Hot off the presses! Aug 21 Cell

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Latest Articles Include:

• In This Issue
  - Cell 138(4):607, 609 (2009)
  
• Philosophies of Funding
  - Cell 138(4):611-615 (2009)
  Successful scientific practice encompasses broader and more varied modes of investigation than can be captured by focusing on hypothesis-driven research. We examine the emphases that major US and UK funding agencies place on particular modes of research practice and suggest that funding agency guidelines should be informed by a more dynamic and multidimensional account of scientific practice.
• iPS Cells: Insights into Basic Biology
  - Cell 138(4):616-618 (2009)
  The discovery that adult somatic cells can be induced to become pluripotent by overexpression of a few key transcription factors provides an exciting new window into the basic biology of pluripotency and differentiation.
• Only Two Ways to Achieve Perfection
  - Cell 138(4):619-621 (2009)
  The functional repertoire of a network is determined by its topology. Ma et al. (2009) analyze enzyme networks with three nodes and take a reverse-engineering approach to ask how many core network topologies can establish perfect adaptation, the ability to reset after perturbation. Surprisingly, the answer is just two.
• Clamping Down on Transposon Targeting
  - Cell 138(4):621-623 (2009)
  The sliding β clamp subunit of the DNA replication machinery in the bacterium Escherichia coli coordinates multiple functions in the cell beyond genome duplication. In this issue, Parks et al. (2009) find that the β clamp interacts with the transposition protein TnsE to target the Tn7 transposon to discontinuously replicating DNA at the replication fork.
• Breast Cancer Stem Cells: Eradication by Differentiation Therapy?
  - Cell 138(4):623-625 (2009)
  During metastasis, migrating breast cancer stem cells undergo a loss of polarity leading to an epithelial-to-mesenchymal transition (EMT). Gupta et al. (2009) use this attribute of cancer stem cells to develop a high-throughput screen, which successfully identifies small molecules that specifically inhibit cancer stem cell proliferation through the induction of differentiation.
• Plant Phase Transitions Make a SPLash
  - Cell 138(4):625-627 (2009)
  During post-embryonic development, plants undergo a series of phase transitions, from juvenile to adult and from the vegetative to the reproductive phase. Recent findings reported in Cell ([Wang et al., 2009] and [Wu et al., 2009]) and Developmental Cell (Yamaguchi et al., 2009) reveal how microRNAs and their transcription factor targets coordinate these phase transitions.
• Importing Mitochondrial Proteins: Machineries and Mechanisms
  - Cell 138(4):628-644 (2009)
  Most mitochondrial proteins are synthesized on cytosolic ribosomes and must be imported across one or both mitochondrial membranes. There is an amazingly versatile set of machineries and mechanisms, and at least four different pathways, for the importing and sorting of mitochondrial precursor proteins. The translocases that catalyze these processes are highly dynamic machines driven by the membrane potential, ATP, or redox reactions, and they cooperate with molecular chaperones and assembly complexes to direct mitochondrial proteins to their correct destinations. Here, we discuss recent insights into the importing and sorting of mitochondrial proteins and their contributions to mitochondrial biogenesis.
• Identification of Selective Inhibitors of Cancer Stem Cells by High-Throughput Screening
  - Cell 138(4):645-659 (2009)
  Screens for agents that specifically kill epithelial cancer stem cells (CSCs) have not been possible due to the rarity of these cells within tumor cell populations and their relative instability in culture. We describe here an approach to screening for agents with epithelial CSC-specific toxicity. We implemented this method in a chemical screen and discovered compounds showing selective toxicity for breast CSCs. One compound, salinomycin, reduces the proportion of CSCs by >100-fold relative to paclitaxel, a commonly used breast cancer chemotherapeutic drug. Treatment of mice with salinomycin inhibits mammary tumor growth in vivo and induces increased epithelial differentiation of tumor cells. In addition, global gene expression analyses show that salinomycin treatment results in the loss of expression of breast CSC genes previously identified by analyses of breast tissues isolated directly from patients. This study demonstrates the ability to identify agents with speci! fic toxicity for epithelial CSCs.
