Imaging functional chromatin architecture in Drosophila

果蝇功能性染色质结构成像

• 批准号: BB/S00758X/1
• 负责人: Robert White
• 金额: $ 48.73万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FS00758X%2F1
• 关键词: Imaging; functional; chromatin; architecture; Drosophila

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.

我们体内的每个细胞都包含在一米的DNA上，与蛋白质包裹在一起形成染色质，并紧紧填充到细胞核中。然而，核不仅是基因的仓库。这是一个工作的工厂，它积极使用DNA中的信息来制造维护细胞并允许它们开发的产品。核中的染色质是如何组织起来使该工厂有效工作的主要问题的主要问题。在细胞核中已经确定了各种组织的水平。大规模的基因组被排列成独特的非活动和主动隔室。在较小的规模上，最近的主要发现是折叠染色质纤维以形成一系列被称为拓扑相关的域（TAD）的簇。这些TAD构成了核中染色质组织的构建基块。 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.我们建议使用最新的超分辨率显微镜的发展来克服这些问题，并研究具有高度核核的细胞类型，使染色质组织更容易看到。在初步研究中，将超分辨率显微镜应用于果蝇精子细胞核，我们看到染色质已组织到群集中。正如我们和其他人以前在果蝇基因组中绘制了Tads，我们将测试簇是否确实与TAD相对应。然后，我们将使用将基因（RNA聚合酶中的信息）转录的酶来标记正在积极转录的基因组区域，以便我们可以比较活性区域与非活动区域中TAD的组织。这将使我们对这两个隔室中染色质域的组织的组织无与伦比，揭示了组织与功能的关联。对于更具体的视图，我们将使用基因组编辑来标记特定基因。我们将专注于两组基因。在我们选择的细胞类型（精子细胞）中专门表达的所有细胞类型中活性和发育调节的基因活性的家具基因。我们先前对基因组序列组织的研究表明，这两个基因集发生在单独的TAD中，因此我们希望它们在促进其不同调节的细胞核中的组织方式不同。此外，对受发展调节基因的分析使我们能够探究TAD组织如何与基因激活相关。每个TAD都包含几个基因，因此，如果打开TAD中的一个基因，则整个TAD会拆开以形成膨胀的染色质环或活化基因的特定区域。这个问题的答案将使我们深入了解基因调节的机制，表明TAD是否仅仅是建筑构建块，或者它们是否也是调节域。果蝇精子细胞具有另一个特征，使其对研究特别有用。当在这些细胞中特别激活y染色体时，一些基因作为巨型染色体环扩展。这些大循环在光学显微镜中很容易看到，并制造了一个非常有吸引力的系统来研究基因激活，染色质环形成和基因转录的组织。我们将使用动态成像方法研究这些过程，并通过鉴定形成这些循环所需的基因来研究所涉及的机制。综上所述，超分辨率显微镜在主要精子细胞特别有利的系统中的应用将在我们对核组织的理解中实现重大进步。

项目成果

The anatomy of transcriptionally active chromatin loops in Drosophila primary spermatocytes using super-resolution microscopy

使用超分辨率显微镜对果蝇原代精母细胞中转录活性染色质环进行解剖

• DOI: 10.1101/2022.07.27.500934
• 发表时间: 2022
• 作者: Ball M

Transcriptionally active chromatin loops contain both 'active' and 'inactive' histone modifications that exhibit exclusivity at the level of nucleosome clusters.

转录活性染色质环包含“活性”和“非活性”组蛋白修饰，在核小体簇水平上表现出排他性。

• DOI: 10.17863/cam.106933
• 发表时间: 2024
• 作者: Koestler S

The anatomy of transcriptionally active chromatin loops in Drosophila primary spermatocytes using super-resolution microscopy.

• DOI: 10.1371/journal.pgen.1010654
• 发表时间: 2023-03
• 期刊: PLoS genetics
• 影响因子: 4.5

Transcriptionally active chromatin loops contain both 'active' and 'inactive' histone modifications that exhibit exclusivity at the level of nucleosome clusters

转录活性染色质环包含“活性”和“非活性”组蛋白修饰，在核小体簇水平上表现出排他性

• DOI: 10.1101/2023.09.03.555774
• 发表时间: 2023
• 作者: Koestler S