Single-cell methylomes identify neuronal subtypes and regulatory elements in mammalian cortex

Abstract

The mammalian brain contains diverse neuronal types, yet we lack single-cell epigenomic assays that are able to identify and characterize them. DNA methylation is a stable epigenetic mark that distinguishes cell types and marks regulatory elements. We generated >6000 methylomes from single neuronal nuclei and used them to identify 16 mouse and 21 human neuronal subpopulations in the frontal cortex. CG and non-CG methylation exhibited cell type-specific distributions, and we identified regulatory elements with differential methylation across neuron types. Methylation signatures identified a layer 6 excitatory neuron subtype and a unique human parvalbumin-expressing inhibitory neuron subtype. We observed stronger cross-species conservation of regulatory elements in inhibitory neurons than in excitatory neurons. Single-nucleus methylomes expand the atlas of brain cell types and identify regulatory elements that drive conserved brain cell diversity.

Fig. 1. High-throughput single nucleus methylome sequencing (snmC-seq) of mouse and human frontal cortex (FC) neurons

(A) Workflow of snmC-seq. (B,C) Number of single neuron methylomes (B) and distribution of genomic coverage per dataset (C).

Fig. 2. Non-CG methylation (mCH) signatures identify distinct neuron populations in mouse and human FC

(A,B) Hierarchical clustering of neuron types using gene body mCH level. (C,D) Two-dimensional visualization of single neuron clusters (tSNE (9)). Mouse and human homologous clusters are labeled with similar colors. (E,F) Gene body mCH at Rorb for each single neuron (top), and the distribution for each cluster (bottom) with hyper/hypo-methylated clusters highlighted in red/blue. (G) Comparison of human neuron clusters defined by mCH with clusters from single nucleus RNA-Seq (4, 9). (H) Fraction of cells in each human cluster assigned to each mouse cluster based on mCH correlation at orthologous genes (9). Mutual best matches are highlighted with black rectangles.

Fig. 3. Conserved and divergent neuron type-specific gene regulatory elements

(A) Heatmap showing differentially methylated regions (CG-DMRs) hypomethylated in one or two neuron clusters; categories of DMRs containing >1,000 regions are shown. (B) Transcription factor binding motif enrichment in CG-DMRs of homologous mouse and human clusters (FDR<10⁻¹⁰). (C) Mouse or human specific enrichment/depletion of TF binding motifs. Asterisks indicate TF binding motifs that are significantly enriched in one species but depleted in the other.

Fig. 4. Conserved gene body mCH and CG-DMRs between mouse and human

(A) Global mCH and mCG levels are strongly conserved within homologous cell types between mouse and human. (B) Cross-species correlation of gene body mCH at orthologous genes shows cell type-specific conservation. Black boxes denote homologous neuron clusters. (C) The median correlation of gene body mCH for homologous clusters is higher than the within-species correlation for distinct clusters. (D) Cross-species correlation of mCG at neuron-type-specific CG-DMRs. (E) Sequence conservation at neuron-type-specific DMRs.