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Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain

Abstract

DNA methylation has critical roles in the nervous system and has been traditionally considered to be restricted to CpG dinucleotides in metazoan genomes. Here we show that the single base–resolution DNA methylome from adult mouse dentate neurons consists of both CpG (75%) and CpH (25%) methylation (H = A/C/T). Neuronal CpH methylation is conserved in human brains, enriched in regions of low CpG density, depleted at protein-DNA interaction sites and anticorrelated with gene expression. Functionally, both methylated CpGs (mCpGs) and mCpHs can repress transcription in vitro and are recognized by methyl-CpG binding protein 2 (MeCP2) in neurons in vivo. Unlike most CpG methylation, CpH methylation is established de novo during neuronal maturation and requires DNA methyltransferase 3A (DNMT3A) for active maintenance in postmitotic neurons. These characteristics of CpH methylation suggest that a substantially expanded proportion of the neuronal genome is under cytosine methylation regulation and provide a new foundation for understanding the role of this key epigenetic modification in the nervous system.

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Figure 1: Pervasive CpH methylation in the in vivo DNA methylome of adult dentate granule neurons.
Figure 2: Conserved CpH methylation in orthologous regions of the human brain DNA.
Figure 3: Genomic features of the neuronal CpH methylation.
Figure 4: Relationship between CpH methylation and protein-DNA interaction or gene expression in vivo.
Figure 5: Repression of reporter gene expression by CpH methylation in neurons.
Figure 6: Recognition of CpH methylation by MeCP2 in vitro and in vivo.
Figure 7: Establishment of CpH methylation during neuronal maturation.
Figure 8: Neuronal CpH methylation is actively maintained by DNMT3A and regulates endogenous gene expression in vivo.

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Acknowledgements

We thank S. Baylin, D. Ginty and members of the Song and Ming laboratories for comments and suggestions and Y. Cai and L. Liu for technical support. This work was supported by the US National Institutes of Health (NIH) (NS047344, ES021957 and MH087874), the Simons Foundation Autism Research Initiative and NARSAD to H.S., by the NIH (HD069184 and NS048271), the Maryland Stem Cell Research Fund (MSCRF), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and NARSAD to G.-l.M., by the Lieber Institute fund to Y.G., by NIH (HD064743, HD066560) to Q.C. and by NIH (NS072924) to G.F.; J.U.G. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation. Y.S. and C.Z. were supported by MSCRF postdoctoral fellowships; J.S. was supported by a Samsung Scholarship.

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Authors and Affiliations

Authors

Contributions

J.U.G. and Y.S. conducted most of the experiments. Y.S. constructed the libraries, and J.U.G. performed the bioinformatic analyses. J.H.S., B.X. and Y.G. assisted with high-throughput sequencing. J.S. contributed to the EMSA and ChIP experiments. H.L. and Q.C. provided the MeCP2-ChIP samples. C.Z. performed the shRNA experiment. S.H. and H.Z. assisted with the EMSA experiments. T.L. and G.F. provided the DNMT-cTKO samples. Y.G., G.-l.M. and H.S. supervised the project. J.U.G., G.-l.M. and H.S. wrote the manuscript. All of the authors discussed results and commented on the manuscript.

Corresponding author

Correspondence to Hongjun Song.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Global levels of CpG and CpH methylation across mouse chromosomes.

Interquartile boxplots of 1 Kb-bin-averaged methylation levels of each mouse chromosome are shown. Note that chrY showed lower levels of CpG methylation, whereas both sex chromosomes exhibited lower levels of CpH methylation.

Supplementary Figure 2 Inter-sample correlation of CpG and CpH methylation.

Methylation levels of all individual CpG (left) and CpH (right) loci were compared between two biological replicates. Both classes of methylation exhibit high correlation between individuals. Pearson's correlation coefficients (r) are shown.

Supplementary Figure 3 CpH methylation is spatially associated with CpG methylation.

(a) Methylation levels of neighboring CpGs of all mCpHs with ≥ 20% methylation. Note the anti-correlation between CpG methylation and its distance from mCpH. (b) Scatter plots of three classes of cytosine methylation. Pearson's correlations (r) calculated using non-overlapping 100 Kb bins are shown.

Supplementary Figure 4 Mouse neuronal CpG and CpH methylation on two opposite DNA strands.

