Nuclear matrix support of DNA replication and transcription

Lead Research Organisation: University of York
Department Name: Biology

Abstract

Background Visualization of the structure of nuclei has been the main method of cancer diagnosis since light microscopy allowed. However, very little is known about the underlying molecular alterations that give rise to aberrant nuclear architecture, or the effect these have on the organisation of nuclear processes. The epigenetic information inherent in nuclear organisation is also likely to be relevant to our understanding of nuclear decay during aging, and the onset of senescence.

Objectives Some proteins that are involved in the regulation of DNA replication are immobilized by attachment to the nuclear matrix in normal cells, but not in cancer cells (or in undifferentiated cells). This project will ask whether the same is true for proteins that regulate transcription, in order to test the extent to which published observations can be generalized beyond DNA replication. This will be important to test further the idea that part of the process of differentiation is to fix processes of DNA metabolism in nuclear space to enable pattern formation.

It will also look in detail at the CIZ1 protein to identify the proteins that it normally binds to in the nuclear matrix. Unlike the cytoskeleton, there is currently no consensus on the main protein components of the nuclear matrix.

Novelty and Timeliness CIZ1 has been implicated in several common human cancer types, as well as chronic age-related disorders, and is being developed as the basis for a blood test for cancer. Its interaction partners are likely to offer similar opportunities for exploitation either as markers of nuclear integrity (for application in regenerative medicine strategies or analysis of cancer cells), and might offer new opportunities for therapeutic drug targets. There are very few molecules that can be used for function-related studies of the nuclear matrix making this work both novel and timely.

The project combines mammalian cell biology with analytical biochemistry, and combines supervisors with related but complementary expertise in the two core process of DNA replication and transcription. Extensive evidence shows that the organization of DNA replication is intrinsically linked to template usage, making this collaboration one that could yield new insight into the mechanistic relevance of the nuclear matrix. The project will involve mammalian cell culture and synchrony techniques, sub-cellular fractionation and stability assays to isolate nuclear matrix fractions using a range of techniques, fluorescence microscopy and protein interaction studies, including 2D-LC-MS to identify new components of the nuclear matrix.

This is an excellent opportunity for a student to work across the cell biology/biochemistry interface on a potentially high impact project. The student will attend lab meetings in both labs as appropriate, and will therefore gain experience in presenting their work to audiences with different expertise/interests. There will also be joint lab meetings between the two groups, which are likely to trigger further collaborations and opportunities. Such approaches are becoming the norm and it is essential that early career scientists gain experience in this new way of working.

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 30/09/2015 29/09/2023
1643825 Studentship BB/M011151/1 30/09/2015 29/09/2019 Emma Rachel Stewart
 
Description I have developed a method allowing me to see where proteins are in the cell and what they are attached to. This allows me to see how proteins may behave differently in different places in the cell or in different cell types (e.g. normal vs cancer). I have used this method and others to look at key players in an important cell process which results in disease when not carried out correctly. This has allowed me to work out some key differences between normal and cancer cells which will inform further work.
Exploitation Route These findings are of use to other researchers in deciding the most relevant models for their research. I have been able to take findings forward as basis of fellowship applications.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description High-fidelity epigenetic maintenance in somatic cells: CIZ1 assemblies as molecular shield
Amount £605,669 (GBP)
Funding ID MR/V029088/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start  
 
Description Honor Fell Travel Awards
Amount £600 (GBP)
Organisation British Society for Cell Biology 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2018 
End 03/2018
 
Description PhD Facilities Awards
Amount £5,000 (GBP)
Organisation University of York 
Sector Academic/University
Country United Kingdom
Start 07/2017 
End 01/2018
 
Description University of York Priming Funds
Amount £5,515 (GBP)
Organisation University of York 
Sector Academic/University
Country United Kingdom
Start 09/2019 
End 02/2020
 
Title Deposition of RNA-Sequencing Data in GEO 
Description Transcriptome analysis of primary (before passage 5) and culture-adapted derivative cell lines of murine embryonic fibroblasts in female WT (6 replicates) and female CIZ1 null (6 replicates) by RNA-seq. Additional samples include 3 inducible CIZ1-null primary murine embryonic fibroblast, 2 of which were induced with doxycycline. Accession Number: GSE122235 DOI of paper in which data used: 10.1038/s41467-018-08072-2 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact This data was used in a publication (DOI 10.1038/s41467-018-08072-2) 
 
Description Publication collaboration 
Organisation University of Oxford
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Experiments and materials towards publication
Collaborator Contribution Experiments and materials towards publication
Impact A scientific publication in Genes and Development
Start Year 2015
 
Description Visit by Rotary Club 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Supporters
Results and Impact Approximately 25 members of the York Rotary Club visited the laboratory and were shown around, with questions and discussions afterwards. Possible further funding for the laboratory including equipment useful to my award.
Year(s) Of Engagement Activity 2017