ALBERT

Description: Publication date: Available online 7 September 2013 Source: FEBS Open Bio Author(s): Veronika Temml , Susanne Kuehnl , Daniela Schuster , Stefan Schwaiger , Hermann Stuppner , Dietmar Fuchs Mediterranean Carthamus tinctorius (Safflower) is used for treatment of inflammatory conditions and neuropsychiatric disorders. Recently C. tinctorius lignans arctigenin and trachelogenin but not matairesinol were described to interfere with the activity of tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) in peripheral blood mononuclear cells in vitro . We examined a potential direct influence of compounds on IDO enzyme activity applying computational calculations based on 3D geometry of the compounds. The interaction pattern analysis and force field-based minimization was performed within LigandScout 3.03, the docking simulation with MOE 2011.10 using the X-ray crystal structure of IDO. Results confirm the possibility of an intense interaction of arctigenin and trachelogenin with the binding site of the enzyme, while matairesinol had no such effect.

Description: Publication date: Available online 7 September 2013 Source: FEBS Open Bio Author(s): Mitsuru Ishikawa , Jun Shiota , Yuta Ishibashi , Tomoyuki Hakamata , Shizuku Shoji , Mamoru Fukuchi , Masaaki Tsuda , Tomoaki Shirao , Yuko Sekino , Toshihisa Ohtsuka , Jay M. Baraban , Akiko Tabuchi Megakaryoblastic leukemia 1 (MKL1) is a member of the MKL family of serum response factor (SRF) coactivators. Here we have identified three rat MKL1 transcripts: two are homologues of mouse MKL1 transcripts, full-length MKL1 (FLMKL1) and basic, SAP, and coiled-coil domains (BSAC), the third is a novel transcript, M KL1- elo ngated d erivative of y ield (MELODY). These rat MKL1 transcripts are differentially expressed in a wide variety of tissues with highest levels in testis and brain. During brain development, these transcripts display differential patterns of expression. The FLMKL1 transcript encodes two isoforms that utilize distinct translation start sites. The longer form possesses three actin-binding RPXXXEL (RPEL) motifs and the shorter form, MKL1met only has two RPEL motifs. All four rat MKL1 isoforms, FLMKL1, BSAC, MKL1met and MELODY increased SRF-mediated transcription, but not CREB-mediated transcription. Accordingly, the differential expression of MKL1 isoforms may help fine-tune gene expression during brain development.

Description: Publication date: Available online 11 September 2013 Source: FEBS Open Bio Author(s): Gisele Castro , Maria Fernanda C. Areias , Lais Weissmann , Paula G.F. Quaresma , Carlos K. Katashima , Mario J.A. Saad , Patricia O. Prada Insulin acts in the hypothalamus, decreasing food intake (FI) by the IR/PI3K/Akt pathway. This pathway is impaired in obese animals and endoplasmic reticulum (ER) stress and low-grade inflammation are possible mechanisms involved in this impairment. Here, we highlighted the amygdala as an important brain region for FI regulation in response to insulin. This regulation was dependent on PI3K/AKT pathway similar to the hypothalamus. Insulin was able to decrease neuropeptide Y (NPY) and increase oxytocin mRNA levels in the amygdala via PI3K, which may contribute to hypophagia. Additionally, obese rats did not reduce FI in response to insulin and AKT phosphorylation was decreased in the amygdala, suggesting insulin resistance. Insulin resistance was associated with ER stress and low-grade inflammation in this brain region. The inhibition of ER stress with PBA reverses insulin action/signaling, decreases NPY and increases oxytocin mRNA levels in the amygdala from obese rats, suggesting that ER stress is probably one of the mechanisms that induce insulin resistance in the amygdala.

Description: Publication date: Available online 12 September 2013 Source: Cell Reports Author(s): Debra A. Mayes , Tilat A. Rizvi , Haley Titus-Mitchell , Rachel Oberst , Georgianne M. Ciraolo , Charles V. Vorhees , Andrew P. Robinson , Stephen D. Miller , Jose A. Cancelas , Anat O. Stemmer-Rachamimov , Nancy Ratner Patients with neurofibromatosis type 1 (NF1) and Costello syndrome Rasopathy have behavioral deficits. In NF1 patients, these may correlate with white matter enlargement and aberrant myelin. To model these features, we induced Nf1 loss or HRas hyperactivation in mouse oligodendrocytes. Enlarged brain white matter tracts correlated with myelin decompaction, downregulation of claudin-11, and mislocalization of connexin-32. Surprisingly, non-cell-autonomous defects in perivascular astrocytes and the blood-brain barrier (BBB) developed, implicating a soluble mediator. Nitric oxide (NO) can disrupt tight junctions and gap junctions, and NO and NO synthases (NOS1–NOS3) were upregulated in mutant white matter. Treating mice with the NOS inhibitor NG-nitro-L-arginine methyl ester or the antioxidant N-acetyl cysteine corrected cellular phenotypes. CNP-HRasG12V mice also displayed locomotor hyperactivity, which could be rescued by antioxidant treatment. We conclude that Nf1/Ras regulates oligodendrocyte NOS and that dysregulated NO signaling in oligodendrocytes can alter the surrounding vasculature. The data suggest that antioxidants may improve some behavioral deficits in Rasopathy patients. Graphical abstract Teaser In this study, Ratner and colleagues show that altering intracellular signaling in oligodendrocytes affects brain astrocytes and blood vessels that together make up the blood-brain barrier. Increasing oligodendrocyte Ras-GTP, mimicking neurofibromatosis type 1 and Costello syndrome, disrupted astrocyte and endothelial cell tight junctions and gap junctions and caused a leaky blood-brain barrier. Exposure to a nitric oxide synthase inhibitor or an antioxidant reversed cellular phenotypes and behavioral hyperactivity. Thus, oligodendrocytes contribute to brain homeostasis, and antioxidant therapy may be beneficial when homeostasis is disrupted.

Description: Publication date: Available online 12 September 2013 Source: Cell Reports Author(s): Dalit Ben-Yosef , Francesca S. Boscolo , Hadar Amir , Mira Malcov , Ami Amit , Louise C. Laurent Given the association between mutational load and cancer, the observation that genetic aberrations are frequently found in human pluripotent stem cells (hPSCs) is of concern. Prior studies in human induced pluripotent stem cells (hiPSCs) have shown that deletions and regions of loss of heterozygosity (LOH) tend to arise during reprogramming and early culture, whereas duplications more frequently occur during long-term culture. For the corresponding experiments in human embryonic stem cells (hESCs), we studied two sets of hESC lines: one including the corresponding parental DNA and the other generated from single blastomeres from four sibling embryos. Here, we show that genetic aberrations observed in hESCs can originate during preimplantation embryo development and/or early derivation. These early aberrations are mainly deletions and LOH, whereas aberrations arising during long-term culture of hESCs are more frequently duplications. Our results highlight the importance of close monitoring of genomic integrity and the development of improved methods for derivation and culture of hPSCs. Graphical abstract Teaser Human embryonic stem cells (hESCs) are potential sources of cells for transplantation therapy. However, given the association between mutations and cancer, the frequency of genetic aberrations observed in hESCs is concerning. Using unique pedigrees of hESC lines, Laurent and colleagues now find that aberrations that occur during cell-line derivation are mainly deletions and loss of heterozygosity, whereas duplications arise more commonly during long-term culture. These results highlight the need for improved methods for derivation and culture that preserve the genetic integrity of hESCs.

Description: Publication date: Available online 12 September 2013 Source: Cell Reports Author(s): Jason Karpac , Benoit Biteau , Heinrich Jasper Loss of metabolic homeostasis is a hallmark of aging and is commonly characterized by the deregulation of adaptive signaling interactions that coordinate energy metabolism with dietary changes. The mechanisms driving age-related changes in these adaptive responses remain unclear. Here, we characterize the deregulation of an adaptive metabolic response and the development of metabolic dysfunction in the aging intestine of Drosophila . We find that activation of the insulin-responsive transcription factor Foxo in intestinal enterocytes is required to inhibit the expression of evolutionarily conserved lipases as part of a metabolic response to dietary changes. This adaptive mechanism becomes chronically activated in the aging intestine, mediated by changes in Jun-N-terminal kinase (JNK) signaling. Age-related chronic JNK/Foxo activation in enterocytes is deleterious, leading to sustained repression of intestinal lipase expression and the disruption of lipid homeostasis. Changes in the regulation of Foxo-mediated adaptive responses thus contribute to the age-associated breakdown of metabolic homeostasis. Graphical abstract Teaser Aging is associated with a loss of metabolic homeostasis, which is a risk factor for various human pathologies. Using Drosophila , Karpac, Biteau, and Jasper show that the transcription factor Foxo regulates intestinal lipid homeostasis as part of an adaptive response to dietary changes and that chronic intestinal activation of Foxo with age leads to the disruption of lipid metabolism. These results demonstrate that changes in the regulation of adaptive signaling mechanisms can contribute to the age-associated breakdown of metabolic homeostasis.

