A detailed network of transcriptome regulation associated with life-span extension in model organisms

Lead Research Organisation: University College London
Department Name: Genetics Evolution and Environment

Abstract

In worms, flies and mice, it has been known for a long time that reduced feeding can result in longer life-spans, and in recent years, simple manipulations in the way these animals sense and response to sugars through insulin have also been shown to increase life-span. However, we are still a long way from understanding the mechanisms involved in food restriction and disruption of insulin signalling. To make this even more difficult, recent discoveries have shown that the cells that make up worms, flies, mice and even ourselves are much more complicated than we ever imagined. The work proposed would use leading-edge technologies to try to understand the mechanisms associated with longer life-spans in animals such as worms and flies where experiments would take months instead of years (mice) or a lifetime (humans). These new technologies generate huge amounts of data and will give the most detailed view possible of the changes that happen in worms and flies to make them long lived. The identification of similar changes in worms and flies (animals that are separated by millions of years of evolution) would suggest a mechanism that is highly conserved and may exist in other animals like mice or humans.

Technical Summary

Recent work has shown the remarkable conservation of mechanisms that can extend life-span in model organisms. Work in worms, flies and mice has shown that single mutations in the insulin/insulin-like growth factor signalling pathways can result in long-lived animals. Similarly, long standing work has shown that dietary restriction also increases life-span in a range of species. I propose the following related projects to understand the mechanisms that extend life-span:

1. Use tiling array and sequencing technology to give a complete view of the transcriptome in model organisms

2. Use DAM-ID technology to understand the DNA-protein and protein-protein interactions of transcription factors (TFs) that are known to be involved in life-span extension

Most transcriptome data collected on long-lived animals has been limited to detecting changes at the 3‘ end of genes. Whole genome tiling arrays would report the expression of different exons and non-coding RNAs. To understand the role of these elements in longevity, total RNA hybridization to tiling arrays from long-lived worms and flies would be compared to hybridizations using wildtype worms and flies. In addition, little is known about the tissue specificity of changes to exon or non-coding expression, and tissue specific samples in flies would be taken for comparison. It is hoped that given such a detailed view of the transcriptome, we will be able to build a network showing the possible interactions between coding and non-coding transcripts that lead to transcriptional and post-transcriptional changes.

Current knowledge of transcription factor binding is also very limited and methods that report interactions between TFs and DNA do not distinguish between regulation that exists because of DNA-protein or protein-protein interactions. Chimeric proteins containing TFs essential for longevity fused to the Escherichia coli DNA adenine methyltransferase (Dam) domain would be used to identify regions where TFs aociate with DNA. These regions are methylated by the Dam domain and can be isolated from unmethylated DNA by restriction enzymes. Chimeric proteins using wildtype TFs would show all interactions, and comparison of DNA methylation profiles of wildtype TFs and mutant TFs that lack DNA binding capability or deletions of domains essential for protein-protein interaction would allow one to distinguish between regulation that exists because of direct DNA-protein interactions or because the TF interacts with another protein that is bound to the DNA. The TF interactions would then be integrated into the regulatory network created for the tiling arrays.
 
Description PhD Studentship
Amount £85,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 01/2013 
End 12/2016
 
Description UCL Postdoctoral Mobility Award
Amount £14,897 (GBP)
Organisation University College London 
Sector Academic/University
Country United Kingdom
Start 01/2013 
End 03/2013
 
Title Additional C. elegans strains expressing transcription-factor::DAM fusions 
Description We have generated additional C. elegans strains that express a transcription factor fused to a DNA methyltransferase and these strains can be used to identify locations of DNA-protein interactions. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The new transcription-factor::DAM fusion protein will allow us to understand the binding and function of the a transcription factor which is known to be involved in regulation of genes involved in detoxification and also is known to be involved in the regulation of lifespan. 
 
Title C. elegans strains expressing DAM-ID and daf-16::DAM fusion 
Description We have generated C. elegans strains that express a transcription factor fused to a DNA methyltransferase and these strains can be used to identify locations of DNA-protein interactions. 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact We are the first to show that DNA methyltransferase technology is a viable alternative to chromatin immunoprecipitation in C. elegans and will make our strain available to other researchers. Data generated from this fusion protein has resulted in a publication in a high impact journal. 
 
