Non-CG DNA methylation and MeCP2 stabilize repeated tuning of long genes that distinguish closely related neuron types [WGBS]

The extraordinary diversity of neuron types in the mammalian brain is delineated at the highest resolution by subtle gene expression differences that may require specialized molecular mechanisms to be maintained. Neurons uniquely express the longest genes in the genome and utilize neuron-enriched non-CG DNA methylation (mCA) together with the Rett syndrome protein, MeCP2, to control gene expression, but the function of these unique gene structures and machinery in regulating finely resolved neuron type-specific gene programs has not been explored. Here, we employ epigenomic and spatial transcriptomic analyses to discover a major role for mCA and MeCP2 in maintaining neuron type-specific gene programs that define high resolution cell types. We uncover differential susceptibility to MeCP2 loss in neuronal populations depending on global mCA levels and dissect methylation patterns and intragenic enhancer repression that drive overlapping and distinct gene regulation between neuron types. Strikingly, we show that mCA and MeCP2 regulate genes that are repeatedly tuned to differentiate neuron types at high cellular resolution, including spatially resolved, vision-dependent gene programs in the visual cortex. These repeatedly tuned genes display genomic characteristics, including long length, numerous intragenic enhancers, and enrichment for mCA, that predispose them to regulation by MeCP2. Thus, long gene regulation by the MeCP2 pathway maintains differential gene expression between closely-related neurons to facilitate the exceptional cellular diversity in the brain. Overall design: Cerebellum, striatum, and hypothalamus tissues and INTACT-isolated subclasses of neurons from the cortex were profiled by whole-genome bisulfite-seq.

哺乳动物大脑神经元类型的极致多样性，可通过细微的基因表达差异以最高分辨率得以界定，而维持此类差异或需专属的分子机制。神经元特异性表达基因组中最长的基因，并借助神经元富集的非CG DNA甲基化（non-CG DNA methylation, mCA）联合雷特综合征蛋白MeCP2调控基因表达，但这类独特的基因结构与调控系统在调控精细分辨率的神经元类型特异性基因程序方面的功能，迄今尚未被深入探究。
本研究通过表观基因组学与空间转录组学分析，揭示了mCA与MeCP2在维持界定高分辨率细胞类型的神经元类型特异性基因程序中的核心作用。我们发现，神经元群体对MeCP2缺失的易感性存在差异，且该差异取决于全局mCA水平；同时我们解析了调控神经元类型间重叠与差异化基因调控的甲基化模式与基因内增强子抑制机制。
值得注意的是，我们证实mCA与MeCP2所调控的基因，会反复被优化以在高细胞分辨率下区分神经元类型，其中包括视觉皮层中经空间解析的视觉依赖型基因程序。这类经反复优化的基因具有特定基因组特征：基因长度较长、拥有大量基因内增强子且mCA富集，这些特征使其易受MeCP2的调控。
因此，经由MeCP2通路实现的长基因调控，可维持密切相关神经元之间的差异基因表达，从而促成大脑极致的细胞多样性。
实验整体设计：采用全基因组亚硫酸氢盐测序（whole-genome bisulfite-seq）技术，对小脑、纹状体、下丘脑组织以及皮层来源的INTACT分离神经元亚类完成了分子谱型分析。