The maintenance and preservation of distinct phases during the cell cycle is a highly complex and coordinated process. It is regulated by phosphorylation--through the activity of cyclin-dependent kinases (CDKs)--and protein degradation, which occurs through ubiquitin ligases such as SCF (SKP1-CUL1-F-box protein) complexes and APC/C (anaphase-promoting complex/cyclosome). Here, we explore the functionality and biology of the F-box proteins, SKP2 (S-phase kinase-associated protein 2) and beta-TrCP (beta-transducin repeat-containing protein), which are emerging as important players in cancer biogenesis owing to the deregulated proteolysis of their substrates.

Summary In response to DNA damage in G2, mammalian cells must avoid entry into mitosis and instead initiate DNA repair. Here we show that in response to genotoxic stress in G2, the phosphatase Cdc14B translocates from the nucleolus to the nucleoplasm and induces the activation of the ubiquitin ligase APC/CCdh1, with the consequent degradation of Plk1, a prominent mitotic kinase. This process induces the stabilization of Claspin and Wee1, as the proteolysis of these two proteins requires phosphorylation by Plk1, and allows an efficient G2 checkpoint. As a by-product of APC/CCdh1 reactivation in DNA-damaged G2 cells, Claspin, which we show to be a novel substrate of APC/CCdh1 in G1, is targeted for degradation. However, this process is counteracted by the deubiquitylating enzyme Usp28 to permit Claspin-mediated activation of Chk1 in response to DNA damage. These findings define a novel pathway that is crucial for the G2 DNA damage response checkpoint.

Activation of NAD-dependent deacetylases, or Sirtuins, prolongs life span and mimics the effects of caloric restriction in yeast. The FoxO subfamily of forkhead transcription factors has been shown to mediate some of the effects of Sirtuins. Here we have shown that Sirtuin activation or hydrogen peroxide treatment overrides the phosphorylation-dependent nuclear exclusion of FoxO1 caused by growth factors and causes nuclear translocation of FoxO1 in hepatocytes. Kinetic measurements of nuclear fluorescence recovery after photobleaching show that FoxO1 is readily diffusible within the nucleus under normal conditions but becomes restricted within a nuclear subdomain following treatment with the prototypical Sirtuin agonist resveratrol or oxidative stress. Expression of FoxO1 target genes is accordingly increased, leading to activation of gluconeogenesis and increased glucose release from hepatocytes. Selective modulation of the FoxO/Sirtuin interaction represents a promising therapeutic modality for metabolic disorders.The long-standing observation that caloric restriction is associated with longevity has led to a widely held theory that metabolism and life span share common cellular pathways (1, 2). One such pathway has been proposed to involve forkhead transcription factors of the FoxO subfamily (3-5). Genetic epistasis in Caenorhabditis elegans and metabolic studies in mice indicate that FoxO genes regulate cell differentiation, transformation, and metabolism (6). In C. elegans, mutations of the FoxO ortholog Daf16 rescue the dauer state caused by mutations of the insulin/insulin-like growth factor receptor ortholog Daf2 (7,8). Moreover, extra copies of the gene encoding the NAD-dependent deacetylase Silent Information Regulator (Sir) 2.1 prolong life span in a Daf16-dependent fashion (9), suggesting that FoxO activity is regulated via deacetylation. These twin observations provide the underpinning for investigations of the role of FoxO proteins in mammalian metabolism and life span.FoxO activity is subject to complex regulation by growth factors and cellular stress. The former inhibit FoxO via serinethreonine phosphorylation and nuclear exclusion (10). The latter causes FoxO acetylation, thus promoting the interaction between FoxO and Sirt1, the mammalian ortholog of Sir2.1 (4,5,11,12). However, the effect of Sirt1-dependent deacetylation on FoxO function remains somewhat controversial, with most (4, 5, 12), but not all (11), studies suggesting that deacetylation increases FoxO-dependent transcription.In this study, we sought to uncover the mechanism by which stress-induced deacetylation affects FoxO activity. To this end, we studied FoxO translocation using live cell imaging, coupled to measurements of protein kinetics with fluorescence recovery after photobleaching (FRAP) 1 and fluorescence loss in photobleaching (FLIP) experiments (13). We also measured expression of FoxO1 target genes and glucose production in hepatocyte cultures. Our findings are consistent with a model in which deacetylation promotes Fo...

JHDM1B is an evolutionarily conserved and ubiquitously expressed member of the JHDM (JmjC-domain-containing histone demethylase) family. Because it contains an F-box motif, this protein is also known as FBXL10 (ref. 4). With the use of a genome-wide RNAi screen, the JHDM1B worm orthologue (T26A5.5) was identified as a gene that regulates growth. In the mouse, four independent screens have identified JHDM1B as a putative tumour suppressor by retroviral insertion analysis. Here we identify human JHDM1B as a nucleolar protein and show that JHDM1B preferentially binds the transcribed region of ribosomal DNA to repress the transcription of ribosomal RNA genes. We also show that repression of ribosomal RNA genes by JHDM1B is dependent on its JmjC domain, which is necessary for the specific demethylation of trimethylated lysine 4 on histone H3 in the nucleolus. In agreement with the notion that ribosomal RNA synthesis and cell growth are coupled processes, we show a JmjC-domain-dependent negative effect of JHDM1B on cell size and cell proliferation. Because aberrant ribosome biogenesis and the disruption of epigenetic control mechanisms contribute to cellular transformation, these results, together with the low levels of JHDM1B expression found in aggressive brain tumours, suggest a role for JHDM1B in cancer development.

