Abstract

DNA modification is known to regulate experience-dependent gene expression. However, beyond cytosine methylation and its oxidated derivatives, very little is known about the functional importance of chemical modifications on other nucleobases in the brain. Here we report that in adult mice trained in fear extinction, the DNA modification N6-methyl-2’-deoxyadenosine (m6dA) accumulates along promoters and coding sequences in activated prefrontal cortical neurons. The deposition of m6dA is associated with increased genome-wide occupancy of the mammalian m6dA methyltransferase, N6amt1, and this correlates with extinction-induced gene expression. The accumulation of m6dA is associated with transcriptional activation at the brain-derived neurotrophic factor (Bdnf) P4 promoter, which is required for Bdnf exon IV messenger RNA expression and for the extinction of conditioned fear. These results expand the scope of DNA modifications in the adult brain and highlight changes in m6dA as an epigenetic mechanism associated with activity-induced gene expression and the formation of fear extinction memory.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

All sequencing raw fastq files have been deposited at the Sequence Read Archive (accession SRP110529) and BioProject (accession PRJNA391201). All customized code is free and accessible at Github for download at https://github.com/Qiongyi/2018_DPNI-Seq_study.

Additional information

Journal peer review information Nature Neuroscience thanks Ian Maze and 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.

References

  1. 1.

    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).

  2. 2.

    Li, X. et al. Neocortical Tet3-mediated accumulation of 5-hydroxymethylcytosine promotes rapid behavioral adaptation. Proc. Natl Acad. Sci. USA 111, 7120–7125 (2014).

  3. 3.

    Wei, W. et al. p300/CBP-associated factor selectively regulates the extinction of conditioned fear. J. Neurosci. 32, 11930–11941 (2012).

  4. 4.

    Miller, C. A., Campbell, S. L. & Sweatt, J. D. DNA methylation and histone acetylation work in concert to regulate memory formation and synaptic plasticity. Neurobiol. Learn. Mem. 89, 599–603 (2008).

  5. 5.

    Baker-Andresen, D., Ratnu, V. S. & Bredy, T. W. Dynamic DNA methylation: a prime candidate for genomic metaplasticity and behavioral adaptation. Trends Neurosci. 36, 3–13 (2013).

  6. 6.

    Gapp, K., Woldemichael, B. T., Bohacek, J. & Mansuy, I. M. Epigenetic regulation in neurodevelopment and neurodegenerative diseases. Neuroscience 264, 99–111 (2014).

  7. 7.

    Korlach, J. & Turner, S. W. Going beyond five bases in DNA sequencing. Curr. Opin. Struct. Biol. 22, 251–261 (2012).

  8. 8.

    Lister, R. et al. Global epigenomic reconfiguration during mammalian brain development. Science 341, 1237905 (2013).

  9. 9.

    Guo, J. U., Su, Y., Zhong, C., Ming, G.-L. & Song, H. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145, 423–434 (2011).

  10. 10.

    Shen, L. et al. Tet3 and DNA replication mediate demethylation of both the maternal and paternal genomes in mouse zygotes. Cell Stem Cell 15, 459–471 (2014).

  11. 11.

    Khare, T. et al. 5-hmC in the brain is abundant in synaptic genes and shows differences at the exon-intron boundary. Nat. Struct. Mol. Biol. 19, 1037–1043 (2012).

  12. 12.

    Miller, C. A. et al. Cortical DNA methylation maintains remote memory. Nat. Neurosci. 13, 664–666 (2010).

  13. 13.

    Vanyushin, B. F., Mazin, A. L., Vasilyev, V. K. & Belozersky, A. N. The content of 5-methylcytosine in animal DNA: the species and tissue specificity. Biochim. Biophys. Acta 299, 397–403 (1973).

  14. 14.

    Iyer, L. M., Zhang, D. & Aravind, L. Adenine methylation in eukaryotes: apprehending the complex evolutionary history and functional potential of an epigenetic modification. Bioessays 38, 27–40 (2016).

  15. 15.

    Hattman, S., Kenny, C., Berger, L. & Pratt, K. Comparative study of DNA methylation in three unicellular eucaryotes. J. Bacteriol. 135, 1156–1157 (1978).

  16. 16.

