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G9a is essential for epigenetic silencing of K+ channel genes in acute-to-chronic pain transition

Nature Neuroscience volume 18, pages 1746–1755 (2015)Cite this article

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Abstract

Neuropathic pain is a debilitating clinical problem and difficult to treat. Nerve injury causes a long-lasting reduction in K+ channel expression in the dorsal root ganglion (DRG), but little is known about the epigenetic mechanisms involved. We found that nerve injury increased dimethylation of Lys9 on histone H3 (H3K9me2) at Kcna4, Kcnd2, Kcnq2 and Kcnma1 promoters but did not affect levels of DNA methylation on these genes in DRGs. Nerve injury increased activity of euchromatic histone-lysine N-methyltransferase-2 (G9a), histone deacetylases and enhancer of zeste homolog-2 (EZH2), but only G9a inhibition consistently restored K+ channel expression. Selective knockout of the gene encoding G9a in DRG neurons completely blocked K+ channel silencing and chronic pain development after nerve injury. Remarkably, RNA sequencing analysis revealed that G9a inhibition not only reactivated 40 of 42 silenced genes associated with K+ channels but also normalized 638 genes down- or upregulated by nerve injury. Thus G9a has a dominant function in transcriptional repression of K+ channels and in acute-to-chronic pain transition after nerve injury.

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Figure 1: Nerve injury reduces expression levels of Kcna4, Kcnd2, Kcnq2 and Kcnma1 and increases expression and activity of HDACs, G9a and EZH2 in the DRG.
Figure 2: Cellular distribution of HDACs, G9a and EZH2 in control and injured DRGs.
Figure 3: Nerve injury increases H3K9me2 levels at K+ channel promoters but does not change DNA methylation status at their promoter regions in the DRG.
Figure 4: Inhibition of G9a activity normalizes K+ channel expression in the DRG diminished by nerve injury.
Figure 5: Inhibition of G9a activity restores Kv channel currents in DRG neurons reduced by nerve injury.
Figure 6: Differential effects of inhibition of G9a, HDACs and EZH2 on pain hypersensitivity induced by nerve injury.
Figure 7: Deletion of Ehmt2 in DRG neurons prevents nerve injury–induced pain hypersensitivity and reduction in K+ channel expression in the DRG.
Figure 8: G9a inhibition normalizes the expression of genes encoding K+ channels and a subset of other genes modified by nerve injury.

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Acknowledgements

We wish to thank C.L. Creasy from GlaxoSmithKline Laboratories for generously providing GSK503 and W.N. Hittleman at MD Anderson Cancer Center for assisting with the confocal microscopy. We also thank Z. Pan (MD Anderson Cancer Center) and F. Wang (Duke University) for providing Ehmt2flox/flox and AdvillinCre mice, respectively. This study was supported by the NIH Blueprint for Neuroscience Research–Grand Challenge on Chronic Pain (R01 DE022015) and the N.G. and Helen T. Hawkins endowment (H.-L.P.).

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Authors and Affiliations

  1. Department of Anesthesiology and Perioperative Medicine, Center for Neuroscience and Pain Research, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Geoffroy Laumet, Shao-Rui Chen, Yuhao Zhang, De-Pei Li, Trevor M Smith, Yingchun Dong & Hui-Lin Pan

  2. Fels Institute for Cancer Research and Molecular Biology, Temple University Lewis Katz School of Medicine, Philadelphia, Pennsylvania, USA

    Judit Garriga, Jaroslav Jelinek, Matteo Cesaroni & Jean-Pierre Issa

  3. Department of Anesthesiology, Institute and Hospital of Stomatology, Nanjing University Medical School, Nanjing, China

    Yingchun Dong

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  1. Geoffroy Laumet
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Contributions

G.L. performed biochemical and behavioral experiments and mouse breeding. S.-R.C. performed all surgeries, tissue extractions, and immunocytochemical experiments. Y.Z. performed ChIP-PCR experiments. J.G. and J.J. performed bisulfite sequencing experiments. J.G. and M.C. performed RNA sequencing experiments. D.-P.L. and T.M.S. performed DRG neuron dissociation and electrophysiological recordings. Y.D. performed some immunocytochemical and behavioral experiments. G.L., J.G., S.-R.C., D.-P.L., T.M.S., J.J., M.C., J.-P.I. and H.-L.P. analyzed the data. H.-L.P. and J.-P.I. conceived the project and supervised the study. G.L. and H.-L.P. wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Evidence for a new upstream exon in Kcna4 in the rat DRG.

