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Tet3 regulates synaptic transmission and homeostatic plasticity via DNA oxidation and repair

Nature Neuroscience volume 18, pages 836–843 (2015)Cite this article

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Abstract

Contrary to the long-held belief that DNA methylation of terminally differentiated cells is permanent and essentially immutable, post-mitotic neurons exhibit extensive DNA demethylation. The cellular function of active DNA demethylation in neurons, however, remains largely unknown. Tet family proteins oxidize 5-methylcytosine to initiate active DNA demethylation through the base-excision repair (BER) pathway. We found that synaptic activity bi-directionally regulates neuronal Tet3 expression. Functionally, knockdown of Tet or inhibition of BER in hippocampal neurons elevated excitatory glutamatergic synaptic transmission, whereas overexpressing Tet3 or Tet1 catalytic domain decreased it. Furthermore, dysregulation of Tet3 signaling prevented homeostatic synaptic plasticity. Mechanistically, Tet3 dictated neuronal surface GluR1 levels. RNA-seq analyses further revealed a pivotal role of Tet3 in regulating gene expression in response to global synaptic activity changes. Thus, Tet3 serves as a synaptic activity sensor to epigenetically regulate fundamental properties and meta-plasticity of neurons via active DNA demethylation.

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Figure 1: Synaptic activity–dependent expression of Tet3 regulates glutamatergic synaptic transmission.
Figure 2: DNA oxidation and BER regulates glutamatergic synaptic transmission.
Figure 3: Tet3 signaling mediates homeostatic synaptic scaling-up of glutamatergic synaptic transmission.
Figure 4: Tet3 signaling mediates bicuculline-induced homeostatic synaptic scaling-down of glutamatergic synaptic transmission.
Figure 5: Tet3 signaling regulates neuronal surface GluR1 levels.
Figure 6: Tet3 regulates gene expression in neurons in response to global synaptic activity changes.
Figure 7: Essential role of Tet3 in neuronal activity–induced DNA methylation dynamics at the Bdnf IV promoter region and gene expression.

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Acknowledgements

We thank R. Huganir, P. Worley, G.C. Turrigiano and L. Chen for suggestions, members of the Ming and Song laboratories for help and critical comments, G.L. Xu for Tet3 antibodies, and L. Liu, Q. Hussaini and Y. Cai for technical support. This work was supported by the US National Institutes of Health (NS047344 to H.S., NS048271 and MH105128 to G.-l.M., NS062691 to G.C. and D.H.G.), a grant from the Simons Foundation (SFARI240011 to H.S.), the Brain and Behavior Research Foundation (H.S., G.-l.M. and Y.S.), Maryland Stem Cell Research Foundation (G.-l.M. and H.S.), and by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (G.-l.M., D.H.G. and G.C.). J.S. was supported by a Samsung fellowship.

Author information

Author notes

  1. Junjie U Guo

    Present address: Present address: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.,

Authors and Affiliations

  1. Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Huimei Yu, Yijing Su, Jaehoon Shin, Chun Zhong, Junjie U Guo, Yi-Lan Weng, Guo-li Ming & Hongjun Song

  2. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Huimei Yu, Yijing Su, Chun Zhong, Junjie U Guo, Yi-Lan Weng, Guo-li Ming & Hongjun Song

  3. Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    Fuying Gao, Daniel H Geschwind & Giovanni Coppola

  4. Program in Neurogenetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    Fuying Gao, Daniel H Geschwind & Giovanni Coppola

  5. Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    Fuying Gao, Daniel H Geschwind & Giovanni Coppola

  6. Department of Psychiatry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

    Fuying Gao, Daniel H Geschwind & Giovanni Coppola

  7. Graduate Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Jaehoon Shin, Guo-li Ming & Hongjun Song

  8. The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Guo-li Ming & Hongjun Song

  9. Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Guo-li Ming

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Contributions

H.Y. performed electrophysiological analyses. Y.S. performed biochemical and DNA methylation analyses. J.S. and J.U.G. performed bioinformatics analysis. C.Z. and Y.-L.W. generated AAV. F.G., D.H.G. and G.C. performed RNA-seq. G.-l.M. and H.S. designed the project and wrote the manuscript.

