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. 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 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 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 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. Conclusions: 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.

Consistent specificity and efficiency of tamoxifen-mediated cre induction across ages

“…With this mouse line, there is some labeling of circulating monocytes and border associated macrophages; however, with the long life span 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 22,27–30 . For the present studies, to compare different ages of Tam induction, Cx3cr1 creERT 2 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.…”