    Hattman, S. DNA-[adenine] methylation in lower eukaryotes. Biochemistry 70, 550–558 (2005).

  17. 17.

    Fu, Y. et al. N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas. Cell 161, 879–892 (2015).

  18. 18.

    Zhang, G. et al. N6-methyladenine DNA modification in Drosophila. Cell 161, 893–906 (2015).

  19. 19.

    Ma, C. et al. N6-methyldeoxyadenine is a transgenerational epigenetic signal for mitochondrial stress adaptation. Nat. Cell Biol. https://doi.org/10.1038/s41556-018-0238-5 (2018).

  20. 20.

    Yao, B. et al. DNA N6-methyladenine is dynamically regulated in the mouse brain following environmental stress. Nat. Commun. 8, 1122 (2017).

  21. 21.

    Usheva, A. & Shenk, T. TATA-binding protein-independent initiation: YY1, TFIIB, and RNA polymerase II direct basal transcription on supercoiled template DNA. Cell 76, 1115–1121 (1994).

  22. 22.

    Bredy, T. W. et al. Histone modifications around individual BDNF gene promoters in prefrontal cortex are associated with extinction of conditioned fear. Learn. Mem. 14, 268–276 (2007).

  23. 23.

    Liu, J. et al. Abundant DNA 6 mA methylation during early embryogenesis of zebrafish and pig. Nat. Commun. 7, 13052 (2016).

  24. 24.

    Wu, T. P. et al. DNA methylation on N(6)-adenine in mammalian embryonic stem cells. Nature 532, 329–333 (2016).

  25. 25.

    Ataman, B. et al. Evolution of Osteocrin as an activity-regulated factor in the primate brain. Nature 539, 242–247 (2016).

  26. 26.

    Xiao, C.-L. et al. N6-Methyladenine DNA Modification in the Human Genome. Mol. Cell 71, 306–318.e7 (2018).

  27. 27.

    Xie, Q. et al. N6-methyladenine DNA Modification in Glioblastoma. Cell 175, 1228–1243.e20 (2018).

  28. 28.

    Luo, G.-Z. et al. Characterization of eukaryotic DNA N(6)-methyladenine by a highly sensitive restriction enzyme-assisted sequencing. Nat. Commun. 7, 11301 (2016).

  29. 29.

    Vovis, G. F. & Lacks, S. Complementary action of restriction enzymes endo R-DpnI and Endo R-DpnII on bacteriophage f1 DNA. J. Mol. Biol. 115, 525–538 (1977).

  30. 30.

    Lacks, S. & Greenberg, B. A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA. J. Biol. Chem. 250, 4060–4066 (1975).

  31. 31.

    Birnboim, H. C., Sederoff, R. R. & Paterson, M. C. Distribution of polypyrimidine. Polypurine segments in DNA from diverse organisms. Eur. J. Biochem. 98, 301–307 (1979).

  32. 32.

    Manor, H., Rao, B. S. & Martin, R. G. Abundance and degree of dispersion of genomic d(GA)n.d(TC)n sequences. J. Mol. Evol. 27, 96–101 (1988).

  33. 33.

    Soeller, W. C., Poole, S. J. & Kornberg, T. In vitro transcription of the Drosophila engrailed gene. Genes Dev. 2, 68–81 (1988).

  34. 34.

    Biggin, M. D. & Tjian, R. Transcription factors that activate the Ultrabithorax promoter in developmentally staged extracts. Cell 53, 699–711 (1988).

  35. 35.

    Wallrath, L. L. & Elgin, S. C. Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev. 9, 1263–1277 (1995).

  36. 36.

    Stephens, C., Reisenauer, A., Wright, R. & Shapiro, L. A cell cycle-regulated bacterial DNA methyltransferase is essential for viability. Proc. Natl Acad. Sci. USA 93, 1210–1214 (1996).

  37. 37.

    Liu, P. et al. Deficiency in a glutamine-specific methyltransferase for release factor causes mouse embryonic lethality. Mol. Cell. Biol. 30, 4245–4253 (2010).

  38. 38.

    Ghosh, A., Carnahan, J. & Greenberg, M. E. Requirement for BDNF in activity-dependent survival of cortical neurons. Science 263, 1618–1623 (1994).

