STARR-seq Analysis of Enhancer Function in Mouse Pluripotent Cells

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.

Pluripotency transcription factors (TFs) control embryonic stem cell (ESC) self-renewal. This is achieved by TF binding to sites in the genome and influencing transcription. However, how TFs act at sites of regulation to exert an effect on cell identity is poorly understood. Here we will use a massively paralleled reporter assay termed STARR-seq in combination with ChIP or targeted BAC shredding to identify enhancers active in pluripotent cells and to investigate how TF binding to enhancers governs efficient ESC self-renewal.
The aims of this work are:
1 Identifying functional enhancers bound by key pluripotency transcription factors (TFs).
2 Identifying enhancers that alter activity on response to binding of a specific TF.
3 Defining enhancer activities specific for distinct pluripotent cell states.
In aim 1, we will define the enhancer repertoire bound by NANOG, OCT4, SOX2 and STAT3 by ChIP in three distinct conditions all supporting high efficiency ESC self-renewal. This will identify the common active enhancers driving efficient ESC self-renewal.
The NANOG protein level is directly related to ESC self-renewal efficiency. In aim 2 we will use ChIP-STARR-seq to identify sites of positive and negative action by NANOG globally. We will also use BACs to focus on 1Mb regions around our previously identified 64 protein coding genes responding to NANOG. These approaches will identify sites by which NANOG drives efficient ESC self-renewal at high resolution.
In aim 3, we will identify enhancers active in naïve, formative and primed pluripotent cells. Using ChIP-STARR-seq libraries made from each distinct pluripotent state and testing activities in all three states, we will identify enhancers distinguishing between pluripotent cell states.
The function of selected endogenous enhancers will be tested by elimination with CRISPR/Cas9.
Together, the proposed studies will deliver an unprecedented view of the active enhancers defining pluripotency.

The social beneficiaries of the proposed research are to be found within the wider public.
The general public will benefit from the results of the proposed work mainly in three ways:
1) The proposed research has potential medical implications in the broad field of regenerative medicine. The proposed work has the potential to enhance the current understanding of the principles of gene regulation governing the specification of cell identity in distinct pluripotent cell populations. Since the ability to promote reprogramming to pluripotency of differentiated cells from patients is a key step in most cellular replacement therapies, the direct investigation of the principles and factors governing this process is of immediate relevance to the field.
2) The biological research carried out during the proposed project will contribute towards maintaining the high standard of academic excellence currently enjoyed by the Centre for Regenerative Medicine. This will be reflected in the ability of the Centre for Regenerative Medicine and the University of Edinburgh being able to offer educational opportunities for undergraduate and post-graduate students training in our group.
3) 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 disseminating knowledge and raising awareness of the latest advances in the field of stem cell biology and regenerative medicine.

The immediate academic beneficiaries of the proposed research are new, or early stage researchers at post-doctoral level funded by the proposed research and additional researchers in the Centre for Regenerative Medicine that will benefit from scientific and methodological advances made during the research and disseminated outwith the immediate research group. This will help ensure that young Centre for Regenerative Medicine researchers derive the maximum benefit from the proposed research to apply to their own work.