Splitting STAT Dimers to Understand Interferon Balance: A Strategy to Dissociate Beneficial and Detrimental Interferon Effects in Infection?

Background: Interferon signalling is critical for health, but knowledge of key aspects remains opaque. Interferons (IFNs) constitute a large family of proteins that alter the behaviour of cells in important ways through changes in gene transcription. Discovered more than 60 years ago for their role in the first line of defence against virus infections, these proteins have now more than reached the potential envisioned by the early discovering virologists. Interferons are commonly used in anti-hepatitis B virus therapy, and they have found additional therapeutic applications for oncology and multiple sclerosis. It has been recognized that interferons enhance innate and acquired immune responses and modulate normal and tumour cell survival and death.
Studies of interferons have resulted in fundamental insights into the mechanisms of cellular signalling, gene transcription and the workings of the innate and adaptive immune systems.
Mutations in STAT proteins and other interferon pathway constituents give rise to a spectrum of severe inherited immune system diseases characterised by susceptibility to infections, autoimmunity, and cancers, unambiguously demonstrating the vital importance of IFNs to the health of animals and humans.
Hypothesis and aims: We postulate that preformed STAT dimers are crucial determinants of interferon signalling and cytokine biology at large. To test this hypothesis, naturally occurring STAT protein mutations will be used to split STAT dimers in vitro and in vivo. This is done to ascertain if STAT dimer spitting changes the nature of interferon signalling. Specifically, it is the aim of the research project to determine if the splitting of STAT1/STAT2 heterodimers shifts the IFN-I-induced transcriptome towards IFN-II, and if this in turn boosts cell-autonomous antimicrobial defences. This PhD project will deliver fundamental insight in the assembly of preformed STAT dimers to understand the balance between Interferon-I and Interferon-II pathways and determine if dimer splitting can offer new ways forward to harness interferon's microbicidal potential. The project supports the BBSRC's long-term aims to deliver discovery-led frontier bioscience research that will underpin strategies for innovation in tackling infections and improving health.
Methods: There are particular hurdles in the study of dynamic processes such as IFN signalling that involve transient protein interactions and the rapid translocation of large protein complexes from the cell membrane to the nucleus. The project thus needs access to a diverse range of tools and methods to achieve its goals. These include live cell fluorescence imaging and FACS imaging to track the intracellular movements of STAT proteins. Studies of natural dimer-splitting STAT mutations will use gene-edited immune cell lines generated by CRISPR/Cas9 technology. Recombinant proteins will be produced in bacteria and insect cells for structural studies of STAT dimerization by analytical ultracentrifugation or protein crystallography. Next generation sequencing and quantitative reverse transcriptase PCR will be used to trace transcriptome changes caused by STAT dimer splitting mutations. Consequences for antimicrobial protection are assessed with cell-based infection experiments and various pathogens. Finally, good science thrives where cutting-edge technology operates in a collegial, intellectually stimulating setting. Our labs in the QMC and the Research Complex at Harwell provide such an environment.