How are enhancers activated during early embryonic stem cell differentiation

All of us develop into complex human beings containing millions of cells from a single cell created by fertilization of an egg. To transit from this single cell state, cells must divide and eventually change their identity and gain specialised functions. For example we need specific types of cells to populate our brains, and this project will address the early steps in the creation of these types of cells. One fundamental process that we aim to understand in this context is the mechanism of "enhancer activation". Enhancers are the parts of our genome that direct gene expression ie the conversion of our DNA from our genes into useful information that provides the building blocks that determine the structure and function of our cells. Importantly, different enhancers are active in different cell types, allowing the production of distinct gene products and hence a range of alternative cell types. However, we lack a fundamental understanding of how these enhancers are activated, and hence we do not know the mechanisms determining cell fate. This project aims to bridge this gap in our knowledge and examine how enhancers are activated. Importantly, our studies will be conducted in embryonic stem cells, and hence will contribute to our knowledge of how to manipulate these cells which is important in the context of using these cells in regenerative medicine.

Recent studies have begun to uncover the regulatory complexities of mammalian genomes through uncovering tens of thousands of enhancer regions. However, only a fraction of these are activated in any given cell type and as cells differentiate, enhancer usage changes dramatically. However, while we now know increasing amounts about what defines an active enhancer, we know comparatively little about how the active state is obtained. This knowledge is particularly important in the context of stem cell research, where enhancer usage and gene expression changes substantially during embryonic stem cell differentiation and in reverse engineering iPS cells. In this project we will focus on the mechanisms of enhancer activation as embryonic stem cells exit from ground state pluripotency and differentiate along the neuronal lineage. We will focus on the transcription factor Otx2 and its role in enhancer activation. Otx2 is one of the earliest determinants of embryonic stem cell differentiation and later during development contributes to neuronal cell specification. Loss and overexpression of this transcription factor are associated with developmental defects and cancer respectively. Using this system, we will ask the following questions: When do Otx2-bound enhancers communicate with gene promoters? Which coregulatory partners does Otx2 use in enhancer activation? What are the mechanisms of Otx2-driven enhancer activation and inactivation? Importantly, in addition to answering these fundamental questions about enhancer activation mechanisms, our findings will impact on the understanding of how an important regulatory of cell fate determination, Otx2, functions at the molecular level. These findings could also have important implications in our understanding and ability to treat disease states such as medullablastomas where Otx2 plays an important role.

Academic beneficiaries: As described in the 'Academic beneficiaries' section, these will include UK and international researchers in the following biological science fields: Gene regulation, stem cell research, chromatin regulation and transcription factor function. Academic beneficiaries from other disciplines will include clinical researchers studying disease states involving deregulated enhancer function, including cancer. Finally, bioinformaticians and mathematical modellers studying dynamic transcriptional networks will benefit from our research as we aim to identify novel regulatory nodes. We will publicise our work by publishing in high impact scientific journals, regular presentation at International conferences, and posting on our laboratory and Faculty websites.

General public: In terms of the wider public beneficiaries, the long term impact of this project will likely be for cancer patients or patients requiring stem cell based therapeutic treatments. Our work will uncover fundamental mechanisms involved in enhancer activation, and this knowledge can be used to understand and develop strategies to combat inappropriate enhancer activation in cancer cells and improve stem cell differentiation and dedifferentiation protocols. We aim to encourage the next generation to become engaged in scientific research through hosting school children in the laboratory. Our work will be communicated to the general public through local charity events and science festivals. Important findings will be communicated through the standard media outlets.

Public sector: As this is a basic research project, there will be no immediate impact on the public sector. However, in the long term the biggest impact is likely to be on the NHS. Any new mechanisms that we identify might lead to improved stem cell based therapies and hence will contribute to regenerative medicine. Training will be provided to medically trained MD students who will develop clinically relevant projects on enhancer activation and deregulation in cancer cells based on the findings of this study.

Industry: There is growing interest from Biotechnology companies in developing stem cell based therapies. One of the key elements in developing new cell types from stem cells is establishing the correct gene expression profiles and this is in turn dictated by enhancer activation and decommissioning. Our work may therefore dictate ways in which differentiation and de-differentiation protocols can be enhanced to provide high fidelity production of specific cell types.
This project will also involve the use of many cutting edge technologies, especially related to systems based approaches to research and advanced bioinformatics skills. As such the project will provide an opportunity for training the next generation of researchers through exposing undergraduate, postgraduate and postdoctoral students in the lab to these approaches.