Tooley Kyla B, Chucair-Elliott Ana J, Ocañas Sarah R, Machalinski Adeline H, Pham Kevin D, Stanford David R, Freeman Willard M
Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK USA.
Genes & Human Disease Program, Oklahoma Medical Research Foundation, Oklahoma City, OK USA.
bioRxiv. 2023 Jun 7:2023.06.05.543497. doi: 10.1101/2023.06.05.543497.
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. This is especially true as for DNA modifications where most data are derived from bisulfite sequencing that cannot differentiate between DNA methylation and hydroxymethylation. In this study, we developed an 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 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 to assess the neuronal translatome and epigenome in the hippocampus of young mice (3 months old). These data were then compared to microglial and astrocytic data from NuTRAP models. When comparing the different cell types, 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, with limited differences occurring within 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 mCG with gene expression within the gene body while a positive relationship between distal promoter and gene body hmCG and gene expression was observed. Furthermore, we identified a neuron-specific inverse relationship between mCH and gene expression across promoter and gene body regions.
In this study, we identified differential usage of DNA modifications across CNS cell types, and assessed the relationship between DNA modifications and gene expression in neurons and glia. Despite having different global levels, the general modification-gene expression relationship was conserved across cell types. The enrichment of differential modifications in gene bodies and distal regulatory elements, but not proximal promoters, across cell types highlights epigenomic patterning in these regions as potentially greater determinants of cell identity.
细胞身份部分由调节基因表达的细胞类型特异性表观基因组图谱决定。在神经科学领域，迫切需要分离并表征健康和疾病状态下特定中枢神经系统（CNS）细胞类型的表观基因组。对于DNA修饰而言尤其如此，因为大多数数据来自亚硫酸氢盐测序，而这种方法无法区分DNA甲基化和羟甲基化。在本研究中，我们开发了一种标记小鼠模型（Camk2a-NuTRAP），用于在无需细胞分选的情况下成对分离神经元DNA和RNA，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。
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，以评估幼鼠（3个月大）海马体中的神经元翻译组和表观基因组。然后将这些数据与来自NuTRAP模型的小胶质细胞和星形胶质细胞数据进行比较。比较不同细胞类型时，小胶质细胞的整体mCG水平最高，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基因间远端区域，近端启动子区域的差异有限。在所有细胞类型中，近端启动子处的DNA修饰（mCG、mCH、hmCG）与基因表达之间呈负相关。相比之下，基因体内mCG与基因表达呈负相关，而远端启动子和基因体hmCG与基因表达之间呈正相关。此外，我们在启动子和基因体区域发现了mCH与基因表达之间的神经元特异性负相关关系。
在本研究中，我们确定了中枢神经系统细胞类型之间DNA修饰的差异使用情况，并评估了神经元和神经胶质细胞中DNA修饰与基因表达之间的关系。尽管整体水平不同，但修饰与基因表达之间的一般关系在所有细胞类型中都是保守的。不同细胞类型在基因体和远端调控元件而非近端启动子中差异修饰的富集，突出了这些区域的表观基因组模式作为细胞身份潜在更大决定因素的作用。
