Hot off the presses! May 01 Cell

The May 01 issue of the Cell is now up on Pubget (About Cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• In This Issue
  - Cell 137(3):383, 385 (2009)
  
• Neurobiology Select
  - Cell 137(3):387, 389 (2009)
  Sleep is one of the most fundamental, yet least understood, facets of animal physiology—its universality reflected in the saying, "life is one long process of getting tired." This issue's Neurobiology Select describes the latest progress toward understanding sleep, including its role in maintaining synaptic homeostasis, its role in long-term memory formation, and the causes and consequences of sleep disruption.
• Commonality but Diversity in Cancer Gene Fusions
  - Cell 137(3):391-395 (2009)
  Recent findings of gene fusions in carcinomas recapitulate the discovery of chromosomal abnormalities in leukemias and sarcomas decades ago. A recurring feature of carcinoma gene fusions, in contrast to those in hematopoietic and mesenchymal malignancies, is that they result in aberrant cell signaling. This may reflect differences in the differentiation programs of these tissues.
• Rfu1: Stimulus for the Ubiquitin Economy
  - Cell 137(3):397-398 (2009)
  During cellular stress, monoubiquitin is in demand due to the accumulation of misfolded proteins that require proteasomal degradation. Kimura et al. (2009) now show in yeast that monoubiquitin levels are bolstered during stress conditions by downregulation of the protein Rfu1, an inhibitor of the deubiquitinating enzyme Doa4.
• Specifying Mouse Embryonic Germ Cells
  - Cell 137(3):398-400 (2009)
  Male germ cells are induced to form from the epiblast of the mouse embryo by a combination of WNT and bone morphogenetic protein signals. Ohinata et al. (2009) now clarify the steps of mouse germ cell formation and use this genetic insight to direct the specification and differentiation of germline progenitor cells in vitro.
• Opening Windows to the Genome
  - Cell 137(3):400-402 (2009)
  Recent mapping of nucleosome positioning has added a new dimension to the study of transcriptional regulation. Hartley and Madhani (2009) now demonstrate the power of this approach and show that a chromatin regulator alters nucleosome positioning in the promoters of a large number of genes in the budding yeast Saccharomyces cerevisiae.
• HIV Entry Revisited
  - Cell 137(3):402-404 (2009)
  HIV has long served as a model for viruses that enter cells by direct fusion at the plasma membrane. Miyauchi et al. (2009) now provide compelling evidence that HIV enters cells primarily by endocytosis.
• Ure(k)a! Sirtuins Regulate Mitochondria
  - Cell 137(3):404-406 (2009)
  Increasing evidence suggests that multiple metabolic pathways are regulated by sirtuin-dependent protein deacetylation in the mitochondria. In this issue, Nakagawa et al. (2009) show that the sirtuin SIRT5 deacetylates and activates a mitochondrial enzyme, carbamoyl phosphate synthetase 1, which mediates the first step in the urea cycle.
• Frodos Found: Behold the CENP-A "Ring" Bearers
  - Cell 137(3):409-412 (2009)
  CENP-A is a histone H3-like protein specific to centromeres that is essential for kinetochore formation and accurate chromosome segregation in eukaryotes. Recent studies ([Dunleavy et al., 2009], [Foltz et al., 2009], [Perpelescu et al., 2009], [Pidoux et al., 2009] and [Williams et al., 2009]) analyze CENP-A binding proteins required for the recruitment of CENP-A to centromeres in humans and in fission yeast, bringing us closer to understanding how centromere identity is faithfully propagated.
• Blinded by the Light: The Growing Complexity of p53
  - Cell 137(3):413-431 (2009)
  While the tumor suppressor functions of p53 have long been recognized, the contribution of p53 to numerous other aspects of disease and normal life is only now being appreciated. This burgeoning range of responses to p53 is reflected by an increasing variety of mechanisms through which p53 can function, although the ability to activate transcription remains key to p53's modus operandi. Control of p53's transcriptional activity is crucial for determining which p53 response is activated, a decision we must understand if we are to exploit efficiently the next generation of drugs that selectively activate or inhibit p53.
