Cuomo, Mariella (2021) DNA methylation dynamics at genes specific level during brain development and in schizophrenia. [Tesi di dottorato]

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Defined epigenetic modifications occurring during brain development may play a fundamental role on brain function. An alteration in the establishment of correct DNA methylation at specific genes has been associated with neuropsychiatric disorders. In this regard, during perinatal period DNA methylation may finely control genes regulating brain levels of critical neuromodulators such as D-Serine and D-Aspartate. Since levels of these D- amino acids have been found altered in some mental disorders such as schizophrenia, the lack of an epigenetic control may contribute to the genesis and/or progression of these diseases. Thus, during my PhD, I performed a comprehensive DNA methylation analysis along with mRNA expression at DAO and DDO genes, involved in the degradation of D-Serine and D-Aspartate, respectively. I performed the analyses in mice during development and in post- mortem tissues of patients with schizophrenia. I evaluated DNA methylation using amplicon bisulfite sequencing on Illumina MiSeq platform and I also performed an in-depth single molecule methylation approach in order to assess the cell to cell methylation heterogeneity. I found strong spatiotemporal changes in DNA methylation at the DAO gene during development, especially in cerebellar astrocytes and particularly at two specific CpG sites. These CpGs at DAO gene promoter showed high degree of hydroxymethylation at post-natal day 1 and at, post-natal day 15 the global levels of DNA methylation AND hydroxymethylation dramatically decreased. This demethylation strongly activated DAO gene expression, indirectly promoting the physiological degradation of cerebellar D-serine. The same mechanisms emerged at DDO promoter in cerebellum, where two CpG sites were demethylated during development activating the expression of the gene. Furthermore, both in mouse brain during development and in post-mortem brain tissues, the applied single- molecule methylation approach demonstrated that epiallele distribution was able to detect differences in DNA methylation representing area-specific methylation 6 signatures, which are likely not detectable with targeted or genome-wide methylation analyses. The present study demonstrates that D-Serine and D- Aspartate levels during brain development are indirectly regulated by DNA methylation that govern the expression of DAO and DDO genes. Furthermore, single-molecule methylation approach promises to identify different cell-type composition and function in different brain areas and developmental stages. Overall, these analyses demonstrate that at selected genes, epiallele-based analyses may be very informative and can be successfully utilized in a broad range of applications, including in depth determination of epigenetic origin of brain diseases.

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