Dynamics of global chromatin landscape through the cell cycle and differentiation

所有高等生物的基因组都以染色质的形式包装，染色质是DNA和与之结合的蛋白质的组合。将DNA包装成染色质是必不可少的，因为每个单独的染色体都由一个长达几厘米的DNA分子组成，但它必须包装在直径只有几微米的细胞核中。事实上，正常人类细胞的DNA总长度约为2米。同时，当需要允许基因的受控表达或细胞分裂周期中DNA的复制时，DNA必须是可访问的。染色质包装的基本单位被称为核小体，它由一种被称为组蛋白的特殊高度保守的蛋白质组成，DNA大约包裹在核小体周围两次。核小体似乎位于特定的位置，特别是在基因等重要特征周围。很明显，生物体的许多特征和一些疾病都受到所谓的表观遗传修饰的影响，这种修饰是由于染色质的变化或DNA的修饰，而不是潜在DNA序列本身的变化。因此，了解染色质在许多生物过程中是至关重要的。除了核小体外，还有许多其他蛋白质复合体与DNA结合，用于许多目的，特别是控制基因的表达。核小体和其他蛋白质与DNA结合的模式可以通过使用酶来可视化，这种酶只在没有蛋白质结合的地方切割DNA。受保护的区域不会被切割，用这种酶消化染色质会产生数百万个DNA片段。它们主要是单个或多个核小体的大小。我们已经开发出一种新技术，可以同时确定整个基因组中所有这样的保护区，从而绘制出每个核小体和其他与DNA结合的蛋白质复合体，以产生一幅“染色质景观”。这利用高通量测序来同时从大约1.8亿个这样的片段的两端产生DNA序列。然后，这些数据被用来将片段的端点映射到基因组序列上，从而通过切割末端很少的区域定位结合蛋白的位置。我们已经使用模式植物拟南芥演示了这项技术。在这项提案中，我们将使用这种新的方法来理解染色质结构是如何随着细胞分裂和细胞分化而改变的。这项分析将在细胞培养和完整组织的细胞中进行，从而详细了解在这些过程中染色质是如何发生变化的。具体地说，我们将研究拟南芥的染色质结构如何对光做出反应，以及它在根和地上部细胞中的不同之处。我们还将研究在染色质结构改变和分化受到影响的特定突变体中，这种模式是如何改变的。因此，我们将详细了解高等生物体中染色质的组织，以及这种组织是如何随着细胞在发育过程中命运的变化而动态改变的。这些技术和方法将适用于所有真核生物，并将对促进我们对基因组的理解具有重要意义。

The genomes of all higher organisms are packaged in a form known as chromatin, a combination of the DNA and proteins that are bound to it. This packaging of DNA into chromatin is essential because each individual chromosome consist of a single molecule of DNA up to several centimeters long but which must to be packaged up in a cell nucleus only a few micrometers across. Indeed the total DNA length in a normal human cell is about 2m. At the same time, the DNA must be accessible when required to allow a controlled expression of genes, or the copying of DNA during the cell division cycle. The basic unit of chromatin packaging is known as the nucleosome, which consists of a core of special highly conserved proteins known as histones around which the DNA is wrapped approximately twice. Nucleosomes appear to be located in specific positions, particularly around important features such as genes. It has become clear that many characteristics of organisms, and some diseases, are affected by so-called epigenetic modifications, resulting from changes in the chromatin or modification of the DNA rather than changes in the underlying DNA sequence itself. Hence understanding chromatin is of fundamental importance in many biological processes.In addition to nucleosomes, there are many other complexes of proteins that bind to DNA for a number of purposes, particularly the controlled expression of genes. The pattern of nucleosomes and other proteins binding to DNA can be visualised by using enzymes that cut DNA only where no proteins are bound. Regions that are protected are not cut, and digesting chromatin with such enzymes generates many millions of DNA fragments. These are predominantly the size of single or multiple nucleosomes. We have developed a new technology that can simultaneously determine all such protected regions across the entire genome, and hence map every nucleosome and other DNA-bound protein complex to generate a "chromatin landscape". This makes use of a high throughput sequencing to generate DNA sequence from both ends of around 180 million such fragments simultaneously. This data is then used to map the endpoints of the fragments onto the genome sequence, thus locating the positions of the bound proteins through regions with few cut ends. We have demonstrated this technology using the model plant Arabidopsis. In this proposal will use this new approach to understand how chromatin structure changes both through the cell cycle as cells divide and as cells differentiate to adopt new characteristics. This analysis will be carried out in both cell culture and in cells from intact tissues, allowing a detailed understanding of how chromatin changes occur during these processes. Specifically we will study how chromatin structure in Arabidopsis changes in response to light and how it is different in root and shoot cells. We will also investigate how this pattern is changed in a specific mutant in which chromatin structure is altered and in which differentiation is affected. As a result, we will develop a detailed understanding of the organization of chromatin in a higher organism, and how this is dynamically altered as the fate of cells changes during the processes of development. These techniques and approaches will be applicable to all eukaryotic organisms and will be of great significance in progressing our understanding of the genome.