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Correspondence to Hongjun Song.

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Integrated supplementary information

Supplementary Figure 1 Basic characterization of neurons with Tet3 knockdown, overexpression of Tet1-CD or Tet1-mCD.

(a) Nuclear localization of Tet3 protein. Shown is a sample image of immunostaining of hippocampal neurons for Tet3, synapsin I and DAPI. Scale bar: 10 μm. (b) Schematic diagrams of AAV constructs used to infect cultured hippocampal neurons. (c) Sample images of neuronal cultures infected with AAV co-expressing EYFP and shRNAs. Scale bar: 20 μm. Note that over 99% neurons were EYFP+, indicating very high infection efficacy. Also shown is q-PCR analysis of mRNA expression of Tet1, 2, 3 after AAV-mediated expression of different shRNAs. Value represent mean + s.e.m. (n = 3; *P < 0.01; ANOVA; P = 0.03, sh-control vs sh-Tet1; P = 0.002, sh-control vs sh-Tet2; P = 0.01, sh-control vs sh-Tet3-1; P = 0.01 sh-control vs sh-Tet3-2). (d) Dot blot analysis of 5hmC levels in neurons under different conditions. Different cultures were treated with TTX (1 μM) or bicuculline (20 μM) for 48 hours before analyses. (e) Lack of differences in the excitability of neurons with or without Tet3 KD. Shown are sample current-clamp whole-cell recording traces in response to injection of different amount of currents (top panels) and summary of numbers of action potentials (bottom panel). Values represent mean + s.e.m. (f) Lack of differences in the density of synapsin 1+ boutons in neurons upon Tet3 knockdown or Tet1-CD expression. Shown on the top panel are sample confocal images of Tuj1 and synapsin I immunostaining and DAPI staining. Scale bar: 50 μm. Shown on the bottom panel is a summary of density of synapsin I+ boutons on Tuj1+ neurites. Values represent mean + s.e.m. (n > 3 cultures).

Supplementary Figure 2 Rank plots of mEPSC amplitudes to calculate scaling factors for neurons with Tet3 KD under different conditions.

(a) Tet3 KD scaled up mEPSC amplitudes multiplicatively. Shown are plots of ranked mEPSC amplitudes recorded from neurons expressing sh-Tet3-1 (left) or sh-Tet3-2 (right) against ranked mEPSC amplitudes from neurons expressing sh-control. The data are best described by a multiplicative increase in mEPSC amplitude with a scaling factor of 1.48 (sh-Tet3-1) and 1.98 (sh-Tet3-2). (b-e) Lack of significant TTX-induced scaling-up (b-c) and bicuculline-induced scaling-down (d-e) in Tet3 KD neurons. Similar as in (a), shown are plots of ranked mEPSC amplitudes recorded from neurons expressing different shRNAs with TTX (1 μM; b-c) or bicuculline treatment (20 μM; d-e) for 48 hours against ranked mEPSC amplitudes from neurons with the saline treatment. Scaling factor for each condition is listed at the top left corner. (f-g) RA (1 μm, 2 hour treatment) and Tet3 KD induced scaling-up occludes each other.

Supplementary Figure 3 Rank plots of mEPSC amplitudes to calculate scaling factors for neurons with treatment of BER inhibitors.

(a) Plots of ranked mEPSC amplitudes from neurons with treatment of ABT (50 μM, left) or CRT (50 μM, right) for 48 hours against ranked mEPSC amplitudes from neurons with the vehicle treatment. The data are best described by a multiplicative increase in mEPSC amplitude with a scaling factor of 1.40 (ABT) and 1.45 (CRT). (b-e) Lack of significant TTX-induced scaling up (b-c) and bicuculline-induced scaling-down (d-e) upon inhibition of DNA repair. Similar as in (a), shown are plots of ranked mEPSC amplitudes recorded from neurons under different drug treatments that were concurrently treated with TTX (1 μM; b-c) or bicuculline (20 μM; d-e) for 48 hours against ranked mEPSC amplitudes from those neurons without the TTX or bicuculline treatment. Scaling factor for each condition is listed at the top left corner.