• LSD1 Is a Subunit of the NuRD Complex and Targets the Metastasis Programs in Breast Cancer
  - Cell 138(4):660-672 (2009)
  Lysine-specific demethylase 1 (LSD1) exerts pathway-specific activity in animal development and has been linked to several high-risk cancers. Here, we report that LSD1 is an integral component of the Mi-2/nucleosome remodeling and deacetylase (NuRD) complex. Transcriptional target analysis revealed that the LSD1/NuRD complexes regulate several cellular signaling pathways including TGFβ1 signaling pathway that are critically involved in cell proliferation, survival, and epithelial-to-mesenchymal transition. We demonstrated that LSD1 inhibits the invasion of breast cancer cells in vitro and suppresses breast cancer metastatic potential in vivo. We found that LSD1 is downregulated in breast carcinomas and that its level of expression is negatively correlated with that of TGFβ1. Our data provide a molecular basis for the interplay of histone demethylation and deacetylation in chromatin remodeling. By enlisting LSD1, the NuRD complex expands its chromatin remodeling capac! ity to include ATPase, histone deacetylase, and histone demethylase.
• Widespread Shortening of 3′UTRs by Alternative Cleavage and Polyadenylation Activates Oncogenes in Cancer Cells
  - Cell 138(4):673-684 (2009)
  In cancer cells, genetic alterations can activate proto-oncogenes, thereby contributing to tumorigenesis. However, the protein products of oncogenes are sometimes overexpressed without alteration of the proto-oncogene. Helping to explain this phenomenon, we found that when compared to similarly proliferating nontransformed cell lines, cancer cell lines often expressed substantial amounts of mRNA isoforms with shorter 3′ untranslated regions (UTRs). These shorter isoforms usually resulted from alternative cleavage and polyadenylation (APA). The APA had functional consequences, with the shorter mRNA isoforms exhibiting increased stability and typically producing ten-fold more protein, in part through the loss of microRNA-mediated repression. Moreover, expression of the shorter mRNA isoform of the proto-oncogene IGF2BP1/IMP-1 led to far more oncogenic transformation than did expression of the full-length, annotated mRNA. The high incidence of APA in cancer cells, with c! onsequent loss of 3′UTR repressive elements, suggests a pervasive role for APA in oncogene activation without genetic alteration.
• Transposition into Replicating DNA Occurs through Interaction with the Processivity Factor
  - Cell 138(4):685-695 (2009)
  The bacterial transposon Tn7 directs transposition into actively replicating DNA by a mechanism involving the transposon-encoded protein TnsE. Here we show that TnsE physically and functionally interacts with the processivity factor of the DNA replication machinery in vivo and in vitro. Our work establishes an in vitro TnsABC+E transposition reaction reconstituted from purified proteins and target DNA structures. Using the in vitro reaction we confirm that the processivity factor specifically reorders TnsE-mediated transposition events on target DNAs in a way that matches the bias with active DNA replication in vivo. The TnsE interaction with an essential and conserved component of the replication machinery, and a DNA structure reveals a mechanism by which Tn7, and probably other elements, selects target sites associated with DNA replication.
• TUT4 in Concert with Lin28 Suppresses MicroRNA Biogenesis through Pre-MicroRNA Uridylation
  - Cell 138(4):696-708 (2009)
  As key regulators in cellular functions, microRNAs (miRNAs) themselves need to be tightly controlled. Lin28, a pluripotency factor, was reported to downregulate let-7 miRNA by inducing uridylation of let-7 precursor (pre-let-7). But the enzyme responsible for the uridylation remained unknown. Here we identify a noncanonical poly (A) polymerase, TUTase4 (TUT4), as the uridylyl transferase for pre-let-7. Lin28 recruits TUT4 to pre-let-7 by recognizing a tetra-nucleotide sequence motif (GGAG) in the terminal loop. TUT4 in turn adds an oligouridine tail to the pre-let-7, which blocks Dicer processing. Other miRNAs with the same sequence motif (miR-107, -143, and -200c) are regulated through the same mechanism. Knockdown of TUT4 and Lin28 reduces the level of stem cell markers, suggesting that they are required for stem cell maintenance. This study uncovers the role of TUT4 and Lin28 as specific suppressors of miRNA biogenesis, which has implications for stem cell research ! and cancer biology.
