Investigating retrotransposon-driven gene expression programmes in early development

受精后，单个受精卵经过一系列分裂步骤发育成多细胞胚胎，称为囊胚。囊胚的细胞能够产生所有的成年细胞类型，这种现象被称为多能性。此外，囊胚内细胞团（ICM）可在培养皿中培养为多能胚胎干细胞（ESCs）。ESCs已成为再生医学和研究发展本身的宝贵工具。在英国，每8对夫妇中就有1对患有不孕症，因此了解促进胚胎健康发育的因素变得越来越重要。转座因子（te）是我们DNA中当前或历史上可移动的部分，即具有将自己“粘贴”到基因组中新位置的能力。许多TE序列过去被认为是简单的“垃圾DNA”；然而，我们开始认识到te已经进化到在发育和疾病中发挥新的和意想不到的作用。例如，不受控制的TE活动与神经变性和癌症有关。然而，在正常发育过程中，许多te的表达也很高，这表明它们可能在细胞中也有有益的作用。本研究的重点是探索一种特殊TE的功能和调控，称为小鼠内源性逆转录病毒型L， MERVL。MERVL是最早表达的TE，在小鼠胚胎2细胞期短暂上调。在人类4-8个细胞胚胎中，这个阶段包含了一个叫做合子基因组激活的基本过程，当胚胎第一次开始开启自己的基因时。这些胚胎也被认为是“全能性的”，这意味着它们不仅可以产生胚胎组织，还可以产生胚胎外组织（如胎盘）。有趣的是，一小部分ESCs在正常培养中瞬间变成“2c样”，也具有增强的发育潜能。在这里，我们将使用小鼠ESCs和小鼠胚胎来研究MERVL调控如何以及为什么在早期发育中很重要。使用这些工具，我们将识别和描述激活和抑制MERVL所需的关键因素。反过来，我们将研究这些因子如何调节2细胞阶段，并影响ZGA和全能性。为了了解MERVL和其他te是如何被直接调控的，我们将把基因组编辑系统CRISPR/Cas9与最新的生化工具结合起来，提取出结合MERVL的蛋白质。最后，我们将探讨MERVL在人类细胞中的功能和调控的保守性，其中已知类似的TE HERVL起保守作用。我们的目标是a)了解HERVL如何调节4-8细胞阶段和人类ZGA； b)研究新的HERVL调节因子如何有助于特定疾病的发生。这些研究将大大增加我们对TEs如何促进早期发育的理解，并将揭示这些过程如何在疾病中受到干扰。

After fertilization, a single zygote proceeds through a series of cleavage steps to develop into a multicellular embryo, called a blastocyst. The cells of the blastocyst are capable of generating all adult cell types, a phenomenon known as pluripotency. The inner cell mass (ICM) of the blastocyst can moreover be cultured in a dish as pluripotent embryonic stem cells (ESCs). ESCs have become invaluable tools in regenerative medicine and to study development itself. With 1 in 8 couples experiencing infertility in the UK, it is ever more important to understand the factors contributing to healthy embryo development.Transposable elements (TEs) are parts of our DNA that are currently or historically mobile, -i.e. having the capacity to 'paste' themselves into new places in the genome. Many TE sequences used to be thought of as simply 'junk DNA'; however, we are beginning to understand that TEs have evolved to play new and unexpected roles in development and disease. For example, uncontrolled TE activity has been implicated in neurodegeneration and cancer. However, the expression of many TEs is also high in normal development, suggesting that they may also have beneficial roles in cells. This proposal focuses on exploring the function and regulation of a particular TE, called mouse endogenous retrovirus type L, MERVL. MERVL is the earliest expressed TE, and is transiently upregulated in mouse embryos at the 2-cell stage. This stage, conserved in human in 4-8 cell embryos, encompasses an essential process called Zygotic Genome Activation, when the embryo begins to turn on its own genes for the first time. These embryos are also considered "totipotent", meaning that they can not only generate embryonic tissues but also extra-embryonic tissues (like placenta). Interestingly, a small proportion of ESCs transiently become "2C-like" in normal culture, also possessing enhanced developmental potency. Here, we will use mouse ESCs and mouse embryos to investigate how and why MERVL regulation is important in early development. Using these tools, we will identify and characterize key factors required to activate and repress MERVL. In turn, we will investigate how these factors regulate the 2-cell stage, and affect ZGA and totipotency. To understand how MERVL and other TEs are directly regulated, we will combine genome-editing systems, called CRISPR/Cas9, with recent biochemical tools to pull out sets of proteins that bind MERVL. Lastly, we will explore the conservation of MERVL function and regulation in human cells, where a similar TE, HERVL, is known to play a conserved role. We aim to a) understand how HERVL regulates the 4-8 cell stage and human ZGA b) investigate how new HERVL regulators might contribute to specific cases of disease. These studies will significantly increase our understanding of how TEs contribute to early development, and will shed insight on how such processes are perturbed in disease.