Research ArticleNEUROSCIENCE

A unique role for DNA (hydroxy)methylation in epigenetic regulation of human inhibitory neurons

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Science Advances  26 Sep 2018:
Vol. 4, no. 9, eaau6190
DOI: 10.1126/sciadv.aau6190
  • Fig. 1 Unique distributions of hmC and mCH in Glu, MGE-GABA, and OLIG cells from the human PFC.

    (A) Method for cell type–specific nuclei isolation for transcriptomic and epigenomic analyses. (B) Genome-wide analysis of mC (OxBS) and hmC (BS-OxBS) shows substantial but significantly different levels of hmCG in both neuronal subtypes (Glu and MGE-GABA) and OLIG. Neurons are enriched in mCH, with a small proportion of hmCH. R1 and R2, biological replicates. (C) Examples of marker genes expressed in Glu (NEUROD6), MGE-GABA (LHX6), or OLIG (OLIG2) cells, together with their DNA methylation and histone modification profiles. Glu (GLU), glutamatergic neurons; MGE-GABA (M-GABA), MGE-derived GABAergic interneurons.

  • Fig. 2 Cell type–specific gene body DNA methylation patterns associate with expression and differential expression.

    (A) Gene body hmCG has a stronger positive relation with expression in MGE-GABA than Glu cells. r, Spearman correlation (P < 10−10). (B) Gene body mCH is negatively related to expression and enriched in cell type–specific up- and down-regulated genes. (C) CG methylation is not related to differential expression after controlling for expression, except for mCG in MGE-GABA neurons. Only genes with expression in the range (10 < TPM < 100) were included. (D to F) mCH is nonmonotonically related to differential expression: Both up- and down-regulated genes have increased mCH compared with non-DE genes at the same level of expression. In (D), only genes with expression in the range (10 < TPM < 100) were included. In (E), only genes with expression in the range (50 < TPM < 100) were included. Asterisks indicate significant differences in tmCH for each set of DE genes compared with non-DE genes (*P < 0.05; **P < 0.01; ***P < 0.001, t test, Bonferroni-corrected). (G) Polycomb repression (H3K27me3) is negatively correlated with expression and enriched in cell type–specific up- and down-regulated genes. (H) Polycomb repression and mCH are positively correlated for most genes (yellow line, median).

  • Fig. 3 Increased hmCG at MGE-GABA–specific distal gene regulatory elements.

    (A) H3K27ac ChIP-seq data identify ~15,000 to 25,000 cell type–specific distal peaks in Glu, MGE-GABA, and OLIG cells. (B) DNA methylation at the center of cell type–specific enhancers. MGE-GABA cells are enriched for hmCG at cell type–specific peaks. R1 and R2, biological replicates. Error bars denote SEM. Significant differences of methylation levels are indicated with asterisks (pairwise t test). (C and D) Mean profile of DNA methylation marks at cell type–specific distal H3K27ac peaks. Genome-wide methylation levels (as in Fig. 1B) are shown in horizontal dashed lines. Differences of methylation levels in the center of the regions were tested between common peaks (C), cell type–specific peaks (S), and inactive peaks (I), and significances are indicated with asterisks (pairwise t test).

  • Fig. 4 Enrichment of cell type–specific DMRs in disease-associated variants.

    (A) The number of DMRs (labeled in thousands; methylation difference ≥ 0.3) detected using tmCG (BS-seq) or mCG (OxBS-seq) data (green, Glu < MGE-GABA; blue, MGE-GABA < Glu; red, OLIG < Glu and MGE-GABA). Many Glu DMRs may be driven by lower hmC levels, whereas MGE-GABA DMRs are mainly driven by lower mCG. (B to D) Enrichment of DMRs near disease-associated genetic variants from GWAS. For each disease, the top 50 most significant regions were retained. Schizophrenia loci were strongly enriched in MGE-GABA DMRs. By contrast, other neuropsychiatric diseases such as attention-deficit hyperactivity disorder (ADHD), autism, and Parkinson’s disease were associated with Glu DMRs (tmCG) that may be driven by lower hmCG. (E) Cell type–specific enhancers for all three brain cell types were enriched in many brain disease–associated variants.

