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

这项工作的目的是研究被称为转录因子（TF）的基因调控因子如何在一种称为多能细胞的特定类型的细胞中发挥作用。这些细胞在哺乳动物发育的早期出现，可以分化成所有的成体细胞类型，将它们定义为多能细胞。多能细胞也可以在实验室中在特定的培养条件下培养为胚胎干细胞（ESC）。在培养过程中，ESC广泛分裂以产生相同的子细胞，这一过程称为自我更新。与此同时，胚胎干细胞保留了其多系分化能力，但只有当培养环境从支持自我更新的环境改变时，这种能力才会被发现。由于这些综合特性，ESCs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ESC的生长和身份是如何控制的。ESC在小鼠中的特征最好，因此，我们的研究集中在小鼠ESC上。ESC身份由一组TF控制，包括称为NANOG的主调节因子。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。基因开启的过程启动了一个称为转录的过程，其中DNA被称为RNA聚合酶II（RNAP 2）的酶解码为mRNA。在最近令人兴奋的工作中，我们已经确定了NANOG和RNAP 2之间的直接物理接触。这是序列特异性DNA结合TF和将DNA转录成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，以确定它是如何发生的，并确定其功能。我们将通过分别突变每种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部分。我们对的初步分析使我们对相互作用有了一个广泛的概述，但为了最大限度地利用我们从这些研究中学到的东西，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TF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，相变发生。我们已经表明，NANOG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RNAP 2之间的相互作用是如何调节的。转录由几种磷酸化酶控制，其中一种称为CDK 9，作用于NANOG和RNAP 2。使用一种称为质谱的技术，我们将确定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-RNAP 2复合物。通过这项技术，我们还可以鉴定出与NANOG-RNAP 2复合物特异性结合的其他蛋白质，从而深入了解该复合物的功能。染色体的三维结构对基因转录的调控有着深远的影响。我们将研究当NANOG-RNAP 2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。我们的研究将导致更全面地了解调节转录的过程，并将对理解维持所有细胞类型的基本过程，它们如何被调节以及它们如何在病理状态下被颠覆产生影响。

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 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.