Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

Ana J Chucair-Elliott,Arlan Richardson,Kyla B Buettner,Victor A Ansere,Justin J Reid,Sarah R Ocañas,Amanda L Sharpe,Nicole L Eliason,Michael B Stout,Willard M Freeman,David R Stanford,Benjamin F Miller,Michael J Beckstead,Hunter Porter

doi:10.1038/s42003-020-01418-x

Abstract

Epigenetic regulation of gene expression occurs in a cell type-specific manner. Current cell-type specific neuroepigenetic studies rely on cell sorting methods that can alter cell phenotype and introduce potential confounds. Here we demonstrate and validate a Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) approach for temporally controlled labeling and isolation of ribosomes and nuclei, and thus RNA and DNA, from specific central nervous system cell types. Analysis of gene expression and DNA modifications in astrocytes or microglia from the same animal demonstrates differential usage of DNA methylation and hydroxymethylation in CpG and non-CpG contexts that corresponds to cell type-specific gene expression. Application of this approach in LPS treated mice uncovers microglia-specific transcriptome and epigenome changes in inflammatory pathways that cannot be detected with tissue-level analysis. The NuTRAP model and the validation approaches presented can be applied to any brain cell type for which a cell type-specific cre is available.

Highlights

Epigenetic regulation of gene expression occurs in a cell type-specific manner
Single-cell suspensions of brains immunostained with ACSA-2 antibody, a pan-astrocytic marker[15], revealed a distinct EGFP+ cell population present in the Aldh1l1-Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) brains but not in the crenegative counterparts, consistent with the reported 10–20% astroglial cellularity in the rodent brain[16,17]
This highlights the importance of studying DNA modifications in a cell-type-specific manner and the value of the Cx3cr1-NuTRAP model to study the relationship between microglia genomic methylation and transcriptome

Summary

Results

The distribution of cell-type-specific gene expression showed enrichment of astrocytic genes and depletion of microglial, neuronal, and oligodendrocytic genes in the positive fraction relative to input (Fig. 3c, d). Additional comparisons of enrichment distribution of gene expression between the different ribosomal profiling methods and gene marker lists from cell-sorting studies were performed (Supplementary Fig. 3) Taken together, these comparisons demonstrate a commonality to astrocyte enriched genes with some minor differences in RiboTag versus NuTRAP and Aldh1l1 versus Gfap cre lines. Gene expression between the different microglia ribosomal profiling methods and gene marker lists from cell-sorting studies demonstrated high level of concurrence between our model and approach and that of other research groups (Fig. 7e–g, Supplementary Fig. 8). Transcriptome comparison between Aldh1l1-NuTRAP and Cx3cr1-NuTRAP positive fractions by regulator and pathway analyses confirmed cell-specific enrichments in agreement with brain astrocytes and microglia, respectively (Supplementary Fig. 9).

10 Astrocytic Oligodendrocytic Neuronal

Discussion

Methods