• Synaptotagmin-Mediated Bending of the Target Membrane Is a Critical Step in Ca2+-Regulated Fusion
  - Cell 138(4):709-721 (2009)
  Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca2+, but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined. Previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending i! s a critical step in membrane fusion.
• Nanog Is the Gateway to the Pluripotent Ground State
  - Cell 138(4):722-737 (2009)
  Pluripotency is generated naturally during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Pluripotency can be recreated by somatic cell reprogramming. Here we present evidence that the homeodomain protein Nanog mediates acquisition of both embryonic and induced pluripotency. Production of pluripotent hybrids by cell fusion is promoted by and dependent on Nanog. In transcription factor-induced molecular reprogramming, Nanog is initially dispensable but becomes essential for dedifferentiated intermediates to transit to ground state pluripotency. In the embryo, Nanog specifically demarcates the nascent epiblast, coincident with the domain of X chromosome reprogramming. Without Nanog, pluripotency does not develop, and the inner cell mass is trapped in a pre-pluripotent, indeterminate state that is ultimately nonviable. These findings suggest that Nanog choreographs synthesis of the naive epiblast ground state in the embryo an! d that this function is recapitulated in the culmination of somatic cell reprogramming.
• miR156-Regulated SPL Transcription Factors Define an Endogenous Flowering Pathway in Arabidopsis thaliana
  - Cell 138(4):738-749 (2009)
  The FT gene integrates several external and endogenous cues controlling flowering, including information on day length. A complex of the mobile FT protein and the bZIP transcription factor FD in turn has a central role in activating genes that execute the switch from vegetative to reproductive development. Here we reveal that microRNA156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes not only act downstream of FT/FD, but also define a separate endogenous flowering pathway. High levels of miR156 in young plants prevent precocious flowering. A subsequent day length-independent decline in miR156 abundance provides a permissive environment for flowering and is paralleled by a rise in SPL levels. At the shoot apex, FT/FD and SPLs converge on an overlapping set of targets, with SPLs directly activating flower-promoting MADS box genes, providing a molecular substrate for both the redundant activities and the feed-forward action of the miR156/SPL and FT/FD modules! in flowering control.
• The Sequential Action of miR156 and miR172 Regulates Developmental Timing in Arabidopsis
  - Cell 138(4):750-759 (2009)
  The transition from the juvenile to the adult phase of shoot development in plants is accompanied by changes in vegetative morphology and an increase in reproductive potential. Here, we describe the regulatory mechanism of this transition. We show that miR156 is necessary and sufficient for the expression of the juvenile phase, and regulates the timing of the juvenile-to-adult transition by coordinating the expression of several pathways that control different aspects of this process. miR156 acts by repressing the expression of functionally distinct SPL transcription factors. miR172 acts downstream of miR156 to promote adult epidermal identity. miR156 regulates the expression of miR172 via SPL9 which, redundantly with SPL10, directly promotes the transcription of miR172b. Thus, like the larval-to-adult transition in Caenorhabditis elegans, the juvenile-to-adult transition in Arabidopsis is mediated by sequentially operating miRNAs. miR156 and miR172 are positively regu! lated by the transcription factors they target, suggesting that negative feedback loops contribute to the stability of the juvenile and adult phases.
