Investigating the role of PIP4K2B, nuclear phosphoinositides and TAF3 in transcription and genome organisation during myogenic differentiation

由于身体环境的变化，细胞不断地被指示(编程)调整它们的行为。特别是，肌肉组织的持续修复对我们的日常生活是必不可少的，但不幸的是，这是一个随着年龄的增长而恶化的过程。肌肉功能的恶化不仅会减少我们的运动和功能，还会使我们更容易患上代谢性疾病，如糖尿病和心血管疾病。卫星细胞是一种特殊的细胞，在肌肉组织中少量存在。当肌肉因运动或疾病受损时，卫星细胞会变得活跃，并产生更多的肌肉细胞，这一过程被称为分化，有助于修复肌肉组织。卫星细胞的激活是复杂的，依赖于受损肌肉环境中产生的信号。随着生物年龄的增长，卫星细胞对这些信号做出反应并产生更多肌肉细胞的能力下降，这在一定程度上是年龄导致的肌肉功能恶化的原因。利用肌肉细胞生成的模型，我们发现，通过控制一种名为PIP4K2B的酶，我们可以增加细胞分化产生肌肉细胞的能力。PIP4K2B控制细胞中自然产生的称为磷脂酰肌醇的分子的水平。众所周知，肌醇磷脂存在于细胞的质膜中，在那里它们控制着许多不同的细胞功能。然而，多年来，越来越多的证据表明，肌醇磷脂也存在于细胞核中，这是细胞的控制中心，在那里它们的水平会随着不同的环境而变化。事实上，在肌肉细胞分化过程中产生的信号的反应下，细胞核中的肌醇磷脂水平上升。我们现在知道，核磷脂与细胞核中的特殊蛋白质相互作用并改变其功能，这些蛋白质参与产生控制细胞行为的指令。其中一种蛋白质是TAF3，它是控制细胞编程的不同蛋白质复合体的一部分。TAF3与磷脂酰肌醇的相互作用使细胞重新编程，促进肌肉细胞分化。在这项提案中，我们将使用一种名为CRISPR CAS的新技术来改变细胞的DNA，以便我们可以研究PIP4K2B和核磷脂酰肌醇如何控制TAF3及其各种复合体以促进肌肉分化。我们认为，专门的TAF3复合体在TAF3与磷脂酰肌醇的相互作用中向细胞发出特定的指令，我们可以使用一种名为CHIP-SEQ的技术来研究这一点。我们还认为，核磷脂与TAF3一起作为一个平台，帮助组织给出哪些指令，以及这些指令是如何协调以促进肌肉分化的。我们将使用一种名为启动子捕获HIC的新技术来研究这一点，这将使我们能够理解用于生成这些指令的核中DNA的三维方面。PIP4K2B是一种非常容易下药的蛋白质，磷酸肌醇和TAF3之间的相互作用部位也是如此，我们希望最终我们能够利用我们的知识开发药物，利用细胞核内的这一控制过程，帮助卫星细胞更有效地分化，从而帮助肌肉组织修复过程。

Cells are constantly being instructed (programmed) to modulate their behaviour because of changes to environment of the body. In particular, continual repair of muscle tissue is essential to our daily lives but unfortunately is a process which deteriorates as we age. Deterioration in muscle function not only decreases our movement and function but also makes us more susceptible to metabolic type diseases such as diabetes and cardiovascular disease. Satellite cells are specialised cells that are present in small numbers within muscle tissue. When muscles are damaged either by exercise or during disease, the satellite cells become active and produce more muscle cells, a process called differentiation that helps to repair muscle tissue. The activation of satellite cells is complex and depends on signals generated within the environment of the damaged muscle. As organisms age the ability of satellite cells to respond to these signals and generate more muscle cells decreases and in part is responsible for age-induced deterioration in muscle function. Using a model of muscle cell generation we have found that by controlling an enzyme called PIP4K2B we can increase the ability of a cell to differentiate to produce muscle cells. PIP4K2B controls the levels of naturally occurring molecules called phosphoinositides that are present in the cells. It is well established knowledge that phosphoinositides are present in the plasma membrane of cells where they control many different cellular functions. However, over the years there has been growing evidence that phosphoinositides are also present in the nucleus, the cells' control centre, where their levels change in response to different environments. In fact in response to signals generated during muscle cell differentiation the levels of phosphoinositides in the nucleus go up. We now know that nuclear phosphoinositides interact with and change the function of special proteins in the nucleus that are involved in generating instructions that control the behaviour of the cells. One such protein is TAF3, which is part of different protein complexes that control the programming of cells. Interaction of TAF3 with phosphoinositides reprograms the cell to increase muscle cell differentiation. In this proposal we will use a novel state of the art technology called CRISPR CAS to change the DNA of cells so that we can investigate how PIP4K2B and nuclear phosphoinositides control TAF3 and its various complexes to increase muscle differentiation. We believe that specialised TAF3 complexes direct specific instructions to the cell in response to the interaction of TAF3 with phosphoinositides and we can investigate this using a technique called CHiP-Seq. We also think that nuclear phosphoinositides together with TAF3 act as a platform that helps to organise which instructions are given and how these are coordinated to increase muscle differentiation. We will investigate this using a novel technique called promoter capture HiC which will allow us to understand the three dimensional aspect of DNA in the nucleus that is used to generate these instructions. PIP4K2B is a very druggable protein as is the site of interaction between phosphoinositides and TAF3 and we hope that eventually we might be able to use our knowledge to develop drugs to harness this control process within a cell's nucleus and help satellite cells differentiate more effectively, thus aiding the process of muscle tissue repair.