Description: Publication date: Available online 17 September 2013 Source: FEBS Open Bio Author(s): Rasheda Sultana , Maria A. Theodoraki , Avrom J. Caplan The UBR1 ubiquitin ligase promotes degradation of proteins via the N-end rule and by another mechanism that detects a misfolded conformation. Although UBR1 was shown recently to act on protein kinases whose misfolding was promoted by inhibition of Hsp90, it was unknown whether this ubiquitin ligase targeted other client types of the chaperone. We analyzed the role of UBR1 in the degradation of nuclear receptors that are classical clients of Hsp90. Our results showed that UBR1 deletion results in impaired degradation of the glucocorticoid receptor and the androgen receptor but not the estrogen receptor α. These findings demonstrate specificity in the actions of the UBR1 ubiquitin ligase in the degradation of Hsp90 clients in the presence of small molecule inhibitors that promote client misfolding. Graphical abstract

Description: Publication date: Available online 19 September 2013 Source: Cell Reports Author(s): George E. Gentsch , Nick D.L. Owens , Stephen R. Martin , Paul Piccinelli , Tiago Faial , Matthew W.B. Trotter , Michael J. Gilchrist , James C. Smith The design of effective cell replacement therapies requires detailed knowledge of how embryonic stem cells form primary tissues, such as mesoderm or neurectoderm that later become skeletal muscle or nervous system. Members of the T-box transcription factor family are key in the formation of these primary tissues, but their underlying molecular activities are poorly understood. Here, we define in vivo genome-wide regulatory inputs of the T-box proteins Brachyury, Eomesodermin, and VegT, which together maintain neuromesodermal stem cells and determine their bipotential fates in frog embryos. These T-box proteins are all recruited to the same genomic recognition sites, from where they activate genes involved in stem cell maintenance and mesoderm formation while repressing neurogenic genes. Consequently, their loss causes embryos to form an oversized neural tube with no mesodermal derivatives. This collaboration between T-box family members thus ensures the continuous formation of correctly proportioned neural and mesodermal tissues in vertebrate embryos during axial elongation. Graphical abstract Teaser The development of effective cell replacement therapies requires detailed knowledge of how embryonic stem cells form primary tissues, such as mesoderm or neurectoderm that later become skeletal muscle or spinal cord. Gentsch, Smith, and colleagues now provide mechanistic insight into how T-box transcription factors regulate stem cells to form neural or mesodermal tissues. The authors show how this ensures the harmonious formation of spinal cord, muscle, and notochord as the vertebrate embryo elongates along its anteroposterior axis.

Description: Publication date: Available online 19 September 2013 Source: Cell Reports Author(s): Ivan Zanoni , Roberto Spreafico , Caterina Bodio , Marco Di Gioia , Clara Cigni , Achille Broggi , Tatiana Gorletta , Michele Caccia , Giuseppe Chirico , Laura Sironi , Maddalena Collini , Mario P. Colombo , Natalio Garbi , Francesca Granucci Natural killer (NK) cells have antitumor, antiviral, and antibacterial functions, and efforts are being made to manipulate them in immunotherapeutic approaches. However, their activation mechanisms remain poorly defined, particularly during bacterial infections. Here, we show that upon lipopolysaccharide or E. coli exposure, dendritic cells (DCs) produce three cytokines—interleukin 2 (IL-2), IL-18, and interferon β (IFN-β)—necessary and sufficient for NK cell activation. IFN-β enhances NK cell activation by inducing IL-15 and IL-15 receptor α not only in DCs but, surprisingly, also in NK cells. This process allows the transfer of IL-15 on NK cell surface and its cis presentation. cis -presented NK cell-derived and trans -presented DC-derived IL-15 contribute equally to optimal NK cell activation. Graphical abstract Teaser NK cells depend on IL-15 provided by accessory cells for their survival under steady-state conditions. It has long been believed that a similar requirement is applied to NK cell activation as well. Zanoni, Granucci, and colleagues now show that NK cells express IL-15 and IL-15Rα when stimulated by type I interferons. NK cells cis -present self-produced IL-15, and this is as important to NK cell activation as trans presentation of IL-15 by dendritic cells.

Description: Publication date: Available online 19 September 2013 Source: Cell Reports Author(s): Oren Ben-Ami , Dan Friedman , Dena Leshkowitz , Dalia Goldenberg , Kira Orlovsky , Niv Pencovich , Joseph Lotem , Amos Tanay , Yoram Groner The t(8;21) and inv(16) chromosomal aberrations generate the oncoproteins AML1-ETO (A-E) and CBFβ-SMMHC (C-S). The role of these oncoproteins in acute myeloid leukemia (AML) etiology has been well studied. Conversely, the function of native RUNX1 in promoting A-E- and C-S-mediated leukemias has remained elusive. We show that wild-type RUNX1 is required for the survival of t(8;21)-Kasumi-1 and inv(16)-ME-1 leukemic cells. RUNX1 knockdown in Kasumi-1 cells (Kasumi-1 RX1-KD ) attenuates the cell-cycle mitotic checkpoint, leading to apoptosis, whereas knockdown of A-E in Kasumi-1 RX1-KD rescues these cells. Mechanistically, a delicate RUNX1/A-E balance involving competition for common genomic sites that regulate RUNX1/A-E targets sustains the malignant cell phenotype. The broad medical significance of this leukemic cell addiction to native RUNX1 is underscored by clinical data showing that an active RUNX1 allele is usually preserved in both t(8;21) or inv(16) AML patients, whereas RUNX1 is frequently inactivated in other forms of leukemia. Thus, RUNX1 and its mitotic control targets are potential candidates for new therapeutic approaches. Graphical abstract Teaser The t(8;21) and inv(16) chimeric oncogenes are major etiological drivers of human acute myeloid leukemia. However, the function of native RUNX1 in these leukemias has remained unknown. Groner and colleagues demonstrate that expression of wild-type RUNX1 is essential for t(8;21) and inv(16) leukemogenesis. Reducing RUNX1 activity destines the leukemic cells for apoptosis. Importantly, an active RUNX1 allele is usually preserved in t(8;21) or inv(16) patients, whereas, in other leukemias, it is frequently inactivated, underscoring the significance of this leukemic cell addiction to RUNX1.

Description: Publication date: Available online 19 September 2013 Source: Cell Reports Author(s): Markus Reschke , John G. Clohessy , Nina Seitzer , Daniel P. Goldstein , Susanne B. Breitkopf , Daniel B. Schmolze , Ugo Ala , John M. Asara , Andrew H. Beck , Pier Paolo Pandolfi Increasing evidence points to an important role for the ribosome in the regulation of biological processes and as a target for deregulation in disease. Here, we describe a SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry approach to probing mammalian riboproteomes. Using a panel of cell lines, as well as genetic and pharmacological perturbations, we obtained a comparative characterization of the cellular riboproteome. This analysis identified a set of riboproteome components, consisting of a diverse array of proteins with a strong enrichment for RNA-binding proteins. Importantly, this global analysis uncovers a high incidence of genetic alterations to riboproteome components in cancer, with a distinct bias toward genetic amplification. We further validated association with polyribosomes for several riboproteome components and demonstrate that enrichment at the riboproteome can depend on cell type, genetics, or cellular stimulus. Our results have important implications for the understanding of how ribosomes function and provide a platform for uncovering regulators of translation. Graphical abstract Teaser Increasing evidence points to an important role for the ribosome in regulating biological processes and as a target in disease. Now, Pandolfi and colleagues use mass spectrometry to probe the mammalian riboproteome. They show that the riboproteome displays differential composition in cancer cells and contains an array of proteins, many of which are frequently amplified in cancer. These results have important implications for the understanding of how ribosomes function and provide a platform to broaden our understanding of translational regulation.

Description: Publication date: Available online 19 September 2013 Source: Cell Reports Author(s): Shunqiang Li , Dong Shen , Jieya Shao , Robert Crowder , Wenbin Liu , Aleix Prat , Xiaping He , Shuying Liu , Jeremy Hoog , Charles Lu , Li Ding , Obi L. Griffith , Christopher Miller , Dave Larson , Robert S. Fulton , Michelle Harrison , Tom Mooney , Joshua F. McMichael , Jingqin Luo , Yu Tao , Rodrigo Goncalves , Christopher Schlosberg , Jeffrey F. Hiken , Laila Saied , Cesar Sanchez , Therese Giuntoli , Caroline Bumb , Crystal Cooper , Robert T. Kitchens , Austin Lin , Chanpheng Phommaly , Sherri R. Davies , Jin Zhang , Megha Shyam Kavuri , Donna McEachern , Yi Yu Dong , Cynthia Ma , Timothy Pluard , Michael Naughton , Ron Bose , Rama Suresh , Reida McDowell , Loren Michel , Rebecca Aft , William Gillanders , Katherine DeSchryver , Richard K. Wilson , Shaomeng Wang , Gordon B. Mills , Ana Gonzalez-Angulo , John R. Edwards , Christopher Maher , Charles M. Perou , Elaine R. Mardis , Matthew J. Ellis To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation. Graphical abstract Teaser In this study, Ellis and colleagues compare whole-tumor genomes from drug-resistant breast cancers with paired xenografts. Genomic fidelity upon transplantation was high for structural variants but variable at the single-nucleotide level. Therefore, tumor and xenograft whole-genome comparisons critically assess genetic drift and clonal representation. Additional analysis revealed ESR1 mutations, amplification, and translocations associated with endocrine resistance in lumenal xenografts. Sequenced patient-derived xenografts are an important resource for functional genomics and capture treatment-resistance etiologies that are not observed in standard cell lines.

