STARR-seq Analysis of Enhancer Function in Mouse Pluripotent Cells

小鼠多能细胞增强子功能的 STARR-seq 分析

• 批准号: BB/R019274/1
• 负责人: Ian Chambers
• 金额: $ 90.22万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR019274%2F1
• 关键词: STARR; seq; Analysis; Enhancer; Function

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.

拟议工作的目的是研究称为转录因子（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。这些细胞在哺乳动物发育的早期就可以分化为所有成年细胞类型，从而将其定义为多能。多能细胞也可以在特定培养条件下在实验室中培养。在培养物中，多能细胞在称为自我更新的过程中广泛划分以产生相同的子细胞。同时，这些细胞保留了它们的多限化分化能力，但这只有在培养环境与支持自我更新的情况下改变时才揭露。由于这些结合特性，多能细胞在再生医学中具有巨大的希望。但是，为了有效地意识到潜力我们需要了解多能细胞的生长和身份如何控制。振动细胞在小鼠中最好的特征在于，因此，我们的研究专注于多能小鼠细胞。存在三种不同类型的多能小鼠细胞。从在开发过程中首次出现的开始，它们被称为幼稚，形成性和底漆。在实验室条件下可能存在的等效细胞类型分别称为胚胎干细胞（ESC），层状细胞（EPILCS）和层状干细胞（EPISCS）。其中，到目前为止，最有特色和理解的是ESC.ESC身份由OCT4，SOX2，NANOG和STAT3在内的TFS组控制。这些TF与染色体上的位点结合，在许多情况下，相互结合。 TFS结合的某些DNA位点可以影响附近基因打开的程度。当将DNA的这些段放在人造圆形DNA中时，可以自主起作用，以增强链接基因的水平（例如，一个编码绿色荧光蛋白[GFP]）被打开并因此称为增强子。但是，并非所有TF结合位点都充当增强剂。我们想知道是什么区分TF结合位点，通过将增强子函数与没有的增强子函数更改来响应TF结合。这将加深我们对细胞身份分子控制的理解。在这项工作中，我们将表征结合Oct4，Sox2，Nanog或STAT3的ESC增强剂的活性。我们将通过制备ESC染色体的片段并使用本身结合TFS的抗体结合每个TF的段来做到这一点。附着的DNA将被纯化，并将其放入含有GFP的人造圆形DNA中。这将使数千至数百万的人工DNA成为一个“库”，每个DNA都包含来自其中一个小鼠染色体的不同部分的DNA段。在将圆形DNA引入ESC之后，我们可以通过测量已打开GFP基因的程度来确定增强剂活性。然后，我们可以净化主动增强剂并确定其DNA代码。我们将在ESC中重复此过程，在此过程中，我们可以将这些TF的水平上升或向下倾斜。这将使我们能够通过像调光开关一样响应TF活动来区分哪些主动增强器改变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中很重要，因此对于控制多能细胞的身份很重要。我们将比较在幼稚的，形成性和启动的多能细胞中的增强子曲目，以帮助了解这些多能细胞类型的区别是什么。最后，我们将使用一组酶促的剪刀来切割选择增强剂，从其正常的染色体位置中脱离，从而测试它们在指导细胞识别方面的真正重要性。我们的研究将导致对控制细胞身份的分子电路的更完整，更深入的了解，并因此对理解正常的发育过程和可能会导致癌症等正常的发育过程和癌症具有影响。

项目成果

Differential repression of Otx2 underlies the capacity of NANOG and ESRRB to induce germline entry.

• DOI: 10.1016/j.stemcr.2021.11.013
• 发表时间: 2022-01-11
• 期刊: Stem cell reports
• 影响因子: 5.9
• 作者: Vojtek M;Zhang J;Sun J;Zhang M;Chambers I
• 通讯作者: Chambers I

Differential repression of Otx2 underlies the capacity of NANOG and ESRRB to induce germline entry

Otx2 的差异抑制是 NANOG 和 ESRRB 诱导种系进入能力的基础

• DOI: 10.1101/2021.06.14.448276
• 发表时间: 2021
• 作者: Vojtek M

Loss of Resf1 reduces the efficiency of embryonic stem cell self-renewal and germline entry.

• DOI: 10.26508/lsa.202101190
• 发表时间: 2021-12
• 期刊: Life science alliance
• 影响因子: 4.4
• 作者: Vojtek M;Chambers I
• 通讯作者: Chambers I