UCL Discovery

Abstract

Mutations in the insulin/IGF-1 signalling (IIS) pathway lead to large lifespan extensions in the nematode Caenorhabiditis elegans. The role of IIS in ageing is evolutionarily conserved and requires the forkhead trancription factor DAF-16. In this thesis, I have attempted a novel strategy to identify genes involved in differential gene expression downstream of IIS in C. elegans. Using microarray data, I found that the H-ferritin gene ftn-1 is strongly regulated by IIS in a daf-16 – dependent manner. Ferritins act as intracellular iron-storage proteins. I then induced expression of a fluorescent GFP reporter of ftn-1 by introducing a mutation in daf-2. I subsequently screened a library of RNAi clones targeting transcription factors in order to identify mediators of IIS-dependent gene regulation. Candidate RNAi treatments were subsequently tested for their effects on expression of endogenous ftn-1 and on ageing. None of the genes identified had as important a role in either ftn- 1 expression or lifespan as daf-16, but several weaker determinants, like mdl-1, were identified. During the course of my screen, I found that loss of the hypoxia inducible factor hif-1 and its binding partner aha-1 leads to a large increase in expression from the ftn-1 GFP reporter, indicating that HIF-1 may act as a repressor of ftn-1 expression. Both HIF-1 protein levels and ftn-1 transcription are known to be responsive to iron levels. I found that regulation of the ftn-1 reporter by iron requires hif-1 and that repression of ftn-1 reporter expression occurs via a 63bp iron dependent element. An unexpected role of the upstream prolyl hydroxylase EGL-9 was also identified. I also contributed to a project aimed at identifying the consequences of overexpression of superoxide dismutases (SOD) on C. elegans ageing. Our efforts revealed that SOD over-expression extends lifespan by affecting signalling, not by reducing oxidative damage.

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