(a) Correlation between individual cytosines of three different classes. All top strand CpG and CHG loci were compared to the cytosines in the bottom strand within the motifs, whereas CHH loci were compared to the closest CHH in the opposite strand. Pearson's correlation coefficients are shown. (b) Bisulfite-Seq (top) and Sanger bisulfite sequencing (bottom) results of CpH methylated regions from both strands. Note that CpH methylation is present on both strands in most regions.

Supplementary Figure 5 Distance analysis of CHG and CHH methylation.

Numbers of mCHG (top) and mCHH (bottom) are plotted against the base-pair distance from any mCHG/mCHH in H1 ESCs (left) and in the adult dentate gyrus (right). Note that only CHG methylation in ESCs exhibits the distinct 8, 21, 29 bp spacing pattern. Cubic spline smoothing curves are shown.

Supplementary Figure 6 Neuronal CpG and CpH methylation around ESC-specific transcription factor binding sites.

CpG and CpH methylation levels were averaged across each of the four sets of ESC-specific transcription factor binding sites (Marson et al., Cell 2008). Note that CpH hypomethylation is much less pronounced than that around neuronal transcription factor binding sites (Fig. 4a). Modest hypomethylation is still observed possibly because many of these binding sites map closely to TSSs, which are intrinsically hypomethylated.

Supplementary Figure 7 Anti-correlation between CpG-far CpH methylation in regulatory regions and gene expression.

440, 410 and 395 of 70,364 mCpHs that do not have any CpG in their 500 bp flanking regions are mapped within 2 kb from TSS, intragenic and extragenic enhancers (Kim et al., Nature 2010), respectively. These mCpH-containing regulatory regions exhibit lower nearest gene expression levels (P values are indicated; Mann-Whitney U-tests).

Supplementary Figure 8 In vitro methylated reporter assay in HEK293 cells.

(a) A schematic illustration of experimental design. (b) FACS results (left) of GFP+ cells for each in vitro methylated reporter. In vitro methylation does not alter transfection efficiencies as measured by qPCR (right). Values represent mean ± s.e.m. (n = 3; P values are indicated; ANOVA). (c) Methylation patterns were determined by bisulfite sequencing of plasmids recovered 2 days after transfection.

Supplementary Figure 9 Effects of CpH methylation on MBD2b-DNA interaction.

(a) An EMSA experiment using different amounts of recombinant MBD2b proteins and the same set of synthetically methylated oligos as in Fig. 6b. (b) Quantification of MBD2b-bound oligos. Note that MBD2b exhibits higher selectivity towards mCpGs than MeCP2 does (Fig. 6b).

Supplementary Figure 10 Knock-down efficiency of shRNAs.

AAVs expressing shRNAs targeting Dnmt1 (left) or Dnmt3a (right) or control AAVs expressing a scrambled shRNA were injected into the adult mouse dentate gyrus. Knock-down efficiency was determined by qPCR using Dnmt1- or Dnmt3a-specific primers. Values represent mean ± s.e.m. (n = 3; P values are indicated; Student's t-test).

Supplementary Figure 11 Lack of effects of DNMT3A knock-down on unmethylated and CpG-methylated CpH-unmethylated regions.

(a) Methylation levels of Bdnf IV (unmethylated, left), Bdnf IX (CpG-methylated/CpH-unmethylated, middle) and Fgf1B (CpG-methylated/CpH-unmethylated, right) promoters after DNMT3A knock-down. (b) Expression levels of Bdnf IV, Bdnf IX and Fgf1B transcripts after DNMT3A knock-down (P values are indicated; Student's t-test).

Supplementary Figure 12 DNMT3A binds to CpH-methylated regions in adult dentate gyrus in vivo.

Occupancy of DNMT3A in CpH-methylated regions, measured by the fraction of input neuronal chromatin immuno-precipitated by a DNMT3A antibody, was compared to unmethylated CpG island regions. A rabbit IgG antibody was used to control for unspecific binding.

Supplementary Figure 13 Dnmt gene expression in adult mouse tissues.

Dnmt gene expression was measured by RT-qPCR in multiple adult mouse tissues. Note that the adult brain expresses a similar level of Dnmt1 and Dnmt3a, a lower level of Dnmt3b compared to other tissues, and no Dnmt3a2. Values represent mean ± s.e.m. (n = 3).

Supplementary Figure 14 Summary of similarities and differences between CpG and CpH methylation in ESCs in vitro and neurons in vivo.

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Supplementary Figures 1–14, Supplementary Tables 1 and 2 (PDF 1452 kb)

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Guo, J., Su, Y., Shin, J. et al. Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain. Nat Neurosci 17, 215–222 (2014). https://doi.org/10.1038/nn.3607

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