Neuronal brain region-specific DNA methylation and chromatin accessibility are associated with neuropsychiatric trait heritability [RNA-Seq]

We analyzed differential methylation (via WGBS) between four distinct human brain regions (NAcc-nucleus accumbens, BA9-dorsolateral prefrontal cortex, BA24-anterior cingulate cortex, and HC-hippocampus) in both sorted nuclei and intact tissues. We isolated neuronal and non-neuronal (glial) nuclei from the same six individuals for each tissue via FACS using the neuronal marker, NeuN. Additionally, we performed WGBS from non-sorted tissues from these same brain regions in a total of 12 individuals (BA9 n = 9; BA24 n = 5; HC n = 6; NAcc n = 7). To complement our DNA methylation analyses, we measured gene expression (RNA-seq) and chromatin accessibility (ATAC-seq) in neuronal and non-neuronal nuclei from the nucleus accumbens and dorsolateral prefrontal cortex from six more individuals. We then performed an integrative analysis to understand how the epigenome contributes to brain region-specific function. Overall design: 72 WGBS libraries were sequenced (from intact tissue (27 libraries) and NeuN+ and NeuN- flow sorted nuclei (45 libraries)) from hippocampus, nucleus accumbens, prefrontal cortex (BA9), and anterior cingulate gyrus (BA24). RNA-seq (20 libraries) and ATAC-seq (23 libraries) data from NeuN+ and NeuN- flow sorted nuclei from the nucleus accumbens and prefrontal cortex (BA9) were also obtained.

我们通过全基因组亚硫酸氢盐测序（Whole Genome Bisulfite Sequencing，WGBS），分析了4个不同人类脑区——伏隔核（NAcc）、背外侧前额叶皮层（BA9）、前扣带回皮层（BA24）与海马体（HC）——在分选细胞核与完整组织中的差异甲基化水平。针对每类脑组织，我们利用神经元标志物NeuN，通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting，FACS）从6名相同供体的样本中分离出神经元细胞核与非神经元（神经胶质）细胞核。此外，我们还对上述脑区的未分选组织开展WGBS测序，共纳入12名供体（BA9组9例、BA24组5例、HC组6例、NAcc组7例）。为补充DNA甲基化分析结果，我们额外选取6名供体，对伏隔核与背外侧前额叶皮层的神经元及非神经元细胞核进行了基因表达（RNA测序，RNA-seq）与染色质可及性（转座酶可及性染色质测序，ATAC-seq）检测。随后我们开展整合分析，以探究表观基因组如何调控脑区特异性功能。整体实验设计：我们从海马体、伏隔核、前额叶皮层（BA9）及前扣带回（BA24）中构建并测序了共计72个WGBS文库，其中包含27个完整组织文库，以及45个经NeuN阳性与NeuN阴性流式分选的细胞核文库。此外，我们还获取了来自伏隔核与前额叶皮层（BA9）的NeuN阳性、NeuN阴性流式分选细胞核的RNA-seq文库20个，以及ATAC-seq文库23个。