas, T. J., Gunnia, U. B. & Thomas, T. Polyamine-induced B-DNA to Z-DNA conformational transition of a plasmid DNA with (dG-dC)(n) insert. J. Biol. Chem. 266, 6137–6141 (1991).

    Article  CAS  PubMed  Google Scholar 

  22. Van Helden, P. D. The effect of adriamycin on Z-DNA formation and DNA synthesis. Nucleic Acids Res. 11, 8415–8420 (1983).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Dieci, G., Fiorino, G., Castelnuovo, M., Teichmann, M. & Pagano, A. The expanding RNA polymerase III transcriptome. Trends Genet. 23, 614–622 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Champ, P. C., Maurice, S., Vargason, J. M., Camp, T. & Ho, P. S. Distributions of Z-DNA and nuclear factor I in human chromosome 22: a model for coupled transcriptional regulation. Nucleic Acids Res. 32, 6501–6510 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Naylor, L. H. & Clark, E. M. D(TG)n·d(CA)n sequences upstream of the rat prolactin gene form z-DNA and inhibit gene transcription. Nucleic Acids Res. 18, 1595–1601 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Rich, A. & Zhang, S. Z-DNA: the long road to biological function. Nat. Rev. Genet. 4, 566–572 (2003).

    Article  CAS  PubMed  Google Scholar 

  27. Shin, S. I. et al. Z-DNA-forming sites identified by ChIP-Seq are associated with actively transcribed regions in the human genome. DNA Res. 23, 477–486 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Wang, G. & Vasquez, K. M. Non-B DNA structure-induced genetic instability. Mutat. Res. - Fundam. Mol. Mech. Mutagen. 598, 103–119 (2006).

    Article  CAS  Google Scholar 

  29. Harteis, S. & Schneider, S. Making the bend: DNA tertiary structure and protein–DNA interactions. Int. J. Mol. Sci. 15, 12335–12363 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Rohs, R. et al. The role of DNA shape in protein-DNA recognition. Nature 461, 1248–1253 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Berber, I. et al. Spectroscopic characterization of a DNA-binding domain, Zα, from the editing enzyme, dsRNA adenosine deaminase: evidence for left-handed Z-DNA in the Zα-DNA complex. Biochemistry 37, 13313–13321 (1998).

    Article  Google Scholar 

  32. Bae, S., Kim, D., Kim, K. K., Kim, Y. G. & Hohng, S. Intrinsic Z-DNA is stabilized by the conformational selection mechanism of Z-DNA-binding proteins. J. Am. Chem. Soc. 133, 668–671 (2011).

    Article  CAS  PubMed  Google Scholar 

  33. Bae, S. et al. Energetics of Z-DNA binding protein-mediated helicity reversals in DNA, RNA, and DNA–RNA duplexes. J. Phys. Chem. B 117, 13866–13871 (2013).

    Article  CAS  PubMed  Google Scholar 

  34. Antrup, H. & Seiler, N. On the turnover of polyamines spermidine and spermine in mouse brain and other organs. Neurochem. Res. 5, 123–143 (1980).

    Article  CAS  PubMed  Google Scholar 

  35. Paleček, E. Local supercoil-stabilized DNA structure. Crit. Rev. Biochem. Mol. Biol. 26, 151–226 (1991).

    Article  PubMed  Google Scholar 

  36. Rahmouni, A. R. & Wells, R. D. Stabilization of Z DNA in vivo by localized supercoiling. Science 246, 358–363 (1989).

    Article  CAS  PubMed  Google Scholar 

  37. Madabhushi, R. et al. Activity-induced DNA breaks govern the expression of neuronal early-response genes. Cell 161, 1592–1605 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Li, X. et al. The DNA repair-associated protein Gadd45ɣ regulates the temporal coding of immediate early gene expression within the prelimbic prefrontal cortex and is required for the consolidation of associative fear memory. J. Neurosci. 39, 970–983 (2018).

    Article  PubMed  Google Scholar 

  39. Wang, G., Christensen, L. A. & Vasquez, K. M. Z-DNA-forming sequences generate large-scale deletions in mammalian cells. PNAS 103, 2677–2682 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ditlevson, J. V. et al. Inhibitory effect of a short Z-DNA forming sequence on transcription elongation by T7 RNA polymerase. Nucleic Acids Res. 36, 3163–3170 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Prigogine, I. Time, structure, and fluctuations. Science 201, 777–785 (1978).

    Article  CAS  PubMed  Google Scholar 

  42. Lu, J. Y. et al. L1 and B1 repeats blueprint the spatial organization of chromatin. Preprint at bioRxiv https://doi.org/10.1101/802173 (2019).

