A direct biochemical connection between the pluripotency regulator, NANOG and RNA Polymerase II

多能性调节剂 NANOG 和 RNA 聚合酶 II 之间的直接生化联系

• 批准号: BB/T008644/1
• 负责人: Ian Chambers
• 金额: $ 85.61万
• 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: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT008644%2F1
• 关键词: direct; biochemical; connection; between; pluripotency

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 as embryonic stem cells (ESCs). During culture, ESCs divide extensively to produce identical daughter cells, in a process termed self-renewal. At the same time, ESCs 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, ESCs hold great promise in regenerative medicine. However, to effectively realise that potential we need to understand how ESC growth and identity is controlled. ESCs are best characterised in the mouse and for that reason, our study focusses on mouse ESCs. ESC identity is controlled by a cohort of TFs including a master regulator called NANOG. These TFs bind to sites on chromosomes and some of these DNA sites can influence the extent to which a nearby gene is switched ON. The process of switching a gene on initiates a process known as transcription in which DNA is decoded into mRNA by an enzyme called RNA polymerase II (RNAP2).In recent exciting work we have identified a direct physical contact between NANOG and RNAP2. This is the first example of a direct contact between a sequence specific DNA binding TF and the central enzyme that transcribes DNA into mRNA. We will investigate this interaction to ascertain how it occurs and determine its function. We will do this by mutating specific residues on each protein separately. This will allow us to identify the parts of the molecules that interact. Our initial analysis of has given us a broad overview of the interaction but to maximise what we learn from these studies we will perform more comprehensive mutagenesis to deliver a high resolution view of the interaction. Recently, it has been proposed that when TFs and other proteins interact at regulatory sites such on chromosomes, a physical transition occurs called 'phase change', similar to the distinction between oil and water. Phase change occurs when the interacting molecules reach a very high local concentration. We have shown that NANOG undergoes phase change and we will further investigate this to determine whether phase change plays a role in the function of NANOG. We will also investigate how the interaction between NANOG and RNAP2 is regulated. Transcription is controlled by several phosphorylation enzymes and one of these, called CDK9, acts on both NANOG and RNAP2. Using a technique called mass spectrometry we will identify sites of phosphorylation on NANOG. We will then investigate the effect of phosphorylation on NANOG function by mutagenesis. We will also use mass spectrometry to analyse NANOG-RNAP2 complexes purified from ESCs. Other proteins that bind specifically to the NANOG-RNAP2 complex will be identified by this technique and this will give insights into how the complex functions.The three-dimensional organisation of chromosomes has a profound effect on the regulation of gene transcription. We will study how the spatial organization changes when the NANOG-RNAP2 complex forms on chromosomal DNA and determine the effect of any changes on the regulation of transcription. Our study will lead to a more complete understanding of the processes regulating transcription and will have implications for understanding processes fundamental to maintenance of all cell types, how they are regulated and how they may be subverted in pathological states.

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出现，并可以分化为所有成年细胞类型，从而将其定义为多能。多能细胞也可以在实验室中在特定培养条件下作为胚胎干细胞（ESC）培养。在文化期间，ESC在称为自我更新的过程中广泛分裂以产生相同的子细胞。同时，ESC保留了其多限化差异能力，但这只有在从支持自我更新的文化环境中改变了文化环境时才能揭露。由于这些结合特性，ESC在再生医学方面具有巨大的希望。但是，为了有效地意识到潜力我们需要了解如何控制ESC的增长和身份。 ESC最好在鼠标中表征，因此，我们的研究集中在小鼠ESC上。 ESC身份由TF的同类控制，包括名为Nanog的主调节器。这些TF与染色体上的位点结合，其中一些DNA位点可以影响附近基因打开的程度。切换基因的过程引发了一种称为转录的过程，其中DNA被一种称为RNA聚合酶II（RNAP2）的酶解码为mRNA。在最近的激动人心的工作中，我们已经确定了Nanog和RNAP2之间的直接物理接触。这是序列特异性DNA结合TF与将DNA转录为mRNA的中心酶之间直接接触的第一个例子。我们将研究这种相互作用，以确定其发生的方式并确定其功能。我们将通过分别突变每个蛋白质上的特定残基来做到这一点。这将使我们能够识别相互作用的分子的部分。我们对我们对相互作用的广泛概述，但为了最大程度地概述我们从这些研究中学到的知识，我们将执行更全面的诱变，以提供相互作用的高分辨率视图。最近，有人提出，当TFS和其他蛋白质在染色体上的调节位点相互作用时，就会发生物理过渡，称为“相变”，类似于油和水之间的区别。当相互作用分子达到非常高的局部浓度时，发生相变。我们已经表明，Nanog经历了相变，我们将进一步调查这一点，以确定相位变化是否在Nanog的功能中起作用。我们还将研究纳米与RNAP2之间的相互作用。转录由几种磷酸化酶控制，其中一种称为CDK9，都作用于Nanog和RNAP2上。使用一种称为质谱的技术，我们将识别纳米上磷酸化的位点。然后，我们将通过诱变研究磷酸化对纳米功能的影响。我们还将使用质谱法分析从ESC纯化的Nanog-RNAP2复合物。该技术将确定其他与Nanog-RNAP2复合物专门结合的蛋白质，这将为复合物功能提供见解。染色体的三维组织对基因转录的调节具有深远的影响。我们将研究当纳米RNAP2复合物在染色体DNA上形成并确定任何变化对转录调节的影响时，空间组织如何变化。我们的研究将使人们对调节转录的过程有更全面的了解，并将对了解所有细胞类型的维持，如何调节以及如何在病理状态中颠覆它们的过程具有影响。

项目成果

Phosphorylation of NANOG by casein kinase I regulates embryonic stem cell self-renewal.

• DOI: 10.1002/1873-3468.13969
• 发表时间: 2021-01
• 期刊: FEBS letters
• 影响因子: 3.5
• 作者: Mullin NP;Varghese J;Colby D;Richardson JM;Findlay GM;Chambers I
• 通讯作者: Chambers I