Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Genes & Human Disease Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK, 73104, USA.
Epigenetics Chromatin. 2023 Nov 13;16(1):45. doi: 10.1186/s13072-023-00522-6.
Cellular identity is determined partly by cell type-specific epigenomic profiles that regulate gene expression. In neuroscience, there is a pressing need to isolate and characterize the epigenomes of specific CNS cell types in health and disease. In this study, we developed an in vivo tagging mouse model (Camk2a-NuTRAP) for paired isolation of neuronal DNA and RNA without cell sorting and then used this model to assess epigenomic regulation, DNA modifications in particular, of gene expression between neurons and glia.
After validating the cell-specificity of the Camk2a-NuTRAP model, we performed TRAP-RNA-Seq and INTACT-whole genome oxidative bisulfite sequencing (WGoxBS) to assess the neuronal translatome and epigenome in the hippocampus of young mice (4 months old). WGoxBS findings were validated with enzymatic methyl-Seq (EM-Seq) and nanopore sequencing. Comparing neuronal data to microglial and astrocytic data from NuTRAP models, microglia had the highest global mCG levels followed by astrocytes and then neurons, with the opposite pattern observed for hmCG and mCH. Differentially modified regions between cell types were predominantly found within gene bodies and distal intergenic regions, rather than proximal promoters. Across cell types there was a negative correlation between DNA modifications (mCG, mCH, hmCG) and gene expression at proximal promoters. In contrast, a negative correlation of gene body mCG and a positive relationship between distal promoter and gene body hmCG with gene expression was observed. Furthermore, we identified a neuron-specific inverse relationship between mCH and gene expression across promoter and gene body regions.
Neurons, astrocytes, and microglia demonstrate different genome-wide levels of mCG, hmCG, and mCH that are reproducible across analytical methods. However, modification-gene expression relationships are conserved across cell types. Enrichment of differential modifications across cell types in gene bodies and distal regulatory elements, but not proximal promoters, highlights epigenomic patterning in these regions as potentially greater determinants of cell identity. These findings also demonstrate the importance of differentiating between mC and hmC in neuroepigenomic analyses, as up to 30% of what is conventionally interpreted as mCG can be hmCG, which often has a different relationship to gene expression than mCG.
细胞特性部分取决于调控基因表达的细胞类型特异性表观基因组图谱。在神经科学领域，迫切需要分离和鉴定健康和疾病状态下特定中枢神经系统细胞类型的表观基因组。在这项研究中，我们开发了一种体内标记小鼠模型（Camk2a-NuTRAP），用于在无需细胞分选的情况下对神经元 DNA 和 RNA 进行配对分离，然后使用该模型评估神经元和神经胶质细胞之间基因表达的表观基因组调控，特别是 DNA 修饰。
在验证了 Camk2a-NuTRAP 模型的细胞特异性后，我们进行了 TRAP-RNA-Seq 和 INTACT-全基因组氧化亚硫酸氢盐测序（WGoxBS），以评估年轻小鼠（4 个月大）海马中的神经元转录组和表观基因组。WGoxBS 结果通过酶甲基化测序（EM-Seq）和纳米孔测序进行了验证。将神经元数据与 NuTRAP 模型中的小胶质细胞和星形胶质细胞数据进行比较，小胶质细胞具有最高的全局 mCG 水平，其次是星形胶质细胞，然后是神经元，而 hmCG 和 mCH 则呈现相反的模式。细胞类型之间差异修饰的区域主要位于基因体和远端基因间区，而不是近端启动子。在整个细胞类型中，DNA 修饰（mCG、mCH、hmCG）与近端启动子处的基因表达呈负相关。相反，在基因体 mCG 处观察到负相关，而在远端启动子和基因体 hmCG 与基因表达之间观察到正相关。此外，我们发现神经元中 mCH 与启动子和基因体区域的基因表达之间存在一种特定的负相关关系。
神经元、星形胶质细胞和小胶质细胞表现出不同的全基因组 mCG、hmCG 和 mCH 水平，这些水平在不同的分析方法中是可重复的。然而，修饰-基因表达关系在整个细胞类型中是保守的。在基因体和远端调控元件中，而非在近端启动子中，细胞类型之间的差异修饰富集，突出了这些区域的表观基因组模式可能是细胞特性的更大决定因素。这些发现还表明，在神经表观基因组分析中区分 mC 和 hmC 非常重要，因为多达 30%的传统上被解释为 mCG 的物质实际上可以是 hmCG，它与基因表达的关系通常与 mCG 不同。
