Studying potential interplay between active demethylation and WT1-dependent transcriptional regulation during glial differentiation.

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Cellular differentiation is governed by the switching on or off of certain genes. Methylation of cytosine contributes to the regulation of gene activity. Old patterns of DNA methylation (5-methylcytosine, 5mC) are erased and new ones are established during differentiation but it is still unclear how 5mC is removed from DNA. We showed that 5mC is being specifically modified during early brain development, which leads to its subsequent removal from certain regions of DNA in neurons and glial cells, suggesting a specific mechanism for DNA demethylation. This, most likely, contributes to the reprogramming of undifferentiated cells into mature neurons and glial cells. WT1 (Wilms' tumour 1) is a protein, which, according to several reports and our preliminary data, may interact with this modified form of 5mC and may be important for brain development. The aim of this proposal is to understand the biological significance of potential interplay between WT1 and modified 5mC taking place in embryonic brain. We propose to study the mechanisms of this process using mouse embryonic stem cells (mESCs) as a model. We will differentiate mESCs into glial lineages and will identify the genes which are being modified during glial differentiation. Moreover, we will compare these genes with the genes which are regulated by WT1 during glial differentiation. Finally we will test how the depletion of WT1 and proteins involved into the 5mC modification affect glial differentiation. The proposed research program would have multiple implications for basic science, cancer research and regenerative medicine.