[Abstract unavailable

Longevity regulatory genes include the Forkhead transcription factor FOXO and the NAD-dependent histone deacetylase silent information regulator 2 (Sir2). Genetic studies demonstrate that Sir2 acts to extend lifespan in Caenorhabditis elegans upstream of DAF-16, a member of the FOXO family, in the insulin-like signaling pathway. However, the molecular mechanisms underlying the requirement of DAF-16 activity in Sir2-mediated longevity remain unknown. Here we show that reversible acetylation of Foxo1 (also known as FKHR), the mouse DAF-16 ortholog, modulates its transactivation function. cAMP-response element-binding protein (CREB)-binding protein binds and acetylates Foxo1 at the K242, K245, and K262 residues, the modification of which is involved in the attenuation of Foxo1 as a transcription factor. Conversely, Sir2 binds and deacetylates Foxo1 at residues acetylated by cAMPresponse element-binding protein-binding protein. Sir2 is recruited to insulin response sequence-containing promoter and increases the expression of manganese superoxide dismutase and p27 kip1 in a deacetylase-activity-dependent manner. Our findings establish Foxo1 as a direct and functional target for Sir2 in mammalian systems.

Covalent modification of many transcription factors with SUMO-1 is emerging as a key role of trans-activational regulation. Here, we demonstrate that peroxisome proliferator-activated receptor (PPAR) ␥, which is a ligand-activated nuclear receptor, is modified by SUMO-1. Sumoylation of PPAR␥ mainly occurs at a lysine residue within the activation function 1 domain. Furthermore, we show that the PIAS family proteins, PIAS1 and PIASx␤, function as E3 ligases (ubiquitin-protein isopeptide ligase) for PPAR␥. PPAR␥ interacts directly with PIASx␤ in a ligand-independent manner. Analysis using a PPAR␥ mutant with a disrupted sumoylation site shows that modification of PPAR␥ by SUMO-1 represses its transcriptional activity. Interestingly, PIASx␤ and Ubc9 enhance the transcriptional activity of PPAR␥ independent of PPAR␥ sumoylation. Furthermore, PPAR␥ ligand-induced apoptosis in a human hepatoblastoma cell line, HepG2, is significantly enhanced by ectopic production of the sumoylation-mutant PPAR␥. These results suggest that the PPAR␥-dependent transactivation pathway seems to be modulated by SUMO-1 modification and may serve as a novel target for apoptosis-induction therapy in cancer cells.PPAR␥ 1 is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors (1) which regulates diverse biological functions including cell differentiation, growth inhibition, lipid metabolism, and apoptosis (2-5). Two isoforms of PPAR␥, PPAR␥1 and PPAR␥2, are generated by alternative promoter usage. PPAR␥2, which contains an additional 30 amino acid residues at the amino terminus compared with PPAR␥1, is predominantly expressed in adipose tissue, whereas PPAR␥1 is widely expressed (6).The role of PPAR␥ in adipogenesis has been extensively studied. Many adipocyte-specific genes, such as adipocytokines, contain PPAR␥-responsible elements in their promoter and/or upstream enhancer regions (7-10). PPAR␥ plays a role as a central transcription factor in cellular differentiation and lipid accumulation during adipogenesis. Recent investigations demonstrate that treatment of a variety of human cancer cell lines with PPAR␥ ligands leads to growth inhibition and apoptosis (2, 11-13). The use of PPAR␥ ligands in the treatment of cancer is a potentially promising nontoxic and selective chemotherapeutic approach, and consequently, increased understanding of the mechanisms of PPAR␥ in tumor suppression is needed.Post-translational modifications regulate the function of many proteins. In the case of PPAR␥, transcriptional activity is reduced by mitogen-activated protein kinase-induced phosphorylation of serine residue 112 (14 -16). Knock-in mice expressing PPAR␥ with a Ser 3 Ala mutation at this residue exhibit preserved insulin sensitivity in the setting of diet-induced obesity by changing fat cell size, generation of adiponectin, and increasing the amount of free fatty acid levels in serum (17).Recently, a number of ubiquitin-like proteins (Ubl) have been identified that are covalently linked to lysine residues in t...

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RNA helicase A (RHA) is a member of an ATPase/DNA and RNA helicase family and is a homologue of Drosophila maleless protein (MLE), which regulates X-linked gene expression. RHA is also a component of holo-RNA polymerase II (Pol II) complexes and recruits Pol II to the CREB binding protein (CBP). The ATPase and/or helicase activity of RHA is required for CREB-dependent transcription. To further understand the role of RHA on gene expression, we have identified a 50-amino-acid transactivation domain that interacts with Pol II and termed it the minimal transactivation domain (MTAD). The protein sequence of this region contains six hydrophobic residues and is unique to RHA homologues and well conserved. A mutant with this region deleted from full-length RHA decreased transcriptional activity in CREB-dependent transcription. In addition, mutational analyses revealed that several tryptophan residues in MTAD are important for the interaction with Pol II and transactivation. These mutants had ATP binding and ATPase activities comparable to those of wild-type RHA. A mutant lacking ATP binding activity was still able to interact with Pol II. In CREB-dependent transcription, the transcriptional activity of each of these mutants was less than that of wild-type RHA. The activity of the double mutant lacking both functions was significantly lower than that of each mutant alone, and the double mutant had a dominant negative effect. These results suggest that RHA could independently regulate CREB-dependent transcription either through recruitment of Pol II or by ATP-dependent mechanisms.RNA helicase A (RHA) is a member of the DExH family of ATPases/helicases and catalyzes the displacement of both double-stranded RNA and DNA from 3Ј to 5Ј (32,61,63). Functional domains of RHA include two double-stranded RNA binding domains at the amino terminus known as dsRBD1 and dsRBD2. The catalytic core domain is located within the central region and contains a DExH motif. This core domain contains seven well-conserved motifs; one of them has an ATP binding site with the consensus GCGKT and FILDD, known as the A site the B site, respectively. The carboxyl terminus contains an RGG-rich region that is capable of binding singlestrand nucleic acids (62).RHA was originally isolated as a human homologue of Drosophila maleless protein (MLE), with which it has 50% sequence identity and 90% sequence similarity (33). In Drosophila, MLE colocalizes with acetylated histone H4 (8, 48). MLE is involved in sex-specific gene dosage compensation and elevates the level of transcription derived from a single X chromosome in male flies to a level equivalent to that derived from two X chromosome in the female (25,29). MLE mutants are embryonic lethal to males, indicating that MLE is an essential factor in Drosophila development.In mammals, RHA-knockout mice are embryonic lethal for homozygous RHA mutants (35). Analysis of these mice revealed that RHA is associated with differentiation of the embryonic ectoderm during gastrulation. It is possible that RHA has an i...