Differential usage of DNA modifications in neurons, astrocytes and microglia [RNA-seq]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE228043 | GEO | 2023/09/22
REPOSITORIES: GEO
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Project description:Cellular identity is determined, in part, by cell type-specific epigenomic profiles that govern gene expression. Isolation and epigenomic characterization of specific cell types is greatly needed in neuroscience. While single cell DNA modification studies offer promise to answer this question, these approaches suffer from limited genomic coverage and cannot differentiate between methylation and hydroxymethylation. Thus, approaches to isolate and analyze DNA from specific CNS cell populations are needed. Here we validate a temporally controlled in vivo nuclear tagging mouse model (NuTRAP) and isolation (INTACT) of neuronal nucleic acids. Analysis of differential DNA modifications (methylation and hydroxymethylation) and gene expression in neurons, astrocytes, and microglia demonstrates a common regulatory function for DNA modifications that transcends cell type. Insight into the normal function of DNA modifications in genomic regulation is crucial in order to better understand the epigenomic mechanisms underlying disease.
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Project description:Studies of neuroepigenetic mechanisms in health and disease are hindered by a lack of approaches to analyze both the transcriptome and epigenome of specific cell types isolated from the complex milieu of the CNS. Cell isolation by cell surface markers is complicated by preparation artifacts, changes in markers with experimental conditions, and lack of specific markers. This study validates a Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) approach with tamoxifen (Tam) inducible cell-type specific cre recombination to allow the parallel interrogation of the epigenome and the transcriptome in astrocytes (Aldh1l1-creERT2) or microglia (Cx3cr1-creERT2). The recombined NuTRAP construct labels, in a cell-type specific manner, nuclei (RanGAP1) with biotin and mCherry, and the ribosomes (L10a) with EGFP, enabling INTACT isolation of DNA and TRAP isolation of RNA. Validation experiments by flow cytometry and imaging demonstrate cell-type specific induction of the NuTRAP construct. Transcriptomic studies demonstrate isolation of highly enriched RNA by TRAP and oxidative bisulfite studies of INTACT-isolated DNA demonstrate differential DNA modification patterns in microglia and astrocytes. LPS administration in Cx3cr1 NuTRAP mice demonstrates that microglia-specific transcriptome and epigenome changes are revealed that cannot be detected with tissue-level samples. These experiments demonstrate that the NuTRAP approach can be applied to CNS cell populations and that INTACT approaches can be used to study DNA modifications. These results also provide an approach for generation and validation of NuTRAP neuroscience models crossed to any relevant cell-type specific cre line.
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Project description:Studies of neuroepigenetic mechanisms in health and disease are hindered by a lack of approaches to analyze both the transcriptome and epigenome of specific cell types isolated from the complex milieu of the CNS. Cell isolation by cell surface markers is complicated by preparation artifacts, changes in markers with experimental conditions, and lack of specific markers. This study validates a Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) approach with tamoxifen (Tam) inducible cell-type specific cre recombination to allow the parallel interrogation of the epigenome and the transcriptome in astrocytes (Aldh1l1-creERT2) or microglia (Cx3cr1-creERT2). The recombined NuTRAP construct labels, in a cell-type specific manner, nuclei (RanGAP1) with biotin and mCherry, and the ribosomes (L10a) with EGFP, enabling INTACT isolation of DNA and TRAP isolation of RNA. Validation experiments by flow cytometry and imaging demonstrate cell-type specific induction of the NuTRAP construct. Transcriptomic studies demonstrate isolation of highly enriched RNA by TRAP and oxidative bisulfite studies of INTACT-isolated DNA demonstrate differential DNA modification patterns in microglia and astrocytes. LPS administration in Cx3cr1 NuTRAP mice demonstrates that microglia-specific transcriptome and epigenome changes are revealed that cannot be detected with tissue-level samples. These experiments demonstrate that the NuTRAP approach can be applied to CNS cell populations and that INTACT approaches can be used to study DNA modifications. These results also provide an approach for generation and validation of NuTRAP neuroscience models crossed to any relevant cell-type specific cre line.
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