Imaging functional chromatin architecture in Drosophila

我们体内的每个细胞都含有超过一米长的DNA，这些DNA与蛋白质包裹在一起形成染色质，并紧密地包裹在细胞核中。然而细胞核不仅仅是基因的仓库；它是一个工作的工厂，积极地利用DNA中的信息来制造维持细胞并使其发育的产品。细胞核中的染色质如何组织以使这个工厂有效地工作是当前生物学中的一个主要问题。在细胞核中已经确定了不同层次的组织。在大范围内，基因组被分成不同的非活性和活性区室。在较小的范围内，最近的一项重大发现是染色质纤维折叠形成一系列被称为拓扑相关结构域（TADs）的簇。这些tad构成了细胞核中染色质组织的基石。这就提出了一个问题，即TADs是如何在细胞核中组装形成更大的活性和非活性区室的，以及将染色质包装到TADs中是如何促进基因组功能的。光学显微镜为研究结构提供了一种强有力的方法，但在过去，它用于研究核组织受到分辨率和核中染色质密集堆积的限制。我们建议利用最近发展的超分辨率显微镜来克服这些问题，并研究一种细胞核高度放大的细胞类型，使染色质组织更容易看到。在应用超分辨率显微镜对果蝇精母细胞核的初步研究中，我们看到染色质被组织成簇。正如我们和其他人之前在果蝇基因组中绘制的TADs一样，我们将测试这些簇是否确实与TADs相对应。然后我们将使用转录基因信息的酶，RNA聚合酶，来标记基因组中活跃转录的区域，这样我们就可以比较活跃区域和非活跃区域中tad的组织。这将给我们一个无与伦比的观点，染色质结构域的组织在这两个室揭示组织是如何与功能相关联的。对于更具体的观点，我们将使用基因组编辑来标记特定的基因。我们将关注两组基因；在所有细胞类型中都活跃的管家基因以及在我们选择的细胞类型精母细胞中特别表达的发育调节基因。我们之前对基因组序列组织的研究表明，这两个基因组发生在不同的TADs中，因此我们预计它们在细胞核中的组织方式不同，从而促进它们的不同调控。此外，对发育调控基因的分析使我们能够探索TAD组织如何与基因激活相关联。每个TAD包含几个基因，所以如果TAD中的一个基因被打开，整个TAD会解开形成一个扩展的染色质环，或者只是激活基因的特定区域。这个问题的答案将使我们深入了解基因调控的机制，表明TADs是否只是简单的建筑构件，还是它们也是调控域。果蝇的精母细胞还有另一个特点，这使得它们对研究特别有用。在这些细胞中，当y染色体上的一些基因被特异性激活时，会扩展成巨大的染色体环。这些大环在光学显微镜下很容易看到，是研究基因激活、染色质环形成和基因转录组织过程的一个非常有吸引力的系统。我们将使用动态成像方法来研究这些过程，并将通过识别形成这些环所需的基因来研究所涉及的机制。总的来说，超分辨率显微镜在初级精母细胞特别有利的系统中的应用将使我们对核组织的理解取得重大进展。

Each cell in our body contains over a metre of DNA, wrapped together with proteins to form chromatin, and tightly packed into the cell nucleus. Yet the nucleus is not simply a warehouse of genes; it is a working factory that actively uses the information in the DNA to make the products that maintain cells and allow them to develop. How the chromatin in the nucleus is organised to enable this factory to work efficiently is a major current question in biology. Various levels of organisation have been identified in the nucleus. On a large scale the genome is arranged into distinct inactive and active compartments. On a smaller scale, a major recent discovery is that the chromatin fibre is folded to form a series of clusters that are known as Topologically Associated Domains (TADs). These TADs form the building blocks of chromatin organisation in the nucleus. This raises the questions of how the TADs assemble to form the larger active and inactive compartments in the nucleus and how does the packaging of chromatin into TADs facilitate the function of the genome.Light microscopy provides a powerful approach to investigate structures but, in the past, its use to study nuclear organisation has been limited by resolution and by the dense packing of chromatin in the nucleus. We propose to overcome these problems using the recent development of super-resolution microscopy and studying a cell type that has a highly enlarged nucleus making chromatin organisation easier to see.In preliminary studies applying super-resolution microscopy to the Drosophila spermatocyte nucleus, we see that the chromatin is organised into clusters. As we, and others, have previously mapped TADs in the Drosophila genome we will test whether the clusters indeed correspond to TADs. Then we will use the enzyme that transcribes the information in genes, RNA Polymerase, to mark regions of the genome that are actively being transcribed so that we can then compare the organisation of TADs in the active versus inactive regions. This will give us an unparalleled view of the organisation of chromatin domains in these two compartments revealing how organisation is associated with function.For a more specific view, we will use genome editing to tag particular genes. We will focus on two sets of genes; house-keeping genes that are active in all cell types and developmentally-regulated genes specifically expressed in our chosen cell type, the spermatocyte. Our previous studies on genome sequence organisation have shown that these two gene sets occur in separate TADs so we expect they will be organised differently in the nucleus facilitating their different regulation. In addition, analysis of developmentally regulated genes allows us to probe how TAD organisation is linked to gene activation. Each TAD contains several genes so if one gene in a TAD is switched on does the whole TAD unravel to form an expanded chromatin loop or only the specific region of the activated gene. The answer to this question will give us insight into the mechanism of gene regulation indicating whether TADs are simply architectural building blocks or whether they are also regulatory domains.The Drosophila spermatocytes have another feature that make them specially useful to study. A few genes on the Y-chromosome when activated specifically in these cells expand as giant chromosome loops. These large loops are easy to see in the light microscope and make a very attractive system to study the processes of gene activation, chromatin loop formation and the organisation of gene transcription. We will use dynamic imaging methods to study these processes and will investigate the mechanisms involved by identifying genes required for the formation of these loops.Overall, the application of super-resolution microscopy in the particularly advantageous system of the primary spermatocyte will enable significant advances in our understanding of nuclear organisation.