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Latest Articles Include:

• Attracting women
  - Nat Cell Biol 11(8):915 (2009)
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• Physical Matryoshka of cell biology
  - Nat Cell Biol 11(8):916 (2009)
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• Homologue pairing: getting it right
  - Nat Cell Biol 11(8):917-918 (2009)
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• Axon growth-stimulus package includes local translation
  - Nat Cell Biol 11(8):919-921 (2009)
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• SASPense and DDRama in cancer and ageing
  - Nat Cell Biol 11(8):921-923 (2009)
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• Research highlights
  - Nat Cell Biol 11(8):924 (2009)
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• Histone levels are regulated by phosphorylation and ubiquitylation-dependent proteolysis
  - Nat Cell Biol 11(8):925-933 (2009)
  Histone levels are tightly regulated to prevent harmful effects such as genomic instability and hypersensitivity to DNA-damaging agents due to the accumulation of these highly basic proteins when DNA replication slows down or stops. Although chromosomal histones are stable, excess (non-chromatin bound) histones are rapidly degraded in a Rad53 (radiation sensitive 53) kinase-dependent manner in Saccharomyces cerevisiae. Here we demonstrate that excess histones associate with Rad53 in vivo and seem to undergo modifications such as tyrosine phosphorylation and polyubiquitylation, before their proteolysis by the proteasome. We have identified the Tyr 99 residue of histone H3 as being critical for the efficient ubiquitylation and degradation of this histone. We have also identified the ubiquitin conjugating enzymes (E2) Ubc4 and Ubc5, as well as the ubiquitin ligase (E3) Tom1 (temperature dependent organization in mitotic nucleus 1), as enzymes involved in the ubiquitylatio! n of excess histones. Regulated histone proteolysis has major implications for the maintenance of epigenetic marks on chromatin, genomic stability and the packaging of sperm DNA.
• Identification of chromosome sequence motifs that mediate meiotic pairing and synapsis in C. elegans
  - Nat Cell Biol 11(8):934-942 (2009)
  Caenorhabditis elegans chromosomes contain specialized regions called pairing centres, which mediate homologous pairing and synapsis during meiosis. Four related proteins, ZIM-1, 2, 3 and HIM-8, associate with these sites and are required for their essential functions. Here we show that short sequence elements enriched in the corresponding chromosome regions selectively recruit these proteins in vivo. In vitro analysis using SELEX indicates that the binding specificity of each protein arises from a combination of two zinc fingers and an adjacent domain. Insertion of a cluster of recruiting motifs into a chromosome lacking its endogenous pairing centre is sufficient to restore homologous pairing, synapsis, crossover recombination and segregation. These findings help to illuminate how chromosome sites mediate essential aspects of meiotic chromosome dynamics.
• A SNAIL1–SMAD3/4 transcriptional repressor complex promotes TGF- mediated epithelial–mesenchymal transition
  - Nat Cell Biol 11(8):943-950 (2009)
  Epithelial–mesenchymal transition (EMT) is essential for organogenesis and is triggered during carcinoma progression to an invasive state1. Transforming growth factor- (TGF-) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT2, 3, 4, 5, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT6, 7. The SNAIL1–SMAD3/4 complex was targeted to the gene promoters of CAR, a tight-junction protein, and E-cadherin during TGF--driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts! of human breast cancer. We propose that activation of a SNAIL1–SMAD3/4 transcriptional complex represents a mechanism of gene repression during EMT.
• A regulatory pathway involving Notch1/-catenin/Isl1 determines cardiac progenitor cell fate.