  39. 39.

    Peters, J., Kalivas, P. W. & Quirk, G. J. Extinction circuits for fear and addiction overlap in prefrontal cortex. Learn. Mem. 16, 279–288 (2009).

  40. 40.

    Lubin, F. D., Roth, T. L. & Sweatt, J. D. Epigenetic regulation of BDNF gene transcription in the consolidation of fear memory. J. Neurosci. 28, 10576–10586 (2008).

  41. 41.

    West, A. E. Biological functions of activity-dependent transcription revealed. Neuron 60, 523–525 (2008).

  42. 42.

    Sakata, K. et al. Role of activity-dependent BDNF expression in hippocampal-prefrontal cortical regulation of behavioral perseverance. Proc. Natl Acad. Sci. USA 110, 15103–15108 (2013).

  43. 43.

    Simon, J. M., Giresi, P. G., Davis, I. J. & Lieb, J. D. Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA. Nat. Protoc. 7, 256–267 (2012).

  44. 44.

    Ratel, D., Ravanat, J.-L., Berger, F. & Wion, D. N6-methyladenine: the other methylated base of DNA. Bioessays 28, 309–315 (2006).

  45. 45.

    Low, D. A., Weyand, N. J. & Mahan, M. J. Roles of DNA adenine methylation in regulating bacterial gene expression and virulence. Infect. Immun. 69, 7197–7204 (2001).

  46. 46.

    Wang, Y., Chen, X., Sheng, Y., Liu, Y. & Gao, S. N6-adenine DNA methylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol II-transcribed genes in Tetrahymena. Nucleic Acids Res. 45, 11594–11606 (2017).

  47. 47.

    Kigar, S. L. et al. N6-methyladenine is an epigenetic marker of mammalian early life stress. Sci. Rep. 7, 18078 (2017).

  48. 48.

    Li, X., Baker-Andresen, D., Zhao, Q., Marshall, V. & Bredy, T. W. Methyl CpG binding domain ultra-sequencing: a novel method for identifying inter-individual and cell-type-specific variation in DNA methylation. Genes Brain Behav. 13, 721–731 (2014).

  49. 49.

    Jung, M. et al. Longitudinal epigenetic and gene expression profiles analyzed by three-component analysis reveal down-regulation of genes involved in protein translation in human aging. Nucleic Acids Res. 43, e100 (2015).

  50. 50.

    Song, G. & Wang, L. Nuclear receptor SHP activates miR-206 expression via a cascade dual inhibitory mechanism. PLoS One 4, e6880 (2009).

  51. 51.

    Pan, H. et al. Negative elongation factor controls energy homeostasis in cardiomyocytes. Cell Rep. 7, 79–85 (2014).

  52. 52.

    Chaudhary, P. et al. HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression. Nucleic Acids Res. 44, 1613–1629 (2016).

  53. 53.

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

  54. 54.

    Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

  55. 55.

    Dunham, I. et al. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012). ENCODE Project Consortium.

  56. 56.

    Huang, W., Sherman, B. T. & Lempicki, R. A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).

  57. 57.

    Huang, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

Download references

Acknowledgements

The authors gratefully acknowledge grant support from the NIH (no. 5R01MH105398 to T.W.B. and P.B.; no. 5R01MH109588 to R.C.S. and T.W.B., no. 1R01GM123558 to P.B., no.1DP2GM119164 to R.C.S.; R.C.S. receives support from a Pew Scholar award), the NHMRC (nos. GNT1033127 and GNT1160823 to T.W.B.), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (no. CNPq-CsF-400850/2014-1 to R.G.-O.), the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior – Brasil (CAPES-Finance Code 001 to R.G.O.) and the Research Council of Norway (FRIMEDBIO grant 32222 to M.B.). X.L. has been supported by postgraduate scholarships from the University of Queensland and the ANZ Trustees Queensland, The University of Queensland developmental fellowship and the ARC Discovery Early Career Researcher Award (no. DE190101078). PROMEC is funded by the Faculty of Medicine and Health Sciences at NTNU and the Central Norway Regional Health Authority. The authors also thank R. Tweedale for helpful editing of the manuscript and S. Gandhi for comments and lively discussion.

