Our main goal is to identify and exploit new factors that might serve as targets in cancer therapy or as markers of cell proliferation potential. To achieve this we are investigating the functional organisation of the mammalian cell nucleus, and the transitions that this undergoes during cell differentiation and passage through the cell cycle.
We are focused on the temporal and spatial organization of DNA replication and related processes, by studying the relationship between replication proteins, chromatin, RNA and the insoluble structural framework referred to as the nuclear matrix (Fig. 1). In mammalian somatic cells cyclin-dependent initiation involves spatially constrained assembly of replication proteins inside the nucleus during G1 phase of the cell cycle, followed by their activation to begin DNA synthesis. In recent years we have studied the MCM protein complex, which becomes briefly attached to the nuclear matrix just before DNA replication begins (Hesketh et al 2015), the cyclins themselves (Munkley et al 2011), and the cyclin-interacting protein CIZ1 (Copeland et al 2015). Cyclin E is spatially constrained in fundamentally different ways in cell types that retain the capacity to change (stem and progenitor cells) compared to those that have a defined identity, suggesting a fundamental transition in the way that regulatory factors access their targets during differentiation. We also showed that cancer cells are similar to stem cells in this respect, having spatially unconstrained replication proteins. Heritable changes in gene expression are accompanied by epigenetic modifications of DNA or chromatin, but our data suggests that epigenetic information may also be specified in somatic cells by immobilization of DNA replication on the nuclear-matrix, facilitating the formation of a constrained genomic arrangement. We have found that this level of control is compromised in cancer cells likely contributing to their plasticity and failure to maintain epigenetic control of gene expression.
Fig. 1 A mammalian cell nucleus from which membranes, soluble proteins and histones have been removed, so that chromatin loops emanate from attachment points on the nuclear matrix to form a diffuse halo (left). Also shown are focal sites of DNA replication (right), which are constrained spatially by attachment to the nuclear matrix. A merged image is shown in the centre, where DNA is pink (courtesy of Rose Wilson)
Currently, we are focused on the CIZ1 protein and its interaction partners, and are studying both function and expression. Recent analyses have implicated CIZ1 in the spatial constraint of Xist RNA at the inactivated X chromosome in female somatic cells, most likely by anchoring the RNA to the nuclear matrix, leading to loss of the epigenetic marks that normally help to prevent gene expression (Fig. 2) Our analysis has also revealed multiple transcript variants of CIZ1 that are linked with different types of cancer, most notably b-variant CIZ1 is expressed in tumours of the lung. We can detect b-variant in blood from patients with very early stage lung tumours (Higgins et al 2012), a finding that is being developed as the basis of a blood test for lung cancer by University of York spin-out company, Cizzle Biotech. Our work has received support from Yorkshire Cancer Research, the Lister Institute of Preventive Medicine, Wellcome Trust, Royal Society, BBSRC and Industry.
Fig. 2 Cells from a mouse with no CIZ1 (null) and a normal equivalent, showing CIZ1 (green) and a chromatin mark associated with repression of gene expression (H3K27Me3, red). In the absence of CIZ1, this epigenetic mark is reduced across the nucleus, and completely lost from the inactive X chromosome, which are yellow in these merged images (courtesy R. Ridings-Figueroa, and J.F-X. Ainscough).
|Emma Stewart||PhD student||The nuclear matrix in development and disease|
|PhD student||Nuclear organisation in quiescent cells|
|MRes student||CIZ1 function at the inactive X chromosome in S phase|
|PhD student (Southgate/Coverley)||Nuclear matrix recruitment of nuclear receptors during urothelial cell differentiation|
|Technician||Ultrasensitive detection of lung cancer biomarker CIZ1B|
|Post-doctoral research associate||Role of CIZ1 in maintenance of epigenetic landscape|
|Technician||Primary cell models|
As a teacher I aim to give students a framework into which they can slot the knowledge they acquire through reading, and the skills they develop through the program. I want students to be able to understand how different topics interlace to build a comprehensive picture of my area of biology, out of which appreciation of underpinning principles can emerge. It was not until I felt I had 'filled in the gaps' between topics that were taught in isolation during my own education, that new questions began to occur to me. I aim to help my students get to that stage so that their originality can emerge.
I am a mammalian cell biologist mainly, with background in biochemistry (PhD) genetics (BSc) and developmental biology (post-doc) so I tend to teach my subject area from multiple angles, and also to use a range of different techniques in my research. Both are focussed on the functional organisation of the mammalian cell nucleus, the transitions that the nucleus undergoes during the cell cycle and during differentiation, and disruption of its organisation in diseases such as cancer. My main teaching area is cancer biology, which is a vast and fascinating subject that will touch most of us at some points in our lives.
My tutorials often start with an overview of what we do in the lab, as a way to introduce techniques and concepts in mammalian cell biology. This usually results in questions and discussion, and often identifies an area which we choose to investigate further in subsequent sessions. The overall theme is the structure and function of the mammalian cell nucleus which relates to my research on epigenetic control of gene expression. Later in the tutorial set students are encouraged to pick a topic that they are interested in, within this overall theme, and to bring their new knowledge to the group. Tutorials are the most interesting and rewarding of my teaching activities because they can be quite unpredictable, and by the end of a set I feel that I know my students.
Students taking projects in my lab will be working on something related to our ongoing research. We have a wide array of molecular tools that have been developed over the years, including antibodies that can recognise specific alternatively spliced forms of the CIZ1 protein, which plays a role in organising DNA replication in nuclear space. Many of these forms of CIZ1 are linked with disease yet we know very little about them. Projects might use specific antibodies to uncover the expression patterns, interactions and regulation of variants using techniques that include mammalian cell culture, sub-cellular fractionation, quantitative immunofluorescence microscopy or western blot. We also use quantitative gene expression analysis, bioinformatic techniques and many other approaches dictated by the question we are trying to answer.
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