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Title: Nanog-Tcf15 axis during exit from naïve pluripotency
Author: Tatar, Tülin
ISNI:       0000 0004 7429 4056
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2018
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Pluripotent cells have the dual abilities to self-renewal and to differentiate into all three germ layers. Pluripotent cells can be isolated from two different stages of mouse embryogenesis. Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of the pre-implantation embryo and are considered to be in a naïve state. On the other hand, cells isolated from epiblast of the post-implantation embryo are referred as epiblast stem cells (EpiSC) and are representative of primed pluripotency. ESCs and EpiSCs are distinct from each other in terms of the morphology, the gene regulatory network and the signalling pathways regulating self-renewal. Under certain conditions, ESCs and EpiSCs can be transitioned into each other. However, the mechanism that regulates this transition from naïve to primed pluripotent state remains to be solved. Nanog, Oct4 and Sox2 form the core gene regulatory network of pluripotency. Additionally, the Id protein family is also important in the maintenance of pluripotency in ESCs. Id proteins function by inhibiting the activity of pro-differentiation factors. Tcf15 is identified as one of the targets of Id inhibition in ESCs. Moreover, Tcf15 has been identified as a repression target of Nanog. In this study, to understand the function of Tcf15, the expression of Tcf15 was characterized in differentiating ESCs. The transient upregulation of Tcf15 mRNA and protein was detected at early stages of differentiation before lineage commitment. Furthermore, Tcf15 protein was heterogeneously expressed in differentiating cells. Mutually exclusive expression of Nanog and Tcf15 proteins were demonstrated in both self-renewing and differentiating ESCs. Further characterization of the effect of Nanog on Tcf15 transcription showed that Tcf15 pre-mRNA was downregulated within 20 minute of Nanog induction. A Nanog binding site was identified at +32kb relative to the Tcf15 transcription start site (TSS). Initially, Nanog binding at this region was confirmed by performing ChIP-PCR experiments. Then, this Nanog binding region was further analysed for its enhancer activity related to the Tcf15 gene. Deletion of the Nanog binding region using CRISPR-Cas9 confirmed that this region acts as Tcf15 enhancer; it was shown that this region was required for the activation of Tcf15 transcription during differentiation. Tcf15 induction experiments were performed in order to the check whether Tcf15 affects Nanog transcription. The results indicate that Nanog is not a direct target of Tcf15, but Tcf15 contributes indirectly to the repression of Nanog. Additional analysis with the Tcf15 enhancer deletion cells showed that Tcf15 is not required for efficient downregulation of naïve markers and the upregulation of primed markers. However, the genes related to the regulation of adhesion properties of cells such as Zyc, Itga3 were induced with lower efficiency in the absence of Tcf15 compared to the wild type cells. In summary, I investigated the reciprocal regulation of Tcf15 and Nanog and the role of Tcf15 for the differentiation. My results suggest that Tcf15 is expressed in the cells that have initiated differentiation but are not lineage-committed. Additionally, Tcf15 can contribute to the regulation of adhesion related genes in order to help the epithelisation of the cells required during the differentiation from naïve to the primed pluripotent state. As a conclusion, Nanog is proposed to help to prevent certain aspects of ESCs differentiation by repressing Tcf15.
Supervisor: Chambers, Ian ; Lowell, Sally Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: embryonic stem cells ; pluripotency ; Nanog ; Tcf15 ; transcription factors ; differentiating cells