• HIV Enters Cells via Endocytosis and Dynamin-Dependent Fusion with Endosomes
  - Cell 137(3):433-444 (2009)
  Enveloped viruses that rely on a low pH-dependent step for entry initiate infection by fusing with acidic endosomes, whereas the entry sites for pH-independent viruses, such as HIV-1, have not been defined. These viruses have long been assumed to fuse directly with the plasma membrane. Here we used population-based measurements of the viral content delivery into the cytosol and time-resolved imaging of single viruses to demonstrate that complete HIV-1 fusion occurred in endosomes. In contrast, viral fusion with the plasma membrane did not progress beyond the lipid mixing step. HIV-1 underwent receptor-mediated internalization long before endosomal fusion, thus minimizing the surface exposure of conserved viral epitopes during fusion and reducing the efficacy of inhibitors targeting these epitopes. We also show that, strikingly, endosomal fusion is sensitive to a dynamin inhibitor, dynasore. These findings imply that HIV-1 infects cells via endocytosis and envelope glyc! oprotein- and dynamin-dependent fusion with intracellular compartments.
• Mechanisms that Specify Promoter Nucleosome Location and Identity
  - Cell 137(3):445-458 (2009)
  The chromatin architecture of eukaryotic gene promoters is generally characterized by a nucleosome-free region (NFR) flanked by at least one H2A.Z variant nucleosome. Computational predictions of nucleosome positions based on thermodynamic properties of DNA-histone interactions have met with limited success. Here we show that the action of the essential RSC remodeling complex in S. cerevisiae helps explain the discrepancy between theory and experiment. In RSC-depleted cells, NFRs shrink such that the average positions of flanking nucleosomes move toward predicted sites. Nucleosome positioning at distinct subsets of promoters additionally requires the essential Myb family proteins Abf1 and Reb1, whose binding sites are enriched in NFRs. In contrast, H2A.Z deposition is dispensable for nucleosome positioning. By regulating H2A.Z deposition using a steroid-inducible protein splicing strategy, we show that NFR establishment is necessary for H2A.Z deposition. These studies ! suggest an ordered pathway for the assembly of promoter chromatin architecture.
• RAD6-Mediated Transcription-Coupled H2B Ubiquitylation Directly Stimulates H3K4 Methylation in Human Cells
  - Cell 137(3):459-471 (2009)
  H2B ubiquitylation has been implicated in active transcription but is not well understood in mammalian cells. Beyond earlier identification of hBRE1 as the E3 ligase for H2B ubiquitylation in human cells, we now show (1) that hRAD6 serves as the cognate E2-conjugating enzyme; (2) that hRAD6, through direct interaction with hPAF-bound hBRE1, is recruited to transcribed genes and ubiquitylates chromatinized H2B at lysine 120; (3) that hPAF-mediated transcription is required for efficient H2B ubiquitylation as a result of hPAF-dependent recruitment of hBRE1-hRAD6 to the Pol II transcription machinery; (4) that H2B ubiquitylation per se does not affect the level of hPAF-, SII-, and p300-dependent transcription and likely functions downstream; and (5) that H2B ubiquitylation directly stimulates hSET1-dependent H3K4 di- and trimethylation. These studies establish the natural H2B ubiquitylation factors in human cells and also detail the mechanistic basis for H2B ubiquitylatio! n and function during transcription.
• Centromere-Specific Assembly of CENP-A Nucleosomes Is Mediated by HJURP
  - Cell 137(3):472-484 (2009)
  The centromere is responsible for accurate chromosome segregation. Mammalian centromeres are specified epigenetically, with all active centromeres containing centromere-specific chromatin in which CENP-A replaces histone H3 within the nucleosome. The proteins responsible for assembly of human CENP-A into centromeric nucleosomes during the G1 phase of the cell cycle are shown here to be distinct from the chromatin assembly factors previously shown to load other histone H3 variants. Here we demonstrate that prenucleosomal CENP-A is complexed with histone H4, nucleophosmin 1, and HJURP. Recruitment of new CENP-A into nucleosomes at replicated centromeres is dependent on HJURP. Recognition by HJURP is mediated through the centromere targeting domain (CATD) of CENP-A, a region that we demonstrated previously to induce a unique conformational rigidity to both the subnucleosomal CENP-A heterotetramer and the corresponding assembled nucleosome. We propose HJURP to be a cell-cy! cle-regulated CENP-A-specific histone chaperone required for centromeric chromatin assembly.