(a) The UCSC Genome browser annotation indicating that rat Kcna4 is monoexonic and that the transcriptional start site (TSS) is around Chr3: 92781000. (a) Bisulfite sequencing data showing the annotated promoter region was hypermethylated in the DRG (open circle: unmethylated CpG sites; closed circle: methylated CpG sites). However, this gene is highly expressed in the DRG (Figure 1a). Because many species have an upstream exon and this region is very conserved (see http://genome.ucsc.edu/cgi-bin/hgGateway genome “rat” search “Kcna4”), we hypothesized that the rat genome also has this upstream exon. Using rat-mouse homology, we saw that the rat DNA sequence of Kcna4 was exactly the same as mouse DNA sequence. (a) We designed 3 pairs of primers (A, B, and C) to determine if rat Kv1.4 mRNA is the same as mouse Kv1.4 mRNA in control DRG samples. Two percent agarose gel image showing RT-PCR byproducts after reverse transcription (RT+) or without reverse transcription (RT-). Total RNAs were treated with DNase, and heat-inactivated cDNA were treated with RNase. We were able to amplify cDNA with these three pairs of primers, which suggested that rat and mouse Kcna4 genes have the similar structure within two exons. (a) Gene annotation map showing the location of the exons in rat, mouse, and human Kcna4 in the original database and the three primers used for panel a. (a) Evidence of an upstream exon in Kcna4 in the rat DRG. Kcna4 has a similar structure in the rat and mouse. Moreover, the new promoter region was not hypermethylated according to its mRNA level (Figure 3i).

Supplementary Figure 2 Nerve injury has no effect on expression of Kcna4, Kcnd2, Kcnq2, Kcnma1, H3K9me2 or G9a in the dorsal spinal cord.

(a) Mean data show the mRNA level of Kcna4, Kcnd2, Kcnq2, Kcnma1 and G9a in the dorsal spinal cord of sham control and SNL rats at 21 days after surgery (n = 11 rats in each group). The mRNA level was quantified by using real-time PCR and normalized to a housekeeping gene (Gapdh). (b) Representative gel images and quantification data show the protein level of G9a and H3K9me2 in the dorsal spinal cord of sham control and SNL rats at 21 days after surgery (n = 7 in each group). GAPDH and histone H3 were used as loading controls. Data are shown as mean ± s.e.m. Supplementary Figure S11 contains the uncropped version of the gel images.

Supplementary Figure 3 Changes in the expression of Hdac, Ehmt2, Ezh2 and Pcna in the DRG after nerve injury.

(a-f) Time course of changes in the mRNA levels of G9a (a), Ezh2 (b), Hdac1 (c), Hdac2 (d), Hdac4 (e), and Hdac5 (f) in L5 and L6 DRG tissues obtained from sham and SNL rats before and 5, 10 and 21 days after surgery (n = 6 in each group). mRNA data were quantified using real-time PCR and normalized to Gapdh. (g) mRNA levels of proliferation cell nuclear antigen (Pcna) in L5 and L6 DRGs obtained from sham and SNL rats before and 5, 10 and 21 days after surgery (n = 6 in each group). Data are presented as mean ± SEM. Statistical analysis was performed using repeated measures ANOVA followed by Dunnett’s post hoc test. * P < 0.05, ** P < 0.01, compared with baseline control (time 0).

Supplementary Figure 4 Effects of intrathecal injections of SAHA, UNC0638 or GSK503 on mechanical thresholds of rats treated with SNL or sham surgery.

(a-f) Quantitative data show the effects of intrathecal injection of 1-50 µg SAHA (a,b, n = 6 rats in each group), 0.1-10 µg UNC0638 (c,d, n = 6 rats in each group) or 0.5-25 µg GSK503 (e,f, n = 6 rats in each group) for 8 days on the Randall-Selitto and von Frey thresholds of SNL rats. (g,h) Quantitative data show the lack of effects of intrathecal daily injections of 5 µg GSK503, 50 µg SAHA or 10 µg UNC0638 for 8 days on the Randall-Selitto and von Frey thresholds in sham rats (n = 6 rats in each group). Data are presented as mean ± s.e.m. Statistical analysis was performed using repeated measures ANOVA followed by Dunnett’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, compared with the baseline control (time 0).