  - Nat Cell Biol 11(8):951-957 (2009)
  Regulation of multipotent cardiac progenitor cell (CPC) expansion and subsequent differentiation into cardiomyocytes, smooth muscle or endothelial cells is a fundamental aspect of basic cardiovascular biology and cardiac regenerative medicine. However, the mechanisms governing these decisions remain unclear. Here, we show that Wnt/-catenin signalling, which promotes expansion of CPCs1, 2, 3, is negatively regulated by Notch1-mediated control of phosphorylated -catenin accumulation within CPCs, and that Notch1 activity in CPCs is required for their differentiation. Notch1 positively, and -catenin negatively, regulated expression of the cardiac transcription factors, Isl1, Myocd and Smyd1. Surprisingly, disruption of Isl1, normally expressed transiently in CPCs before their differentiation4, resulted in expansion of CPCs in vivo and in an embryonic stem (ES) cell system. Furthermore, Isl1 was required for CPC differentiation into cardiomyocyte and smooth muscle cells, bu! t not endothelial cells. These findings reveal a regulatory network controlling CPC expansion and cell fate that involves unanticipated functions of -catenin, Notch1 and Isl1 that may be leveraged for regenerative approaches involving CPCs.
• Mitochondrial fission factor Drp1 is essential for embryonic development and synapse formation in mice
  - Nat Cell Biol 11(8):958-966 (2009)
  Mitochondrial morphology is dynamically controlled by a balance between fusion and fission. The physiological importance of mitochondrial fission in vertebrates is less clearly defined than that of mitochondrial fusion. Here we show that mice lacking the mitochondrial fission GTPase Drp1 have developmental abnormalities, particularly in the forebrain, and die after embryonic day 12.5. Neural cell-specific (NS) Drp1-/- mice die shortly after birth as a result of brain hypoplasia with apoptosis. Primary culture of NS-Drp1-/- mouse forebrain showed a decreased number of neurites and defective synapse formation, thought to be due to aggregated mitochondria that failed to distribute properly within the cell processes. These defects were reflected by abnormal forebrain development and highlight the importance of Drp1-dependent mitochondrial fission within highly polarized cells such as neurons. Moreover, Drp1-/- murine embryonic fibroblasts and embryonic stem cells revealed ! that Drp1 is required for a normal rate of cytochrome c release and caspase activation during apoptosis, although mitochondrial outer membrane permeabilization, as examined by the release of Smac/Diablo and Tim8a, may occur independently of Drp1 activity.
• Prolyl isomerase Pin1 acts as a switch to control the degree of substrate ubiquitylation
  - Nat Cell Biol 11(8):967-972 (2009)
  Pin1, a conserved eukaryotic peptidyl-prolyl cis/trans isomerase, has important roles in cellular regulation1, 2. Because of its activity to switch the conformation of peptidyl-proline bonds in polypeptide chains, Pin1 operates as a binary switch, often in fate-determining pathways. Pin1 activity is usually controlled by substrate phosphorylation1, 2, but how Pin1 switches protein fates has been unclear. Here we show that Pin1 controls the degree of substrate ubiquitylation and thereby protein functions. We found that yeast Pin1 (Ess1) is essential for viability because it controls the NF-B-related Spt23 transcription factor involved in unsaturated fatty-acid synthesis3. High Pin1 activity results in low ubiquitylation of Spt23, which triggers Spt23 precursor processing and hence transcription factor activation. By contrast, decreased Pin1 activity leads to robust Spt23 polyubiquitylation and subsequent proteasomal degradation. Inhibition of Pin1 in mammalian cells cha! nges the ubiquitylation status of the tumour suppressor protein p53 from oligoubiquitylation, which is known to trigger nuclear export4, to polyubiquitylation, which causes nuclear p53 degradation. This suggests that the Pin1 activity is often translated into a fate-determining ubiquitylation switch, and that Pin1 may affect the degree of substrate ubiquitylation in other pathways as well.
• Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion
  - Nat Cell Biol 11(8):973-979 (2009)
  Cellular senescence suppresses cancer by stably arresting the proliferation of damaged cells1. Paradoxically, senescent cells also secrete factors that alter tissue microenvironments2. The pathways regulating this secretion are unknown. We show that damaged human cells develop persistent chromatin lesions bearing hallmarks of DNA double-strand breaks (DSBs), which initiate increased secretion of inflammatory cytokines such as interleukin-6 (IL-6). Cytokine secretion occurred only after establishment of persistent DNA damage signalling, usually associated with senescence, not after transient DNA damage responses (DDRs). Initiation and maintenance of this cytokine response required the DDR proteins ATM, NBS1 and CHK2, but not the cell-cycle arrest enforcers p53 and pRb. ATM was also essential for IL-6 secretion during oncogene-induced senescence and by damaged cells that bypass senescence. Furthermore, DDR activity and IL-6 were elevated in human cancers, and ATM-depleti! on suppressed the ability of senescent cells to stimulate IL-6-dependent cancer cell invasiveness. Thus, in addition to orchestrating cell-cycle checkpoints and DNA repair, a new and important role of the DDR is to allow damaged cells to communicate their compromised state to the surrounding tissue.
• The DNA damage response at eroded telomeres and tethering to the nuclear pore complex
  - Nat Cell Biol 11(8):980-987 (2009)
  The ends of linear eukaryotic chromosomes are protected by telomeres, which serve to ensure proper chromosome replication and to prevent spurious recombination at chromosome ends. In this study, we show by single cell analysis that in the absence of telomerase, a single short telomere is sufficient to induce the recruitment of checkpoint and recombination proteins. Notably, a DNA damage response at eroded telomeres starts many generations before senescence and is characterized by the recruitment of Cdc13 (cell division cycle 13), replication protein A, DNA damage checkpoint proteins and the DNA repair protein Rad52 into a single focus. Moreover, we show that eroded telomeres, although remaining at the nuclear periphery, move to the nuclear pore complex. Our results link the DNA damage response at eroded telomeres to changes in subnuclear localization and suggest the existence of collapsed replication forks at eroded telomeres.
• A two-step model for senescence triggered by a single critically short telomere
  - Nat Cell Biol 11(8):988-993 (2009)
  Telomeres protect chromosome ends from fusion and degradation1. In the absence of a specific telomere elongation mechanism, their DNA shortens progressively with every round of replication, leading to replicative senescence2. Here, we show that telomerase-deficient cells bearing a single, very short telomere senesce earlier, demonstrating that the length of the shortest telomere is a major determinant of the onset of senescence. We further show that Mec1p–ATR specifically recognizes the single, very short telomere causing the accelerated senescence. Strikingly, before entering senescence, cells divide for several generations despite complete erosion of their shortened telomeres. This pre-senescence growth requires RAD52 (radiation sensitive) and MMS1 (methyl methane sulfonate sensitive), and there is no evidence for major inter-telomeric recombination. We propose that, in the absence of telomerase, a very short telomere is first maintained in a pre-signalling state b! y a RAD52–MMS1-dependent pathway and then switches to a signalling state leading to senescence through a Mec1p-dependent checkpoint.
• VHL loss causes spindle misorientation and chromosome instability
  - Nat Cell Biol 11(8):994-1001 (2009)
  Error-free mitosis depends on fidelity-monitoring checkpoint systems that ensure correct temporal and spatial coordination of chromosome segregation by the microtubule spindle apparatus. Defects in these checkpoint systems can lead to genomic instability, an important aspect of tumorigenesis. Here we show that the von Hippel-Lindau (VHL) tumour suppressor protein, pVHL, which is inactivated in hereditary and sporadic forms of renal cell carcinoma, localizes to the mitotic spindle in mammalian cells and its functional inactivation provokes spindle misorientation, spindle checkpoint weakening and chromosomal instability. Spindle misorientation is linked to unstable astral microtubules and is supressed by the restoration of wild-type pVHL in pVHL-deficient cells, but not in naturally-occurring VHL disease mutants that are defective in microtubule stabilization. Impaired spindle checkpoint function and chromosomal instability are the result of reduced Mad2 (mitotic arrest ! deficient 2) levels actuated by pVHL-inactivation and are rescued by re-expression of either Mad2 or pVHL in VHL-defective cells. An association between VHL inactivation, reduced Mad2 levels and increased aneuploidy was also found in human renal cancer, implying that the newly identified functions of pVHL in promoting proper spindle orientation and chromosomal stability probably contribute to tumour suppression.
