<br>DNA 修饰在神经元、星形胶质细胞和小胶质细胞中的差异使用,Epigenetics & Chromatin

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DNA 修饰在神经元、星形胶质细胞和小胶质细胞中的差异使用
Epigenetics & Chromatin ( IF 4.2 ) Pub Date : 2023-11-13 , DOI: 10.1186/s13072-023-00522-6
Kyla B Tooley _{1,

2} , Ana J Chucair-Elliott ₂ , Sarah R Ocañas _{1,

2} , Adeline H Machalinski ₂ , Kevin D Pham ₂ , Walker Hoolehan ₂ , Adam M Kulpa ₂ , David R Stanford ₃ , Willard M Freeman _{1,

4,

5}

Affiliation

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。在神经科学中，迫切需要分离和表征健康和疾病中特定 CNS 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），用于在没有细胞分选的情况下配对分离神经元 DNA 和 RNA，然后使用该模型来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）海马中的神经元翻译组和表观基因组。WGoxBS 结果通过酶促甲基测序（EM-Seq）和纳米孔测序进行验证。将神经元数据与来自 NuTRAP 模型的小胶质细胞和星形胶质细胞数据进行比较，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，hmCG 和 mCH 观察到相反的模式。细胞类型之间的差异修饰区域主要存在于基因体和远端基因间区域，而不是近端启动子。在各种细胞类型中，DNA 修饰（mCG、mCH、hmCG）与近端启动子的基因表达呈负相关。相比之下，观察到基因体 mCG 呈负相关，远端启动子与基因体 hmCG 与基因表达呈正相关。此外，我们确定了 mCH 与启动子和基因体区域基因表达之间的神经元特异性负相关。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，这些水平在分析方法中是可重现的。然而，修饰-基因表达关系在细胞类型之间是保守的。基因体和远端调节元件中不同细胞类型的差异修饰的富集，但不是近端启动子，突出了这些区域的表观基因组模式，这可能是细胞身份的更大决定因素。这些发现还证明了在神经表观基因组分析中区分 mC 和 hmC 的重要性，因为在传统上解释为 mCG 的物质中，高达 30% 可能是 hmCG，而 hmCG 通常与 mCG 的基因表达不同。

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Differential usage of DNA modifications in neurons, astrocytes, and microglia

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.

更新日期：2023-11-13

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