Regulation of the longevity associated tumor suppressor FoxO3 by lysine methylation and binding to protein partners [electronic resource]

The FoxO family of transcription factors plays an important role in longevity and tumor suppression by regulating the expression of a wide range of target genes. FoxO3 has recently been found to be associated with extreme longevity in humans and to regulate the homeostasis of adult stem cell pools in mammals, which may contribute to longevity. The activity of FoxO3 is controlled by a variety of post-translational modifications that have been proposed to form a 'code' affecting FoxO3 subcellular localization, DNA binding ability, protein-protein interactions and protein stability. Lysine methylation is a key post-translational modification on histones that regulate chromatin accessibility and is a key part of the 'histone code'. However, whether lysine methylation plays a role in modulating FoxO3 activity has never been examined. I found that the methyltransferase Set9 directly methylates FoxO3 in vitro and in cells. Using a combination of tandem mass spectrometry and methyl-specific antibodies, I find that Set9 methylates FoxO3 at a single residue, lysine 271, a site previously known to be deacetylated by Sirt1. Methylation of FoxO3 by Set9 decreases FoxO3 protein stability, while slightly increasing FoxO3 transcriptional activity. The modulation of FoxO3 stability and activity by methylation may be critical for fine-tuning cellular responses to stress stimuli, which may in turn affect FoxO3's ability to promote tumor suppression and longevity. Post-translational modifications control many aspects of FoxO3 activity, including protein stability, subcellular localization and binding partner association. To analyze whether FoxO3 functions primarily alone, or in a complex with other factors, I used size exclusion chromatography to assess the size of the potential FoxO3 protein complexes in cellular extracts. I found that FoxO3 is present in fractions with a relative molecular weight larger ranging from 250 to 700 kDa. Interestingly, the cytoplasmic fraction of FoxO3 appears to be in a larger complex than the nuclear fraction, suggesting that FoxO3 is present in more than one protein complex in cells. To identify novel binding partners of FoxO3 that could be present in these large molecular-weight protein complexes, I conducted a tandem affinity purification (TAP) of dual tagged FoxO3 in HeLa S3 cells followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) of the elution from the immunoprecipitation. I identified over 1,000 potential FoxO3 binding partners that were not present in the control immunoprecipitation. These candidate binding partners included protein kinases (e.g. mTOR, MAPKs), other tumor suppressors (e.g. p65, p130) and oncogenes (e.g.Myb), as well as proteins that contain a methyl lysine (e.g. WDR17, PHF3) and acetyl lysine (e.g. BAZ2B, BRD1) binding domains. Taken together, these results suggest that FoxO3 functions in large protein complexes that could affect FoxO3 activity. The formation of FoxO3-containing complexes could be triggered by FoxO3 post-translational modifications. By increasing our understanding of the regulation of FoxO3, as well identifying possible interacting proteins, we further our knowledge of the pathways important for both longevity and age related diseases, including cancer.

Advisor: Brunet Anne
Artandi Steven E. advisor. Thesis advisor
Gozani Or Pinchas advisor. Thesis advisor
Lipsick Joseph Steven