A molecular understanding of transposon-based enhancer activation by the ChAHP complex during human cell fate decisions

转座元件或转座子是细胞DNA中的短、重复序列。它们有时被称为“跳跃基因”，因为它们可以复制和整合到新的位置。这些整合天生就是诱变的，所以我们的基因组进化出了抑制它们的机制。这导致了一场进化的军备竞赛，转座子进化以逃避这些防御机制，而我们的防御进化以沉默转座子。其结果是，我们的基因组包含数千个失活的、经常发生突变的转座子，以及数量相对较少的活跃转座子。重复序列可能逃脱失活的一种方法是为细胞提供有用的功能。事实上，有几个例子表明，哺乳动物细胞在发育过程中利用最近进化出的一类短粒状核素(SINE)来增强基因表达。这些序列被称为“eSINE”。细胞必须精确地控制eSINE何时何地激活，以免不受控制的活动破坏正常发育。DNA的活性由它在细胞核内的包装方式控制。DNA在细胞内并不是“裸露的”，而是以染色质的形式存在，也就是说，它包裹在蛋白质周围，所以它是紧凑和稳定的。一组名为染色质重塑的蛋白质控制着基因组不同部分的紧密排列，从而控制它们是“活跃的”还是“不活跃的”。我们花了很多年研究一种名为CHD4的染色质重塑。CHD4是两种不同的多亚基复合体NuRD和ChAHP的组成部分。我们已经证明，NuRD通过控制调控DNA序列的活性，促进小鼠和人类干细胞的发育决定。我们预计NuRD和ChAHP在作用方式上会有一些相似之处，然而这两个复合体有许多不同的组件，虽然NuRD在所有的活性调控序列中都被发现，但ChAHP主要定位于SINE。ChAHP组件之一ADNP的突变会导致一种名为Helmoortel Van der AA综合征(HVDAS)的人类神经发育疾病。HVDAS的特点是面部变形、心血管和胃肠道问题以及自闭症谱系障碍。了解ChAHP在人类发育过程中所做的工作以及它是如何做到的，不仅将为HVDAS提供重要信息，也将为理解自闭症谱系障碍和人类组织发育提供重要信息。我们假设ChAHP使eSINE处于非活性状态，但当细胞需要开始分化时，它可以被激活。我们进一步认为，在eSINE激活过程中，ADNP被取代，允许CHD4招募NuRD组件，帮助eSINE与启动子相互作用并激活基因表达。在这个项目中，我们将使用尖端技术来确定ChAHP在人类细胞命运决定过程中的分子和发育功能。在小鼠干细胞中，ChAHP作用于一类在灵长类动物中不存在的Sine，因此，为了全面了解人类ChAHP的功能，我们需要在人类细胞中进行研究。我们将创造人类多能干细胞，在其中我们可以快速耗尽蛋白质，并评估它们丢失对ChAHP组装和功能的主要后果。我们将定义ChAHP如何被定向到其作用部位，它在那里做什么，它如何与细胞的转录机制相互作用，以及ChAHP如何促进使用eSINE来控制基因表达。我们将准确地定义在一些受HVDAS影响最严重的组织的形成过程中，哪些发育决定显示出对ADNP的依赖，然后确定携带致病ADNP突变的细胞中出了什么问题。这将是一项关于人类干细胞如何能够使用eSINE以及这如何在人类疾病中出错的全面调查。我们的发现不仅对那些受HVDAS或自闭症影响的人有意义，而且对研究转座子、转录控制和人类干细胞生物学的基础科学家也有意义。

Transposable elements, or transposons, are short, repetitive sequences within a cell's DNA. They are sometimes known as "jumping genes" because they can replicate and integrate into new sites. These integrations are inherently mutagenic so our genomes have evolved mechanisms to repress them. This has resulted in an evolutionary arms race where transposons evolve to evade these defence mechanisms, while our defences evolve to silence the transposons. The result is that our genomes contain many thousands of inactivated, often mutated transposons, along with a relatively small number of active ones. One way a repeat might escape inactivation is to deliver useful function for the cell. Indeed, several examples exist where a recently evolved class of short interspersed nuclear element (SINE) is used by mammalian cells to enhance gene expression during development. These sequences have been termed "eSINEs".Cells must precisely control when and where eSINEs are active lest uncontrolled activity derail normal development. The activity of DNA is controlled by how it is packaged within the nucleus. DNA is not "naked" within cells but exists in the form of chromatin, i.e., it is wrapped around proteins so it is compact and stable. A group of proteins called chromatin remodellers control how tightly packed different parts of the genome are, and therefore whether they are "active" or "inactive."We have spent many years working on a chromatin remodeller called CHD4. CHD4 is a component of two different multi-subunit complexes, NuRD and ChAHP. We've shown that NuRD facilitates developmental decisions in mouse and human stem cells by controlling the activity of regulatory DNA sequences. We expect NuRD and ChAHP to share some similarities in how they act, however the two complexes have many different components and while NuRD is found at all active regulatory sequences, ChAHP localises mostly to SINEs.Mutations in one of the ChAHP components, ADNP, give rise to a human neurodevelopmental disorder called Helsmoortel Van der Aa Syndrome (HVDAS). HVDAS is characterised by facial dysmorphisms, cardiovascular and gastrointestinal problems and autism spectrum disorder. Understanding what ChAHP does during human development and how it does it will provide important information not only for HVDAS, but also for understanding autism spectrum disorders and human tissue development generally.We hypothesise that ChAHP holds eSINEs inactive but ready to be activated if/when cells need to start differentiating. We further suggest that ADNP is displaced from eSINEs during their activation, allowing CHD4 to recruit NuRD components which help the eSINEs to interact with promoters and activate gene expression.In this project we will use cutting edge technologies to determine the molecular and developmental functions of ChAHP during human cell fate decisions. In mouse stem cells ChAHP acts on a class of SINEs which does not exist in primates so to fully understand human ChAHP function we need study it in human cells. We will create human pluripotent stem cells in which we can quickly deplete proteins and assess the primary consequences of their loss on ChAHP assembly and function. We will define how ChAHP is directed to its sites of action, what it does there, how it interacts with the cell's transcription machinery, and how ChAHP facilitates the use of eSINEs to control gene expression. We will define exactly which developmental decisions show ADNP dependency during formation of some of the tissues most affected in HVDAS, and then determine what goes wrong in cells harbouring disease-causing ADNP mutations. Together this will be a comprehensive investigation into how human stem cells are able to use eSINEs and how this can go wrong in human disease. Our findings will be of relevance not only to those affected by HVDAS or autism, but also to basic scientists studying transposons, transcriptional control, and human stem cell biology.