Next generation approaches to understand tissue specific regulation of the glucocorticoid response

了解类固醇激素受体的调节因子如何协同工作来处理细胞对糖皮质激素的反应是生物学几个领域的基础，包括基因调控、信号反应和生物异质性。这些系统是高度相关的，因为糖皮质激素不仅是人体自然产生的，而且由于其低成本和抗炎作用，它们也是世界上最常用的处方治疗方法之一。因此，在我们的一生中，细胞不断地与类固醇接触，要么是身体产生的，要么是治疗性的。糖皮质激素受体（GR）是一种几乎存在于人体每个细胞中的蛋白质。GR对身体产生的类固醇激素皮质醇作出反应，并通过激活控制多种细胞反应的基因将这一信号转化为行动，包括发育、代谢和免疫反应。GR在分子水平上得到了广泛的研究。这些研究揭示了皮质醇是如何激活GR并引导其进入细胞核的。一旦进入细胞核，GR就直接与基因的启动子和增强子结合，激活它们。我们还了解到GR不是单独起作用的：受体受一个复杂的相互作用网络的调节，包括共激活因子、共抑制因子和表观遗传修饰因子，以及其他核受体。不同细胞类型中不同调节因子的存在所引起的反应范围可能是巨大的。例如，GR的激活在促进乳腺组织生长的同时，同样的激活会导致血液中的淋巴细胞数量迅速减少。为了精确控制细胞对类固醇的反应，我们必须了解这些反应的差异是如何由这些调节因子控制的。一旦理解，我们将能够重新编程一种细胞类型，使其反应更像另一种。考虑到GR激活化合物无处不在的性质，这具有深远的意义：使我们能够阻断不想要的细胞反应，激活细胞中的新反应以选择性地指导它们生存或死亡，或精确地靶向特定组织以获得特定结果。本项目要解决的正是这种知识差距。通常，研究像GR这样的复杂系统是非常耗时的，因为必须依次识别和研究系统的每个部分。相反，我们将利用单细胞实验技术的发展，使GR研究成为更大系统的一部分。第一步将是在多个组织中检测与GR相互作用以响应糖皮质激素的共同调节因子。这些将定义GR与系统的部分相互作用以及它们如何在组织之间变化。第二步将是删除编码这些部分的基因，即GR的调节基因，然后监测它如何改变细胞对激活GR的反应。最先进的单细胞测序技术使这成为可能，使我们能够增加我们可以靶向的调节基因的数量，并增加我们可以收集的数据量。此外，由于单细胞测序比以前的方法需要更少的细胞，这项技术使我们能够在健康志愿者的细胞中进行工作。我们生成的描述这些基因编辑细胞中GR反应的数据将使用当前和未来的计算方法进行拟合，以构建一个功能和实验可测试的共同调节网络，该网络调节每个组织中的GR信号。这将有效地为我们提供不同组织的接线图。最后，我们将使用我们生成的模型来确定如何精确地改变每个组织中细胞对类固醇的反应。

Understanding how the regulators of steroid hormone receptors work together to process the cells' response to glucocorticoids is fundamental to several fields of biology, including gene regulation, signaling responses and biological heterogeneity. These systems are highly relevant as not only are glucocorticoids naturally produced by the body, they are also some of the most commonly prescribed therapeutics worldwide because of their low-cost and anti-inflammatory effects. Therefore, throughout our lives, cells are constantly coming into contact with steroids, either produced by the body or therapeutically. The Glucocorticoid Receptor (GR) is a protein found in almost every cell in the body. The GR responds to the body's production of the steroid hormone cortisol and translates this signal into action by activating genes that control a diverse range of cellular responses, including development, metabolism and immune response. The GR has been extensively studied at the molecular level. Such research has revealed how cortisol activates the GR and leads it to enter the nucleus. Once in the nucleus the GR binds directly to the promoters and enhancers of genes, activating them. We have also learnt that the GR does not work alone: the receptor is regulated by a complex network of interactions including co-activators, co-repressors and epigenetic modifiers, along with other nuclear receptors. The range of responses caused by the presence of different regulators within the different cell types can be huge. For example, while GR activation promotes the growth of mammary tissue, the same activation of the GR causes lymphocytes within the blood to rapidly reduce in number. In order to precisely control the cellular response to steroids it is vital that we understand how the differences in these responses are controlled by these regulators. Once understood we will be able to reprogram one cell type to respond more like another. Given the ubiquitous nature of GR activating compounds this has far-reaching implications: enabling us to block an unwanted cellular response, activating novel responses in cells to selectively direct them to survive or die, or precisely target a specific tissue to gain a specific outcome. It is this knowledge gap that the present project will address. Typically, studies that focus on complex systems like the GR are extremely time consuming as each part of the system must be identified and studied in turn. Instead, we will apply leverage to the development of single cell experimental techniques to enable the study of the GR as part of a much larger system. The first step will be to detect the co-regulators that interact with the GR in response to glucocorticoids in multiple tissues. These will define the parts of the system that the GR interacts with and how they change between the tissue.The second step will be to delete the genes that encode for these parts, the regulators of the GR, and then monitor how it alters the cells' response to activating the GR. State-of-the-art single cell sequencing technology makes this possible by enabling us to increase the number of regulator genes we can target and increases the amount of data we can collect. Additionally, because single-cell sequencing requires fewer cells than previous methods, the technology enables us to undertake the work in cells from healthy volunteers.The data we generate that describes the GR responses in these gene-edited cells will be fitted, using current and future computational methods, to build a functional and experimentally testable representation of the network of co-regulators that modulate GR signaling in each tissue. This will effectually provide us with a wiring diagram of the different tissues.Finally, we will use the models we have generated to establish how to precisely alter the cells' response to steroids in each tissue.