Transcription factor control of dynamic transitions within and beyond pluripotency

Cell identity depends on the action of transcription factors and environmental signals that together read the genome. The mechanisms by which cell identity changes during development are of particular interest to fundamental developmental and stem cell biology. In particular, pluripotent cells, which can both self-renew and differentiate to give rise to all lineages in vitro and in vivo, are ideally suited to studying maintenance and changes in cell identity. Application of such knowledge is crucial to the design of robust protocols for in vitro differentiation of cells free from germline tumour-initiating cells for use in cell transplantation and drug discovery.
Pluripotent stem cell self-renewal is governed by a pluripotency gene regulatory network centred on the transcription factors OCT4, SOX2 and NANOG. While considerable advances have been made in identifying additional pluripotency gene regulatory network components and in analysing global chromatin binding by transcription factors, these powerful approaches do not tell us how cell decisions are actually made. To better understand the distinction between self-renewal and differentiation we will use precise mechanistic analysis to determine how individual transcription factors affect the operation of the pluripotency gene regulatory network.
Pluripotent cells pass through successive states during differentiation. Segregation of germline and somatic lineages occurs from a transitional 'formative' pluripotent state. A key unanswered question is why many pluripotency transcription factors (including OCT4, SOX2 and NANOG) also operate in the germline gene regulatory network and are critical for primordial germ cell (PGC) function. As these cell types have arguably the most radically divergent potencies, this is paradoxical. By studying OCT4, SOX2 and NANOG and by doing so in cells at distinct stages during the transition from naïve pluripotent embryonic stem cells (ESCs) to formative epiblast-like cells (EpiLCs) and from EpiLCs to PGCs, we aim to resolve this paradox and to reveal principles at the very foundation of phenotypic cell identity. Our studies will extend knowledge from the mouse to address germline entry in humans.
This proposal has three aims, focused on distinct aspects of pluripotent cell function:
1. How is pluripotency specified and lost in vivo?
2. How do transcription factors act in the maintenance and loss of naïve pluripotency in vitro?
3. What regulatory interactions determine the efficiency of entry to the germline?
Delivering the above aims will advance the field by assessing how transcription factors operate as an ensemble to control ESC identity, and by revealing how transcription factors are repurposed at distinct developmental stages. This will break new ground by identifying key mechanisms by which cells exit specific pluripotent states, particularly as they enter the germline, or initiate entry into the primitive streak. Together, this knowledge will provide the insights needed to rigorously command the uniform differentiation of pluripotent cells demanded by future recipients of regenerative medicine strategies.

Pluripotent cell identity is regulated by the transcription factors (TFs) Oct4, Sox2 and Nanog. However, the mechanisms by which TFs control self-renewal and differentiation remain elusive. Paradoxically, unipotent primordial germ cells also express these TFs. We will examine TF function through progression of pluripotent cells from a naïve to a formative state and into either the germline or the soma to reveal how PGRN TFs are repurposed during state transitions.
Aim 1: How is pluripotency specified and lost in vivo?
Single cell RNA-seq will identify specification defects in TF mutant embryos. We will assess NANOG variants and modulation of NANOG-responsive genes to compensate for Nanog in pluripotency specification. The mechanisms driving initial loss of pluripotency in vivo will be investigated and roles of pluripotency TFs in this process identified.
Aim 2: How do TFs act in the maintenance and loss of naïve pluripotency in vitro?
The involvement of direct NANOG target genes in ESC function will be assessed using a novel Cas9 approach that can potentially modulate positively and negatively regulated NANOG targets. Commitment from naïve pluripotency will be analysed using FACS-sorting of reporter lines and manipulation of the intracellular environment. Enhancer regulation by pluripotency TFs will be analysed by modified ChIP-seq techniques.
Aim 3: What regulatory interactions determine the efficiency of entry to the germline?
Formative pluripotent cells are competent for germline entry, a process enhanced by Otx2 mutation. We will investigate germline entry parameters and clarify Blimp1 operation. We will investigate whether entry to the human germline is increased in Otx2 mutants.
This work will deliver mechanistic insight into pluripotent TF function. Application of such knowledge is crucial to design of robust protocols for in vitro differentiation of cells free from germline tumour-initiating cells for use in cell transplantation and drug discovery.

The general public will benefit from the results of the proposed work mainly in four ways:
1) Medical research: the proposed research has potential medical implications in two fields of broad interest that may contribute to enhancing the quality of life: regenerative medicine and reproductive medicine.

Regenerative Medicine: The proposed work has the potential to enhance the current understanding of the principles governing the specification and differentiation of pluripotent cell populations and how these processes are balanced. Since the abilities to promote reprogramming to pluripotency of differentiated cells from patients and the ability to robustly and predictably control the differentiation of the resulting pluripotent cells are key steps in most cellular replacement therapies, the direct investigation of the principles and factors governing these processes are of immediate relevance to the field of regenerative medicine.

Reproductive medicine: understanding how pluripotent cells change identity to specifically enter into the germline is one of the most basic fundamental processes underlying mammalian reproduction. The potential contribution of the outlined experiments to the understanding of the molecular control of germ cell specification cannot be immediately translated into advances of clinical relevance. Nonetheless, a fuller understanding of the molecular control of pluripotent cell function will inform and accelerate parallel studies on the mechanisms of germ cell development. This is of pivotal importance in understanding the process of gamete production, and correcting potential defects observed in this lineage.

2) The proposed research has the potential to allow future development of culture additives that can enhance or eliminate propagation of pluripotent cells in culture. The latter is still a hinderance to the application of pluripotent cells, so potential commercial value exists here. If so, beneficiaries include companies and both scientists and patients using any commercially developed reagents or downstream products.

3) The biological research carried out during the proposed project will contribute towards maintaining the high standard of academic excellence currently enjoyed by the MRC Centre for Regenerative Medicine. This will be reflected in the ability of the MRC Centre for Regenerative Medicine and the University of Edinburgh to offer educational opportunities for undergraduate and post-graduate student training. This in turn will support our outreach activities (point 4) as all our students are encouraged to be, and many are, active in outreach.

4) The conceptual advances and tangible material, such as pictures, diagrams and illustrations generated to present the results of the proposed experiments will add to the resources used by our science communication staff during outreach activities aimed at raising awareness of the latest advances in the field of stem cell biology and regenerative medicine amongst diverse audiences within the general public.