Author information

Author notes

    • Michael R. Emami

    Present address: Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA

  1. These authors contributed equally: Xiang Li, Qiongyi Zhao.

Affiliations

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

    • Xiang Li
    • , Qiongyi Zhao
    • , Wei Wei
    • , Paul R. Marshall
    • , Jiayu Yin
    • , Sachithrani U. Madugalle
    • , Ziqi Wang
    • , Laura J. Leighton
    • , Esmi L. Zajaczkowski
    •  & Timothy W. Bredy
  2. Intellectual Development and Disabilities Research Center, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA

    • Quan Lin
  3. Department of Computer Science and Institute for Genomics and Bioinformatics, University of California Irvine, Irvine, CA, USA

    • Christophe Magnan
    •  & Pierre F. Baldi
  4. Department of Neurobiology and Behavior and Center for the Neurobiology of Learning and Memory, University of California Irvine, Irvine, CA, USA

    • Michael R. Emami
  5. Brain Institute, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil

    • Luis E. Wearick-Silva
    • , Thiago W. Viola
    •  & Rodrigo Grassi-Oliveira
  6. Department of Pharmaceutical Sciences, University of California Irvine, Irvine, CA, USA

    • Sarah Nainar
    • , Ke Ke
    •  & Robert C. Spitale
  7. Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway

    • Cathrine Broberg Vågbø
    •  & Magnar Bjørås

Authors

  1. Search for Xiang Li in:

  2. Search for Qiongyi Zhao in:

  3. Search for Wei Wei in:

  4. Search for Quan Lin in:

  5. Search for Christophe Magnan in:

  6. Search for Michael R. Emami in:

  7. Search for Luis E. Wearick-Silva in:

  8. Search for Thiago W. Viola in:

  9. Search for Paul R. Marshall in:

  10. Search for Jiayu Yin in:

  11. Search for Sachithrani U. Madugalle in:

  12. Search for Ziqi Wang in:

  13. Search for Sarah Nainar in:

  14. Search for Cathrine Broberg Vågbø in:

  15. Search for Laura J. Leighton in:

  16. Search for Esmi L. Zajaczkowski in:

  17. Search for Ke Ke in:

  18. Search for Rodrigo Grassi-Oliveira in:

  19. Search for Magnar Bjørås in:

  20. Search for Pierre F. Baldi in:

  21. Search for Robert C. Spitale in:

  22. Search for Timothy W. Bredy in:

Contributions

X.L. prepared lentiviral constructs carried out the experiments and wrote the manuscript. Q.Z. performed all the bioinformatics analysis and wrote the manuscript. W.W. prepared lentiviral constructs, carried out the chIP assay and qPCR experiments and performed FACS sorting of activated neurons in RNA-seq experiment. Q.L. built the N6amt1 overexpression constructs. C.M. performed bioinformatics analysis. M.R.E., L.E.W. and T.W.B. performed behavioral experiments. P.R.M. prepared the immunohistochemistry. J.Y. performed protein analysis on the in vivo experiments. S.U.M. performed behavioral experiments. Z.W. performed the western blot on N6amt1 overexpression in HEK cells. S.N. performed RNA and DNA extraction. C.B.V. performed mass spectrometry experiments. E.L.Z. performed quantitaitive PCR experiments. K.K. performed protein studies. R.G.-O. contributed reagents and helped write the manuscript. M.B. contributed reagents and helped write the manuscript. P.F.B. contributed access to the bioinformatic analysis pipeline and helped write the manuscript. R.C.S. contributed reagents and helped write the manuscript. T.W.B. conceived the study, designed experiments and wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Xiang Li or Timothy W. Bredy.

Integrated supplementary information

  1. Supplementary Figure 1 Using flow cytometry coupled with DpnI-seq, we generated a genome-wide profile of cell-type-specific, learning-induced m6dA deposition at single-base resolution.

    (A) The schema shows the workflow. (B) Flow cytometry scatterplot represents 30 individual enrichment for activated neurons using Arc and NeuN as tags. (C) FACS reports show there is distinct a population of cells co-expressing Arc and NeuN in the ILPFC (9.84%) after fear extinction training.

