Abstract
DNA methylation plays essential roles in various cellular processes. Next-generation sequencing has enabled us to study the functional implication of DNA methylation across the whole genome. However, this approach usually requires a substantial amount of genomic DNA, which limits its application to defined cell types within a discrete brain region. Here, we applied two separate protocols, Accel-NGS Methyl-Seq (AM-seq) and Enzymatic Methyl-seq (EM-seq), to profile the methylome of D2 dopamine receptor-expressing medium spiny neurons (D2-MSNs) in mouse nucleus accumbens (NAc). Using 40 ng DNA extracted from FACS-isolated D2-MSNs, we found that both methods yielded comparably high-quality methylome data. Additionally, we identified numerous unmethylated regions (UMRs) as cell type-specific regulatory regions. By comparing the NAc D2-MSN methylome with the published methylomes of mouse prefrontal cortex excitatory neurons and neural progenitor cells (NPCs), we identified numerous differentially methylated CpG and non-CpG regions. Our study not only presents a comparison of these two low-input DNA whole genome methylation profiling protocols, but also provides a resource of DNA methylome of mouse accumbal D2-MSNs, a neuron type that has critical roles in addiction and other neuropsychiatric disorders.
Highlights
DNA methylation is an epigenetic mechanism that plays important roles in gene regulation and genome stability [1]
We found that the 13,550 genomic regions in the “CpG islands (CGIs) within unmethylated regions (UMRs)” category covered a broad range of genes and biological pathways (Figure S4b, Table S7), whereas the other three categories were enriched with limited numbers of specific pathways
By comparing the methylation-related regulatory regions that we identified as UMRs and CpG Differential Methylation Region (DMR) with the curated set of mouse candidate cis-regulatory elements from the ENCODE project [86], we found that almost all UMRs overlapped with one or more cCRE
Summary
DNA methylation is an epigenetic mechanism that plays important roles in gene regulation and genome stability [1]. DNA cytosine methylation is vital for development and cell fate commitment, cell identity maintenance, and various other cellular functions [2–9]. Methylation-sensitive restriction enzymes are used to infer the methylation status of genomic loci based on their differential enzymatic cutting activity at DNA motifs containing methylated or unmethylated cytosines [12]. Antibodies against methylated cytosines have been developed to examine methylation status by immunostaining or by immunoprecipitation followed by sequencing [8,13,14]. Sodium bisulfitebased techniques can be used to differentiate methylated and unmethylated states of DNA, as sodium bisulfite deaminates unmethylated cytosines into uracils while leaving methylated ones intact [15,16]. Combining bisulfite treatment and next-generation sequencing, allows for DNA methylation profiling across the entire genome with single-base resolution and in a quantitative manner [17–21]
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