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

这项拟议工作的目的是研究被称为转录因子(TF)的基因调节器如何在一种称为多能细胞的特定类型的细胞中发挥作用。这些细胞出现在哺乳动物发育的早期，可以分化为所有成体细胞类型，从而定义它们为多能性细胞。多能细胞也可以在特定的培养条件下在实验室培养，如胚胎干细胞(ESCs)。在培养过程中，胚胎干细胞广泛分裂，产生相同的子细胞，这一过程被称为自我更新。同时，胚胎干细胞保持其多谱系分化能力，但只有当培养环境从支持自我更新的环境改变时，这种能力才会被揭示出来。由于这些特性的结合，胚胎干细胞在再生医学中有着很大的前景。然而，为了有效地实现这一潜力，我们需要了解ESC的增长和身份是如何控制的。胚胎干细胞最适合小鼠，因此，我们的研究集中在小鼠胚胎干细胞上。ESC身份由一群TF控制，其中包括一个名为NANOG的主监管机构。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的开启程度。打开一个基因的过程启动了一个被称为转录的过程，在这个过程中，DNA被一种名为RNA聚合酶II(RNAP2)的酶解码成mRNA。在最近令人兴奋的工作中，我们发现NANOG和RNAP2之间存在直接的物理接触。这是序列特异性DNA结合转运蛋白与将DNA转录成信使核糖核酸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分子中相互作用的部分。我们对相互作用的初步分析让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突变，以提供对相互作用的高分辨率视图。最近，有人提出，当转铁蛋白和其他蛋白质在染色体上的调控部位相互作用时，会发生一种物理转变，称为相变，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。我们已经证明了NANOG经历了相变，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。我们还将调查NANOG和RNAP2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CDK9，作用于NANOG和RNAP2。使用一种名为质谱学的技术，我们将识别NANOG上的磷酸化位点。然后我们将通过突变来研究磷酸化对NANOG功能的影响。我们还将使用质谱仪分析从ESCs中纯化的NANOG-RNAP2复合体。其他专门与NANOG-RNAP2复合体结合的蛋白质将通过这项技术被鉴定出来，这将使人们对该复合体的功能有更深入的了解。染色体的三维组织对基因转录的调控有深远的影响。我们将研究当NANOG-RNAP2复合体在染色体DNA上形成时，空间组织是如何变化的，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控过程的更完整的理解，并将对理解维持所有细胞类型的基本过程、它们是如何调节的以及它们在病理状态下可能如何被颠覆具有重要意义。

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.