  2. Supplementary Figure 2 Motifs of m6dA sites in the mouse neuronal genome identified by DpnI-seq.

    (A) De novo motif result generated by HOMER Motif Analysis. The height represents the significance of enrichment of each nucleotide at that position. (B) List of all motifs that were identified by DpnI-seq followed by number of sites.

  3. Supplementary Figure 3 An independent cohort of animals was used to validate the DpnI-seq data on selected candidate genes.

    (A) There was no effect of extinction learning on m6dA in the BDNF P1 promoter (two-tailed, unpaired Student’s t test, t=0.8238, df=8,*p=.4339, RC: median=1.773, data range: 0.256 to 2.758 and EXT: median=1.092, data range: 0.017 to 2.956), (B) Extinction learning led to an increase in m6dA accumulation in the Rab3a (two-tailed, unpaired Student’s t test, t=2.557, df=8,*p=.0338, RC: median=0.097, data range: 0.038 to 0.224 and EXT: median=0.274, data range: 0.186 to 0.515) and (C) Gabrr3 (two-tailed, unpaired Student’s t test, t=2.496, df=8,*p=0.0372, RC: median=0.481, data range: 0.326 to 0.587 and EXT: median=0.727, data range: 0.436 to 0.929), (D) Gabrd (two-tailed, unpaired Student’s t test, t=4.495, df=8,**p=.0020, RC: median=0.096, data range: 0.026 to 0.136 and EXT: median=0.196, data range: 0.164 to 0.224), (E) There was no detectable change in m6dA in the Homer2 promoter following extinction learning (two-tailed, unpaired Student’s t test, t=1.56, df=8, p=.1575, RC: median=0.737, data range: 0.382 to 1.313 and EXT: median=1.109, data range: 0.897 to 1.448). (all n=5 biologically independent animals per group).

  4. Supplementary Figure 4 Learning-induced accumulation of m6dA only occurs in activated neurons.

    (A, C, E G and I) Raw reads image of DpnI-seq data from IGV browser. DpnI-qPCR results reveal cell-type specific accumulation of m6dA at (B) BDNF P4 proximal GATC site (one-way ANOVA F1,28 = 11.52, p<.01; Dunnett’s posthoc test: RC within quiescent neurons vs. EXT within activated neurons, **p=.0032, RC within quiescent neurons: median=1.028, data range: 0.922 to 1.152 and EXT within activated neurons: median=0.463, data range: 0.150 to 0.807), (D) Homer2 (one-way ANOVA F1,28 = 11.38, p<.01; Dunnett’s posthoc test: RC within quiescent neurons vs. EXT within activated neurons, ***p=.0002, RC within quiescent neurons: median=1.035, data range: 0.463 to 1.206 and EXT within activated neurons: median=0.436, data range: 0.213 to 0.634); (F) Gabrr3 (one-way ANOVA F1,27 = 6.703, p<.05; Dunnett’s posthoc test: RC within quiescent neurons vs. EXT within activated neurons, *p=.034, RC within quiescent neurons: median=1.049, data range: 0.576 to 1.584 and EXT within activated neurons: median=0.536, data range: 0.335 to 0.675); (H) Gabrd (one-way ANOVA F1,27 = 14.21, p<.001; Dunnett’s posthoc test: RC within quiescent neurons vs. EXT within activated neurons, ****p<.0001, RC within quiescent neurons: median=0.8615, data range: 0.760 to 1.039 and EXT within activated neurons: median=0.414, data range: 0.229 to 0.578) and (J) Rab3a (one-way ANOVA F1,28 = 17.21, p<.001; Dunnett’s posthoc test: RC within quiescent neurons vs. EXT within activated neurons, ****p<.0001, RC within quiescent neurons: median=1.025, data range: 0.923 to 1.201 and EXT within activated neurons: median=0.393, data range: 0.212 to 0.537). (all n=8 biologically independent animals per group).

