Prioritised expression of stress-related proteins in environmental thermoadaptive responses of animals
Lead Research Organisation:
University of Liverpool
Department Name: Institute of Integrative Biology
Abstract
Underpinning the assumptions and models of how climate will affect natural populations are expectations of how the predicted temperature change affects the life history, fecundity, survival etc. of species in a given habitat, region or climatic zone. An important component of such models is knowledge of how well animal species can modify their bodies in response to persistent environmental signals, such as seasons or daily temperature fluctuations, and thereby to protect themselves from the debilitation by unexpected cold and heat snaps. Understanding the potential for protection requires an understanding of the fundamental mechanisms induced at the level of cell, tissue and whole body and which correlate with altered resistance to environmental stress.
Research into how animals interact with their thermal environment has progressed enormously in the past 15 years. Advances in genomic science now offers 'globalised' measurement of thermal properties of animals, meaning simultaneous assessment of all of its constituent genes and proteins. This whole-system profiling has emerged as a key tool for analysing the properties and responses of biological systems, and the outcomes have informed many different areas of biology. But the technology continues to evolve and advance. Earlier techniques have given way to new approaches that give the whole picture, with more detail, faster, and for less cost. These new techniques, mainly based on very high-throughput DNA sequencing, have recently been adapted to provide many different kinds of information.
One of these techniques allows us to quantify the number of proteins being made in tissues that are responding to sudden changes in their circumstances, such as temperature, and which improve the ability of animals to survive difficult times. Identifying the genes and proteins that respond tells us a lot about how the animal improves its life chances, when otherwise it might not survive. It also tells us which problems are caused by temperature shock and how these problems are resolved or mitigated.
Our project will apply a new technique to this well-understood environmental problem, which is how a fish survives extreme cold snaps, which can be so severe in freshwater habitats that fish are killed, sometimes in large numbers. Yet, if the fish were given a 'taste' of cool waters signalling the likely occurrance of more extreme temperatures it can prepare its body and tissues such that extreme cold isn't so damaging. So we wish to define exactly what causes debilitation and death, and how the animals protects itself from extreme, debilitating cold by preconditioning to cool. But understanding the fundamental mechanisms operating in these animals will provide information of much broader significance. The responses to cold include genes and proteins working in most, if not all, animals, which gives them an ability to respond to stressors other than cold. A good example is how a cold shock protein in fish and hibernating mammals has been shown to protect the central nervous system in mouse models from disruption caused by neurodegenerative disease.
We will extract tissues of the common carp before and after they have been cooled from a summer temperature to a winter temperature, and 'globally' measure the number and identity of thousands of new proteins created immediately after cooling. This new knowledge will point directly to responding genes and the proteins they encode. We will also focus on a particular group of genes whose proteins are already known to be highly expressed in the cold. We will test the idea that these proteins help protect the DNA and RNA of cells from structural imperfections caused by cold.
Research into how animals interact with their thermal environment has progressed enormously in the past 15 years. Advances in genomic science now offers 'globalised' measurement of thermal properties of animals, meaning simultaneous assessment of all of its constituent genes and proteins. This whole-system profiling has emerged as a key tool for analysing the properties and responses of biological systems, and the outcomes have informed many different areas of biology. But the technology continues to evolve and advance. Earlier techniques have given way to new approaches that give the whole picture, with more detail, faster, and for less cost. These new techniques, mainly based on very high-throughput DNA sequencing, have recently been adapted to provide many different kinds of information.
One of these techniques allows us to quantify the number of proteins being made in tissues that are responding to sudden changes in their circumstances, such as temperature, and which improve the ability of animals to survive difficult times. Identifying the genes and proteins that respond tells us a lot about how the animal improves its life chances, when otherwise it might not survive. It also tells us which problems are caused by temperature shock and how these problems are resolved or mitigated.
Our project will apply a new technique to this well-understood environmental problem, which is how a fish survives extreme cold snaps, which can be so severe in freshwater habitats that fish are killed, sometimes in large numbers. Yet, if the fish were given a 'taste' of cool waters signalling the likely occurrance of more extreme temperatures it can prepare its body and tissues such that extreme cold isn't so damaging. So we wish to define exactly what causes debilitation and death, and how the animals protects itself from extreme, debilitating cold by preconditioning to cool. But understanding the fundamental mechanisms operating in these animals will provide information of much broader significance. The responses to cold include genes and proteins working in most, if not all, animals, which gives them an ability to respond to stressors other than cold. A good example is how a cold shock protein in fish and hibernating mammals has been shown to protect the central nervous system in mouse models from disruption caused by neurodegenerative disease.