• BCOR regulates mesenchymal stem cell function by epigenetic mechanisms
  - Nat Cell Biol 11(8):1002-1009 (2009)
  The BCL-6 co-repressor (BCOR) represses gene transcription by interacting with BCL-6 (Refs 1, 2). BCOR mutation is responsible for oculo-facio-cardio-dental (OFCD) syndrome, which is characterized by canine teeth with extremely long roots, congenital cataracts, craniofacial defects and congenital heart disease3, 4, 5. Here we show that BCOR mutation increased the osteo-dentinogenic potential of mesenchymal stem cells (MSCs) isolated from a patient with OFCD, providing a molecular explanation for abnormal root growth. AP-2 was identified as a repressive target of BCOR, and BCOR mutation resulted in abnormal activation of AP-2. Gain- and loss-of-function assays suggest that AP-2 is a key factor that mediates the increased osteo-dentinogenic capacity of MSCs. Moreover, we found that BCOR maintained tissue homeostasis and gene silencing through epigenetic mechanisms. BCOR mutation increased histone H3K4 and H3K36 methylation in MSCs, thereby reactivating transcription of s! ilenced target genes. By studying a rare human genetic disease, we have unravelled an epigenetic mechanism for control of human adult stem cell function.
• Reversible acetylation of the chromatin remodelling complex NoRC is required for non-coding RNA-dependent silencing
  - Nat Cell Biol 11(8):1010-1016 (2009)
  The SNF2h (sucrose non-fermenting protein 2 homologue)-containing chromatin-remodelling complex NoRC silences a fraction of ribosomal RNA genes (rDNA) by establishing a heterochromatic structure at the rDNA promoter1, 2, 3. Here we show that the acetyltransferase MOF (males absent on the first) acetylates TIP5, the largest subunit of NoRC, at a single lysine residue, K633, adjacent to the TIP5 RNA-binding domain, and that the NAD+-dependent deacetylase SIRT1 (sirtuin-1) removes the acetyl group from K633. Acetylation regulates the interaction of NoRC with promoter-associated RNA (pRNA), which in turn affects heterochromatin formation, nucleosome positioning and rDNA silencing. Significantly, NoRC acetylation is responsive to the intracellular energy status and fluctuates during S phase. Activation of SIRT1 on glucose deprivation leads to deacetylation of K633, enhanced pRNA binding and an increase in heterochromatic histone marks. These results suggest a mechanism that! links the epigenetic state of rDNA to cell metabolism and reveal another layer of epigenetic control that involves post-translational modification of a chromatin remodelling complex.
• The CDK4–pRB–E2F1 pathway controls insulin secretion
  - Nat Cell Biol 11(8):1017-1023 (2009)
  CDK4–pRB–E2F1 cell-cycle regulators are robustly expressed in non-proliferating cells, suggesting that besides the control of -cell number the CDK4–pRB–E2F1 pathway has a role in -cell function. We show here that E2F1 directly regulates expression of Kir6.2, which is a key component of the KATP channel involved in the regulation of glucose-induced insulin secretion. We demonstrate, through chromatin immunoprecipitation analysis from tissues, that Kir6.2 expression is regulated at the promoter level by the CDK4–pRB–E2F1 pathway. Consistently, inhibition of CDK4, or genetic inactivation of E2F1, results in decreased expression of Kir6.2, impaired insulin secretion and glucose intolerance in mice. Furthermore we show that rescue of Kir6.2 expression restores insulin secretion in E2f1-/- cells. Finally, we demonstrate that CDK4 is activated by glucose through the insulin pathway, ultimately resulting in E2F1 activation and, consequently, increased expression o! f Kir6.2. In summary we provide evidence that the CDK4–pRB–E2F1 regulatory pathway is involved in glucose homeostasis, defining a new link between cell proliferation and metabolism.