  5. Supplementary Figure 5 N6amt1 mRNA expression is extinction-learning-induced in primary cortical neurons.

    (A) Activity-induced N6amt1 mRNA expression in primary cortical neurons, in vitro (two-tailed, unpaired student’s t test, t=4.411, df=6,**p=.0031, KCl-: median=1.000, data range: 0.982 to 1.017 and KCl+: median=1.295, data range: 1.128 to 1.473). (B) No effect of neuronal stimulation on N6amt2 mRNA expression (two-tailed, unpaired student’s t test, t=0.4867, df=6, p=.6348, KCl-: median=0.999, data range: 0.989 to 1.027 and KCl+: median=1.039, data range: 0.823 to 1.201). (all n=4 biologically independent experiments per group).

  6. Supplementary Figure 6 Pseudoconditioning followed by extinction training has no effect on N6amt1 expression, m6dA and N6amt1 accumulation at the BDNF P4 promoter.

    N6amt1 expression (one-way ANOVA F2,13 = 21.97, p<.0001; Dunnett’s posthoc test: RC vs. EXT, ***p=.0001, RC: median=1.003, data range: 0.928 to 1.150 and EXT: median=2.015, data range: 1.695 to 2.499), (B) m6dA accumulation (one-way ANOVA F2,15 = 8.122, p<.01; Dunnett’s posthoc test: RC vs. EXT, **p=.0065, RC: median=1.023, data range: 0.766 to 1.316 ; EXT: median=0.677, data range: 0.569 to 0.804) or (C) N6amt1 occupancy at BDNF P4 (one-way ANOVA F2,14 = 6.209, p<.05; Dunnett’s posthoc test: RC vs. EXT, *p=.0344, RC: median=0.175, data range: 0.082 to 0.156; EXT: median=0.314, data range: 0.205 to 0.367). (D) Pseudoconditioning followed by extinction training leads to a decrease on Bdnf exon IV expression (one-way ANOVA F2,13 = 30.48, p<.0001; Dunnett’s posthoc test: RC vs. EXT, **p=.0016; RC vs. PseudoCon+EXT **p=.0038, RC: median=1.004, data range: 0.886 to 1.138; EXT: median=1.523, data range: 1.211 to 2.009 and PesudoCon+EXT: median=0.54, data range: 0.311 to 0.676). (all n=6 biologically independent animals per group).

  7. Supplementary Figure 7 N6amt1 overexpression leads to a global increase in m6dA, and n6amt1 knockdown reduces global level of m6dA in vitro.

    (A) N6amt1 protein levels in HEK293t cells shows N6amt1 overexpression construct can increased N6amt1 expression two-tailed, unpaired student’s t test, t=13.08, df=4, ***p=.0002, empty vector: median=1, data range: 0.968 to 1.062 and N6amt1 OX: median=2.776, data range: 2.515 to 2.944). (B) N6amt1 overexpression leads to an increase in the global level of m6dA in primary cortical neurons (two-tailed, unpaired student’s t test, t=12.75, df=4, ***p=.0002, empty vector: median=1, data range: 0.792 to 1.122 and N6amt1 OX: median=3.132, data range: 2.896 to 3.348). (C) N6amt1 shRNA knockdown leads to a reduction of protein level in primary cortical neurons (two-tailed, unpaired student’s t test, t=2.845, df=4, *p=.047, Scrambled control: median=1, data range: 0.922 to 1.135 and N6amt1 shRNA: median=0.741, data range: 0.647 to 0.854). (All n=3 biological independent experiments per group, Error bars represent S.E.M.)., which leads to (D) reduction of global level of m6dA (n=1 per group).

  8. Supplementary Figure 8 ChIP-qPCR with IgG controls of selected antibodies against N6amt1, H3K4me3, YY1, PolII and m6dA.

    There is no significant difference between retention control (RC) and extinction (EXT) at the proximal GATC site in the BDNF P4 promoter with (A) IgG control for N6amt1 (two-tailed, unpaired student’s t test, t=0.8583, df=8, p=.4157), (B) IgG control for H3K4me3 (two-tailed, unpaired student’s t test, t=0.3407, df=8, p=.7421), (C) IgG control for YY1 (two-tailed, unpaired student’s t test, t=0.4786, df=8, p=.6450), (D) IgG control for TFIIB, (E) IgG control for Pol II (two-tailed, unpaired student’s t test, t=0.7937, df=8, p=.4503) and (F) IgG control for m6dA (two-tailed, unpaired student’s t test, t=0.578, df=8, p=.5792). (all n=5 biologically independent animals per group).