  • Fig. 5 CGI-associated regions of elevated tmCG near promoters of key neuron subtype–specific genes are enriched in hmCG and depleted of H3K27me3.

    (A) Glu versus MGE-GABA tmCG DMR density around CpG islands. CpG shores and shelves (<4 kb away from CpG islands) are indicated in the shaded area. The enrichment of DMRs was tested using DMR density in the flanking region (>10 kb away) as a background control. Asterisks indicate significant enrichments (P < 0.05). (B) Cell type–specific tmCG DMRs within CGIs, shores, and shelves (CGI ± 4 kb) (gray dots). Most tmCG DMRs are negatively related to expression of the nearest gene. A subset of genes has tmCG DMRs with positive association with gene expression (green, Glu-enriched genes; blue, MGE-GABA–enriched genes). (C) tmCG DMRs that demonstrate positive association with gene expression have low tmCG levels in the fetal cortex. These tmCG DMRs demonstrate neuron subtype–specific enrichment in hmCG (D) and H3K27me3 depletion in the gene body of the nearest gene (E). (F and G) Examples of DMRs positively associated with gene expression of nearest MGE-GABA–specific (F) (GAD2, DLX2, and VAX1) or Glu-specific (G) (NEUROD2, EMX1, CBLN1, and SLC17A6) genes. MB, million base pairs or megabase.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/9/eaau6190/DC1

    Fig. S1. RNA-seq validates the identities of FANS-sorted populations.

    Fig. S2. Cell type–specific gene body DNA methylation patterns in neurons and OLIG associate with expression and differential expression.

    Fig. S3. Association of epigenetic modifications at promoters with gene expression in different brain cell types.

    Fig. S4. DNA methylation profiles at cell type–specific distal gene regulatory elements.

    Fig. S5. DNA methylation profiles at cell type–specific distal H3K27ac peaks situated at different distances from TSS.

    Fig. S6. Comparison of adult enhancers (H3K27ac peaks) with regions of open chromatin (ATAC-seq) in the developing human fetal brain.

    Fig. S7. Comparison of cell type–specific DMRs with DMRs from single-nucleus methylC-seq data.

    Table S1. Sample information and demographics.

    Table S2. Results of RNA-seq analysis (TPMs of all genes in each sample and results of differential expression analyses).

    Table S3. Sequencing metrics for BS-seq, OX-BS-seq, and RNA-seq analyses.

    Table S4. DMRs for tmCG, mCG, and hmCG.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. RNA-seq validates the identities of FANS-sorted populations.
    • Fig. S2. Cell type–specific gene body DNA methylation patterns in neurons and OLIG associate with expression and differential expression.
    • Fig. S3. Association of epigenetic modifications at promoters with gene expression in different brain cell types.
    • Fig. S4. DNA methylation profiles at cell type–specific distal gene regulatory elements.
    • Fig. S5. DNA methylation profiles at cell type–specific distal H3K27ac peaks situated at different distances from TSS.
    • Fig. S6. Comparison of adult enhancers (H3K27ac peaks) with regions of open chromatin (ATAC-seq) in the developing human fetal brain.
    • Fig. S7. Comparison of cell type–specific DMRs with DMRs from single-nucleus methylC-seq data.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Sample information and demographics.
    • Table S2 (Microsoft Excel format). Results of RNA-seq analysis (TPMs of all genes in each sample and results of differential expression analyses).
    • Table S3 (Microsoft Excel format). Sequencing metrics for BS-seq, OX-BS-seq, and RNA-seq analyses.
    • Table S4 (Microsoft Excel format). DMRs for tmCG, mCG, and hmCG.

    Files in this Data Supplement:

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