Cell culture-generated neurons show non-physiological DNA methylation signatures

Patterns of cytosine methylation in genomic DNA occur in a cell-specific manner and thereby are highly effective markers for a cell’s identity, with DNA methylation being associated with transcriptional repression in certain contexts. In most cells in mammals, DNA methylation is predominantly found in the CG sequence context (mCG), where the majority of CG dinucleotides are methylated. Interestingly, in the central nervous system, especially in neurons, DNA methylation is also found in non-CG context (mCH, where H = A, C or T), which increases from birth to adulthood and ultimately becomes the most abundant form of methylcytosine in the genome of adult neurons. Since these brain-specific mCH patterns are established in the first weeks after birth, coinciding with a wave of synaptogenesis, we queried whether mCH would be a suitable indicator for the maturation status of neurons. As neuronal cell culture systems are widely used to study brain physiology and disease mechanisms, a consistent marker for mature neurons would greatly improve the usability of such cell culture systems. We used whole genome bisulfite sequencing to define the methylation state of mouse cell cultures, starting from ES cells to mature neurons, and compared it to in vivo datasets of different developmental stages. Levels of mCH similar to adult brain were first seen in cell culture-generated neurons at day 38. Deep sequencing revealed that although global methylation values are comparable, mCH patterns are dramatically different throughout the genome. While neurons generated in cell culture are similar to embryonic brain neurons in the CG context, their CH methylation patterns do not resemble any population or time point found in vivo. Differentially methylated regions are enriched for genes involved in neurodevelopment and maturation or are associated with disorders of the autism-spectrum. We postulate that differences in CH methylation may affect the ability of cell-culture generated neurons to fully mature to a state comparable to the situation in vivo.