Imaging functional chromatin architecture in Drosophila

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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.