SUMMARY Genetically unstable tetraploid cells can promote tumorigenesis. Recent estimates suggest that ~37% of human tumors have undergone a genome-doubling event during their development. This potentially oncogenic effect of tetraploidy is countered by a p53-dependent barrier to proliferation. However, the cellular defects and corresponding signaling pathways that trigger growth suppression in tetraploid cells are not known. Here we combine RNAi screening and in vitro evolution approaches to demonstrate that cytokinesis failure activates the Hippo tumor suppressor pathway in cultured cells as well as in naturally occurring tetraploid cells in vivo. Induction of the Hippo pathway is triggered in part by extra centrosomes, which alter small G-protein signaling and activate LATS2 kinase. LATS2 in turn stabilizes p53 and inhibits the transcriptional regulators YAP and TAZ. These findings define an important tumor suppression mechanism and uncover adaptive mechanisms potentially available to nascent tumor cells that bypass this inhibitory regulation.

Nonsense-mediated mRNA decay (NMD) in mammalian cells depends on phosphorylation of Upf1, an RNA-dependent ATPase and 5-to-3 helicase. Upf1 phosphorylation is mediated by Smg1, a phosphoinositol 3-kinase-related protein kinase. Here, we describe a human protein, which we call hSmg5/7a, that manifests similarity to Caenorhabditis elegans NMD factors CeSMG5 and CeSMG7, as well as two Drosophila melanogaster proteins that are also similar to the C. elegans NMD factors. Results indicate that hSmg5/7a functions in the dephosphorylation of Upf1. Furthermore, hSmg5/7a copurifies with Upf1, Upf2, Upf3X, Smg1, and the catalytic subunit of protein phosphatase 2A. We also demonstrate that Upf2, another factor involved in NMD, is a phosphoprotein. However, hSmg5/7a plays no role in the dephosphorylation of Upf2. These data indicate that hSmg5/7a targets protein phosphatase 2A to Upf1 but not Upf2. Results of Western blotting reveal that hSmg5/7a is mostly cytoplasmic in HEK293T cells.

We have developed an advanced method for conditional gene expression in mice that integrates the Cre-mediated and tetracycline-dependent expression systems. An rtTA gene, preceded by a loxP-flanked STOP sequence, was inserted into the ROSA26 locus to create a R26STOPrtTA mouse strain. When the STOP sequence is excised by Cre-mediated recombination, the rtTA is expressed in the Cre-expressing cells and all of their derivatives. Therefore, cell type-, tissue-, or lineage-specific expression of rtTA is achieved by the use of an appropriate Cre transgenic strain. In mice also carrying a target gene under the control of the tetracycline response element, inducible expression of the target gene is temporally regulated by administration of doxycycline. Our results demonstrate that this universal system is uniquely suited for spatiotemporal and lineage-specific gene expression in an inducible fashion. Gene expression can be manipulated in specific cell types and lineages with a flexibility that is difficult to achieve with conventional methods.conditonal gene expression ͉ Cre ͉ ROSA26 locus ͉ rtTA D evelopment of advanced transgenic systems for conditional gene expression is essential for studying development and modeling human diseases in mice. Two powerful systems that have been used extensively are the Cre-loxP system, which allows genes to be permanently activated or inactivated in specific lineages (1), and the tetracycline-dependent system, which permits temporal as well as cell type-or tissue-specific control of transgene expression (2). The basic strategy for the Cre-loxP system is to delete the DNA sequence flanked by two loxP sites, thereby achieving conditional gene activation and inactivation. Manipulating the expression of Cre in a specific place and at a specific time enables conditional regulation. In the tetracyclinedependent system, three essential elements are required. First is the transcription factor tTA or rtTA, which can be expressed in a spatial specific manner. Second, the tetracycline response element (TRE) is used to control the expression of target genes. The presence of the third element, doxycycline (Dox), which can be administrated in a temporal specific fashion, activates target gene expression. We have developed a mouse strain, R26STOPrtTA, which combines features of these two systems and permits greater flexibility of conditional gene expression in the mouse. An rtTA gene, preceded by a loxP-flanked STOP sequence, was inserted into the ubiquitously active ROSA26 locus. When R26STOPrtTA is crossed with Cre-expressing strains, the STOP sequence is excised, yielding the R26rtTA allele, which expresses rtTA in the Cre-expressing cells and all of their descendants. Thus, the cell type, tissue, or lineage specificity of rtTA expression is dictated by selection of an appropriate Cre transgenic strain. In mice also carrying a target gene under the control of TRE, expression of the target gene is temporally regulated by administration of Dox. Using this approach, we have successfully achieved tr...

Post-translational modification of proteins by small ubiquitin-related modifier (SUMO) is reversible and highly evolutionarily conserved from yeasts to humans. Unlike ubiquitination with a well-established role in protein degradation, sumoylation may alter protein function, activity, stability and subcellular localization. Members of SUMO-specific protease (SENP) family, capable of SUMO removal, are involved in the reversed conjugation process. Although SUMO-specific proteases are known to reverse sumoylation in many well-defined systems, their importance in mammalian development and pathogenesis remains largely elusive. In patients with neurodegenerative diseases, aberrant accumulation of SUMO-conjugated proteins has been widely described. Several aggregation-prone proteins modulated by SUMO have been implicated in neurodegeneration, but there is no evidence supporting a direct involvement of SUMO modification enzymes in human diseases. Here we show that mice with neural-specific disruption of SENP2 develop movement difficulties which ultimately results in paralysis. The disruption induces neurodegeneration where mitochondrial dynamics is dysregulated. SENP2 regulates Drp1 sumoylation and stability critical for mitochondrial morphogenesis in an isoform-specific manner. Although dispensable for development of neural cell types, this regulatory mechanism is necessary for their survival. Our findings provide a causal link of SUMO modification enzymes to apoptosis of neural cells, suggesting a new pathogenic mechanism for neurodegeneration. Exploring the protective effect of SENP2 on neuronal cell death may uncover important preventive and therapeutic strategies for neurodegenerative diseases.