Imaging chromatin transitions using high performance microscopy

The DNA sequence that encodes the information required to specify many organisms, including humans is known. This information exists as functional units referred to as genes, and it is normally the case that in a specific context only a subset of genes are used. Understanding how genes are activated and switched off at the appropriate times represents a key challenge to understanding how healthy organisms develop normally and what goes wrong during disease.

A key step in the activation of genes is the generation of an RNA copy of the DNA gene by a protein called RNA polymerase. The ability of RNA polymerase to gain access to DNA is normally prevented by packaging of DNA with proteins to for a condensed structure termed chromatin. It is becoming apparent that the unfolding of chromatin during activation and the subsequent refolding represent important points of control. However, understanding what occurs during the regulation of individual genes is to some extent limited by the experimental techniques that are available.

In this proposal we propose to use very recent developments in high performance microscopy to directly track the motion of a single gene both when it is switched off and when it is on. This type of visual information is very informative, providing a measurement of the extent that genes are unfolded and for how long they are unfolded during the course of RNA synthesis.

We hope that the observations we make will help to fill gaps in our understanding of a process which is fundamental to most biological systems.

The genomes of eukaryotes exist predominantly as chromatin. It's clear that the structure of chromatin is not static but subject to dynamic regulation. Major insight into chromatin organisation has been provided by biochemical and structural studies performed with isolated components. However, for complex rearrangements, such as those occurring during transcription, it is difficult to know whether effects observed in vitro truly reflect a process that occurs within cells. While it is possible to determine the positions of factors such as polymerases and nucleosomes along a DNA sequence using genomic approaches, they do not provide high resolution structural information in real time. Advances in the spatial and temporal resolution of fluorescent light microscopy make this approach far better suited for the study of chromatin rearrangements. Here we propose to adopt these approaches to study the changes in the conformation of a gene during the course of transcription. This will provide new insight into the folding of nucleosome arrays and the extent to which nucleosomes are removed from DNA during the course of transcription. As such it will provide a new route to addressing the long standing issue of how polymerases transit chromatin.

It is envisioned that the major beneficiaries of this work will be academics. These will fall into those interested in genome organisation, transcriptional regulation and chromatin structure. In order to ensure that these groups of academics have the opportunity to engage with this research our work will be pretend at national and international research symposia. We will publish our work in open access publications and ensure a description of our research is kept up to date on our laboratory web site, both at a level understandable by experts and the general public.

There is also a technological aspect to our work in that it pushes in vivo 3D tracking to the limits of what is attainable. This involves iterative cycles of adjusting hardware and data analysis tools. The experience we gain is likely to be of interest to organisations developing subcellular imaging for a broad range of approaches. Our ethos will not be to restrict access to such developments in order to commercially exploit them, but to openly divulge what we learn. The involvement of the OMX technologist Markus Posche in the project provides one way of passing on this expertise to other users of the OMX in Dundee (which include users from other Scottish Universities). In addition, these technological developments will be passed on by publication and presentation at meetings. In this respect Jason Swedlow is well placed to pass on knowledge as an instructor at international workshops on microscopy.

The application of new systems for analysing high resolution images has relevance to community based projects such as the Open Microscopy Environment (Nature Methods 9:245) a collaborative effort to produce tools to support data management for light microscopy. We will interact with the developers of this project to enable new approaches to be incorporated. Open disclosure does preclude commercial development and this is illustrated by the activities of companies such as Glencoe Software, which has strong links to Dundee, specialising in the application of expertise in data management and analysis.

We have a track record of communicating our results to the public through participation in open days, visiting schools, producing movies that describe our research