Biological physics of protein clustering in epigenetic memory and transcriptional control

近年来，已经清楚的是，许多蛋白质在单个细胞内通过大量组合成密集的分子簇而集体起作用。然而，这些簇如何形成以及它们执行什么生物功能通常仍然是一个谜。在这个提议中，我们的目标是通过研究两种类型的集群在一个单一的目标，一个基因称为FLOWARDINLOCUS C在植物拟南芥解开这些谜团。第一种类型的聚类是由蛋白质以有序的方式聚集在一起引起的，这样，临界数量的蛋白质的紧密结合将刺激进一步的反馈，以将更多的蛋白质招募到聚类中。这种现象称为低聚化。我们的初步证据表明，这种类型的聚类发生在FLC，并在建立一个记忆的基因是多么活跃的关键功能。具体地说，这种附着在FLC DNA上的蛋白质簇的存在可以导致转录被关闭，这种状态然后通过DNA复制、细胞分裂和许多随后的细胞周期遗传。这种记忆对于控制细胞的行为至关重要，有时也被称为表观遗传记忆。第二种蛋白质聚集有不同的物理起源：许多蛋白质经历所谓的液-液相分离，在那里它们会自发地从周围的介质中分离出来，形成一个自组装的隔间。这个过程类似于水中的油自发分离成液滴。我们的初步证据表明，这种类型的集群也存在于FLC，虽然在不同的时间段在植物发育的寡聚集群。我们相信这些相分离的簇也是基因表达的关键调节因子。在这个项目中，我们的目标是机械地理解这两种类型的集群的形成和生物功能。要做到这一点，需要生物学和物理学的各种技术和专业知识。物理思维是特别需要的，因为据信形成团簇的机制，低聚和相分离，是内在的物理现象。我们将使用分子生物学和遗传学来干扰簇的组成部分，并检查它们对基因表达的影响。我们将使用先进的单分子成像技术来观察团簇，测量它们的动力学并计算所涉及的分子数量。最后，我们将发展详细的理论物理模型的两种类型的集群结合实验的结果。这些实验和模型可能会揭示生物学如何利用新的生物物理学来提供转录调控和记忆所需的精细控制。

In recent years it has become clear that many proteins act collectively inside single cells by teaming-up in large numbers into dense molecular clusters. However, how these clusters form and what biological function they perform often remains a mystery. In this proposal, we aim to unlock these mysteries by investigating two types of clustering at a single target, a gene called FLOWERING LOCUS C in the plant Arabidopsis. The first type of clustering is caused by proteins gathering together in an ordered way, such that close association of a critical number of proteins will then stimulate further feedback to recruit more proteins into the cluster. This phenomenon is called oligomerization. Our preliminary evidence indicates that this type of clustering occurs at FLC and has a critical function in establishing a memory of how active the gene is. Specifically, the presence of this cluster of proteins attached to the FLC DNA can cause transcription to be switched off, a state that is then inherited through DNA replication, cell division and through many subsequent cell cycles. Such memory is vitally important in controlling how cells behave and is sometimes called epigenetic memory.The second type of protein clustering has a different physical origin: many proteins undergo what is called liquid-liquid phase separation, where they will spontaneously separate themselves from the surrounding medium and form a self-assembling compartment. This process is analogous to the spontaneous separation of oil in water into droplets. Our preliminary evidence demonstrates that this type of clustering is also present at FLC, though at a different time period in plant development to the oligomeric clustering. We believe that these phase-separated clusters are also critical regulators of gene expression. In this project, we aim to mechanistically understand the formation and biological function of both types of cluster. To do this will require a wide diversity of techniques and expertise from both biology and physics. Physics thinking is specifically needed because the mechanisms by which the clusters are believed to form, oligomerization and phase separation, are intrinsic physics phenomena. We will use molecular biology and genetics to perturb the components of the clusters and examine their effects on gene expression. We will use advanced single-molecule imaging techniques to observe the clusters, measure their dynamics and count the number of molecules involved. Finally, we will develop detailed theoretical physics models of the two types of clusters incorporating the results from the experiments. These experiments and models may potentially reveal how new kinds of biological physics have been exploited by biology to provide the exquisite control needed for transcriptional regulation and memory.