DNA methylation and CpG sites just go together, so in fact well that sometimes we forget that DNA methylation can happen in other cytosine contexts too! With new advances in sequencing technologies, non-CpG methylation is stepping out of the shadows and being seen for the important role it plays. DNA methylation is particularly important in neurons during brain development. Large-scale studies of DNA methylation during brain development have usually involved using homogenate tissue, mixing brain cell types, and diluting neuron-specific changes. Studies that have purified human neurons have typically used microarrays, limiting the sites that are investigated and not giving a full picture of the methylome.
The lab of Andrew Jaffe at Johns Hopkins (USA) wanted to investigate the full extent of cytosine methylation changes during human cortical neuron development. They used whole genome bisulfite sequencing (WGBS) to characterized both CpG and non-CpG (CpH) methylation. Unlike most other tissues, CpH methylation is known to be abundant in neuronal cells. They profiled neurons (NeuN+) and glia (NeuN–) isolated from human dorsolateral prefrontal cortex (DLPFC) of postnatal samples and compared to whole-brain homogenate from prenatal cortical samples. [BL1] [EC2] They compared the diverse sample types by putting them into six groups based on age[BL3] [EC4] . In total, they assayed 58.1 million CpH cytosine sites with a coverage of at least 5x across all samples. Here’s what they found:
- Most CpH sites show low methylation levels, but this is higher than glia at 99% of sites.
- Most CpH methylation (mCpH) occurs in the CAG or
CAC context over the first 5 years of postnatal life, which then tapers into a
global DNA methylation increase in adulthood.
- K-means clustering identified six distinct, cell-type specific mCpH developmental trajectories
- During prenatal development, total mCAG increases 40% faster than mCAC in neurons, while in glia mCAG accumulates 50% slower
- In contrast to CpG methylation, CpH methylation is not highly correlated between neighbouring sites; however, when mCpG is included methylation levels across all nearby mC’s are correlated
- Gene expression in NeuN+ sorted [BL5] nuclear
RNA-seq data is negatively[BL6]
correlated with mCpH in promoters, gene bodies, and especially exons, all of which increase
in quantity with age[BL7]
- mCpH (and not mCpG) is associated with splicing events
- mCpG and mCpH differentially methylated regions (DMRs) associated with developmental time are enriched in genes involved in several neuropsychiatric disorders
CpH and CpG cytosine methylation appear to have distinct but converging roles in neurodevelopment. These analyses demonstrate the importance of cell-type specific assays to understand developmental DNA methylation dynamics. The expanded relationship between CpG methylation, CpH methylation, and gene expression described here should fuel further studies into these phenomena.
Go let your brain watch the full story develop in Genome Biology, September 2019