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

Abstract

Background 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. Results 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. Conclusions 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.

Specificity and efficiency of tamoxifen-mediated Cre induction is equivalent regardless of age

“… 27 , 28 With this mouse line, there is some labeling of circulating monocytes and border associated macrophages; however, with the long lifespan of microglial cells, and high turnover of peripheral monocytes, labeled circulating macrophages are cleared 2–4 weeks after Cre induction and the bone marrow progenitors do not demonstrate recombination. 22 , 27 , 28 , 29 , 30 For the present studies, to compare different ages of Tam induction, Cx3cr1 creERT2 22 mice were crossed to mice with a floxed NuTRAP allele (nuclear tagging and translating ribosome affinity purification) 31 which tags nuclei with biotin/mCherry and ribosomes with eGFP 32 , 33 similar to Ribotag protocols. 34 These tags can be used for INTACT (Isolation of Nuclei TAgged in specific Cell Types) 35 isolation of nuclei or TRAP (translating ribosome affinity purification) 36 isolation of ribosomes to obtain cell type-specific nucleic acids (DNA and RNA, respectively) from cellularly heterogeneous tissues.…”