Defining and exploiting the role of PML protein in innate immune responses to pathogens
Lead Research Organisation:
University of Warwick
Department Name: School of Life Sciences
Abstract
Just as we swap our lightweight summer clothes for winter ones when it gets cold, so our cells need to make different proteins to meet changing circumstances, such as when they are invaded by a virus. This project is about understanding how our cells change in response to virus infection, in particular, how they make what is termed an innate immune response. This response involves the cell making new proteins that act to limit the growth and spread of the virus. Proteins are produced from instructions carried in RNA molecules. These in turn are made by copying the information held in the cell's genes, a process known as transcription. So, when a cell is stressed by a virus infection, it responds by changes in its pattern of gene transcription.
We have discovered that a protein known as PML-II behaves like a master regulator of genes whose transcription is turned on rapidly after a virus infection. A major aim of this project is to find out just how broad is the set of genes that responds to the presence of this protein and, most importantly, what distinguishes these genes from those that are independent of it. We plan to use the most modern techniques to examine events across all the 20,000 or more genes in the human genome when an innate immune response is launched. In this way, we will get a system-wide view of the effects of the PML-II protein that is not limited by any preconceptions.
Going on from this, we intend to find out how differences in PML-II between individuals may influence how they respond to virus infection, since we know that there are major differences in the outcome of apparently similar infections between one person and another. We will be testing two types of difference in PML-II: first, differences at the gene level, as a result of the different genes we inherit; and second, differences that arise in cells as a result of the environment they experience. Here we will be looking particularly at heat stress - the kind of effect that can arise naturally when someone experiences a fever - and asking whether changes in PML that result from such stress are the basis for differences in innate responses and susceptibility to virus infection that we have already seen.
It is really important to unravel the ways in which innate immune responses are generated in the body. Cells being unable to respond sufficiently could mean that an infection spreads further and faster than it would have done - making you more sick. Alternatively, an innate response that becomes overly prolonged can lead to chronic inflammation, a condition which is associated with significant human diseases such as rheumatoid arthritis. Building on the work we will do to understand the involvement of PML-II in innate responses, this project will also evaluate a new strategy aimed at controlling these responses.
We have discovered that a protein known as PML-II behaves like a master regulator of genes whose transcription is turned on rapidly after a virus infection. A major aim of this project is to find out just how broad is the set of genes that responds to the presence of this protein and, most importantly, what distinguishes these genes from those that are independent of it. We plan to use the most modern techniques to examine events across all the 20,000 or more genes in the human genome when an innate immune response is launched. In this way, we will get a system-wide view of the effects of the PML-II protein that is not limited by any preconceptions.
Going on from this, we intend to find out how differences in PML-II between individuals may influence how they respond to virus infection, since we know that there are major differences in the outcome of apparently similar infections between one person and another. We will be testing two types of difference in PML-II: first, differences at the gene level, as a result of the different genes we inherit; and second, differences that arise in cells as a result of the environment they experience. Here we will be looking particularly at heat stress - the kind of effect that can arise naturally when someone experiences a fever - and asking whether changes in PML that result from such stress are the basis for differences in innate responses and susceptibility to virus infection that we have already seen.
It is really important to unravel the ways in which innate immune responses are generated in the body. Cells being unable to respond sufficiently could mean that an infection spreads further and faster than it would have done - making you more sick. Alternatively, an innate response that becomes overly prolonged can lead to chronic inflammation, a condition which is associated with significant human diseases such as rheumatoid arthritis. Building on the work we will do to understand the involvement of PML-II in innate responses, this project will also evaluate a new strategy aimed at controlling these responses.
Technical Summary
This project will define and elucidate the role of PML-II protein in the transcription of genes induced during interferon (IFN) and inflammatory responses, determine whether this role represents a source of natural variation in these responses and whether it can be targeted to tune their potency.
We will use the newly emergent CRISPR/Cas9 approach to generate isogenic cells that are either capable or incapable of expressing PML-II. These cells will be compared, using state-of-the-art RNAseq to analyse mRNA populations following treatment with IFN inducers or IFN itself. Similarly, we will use ChIP-seq to analyse the binding of key transcription factors (TFs) to chromatin that follows the application of these inducers, with or without PML-II. Thirdly, we will use stable isotope labelling (SILAC)-based quantitative proteomics to determine which proteins interact with the unique C-terminal domain of PML-II, which we have shown previously to be necessary for its function in transcription and for its association with specific TFs and co-activators. We will then correlate these data sets using powerful systems analysis tools to determine which TFs mediate PML-II-dependent responses.
Natural variants of PML-II will be cloned by standard techniques and their ability to support robust innate immune response gene activation assessed by RT-qPCR analysis of mRNA from transfected cell cultures subjected to innate immune stimulation, an established assay of PML-II function. Variation in PML isoform expression and assembly in the cell will also be induced by short and long-term mild heat stress and the effects on the ability of cells to support virus infection and to mount innate immune responses assessed using immunofluorescence, RT-qPCR, western blotting and cytokine ELISA. Finally, short segments of PML-II will be generated by cDNA cloning and expression, and tested as potential inhibitors of the innate response, measured at the mRNA level using RT-qPCR.
We will use the newly emergent CRISPR/Cas9 approach to generate isogenic cells that are either capable or incapable of expressing PML-II. These cells will be compared, using state-of-the-art RNAseq to analyse mRNA populations following treatment with IFN inducers or IFN itself. Similarly, we will use ChIP-seq to analyse the binding of key transcription factors (TFs) to chromatin that follows the application of these inducers, with or without PML-II. Thirdly, we will use stable isotope labelling (SILAC)-based quantitative proteomics to determine which proteins interact with the unique C-terminal domain of PML-II, which we have shown previously to be necessary for its function in transcription and for its association with specific TFs and co-activators. We will then correlate these data sets using powerful systems analysis tools to determine which TFs mediate PML-II-dependent responses.