Description: Publication date: Available online 26 September 2013 Source: Cell Reports Author(s): Jun Ding , Ursula Loizides-Mangold , Gianpaolo Rando , Vincent Zoete , Olivier Michielin , Janardan K. Reddy , Walter Wahli , Howard Riezman , Bernard Thorens Specific metabolic pathways are activated by different nutrients to adapt the organism to available resources. Although essential, these mechanisms are incompletely defined. Here, we report that medium-chain fatty acids contained in coconut oil, a major source of dietary fat, induce the liver ω-oxidation genes Cyp4a10 and Cyp4a14 to increase the production of dicarboxylic fatty acids. Furthermore, these activate all ω- and β-oxidation pathways through peroxisome proliferator activated receptor (PPAR) α and PPARγ, an activation loop normally kept under control by dicarboxylic fatty acid degradation by the peroxisomal enzyme L-PBE. Indeed, L-pbe −/− mice fed coconut oil overaccumulate dicarboxylic fatty acids, which activate all fatty acid oxidation pathways and lead to liver inflammation, fibrosis, and death. Thus, the correct homeostasis of dicarboxylic fatty acids is a means to regulate the efficient utilization of ingested medium-chain fatty acids, and its deregulation exemplifies the intricate relationship between impaired metabolism and inflammation. Graphical abstract Teaser Specific metabolic pathways are activated by different nutrients to adapt the organism to available resources. Riezman, Thorens, and colleagues find that mice lacking the peroxisomal L-bifunctional enzyme (L-pbe) die of liver failure when fed coconut oil but not lard. Medium-chain fatty acids in coconut oil induce the liver ω-oxidation to increase the production of dicarboxylic fatty acids (DCAs). Furthermore, these activate all ω- and β-oxidation pathways through peroxisome proliferator activated receptors, an activation loop normally fine tuned by L-PBE degrading DCAs. Their work demonstrates the physiological role of mouse L-PBE in hepatic adaptation to medium-chain fatty acids.

Description: Publication date: Available online 26 September 2013 Source: Cell Reports Author(s): Tae Kyung Kim , Jai-Yoon Sul , Henrik Helmfors , Ulo Langel , Junhyong Kim , James Eberwine Protein synthesis in neuronal dendrites underlies long-term memory formation in the brain. Local translation of reporter mRNAs has demonstrated translation in dendrites at focal points called translational hotspots. Various reports have shown that hundreds to thousands of mRNAs are localized to dendrites, yet the dynamics of translation of multiple dendritic mRNAs has remained elusive. Here, we show that the protein translational activities of two dendritically localized mRNAs are spatiotemporally complex but constrained by the translational hotspots in which they are colocalized. Cotransfection of glutamate receptor 2 (GluR2) and GluR4 mRNAs (engineered to encode different fluorescent proteins) into rat hippocampal neurons demonstrates a heterogeneous distribution of translational hotspots for the two mRNAs along dendrites. Stimulation with s -3,5-dihydroxy-phenylglycine modifies the translational dynamics of both of these RNAs in a complex saturable manner. These results suggest that the translational hotspot is a primary structural regulator of the simultaneous yet differential translation of multiple mRNAs in the neuronal dendrite. Graphical abstract Teaser Local translation of dendritic mRNAs plays a crucial role in synaptic plasticity and formation of long-term memory. However, the dynamics of simultaneous translation of multiple dendritic mRNAs has remained elusive. In this study, Eberwine and colleagues show that the translational activities of two dendritically localized mRNAs are spatiotemporally complex but constrained by the translational hotspots in which they are colocalized. The results suggest structural constraints and stochastic regulation of synaptic plasticity.

Description: Publication date: Available online 26 September 2013 Source: Cell Reports Author(s): Sara Ribeiro , Ilaria Napoli , Ian J. White , Simona Parrinello , Adrienne M. Flanagan , Ueli Suter , Luis F. Parada , Alison C. Lloyd Schwann cells are highly plastic cells that dedifferentiate to a progenitor-like state following injury. However, deregulation of this plasticity, may be involved in the formation of neurofibromas, mixed-cell tumors of Schwann cell (SC) origin that arise upon loss of NF1. Here, we show that adult myelinating SCs (mSCs) are refractory to Nf1 loss. However, in the context of injury, Nf1-deficient cells display opposing behaviors along the wounded nerve; distal to the injury, Nf1 −/− mSCs redifferentiate normally, whereas at the wound site Nf1 −/− mSCs give rise to neurofibromas in both Nf1 +/+ and Nf1 +/− backgrounds. Tracing experiments showed that distinct cell types within the tumor derive from Nf1-deficient SCs. This model of neurofibroma formation demonstrates that neurofibromas can originate from adult SCs and that the nerve environment can switch from tumor suppressive to tumor promoting at a site of injury. These findings have implications for both the characterization and treatment of neurofibromas. Graphical abstract Teaser Neurofibromas are mixed-cell tumors of Schwann cell (SC) origin that arise upon loss of NF1. Here, Lloyd and colleagues show that adult myelinating SCs (mSCs) are insensitive to NF1 loss. However, when nerves are injured, NF1-deficient mSCs display opposing behavior along the wounded nerve, forming tumors at the injury site while redifferentiating normally along the rest of the nerve. This demonstrates that the nerve environment can switch from tumor suppressive to tumor promoting at a site of injury.

Description: Publication date: Available online 26 September 2013 Source: Cell Reports Author(s): Hui Zheng , Vibhor Gupta , Jeffrey Patterson-Fortin , Sabyasachi Bhattacharya , Kanstantsin Katlinski , Junmin Wu , Bentley Varghese , Christopher J. Carbone , Bernadette Aressy , Serge Y. Fuchs , Roger A. Greenberg Lysine63-linked ubiquitin (K63-Ub) chains represent a particular ubiquitin topology that mediates proteasome-independent signaling events. The deubiquitinating enzyme (DUB) BRCC36 segregates into distinct nuclear and cytoplasmic complexes that are specific for K63-Ub hydrolysis. RAP80 targets the five-member nuclear BRCC36 complex to K63-Ub chains at DNA double-strand breaks. The alternative four-member BRCC36 containing complex (BRISC) lacks a known targeting moiety. Here, we identify serine hydroxymethyltransferase (SHMT) as a previously unappreciated component that fulfills this function. SHMT directs BRISC activity at K63-Ub chains conjugated to the type 1 interferon (IFN) receptor chain 1 (IFNAR1). BRISC-SHMT2 complexes localize to and deubiquitinate actively engaged IFNAR1, thus limiting its K63-Ub-mediated internalization and lysosomal degradation. BRISC-deficient cells and mice exhibit attenuated responses to IFN and are protected from IFN-associated immunopathology. These studies reveal a mechanism of DUB regulation and suggest a therapeutic use of BRISC inhibitors for treating pathophysiological processes driven by elevated IFN responses. Graphical abstract Teaser Fuchs, Greenberg, and colleagues describe a deubiquitinating (DUB) enzyme complex consisting of an interaction between BRISC and SHMT enzymes. This lysine63-ubiquitin-specific DUB complex deubiquitinates the actively engaged type I interferon receptor, resulting in receptor stabilization and activation of interferon signaling pathways. BRISC-deficient cells and mice have attenuated interferon responses and are resistant to bacterial lipopolysaccharide. These findings suggest strategies for treating disease states that arise from elevated interferon signals.

Description: Publication date: Available online 3 October 2013 Source: Cell Reports Author(s): Tamás Schauer , Petra C. Schwalie , Ava Handley , Carla E. Margulies , Paul Flicek , Andreas G. Ladurner Chromatin organization and gene activity are responsive to developmental and environmental cues. Although many genes are transcribed throughout development and across cell types, much of gene regulation is highly cell-type specific. To readily track chromatin features at the resolution of cell types within complex tissues, we developed and validated chromatin affinity purification from specific cell types by chromatin immunoprecipitation (CAST-ChIP), a broadly applicable biochemical procedure. RNA polymerase II (Pol II) CAST-ChIP identifies ∼1,500 neuronal and glia-specific genes in differentiated cells within the adult Drosophila brain. In contrast, the histone H2A.Z is distributed similarly across cell types and throughout development, marking cell-type-invariant Pol II-bound regions. Our study identifies H2A.Z as an active chromatin signature that is refractory to changes across cell fates. Thus, CAST-ChIP powerfully identifies cell-type-specific as well as cell-type-invariant chromatin states, enabling the systematic dissection of chromatin structure and gene regulation within complex tissues such as the brain. Graphical abstract Teaser Much gene regulation is cell-type specific, but there are few tools that allow the genome-wide tracking of chromatin features and transcriptional states at the resolution of cell types within complex tissues. Margulies, Flicek, Ladurner, and colleagues developed and validated CAST-ChIP, a biochemical procedure that profiles chromatin-associated proteins in cell types of the Drosophila brain. CAST-ChIP identified 1,500 neuron- and glia-specific genes and revealed that histone H2A.Z marks cell-type-invariant domains. CAST-ChIP enables the systematic dissection of gene regulation in cell types.

