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

Abstract

23Epigenetic regulation of gene expression occurs in a cell type-specific manner. Current cell-type 24 specific neuroepigenetic studies rely on cell sorting methods that can alter cell phenotype and 25 introduce potential confounds. Here we demonstrate and validate a Nuclear Tagging and 26Translating Ribosome Affinity Purification (NuTRAP) approach for temporally controlled labeling 27 and isolation of ribosomes and nuclei, and thus RNA and DNA, from specific CNS cell types. 28Paired analysis of the transcriptome and DNA modifications in astrocytes and microglia 29 demonstrates differential usage of DNA methylation and hydroxymethylation in CG and non-CG 30 contexts that corresponds to cell type-specific gene expression. Application of this approach in 31 LPS treated mice uncovers microglia-specific transcriptome and epigenome changes in 32 inflammatory pathways that cannot be detected with tissue-level analysis. The NuTRAP model 33 and the validation approaches presented can be applied to any CNS cell type for which a cell 34 type-specific cre is available. 36Significant advances are being made in understanding the epigenome and its relationship with 37 gene expression in the brain 1-3 . However, the lack of approaches for paired analysis of DNA and 38RNA profiles at the cell type-specific level within the same animal is a significant limitation for the 39 field, given that epigenetic processes differ across CNS cell types at the level of chromatin 40 organization and DNA modifications 1,4 . Obtaining enriched cell populations by flow sorting 41 requires cell surface markers but these markers can change with experimental conditions and cell 42 sorting causes molecular, morphological, and functional changes, such as cell activation, that 43 could confound studies 3,5,6 . Single cell approaches 7 may overcome some of the challenges of 44 cell sorting but the scale of such studies, partial genomic coverage, restriction to only certain types 45 of endpoints, and continued potential for brain dissociation artifacts are limitations. 46This has led to development of transgenic labeling approaches to isolate RNA or DNA from 47 specific cell types. Ribosome labeling and RNA isolation methods, such as Translating Ribosome 48Affinity Purification (TRAP 8 ), and ribosome tagging (RiboTag 9 ), are gaining acceptance across 49 neuroscience studies examining the transcriptome. Similar approaches have been developed to 50 transgenically tag and allow isolation of nuclei and thus DNA (Isolation of Nuclei TAgged in 51 Specific Cell Types, INTACT) 10 . However, using separate transgenic mouse strains for DNA and 52RNA endpoints is a complicated and resource intensive approach. 53Here we describe an approach where Nuclear Tagging and Translating Ribosome Affinity 54Purification (NuTRAP) 11 is combined with well-established cell-specific inducible cre-recombinase 55 expressing systems 12,13 to perform paired transcriptomic and epigenomic analyses of specific 56 CNS cell types in a temporally controllable manner from a single mouse. ...

The Expression of Interleukin-1β Is Regulated by DNA Methylation in Microglia of Hippocampus to Participate Postoperative Cognitive Dysfunction in Aged Mice

“…A study using microglia isolated from LPStreated mice to explore the changes of IL-1β and its DNA methylation is consistent with ours [18]. Further study by using of transgenic inducible cre mouse models that allow for manipulation of speci c oxed genes, or tagging of cell-speci c nuclei/and or polysomes represent valuable research tools is needed [75]. Interestingly, we also found that global DNA methylation of dorsal hippocampus was unchanged, suggesting that perhaps DNA hypomethylation is not a global phenomenon.…”

Early life exercise primes the neural epigenome to facilitate gene expression and hippocampal memory consolidation

“…Previous methods for performing INTACT and TRAP using NuTRAP mice have taken separate tissue homogenates for each procedure [28, 38]. In this study, we performed simultaneous isolation of nuclei and RNA from a single tissue homogenate containing bilateral hippocampi (referred to as “simultaneous isolations”).…”

“…Roh et al first developed the NuTRAP mouse to characterize adipocyte-specific and hepatocyte-specific epigenetic and transcriptional profiles [29]. The NuTRAP mouse has since been used to perform paired transcriptome and DNA modification analysis in astrocytes and microglia, demonstrating its utility in CNS cell-types [38]. The Emx1-NuTRAP mouse described here provides rapid, cost-effective characterization of neuron-specific changes to epigenetic state and gene expression using whole-tissue homogenates with heterogeneous cell types.…”

“…A common approach to paired transcriptomic and epigenomic analyses is to use separate brain tissue from one hemisphere for gene expression studies and the other side for epigenetic analyses [74]. A previous study utilizing the NuTRAP allele in non-neuronal brain cells used separate brain hemispheres to perform INTACT and TRAP [75]. However, this assumes that cells of the same type from each half of the tissue will show the same epigenetic and transcriptional profile.…”

Minimizing the Ex Vivo Confounds of Cell-Isolation Techniques on Transcriptomic and Translatomic Profiles of Purified Microglia

“…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/ERT2) have been combined with various floxed ribosomal tagging models: 1) ribosome tagging (RiboTag) (Haimon et al, 2018), 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.…”