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. Single Cell Methylome analysis of mouse and human frontal cortex

哺乳动物大脑包含多种具有独特生理、形态及分子特征的神经元细胞类型。然而，目前仍缺乏针对表观遗传层面差异显著的神经元类别的全面评估。胞嘧啶DNA甲基化（cytosine DNA methylation）是一种稳定的表观遗传标记，可区分神经元类型并标记基因调控元件。我们开发了一种高效的单细胞核甲基化组测序（single-nucleus methylome sequencing）方法，能够实现稳定可靠的高通量神经元类型分类。我们获取了超过6000个单细胞核甲基化组数据，并在额叶皮层（frontal cortex）中鉴定出16种小鼠神经元亚群与21种人类神经元亚群。CG甲基化与非CG甲基化均呈现细胞类型特异性分布，既印证了单神经元转录组分析（single neuron transcriptome profiling）的研究发现，又进一步拓展了相关认知。此外，我们在小鼠与人类皮层中发现了约50万个神经元类型特异性调控元件，这些元件在两类皮层中均表现出显著的甲基化差异。独特的甲基化特征分别鉴定出了一种独特的人类表达小白蛋白（Parvalbumin）的抑制性神经元亚群，以及小鼠皮层中第6层特异性的兴奋性神经元亚群。比较表观基因组分析显示，与兴奋性神经元相比，抑制性神经元的基因调控元件具有更强的保守性。上述研究结果证实了单细胞核甲基化组分析在拓展脑细胞类型图谱以及鉴定潜在驱动这些细胞类型差异的调控元件两方面的应用价值。小鼠与人类额叶皮层的单细胞核甲基化组分析