Description: Publication date: Available online 3 October 2013 Source: Cell Reports Author(s): Per Nilsson , Krishnapriya Loganathan , Misaki Sekiguchi , Yukio Matsuba , Kelvin Hui , Satoshi Tsubuki , Motomasa Tanaka , Nobuhisa Iwata , Takashi Saito , Takaomi C. Saido Alzheimer’s disease (AD) is a neurodegenerative disease biochemically characterized by aberrant protein aggregation, including amyloid beta (Aβ) peptide accumulation. Protein aggregates in the cell are cleared by autophagy, a mechanism impaired in AD. To investigate the role of autophagy in Aβ pathology in vivo, we crossed amyloid precursor protein (APP) transgenic mice with mice lacking autophagy in excitatory forebrain neurons obtained by conditional knockout of autophagy-related protein 7. Remarkably, autophagy deficiency drastically reduced extracellular Aβ plaque burden. This reduction of Aβ plaque load was due to inhibition of Aβ secretion, which led to aberrant intraneuronal Aβ accumulation in the perinuclear region. Moreover, autophagy-deficiency-induced neurodegeneration was exacerbated by amyloidosis, which together severely impaired memory. Our results establish a function for autophagy in Aβ metabolism: autophagy influences secretion of Aβ to the extracellular space and thereby directly affects Aβ plaque formation, a pathological hallmark of AD. Graphical abstract Teaser In this study, Nilsson, Saido, and colleagues show that autophagy influences secretion of Alzheimer’s disease (AD) amyloid beta (Aβ) peptide. Autophagy deficiency, achieved by genetic deletion of autophagy-related gene 7 in excitatory neurons in mouse brain, reduced Aβ plaque load and caused intracellular Aβ accumulation. In addition, amyloidosis exacerbated autophagy-deficiency-induced neurodegeneration and caused severe memory impairment. Thus, autophagy has a key role in the two main characteristics of AD: intracellular Aβ accumulation and extracellular Aβ plaque formation.

Description: Publication date: Available online 3 October 2013 Source: Cell Reports Author(s): Antonia Borovina , Brian Ciruna The role for cilia in establishing planar cell polarity (PCP) is contentious. Although knockdown of genes known to function in ciliogenesis has been reported to cause PCP-related morphogenesis defects in zebrafish, genetic mutations affecting intraflagellar transport (IFT) do not show PCP phenotypes despite the requirement for IFT in cilia formation. This discrepancy has been attributed to off-target effects of antisense morpholino oligonucleotide (MO) injection, confounding maternal effects in zygotic mutant embryos, or an inability to distinguish between cilia-dependent versus cilia-independent protein functions. To determine the role of cilia in PCP, we generated maternal + zygotic IFT88 (MZ ift88 ) mutant zebrafish embryos, which never form cilia. We clearly demonstrate that cilia are not required to establish PCP. Rather, IFT88 plays a cilia-independent role in controlling oriented cell divisions at gastrulation and neurulation. Our results have important implications for the interpretation of cilia gene function in normal development and in disease. Graphical abstract Teaser The role of cilia in establishing planar cell polarity (PCP) remains contentious, confounded by off-target effects of antisense morpholino oligonucleotide use, maternal effects in zygotic mutant backgrounds, and difficulties distinguishing between cilia-dependent versus cilia-independent protein function. Here, Borovina and Ciruna clearly demonstrate that PCP is established normally in cilia-deficient maternal-zygotic IFT88 mutant zebrafish. Furthermore, by analyzing morphogenic events that occur prior to cilia formation, they identify a cilia- and PCP-independent role for IFT88 in controlling polarized cell divisions.

Description: Publication date: Available online 3 October 2013 Source: Cell Reports Author(s): Tian Xie , Wei Peng , Chuangye Yan , Jianping Wu , Xinqi Gong , Yigong Shi RIP3 is an essential upstream kinase in necroptosis. The pseudokinase MLKL functions as a substrate of RIP3 to mediate downstream signaling. The molecular mechanism by which RIP3 recognizes and phosphorylates MLKL remains unknown. Here, we report the crystal structures of the mouse RIP3 kinase domain, the MLKL kinase-like domain, and a binary complex between the two. Both RIP3 and MLKL adopt the canonical kinase fold. Free RIP3 exists in an active conformation, whereas MLKL-bound RIP3 is stabilized by AMP-PNP to adopt an inactive conformation. The formation of the RIP3-MLKL complex, involving their respective N- and C-lobes, is accompanied by pronounced conformational changes of the αC helix and activation loop in RIP3 and the corresponding structural elements in MLKL. RIP3-mediated MLKL phosphorylation, though important for downstream signaling, is dispensable for stable complex formation between RIP3 and MLKL. Our study serves as a framework for mechanistic understanding of RIP3-mediated necroptotic signaling. Graphical abstract Teaser RIP3 is an essential upstream kinase in necroptosis. The pseudokinase MLKL functions as a substrate of RIP3 to mediate downstream signaling. In this study, Shi and colleagues report the crystal structures of the mouse RIP3 kinase domain, the MLKL kinase-like domain, and a binary complex between the two, which reveals the structural basis of MLKL recognition by RIP3 and gives structural insights into RIP3-mediated necroptotic signaling. Their structural analysis serves as a framework for understanding RIP3-mediated necroptosis.

Description: Publication date: Available online 3 October 2013 Source: Cell Reports Author(s): Albert Ribes-Zamora , Sandra M. Indiviglio , Ivana Mihalek , Christopher L. Williams , Alison A. Bertuch Telomeres are protected from nonhomologous end-joining (NHEJ) to avoid deleterious chromosome fusions, yet they associate with the Ku heterodimer that is principal in the classical NHEJ (c-NHEJ) pathway. T-loops have been proposed to inhibit Ku’s association with telomeric ends, thus inhibiting c-NHEJ; however, deficiencies in the t-loop model suggest additional mechanisms are in effect. We demonstrate that TRF2 interacts with Ku at telomeres and via residues in Ku70 helix 5 (α5), which are vital for NHEJ. We show that Ku’s interaction with a TRF2 mutant that induces telomeric fusions is significantly impaired. Additionally, we demonstrate that Ku70 α5 is required for Ku self-association in live cells, which can bridge DNA ends. Together, these findings lead us to propose a model in which telomeres are directly protected from c-NHEJ via TRF2 impeding Ku’s ability to synapse telomere ends. Graphical abstract Teaser The protection of chromosomal termini from fusions is an essential function of telomeres. Nonetheless, the Ku heterodimer, a factor required for nonhomologous end-joining (NHEJ), is associated with functional telomeres. In this study, Bertuch and colleagues show that the telomere end protection factor TRF2 interacts with a region of Ku required for NHEJ. They further show that this region promotes Ku-Ku interactions. These data lead the authors to propose a model for the protection of telomeres from NHEJ.

Description: Publication date: Available online 3 October 2013 Source: Cell Reports Author(s): Hong Wu , Nikolas Mathioudakis , Boubou Diagouraga , Aiping Dong , Ludmila Dombrovski , Frédéric Baudat , Stephen Cusack , Bernard de Massy , Jan Kadlec PRDM9, a histone lysine methyltransferase, is a key determinant of the localization of meiotic recombination hot spots in humans and mice and the only vertebrate protein known to be involved in hybrid sterility. Here, we report the crystal structure of the PRDM9 methyltransferase domain in complex with a histone H3 peptide dimethylated on lysine 4 (H3K4me2) and S-adenosylhomocysteine (AdoHcy), which provides insights into the methyltransferase activity of PRDM proteins. We show that the genuine substrate of PRDM9 is histone H3 lysine 4 (H3K4) and that the enzyme possesses mono-, di-, and trimethylation activities. We also determined the crystal structure of PRDM9 in its autoinhibited state, which revealed a rearrangement of the substrate and cofactor binding sites by a concerted action of the pre-SET and post-SET domains, providing important insights into the regulatory mechanisms of histone lysine methyltransferase activity. Graphical abstract Teaser The histone methyltransferase PRDM9 is a key determinant of meiotic recombination hot spots in humans and mice and the only vertebrate protein known to be involved in hybrid sterility. In this study, de Massy, Kadlec, and colleagues analyzed PRDM9 substrate specificity and report the crystal structures of its methyltransferase domain in an autoinhibited state and in complex with an H3K4me2 peptide and AdoHcy. These data provide important insights into the regulatory mechanisms of histone lysine methyltransferase activity.

Description: Publication date: Available online 3 October 2013 Source: Cell Reports Author(s): Gwynneth Thomas , Jenna L. Betters , Caleb C. Lord , Amanda L. Brown , Stephanie Marshall , Daniel Ferguson , Janet Sawyer , Matthew A. Davis , John T. Melchior , Lawrence C. Blume , Allyn C. Howlett , Pavlina T. Ivanova , Stephen B. Milne , David S. Myers , Irina Mrak , Vera Leber , Christoph Heier , Ulrike Taschler , Jacqueline L. Blankman , Benjamin F. Cravatt , Richard G. Lee , Rosanne M. Crooke , Mark J. Graham , Robert Zimmermann , H. Alex Brown , J. Mark Brown The serine hydrolase α/β hydrolase domain 6 (ABHD6) has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG) in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this, we utilized targeted antisense oligonucleotides (ASOs) to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and understand ABHD6’s role in energy metabolism. Here, we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches, we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes. Graphical abstract Teaser Metabolic syndrome has become a leading health concern. Now, Brown and colleagues show that that serine hydrolase ABHD6 is a key driver of the metabolic syndrome and that inhibition of ABHD6 protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic and in vitro enzymology approaches, the authors show that ABHD6 is a promiscuous lipase that hydrolyzes monoacylglycerols and lysophospholipids and argue that ABHD6 inhibitors may hold therapeutic promise.