  9. Supplementary Figure 9 N6amt1-mediated and m6dA-related changes in chromatin and transcriptional machinery do not occur at a distal GATC sequence in the BDNF P4 promoter in ILPFC after learning.

    Extinction-induced effect on (A) N6amt1 occupancy (two-tailed, unpaired student’s t test, t=1.77, df=6, p=.1272, RC: median=0.684, data range: 0.519 to 0.891 and EXT: median=0.902, data range: 0.680 to 1.075) (B) the deposition of m6dA, (two-tailed, unpaired student’s t test, t=0.0282, df=6, p=.978, RC: median=0.329, data range: 0.303 to 0.356 and EXT: median=0.332, data range: 0.213 to 0.562) or (C-F) the presence of open chromatin structure, H3K4me3 (two-tailed, unpaired student’s t test, t=0.3462, df=6, p=.7410, RC: median=0.387, data range: 0.272 to 0.572 and EXT: median=0.359, data range: 0.219 to 0.419), TFIIB, (two-tailed, unpaired student’s t test, t=2.092, df=6, p=.0629, RC: median=0.155, data range: 0.017 to 0.427 and EXT: median=0.3374, data range: 0.099 to 0.480), YY1(two-tailed, unpaired student’s t test, t=0.1296, df=6, p=.9011, RC: median=0.470, data range: 0.424 to 0.564 and EXT: median=0.480, data range: 0.294 to 0.613) and RNA Pol II (two-tailed, unpaired student’s t test, t=0.03763, df=6, p=.971, RC: median=0.420, data range: 0.261 to 0.423 and EXT: median=0.9965, data range: 0.312 to 0.507) at the distal GATC site within the BDNF P4 promoter. (all n=5 biologically independent animals per group).

  10. Supplementary Figure 10 N6amt1-mediated and m6dA-related changes in chromatin and transcriptional machinery do not occur at the BDNF P1 promoter in ILPFC after learning.

    Extinction-induced effect on (A) N6amt1 occupancy (two-tailed, unpaired student’s t test, t=1.346, df=8, p=.2153, RC: median=0.222, data range: 0.123 to 0.292 and EXT: median=0.333, data range: 0.197 to 0.615) (B) the deposition of m6dA (two-tailed, unpaired student’s t test, t=0.8238, df=8, p=.4339, RC: median=1.773, data range: 0.255 to 2.758 and EXT: median=1.092, data range: 0.017 to 2.956), or (C) the presence of open chromatin structure (two-tailed, unpaired student’s t test, t=0.3436, df=8, p=.740, RC: median=1.238, data range: 0.405 to 2.706 and EXT: median=1.438, data range: 0.135 to 2.559), (D) H3K4me3 and (two-tailed, unpaired student’s t test, t=2.198, df=8, p=.0592, RC: median=0.027, data range: 0.019 to 0.033 and EXT: median=0.173, data range: 0.024 to 0.401) (E) TFIIB (two-tailed, unpaired student’s t test, t=0.2088, df=8, p=.8398, RC: median=0.199, data range: 0.075 to 0.404 and EXT: median=0.223, data range: 0.023 to 0.573); (F) observed a reduction on YY1 occupancy (two-tailed, unpaired student’s t test, t=8.24, df=8, ****p<.0001, RC: median=1.657, data range: 1.180 to 2.056 and EXT: median=0.258, data range: 0.158 to 0.328). Also, no significant induction is detected on (G) RNA Pol II (two-tailed, unpaired student’s t test, t=0.5153, df=8, p=.6203, RC: median=0.067, data range: 0.025 to 0.078 and EXT: median=0.079, data range: 0.028 to 0.154) at the distal GATC site within the BDNF P4 promoter. (H) BDNF exon I expression (two-tailed, unpaired student’s t test, t=1.9, df=8, p=.0839, RC: median=1.329, data range: 0.734 to 2.179 and EXT: median=1.897, data range: 1.304 to 2.592) (all n=5 biologically independent animals per group).

  11. Supplementary Figure 11 Extinction learning induces N6amt1 occupancy and active chromatin structures that are associated with increased Rab3a mRNA expression.