Supplementary Figure 5 Effects of UNC0638, SAHA and GSK503 treatment on substrate levels of G9a, HDACs and EZH2 in the injured DRG.

(a,b) Western blot images and quantitative data showing the effects of intrathecal injections of vehicle, SAHA (50 µg), UNC0638 (10 µg), or GSK503 (5 µg) for 8 days on the protein levels of histone H3 in the DRG obtained from SNL rats (n = 6 in each group). GAPDH was used as a loading control. (c,d) Gel images and quantification data showing that intrathecal SAHA treatment increased the level of acetyl-H3, whereas treatment with UNC0638 and GSK503 decreased the level of H3K9me2 and H3K27me3, respectively, in the DRG obtained from SNL rats 28 days after surgery (n = 6 in each group). (e,f) Original gel images and quantification data showing the effects of intrathecal injections of SAHA, UNC0638, and GSK503 on the protein levels of H3K27me3, H3K9me2, H3K9ac and acetyl-H3 in the DRG obtained from SNL rats 28 days after surgery (n = 6 in each group). Histone H3 was used as a loading control. Data are shown as mean ± s.e.m. Statistical analysis was performed using the Mann-Whitney test or one-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001, compared with the vehicle group. Supplementary Figure S12 contains the uncropped version of gel images.

Supplementary Figure 6 Nerve injury and UNC0638 treatment do not change the distribution of IB4-positive neurons in the DRG.

Representative confocal images show the distribution of IB4-positive and NeuN-immunoreactive neurons in the DRG. Double immunocytochemical labeling was performed on the left L5 DRG removed from sham-operated and SNL rats treated with vehicle or UNC0638 four weeks after surgery. The percentage of IB4-positive neurons over the total of NeuN-immunoreactive neurons in sham-operated rats, SNL rats treated with vehicle, and SNL rats treated with UNC0638 was 69.7 ± 6.5, 38.3 ± 5.7 and 38.5 ± 5.9, respectively (n = 12 DRG sections from 3 separate rats in each group).

Supplementary Figure 7 Nerve injury changes expression levels of multiple genes in the DRGs.

(a) Principal component analysis representation of the correlation among the expression values for all mapped genes (n = 16,876) for the different samples in each of the two experimental conditions (SNL plus vehicle, or SNL plus UNC0638) and the control (Sham). (b) Heat map generated with the expression values of the 2,035 genes with a change in expression level of at least two fold after nerve injury. The small dendrogram at top shows the clustering of the different samples based on the expression values (4 rats in the sham group, 4 rats in the SNL group, and 3 SNL rats treated with UNC0638). (c) Ingenuity Pathway Analysis showing the principal canonical pathways for the genes with changes in expression after nerve injury. The raw RNA sequence data have been deposited in the database repository of Gene Expression Omnibus (GEO accession #GSE59043).

Supplementary Figure 8 Uncropped gel images for Figure 1.

Original full-length western blotting gel images used for Figure 1b,d.

Supplementary Figure 9 Uncropped gel images for Figure 4.

Original full-length western blotting gel images used for Figure 4e,g.

Supplementary Figure 10 Uncropped gel images for Figure 7.

Original full-length western blotting gel images used for Figure 7b.

Supplementary Figure 11 Uncropped gel images for Supplementary Figure 2.

Original full-length western blotting gel images used for Supplementary Figure 2b.

Supplementary Figure 12 Uncropped gel images for Supplementary Figure 5.

Original full-length western blotting gel images used for Supplementary Figure 5a,c,e.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Table 4 (PDF 3513 kb)

Supplementary Methods Checklist (PDF 567 kb)

Supplementary Table 1

List of DNA methylation levels of 80 CpG sites within 1 kb of TSSs in 30 K+ channel genes of DRGs obtained from sham-treated and SNL rats. (XLSX 11 kb)

Supplementary Table 2

List of 105 K+ channel-related genes and their expression levels in the DRGs from sham control, vehicle-treated SNL and UNC0638-treated SNL rats. (XLSX 22 kb)

Supplementary Table 3

List of 638 genes in the DRG that are altered by nerve injury and normalized by treatment with UNC06838. (XLSX 86 kb)

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Laumet, G., Garriga, J., Chen, SR. et al. G9a is essential for epigenetic silencing of K+ channel genes in acute-to-chronic pain transition. Nat Neurosci 18, 1746–1755 (2015). https://doi.org/10.1038/nn.4165

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