Description: Publication date: Available online 3 October 2013 Source: Cell Reports Author(s): Kinyui Alice Lo , Adam Labadorf , Norman J. Kennedy , Myoung Sook Han , Yoon Sing Yap , Bryan Matthews , Xiaofeng Xin , Lei Sun , Roger J. Davis , Harvey F. Lodish , Ernest Fraenkel Diet-induced obesity (DIO) predisposes individuals to insulin resistance, and adipose tissue has a major role in the disease. Insulin resistance can be induced in cultured adipocytes by a variety of treatments, but what aspects of the in vivo responses are captured by these models remains unknown. We use global RNA sequencing to investigate changes induced by TNF-α, hypoxia, dexamethasone, high insulin, and a combination of TNF-α and hypoxia, comparing the results to the changes in white adipose tissue from DIO mice. We found that different in vitro models capture distinct features of DIO adipose insulin resistance, and a combined treatment of TNF-α and hypoxia is most able to mimic the in vivo changes. Using genome-wide DNase I hypersensitivity followed by sequencing, we further examined the transcriptional regulation of TNF-α-induced insulin resistance, and we found that C/EPBβ is a potential key regulator of adipose insulin resistance. Graphical abstract Teaser Obesity-related insulin resistance is an increasingly common medical problem. Lodish, Fraenkel, and colleagues examine in vitro models for adipose insulin resistance using next-generation sequencing to analyze mRNA levels and chromatin states. They demonstrate that common models capture dramatically different aspects of the in vivo changes, with a combined TNF-α-hypoxia treatment most able to mimic diet-induced obesity. Their approach reveals C/EPBβ as a potential regulator of adipose insulin resistance. AdipoSight, a web portal, provides access to their data and algorithms.

Description: Publication date: Available online 3 October 2013 Source: Cell Reports Author(s): Ricardo Weinlich , Andrew Oberst , Christopher P. Dillon , Laura J. Janke , Sandra Milasta , John R. Lukens , Diego A. Rodriguez , Prajwal Gurung , Chandra Savage , Thirumala D. Kanneganti , Douglas R. Green Caspase-8 or cellular FLICE-like inhibitor protein (cFLIP) deficiency leads to embryonic lethality in mice due to defects in endothelial tissues. Caspase-8 −/− and receptor-interacting protein kinase-3 (RIPK3) −/− , but not cFLIP −/− and RIPK3 −/− , double-knockout animals develop normally, indicating that caspase-8 antagonizes the lethal effects of RIPK3 during development. Here, we show that the acute deletion of caspase-8 in the gut of adult mice induces enterocyte death, disruption of tissue homeostasis, and inflammation, resulting in sepsis and mortality. Likewise, acute deletion of caspase-8 in a focal region of the skin induces local keratinocyte death, tissue disruption, and inflammation. Strikingly, RIPK3 ablation rescues both phenotypes. However, acute loss of cFLIP in the skin produces a similar phenotype that is not rescued by RIPK3 ablation. TNF neutralization protects from either acute loss of caspase-8 or cFLIP. These results demonstrate that caspase-8-mediated suppression of RIPK3-induced death is required not only during development but also for adult homeostasis. Furthermore, RIPK3-dependent inflammation is dispensable for the skin phenotype. Graphical abstract Teaser In this study, Green and colleagues show that acute loss of caspase-8 in the gut or the skin can induce a TNF-dependent, RIPK3-mediated loss of tissue homeostasis and inflammation, demonstrating that RIPK3 function is tightly regulated in adult tissues. Strikingly, the authors show that loss of cFLIP in RIPK3-deficient background induces a similar phenotype, suggesting that loss of tissue barrier function, rather than the type of cell death (necroptosis or apoptosis), defines the onset of disease.

Description: Publication date: Available online 3 October 2013 Source: Cell Reports Author(s): Todd C. Metzger , Imran S. Khan , James M. Gardner , Maria L. Mouchess , Kellsey P. Johannes , Anna K. Krawisz , Katarzyna M. Skrzypczynska , Mark S. Anderson Thymic epithelial cells in the medulla (mTECs) play a critical role in enforcing central tolerance through expression and presentation of tissue-specific antigens (TSAs) and deletion of autoreactive thymocytes. TSA expression requires autoimmune regulator (Aire), a transcriptional activator present in a subset of mTECs characterized by high CD80 and major histocompatibility complex II expression and a lack of potential for differentiation or proliferation. Here, using an Aire-DTR transgenic line, we show that short-term ablation specifically targets Aire + mTECs, which quickly undergo RANK-dependent recovery. Repeated ablation also affects Aire − mTECs, and using an inducible Aire-Cre fate-mapping system, we find that this results from the loss of a subset of mTECs that showed prior expression of Aire, maintains intermediate TSA expression, and preferentially migrates toward the center of the medulla. These results clearly identify a distinct stage of mTEC development and underscore the diversity of mTECs that play a key role in maintaining tolerance. Graphical abstract Teaser In this study, Anderson and colleagues investigate the recovery and developmental potential of Aire + thymic epithelial cells (TECs), a cell population with unique roles in limiting self-reactivity of developing T cells. The authors find that this population is capable of rapid recovery following targeted ablation and that such ablation leads to loss of tolerance to self. The authors also find that Aire + TECs progress to a terminal Aire − stage, which may have a unique role in driving central tolerance.

Description: Publication date: Available online 3 October 2013 Source: Cell Reports Author(s): Anastazja Grabarz , Josée Guirouilh-Barbat , Aurélia Barascu , Gaëlle Pennarun , Diane Genet , Emilie Rass , Susanne M. Germann , Pascale Bertrand , Ian D. Hickson , Bernard S. Lopez The choice of the appropriate double-strand break (DSB) repair pathway is essential for the maintenance of genomic stability. Here, we show that the Bloom syndrome gene product, BLM, counteracts CtIP/MRE11-dependent long-range deletions (>200 bp) generated by alternative end-joining (A-EJ). BLM represses A-EJ in an epistatic manner with 53BP1 and RIF1 and is required for ionizing-radiation-induced 53BP1 focus assembly. Conversely, in the absence of 53BP1 or RIF1, BLM promotes formation of A-EJ long deletions, consistent with a role for BLM in DSB end resection. These data highlight a dual role for BLM that influences the DSB repair pathway choi (1) protection against CtIP/MRE11 long-range deletions associated with A-EJ and (2) promotion of DNA resection. These antagonist roles can be regulated, according to cell-cycle stage, by interacting partners such as 53BP1 and TopIII, to avoid unscheduled resection that might jeopardize genome integrity. Graphical abstract Teaser The choice of the appropriate double-strand break (DSB) repair pathway is essential for the maintenance of genomic stability. Here, Lopez and colleagues show a dual role for the Bloom syndrome gene product, BLM, that influences the DSB repair pathway choice in two ways: (1) protection against long-range deletions associated with DSB end-joining and (2) promotion of DNA resection. These antagonistic roles can be regulated, according to cell-cycle stage, by interacting partners, to avoid unscheduled resection that might jeopardize genome integrity.

Description: Publication date: Available online 3 October 2013 Source: Cell Reports Author(s): Samantha B. Foley , Zacariah L. Hildenbrand , Abigail A. Soyombo , Jeffery A. Magee , Yipin Wu , Katherine I. Oravecz-Wilson , Theodora S. Ross Chronic myeloid leukemia (CML) and some acute lymphoblastic leukemias are characterized by the t(9;22) chromosome, which encodes the BCR/ABL oncogene. Multiple mouse models of CML express BCR/ABL at high levels from non- Bcr promoters, resulting in the development of leukemias. In contrast, a significant fraction of healthy humans have been found to have BCR/ABL-positive hematopoietic cells. To bridge the gap between the information derived from current mouse models and nonleukemic humans with the BCR/ABL oncogene, we generated a knockin model with BCR/ABL p210 expressed from the Bcr locus. Unlike previous models, expression of BCR/ABL from the knockin allele did not induce leukemia. BCR/ABL mutant cells did exhibit favorable bone marrow engraftment compared to control cells. These data suggest that BCR/ABL expression alone is insufficient to induce disease. This model allows for inducible spatial and temporal control of BCR/ABL expression for analysis of early steps in the pathogenesis of BCR/ABL-expressing leukemias. Graphical abstract Teaser In this study, Ross and colleagues show that when the BCR/ABL oncogene is placed in the mouse genome as a single-copy knockin mutation, the mice, like some humans with the BCR/ABL mutation, are leukemia free. The combination of this BCR/ABL allele with an AML1/ETO knockin allele led to myeloid neoplasia. This mouse mutation will aid in understanding BCR/ABL-associated predisease, which many predict is the precursor to florid chronic myeloid leukemia.

