STARR-seq Analysis of Enhancer Function in Mouse Pluripotent Cells

这项工作的目的是研究被称为转录因子（TFs）的基因调节因子如何在称为多能细胞的特定类型的细胞中起作用。这些细胞出现在哺乳动物发育的早期，可以分化成所有的成年细胞类型，定义它们为多能性。多能细胞也可以在实验室特定的培养条件下培养。在培养过程中，多能细胞广泛分裂产生相同的子细胞，这一过程被称为自我更新。同时，这些细胞保留了它们的多系分化能力，但只有当培养环境从支持自我更新的环境中改变时，这种能力才会被揭示。由于这些综合特性，多能细胞在再生医学中具有很大的前景。然而，为了有效地实现这一潜力，我们需要了解多能细胞的生长和身份是如何被控制的。多能性细胞在小鼠中得到了最好的表征，因此，我们的研究集中在多能性小鼠细胞上。存在三种不同类型的多能小鼠细胞。从第一个在发展过程中出现的开始，这些被称为naïve，形成和启动。可以在实验室条件下存在的等效细胞类型分别被称为胚胎干细胞（ESCs），上皮样细胞（EpiLCs）和上皮干细胞（EpiSCs）。其中，迄今为止最具特征和理解的是ESCs。ESC身份由一系列tf控制，包括OCT4、SOX2、NANOG和STAT3。这些tf与染色体上的位点结合，并且在许多情况下彼此结合。tf结合的一些DNA位点可以影响附近基因开启的程度。当将这些DNA片段置于人造环状DNA中时，这些片段可以自主地发挥作用，以提高连接基因（例如编码绿色荧光蛋白的基因[GFP]）的开启水平，因此被称为增强子。然而，并不是所有的TF结合位点都起到增强子的作用。我们想知道通过改变增强子功能来响应TF结合的TF结合位点与不响应增强子功能的TF结合位点的区别。这将加深我们对细胞身份的分子控制的理解。在这项工作中，我们将描述结合OCT4、SOX2、NANOG或STAT3的ESC增强子的活性。我们将通过准备ESC染色体片段，并使用自身结合tf的抗体纯化这些结合tf的片段来做到这一点。附着的DNA将被纯化并放置在一个人工的环状DNA中，该环状DNA含有处于OFF状态的GFP。这将形成一个由数千到数百万个这样的人造DNA组成的“文库”，每个DNA都包含来自小鼠染色体不同部分的DNA片段。在将环状dna引入ESCs后，我们可以通过测量GFP基因被打开的程度来确定增强子的活性。然后我们可以纯化活性增强子并确定它们的DNA密码。我们将在ESCs中重复这个过程，我们可以提高或降低这些tf的水平。这将使我们能够区分哪些活性增强剂通过像调光开关一样响应TF活性来改变GFP亮度。这些实验将告诉我们DNA的哪些部分在打开或关闭基因方面是重要的，因此在控制多能细胞身份方面是重要的。我们将比较naïve、形成型和引物型多能细胞中的增强子库，以帮助理解这些多能细胞类型之间的区别。最后，我们将使用一套酶剪刀将选择的增强子从其正常染色体位置剪切下来，从而测试它们在指导细胞身份方面的真正重要性。我们的研究将导致对控制细胞身份的分子电路的更完整和更深入的理解，因此将对理解正常的发育过程以及它们如何出错（例如在癌症等病理状态下）产生影响。

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.