We will extract tissues of the common carp before and after they have been cooled from a summer temperature to a winter temperature, and 'globally' measure the number and identity of thousands of new proteins created immediately after cooling. This new knowledge will point directly to responding genes and the proteins they encode. We will also focus on a particular group of genes whose proteins are already known to be highly expressed in the cold. We will test the idea that these proteins help protect the DNA and RNA of cells from structural imperfections caused by cold.
Planned Impact
A. The commercial private sector?
Pharma might become interested in the commercial value of newly discovered systems for coping with environmental stress. This is because such systems are likely to be present in most if not all species, including farm animals and humans; it is their presentation in chill-stressed animals that makes their activity known to researchers. The problems caused by cold and warm are not dissimilar to problems faced at more normal temperatures in diseases; good examples are proteins folding diseases and prions, or problems in ensuring proper secondary structures of nuclei acids.
A very recent and possibly important example of the serendipitous biomedical application of chill research is the publication in Nature of a potential means of protecting against neurodegenerative disease through the activation of RBM3, a gene shown by us to be activated in the tissues of torpid small mammals (Nature: 15th Jan 2015). This received extensive coverage in the national press and BBC. To this extent the proposed project on chill stress can in the long term contribute to the nation's health. It is worth noting that the discovery of stress proteins originated in work on chromosomes of insects subjected to heat shock, and several decades of subsequent science now provides a very good understanding of a major element of cell biology.
B. Policy-makers
Policy makers at all levels might benefit in the longer term from a much better understanding of the potential for protection against thermal debilitation. Understanding the extent of protection, and its distribution across taxonomic grades and different geographic and climatic zones is needed to indicate the likely problems of changing climates. Understanding the extent to which protective responses work in different climatic zones (i.e. Antarctic, tropical reef etc.) is key to identifying the species that are most sensitive to climate change, both to the long-term averaged temperatures but also to the daily and weekly fluctuations that are projected to become greater as global warming progresses. This project is a discovery project that will likely form part of a major international research effort leading to these effects, and providing novel biomarkers of potential value for regulators. Thus, it will not have its full impact for some years, though we do expect that the global nature of our search for responding genes/proteins will more rapidly identify the major elements of the stress protection process.
C. The wider public
Carefully proscribing the way in which fish can be protected from chill experiences can be useful to the lay public who own amenity fish ponds or for the local ponds used in coarse fisheries throughout the UK. Thus, carp are frequently stocked in gardens (koi) or in coarse fisheries that are likely to experience extreme stress in the winter, as in December 2010 and subsequently as diseases cause some mortality due to poor condition of the fish. It is possible to define the best way of ameliorating these problems by pre-conditioning animals, and by genetic selection and stocking of resistant lines (as in the Amur strain in Asia).
D. Staff training
This project will provide high-level scientific training for two high quality postdoctoral researchers, as well as opportunities for any MSc/MRes students who wish to engage in the programme. They will experience the very latest techniques in both genomic (Hall/Cossins) and proteomic (Beynon/Eyers) research methodology, at one of 4 major sequencing hubs in the UK, core-funded by NERC and MRC, and within a well-developed and vibrant research infrastructure. They will have the opportunity for attending international meetings and to present their work.
Pharma might become interested in the commercial value of newly discovered systems for coping with environmental stress. This is because such systems are likely to be present in most if not all species, including farm animals and humans; it is their presentation in chill-stressed animals that makes their activity known to researchers. The problems caused by cold and warm are not dissimilar to problems faced at more normal temperatures in diseases; good examples are proteins folding diseases and prions, or problems in ensuring proper secondary structures of nuclei acids.