• Axonal elongation triggered by stimulus-induced local translation of a polarity complex protein
  - Nat Cell Biol 11(8):1024-1030 (2009)
  During development, axon growth rates are precisely regulated to provide temporal control over pathfinding1, 2. The precise temporal regulation of axonal growth is a key step in the formation of functional synapses and the proper patterning of the nervous system. The rate of axonal elongation is increased by factors such as netrin-1 and nerve growth factor (NGF), which stimulate axon outgrowth using incompletely defined pathways. To clarify the mechanism of netrin-1- and NGF-stimulated axon growth, we explored the role of local protein translation. We found that intra-axonal protein translation is required for stimulated, but not basal, axon outgrowth. To identify the mechanism of translation-dependent outgrowth, we examined the PAR complex, a cytoskeleton regulator3. We found that the PAR complex, like local translation, is required for stimulated, but not basal, outgrowth. Par3 mRNA is localized to developing axons, and NGF and netrin-1 trigger its local translation.! Selective ablation of Par3 mRNA from axons abolishes the outgrowth-promoting effect of NGF. These results identify a new role for local translation and the PAR complex in axonal outgrowth.
• MicroRNA MiR-17 retards tissue growth and represses fibronectin expression
  - Nat Cell Biol 11(8):1031-1038 (2009)
  MicroRNAs (miRNAs) are single-stranded regulatory RNAs, frequently expressed as clusters. Previous studies have demonstrated that the six-miRNA cluster miR-1792 has important roles in tissue development and cancers. However, the precise role of each miRNA in the cluster is unknown. Here we show that overexpression of miR-17 results in decreased cell adhesion, migration and proliferation. Transgenic mice overexpressing miR-17 showed overall growth retardation, smaller organs and greatly reduced haematopoietic cell lineages. We found that fibronectin and the fibronectin type-III domain containing 3A (FNDC3A) are two targets that have their expression repressed by miR-17, both in vitro and in transgenic mice. Several lines of evidence support the notion that miR-17 causes cellular defects through its repression of fibronectin expression. Our single miRNA expression assay may be evolved to allow the manipulation of individual miRNA functions in vitro and in vivo. We antici! pate that this could serve as a model for studying gene regulation by miRNAs in the development of gene therapy.
• Differential requirements for actin during yeast and mammalian endocytosis
  - Nat Cell Biol 11(8):1039-1042 (2009)
  Key features of clathrin-mediated endocytosis have been conserved across evolution. However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis. We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure. By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis. Conversely, increased turgor pressure in specific yeast mutants correlated with a decreased rate of endocytic patch invagination.
• Corrigendum
  - Nat Cell Biol 11(8):1042 (2009)
  Introduction In the letter by Tian et al. (Nature Cell Biology 11; 211–218, 2009), the reported sequence of the synthetic 16mer phosphopeptide used to generate the phospho-specific antibody against ATM Ser794 in the Methods section 'Generation of anti-phospho-Ser794 ATM antibody' contained an error. The correct sequence should be acetyl-CLSNATKKpSPNKIASG-amide.
• Epidermal growth factor-like domain 7 (EGFL7) modulates Notch signalling and affects neural stem cell renewal
  - Nat Cell Biol 11(8):1043 (2009)
  Introduction In the version of this article initially published online, the labelling of the boxes in Fig. 1b was incorrect. In Fig. 1d the mNotch 2 values were missing, in Fig. 1g the Anti-EGFL7 was incorrectly labelled. In Fig 2c there was an extra '+' sign. In Fig 3d the title was incorrect. In Fig 5b the BSA label was missing from under the fifth bar. The correct versions of these figures are shown below. These errors have been corrected in the HTML and PDF versions of the article