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

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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.