STARR-seq Analysis of Enhancer Function in Mouse Pluripotent Cells

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The aim of the proposed work is to study how gene regulators known as transcription factors (TFs) work in a specific type of cell termed a pluripotent cell. These cells arise early in mammalian development and can differentiate into all adult cell types, defining them as pluripotent. Pluripotent cells can also be cultured in the lab in specific culture conditions. During culture pluripotent cells divide extensively to produce identical daughter cells, in a process termed self-renewal. At the same time, these cells retain their multilineage differentiation capacity but this is only unmasked if the culture environment is altered from that supporting self-renewal. Due to these combined properties, pluripotent cells hold great promise in regenerative medicine. However, to effectively realise that potential we need to understand how pluripotent cell growth and identity is controlled.Pluripotent cells are best characterised in the mouse and for that reason, our study focusses on pluripotent mouse cells. Three distinct types of pluripotent mouse cells exist. Starting with the first to emerge during development, these are termed naïve, formative and primed. The equivalent cell types that can exist in lab conditions are respectively known as embryonic stem cells (ESCs), epiblast-like cells (EpiLCs) and epiblast stem cells (EpiSCs). Of these, by far the best characterised and understood are ESCs.ESC identity is controlled by a cohort of TFs including OCT4, SOX2, NANOG and STAT3. These TFs bind to sites on chromosomes and in many cases bind near one another. Some of the DNA sites that TFs bind can influence the extent to which a nearby gene is switched ON. These segments of DNA can act autonomously when placed in an artificial circular DNA to enhance the level that a linked gene (e.g., one encoding a green fluorescent protein [GFP]), is turned ON and are therefore referred to as enhancers. However, not all TF binding sites act as enhancers. We want to know what distinguishes TF binding sites that respond to TF binding by altering enhancer function from those that do not. This will deepen our understanding of the molecular control of cell identity. In this work we will characterise the activity of ESC enhancers that bind either OCT4, SOX2, NANOG or STAT3. We will do this by preparing segments of ESC chromosomes and purifying those segments that bind each of these TFs using antibodies that themselves bind the TFs. The attached DNA will be purified and placed into an artificial circular DNA containing a GFP in an OFF state. This will make a 'library' of thousands to millions of such artificial DNAs, each one of which contains a DNA segment from a different part of one of the mouse chromosomes. After introducing the circular DNAs to ESCs we can determine the enhancer activity by measuring the extent to which the GFP gene has been turned ON. We can then purify the active enhancers and determine their DNA code. We will repeat this process in ESCs in which we can turn the level of these TFs up or down. This will allow us to distinguish which active enhancers alter the GFP brightness by responding to TF activity like a dimmer switch. These experiments will tell us what parts of the DNA are important in turning genes ON or OFF and that are therefore important in controlling pluripotent cell identity.We will compare the enhancer repertoire in naïve, formative and primed pluripotent cells, to help understand what distinguishes these pluripotent cell types from one another. Finally, we will use an enzymatic set of scissors to cut chosen enhancers out of their normal chromosomal locations to thereby test how important they really are in directing cell identity.Our study will lead to a more complete and deeper understanding of the molecular circuitry controlling cell identity and will therefore have implications for understanding the normal processes of development and how they may go awry, for example in pathological states such as cancer.