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

胚胎干细胞的定向分化依赖于种群内的异质性，在这种异质性中，细胞状态的自然波动可以通过分化线索来增强或抑制。在拟议的项目中，我想研究从小鼠ESCs到EpiLCs(表皮样细胞)过渡过程中的细胞群体异质性，EpiLCs是一种植入发育的细胞培养模型。在这一发育阶段，多能细胞为分化做好准备，基因组DNA甲基化模式建立。初步数据表明，哺乳动物DNA去甲基化机制的关键成员，双加氧酶TET1和TET2以及DNA糖基酶TDG，在建立和维持细胞群体异质性方面发挥着关键作用。将在可诱导的TDG基因敲除细胞系中研究细胞间的变异，其中TDG基因可以通过在细胞培养基中添加他莫昔芬来移除。此外，Tet1/TET2/TDG缺陷的ESCs将被包括在分析中，以剖析DNA甲基化和修复活动的氧化影响。将研究种群异质性的几个方面，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测序来分析转录异质性。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类(FACS)进行评估。第三，将识别和隔离总种群的同质亚群。在这些亚群中，DNA甲基化含量将通过RRBS(简化表示亚硫酸氢盐测序)进行定位和量化。最后，细胞培养系统的结果将在体内得到验证，以研究它们在早期胚胎发育中的作用。上述实验的结果将阐明Tet/TDG介导的DNA去甲基化如何促进细胞群体的异质性，以及这种异质性是否对ESC到EpiLC的转变和随后的分化是必要的。总而言之，所获得的数据将极大地加强目前对干细胞表观遗传异质性的调节和动态及其在分化中的作用的理解。

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.