Imaging functional chromatin architecture in 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.

How chromatin is organised in the nucleus to facilitate genome function is a major current topic in biology. The discovery of Topologically Associated Domains (TADs) is a key recent advance providing an architectural foundation for genome function. However, many questions remain concerning how TADs assemble to form functional nuclear compartments and how TAD structure reorganises in different states of gene activity. Imaging approaches have great potential to solve these questions particularly with advances in super-resolution microscopy. However, the dense packing of chromatin in the nucleus still presents a major challenge. In this proposal we will exploit a particular cell, the Drosophila spermatocyte, since in their giant nuclei decreased chromatin density greatly facilitates visualisation of domain organisation. Using super-resolution microscopy, we will compare active and inactive chromatin to determine the domain organisation associated with gene activity. We will use locus-tagging to image individual genes enabling us to separately determine the organisation of constitutively-active versus regulated genes. The latter will enable us to establish how domain structure alters upon gene activation and whether activation operates at the level of whole domains or in the looping-out of individual genes. The enormous (1-4Mb) Y-loops of the spermatocyte offer a powerful model for studying the formation and transcription of active chromatin loops. We will exploit the Y-loops to study the linkage between loop formation and transcription and the spatial organisation of transcription, determining whether polymerases are fixed or mobile in the nucleus and, using RNAi, to identify key mechanisms involved in the initiation and expansion of active chromatin. 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.

Outside of our immediate professional circle and the wider academic community described above, we believe there is potential impact for the biotech/biomedical communities in the medium to long term. Firstly our work is at the frontier of the application of super-resolution microscopy to biological imaging and, with the collaboration of CAIC, we will be extending and developing methodology in super-resolution imaging. As super-resolution becomes more generally available these examples of super-resolution imaging and developed methods will potentially have wide-ranging application. Also our work investigating the functional organization of chromatin in the nucleus offers a path to characterizing "nuclear phenotypes" of different cell states. In line with the mounting evidence for the importance of epigenetic changes affecting chromatin structure in cancer such a "nuclear phenotype" may define disease states and also contribute to cell state identification in reprogramming in regenerative medicine. We will deliver these potential impacts by the usual route of peer-reviewed publication and conference presentation. Any published work will be flagged to our Press office for possible press release.

Supporting the need for a trained workforce, we will deliver training to the researchers and any graduate students associated with this work in the BBSRC priority area of bioimaging in the "new ways of working" strategic theme. This will make a contribution to increasing the UK skill base in bioimaging. In particular, the analysis of large scale imaging data requires a considerable degree of IT skills and an understanding of statistical methods. Training in these areas will be provided in the course of this project and will be general enough to be applicable also in other work areas. This impact will be delivered over the course of the funding period.

More generally, we believe our international reputation in the field of genome architecture is of wider benefit to UK science. Our previous impacts in this area have included increased international collaborations, including participating in major international efforts such as modENCODE. Maintaining a UK profile in modern bioscience research is important for attracting research to the UK. Our publications and presentations will continue to provide these impact benefits over the course of the grant.

Our work will also have public impact. How the vast information in the genome is decoded is a topic of wide public interest. How this information is densely packaged into the cell nucleus and yet is also available for use is a biological mystery that is easily grasped and appreciated. Our work directly addresses this mystery and combined with the exciting new possibilities of super-resolution microscopy there is much to communicate with the public. Our efforts to present this work at University open events will progress throughout the period of the funding and continue beyond.