No abstract

Members of the FOXO (forkhead O) class of transcription factors are tumor suppressors that also control aging and organismal life span. Mammalian FOXO degradation is proteasomemediated, although the ubiquitin E3 ligase for FOXO factors remains to be defined. We show that MDM2 binds to FOXO1 and FOXO3A and promotes their ubiquitination and degradation, a process apparently dependent on FOXO phosphorylation at AKT sites and the E3 ligase activity of MDM2. Binding of MDM2 to FOXO occurs through the region of MDM2 that directs its cellular trafficking and the forkhead box of FOXO1. MDM2 promotes the ubiquitination of FOXO1 in a cell-free system, and its knockdown by small interfering RNA causes accumulation of endogenous FOXO3A protein in cells and enhances the expression of FOXO target genes. In cells stably expressing a temperature-sensitive p53 mutant, activation of p53 by shifting to permissive temperatures leads to MDM2 induction and degradation of endogenous FOXO3A. These data suggest that MDM2 acts as an ubiquitin E3 ligase, downstream of p53, to regulate the degradation of mammalian FOXO factors.FOXO (forkhead O) proteins belong to the family of forkhead transcriptional factors, which are characterized by a conserved DNA binding domain termed the "Forkhead box" (1). Mammalian FOXO factors include FOXO1 (previously known as FKHR), FOXO3A (previously known as FKHRL1), FOXO4 (previously known as AFX), and FOXO6. These factors control the expression of a variety of genes that regulate essential cellular processes, such as cell cycle (2-4), apoptosis (5), oxidative stress (6, 7), atrophy (8), energy homeostasis, and glucose metabolism (9, 10). Whole organism studies in worms and flies show that FOXO factors have conserved ability to increase the organismal longevity (11). Genetic and functional analysis identifies FOXO1 as a tumor suppressor in the prostate (12). Knock-out studies show that mammalian FOXO factors act redundantly to suppress tumorigenesis in a lineage-specific fashion (13) and to maintain the long term regenerative potential of hematopoietic stem cells by regulating genes involved in the cellular response to physiological oxidative stresses (14).The transcription of FOXO factors is regulated by posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. Multiple kinases, including AKT (15, 16), serum-and glucocorticoid-induced kinase (17), casein kinase 1 (18), mammalian Ste20-like kinase 1 (19), I B kinase (20), and cyclin-dependent kinase 2 (21), catalyze FOXO phosphorylation and often promote FOXO nuclear exportation. In response to insulin and growth factors, FOXO1 and FOXO3A are ubiquitinated and degraded by the proteasome pathway after phosphorylation at known AKT sites (15,22,23). Acetyltransferases, p300 (24) and CBP (25), and SIRT1 deacetylase (26, 27) regulate the activity of FOXO through acetylation/deacetylation. The role of FOXO acetylation is controversial, but it could affect their nuclear retention (28), phosphorylation (25), and ubiquitination-medi...

Epigenetic maintenance of the expression state of the genome is critical for development. Drosophila GAGA factor interacts with FACT and modulates chromatin structure for the maintenance of gene expression. Here we show that the GAGA factor-FACT complex and its binding site just downstream from the white gene are crucial for position effect variegation. Interestingly there is a dip of histone H3 Lys 9 methylation and a peak of H3 Lys 4 methylation at this site. The GAGA factor and FACT direct replacement of histone H3 by H3.3 through association of HIRA at this site, and maintain white expression under the heterochromatin environment. Based on these findings we propose that the GAGA factor and FACT-dependent replacement of Lys 9-methylated histone H3 by H3.3 counteracts the spreading of silent chromatin.[Keywords: GAGA factor; FACT; histone H3.3; position effect variegation; heterochromatin] Supplemental material is available at http://www.genesdev.org.

Chromatin structure plays a critical role in the regulation of transcription. Drosophila GAGA factor directs chromatin remodeling to its binding sites. We show here that Drosophila FACT (facilitates chromatin transcription), a heterodimer of dSPT16 and dSSRP1, is associated with GAGA factor through its dSSRP1 subunit, binds to a nucleosome, and facilitates GAGA factor-directed chromatin remodeling. Moreover, genetic interactions between Trithorax-like encoding GAGA factor and spt16 implicate the GAGA factor-FACT complex in expression of Hox genes Ultrabithorax, Sex combs reduced, and Abdominal-B. Chromatin immunoprecipitation experiments indicated the presence of the GAGA factor-FACT complex in the regulatory regions of Ultrabithorax and Abdominal-B. These data illustrate a crucial role of FACT in the modulation of chromatin structure for the regulation of gene expression.

SUMMARYEstablishment and maintenance of epigenetic memories are essential for development. Replacement of canonical histone H3 by its variant H3.3 has been implicated in cellular memory. Drosophila sequence-specific DNA-binding protein GAGA factor and a chromatin factor FACT direct H3.3 replacement in conjunction with H3.3-specific chaperone HIRA at chromatin boundaries to counteract the spreading of silent chromatin. However, little is known about which ATP-driven chromatin remodeling factor is responsible for the H3.3 replacement at chromatin boundaries. Here, we report that GAGA factor associates with the Polybromoassociated Brm (PBAP) remodeling complex, which consists of many Trithorax group proteins, and recruits this complex to chromatin boundaries d1 (which is downstream of w), the Fab-7 DNase-hypersensitive site (HS) 1 of Abd-B and the bxd region of Ubx. Trlencoding GAGA factor, brm and polybromo/bap180 mutations compromise the H3.3 replacement and boundary functions in a synergistic manner. Furthermore, Polybromo is necessary for generation of the DNase HS at d1, and HIRA functions to restore the alteration. Taken together, we propose that FACT and PBAP complexes are recruited to chromatin boundaries in a GAGA factordependent manner, and are needed for H3.3 replacement to execute boundary functions. Our results provide new insight into the function of the trithorax group during development.