IDENTIFICATION AND CHARACTERIZATION OF REGULATORS OF THE LONGEVITY FACTOR DAF-16 IN C. ELEGANS
The transcription factor DAF-16/FOXO is a critical longevity determinant in diverse organisms. It is the major effector of the insulin/IGF-1 signaling (IIS) cascade which is critical for regulating development, longevity, metabolism and stress resistance. However the molecular basis of how its transcriptional activity is regulated remains largely unknown. The aim of my research is to better understand the regulation of DAF-16 using C. elegans as a model system. My work reveals that the 14-3-3 protein FTT-2 is a new regulatory factor of DAF-16 in response to IIS. I found that RNAi knock down of ftt-2 specifically enhanced the IIS-mediated dauer formation. Furthermore, ftt-2 knock down caused the nuclear accumulation of DAF-16 and enhanced its transcriptional activities. In contrast to ftt-2, RNAi knock down of par-5/ftt-1, the only other 14-3-3 gene in C. elegans, did not show any notable effect on DAF-16 regulation, underscoring the functional specification of FTT-2 and PAR-5 despite their high sequence similarity. Using co-immunoprecipitation, I showed that FTT-2 formed a complex with DAF-16. My work indicates that FTT-2 binds DAF-16 in C. elegans and regulates DAF-16 by sequestering it in the cytoplasm. A similar mechanism of regulation of FOXO by 14-3-3 has been reported in mammalian cells, highlighting the high degree of conservation of DAF-16/FOXO regulation. My work also shows that the C. elegans homolog of host cell factor 1 (HCF-1) represents a new longevity modulator and functions as a negative regulator of DAF-16. In C. elegans, hcf-1 inactivation caused a daf-16-dependent lifespan extension up to 40% and heightened resistance to specific stress stimuli. HCF-1 showed ubiquitous nuclear localization and physically associated with DAF-16 in worms. Furthermore, loss of hcf-1 resulted in elevated DAF-16 recruitment to the promoters of its target genes and altered expression of a subset of DAF-16-regulated genes. We propose that HCF-1 modulates C. elegans longevity and stress response by forming a complex with DAF-16. This complex limits a fraction of DAF-16 from accessing its target gene promoters, and thereby regulating DAF-16-mediated transcription of selective target genes. As HCF-1 is highly conserved, my results have important implication for aging and FOXO regulation in mammals.
This is the resubmission of my dissertation according to the comments of Pattie.
DAF-16; Longevity; Aging; HCF-1; FTT-2; Insulin signaling; FOXO; 14-3-3
Dissertation or Thesis