Description: Publication date: Available online 12 September 2013 Source: Cell Reports Author(s): Ke Zhao , Juan Du , Xue Han , John L. Goodier , Peng Li , Xiaohong Zhou , Wei Wei , Sean L. Evans , Linzhang Li , Wenyan Zhang , Ling E. Cheung , Guanjun Wang , Haig H. Kazazian Jr. , Xiao-Fang Yu Long interspersed elements 1 (LINE-1) occupy at least 17% of the human genome and are its only active autonomous retrotransposons. However, the host factors that regulate LINE-1 retrotransposition are not fully understood. Here, we demonstrate that the Aicardi-Goutières syndrome gene product SAMHD1, recently revealed to be an inhibitor of HIV/simian immunodeficiency virus (SIV) infectivity and neutralized by the viral Vpx protein, is also a potent regulator of LINE-1 and LINE-1-mediated Alu/SVA retrotransposition. We also found that mutant SAMHD1s of Aicardi-Goutières syndrome patients are defective in LINE-1 inhibition. Several domains of SAMHD1 are critical for LINE-1 regulation. SAMHD1 inhibits LINE-1 retrotransposition in dividing cells. An enzymatic active site mutant SAMHD1 maintained substantial anti-LINE-1 activity. SAMHD1 inhibits ORF2p-mediated LINE-1 reverse transcription in isolated LINE-1 ribonucleoproteins by reducing ORF2p level. Thus, SAMHD1 may be a cellular regulator of LINE-1 activity that is conserved in mammals. Graphical abstract Teaser Long interspersed elements 1 (LINE-1) are active autonomous retrotransposons that occupy 17% of the human genome. The host factors that regulate LINE-1 retrotransposition are not fully understood. In this study, Yu and colleagues show that the Aicardi-Goutières syndrome (AGS) gene product, SAMHD1, is a potent regulator of LINE-1 retrotransposition. SAMHD1 mutants linked to AGS are defective in LINE-1 inhibition. Although SAMHD1 has also recently been shown to suppress HIV-1, the authors find that suppression of LINE-1 occurs through a distinct mechanism.

Description: Publication date: Available online 12 September 2013 Source: Cell Reports Author(s): Runsheng He , Ning Huang , Yitian Bao , Haining Zhou , Junlin Teng , Jianguo Chen During interphase, centrosomes are connected by a proteinaceous linker between the proximal ends of the centrioles, which is important for the centrosomes to function as a single microtubule-organizing center. However, the composition and regulation of centrosomal linker remain largely unknown. Here, we show that LRRC45 is a centrosome linker that localizes at the proximal ends of the centrioles and forms fiber-like structures between them. Depletion of LRRC45 results in centrosome splitting during interphase. Moreover, LRRC45 interacts with both C-Nap1 and rootletin and is phosphorylated by Nek2A at S661 during mitosis. After phosphorylation, both LRRC45 centrosomal localization and fiber-like structures are significantly reduced, which subsequently leads to centrosome separation. Thus, LRRC45 is a critical component of the proteinaceous linker between two centrioles and is required for centrosome cohesion. Graphical abstract Teaser During interphase, the centrosomes are connected by a loose proteinaceous linker. Here, Teng, Chen, and colleagues identify LRRC45 as a centrosome linker required for centrosome cohesion. It is recruited by C-Nap1 at the proximal ends of the centrioles and forms fiber-like structures, together with rootletin. Also, LRRC45 is phosphorylated by Nek2A, which induces centrosome separation during mitosis. These findings facilitate a better understanding of the composition and regulation of centrosome linkers.

Description: Publication date: Available online 12 September 2013 Source: Cell Reports Author(s): Ying Tan , Dinghui Yu , Germain U. Busto , Curtis Wilson , Ronald L. Davis Wnt signaling regulates synaptic plasticity and neurogenesis in the adult nervous system, suggesting a potential role in behavioral processes. Here, we probed the requirement for Wnt signaling during olfactory memory formation in Drosophila using an inducible RNAi approach. Interfering with β-catenin expression in adult mushroom body neurons specifically impaired long-term memory (LTM) without altering short-term memory. The impairment was reversible, being rescued by expression of a wild-type β-catenin transgene, and correlated with disruption of a cellular LTM trace. Inhibition of wingless , a Wnt ligand, and arrow , a Wnt coreceptor, also impaired LTM. Wingless expression in wild-type flies was transiently elevated in the brain after LTM conditioning. Thus, inhibiting three key components of the Wnt signaling pathway in adult mushroom bodies impairs LTM, indicating that this pathway mechanistically underlies this specific form of memory. Graphical abstract Teaser Wnt signaling is crucial for many aspects of embryonic development, including cell proliferation, cell movement, and cell-fate decisions. In this report, Davis and colleagues show that Wnt signaling is required for the formation of protein-synthesis-dependent, long-term memory. Using RNAi approaches that target Wnt signaling components in the adult fly mushroom body, they show that knockdown of the Wnt ligand wingless , the Wnt coreceptor arrow , and the effector molecule β-catenin all impair the formation of long-term behavioral memory as well as a cellular memory trace representing this form of memory.

Description: Publication date: Available online 19 September 2013 Source: FEBS Open Bio Author(s): Wei Chi , Xiaoni Gan , Wuhan Xiao , Wen Wang , Shunping He Hypoxia-inducible factor (HIF) is a crucial regulator of cellular and systemic responses to low oxygen levels. Here we firstly cloned three HIF-α isoforms from the basal branches of Osteichthyes and used computational tools to characterize the molecular change underlying the functional divergence of HIF-α isoforms in different lineages. Only the HIF-1α and HIF-2α in African lungfish and amphibians were found under positive selection. HIF-1α and -2α were less functionally divergent in basal ray-finned fish than in teleosts, and showed conserved but different transcriptional activity toward specific target genes.

Description: Publication date: Available online 19 September 2013 Source: Cell Reports Author(s): Hongxia Zhao , Alphée Michelot , Essi V. Koskela , Vadym Tkach , Dimitrios Stamou , David G. Drubin , Pekka Lappalainen Bin-Amphiphysin-Rvs (BAR) domain proteins are central regulators of many cellular processes involving membrane dynamics. BAR domains sculpt phosphoinositide-rich membranes to generate membrane protrusions or invaginations. Here, we report that, in addition to regulating membrane geometry, BAR domains can generate extremely stable lipid microdomains by “freezing” phosphoinositide dynamics. This is a general feature of BAR domains, because the yeast endocytic BAR and Fes/CIP4 homology BAR (F-BAR) domains, the inverse BAR domain of Pinkbar, and the eisosomal BAR protein Lsp1 induced phosphoinositide clustering and halted lipid diffusion, despite differences in mechanisms of membrane interactions. Lsp1 displays comparable low diffusion rates in vitro and in vivo, suggesting that BAR domain proteins also generate stable phosphoinositide microdomains in cells. These results uncover a conserved role for BAR superfamily proteins in regulating lipid dynamics within membranes. Stable microdomains induced by BAR domain scaffolds and specific lipids can generate phase boundaries and diffusion barriers, which may have profound impacts on diverse cellular processes. Graphical abstract Teaser Bin-Amphiphysin-Rvs (BAR) domain superfamily proteins are central membrane-sculpting proteins in all eukaryote cells. Here, Lappalainen and colleagues demonstrate that BAR domain scaffolds not only bend membranes but also affect lipid distribution and dynamics by dramatically inhibiting the lateral diffusion of phosphoinositides. The extremely stable BAR domain-induced phosphoinositide microdomains can generate lipid phase boundaries and diffusion barriers, which are likely to have profound impacts on a wide variety of cellular processes, including endocytosis.

Description: Publication date: Available online 25 September 2013 Source: FEBS Open Bio Author(s): Michal Slutzki , Maroor K. Jobby , Seth Chitayat , Alon Karpol , Bareket Dassa , Yoav Barak , Raphael Lamed , Steven P. Smith , Edward A. Bayer The cellulosome is a large extracellular multi-enzyme complex that facilitates the efficient hydrolysis and degradation of crystalline cellulosic substrates. During the course of our studies on the cellulosome of the rumen bacterium Ruminococcus flavefaciens, we focused on the critical ScaA dockerin (ScaADoc), the unique dockerin that incorporates the primary enzyme-integrating ScaA scaffoldin into the cohesin-bearing ScaB adaptor scaffoldin. In the absence of a high-resolution structure of the ScaADoc module, we generated a computational model, and, upon its analysis, we were surprised to discover a putative stacking interaction between an N-terminal Trp and a C-terminal Pro, which we termed intramolecular clasp. In order to verify the existence of such an interaction, these residues were mutated to alanine. Circular dichroism spectroscopy, intrinsic tryptophan and ANS fluorescence, and NMR spectroscopy indicated that mutation of these residues has a destabilizing effect on the functional integrity of the Ca 2+ -bound form of ScaADoc. Analysis of recently determined dockerin structures from other species revealed the presence of other well-defined intramolecular clasps, which consist of different types of interactions between selected residues at the dockerin termini. We propose that this thematic interaction may represent a major distinctive structural feature of the dockerin module. Graphical abstract

Description: Publication date: 26 September 2013 Source: Cell Reports, Volume 4, Issue 6 Author(s): Steffi Oesterreich , Adam M. Brufsky , Nancy E. Davidson In this issue of Cell Reports , Li et al. show that the analysis of genetic changes in patient-derived xenografts can reveal crucial details of tumor evolution, such as the emergence of functional estrogen receptor mutations in endocrine-resistant breast cancer.

