The impact of TDG-dependent active DNA demethylation on establishing population heterogeneity in embryonic stem cells and differentiation

胚胎干细胞（ESCs）的定向分化依赖于群体内的异质性，其中细胞状态的自然波动可以被分化线索重新加强或抑制。在拟议的项目中，我想研究从小鼠胚胎干细胞向上皮样细胞（epiblast-like cells，一种着床发育的细胞培养模型）转化过程中的细胞群体异质性。在这个发育步骤中，多能细胞开始分化，并建立基因组DNA甲基化模式。初步数据表明，哺乳动物DNA去甲基化机制的关键参与者，双加氧酶TET 1和TET 2以及DNA糖基化酶TDG，在建立和维持细胞群体异质性中起着关键作用。将在诱导型Tdg敲除细胞系中研究细胞间的变异，其中可以通过向细胞培养基中添加他莫昔芬来去除Tdg基因。此外，Tet 1/Tet 2/Tdg缺陷型ESC将被纳入分析，以剖析DNA甲基化氧化和修复活性的影响。研究群体异质性的几个方面，以全面了解TDG和DNA去甲基化系统的具体作用。首先，将通过单细胞RNA测序分析转录异质性。其次，蛋白质表达水平的变化将通过免疫荧光染色和荧光激活细胞分选（FACS）来评估。第三，总群体的同质亚群将被识别和分离。在这些亚群中，DNA甲基化含量将通过RRBS（简化代表性亚硫酸氢盐测序）进行定位和定量。最后，将在体内验证所获得的细胞培养系统的结果，以研究它们在早期胚胎发育过程中的作用，所描述的实验的结果将阐明泰特/TDG介导的DNA去甲基化是如何导致细胞群体异质性的，以及这种异质性是否是ESC向EpiLC转化和随后分化所必需的。总之，所获得的数据将显着提高目前的理解有关的调控和干细胞的表观遗传异质性及其在分化中的作用。

Directed differentiation of embryonic stem cells (ESCs) has been proposed to rely on intra-population heterogeneity, where natural fluctuations of cellular states can be re-inforced or inhibited by differentiation cues. In the proposed project, I want to study cell population heterogeneity during transition from murine ESCs to EpiLCs (epiblast-like cells), a cell culture model of implantation development. At this developmental step, pluripotent cells become primed towards differentiation and genomic DNA methylation patterns are established. Preliminary data suggest that key players of the mammalian DNA demethylation machinery, the dioxygenases TET1 and TET2 and the DNA glycosylase TDG, play a pivotal role in establishment and maintenance of cell population heterogeneity. Cell-to-cell variations will be studied in an inducible Tdg knock out cell line, where the Tdg gene can be removed by addition of tamoxifen to the cell culture medium. Additionally, Tet1/Tet2/Tdg-deficient ESCs will be included in the analysis to dissect the impacts of oxidation of DNA methylation and repair activities. Several aspects of population heterogeneity will be studied to gain a comprehensive understanding about the specific role of TDG and the DNA demethylation system. First, transcriptional heterogeneity will be analysed by single-cell RNA sequencing. Second, variations in protein expression levels will be assessed by immunofluorescence stainings and fluorescence-activated cell sorting (FACS). Third, homogenous subpopulations of the total population will be identified and isolated. In these subpopulations, DNA methylation content will be mapped and quantified by RRBS (reduced-representation bisulfite sequencing). Finally, the obtained results of the cell culture system will be verified in vivo to study their role during early embryonic development.The outcome of the described experiments will shed light onto the question how TET/TDG-mediated DNA demethylation contributes to cell population heterogeneity and if this heterogeneity is essential for the ESC-to-EpiLC transition and subsequent differentiation. In summary, the obtained data will significantly enhance the current understanding about the regulation and dynamics of epigenetic heterogeneity of stem cells and its role in differentiation.