Higher-order chromatin structure and regulatory sequence variation in human induced pluripotent stem cell (iPSC) self-renewal and differentiation

人体包含200多种不同类型的细胞，它们在不同的器官（如肝脏、大脑、心脏和皮肤）中实现高度专业化的功能。多能干细胞（PSCs）令人着迷，因为它们可以在称为细胞分化的过程中产生每种特化的细胞类型。这种独特的性质（多能性）使psc不仅具有极大的研究开发价值，而且在医学应用方面也具有很大的前景。一项获得2012年诺贝尔奖的发现发现，将PSCs分化为特化细胞的过程可以逆转：成熟的皮肤细胞可以被重新编程，成为所谓的诱导PSCs （iPSCs）。在临床上，这种重编程使得从患者的皮肤细胞（通过iPSCs）中获得一整套定制细胞成为可能，例如替换受损细胞甚至整个器官。这是再生医学的一个关键目标，它有可能解决可供捐赠的器官短缺和器官移植排斥的问题。所有的细胞都含有相同的基因；使细胞类型不同的是特定细胞类型中哪组基因是“开启”和“关闭”的。这决定了细胞中存在哪些蛋白质（细胞的主力）；只有“开启”的基因被表达并翻译成蛋白质（例如，在肝细胞中，“开启”的一组基因与在psc中不同）。有趣的是，虽然基因控制着细胞的功能，但它们只占我们细胞DNA的一小部分（约2%）。我们的绝大多数DNA（约98%）是非编码的，即不携带制造蛋白质的信息。曾经被认为是没有功能的“垃圾”DNA，我们现在知道这种非编码DNA包含大量所谓的调节元件。这些调节元件的功能就像分子开关一样，通过控制哪些基因何时“开启”。调控元件通过与目标基因的物理接触来开启它们。这是通过DNA环来实现的；基因组以一种特定细胞类型的方式以三维（3D）折叠，将适当的开关和基因聚集在一起。我们经常谈论人类基因组，但这是误导，因为有数十亿的人类基因组-我们都是不同的基因（除了同卵双胞胎）。我们基因组中的序列差异主要局限于非编码基因组，这不仅可以解释为什么我们看起来都不一样，还可以解释为什么我们体内的细胞以一种略微不同的方式运作，例如在疾病的背景下。在诱导多能干细胞中，这种自然发生的序列变异被认为是功能异质性的原因：一些诱导多能细胞系可以更有效地分化为肝细胞，而另一些诱导多能细胞系可以更有效地分化为脑细胞，而另一些诱导多能细胞系向所有更特化的细胞类型表现出普遍较差的分化效率。从机制上理解这一现象对临床很重要，因为它将在未来帮助定制再生医学治疗方案，特别是针对个体患者的要求；这是个性化医疗的一个关键目标。由于它们在基础研究和再生医学方面的巨大潜力，我们必须了解赋予多能干细胞独特特性的分子机制。因此，该项目的目的是i)绘制iPSCs中的3D基因组折叠图，以揭示它们使用哪些调控元件，ii)阐明这些调控元件如何随着iPSCs的分化而变化，以及iii)确定这些调控元件中的遗传变异，这些遗传变异导致iPS细胞系更有效或更低效地分化为特化细胞类型。这些知识在psc以外的研究中也很重要，因为异常的3D基因组折叠可以将错误的调控元件和基因对聚集在一起，从而导致包括发育畸形和癌症在内的疾病。

The human body contains over 200 different cell types which fulfil highly specialized functions in different organs such as the liver, brain, heart, and skin. Pluripotent stem cells (PSCs) are fascinating because they can give rise to each of these specialized cell types, in a process called cell differentiation. This unique property (pluripotency) makes PSCs not only immensely valuable to study development, but also endows them with great promise for medical applications. A discovery honoured with the 2012 Nobel prize found that the process of differentiating PSCs into specialized cells can be reversed: mature skin cells can be reprogrammed to become so-called induced PSCs (iPSCs). In the clinic, this reprogramming makes it possible to derive an entire palette of bespoke cells from a patient's skin cells (via iPSCs), for example to replace damaged cells or even entire organs. This is a key aim of regenerative medicine, and it has the potential to solve the problems of the shortage of organs available for donation, and of organ transplant rejection.All cells contain the same genes; what makes cell types different is which sets of genes are 'on' and 'off' in a particular cell type. This determines which proteins (the cell's workhorses) are present in a cell; only 'on' genes are expressed and translated into proteins (for example, in liver cells a different set of genes is 'on' than in PSCs). Interestingly, although genes control cell function, they make up only a small amount of the DNA in our cells (~2%). The vast majority of our DNA (~98%) is non-coding, i.e. does not carry the information to make proteins. Once thought to be 'junk' DNA without a function, we now know that this non-coding DNA contains a plethora of so-called regulatory elements. These regulatory elements function like molecular switches by controlling which genes are 'on' and when. Regulatory elements make physical contacts with their target genes to switch them on. This is achieved by DNA looping; the genome folds in three dimensions (3D) in a cell-type specific manner to bring the appropriate switches and genes together.We often talk of THE human genome, but this is misleading as there are billions of human genomes - we are all different genetically (apart from identical twins). The sequence differences in our genomes are largely confined to the non-coding genome, and can explain not only why we all look different, but also why the cells in our bodies function in a slightly different way, for example in the context of disease. In iPS cells, this naturally occurring sequence variation is thought to be the reason for functional heterogeneity: some iPS cell lines can be differentiated more efficiently into liver cells, whereas other iPS cell lines can be differentiated more efficiently into brain cells, and yet others show a generally poor differentiation efficiency towards all more specialised cell types. Understanding this phenomenon mechanistically is important for the clinic, as it will in the future help to tailor treatment options in regenerative medicine specifically to the requirements of individual patients; this is a key aim of personalised medicine.Because of their immense potential for basic research and regenerative medicine, it is crucial that we understand the molecular mechanisms that endow iPSCs with their unique properties. The aim of this project therefore is to i) map the 3D genome folding in iPSCs to uncover which regulatory elements they use, ii) elucidate how this repertoire of regulatory elements changes as iPSCs differentiate, and iii) identify the genetic variants in these regulatory elements that cause iPS cell lines to differentiate more/less efficiently into specialised cell types.This knowledge is also important in research outside of PSCs, as aberrant 3D genome folding can bring together the wrong pairs of regulatory elements and genes, which can lead to disease including developmental malformations and cancer.