A very recent and possibly important example of the serendipitous biomedical application of chill research is the publication in Nature of a potential means of protecting against neurodegenerative disease through the activation of RBM3, a gene shown by us to be activated in the tissues of torpid small mammals (Nature: 15th Jan 2015). This received extensive coverage in the national press and BBC. To this extent the proposed project on chill stress can in the long term contribute to the nation's health. It is worth noting that the discovery of stress proteins originated in work on chromosomes of insects subjected to heat shock, and several decades of subsequent science now provides a very good understanding of a major element of cell biology.
B. Policy-makers
Policy makers at all levels might benefit in the longer term from a much better understanding of the potential for protection against thermal debilitation. Understanding the extent of protection, and its distribution across taxonomic grades and different geographic and climatic zones is needed to indicate the likely problems of changing climates. Understanding the extent to which protective responses work in different climatic zones (i.e. Antarctic, tropical reef etc.) is key to identifying the species that are most sensitive to climate change, both to the long-term averaged temperatures but also to the daily and weekly fluctuations that are projected to become greater as global warming progresses. This project is a discovery project that will likely form part of a major international research effort leading to these effects, and providing novel biomarkers of potential value for regulators. Thus, it will not have its full impact for some years, though we do expect that the global nature of our search for responding genes/proteins will more rapidly identify the major elements of the stress protection process.
C. The wider public
Carefully proscribing the way in which fish can be protected from chill experiences can be useful to the lay public who own amenity fish ponds or for the local ponds used in coarse fisheries throughout the UK. Thus, carp are frequently stocked in gardens (koi) or in coarse fisheries that are likely to experience extreme stress in the winter, as in December 2010 and subsequently as diseases cause some mortality due to poor condition of the fish. It is possible to define the best way of ameliorating these problems by pre-conditioning animals, and by genetic selection and stocking of resistant lines (as in the Amur strain in Asia).
D. Staff training
This project will provide high-level scientific training for two high quality postdoctoral researchers, as well as opportunities for any MSc/MRes students who wish to engage in the programme. They will experience the very latest techniques in both genomic (Hall/Cossins) and proteomic (Beynon/Eyers) research methodology, at one of 4 major sequencing hubs in the UK, core-funded by NERC and MRC, and within a well-developed and vibrant research infrastructure. They will have the opportunity for attending international meetings and to present their work.
Publications
Forootan SS
(2016)
Transcriptome sequencing of human breast cancer reveals aberrant intronic transcription in amplicons and dysregulation of alternative splicing with major therapeutic implications.
in International journal of oncology
Liu X
(2017)
Transcriptomic signatures differentiate survival from fatal outcomes in humans infected with Ebola virus.
in Genome biology
Description | We have finally developed techniques for preparing ribosomal footprints from fish tissues by adaptation of methods published by Ingolia et al. This proved to be an exhausting process due to preparative differences between fish & mammals. We have undertaken a pilot study of 20 animals subjected to a simple cooling experience, within the Home Office licencing. This revealed a very orderly change of the kidney translatome as a function of time, which has been presented at 2 academic meetings in the UK. We have subsequently conducted a large-scale experiment comprising 96 individual animals, both uncooled controls, and cooled experimental animals from 25°C down to 11°C. Initial transcriptomic results are very encouraging, and we await the corresponding footprint outcomes. This project has proved very difficult to replicate the experiences of other labs, but we are now confident that our technical issues have been resolved and we are now in the informatic phase of the project. This experiment offers an entirely new understanding of how best to screen the whole genome in order to identify genes that contribute to phenotypic adaptations, or to disease processes. It indicates a straightforward, if tedious, approach to relate gene responses more discretely to functional outcomes. Previous work indicate use just of transcript abundance, but it is now clear that some genes respond to change without changing transcript abundance. |
