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

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.

Our previous results suggest that both active demethylation and 5caC-dependent transcriptional regulation are involved in epigenetic reprogramming taking place during lineage specification of NSCs. WT1 protein has also been recently implicated into both recruiting Tet proteins to their target sequences and specific recognition of 5caC-containing DNA substrates. Moreover, our pilot experiments revealed that WT1 regulates the same key glial markers which transiently accumulate 5caC in differentiating NSCs. There may be 2 main modes of WT1 interaction with the DNA methylome in embryonic brain: (1) WT1 may participate in recruiting TET2 and/or TET3, which both are expressed in embryonic brain, to their respective target sequences and/or (2) WT1 may directly bind to 5caC-enriched promoters causing activation or repression of corresponding genes.

The aim of this proposal is to acquire a mechanistic insight into the roles of Tet-dependent DNA demethylation and WT1 transcriptional regulation and their potential interplay during glial differentiation.

Specifically, we propose (1) to determine the distribution of oxi-mCs during glial differentiation and potential dependence of active demethylation on recruiting Tet proteins by WT1, (2) to compare the patterns of genomic distribution of WT1 with those of oxi-mCs in differentiating NSCs addressing the possibility that this transcriptional factor directly interacts with 5caC-enriched DNA and (3) to identify potential impacts of Tet-dependent 5mC oxidation and WT1 transcriptional regulation on glial differentiation.

Understanding the roles of both DNA demethylation and WT1 in glial differentiation would have multiple implications for basic research in the fields of epigenetics, developmental biology, neuroscience, regenerative medicine and cancer biology.

This work will directly address the BBSRC priorities of "Healthy ageing across the lifecourse", "Data driven biology" and "Systems approaches to the biosciences".

Our work will contribute to advancing the areas of regenerative medicine, health science, pharmaceutical industry and biotechnology. We identify the following groups of stakeholders who will directly benefit from the outcomes of our research:

1. Stem cell technology and biotechnology companies. Our research will get an insight into epigenetic reprogramming taking place during lineage specification in embryonic brain. This will expand understanding of the mechanisms of neural and glial lineage specification in mammlian brain, which will lead to modification and refinement of existing protocols of ESCs differentiation and potentially will provide new strategies for differentiation of ESCs into specific lineages.

2. Clinicians working in the field of cancer, particularly in the field of pathogenesis of brain tumours. Since both the levels of oxi-mCs and expression of WT1 are altered in a number of cancers, including gliomas, our findings will facilitate the development of novel cancer treatments and diagnostic approaches for brain tumours.

3. Clinical neurologists and psychiatrists. As emerging evidence suggests important roles for 5hmC and Tet proteins in the development of certain psychiatric conditions (e. g. schizophrenia, bipolar disorder) our work will contribute to decoding the mechanisms of the pathogenesis of these disorders and will advance the establishment of novel approaches for their identification and therapy.