• HJURP Is a Cell-Cycle-Dependent Maintenance and Deposition Factor of CENP-A at Centromeres
  - Cell 137(3):485-497 (2009)
  The histone H3 variant CenH3, called CENP-A in humans, is central in centromeric chromatin to ensure proper chromosome segregation. In the absence of an underlying DNA sequence, it is still unclear how CENP-A deposition at centromeres is determined. Here, we purified non-nucleosomal CENP-A complexes to identify direct CENP-A partners involved in such a mechanism and identified HJURP. HJURP was not detected in H3.1- or H3.3-containing complexes, indicating its specificity for CENP-A. HJURP centromeric localization is cell cycle regulated, and its transient appearance at the centromere coincides precisely with the proposed time window for new CENP-A deposition. Furthermore, HJURP downregulation leads to a major reduction in CENP-A at centromeres and impairs deposition of newly synthesized CENP-A, causing mitotic defects. We conclude that HJURP is a key factor for CENP-A deposition and maintenance at centromeres.
• An Effector of RNA-Directed DNA Methylation in Arabidopsis Is an ARGONAUTE 4- and RNA-Binding Protein
  - Cell 137(3):498-508 (2009)
  DNA methylation is a conserved epigenetic mark in plants and mammals. In Arabidopsis, DNA methylation can be triggered by small interfering RNAs (siRNAs) through an RNA-directed DNA methylation (RdDM) pathway. Here, we report the identification of an RdDM effector, KTF1. Loss-of-function mutations in KTF1 reduce DNA methylation and release the silencing of RdDM target loci without abolishing the siRNA triggers. KTF1 has similarity to the transcription elongation factor SPT5 and contains a C-terminal extension rich in GW/WG repeats. KTF1 colocalizes with ARGONAUTE 4 (AGO4) in punctate nuclear foci and binds AGO4 and RNA transcripts. Our results suggest KTF1 as an adaptor protein that binds scaffold transcripts generated by Pol V and recruits AGO4 and AGO4-bound siRNAs to form an RdDM effector complex. The dual interaction of an effector protein with AGO and small RNA target transcripts may be a general feature of RNA-silencing effector complexes.
• Collapse of Germline piRNAs in the Absence of Argonaute3 Reveals Somatic piRNAs in Flies
  - Cell 137(3):509-521 (2009)
  Piwi-interacting RNAs (piRNAs) silence transposons in animal germ cells. piRNAs are thought to derive from long transcripts spanning transposon-rich genomic loci and to direct an autoamplification loop in which an antisense piRNA, bound to Aubergine or Piwi protein, triggers production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to produce a second antisense piRNA. Here, we describe strong loss-of-function mutations in ago3, allowing a direct genetic test of this model. We find that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. We also detect a second, Ago3-independent piRNA pathway centered on Piwi. Transposons targeted by this second pathway often reside in the flamenco locus, which is expressed in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germline.
• Specialized piRNA Pathways Act in Germline and Somatic Tissues of the Drosophila Ovary
  - Cell 137(3):522-535 (2009)
  In Drosophila gonads, Piwi proteins and associated piRNAs collaborate with additional factors to form a small RNA-based immune system that silences mobile elements. Here, we analyzed nine Drosophila piRNA pathway mutants for their impacts on both small RNA populations and the subcellular localization patterns of Piwi proteins. We find that distinct piRNA pathways with differing components function in ovarian germ and somatic cells. In the soma, Piwi acts singularly with the conserved flamenco piRNA cluster to enforce silencing of retroviral elements that may propagate by infecting neighboring germ cells. In the germline, silencing programs encoded within piRNA clusters are optimized via a slicer-dependent amplification loop to suppress a broad spectrum of elements. The classes of transposons targeted by germline and somatic piRNA clusters, though not the precise elements, are conserved among Drosophilids, demonstrating that the architecture of piRNA clusters has coevol! ved with the transposons that they are tasked to control.