Exploitation Route | We have developed suitable techniques for quantifying ribosomal 'footprints' by careful adaptation of methods originally published by Ingolia et al. This project has proved very difficult to replicate the experiences of other labs, but we are now confident that our technical issues have been resolved and we undertook a successful preliminary study of 20 fish in 2018, followed in 2019 by a much greater analysis of 96 carp, previously acclimated for several months to 25°C, then subjected to a cooling experience down to 11°C over 2 days, at which they held over a 3-week period, all within the limits specified by our Home Office licence. On each of 13 occasions we sampled 6 replicate fish, including from controls, and rapidly excised multiple tissues for storage at -80°C. Statistical analysis of the data revealed an orderly change, relative to un-cooled controls, of the kidney transcriptome and also of its associated translatome (i.e. footprint) analysis, employing ~50% of the entire 44K tetraploid genome, all as a function of time after transfer. This massive response shows that the cooling response of genes comprises genes displaying the full range of correlation coefficients between transcript and footprint analyses from +1.0 down to -0.5, which points to the activation of multiple post-transcriptional mechanisms for a substantial fraction of genes. This project now provides an entirely new approach to screen whole vertebrate genomes to identify gene responses that contribute to phenotypic adaptations. It also provides a more straightforward, if tedious, approach to relate gene responses to specific functional outcomes, thereby to improve our mechanistic understanding of processes contributing to environmental adaptation. Early work in this field focused solely on transcript abundance as a measure of effect, but it is now clear that this provides an inadequate understanding of induced responses, given that many genes respond via translational as well as transcriptional processes on exposure to changed circumstances, rather than solely using the transcript response. Our approach provides findings that relate more specifically to the list of KEGG metabolic pathways that display statistically significant responses to cooling. These include up-regulated KEGG pathways such as ribosome biogenesis, ribosome function, oxidative metabolism, glycolysis/gluconeogenesis, citrate metabolism, pyruvate metabolism, protein export, proteosome, mRNA surveillance, and biosynthesis of amino-acids. We also showed that cooling caused down-regulation of multiple (i.e. 10) signalling pathways, cytoskeletal regulation, intercellular connection, endocytosis, cell senescence and necroptosis. This allows a much more detailed analysis of and tissue processes responding to a change in environmental circumstances. Finally, we also showed that the acyl-CoA desaturase enzyme displays the largest up-regulation of any gene, a response that leads to increased levels of membrane lipid unsaturation, by which membrane function is maintain within the normal zone. To date this outcomes of this programme of work has been presented at 2 academic meetings in the U.K., and at the 2019 S.E.B. meeting in Seville, Spain. In the past 18 months we have assisted colleagues at the University of Oslo to undertake a similar hypoxia experiment using crucian carp. The same technologies can be used in assessing tissue responses of any animals and plants to any of a wide range of environmental situations, . Regarding the issue of Covid-19 and its potential influence on progression of our project, we were affected during the period March 2020 to March 2021, after we had formally completed the laboratory work. This Covid period covered the period of time when we focused entirely on the workup of data and preparation of the resulting manuscript. All of the associated staff undertook home-working, one of whom moved back to their home country of Finland, with interactions between the partners via telephone, Zoom and email. In principal, this should reasonably sufficient, but we experienced problems in resolving some statistical and design issues which proved frustrating. However, the 'Centre for Genome Research' at the 'University of Liverpool' provided continual services of an expert statistician throughout this period, and other colleagues provided feedback during their non-working time. We are now evangelising the benefits of combining the RNA and Riboseq techniques within the research community in Liverpool, focusing particularly on cancer studies. |
Sectors | Agriculture Food and Drink Environment |
Description | Recent analyses of our base data have indicated that cooling treatment of fish induces tissue responses both at the level of mRNA and also of ribosomal activity. We show that integration of both aspects leads to enhanced tissue capability complex in the cold. We also recognise the constraints caused by rapid changes in water temperature, and the need to deploy gentle cooling processes. Our project has identified the base cellular processes that generate improved physiological resilience to rapid temperature changes. |
First Year Of Impact | 2002 |
Sector | Agriculture, Food and Drink,Environment,Other |
Impact Types | Societal Economic |
Title | Development of a new machine learning classification technique |
Description | This technique was developed by statistician Yongxiang Fang. It uses a ratio technique based on selecting gene pairs that can most clearly discriminate in a two class prediction model, avoiding problems caused by systematic bias in new datasets being applied to the training pre-existing model. The predictive power of this technique was consistently better than the more conventional techniques of random forests and SVMs. |
Type Of Material | Technology assay or reagent |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | A method was developed to identify the optimal combination of gene pairs with the successful application to a typical two-class problem, namely predicting the outcome (survival or fatality) in Ebola patients. |
