DNA methylation and hydroxymethylation characterize the identity of D1 and D2 striatal projection neurons

Marion‐Poll, Lucile; Roussarie, Jean-Pierre; Taing, Lieng; Dard-Dascot, Cloelia; Servant, Nicolas; Jaszczyszyn, Yan; Jordi, Emmanuelle; Mulugeta, Eskeatnaf; Hervé, Denis; Bourc’his, Déborah; Greengard, Paul; Thermes, Claude; Girault, Jean‐Antoine

doi:10.1038/s42003-022-04269-w

Cited by 6 publications

(3 citation statements)

References 94 publications

(108 reference statements)

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“…hmCG then demonstrated a positive association with expression in the gene body. While this relationship has been described in neurons [3,4,102], the relationship of hmCG to expression has not been characterized in astrocytes and microglia and provides strong evidence that hmCG is a genome regulator in glial cell types of the CNS as well as neurons. While no discernable associations between astrocytic and microglial mCH were clear, greater sequencing depth could help resolve this.…”

Section: Discussionmentioning

confidence: 99%

Differential usage of DNA modifications in neurons, astrocytes, and microglia

Tooley

Chucair-Elliott

Ocañas

et al. 2023

Preprint

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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.

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Section: Discussionmentioning

confidence: 99%

Differential usage of DNA modifications in neurons, astrocytes, and microglia

Tooley

Chucair-Elliott

Ocañas

et al. 2023

Preprint

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“…hmCG then demonstrated a positive association with expression in the gene body. While this relationship has been described in neurons [ 3 , 4 , 112 ], the relationship of hmCG to expression has not been characterized in astrocytes and microglia and provides strong evidence that hmCG is a genome regulator in glial cell types of the CNS as well as neurons. While no discernable associations between astrocytic and microglial mCH were clear, greater sequencing depth could help resolve this.…”

Section: Discussionmentioning

confidence: 99%

Differential usage of DNA modifications in neurons, astrocytes, and microglia

Tooley,

Chucair-Elliott,

Ocañas

et al. 2023

Epigenetics & Chromatin

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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. 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, DNA modifications in particular, 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 (WGoxBS) to assess the neuronal translatome and epigenome in the hippocampus of young mice (4 months old). WGoxBS findings were validated with enzymatic methyl-Seq (EM-Seq) and nanopore sequencing. Comparing neuronal data to microglial and astrocytic data from NuTRAP models, 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, rather than 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 gene body mCG and a positive relationship between distal promoter and gene body hmCG with gene expression was observed. Furthermore, we identified a neuron-specific inverse relationship between mCH and gene expression across promoter and gene body regions. Conclusions Neurons, astrocytes, and microglia demonstrate different genome-wide levels of mCG, hmCG, and mCH that are reproducible across analytical methods. However, modification-gene expression relationships are conserved across cell types. Enrichment of differential modifications across cell types in gene bodies and distal regulatory elements, but not proximal promoters, highlights epigenomic patterning in these regions as potentially greater determinants of cell identity. These findings also demonstrate the importance of differentiating between mC and hmC in neuroepigenomic analyses, as up to 30% of what is conventionally interpreted as mCG can be hmCG, which often has a different relationship to gene expression than mCG.

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“…These three studies also consistently reported differences in pathways related to nervous system development and synapse function, even in non-neural tissues. This link should be further explored, as small, sparse, and local differences in hydroxymethylation have been associated with gene expression ( Marion-Poll et al, 2022 ), potentially representing a “fine tuning” mechanism of gene regulation that is linked with transcription factor recruitment ( Lercher et al, 2014 ). Because the brain is the primary target organ of Pb toxicity, collective results may be revealing differences in hydroxymethylation patterns that underlie the link between developmental Pb exposure and later life neurotoxic effects.…”

Section: Discussionmentioning

confidence: 99%

Developmental exposures to common environmental contaminants, DEHP and lead, alter adult brain and blood hydroxymethylation in mice

Petroff

Cavalcante

Colacino

et al. 2023

Front. Cell Dev. Biol.

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Introduction: The developing epigenome changes rapidly, potentially making it more sensitive to toxicant exposures. DNA modifications, including methylation and hydroxymethylation, are important parts of the epigenome that may be affected by environmental exposures. However, most studies do not differentiate between these two DNA modifications, possibly masking significant effects.Methods: To investigate the relationship between DNA hydroxymethylation and developmental exposure to common contaminants, a collaborative, NIEHS-sponsored consortium, TaRGET II, initiated longitudinal mouse studies of developmental exposure to human-relevant levels of the phthalate plasticizer di(2-ethylhexyl) phthalate (DEHP), and the metal lead (Pb). Exposures to 25 mg DEHP/kg of food (approximately 5 mg DEHP/kg body weight) or 32 ppm Pb-acetate in drinking water were administered to nulliparous adult female mice. Exposure began 2 weeks before breeding and continued throughout pregnancy and lactation, until offspring were 21 days old. At 5 months, perinatally exposed offspring blood and cortex tissue were collected, for a total of 25 male mice and 17 female mice (n = 5–7 per tissue and exposure). DNA was extracted and hydroxymethylation was measured using hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq). Differential peak and pathway analysis was conducted comparing across exposure groups, tissue types, and animal sex, using an FDR cutoff of 0.15.Results: DEHP-exposed females had two genomic regions with lower hydroxymethylation in blood and no differences in cortex hydroxymethylation. For DEHP-exposed males, ten regions in blood (six higher and four lower) and 246 regions (242 higher and four lower) and four pathways in cortex were identified. Pb-exposed females had no statistically significant differences in blood or cortex hydroxymethylation compared to controls. Pb-exposed males, however, had 385 regions (all higher) and six pathways altered in cortex, but no differential hydroxymethylation was identified in blood.Discussion: Overall, perinatal exposure to human-relevant levels of two common toxicants showed differences in adult DNA hydroxymethylation that was specific to sex, exposure type, and tissue, but male cortex was most susceptible to hydroxymethylation differences by exposure. Future assessments should focus on understanding if these findings indicate potential biomarkers of exposure or are related to functional long-term health effects.

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DNA methylation and hydroxymethylation characterize the identity of D1 and D2 striatal projection neurons

Cited by 6 publications

References 94 publications

Differential usage of DNA modifications in neurons, astrocytes, and microglia

Differential usage of DNA modifications in neurons, astrocytes, and microglia

Differential usage of DNA modifications in neurons, astrocytes, and microglia

Developmental exposures to common environmental contaminants, DEHP and lead, alter adult brain and blood hydroxymethylation in mice

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