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

细胞分化是由某些基因的开启或关闭所控制的。胞嘧啶的甲基化有助于基因活性的调节。在分化过程中，旧的DNA甲基化模式（5-甲基胞嘧啶，5mC）被清除，新的模式被建立，但5mC是如何从DNA中去除的尚不清楚。我们发现5mC在早期大脑发育过程中被特异性修饰，导致其随后从神经元和神经胶质细胞的某些DNA区域移除，这表明DNA去甲基化的特定机制。这很可能有助于未分化细胞重编程为成熟的神经元和神经胶质细胞。WT1 （Wilms' tumor 1）是一种蛋白质，根据一些报道和我们的初步数据，它可能与5mC的这种修饰形式相互作用，可能对大脑发育很重要。本提案的目的是了解胚胎脑中WT1与修饰5mC之间潜在相互作用的生物学意义。我们建议以小鼠胚胎干细胞（mESCs）为模型研究这一过程的机制。我们将把mESCs分化成胶质细胞谱系，并将鉴定在胶质细胞分化过程中被修饰的基因。此外，我们将这些基因与在胶质细胞分化过程中受WT1调控的基因进行比较。最后，我们将测试WT1和参与5mC修饰的蛋白质的消耗如何影响胶质细胞分化。拟议中的研究项目将对基础科学、癌症研究和再生医学产生多重影响。

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.