• Defining Network Topologies that Can Achieve Biochemical Adaptation
  - Cell 138(4):760-773 (2009)
  Many signaling systems show adaptation—the ability to reset themselves after responding to a stimulus. We computationally searched all possible three-node enzyme network topologies to identify those that could perform adaptation. Only two major core topologies emerge as robust solutions: a negative feedback loop with a buffering node and an incoherent feedforward loop with a proportioner node. Minimal circuits containing these topologies are, within proper regions of parameter space, sufficient to achieve adaptation. More complex circuits that robustly perform adaptation all contain at least one of these topologies at their core. This analysis yields a design table highlighting a finite set of adaptive circuits. Despite the diversity of possible biochemical networks, it may be common to find that only a finite set of core topologies can execute a particular function. These design rules provide a framework for functionally classifying complex natural networks and a ma! nual for engineering networks. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.
• Protein Sectors: Evolutionary Units of Three-Dimensional Structure
  - Cell 138(4):774-786 (2009)
  Proteins display a hierarchy of structural features at primary, secondary, tertiary, and higher-order levels, an organization that guides our current understanding of their biological properties and evolutionary origins. Here, we reveal a structural organization distinct from this traditional hierarchy by statistical analysis of correlated evolution between amino acids. Applied to the S1A serine proteases, the analysis indicates a decomposition of the protein into three quasi-independent groups of correlated amino acids that we term "protein sectors." Each sector is physically connected in the tertiary structure, has a distinct functional role, and constitutes an independent mode of sequence divergence in the protein family. Functionally relevant sectors are evident in other protein families as well, suggesting that they may be general features of proteins. We propose that sectors represent a structural organization of proteins that reflects their evolutionary hist! ories.
• The Localization of the Golgin GCC185 Is Independent of Rab6A/A' and Arl1
  - Cell 138(4):787-794 (2009)
  Mammalian golgins of the trans-Golgi network (TGN) are small G protein effectors that are required for membrane transport and contain a Golgi targeting C-terminal GRIP domain. The localization of two TGN golgins, p230/golgin-245 and golgin-97, is mediated by the small GTPase Arl1, whereas recruitment of the TGN golgin GCC185 is controversial. Recently, GCC185 was proposed to localize to the Golgi by the co-operation of two small GTPases, Rab6A/A' and Arl1 (Burguete et al., 2008), a model based predominantly on in vitro interactions. Here we demonstrate that Golgi recruitment of endogenous GCC185 does not involve Rab6A/A' and Arl1. We find minimal colocalization between Rab6A/A' and endogenous GCC185 on Golgi membranes and failed to detect an interaction between Rab6A/A' and C-terminal domains of GCC185 by yeast two-hybrid analyses. Moreover, depletion of both Rab6A/A' and Arl1 also had no effect on the localization of endogenous GCC185 or the isolated GRIP domain of GC! C185.
• Full Dynamic Range Proteome Analysis of S. cerevisiae by Targeted Proteomics
  - Cell 138(4):795-806 (2009)
  The rise of systems biology implied a growing demand for highly sensitive techniques for the fast and consistent detection and quantification of target sets of proteins across multiple samples. This is only partly achieved by classical mass spectrometry or affinity-based methods. We applied a targeted proteomics approach based on selected reaction monitoring (SRM) to detect and quantify proteins expressed to a concentration below 50 copies/cell in total S. cerevisiae digests. The detection range can be extended to single-digit copies/cell and to proteins undetected by classical methods. We illustrate the power of the technique by the consistent and fast measurement of a network of proteins spanning the entire abundance range over a growth time course of S. cerevisiae transiting through a series of metabolic phases. We therefore demonstrate the potential of SRM-based proteomics to provide assays for the measurement of any set of proteins of interest in yeast at high-thr! oughput and quantitative accuracy.
• Principles of Cancer Therapy: Oncogene and Non-oncogene Addiction
  - Cell 138(4):807 (2009)
  
• SnapShot: Import and Sortingof Mitochondrial Proteins
  - Cell 138(4):808-808.e1 (2009)