Development of a chromatin immunoprecipitation protocol applicable to small cell populations and its application to embryo research.

构成我们身体的不同类型的细胞都含有相同的一组基因，一半遗传自母亲，一半遗传自父亲。这个简单的事实提出了一个有趣的问题：为什么不同类型的细胞差异如此之大？例如，肌肉细胞，皮肤细胞和白细胞，不仅看起来不同，而且在我们的身体中承担着非常不同的任务。这个问题的答案是，尽管所有细胞都有相同的一组基因，但它们以不同的方式使用它们。在所有细胞类型中，都有少数基因被“开启”或“表达”。这些基因编码对基本细胞功能至关重要的rna和蛋白质，如产生能量或制造基本细胞成分。它们被称为“管家”基因。其他基因仅在特定的细胞类型中才需要。负责制造携带氧气的蛋白质血红蛋白的基因只在那些变成红细胞的血细胞中表达。在我们体内所有不同类型的细胞中，都有一组不同的关键“组织特异性”基因被表达，这些基因制造蛋白质和rna，使细胞能够采用自己特定的形状，并执行自己的特定功能。如果我们能够理解基因开启和关闭的控制机制，那么我们就可以开始进行干预，将一种细胞类型变成另一种。例如，癌症的发生通常是由于基因表达异常，因此了解如何改变关键基因的表达，就有可能阻止或逆转肿瘤细胞的生长。基因可以通过几种方式调节，但一个适用于所有或绝大多数基因的关键因素是，DNA被包装成一种称为染色质的DNA-蛋白质复合体，主要由一小群蛋白质组蛋白组成。如果含有特定基因的DNA被组蛋白紧紧捆绑在一起，那么合成RNA所需的酶和其他因子就无法进入这些基因，这些基因就会被关闭。相反，如果DNA被解开，打开并可以接近，那么基因就可以表达。早期胚胎代表了生命中的一个阶段，在这个阶段，基因表达模式的变化尤为重要。受精卵能够制造我们体内所有不同类型的细胞。据说它是“全能的”。然而，随着胚胎的分裂，可能早在四细胞阶段，单个细胞就开始改变它们的基因表达模式，并致力于转变成一种特定的细胞类型。在胚胎生命的最初几天和几周内，随着细胞数量的增加，细胞继续改变它们的基因表达模式，因为它们变得更加特化，它们能成为的细胞种类也更加有限。了解控制这些基因表达早期变化的机制对于理解环境因素如何改变生命脆弱阶段的胚胎发育至关重要。到目前为止，研究早期胚胎中组蛋白对DNA的包装是不可能的，因为可用的细胞数量太少了。即使将多个胚胎结合起来，其数量也只有几百个，比目前实验技术所需的数量少了一万倍。我们开发了一种改进的“染色质免疫沉淀”方案，其中我们使用抗体从少至50-100个细胞中分离出由特定修饰组蛋白包装的基因。这项新技术使我们第一次能够研究早期胚胎细胞中DNA包装和关键基因调控的机制。为了充分利用这种新方法的巨大潜力，我们希望确定，首先，它如何适应于研究各种非组蛋白，其中一些转录因子，在基因表达中起关键作用，其次，它是否可以应用于最早的胚胎（2 -16细胞阶段），以研究环境影响（毒素，饮食成分等）对基因调控和胚胎发育

The different types of cells that make up our bodies all contain the same set of genes, half inherited from our mothers and half from our fathers. This simple fact raises the interesting question of why different types of cell are so very different; a muscle cells, skin cells and white blood cells, for example, not only look different, but do very different tasks in our bodies. The answer to this question is that although all cells have the same set of genes, they use them in different ways. A small number of genes are 'switched on', or 'expressed', in all cell types. These genes encode crucial RNAs and proteins that are essential for basic cell functions, such as generating energy or making essential cell components. They are called 'housekeeping' genes. Other genes are required only in specific cell types. Genes responsible for making the oxygen carrying protein haemoglobin are expressed only in those blood cells that become red blood cells. In all the different cell types in our bodies a different set of key 'tissue-specific' genes are expressed, making proteins and RNAs that allow that cell to adopt its own particular shape and carry out its own specific function. If we can understand the control mechanisms by which genes are switched on and off, then we can begin to intervene to turn one cell type into another. For example, cancers often occur due to abnormal gene expression so understanding how to alter the expression of key genes opens up the possibility of halting, or reversing the growth of tumour cells. Genes can be regulated in several ways, but a key factor applying to all, or the great majority of genes, is the packaging of DNA into a DNA-protein complex called chromatin, primarily by a small group of proteins, the histones. If the DNA containing specific genes is tightly bundled up by histones, then those genes will be inaccessible to the enzymes and other factors required to make RNA and will switched off. Conversely, if the DNA is unwrapped, opened up and made accessible, then the gene can be expressed. The early embryo represents a stage in life at which changing patterns of gene expression are particularly crucial. The fertilised egg is capable of making all the different cell types in our bodies. It is said to be 'totipotent'. However, as the embryo divides, possibly as soon as the four cell stage, then individual cells begin to change their patterns of gene expression and become committed to turning into a particular cell type. As cell numbers increase during the first few days and weeks of embryonic life, cells continue to change their patterns of gene expression as they become more specialised, and more limited in what sort of cell they can become. Understanding the mechanisms that control these early changes in gene expression is crucial in understanding how environmental factors can alter embryonic development at this vulnerable stage of life. Until now it has been impossible to study the packaging of DNA by histones in early embryos because the number of cells available is so small. Even by combining multiple embryos, the numbers are measured in hundreds, ten thousand times less than what is needed for current experimental techniques. We have developed a modified 'chromatin immunoprecipitation' protocol in which we use antibodies to isolate genes packaged by particular modified histones from as few as 50-100 cells.This new technique allows us, for the first time, to study mechanisms of DNA packaging and regulation of key genes in cells of the early embryo. In order to fully exploit the enormous potential of this new approach, we wish to determine, firstly how it can be adapted to study a variety of non-histone proteins, some of which, the 'transcription factors, play key roles in gene expression and secondly whether it can be applied to the very earliest embryos (2 -16 cell stages) in order to study environmental effects (toxins, dietary components etc) on gene regulation and embryonic development