  43. Oh, D.-B., Kim, Y.-G. & Rich, A. Z-DNA-binding proteins can act as potent effectors of gene expression in vivo. Proc. Natl Acad. Sci. USA 99, 16666–16671 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Rothenburg, S. et al. A PKR-like eukaryotic initiation factor 2 kinase from zebrafish contains Z-DNA binding domains instead of dsRNA binding domains. Proc. Natl Acad. Sci. USA 102, 1602–1607 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wölfl, S., Martinez, C., Rich, A. & Majzoub, J. A. Transcription of the human corticotropin-releasing hormone gene in NPLC cells is correlated with Z-DNA formation. Proc. Natl Acad. Sci. USA 93, 3664–3668 (1996).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Kang, H. J. et al. Novel interaction of the Z-DNA binding domain of human ADAR1 with the oncogenic c-myc promoter G-quadruplex. J. Mol. Biol. 426, 2594–2604 (2014).

    Article  CAS  PubMed  Google Scholar 

  47. Liddicoat, B. J. et al. RNA editing by ADAR1 prevents MDA5 sensing of endogenous dsRNA as nonself. Science 349, 1115–1120 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Lin, Q. et al. The brain-specific microRNA miR-128b regulates the formation of fear-extinction memory. Nat. Neurosci. 14, 1115–1117 (2011).

    Article  CAS  PubMed  Google Scholar 

  49. O’Neill, L. P., VerMilyea, M. D. & Turner, B. M. Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations. Nat. Genet. 38, 835–841 (2006).

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors gratefully acknowledge grant support from the National Institutes of Health (R01MH105398-TWB) and the National Health and Medical Research Council (GNT1145172 and GNT1160823-TWB), the ARC (GNT190101078-XL), the Westpac Future Scholars program (E.L.Z., L.J.L. and S.U.M.) and postgraduate scholarships from the Natural Sciences and Engineering Research Council (P.R.M.) and the University of Queensland (P.R.M., E.L.Z., L.J.L., D.B., J.Y. and S.U.M.). We would also like to thank R. Tweedale for helpful editing of the manuscript.

Author information

Authors and Affiliations

  1. Cognitive Neuroepigenetics Laboratory, Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia

    Paul R. Marshall, Qiongyi Zhao, Xiang Li, Wei Wei, Ambika Periyakaruppiah, Esmi L. Zajaczkowski, Laura J. Leighton, Sachithrani U. Madugalle, Dean Basic, Ziqi Wang, Jiayu Yin, Wei-Siang Liau & Timothy W. Bredy

  2. Cancer and RNA Biology Laboratory, St Vincent’s Institute; Department of Medicine, St. Vincent’s Hospital, The University of Melbourne; Mary MacKillop Institute for Health Research, Australian Catholic University, Fitzroy, VIC, Australia

    Ankita Gupte & Carl R. Walkley

Authors
  1. Paul R. Marshall
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qiongyi Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wei Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ambika Periyakaruppiah
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Esmi L. Zajaczkowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Laura J. Leighton
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sachithrani U. Madugalle
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Dean Basic
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ziqi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jiayu Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Wei-Siang Liau
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Ankita Gupte
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Carl R. Walkley
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Timothy W. Bredy
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.R.M designed and performed all wet lab and behavioral experiments and wrote the manuscript with T.W.B. Q.Z. performed all the bioinformatics analysis. X.L. and W.W. assisted in experimental design and ChIP-seq experiments. A.P. assisted in the production of lentivirus. E.Z, L.J.L. and S.U.M. assisted with behavioral experiments. D.B. assisted with bioinformatic analysis. Z.W., J.Y. and W.S.L. assisted with western blots and qPCR. A.G. assisted in producing and validating the ADAR1 mutant constructs. C.W. contributed reagents and helped write the manuscript. T.W.B. conceived the study, designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Timothy W. Bredy.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Neuroscience thanks Alan Herbert, Larry Zweifel and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Data from this paper can be accessed at PRJNA545193.

Supplementary information

Supplemental Information

Supplementary Figs. 1–9 and Supplementary Tables 1–3.

Reporting Summary

.Supplementary Table

Supplementary Table 1. Genome-wide ADAR1 significant peaks in Excel file.

Source data

Source Data Fig. 1

Unprocessed western blots for Fig. 1

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Marshall, P.R., Zhao, Q., Li, X. et al. Dynamic regulation of Z-DNA in the mouse prefrontal cortex by the RNA-editing enzyme Adar1 is required for fear extinction. Nat Neurosci 23, 718–729 (2020). https://doi.org/10.1038/s41593-020-0627-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41593-020-0627-5

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing