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

TDG依赖性主动DNA去甲基化对建立胚胎干细胞群体异质性和分化的影响

• 批准号: 396808758
• 负责人: Dr. Christina Bauer
• 金额: --
• 依托单位: Research Group Genome Plasticity (Schaer Lab)
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/396808758?language=en
• 关键词: impact; TDG; dependent; active; DNA

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.

胚胎干细胞（ESC）的定向分化被认为依赖于群体内的异质性，其中细胞状态的自然波动可以通过分化线索得到加强或抑制。在拟议的项目中，我想研究从小鼠 ESC 向 EpiLC（外胚层样细胞）（一种植入发育的细胞培养模型）转变过程中的细胞群异质性。在这个发育步骤中，多能细胞已做好分化准备，并建立了基因组 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 TET1 和 TET2 以及 DNA 糖基化酶 TDG 在细胞群异质性的建立和维持中发挥着关键作用。将在可诱导的 Tdg 敲除细胞系中研究细胞间的变异，其中可以通过在细胞培养基中添加他莫昔芬来去除 Tdg 基因。此外，Tet1/Tet2/Tdg 缺陷的 ESC 将被纳入分析中，以剖析 DNA 甲基化氧化和修复活动的影响。将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，通过单细胞 RNA 测序分析转录异质性。其次，将通过免疫荧光染色和荧光激活细胞分选（FACS）来评估蛋白质表达水平的变化。第三，将识别并隔离总人口中的同质亚群。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）进行绘制和定量。最后，细胞培养系统获得的结果将在体内得到验证，以研究它们在早期胚胎发育过程中的作用。所描述的实验结果将揭示TET/TDG介导的DNA去甲基化如何促进细胞群异质性以及这种异质性是否对于ESC向EpiLC转变和随后的分化至关重要的问题。总之，所获得的数据将显着增强目前对干细胞表观遗传异质性的调控和动态及其在分化中的作用的理解。