Description: Publication date: Available online 26 September 2013 Source: Cell Reports Author(s): Lingjie Li , Bo Liu , Orly L. Wapinski , Miao-Chih Tsai , Kun Qu , Jiajing Zhang , Jeff C. Carlson , Meihong Lin , Fengqin Fang , Rajnish A. Gupta , Jill A. Helms , Howard Y. Chang Long noncoding RNAs (lncRNAs) are thought to be prevalent regulators of gene expression, but the consequences of lncRNA inactivation in vivo are mostly unknown. Here, we show that targeted deletion of mouse Hotair lncRNA leads to derepression of hundreds of genes, resulting in homeotic transformation of the spine and malformation of metacarpal-carpal bones. RNA sequencing and conditional inactivation reveal an ongoing requirement of Hotair to repress HoxD genes and several imprinted loci such as Dlk1-Meg3 and Igf2-H19 without affecting imprinting choice. Hotair binds to both Polycomb repressive complex 2, which methylates histone H3 at lysine 27 (H3K27), and Lsd1 complex, which demethylates histone H3 at lysine 4 (H3K4) in vivo. Hotair inactivation causes H3K4me3 gain and, to a lesser extent, H3K27me3 loss at target genes. These results reveal the function and mechanisms of Hotair lncRNA in enforcing a silent chromatin state at Hox and additional genes. Graphical abstract Teaser A targeted and conditional knockout of the lncRNA, Hotair , is now presented by Chang and colleagues. The authors find that Hotair disruption causes homeotic transformation and skeletal malformation during mouse development. Hotair knockout leads to derepression of multiple genes, including HoxD and several imprinted genes. Furthermore, Hotair interacts with PRC2 and LSD1, and Hotair deletion altered chromatin states at specific targets.

Description: Publication date: Available online 26 September 2013 Source: Cell Reports Author(s): Saskia Delpretti , Thomas Montavon , Marion Leleu , Elisabeth Joye , Athanasia Tzika , Michel Milinkovitch , Denis Duboule Hox genes are required for the development of the intestinal cecum, a major organ of plant-eating species. We have analyzed the transcriptional regulation of Hoxd genes in cecal buds and show that they are controlled by a series of enhancers located in a gene desert flanking the HoxD cluster. The start site of two opposite long noncoding RNAs (lncRNAs), Hotdog and Twin of Hotdog , selectively contacts the expressed Hoxd genes in the framework of a topological domain, coinciding with robust transcription of these genes during cecum budding. Both lncRNAs are specifically transcribed in the cecum, albeit bearing no detectable function in trans . Hedgehogs have kept this regulatory potential despite the absence of the cecum, suggesting that these mechanisms are used in other developmental situations. In this context, we discuss the implementation of a common “budding toolkit” between the cecum and the limbs. Graphical abstract Teaser The intestinal cecum is a major organ for plant-eating species. Duboule and colleagues report that a series of enhancers, along with the Hotdog lncRNA, selectively contact a subset of HoxD genes and form a 3D regulatory structure, which coincides with a topological domain and elicits robust transcription. Hedgehogs have kept this regulatory potential despite absence of the cecum, suggesting that these mechanisms are part of a common “budding toolkit” also used during limb bud development.

Description: Publication date: Available online 26 September 2013 Source: Cell Reports Author(s): Inna Shcherbakova , Aaron A. Hoskins , Larry J. Friedman , Victor Serebrov , Ivan R. Corrêa Jr. , Ming-Qun Xu , Jeff Gelles , Melissa J. Moore Removal of introns from nascent transcripts (pre-mRNAs) by the spliceosome is an essential step in eukaryotic gene expression. Previous studies have suggested that the earliest steps in spliceosome assembly in yeast are highly ordered and the stable recruitment of U1 small nuclear ribonucleoprotein particle (snRNP) to the 5′ splice site necessarily precedes recruitment of U2 snRNP to the branch site to form the “prespliceosome.” Here, using colocalization single-molecule spectroscopy to follow initial spliceosome assembly on eight different S. cerevisiae pre-mRNAs, we demonstrate that active yeast spliceosomes can form by both U1-first and U2-first pathways. Both assembly pathways yield prespliceosomes functionally equivalent for subsequent U5⋅U4/U6 tri-snRNP recruitment and for intron excision. Although fractional flux through the two pathways varies on different introns, both are operational on all introns studied. Thus, multiple pathways exist for assembling functional spliceosomes. These observations provide insight into the mechanisms of cross-intron coordination of initial spliceosome assembly. Graphical abstract Teaser Intron excision by the spliceosome is an essential process in eukaryotic gene expression. Gelles, Moore, and colleagues use single-molecule colocalization to monitor early spliceosome assembly events in yeast whole-cell extract. They demonstrate that pre-mRNAs can initiate the formation of functional spliceosomes by first binding either U1 or U2 snRNP. This branched pathway has important implications for mechanisms of cross-intron coordination during early spliceosome assembly.

Description: Publication date: Available online 26 September 2013 Source: Cell Reports Author(s): Naif Zaman , Lei Li , Maria Luz Jaramillo , Zhanpeng Sun , Chabane Tibiche , Myriam Banville , Catherine Collins , Mark Trifiro , Miltiadis Paliouras , Andre Nantel , Maureen O’Connor-McCourt , Edwin Wang Individual cancer cells carry a bewildering number of distinct genomic alterations (e.g., copy number variations and mutations), making it a challenge to uncover genomic-driven mechanisms governing tumorigenesis. Here, we performed exome sequencing on several breast cancer cell lines that represent two subtypes, luminal and basal. We integrated these sequencing data and functional RNAi screening data (for the identification of genes that are essential for cell proliferation and survival) onto a human signaling network. Two subtype-specific networks that potentially represent core-signaling mechanisms underlying tumorigenesis were identified. Within both networks, we found that genes were differentially affected in different cell lines; i.e., in some cell lines a gene was identified through RNAi screening, whereas in others it was genomically altered. Interestingly, we found that highly connected network genes could be used to correctly classify breast tumors into subtypes on the basis of genomic alterations. Further, the networks effectively predicted subtype-specific drug targets, which were experimentally validated. Graphical abstract Teaser In this study, Wang and colleagues examine cancer genome sequencing data in a framework of a signaling network integrated with genome-wide RNAi knockdown information. They show that cancer cell survival network genes recurrently switch roles, between essential and cancer driving, among cancer subtypes. Mutations and copy number variations driving tumorigenesis are selected to be convergent onto cell survival networks. Genomic alterations of cancer cell survival subtype-specific network genes successfully predicted cancer subtype-specific drug targets and accurately classified cancer subtypes.

Description: Publication date: Available online 27 September 2013 Source: Cell Reports Author(s): Elizabeth Garner , Yonghwan Kim , Francis P. Lach , Molly C. Kottemann , Agata Smogorzewska Holliday junctions (HJs), the DNA intermediates of homologous recombination, need to be faithfully processed in order to preserve genome integrity. In human cells, the BLM helicase complex promotes nonnucleolytic dissolution of double HJs. In vitro, HJs may be nucleolytically processed by MUS81-EME1, GEN1, and SLX4-SLX1. Here, we exploit human SLX4 -null cells to examine the requirements for HJ resolution in vivo. Lack of BLM and SLX4 or GEN1 and SLX4 is synthetically lethal in the absence of exogenous DNA damage, and lethality is a consequence of dysfunctional mitosis proceeding in the presence of unprocessed HJs. Thus, GEN1 activity cannot be substituted for the SLX4-associated nucleases, and one of the HJ resolvase activities, either of those associated with SLX4 or with GEN1, is required for cell viability, even in the presence of BLM. In vivo HJ resolution depends on both SLX4-associated MUS81-EME1 and SLX1, suggesting that they are acting in concert in the context of SLX4. Graphical abstract Teaser The requirements for Holliday junction (HJ) processing in human mitotic cells are now dissected by Smogorzewska and colleagues. The authors demonstrate that nuclease-mediated resolution of HJs, provided by SLX4-associated nucleases and GEN1, is a requirement for cell viability rather than a backup pathway for nonnucleolytic dissolution. When HJs do not get processed, dramatic chromosomal abnormalities appear and cells cannot complete division. The study also finds that two SLX4-associated nucleases, MUS81-EME1 and SLX1, are necessary for HJ resolution.

Description: Publication date: Available online 29 September 2013 Source: FEBS Open Bio Author(s): Chantel N. Jensen , Sohail T. Ali , Michael J. Allen , Gideon Grogan The flavoprotein monooxygenase (FPMO) from Stenotrophomonas maltophilia (SMFMO, Uniprot: B2FLR2) catalyses the asymmetric oxidation of thioethers and is unusual amongst FPMOs in its ability to use the non-phosphorylated cofactor NADH, as well as NADPH, for the reduction of the FAD coenzyme. In order to explore the basis for cofactor promiscuity, structure-guided mutation of two residues in the cofactor binding site, Gln193 and His194, in SMFMO were performed in an attempt to imitate the cofactor binding site of the NADPH-dependent FMO from Methylophaga aminisulfidivorans sp. SK1 (mFMO), in which structurally homologous residues Arg234 and Thr235 bind the NADPH 2’-ribose phosphate. Mutation of His194 to threonine proved most significant, with a switch in specificity from NADH to NADPH [( k cat / K m NADH)/ k cat / K m NADPH) from 1.5:1 to 1:3.5, mostly as a result of a reduced K m for NADPH of approximately seven-fold in the His194Thr mutant. The structure of the Gln193Arg/His194Thr mutant revealed no substantial changes in the backbone of the enzyme or orientation of side chains resulting from mutation. Mutation of Phe52, in the vicinity of FAD, and which in mFMO is an asparagine thought to be responsible for flavin hydroperoxide stabilisation, is, in SMFMO, a determinant of enantioselectivity in sulfoxidation. Mutation of Phe52 to valine resulted in a mutant that transformed para -tolyl methyl sulfide into the ( S )-sulfoxide with 32% e.e., compared to 25% ( R )- for the wild type. These results shed further light both on the cofactor specificity of FPMOs, and their determinants of enantioselectivity, with a view to informing engineering studies of FPMOs in the future.

