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

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. To examine the impact of genome topology on CA methylation in a specific cell type, we conducted Hi-C analysis on PV nuclei isolated from the mouse cortex through the INTACT method.哺乳动物大脑中神经元类型的极端多样性，可通过精细的基因表达差异以最高分辨率得以刻画，而这类差异或许需要特殊的分子机制来维持。神经元独有地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）与雷特综合征蛋白MeCP2协同调控基因表达；然而，这类独特的基因结构与调控机制，在调控高精度神经元类型特异性基因程序中的功能尚未得到探索。本研究借助表观基因组学（epigenomics）与空间转录组学（spatial transcriptomics）分析，揭示了mCA与MeCP2在维持定义高精度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，该差异取决于整体mCA水平；同时解析了驱动不同神经元类型间重叠与差异化基因调控的甲基化模式及基因内增强子抑制机制。值得注意的是，我们证实mCA与MeCP2可调控那些反复被用于以高细胞分辨率区分神经元类型的基因，其中包括视觉皮层中具备空间分辨率的视觉依赖性基因程序。这类反复参与神经元分型的基因具备一系列基因组特征：基因长度较长、存在大量基因内增强子，且富集mCA，这些特征使其易于受到MeCP2的调控。因此，经由MeCP2通路介导的长基因调控，可维持密切相关神经元间的差异基因表达，进而助力大脑实现卓越的细胞多样性。为探究基因组拓扑结构对特定细胞类型中CA甲基化的影响，我们通过INTACT方法分离小鼠皮层中的PV细胞核，并对其开展Hi-C分析。