    Extinction training leads to (A) an increase in N6amt1 (n=6 biologically independent animals per group, two-tailed, unpaired student’s t test, t=7.252, df=10, ****p<.0001, RC: median=0.285, data range: 0.117 to 0.385 and EXT: median=1.214, data range: 0.838 to 1.573), (B) increased open chromatin structure (n=5 biologically independent animals per group, two-tailed, unpaired student’s t test, t=2.372, df=8, *p=.0451, RC: median=1.102, data range: 0.482 to 1.667 and EXT: median=1.848, data range: 1.255 to 2.358), (C) increased occupancy of the chromatin mark H3K4me3 (n=5 biologically independent animals per group, two-tailed, unpaired student’s t test, t=2.334, df=8, *p=.0478, RC: median=0.027, data range: 0.019 to 0.033 and EXT: median=0.177, data range: 0.044 to 0.402), however, no significant induction of (D) TFIIB recruitment (n=5 biologically independent animals per group, two-tailed, unpaired student’s t test, t=0.2881, df=8, p=.7806, RC: median=1.988, data range: 1.064 to 3.341 and EXT: median=1.763, data range: 0.624 to 4.190). On the other hand, (E) an increase in the presence of Pol II was observed (two-tailed, unpaired student’s t test, n=5 biologically independent animals per group, t=3.111, df=8, *p=.0144, RC: median=0.067, data range: 0.025 to 0.085 and EXT: median=0.113, data range: 0.098 to 0.154), and (F) a correlated increase in the induction of Rab3a mRNA expression (n=6 biologically independent animals per group, two-tailed, unpaired student’s t test, t=3.471, df=10, **p<.006, RC: median=0.983, data range: 0.725 to 1.375 and EXT: median=1.654, data range: 1.185 to 2.252).

  12. Supplementary Figure 12 Representative data of FACS based on the GFP signal that is mediated by lentiviral infection.

    Flow cytometry scatterplot represents 10 individual’s enrichment for lentiviral infected neurons using GFP as tag and FACS report.

  13. Supplementary Figure 13 N6amt1 knockdown in the PLPFC has no effect on the formation of fear extinction memory.

    (A) The schema describes the behavioral test protocol. (B) knockdown of N6amt1 in PLPFC has no effect during extinction training (two-way ANOVA F4,50 =0.6444, p=0.6344) and on the formation of extinction formation (one-way ANOVA F8,34 = 16.71, p<.0001; Tukey’s posthoc test: scrambled control RC vs. scrambled control EXT, **p=.00944, scrambled control RC: median=72.42, data range: 71.12 to 74.386 and scrambled control EXT: median=35.05, data range: 9.05 to 67.35; N6amt1 shRNA RC vs. N6amt1 shRNA EXT, *p=.0124, N6amt1 shRNA RC: median=62.1, data range: 47.81 to 76.69 and N6amt1 shRNA EXT: median=34.22, data range: 0.96 to 58.85). (n=5 biologically independent animals per group for RC scramble and RC N6amt1 shRNA, n=6 biologically independent animals per group for EXT scramble control and EXT N6amt1 shRNA).

  14. Supplementary Figure 14 N6amt1 shRNA blocks the induction of N6amt1 mRNA expression following extinction learning.

    N6amt1 mRNA expression was not increased post extinction training in mice treated with N6amt1 shRNA (n=7 biologically individual animals per group, two-way ANOVA, F1,24=31.46, p<.0001; Dunnett’s posthoc test: Scrambled control RC vs. Scrambled control EXT, ****p<.0001, scrambled control RC: median=1.005, data range: 0.754 to 1.248 and scrambled control EXT: median=1.57, data range: 1.146 to 1.901).

  15. Supplementary Figure 15 A schematic of the hypothesized role of m6dA within the BDNF P4 promoter of neurons activated by extinction learning.

    The dynamic accumulation of m6dA drives activity-dependent Bdnf exon IV gene expression by facilitating an active and open chromatin state, and the recruitment of essential components of the transcriptional machinery, including YY1, TFIIb and PolII.

Supplementary information

  1. Supplementary information

    Supplementary Figs. 1–15, Supplementary Tables 1 and 2, and Supplementary Note.

  2. Reporting Summary

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41593-019-0339-x