Supplementary Figure 4 Rank plots of mEPSC amplitudes to calculate scaling factors for neurons with overexpression of Tet3.

(a) Plot of ranked mEPSC amplitudes from neurons co-expressing EYFP and Tet3 against ranked mEPSC amplitudes from neurons expressing EYFP alone. The data are best described by a multiplicative decrease in mEPSC amplitude with a scaling factor of 0.47. (b-e) Lack of significant TTX-induced scaling up (b-c) and bicuculline-induced scaling-down (d-e) upon Tet3 overexpression. Similar as in (a), shown are plots of ranked mEPSC amplitudes recorded from neurons over-expressing Tet3 (b, d) or EYFP (c, e) with TTX (1 μM; b-c) or bicuculline treatment (20 μM; d-e) for 48 hours against ranked mEPSC amplitudes from those neurons with the saline treatment. Scaling factor for each condition is listed at the top left corner.

Supplementary Figure 5 Rank plots of mEPSC amplitudes to calculate scaling factors for neurons with overexpression of Tet1-CD or Tet1-mCD.

(a) Plot of ranked mEPSC amplitudes from neurons co-expressing EYFP and Tet1-CD against ranked mEPSC amplitudes from neurons expressing EYFP alone with a scaling factor of 0.77. (b-e) Lack of significant TTX-induced scaling up (b-c) and bicuculline-induced scaling-down (d-e) upon Tet1-CD expression. Similar as in (a), shown are plots of ranked mEPSC amplitudes recorded from neurons over-expressing Tet1-CD (b, d) or EYFP (c, e) with TTX (1 μM; b-c) or bicuculline treatment (20 μM; d-e) for 48 hours against ranked mEPSC amplitudes from those neurons with the saline treatment. Scaling factor for each condition is listed at the top left corner.

Supplementary Figure 6 Total GluR1 and Arc protein levels under different conditions.

Shown are sample cropped Western blot images and quantification of total GluR1 (a) or Arc (b) levels under different conditions. The same biological samples as in Fig. 5c were used for a. Values represent mean + s.e.m. (n = 3 experiments; *P < 0.05; #P > 0.1; ANOVA; b: P = 0.002, sh-control vs sh-control + TTX; P = 0.007, sh-control vs sh-control + Bicu; P = 0.03, sh-control vs sh-Tet3-2). Full-length blots are presented in Supplementary Figure 11.

Supplementary Figure 7 Functional change of GluR2-lacking AMPA receptors upon changes in Tet3 expression and DNA demethylation signalling pathway.

(a) Lack of changes of surface or total GluR2 levels upon Tet3 KD or Tet1-CD expression. Shown are sample cropped Western blot images and quantification of surface and total GluR2 levels under different conditions. The same biological samples were used. Values represent mean + s.e.m. (n = 3). Full-length blots are presented in Supplementary Figure 11. (b) Effect of NASPM treatment on the mEPSC amplitude of neurons expressing sh-control or sh-Tet3-2. Values represent mean + s.e.m. (*P < 0.05; **P < 0.01; ANOVA; P = 0.03, before vs after in sh-control group; P = 0.003, before vs after in sh-Tet3-2 group). (c) Summary of decay time of mEPSCs recorded under different conditions. Same cells as in Fig. 1c-d & 2b-c. Values represent mean + s.e.m. (***P < 0.001; **P < 0.01; *P < 0.05; ANOVA; Left: P = 0.000006, sh-control vs sh-Tet3-1; P = 0.00005, sh-control vs sh-Tet3-2; Middle: P = 0.001, EYFP vs EYFP/Tet3 OE; P = 0.03, EYFP vs Tet1-CD; P = 0.02, Tet1-CD vs Tet1-mCD; right: P = 0.02, vehicle vs ABT; P = 0.002, vehicle vs CRT).

