Single Cell Methylome Signatures Reveal Distinct Neuronal Populations and Regulatory Elements in the Mammalian Cortex

The mammalian brain contains diverse neuron cell types with distinct physiological, morphological and molecular characteristics. However, a comprehensive assessment of the epigenetically distinct neuronal classes is currently missing. Cytosine DNA methylation is a stable epigenetic mark that distinguishes neuron types and marks gene regulatory elements. We developed an efficient single-nucleus methylome sequencing approach that allows robust high-throughput neuron-type classification. We generated >6,000 single-nucleus methylomes and identified 16 mouse and 21 human neuronal subpopulations in the frontal cortex. Both CG and non-CG methylation exhibited cell type-specific distributions that recapitulate and extend findings from single neuron transcriptome profiling. Moreover, we found approximately 500,000 neuron-type-specific regulatory elements showing strong differential methylation in mouse and human cortex. Distinct methylation signatures identified an unique human Parvalbumin-expressing inhibitory sub-type and a layer 6 specific excitatory sub-type in mouse. Comparative epigenomic analysis showed stronger conservation of gene regulatory elements in inhibitory compared with excitatory neurons. These findings demonstrate the utility of single nucleus methylome profiling for both expanding the atlas of brain cell types and identifying regulatory elements that potentially drive these differences.