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Title: Mechanisms of Oct4 in the entry to, maintenance of, and exit from pluripotency
Author: Bates, Lawrence E.
ISNI:       0000 0004 8501 3061
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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Pluripotency is defined as the capacity to give rise to all cell types of the embryo proper. It arises in the early mammalian embryo but is lost after a short period of time as cells differentiate and become committed to different lineages. Prior to implantation, mouse epiblast cells enter the pluripotent naïve state, which can be captured in vitro in the form of embryonic stem cells. These cells are characterized by a capacity for indefinite self-renewal, and the ability to re-enter normal development upon being returned to the naïve epiblast. A complex transcription factor network promotes this state. Overexpression of one of many of these factors leads to stabilisation of the naïve state, with enhanced self-renewal and reduced spontaneous differentiation. However, the transcription factor Oct4 must be maintained within a tight window of expression; depletion results in extraembryonic differentiation, while overexpression also results in exit from pluripotency. Oct4 was identified as a protein expressed in the early embryo and in germ cells, and was subsequently discovered to be essential for the establishment of the naïve epiblast. In vitro studies determined that loss of Oct4 in ESCs induced trophoblast differentiation. Meanwhile, overexpression of Oct4 led to differentiation of ESCs, and constitutive expression of Oct4 was not sufficient to replace any of the extrinsic factors required for ESC self-renewal. Despite these dramatic phenotypes, the essential role of Oct4 remains unclear, further complicated by the finding that a reduced level of Oct4 promotes self-renewal at the expense of differentiation capacity. In this work, I generated a novel Oct4 fusion protein capable of rapid inducible degradation in order to study the immediate responses to removal of Oct4. This system utilizes the auxin responsive degradation domain of the Arabidopsis thaliana IAA17 protein to recruit a transgenic F-box protein Tir1 on addition of the small molecule auxin to the culture medium. Subsequent ubiquitination by the endogenous SCF complex leads to rapid proteolytic degradation of the Oct4 fusion protein, resulting in loss of detectable protein in as little as two hours. This system allows the study of immediate responses to loss of Oct4 in contrast to conventional depletion systems in which Oct4 levels decay over a protracted period making it difficult to disentangle direct and indirect effects. I established that several pluripotency-associated genes require Oct4 for their transcription. RNA levels of these factors decrease rapidly on depletion of Oct4, prior to significant changes in the expression of other key pluripotency factors such as Nanog and before protein levels of other factors can change dramatically. Furthermore, I established that the presence of Oct4 antagonises chromatin binding by a naïve transcription factor, revealing a possible mechanism by which increased Oct4 levels are detrimental to the naïve state. Together, these findings may be sufficient to explain the simultaneous requirement for, and antagonistic activity of, Oct4 in naïve pluripotent cells. I also found that an ESC level of Oct4 facilitates cell identity transitions. Cells constitutively expressing Oct4 and differentiated in vivo could be reverted to naïve pluripotency in vitro through the application of defined naïve pluripotency growth conditions. Additionally, in the course of these experiments I examined the phenotypic abnormalities that occur in mouse embryonic development under continuous expression of Oct4. In keeping with previous work, we observed abnormalities in limb development and in the skin. We also observed exencephaly in a number of embryos. In conventional exit from pluripotency, female cells must inactive an X chromosome in order to balance X-linked gene expression between males and females. This is achieved via expression of Xist from a single X chromosome, where it orchestrates chromosome-wide silencing. I show that Oct4 plays an important role in the regulation of Xist during the exit from pluripotency. Normally, Oct4 expression persists after the downregulation of most naïve transcription factors during early differentiation. I propose that this allows Oct4 to antagonise expression of the Xist, and thus ensure proper control over X chromosome inactivation. Together this work focuses on the dual roles of Oct4 in regulating both pluripotency and differentiation. I address the contradictory phenotypes relating to altered expression of Oct4, and establish a unifying theory to explain them. I put forward evidence that Oct4 promotes cell fate transitions by regulating naïve transcription factors. I propose that the environment plays a key role in determining cell identity, while Oct4 acts to maintain plasticity by preventing cells from being trapped within otherwise stable states.
Supervisor: Silva, José C. R. Sponsor: MRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: naive pluripotency ; mouse ESC ; Oct4 ; Embryonic stem cell