Supplementary Figure 8 RNA-seq analysis of neurons under different conditions.

(a) Confirmation of Tet3 knockdown efficacy upon AAV-mediated expression of sh-Tet3-2 in samples for RNA-seq. FPKM reads from three samples each are shown. (b) Heat-map of Pearson correlations among different RNA-seq samples. A total of 17 samples were subject to RNA-seq analysis and the top 2000 highly expressed genes were used for calculation of Pearson correlation. (c) Multidimensional scaling plot. edgeR was used for the analysis of 17 RNA-seq samples. Note clustering of biological replicates, but clear segregation of gene expression from neurons expressing sh-control and neurons expressing sh-Tet3-2. For neurons expressing sh-control, TTX and bicuculline treatment induced the opposite direction of gene expression alterations. Importantly, in Tet3-KD neurons, such changes were either attenuated (for bicuculline treatment) or abolished (for TTX treatment).

Supplementary Figure 9 Tet3 regulates expression of genes associated with synapses and synaptic transmission.

Pathway diagrams are modified from Kyoto Encyclopedia of Genes and Genomes (KEGG). Genes that exhibited significant changes of expression in Tet3-KD neurons (FDR < 0.05) are indicated by stars (red: genes activated by Tet3; blue: genes inhibited by Tet3). Data is based on RNA-seq analyses of neurons expressing sh-Tet3-2 compared to those expressing sh-control (n = 3 samples each).

Supplementary Figure 10 Lack of effect of Tet3 KD on immediate early genes.

(a) Q-PCR analysis of expression of immediate early genes in neurons expressing sh-control or sh-Tet3-2 at 4 hours after bicuculline treatment (20 μM). Values represent mean + s.e.m. (n = 3; *** P < 0.001; **P < 0.01; *P < 0.05; #P > 0.1; Student’s t-test; Arc: P = 0.02, vehicle vs Bicu in sh-control group; P = 0.01, vehicle vs Bicu in sh-Tet3-2 group; Egr4: P = 0.02, vehicle vs Bicu in sh-control group; P = 0.03, vehicle vs Bicu in sh-Tet3-2 group; c-fos: P = 0.006, vehicle vs Bicu in sh-control group; P = 0.00001, vehicle vs Bicu in sh-Tet3-2 group; Npas4: P = 0.002, vehicle vs Bicu in sh-control group; P = 0.02, vehicle vs Bicu in sh-Tet3-2 group). (b) Methylation status of Arc and Npas4 promoters and intragenic regions in neurons expressing sh-control or sh-Tet3-2. Each row represents one allele showing methylation status of individual CpG sites (open circle: unmethylated; closed circle methylated).

Supplementary Figure 11 Full length blots sample blots shown in other figures.

Shown are full blots for cropped sample gel images shown in Figures 1b, 5c, 7c and Supplementary Figures 6a, 6b and 7a.

Supplementary information

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Supplementary Methods Checklist (PDF 387 kb)

Supplementary Table 1

Summary of shRNA and primer sequences used in the current study. (a) Primer sequences used for qPCR analysis; (b) shRNA sequences; (c) Primer sequences for bisulfite sequencing analysis; (d) Primer sequences for ChIP-PCR analyses. (XLS 41 kb)

Supplementary Table 2

Summary of RNA-seq analysis. (a) RNA-seq run and mapping information; (b) Gene list and information on differentially expressed genes upon Tet3 knockdown. (c-d) Gene lists and information on bicuculline-regulated genes in neurons expressing sh-control (c) and those expressing sh-Tet3-2 (d). (e-f) Gene lists and information on TTX-regulated genes in neurons expressing shRNA-control (e) and those expressing sh-Tet3-2 (f). (g) KEGG pathway analysis. (XLS 904 kb)

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Yu, H., Su, Y., Shin, J. et al. Tet3 regulates synaptic transmission and homeostatic plasticity via DNA oxidation and repair. Nat Neurosci 18, 836–843 (2015). https://doi.org/10.1038/nn.4008

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