FoxO transcription factors mediate longevity downstream of the insulin pathway in worms and flies [1
]. In mammals, the role of FoxO factors in longevity has not been investigated yet, but mice with mutations in the insulin or IGF-1 receptors are long-lived [6
]. In addition, recent genetic studies on long-lived humans have shown that single nucleotide polymorphisms (SNPs) in the FoxO3 gene, one of the four human FoxO family members, are closely linked to extreme longevity and the reduction in age-related diseases [8
]. These studies are consistent with the notion that mammalian FoxO3 may also be involved in regulating longevity. Importantly, FoxO transcription factors also act as tumor suppressors in mammals [13
]. Indeed, mice in which the FoxO1, FoxO3, and FoxO4 genes are conditionally deleted in the adults develop thymic lymphomas and hemangiomas [14
]. In humans, inactivation of FoxO3 correlates with poor prognosis in estrogen-dependent breast cancer [15
], illustrating the conserved role of the FoxO family in tumor suppression.
In mammalian cells, FoxO factors act as potent transcriptional activators that upregulate the expression of programs of genes involved in stress resistance, cell cycle arrest, differentiation, apoptosis, autophagy, and metabolism [17
]. Recent evidence indicates that FoxO factors, and in particular FoxO3, is important for the maintenance of adult neural and hematopoietic stem cells [31
]. However, much remains unknown about how FoxO transcription factors integrate extracellular stimuli to regulate different programs of gene expression and cellular responses that ultimately lead to lifespan extension and tumor suppression.
FoxO transcription factors are regulated by a wide array of external stimuli, ranging from insulin and growth factors to oxidative and nutrient stresses [36
], as well as a variety of chemical activators such as LY294002 [37
] and hypomethylating agents such as azacitidine and decitabine [38
]. The principal mode of regulation of FoxO factors is via post-translational modifications (PTMs). Phosphorylation by the insulin-activated protein kinases Akt and SGK at three conserved sites on FoxO factors leads to the sequestration of FoxO in the cytoplasm, thereby preventing FoxO factors from transactivating their target genes [18
]. Phosphorylation of FoxO factors in response to insulin pathway stimulation and to cytokines also promotes the degradation of FoxO factors by the ubiquitin-proteasome pathway [15
FoxO factors are post-translationally modified at a number of lysine residues by ubiquitination and acetylation. For example, FoxO1 is polyubiquitinated by the SCFskp2 complex [46
], and FoxO3 is polyubiquitinated by MDM2 and Skp-2 [47
], prior to degradation. FoxO factors can also be mono-ubiquitinated, which promotes FoxO nuclear translocation and regulates FoxO transcriptional activity [49
]. Acetylation/deacetylation of FoxO factors at a number of lysine residues also regulates FoxO subcellular localization, DNA binding capacity and transcriptional activity towards specific target genes [51
]. The large number and diversity of PTMs on FoxO factors have lead us to hypothesize that these PTMs form a ‘FoxO code’ that could regulate several aspects of FoxO activity [62
Methylation of lysine residues is a critical PTM for the regulation of histone proteins in chromatin [63
]. There are about 50 lysine methyltransferases in mammals [64
]. The Su(var), Enhancer of zeste, and Trithorax [28
] -domain containing lysine methyltransferases are conserved throughout species and catalyze a variety of histone methylations that are critical in regulating chromatin structure. Interestingly, some lysine methyltransferases can also methylate non-histone proteins. For example, Set9 (also known as Set7/9 and KMT7) was first identified as a mono-methyltransferase for lysine 4 in Histone H3 (H3K4) [66
], but Set9 also methylates several non-histone proteins, including transcription factors (RelA (p65), p53, ERa), enzymes (DNMT1, PCAF), and transcription-initiation proteins (TAF10) [67
]. Set9 methylation has been shown to affect the protein stability of Rel, p53, ERa and DNMT1 [67
], and to alter the recruitment of transcription factors and transcriptional machinery such as RelA and TAF10 to different promoters [71
]. These observations suggest that Set9 has a complex role beyond chromatin regulation.
The importance of Set9 in the organism is underscored by the observation that half of the Set9 knockout mice die prior to birth [75
]. Mouse embryonic fibroblasts (MEFs) from Set9 heterozygous and null mice are more susceptible to transformation than wildtype MEFs, suggesting that Set9, like FoxO3, acts as a tumor suppressor [75
]. This observation, coupled with the role of Set9 in a variety of cellular processes also regulated by FoxO3, including cell cycle arrest [69
] and apoptosis [69
], and the activation of Set9 by stress stimuli [69
], raised the possibility of a connection between Set9 and FoxO3.
Here we find that the lysine methyltransferase Set9 methylates FoxO3 directly in vitro and in cells. We identify a single lysine residue methylated by Set9 on FoxO3. This residue is important in modulating the transcriptional activity of FoxO3 and its stability. In addition to uncovering a novel non-histone substrate for Set9, our study identifies lysine methylation as an additional post-translational modification of FoxO3 that is likely part of the ‘code’ that modulates FoxO3's activity in response to environmental stimuli. Our findings further our understanding of the regulation of a critical transcription factor involved in longevity and cancer, and expand our knowledge of the role of Set9 in cells.