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

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.

Minimizing the ex vivo confounds of cell-isolation techniques on transcriptomic -profiles of purified microglia

“…While in vitro cell culture models are useful for mechanistic studies, they fail to recapitulate the complexity of the nervous system milieu. As such, models and methods to 'debulk' microglia from brain tissue have become a focus in the field (Chucair-Elliott et al, 2020;McKinsey et al, 2020). The most common methods for isolating microglia include enzymatic and/or mechanical dissociation of brain tissue followed by immunolabeling with magnetic beads (Bordt et al, 2020) or fluorescent-conjugated antibodies for FACS-based enrichment (Bohlen, Bennett, & Bennett, 2019).…”

“…The copyright holder for this preprint (which this version posted July 15, 2021. ; https://doi.org/10.1101/2021.07.15.452509 doi: bioRxiv preprint Following cell preparation and isolation using methods displayed in Figure 1A, RNA was isolated from cells for preparation of stranded RNA-Seq libraries. We first examined enrichment/depletion of previously published microglial, astrocytic, oligodendrocytic, neuronal, and endothelial markers in the transcriptomic profiles (Chucair-Elliott et al, 2020;McKenzie et al, 2018) (Supplemental Table 1). Each of the four sort methods showed similar levels of enrichment of microglial marker genes (Figure 2A) and depletion of astrocytic, oligodendrocytic, neuronal, and endothelial marker genes (Figure 2B-E) when compared to cell input.…”

“…The copyright holder for this preprint (which this version posted July 15, 2021. ; https://doi.org/10.1101/2021.07.15.452509 doi: bioRxiv preprint Directional RNA-Seq libraries were made from 5-100 ng RNA, as previously described (Chucair-Elliott et al, 2020;Chucair-Elliott et al, 2019). Briefly, poly-adenylated RNA was captured using NEBNext Poly(A) mRNA Magnetic Isolation Module (#NEBE7490L; New England Biolabs, Ipswich, MA) and then processed using NEBNext Ultra II Directional Library Prep Kit for Illumina (#NEBE7760L; New England Biolabs) for the creation of cDNA libraries, according to the manufacturer's instruction.…”

“…Ex vivo microglial activation has the potential of introducing confounds that may mask endogenously induced activation, such as in a pathological state. To avoid cell-isolation confounds, microglial-specific cre-lines (Cx3cr1-Cre) have been combined with various floxed ribosomal tagging models: 1) ribosome tagging (RiboTag) , 2) translating ribosome affinity purification (TRAP) (Ayata et al, 2018), and 3) nuclear tagging and translating ribosome affinity purification (NuTRAP) (Chucair-Elliott et al, 2020), allowing the immunoprecipitation (IP) of tagged polysomes to isolate microglial-specific translatomes without the need for cell isolation. Although transgenic ribosome IP-approaches overcome many of the potential confounds of ex vivo activation, experimental endpoints such as proteomics and single cell heterogeneity still require cell isolation of intact microglial cells.…”

“…The previously described Cx3cr1-NuTRAP line (Chucair-Elliott et al, 2020) was used in all comparisons of isolation efficacy of various cell sorting techniques including high-and lowpressure fluorescence-activated cell sorting (FACS) as well as magnetic-activated cell sorting (MACS) isolation. We compared artifacts induced through three cell sorting techniques via transcriptomic profiling of bulk tissue, sorted cells, and immunoprecipitated translatomes and found similar performance with ex vivo activational signatures principally occurring during the enzymatic digestion and mechanical dissociation during initial cell preparation.…”