SUMMARY To prevent ATR activation, telomeres deploy the single-stranded DNA binding activity of TPP1/POT1a. POT1a blocks the binding of RPA to telomeres, suggesting that ATR is repressed through RPA exclusion. However, comparison on the DNA binding affinities and abundance of TPP1/POT1a and RPA indicates that TPP1/POT1a by itself is unlikely to exclude RPA. We therefore analyzed the central shelterin protein TIN2, which links TPP1/POT1a (and POT1b) to TRF1 and TRF2 on the double-stranded telomeric DNA. Upon TIN2 deletion, telomeres lost TPP1/POT1a, accumulated RPA, elicited an ATR signal, and showed all other phenotypes of POT1a/b deletion. TIN2 also affected the TRF2-dependent repression of ATM kinase signaling but not to TRF2-mediated inhibition of telomere fusions. Thus, while TIN2 has a minor contribution to the repression of ATM by TRF2, its major role is to stabilize TPP1/POT1a on the ss telomeric DNA, thereby allowing effective exclusion of RPA and repression of ATR signaling.

REST/NRSF (repressor-element-1-silencing transcription factor/neuron-restrictive silencing factor) negatively regulates the transcription of genes containing RE1 sites 1,2 . REST is expressed in nonneuronal cells and stem/progenitor neuronal cells, in which it inhibits the expression of neuronspecific genes. Overexpression of REST is frequently found in human medulloblastomas and neuroblastomas 3-7 , in which it is thought to maintain the stem character of tumour cells. Neural stem cells forced to express REST and c-Myc fail to differentiate and give rise to tumours in the mouse cerebellum 3 . Expression of a splice variant of REST that lacks the carboxy terminus has been associated with neuronal tumours and small-cell lung carcinomas 8-10 , and a frameshift mutant (REST-FS), which is also truncated at the C terminus, has oncogenic properties 11 . Here we show, by using an unbiased screen, that REST is an interactor of the F-box protein β-TrCP. REST is degraded by means of the ubiquitin ligase SCF β-TrCP during the G2 phase of the cell cycle to allow transcriptional derepression of Mad2, an essential component of the spindle assembly checkpoint. The expression in cultured cells of a stable REST mutant, which is unable to bind β-TrCP, inhibited Mad2 expression and resulted in a phenotype analogous to that observed in Mad2 +/− cells. In particular, we observed defects that were consistent with faulty activation of the spindle checkpoint, such as shortened mitosis, premature sister-chromatid separation, chromosome bridges and missegregation in anaphase, tetraploidy, and faster mitotic slippage in the presence of a spindle inhibitor. An indistinguishable phenotype was observed by expressing the oncogenic REST-FS mutant 11 , which does not bind β-TrCP. Thus, SCF β-TrCP -dependent degradation of REST during G2 permits the optimal activation of the spindle checkpoint, and consequently it is required for the fidelity of mitosis. The high levels of REST or its truncated variants found in certain human tumours may contribute to cellular transformation by promoting genomic instability.

Heterochromatin plays an essential role in the preservation of epigenetic information, the transcriptional repression of repetitive DNA elements and inactive genes and the proper segregation of chromosomes during mitosis. Here we identify KDM2A, a JmjC-domain containing histone demethylase, as a heterochromatin-associated protein that is required to maintain the heterochromatic state, as determined using both a candidate-based approach and an unbiased siRNA library screen. Moreover, we demonstrate that KDM2A represses transcription of small non-coding RNAs that are encoded by clusters of satellite repeats at the centromere. Finally, we show that KDM2A is required to sustain centromeric integrity and genomic stability, particularly during mitosis. Since the disruption of epigenetic control mechanisms contributes to cellular transformation, these results, together with the low levels of KDM2A found in prostate carcinomas, suggest a role for KDM2A in cancer development.

Drosophila melanogaster embryogenesis begins with 13 nuclear division cycles within a syncytium. This produces >6,000 nuclei that, during the next division cycle, become encased in plasma membrane in the process known as cellularization. In this study, we investigate how the secretory membrane system becomes equally apportioned among the thousands of syncytial nuclei in preparation for cellularization. Upon nuclear arrival at the cortex, the endoplasmic reticulum (ER) and Golgi were found to segregate among nuclei, with each nucleus becoming surrounded by a single ER/Golgi membrane system separate from adjacent ones. The nuclear-associated units of ER and Golgi across the syncytial blastoderm produced secretory products that were delivered to the plasma membrane in a spatially restricted fashion across the embryo. This occurred in the absence of plasma membrane boundaries between nuclei and was dependent on centrosome-derived microtubules. The emergence of secretory membranes that compartmentalized around individual nuclei in the syncytial blastoderm is likely to ensure that secretory organelles are equivalently partitioned among nuclei at cellularization and could play an important role in the establishment of localized gene and protein expression patterns within the early embryo.