Title DAF-16 regulation of lifespan 
Description We have generated a new model for how the DAF-16 transcription factor regulates lifespan in C. elegans. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2010 
Provided To Others? Yes  
Impact The human ortholog of DAF-16 is FoxO3. FoxO3 has been shown in several studies to be associated with longevity in humans and the deregulation of FoxO3 is known to be involved in tumorigenesis. Our work in C. elegans suggests that a specific set of kinases and transcription factors are regulated by DAF-16 and the orthologs of these genes are prime targets for study in mice and humans. 
 
Description DNA methylation changes during ageing 
Organisation University College London
Department Department of Genetics, Evolution and Environment
Country United Kingdom 
Sector Academic/University 
PI Contribution I provided bioinformatics analysis of transcriptome and methylome data.
Collaborator Contribution Partner provides next generation sequencing data for analysis.
Impact Collaboration recently generated a publication (Pubmed ID 23949429) and should provide further publications. Collaboration is a mix of experimental and computational analyses.
Start Year 2012
 
Description Network Analysis 
Organisation University College London
Department Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We are providing large datasets for a meta-analysis of C. elegans gene expression data.
Collaborator Contribution Partner is providing network interactions and kernel based methods to identify genes that are co-regulated in our dataset.
Impact Multi-disciplinary (experimental and computational) collaboration. Currently working on a paper on gene regulation in C. elegans.
Start Year 2012
 
Description New model organisms for the studying of ageing 
Organisation University of Exeter
Department College of Life and Environmental Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided bioinformatics support for de novo transcriptome assembly and analysis
Collaborator Contribution Provided RNA-seq data for analysis
Impact The collaboration is multi-disciplinary and has both a computational and experimental component.
Start Year 2013
 
Description Regulation of Ageing in C. elegans 
Organisation University College London
Department Institute of Healthy Ageing
Country United Kingdom 
Sector Academic/University 
PI Contribution My team is currently helping generate new transcription factors for analysis with DNA adenine methyltransferase (DAM) and will also help the the bioinformatics analysis of any data produced. We are currently collaborating on generating new transcription-factor/dna-adenine-methylase fusion proteins to better understand the relationship between transcription factor binding and changes to mRNA expensive in long lived worms.
Collaborator Contribution Partner will provide experimental evidence of protein-DNA interactions.
Impact The expected outputs of this collaboration will be the generation of several transcription factor/DAM fusion proteins that my lab can use for detailed analysis of transcription factor binding in a variety of genetic and environmental backgrounds. Currently one additional transcription factor/DAM fusion has been generated. The collaboration is multi-discipinary as it involves experimental work, computional analysis of the data followed by building of empirical models of transcription factor binding. The collaboration also involves the understanding of several mechanisms that extend lifespan in C. elegans and recently a publication has been generated in this collaboration (Pubmed ID 23540700).
Start Year 2009
 
Description Transcription Repressor 
Organisation University College London
Department UCL Cancer Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration to look into transcription factors that repress gene expression. My group will provide the analysis of the data.
Collaborator Contribution Collaborator will produce next-generation ChIP-seq data.
Impact We expect there to be more than one publication produced from this collaboration and if initial experiments and analysis are successful then it is expected that a joint application for funding would be made in the next year to expand the collaboration. Collaboration is multi-disciplinary, as it will combine cell biology (from collaborator) and my group will provide computational analysis of the data and development of computational models to describe how this gene functions (e.g. identification of binding motifs and co-factors). One paper has already been generated in this collaboration (Pubmed ID 23894613).
Start Year 2011
 
Description Royal Society Ageing and Nutrition workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact 20 internationally known Principle Investigators attended my talk and engaged in questions and discussions about future work and collaborations

a collaboration with a Royal Society fellow
Year(s) Of Engagement Activity 2013
 
Description Systems Biology Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact over 100 researchers (PhDs, postdocts and Principle Investigators) attended a talk on Systems Biology of Ageing and had questions and discussions after the presentation.

A received several inquires about collaborating on projects
Year(s) Of Engagement Activity 2014
URL https://www.dur.ac.uk/bsi/isgsb/