Natural variants of PML-II will be cloned by standard techniques and their ability to support robust innate immune response gene activation assessed by RT-qPCR analysis of mRNA from transfected cell cultures subjected to innate immune stimulation, an established assay of PML-II function. Variation in PML isoform expression and assembly in the cell will also be induced by short and long-term mild heat stress and the effects on the ability of cells to support virus infection and to mount innate immune responses assessed using immunofluorescence, RT-qPCR, western blotting and cytokine ELISA. Finally, short segments of PML-II will be generated by cDNA cloning and expression, and tested as potential inhibitors of the innate response, measured at the mRNA level using RT-qPCR.
Planned Impact
Innate immunity is a critical first line of defence against virus infection. The speed and extent of these responses is an important determinant of the outcome of virus infection, which can vary greatly between individuals. This project focuses on how innate immune responses are regulated and how natural variation in the activity of PML-II may affect the quality of these responses. The outcome will be an understanding of how PML-II is required for cells to activate and then regulate the innate immune response they make to virus infection. This research topic is a key area relevant to Natural Protection (MRC Strategic Aim 1: Theme 1) while the investigation of natural variation in innate immune function will contribute to the Molecular Datasets & Disease area (MRC Strategic Aim 1: Theme 2). By delivering the project objectives, we will therefore make a significant contribution to MRC's strategic plan.
Wider Research Communities:
As well as benefitting other researchers working in the area of innate immune and inflammatory responses, the work will also to have potential impact in other research communities, specifically those interested in cellular stress responses more generally and those researching chronic inflammatory diseases such as rheumatoid arthritis where innate immune responses have become disregulated. The data from the project will open new avenues of research for these groups.
General Public:
Although the details of the work being done in this project will seem esoteric to non-scientists, the project relates to an overarching question to which they will readily relate. This can be stated as: 'How do our bodies respond to viruses and why do people differ in how ill a virus makes them?' This question can capture the imagination and engage people in thinking about how our bodies work as a system when a virus is encountered, creating a societal benefit through improved understanding of this key aspect of human health and disease. This benefit can be realised both at the individual level and through broadcast, print and digital media, which are vehicles for delivering impact to the public and can be seen as beneficiaries in their own right.
Industry:
The planned project outcomes will make a significant contribution to our enhanced understanding of innate immune responses in humans. Since this topic is an important commercial biomedical research area, with considerable interest in therapies for diseases associated with chronic inflammation, such as rheumatoid arthritis, the sector will benefit from full awareness of the project and its outcomes. More specifically, the project will test a new potential drug target for intervention in such diseases, which will be an opportunity for future industry links and development if results are positive.
Medical Practitioners and Patients:
Although delivering the project aims is not expected to have an immediate impact on clinical practice, it will bear on future practice through the knowledge gained in the area of individual variation in innate immunity, which has the potential to affect personalized medicine in many ways. The work may also, with a 5-10 year horizon, be the beginning of development of a new drug target in chronic inflammatory disease.
Training and Skills:
The project provides an excellent training opportunity at the interface between wet biology and bioinformatics which is key to modern biomedical research. It will therefore positively impact the UK skills base, producing a PDRA who, by the conclusion of the project, will have gained considerable new technical and analytical experience in a key area. They will also gain transferable skills in science communication, research planning and administration. These skills will provide a positive impact first to the PDRA in their future career and second to their future employers in either the industrial or academic sectors.
Wider Research Communities:
As well as benefitting other researchers working in the area of innate immune and inflammatory responses, the work will also to have potential impact in other research communities, specifically those interested in cellular stress responses more generally and those researching chronic inflammatory diseases such as rheumatoid arthritis where innate immune responses have become disregulated. The data from the project will open new avenues of research for these groups.
General Public:
Although the details of the work being done in this project will seem esoteric to non-scientists, the project relates to an overarching question to which they will readily relate. This can be stated as: 'How do our bodies respond to viruses and why do people differ in how ill a virus makes them?' This question can capture the imagination and engage people in thinking about how our bodies work as a system when a virus is encountered, creating a societal benefit through improved understanding of this key aspect of human health and disease. This benefit can be realised both at the individual level and through broadcast, print and digital media, which are vehicles for delivering impact to the public and can be seen as beneficiaries in their own right.
Industry:
The planned project outcomes will make a significant contribution to our enhanced understanding of innate immune responses in humans. Since this topic is an important commercial biomedical research area, with considerable interest in therapies for diseases associated with chronic inflammation, such as rheumatoid arthritis, the sector will benefit from full awareness of the project and its outcomes. More specifically, the project will test a new potential drug target for intervention in such diseases, which will be an opportunity for future industry links and development if results are positive.
Medical Practitioners and Patients:
Although delivering the project aims is not expected to have an immediate impact on clinical practice, it will bear on future practice through the knowledge gained in the area of individual variation in innate immunity, which has the potential to affect personalized medicine in many ways. The work may also, with a 5-10 year horizon, be the beginning of development of a new drug target in chronic inflammatory disease.
Training and Skills:
The project provides an excellent training opportunity at the interface between wet biology and bioinformatics which is key to modern biomedical research. It will therefore positively impact the UK skills base, producing a PDRA who, by the conclusion of the project, will have gained considerable new technical and analytical experience in a key area. They will also gain transferable skills in science communication, research planning and administration. These skills will provide a positive impact first to the PDRA in their future career and second to their future employers in either the industrial or academic sectors.