• Disassembly of Exon Junction Complexes by PYM
  - Cell 137(3):536-548 (2009)
  Exon junction complexes (EJCs) are deposited onto mRNAs during splicing, serve as positional landmarks for the intron exon structure of genes, and direct posttranscriptional processes in the cytoplasm. EJC removal and recycling by translation are ill understood and have been attributed to ribosomal passage. This work identifies the ribosome-associated protein PYM as an EJC disassembly factor and defines its mechanism of function. Whereas EJC assembly intermediates are resistant to PYM, fully assembled EJCs are dissociated from spliced mRNAs by PYM. This disassembly involves PYM binding to the EJC proteins MAGOH-Y14. PYM overexpression in cells disrupts EJC association with spliced mRNA and inhibits nonsense-mediated mRNA decay. In cells depleted of PYM, EJCs accumulate on spliced mRNAs and EJC protein recycling is impaired. Hence, PYM is an EJC disassembly factor that acts both in vitro and in living cells, and that antagonizes important EJC functions.
• An Inhibitor of a Deubiquitinating Enzyme Regulates Ubiquitin Homeostasis
  - Cell 137(3):549-559 (2009)
  The dynamic and reversible process of ubiquitin modification controls various cellular activities. Ubiquitin exists as monomers, unanchored chains, or protein-conjugated forms, but the regulation of these interconversions remains largely unknown. Here, we identified a protein designated Rfu1 (regulator of free ubiquitin chains 1), which regulates intracellular concentrations of monomeric ubiquitins and free ubiquitin chains in Saccharomyces cerevisiae. Rfu1 functions as an inhibitor of Doa4, a deubiquitinating enzyme. Rapid loss of free ubiquitin chains upon heat shock, a condition in which more proteins require ubiquitin conjugation, was mediated in part by Doa4 and Rfu1. Thus, regulation of ubiquitin homeostasis is controlled by a balance between a deubiquitinating enzyme and its inhibitor. We propose that free ubiquitin chains function as a ubiquitin reservoir that allows maintenance of monomeric ubiquitins at adequate levels under normal conditions and rapid supply! for substrate conjugation under stress conditions.
• SIRT5 Deacetylates Carbamoyl Phosphate Synthetase 1 and Regulates the Urea Cycle
  - Cell 137(3):560-570 (2009)
  Sirtuins are NAD-dependent protein deacetylases that connect metabolism and aging. In mammals, there are seven sirtuins (SIRT1-7), three of which are associated with mitochondria. Here, we show that SIRT5 localizes in the mitochondrial matrix and interacts with carbamoyl phosphate synthetase 1 (CPS1), an enzyme, catalyzing the initial step of the urea cycle for ammonia detoxification and disposal. SIRT5 deacetylates CPS1 and upregulates its activity. During fasting, NAD in liver mitochondria increases, thereby triggering SIRT5 deacetylation of CPS1 and adaptation to the increase in amino acid catabolism. Indeed, SIRT5 KO mice fail to upregulate CPS1 activity and show elevated blood ammonia during fasting. Similar effects occur during long-term calorie restriction or a high protein diet. These findings demonstrate SIRT5 plays a pivotal role in ammonia detoxification and disposal by activating CPS1.
• A Signaling Principle for the Specification of the Germ Cell Lineage in Mice
  - Cell 137(3):571-584 (2009)
  Specification of the germ cell lineage is vital to development and heredity. In mice, the germ cell fate is induced in pluripotent epiblast cells by signaling molecules, yet the underlying mechanism remains unknown. Here we demonstrate that germ cell fate in the epiblast is a direct consequence of Bmp4 signaling from the extraembryonic ectoderm (ExE), which is antagonized by the anterior visceral endoderm (AVE). Strikingly, Bmp8b from the ExE restricts AVE development, thereby contributing to Bmp4 signaling. Furthermore, Wnt3 in the epiblast ensures its responsiveness to Bmp4. Serum-free, defined cultures revealed that, in response to Bmp4, competent epiblast cells uniformly expressed key transcriptional regulators Blimp1 and Prdm14 and acquired germ-cell properties, including genome-wide epigenetic reprogramming, in an orderly fashion. Notably, the induced cells contributed to both spermatogenesis and fertility of offspring. By identifying a signaling principle in ger! m cell specification, our study establishes a robust strategy for reconstituting the mammalian germ cell lineage in vitro.
• SnapShot: MicroRNAs in Cancer
  - Cell 137(3):586-586.e1 (2009)