Title | Separation of genes displaying either 'forwarded' or 'DTE, differential translational efficiency' behaviour in environmental responses |
Description | Carp fish are interesting in that they possess a duplicated genome with twice the number of genes that the vast majority of vertebrates possess. They also possess a particularly resilient physiology when facing seasonally altered water temperatures, lack of oxygen, starvation, etc. Our £700K NERC project has determined how their tissues re-adjust their cellular activities in response to a significant (13°C) environmental cooling imposed over a 3-week period. We quantified how this affected the expression of 44K genes AND their protein products within a single organ, the kidney. We defined two separate basic mechanisms, one which was based on the generation of similar transcript and protein products (the so-called 'forwarded' response, approx. 60% of all genes), whilst the other either suppressed or amplified protein production relative to transcript abundance (the so-called 'translational efficiency' or DTE, approx. 40% of all genes). Deep statistical analysis of this data has identified the adaptive features of the latter mechanism during a cooling event, initiated by the binding of 'RNA-binding proteins' to transcripts in order to influence the rate of production of proteins for a given stock of active transcripts. To our knowledge, this is the first demonstration of this mechanism in poikilothermic animals subjected to altered environmental conditions, but other groups have identified not dissimilar mechanisms in mammals subjected to disease states. |
Type Of Material | Data analysis technique |
Year Produced | 2021 |
Provided To Others? | No |
Impact | Back in the year 2000, when global gene expression was first assessed by transcript screening, it was common to use transcript abundance as the defining feature in identifying the underpinning mechanisms that restore function when normal cellular processes are disturbed. But our new analysis makes it clear that this rigid expectation is entirely wrong, in that post-transcriptional 'translational' mechanisms have substantial effects upon the outcome of environmental transitions. We have shown that this applies to the activity of 'RNA binding proteins', as well as to 'zinc finger proteins' and 'eukaryotic translation initiation factors', of which there are several hundred genes. We used 'over-representational' statistical analysis to determine which of the KEGG pathways displayed the most significant responses to either of these mechanisms, and this has pointed to a much more complete understanding of the cellular mechanisms by which cells and tissues respond to dietary, disease, environmental or other tissue disturbances. |
Description | Developing the aquaculture potential of the common and crucian carp |
Organisation | Chinese Academy of Social Sciences |
Department | Heilongjiang River Fishery Research Institute (HRFRI) |
Country | China |
Sector | Academic/University |
PI Contribution | Provision of genomic techniques and gene-0specific information on responses to environmental treatment of fish, including disease models |
Collaborator Contribution | Provision of genomic sequences that incorporate gene annotations in a suitable informatics package for us to use in Liverpool |
Impact | This collaboration is key to energising genomic approaches to these important aquacultural species. Visits by UK partner are scheduled for April 2016. |
Start Year | 2013 |
Description | Attendance at Biochemical Society Workshop on "Ribosome Profiling". |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | This one day event was a combination of training and round-table discussions. Many scientists are trying to use these methods and a discussion of what experts in the field are doing and alternative experimental and bioinformatic pipelines will allow scientists to learn about the tools available and share experiences of data analysis. This was ideal for those just starting to plan ribosome profiling experiments and those in the middle of doing/analysing them. Both of my postdocs (Dr Williams (lab biochemistry) and Dr Yongxiang Fang (informatics and statistics) attended and gained much more confidence with their projects. |
Year(s) Of Engagement Activity | 2016 |
URL | https://www.biochemistry.org/Events/tabid/379/View/Programme/MeetingNo/WS020/Default.aspx |
Description | Attendance at the Annual Symposium of the Society of Experimental Biology, Sevilla, Spain. Presentation on the outcome of NERC grant. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Attended the annual meeting of the Society for Experimental Biology in Sevilla, Spain, and presented a paper based on the outcomes of this funded research grant. The audience was 50-70 persons all of whom were expert in the field of environmental responses of animals. |
Year(s) Of Engagement Activity | 2018 |
Description | Research Visit to the Chinese Academy of Fisheries Science |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Attended two centres in the Chinese Academy of Fisheries Science, in Harbin and Beijing. In both I gave research seminars and engaged in collaborative research discussions. The outcome was a series of research proposals and interactions around the issue of protecting carp aquaculture from losses caused by koi herpes virus. |
Year(s) Of Engagement Activity | 2016 |