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

转座因子，或转座子，是细胞DNA中的短而重复的序列。它们有时被称为“跳跃基因”，因为它们可以复制并整合到新的位点。这些整合本身就具有诱变性，因此我们的基因组进化出了抑制它们的机制。这导致了一场进化军备竞赛，转座子进化为逃避这些防御机制，而我们的防御机制进化为沉默转座子。结果是，我们的基因组包含成千上万个失活的、经常发生突变的转座子，以及相对少量的活跃转座子。重复序列避免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的一类短穿插核元素（sin）来增强基因表达。这些序列被称为“eSINEs”。细胞必须精确控制eSINEs的活动时间和地点，以免不受控制的活动破坏正常的发育。DNA的活性是由它在细胞核内的包装方式控制的。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，它被包裹在蛋白质周围，所以它是紧密而稳定的。一组被称为染色质重塑分子的蛋白质控制着基因组不同部分的紧密程度，从而决定它们是“活跃”还是“不活跃”。“我们花了很多年研究一种叫做CHD4的染色质重塑剂。CHD4是两个不同的多亚基复合物NuRD和ChAHP的组成部分。我们已经证明NuRD通过控制调控DNA序列的活性来促进小鼠和人类干细胞的发育决策。我们预计NuRD和ChAHP在作用方式上有一些相似之处，然而这两个复合物有许多不同的成分，而NuRD在所有活跃的调控序列中都有发现，而ChAHP主要定位于sin。ChAHP的一种成分ADNP发生突变，导致一种叫做Helsmoortel Van der Aa综合征（HVDAS）的人类神经发育障碍。HVDAS的特点是面部畸形、心血管和胃肠道问题以及自闭症谱系障碍。了解ChAHP在人类发育过程中起什么作用，以及它是如何起作用的，不仅将为HVDAS提供重要信息，还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eSINEs失活，但当细胞需要开始分化时，ChAHP准备激活eSINEs。我们进一步认为，ADNP在eSINEs激活过程中从eSINEs中转移，使CHD4招募NuRD成分，帮助eSINEs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，因此为了充分了解人类ChAHP的功能，我们需要研究人类细胞中的ChAHP。我们将创造人类多能干细胞，我们可以快速消耗蛋白质，并评估其损失对ChAHP组装和功能的主要后果。我们将定义ChAHP如何被引导到它的作用位点，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eSINEs来控制基因表达。我们将准确定义在HVDAS中受影响最严重的一些组织形成过程中哪些发育决定显示ADNP依赖性，然后确定在携带致病ADNP突变的细胞中出现了什么问题。总之，这将是对人类干细胞如何能够使用eSINEs以及如何在人类疾病中出错的全面调查。我们的发现不仅与那些受HVDAS或自闭症影响的人有关，而且与研究转座子、转录控制和人类干细胞生物学的基础科学家有关。

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.