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Identification and functional characterization of genes associated with cellular biomarkers of longevity

Final Report Summary - LONGENES (Identification and functional characterization of genes associated with cellular biomarkers of longevity)

In this project, we employed cutting edge genomic techniques to identify new candidate mammalian genes for a role in ageing and longevity. Specifically, we identified genes regulating susceptibility to stress, a major biomarker of longevity, using RNA interference techniques and by studying candidate genes from the long-lived naked mole-rat (Heterocephalus glaber). A large-scale, RNAi-based pooled screen for stress resistance was carried out in mouse embryonic stem cells and several candidate genes identified. We developed and employed a method using flow cytometry that consists of mixing normal cells with cells transfected with a lentivirus expressing the shRNA coupled to a fluorescent protein. This allowed us to detect shRNAs with subtle effects. We also prioritized candidate genes by incorporating functional enrichment analysis, interaction networks and gene expression information. A bioinformatics pipeline was developed for this purpose. These methods will be useful for candidate gene prioritization of large-scale RNAi-based screens. Of note, we demonstrated a detrimental role of Edd1 silencing in cell growth which serves as a proof-of-concept of our pipeline. Many other interesting genes have emerged from our results, including C1orf112, an unstudied gene. Both Edd1 and C1orf112 affect cell proliferation when silenced and also appear to affect DNA damage responses, and thus we hypothesize that C1orf112 is related to cell cycle regulation under DNA damage. In parallel, candidate naked mole-rat genes were cloned and sequenced. Sequence analyses revealed a number of putative functional changes in naked mole-rat genes and proteins which may be related to the exceptional longevity of these animals. Further studies of these genes may be important in the context of ageing and cancer. Mouse cell lines with the mouse gene replaced by the naked mole-rat gene were generated. We also analyzed RNA sequencing data from naked mole-rats which provides new research foci and methods for studying the naked mole-rat: Our results revealed possible genes and processes involved in ageing, in particular related to mitochondria and oxidation reduction processes, that may help explain the longevity of the naked mole-rat. To facilitate studies by other researchers, we developed the Naked Mole-Rat Genome Resource (http://www.naked-mole-rat.org/) arguably the leading online resource on the naked mole-rat. Overall, this project will have a strong impact in the field by providing new genes and paradigms to study ageing. The genes identified will serve as new foci for additional studies to understand and maybe even treat specific age-related diseases.