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.

这项工作的目的是研究被称为转录因子 (TF) 的基因调节因子如何在称为多能细胞的特定类型细胞中发挥作用。这些细胞出现在哺乳动物发育的早期，可以分化成所有成体细胞类型，将它们定义为多能细胞。多能细胞也可以在实验室的特定培养条件下作为胚胎干细胞（ESC）进行培养。在培养过程中，ESC 广泛分裂，产生相同的子细胞，这一过程称为自我更新。与此同时，ESC 保留了其多谱系分化能力，但只有当培养环境改变，不再支持自我更新时，这种能力才会被揭露。由于这些综合特性，ESC 在再生医学领域具有广阔的前景。然而，为了有效地实现这一潜力，我们需要了解 ESC 的生长和特性是如何控制的。 ESC 在小鼠中得到了最好的表征，因此，我们的研究重点是小鼠 ESC。 ESC 身份由一组 TF 控制，其中包括一个名为 NANOG 的主调节器。这些转录因子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开启的程度。开启基因的过程会启动一个称为转录的过程，其中 DNA 被一种称为 RNA 聚合酶 II (RNAP2) 的酶解码为 mRNA。在最近令人兴奋的工作中，我们发现了 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 的中心酶直接接触的第一个例子。我们将研究这种相互作用，以确定它是如何发生的并确定其功能。我们将通过分别突变每个蛋白质上的特定残基来实现这一点。这将使我们能够识别相互作用的分子部分。我们的初步分析使我们对相互作用有了一个广泛的概述，但为了最大限度地提高我们从这些研究中学到的知识，我们将进行更全面的诱变，以提供相互作用的高分辨率视图。最近，有人提出，当转录因子和其他蛋白质在染色体等调控位点相互作用时，会发生称为“相变”的物理转变，类似于油和水之间的区别。当相互作用的分子达到非常高的局部浓度时，就会发生相变。我们已经证明 NANOG 会经历相变，我们将进一步研究这一点，以确定相变是否在 NANOG 的功能中发挥作用。我们还将研究 NANOG 和 RNAP2 之间的相互作用是如何调节的。转录由多种磷酸化酶控制，其中一种称为 CDK9，作用于 NANOG 和 RNAP2。使用质谱技术，我们将识别 NANOG 上的磷酸化位点。然后我们将通过诱变研究磷酸化对 NANOG 功能的影响。我们还将使用质谱分析从 ESC 纯化的 NANOG-RNAP2 复合物。该技术将鉴定与 NANOG-RNAP2 复合物特异性结合的其他蛋白质，这将深入了解该复合物的功能。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 NANOG-RNAP2 复合物在染色体 DNA 上形成时，空间组织如何变化，并确定任何变化对转录调控的影响。我们的研究将导致对转录调节过程的更全面的理解，并将对理解所有细胞类型的维持的基本过程、它们如何被调节以及它们在病理状态下如何被颠覆具有重要意义。

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