Description: Publication date: Available online 30 September 2013 Source: FEBS Open Bio Author(s): Yoshichika Taira , Yuki Okegawa , Kazuhiko Sugimoto , Masato Abe , Hideto Miyoshi , Toshiharu Shikanai Antimycin A 3 (AA) is used as an inhibitor of cyclic electron transport around photosystem I. However, the high concentrations of AA that are needed for inhibition have secondary effects, even in chloroplasts. Here, we screened for chemicals that inhibited ferredoxin-dependent plastoquinone reduction in ruptured chloroplasts at lower concentrations than those required for AA. We identified two AA-like compounds: AAL1 and AAL2. AAL1 likely shares an inhibitory site with AA, most probably in the PGR5–PGRL1 protein complex, and enhances O 2 evolution in photosystem II, most likely via an uncoupler-like effect. AAL1 and AAL2 are unlikely to penetrate intact leaves. In ruptured chloroplasts, AALs are superior to AA as inhibitors of cyclic electron transport.

Description: Publication date: Available online 6 June 2013 Source: Cell Reports Author(s): Guillaume Diss , Alexandre K. Dubé , Joël Boutin , Isabelle Gagnon-Arsenault , Christian R. Landry Cells contain many important protein complexes involved in performing and regulating structural, metabolic, and signaling functions. One major challenge in cell biology is to elucidate the organization and mechanisms of robustness of these complexes in vivo. We developed a systematic approach to study structural dependencies within complexes in living cells by deleting subunits and measuring pairwise interactions among other components. We used our methodology to perturb two conserved eukaryotic complexes: the retromer and the nuclear pore complex. Our results identify subunits that are critical for the assembly of these complexes, reveal their structural architecture and uncover…, and uncover mechanisms by which protein interactions are modulated. Our results also show that paralogous proteins play a key role in the robustness of protein complexes and shape their assembly landscape. Our approach paves the way for studying the response of protein interactomes to mutations and enhances our understanding of genotype-phenotype maps. Graphical abstract

Description: Publication date: Available online 6 June 2013 Source: Cell Reports Author(s): Christopher R. Faehnle , Elad Elkayam , Astrid D. Haase , Gregory J. Hannon , Leemor Joshua-Tor Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1–hAgo4) only hAgo2 is an active slicer. We determined the structure of hAgo1 bound to endogenous copurified RNAs to 1.75 Å resolution and hAgo1 loaded with let-7 microRNA to 2.1 Å. Both structures are strikingly similar to the structures of hAgo2. A conserved catalytic tetrad within the PIWI domain of hAgo2 is required for its slicing activity. Completion of the tetrad, combined with a mutation on a loop adjacent to the active site of hAgo1, results in slicer activity that is substantially enhanced by swapping in the N domain of hAgo2. hAgo3, with an intact tetrad, becomes an active slicer by swapping the N domain of hAgo2 without additional mutations. Intriguingly, the elements that make Argonaute an active slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme. Graphical abstract

Description: Publication date: Available online 11 June 2013 Source: FEBS Open Bio Author(s): Jun Kawaguchi , Kumino Maejima , Hiroyuki Kuroiwa , Masumi Taki The introduction of non-natural amino acids at the N-terminus of peptides/proteins using leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) is a useful technique for protein engineering. To accelerate the chemoenzymatic reaction, here we systematically optimized the N-terminal penultimate residue of the acceptor peptide. Positively charged, small, or hydrophilic amino acids at this position show positive effects for the reaction. Kinetic analysis of peptides possessing different penultimate residues suggests that the side chain of the residue affects peptide-binding affinity towards the L/F-transferase. These findings also provide biological insight into the effect of the penultimate amino acid on substrate specificity of natural proteins to be degraded via the N-end rule pathway. Graphical abstract

Description: Publication date: Available online 6 June 2013 Source: Cell Reports Author(s): Mohamed A. Sobhy , Luay I. Joudeh , Xiaojuan Huang , Masateru Takahashi , Samir M. Hamdan Human flap endonuclease 1 (FEN1), one of the structure-specific 5′ nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5′ nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5′ end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5′ nuclease mechanisms. This is achieved by coordinating threading of the 5′ flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5′ nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection of DNA junctions from nonspecific bending and cleavage. Graphical abstract

Description: Publication date: Available online 6 June 2013 Source: Cell Reports Author(s): Robert D.S. Pitceathly , Shamima Rahman , Yehani Wedatilake , James M. Polke , Sebahattin Cirak , A. Reghan Foley , Anna Sailer , Matthew E. Hurles , Jim Stalker , Iain Hargreaves , Cathy E. Woodward , Mary G. Sweeney , Francesco Muntoni , Henry Houlden , Jan-Willem Taanman , Michael G. Hanna The molecular basis of cytochrome c oxidase (COX, complex IV) deficiency remains genetically undetermined in many cases. Homozygosity mapping and whole-exome sequencing were performed in a consanguineous pedigree with isolated COX deficiency linked to a Leigh syndrome neurological phenotype. Unexpectedly, affected individuals harbored homozygous splice donor site mutations in NDUFA4 , a gene previously assigned to encode a mitochondrial respiratory chain complex I (NADH:ubiquinone oxidoreductase) subunit. Western blot analysis of denaturing gels and immunocytochemistry revealed undetectable steady-state NDUFA4 protein levels, indicating that the mutation causes a loss-of-function effect in the homozygous state. Analysis of one- and two-dimensional blue-native polyacrylamide gels confirmed an interaction between NDUFA4 and the COX enzyme complex in control muscle, whereas the COX enzyme complex without NDUFA4 was detectable with no abnormal subassemblies in patient muscle. These observations support recent work in cell lines suggesting that NDUFA4 is an additional COX subunit and demonstrate that NDUFA4 mutations cause human disease. Our findings support reassignment of the NDUFA4 protein to complex IV and suggest that patients with unexplained COX deficiency should be screened for NDUFA4 mutations. Graphical abstract

Description: Publication date: Available online 6 June 2013 Source: Cell Reports Author(s): Rémy Bétous , Frank. B. Couch , Aaron C. Mason , Brandt F. Eichman , Maria Manosas , David Cortez Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases. Graphical abstract

Description: Publication date: Available online 6 June 2013 Source: Cell Reports Author(s): Annalisa Izzo , Kinga Kamieniarz-Gdula , Fidel Ramírez , Nighat Noureen , Jop Kind , Thomas Manke , Bas van Steensel , Robert Schneider Human cells contain five canonical, replication-dependent somatic histone H1 subtypes (H1.1, H1.2, H1.3, H1.4, and H1.5). Although they are key chromatin components, the genomic distribution of the H1 subtypes is still unknown, and their role in chromatin processes has thus far remained elusive. Here, we map the genomic localization of all somatic replication-dependent H1 subtypes in human lung fibroblasts using an integrative DNA adenine methyltransferase identification (DamID) analysis. We find in general that H1.2 to H1.5 are depleted from CpG-dense regions and active regulatory regions. H1.1 shows a DamID binding profile distinct from the other subtypes, suggesting a unique function. H1 subtypes can mark specific domains and repressive regions, pointing toward a role for H1 in three-dimensional genome organization. Our work integrates H1 subtypes into the epigenome maps of human cells and provides a valuable resource to refine our understanding of the significance of H1 and its heterogeneity in the control of genome function. Graphical abstract

Description: Publication date: Available online 6 June 2013 Source: Cell Reports Author(s): Sophie M. Morgani , Maurice A. Canham , Jennifer Nichols , Alexei A. Sharov , Rosa Portero Migueles , Minoru S.H. Ko , Joshua M. Brickman Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought to represent an embryonically restricted ground state. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. Similarly, 2i ESC cultures were heterogeneous and contained a Hex -positive fraction primed to differentiate into trophoblast and extraembryonic endoderm. Single Hex -positive ESCs coexpressed epiblast and extraembryonic genes and contributed to all lineages in chimeras. The cytokine LIF, necessary for ESC self-renewal, supported the expansion of this population but did not directly support Nanog -positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants. Graphical abstract

Description: Publication date: Available online 6 June 2013 Source: Cell Reports Author(s): James E. Haber , Hannes Braberg , Qiuqin Wu , Richard Alexander , Julian Haase , Colm Ryan , Zach Lipkin-Moore , Kathleen E. Franks-Skiba , Tasha Johnson , Michael Shales , Tineke L. Lenstra , Frank C.P. Holstege , Jeffrey R. Johnson , Kerry Bloom , Nevan J. Krogan Genetic interactions reveal the functional relationships between pairs of genes. In this study, we describe a method for the systematic generation and quantitation of triple mutants, termed triple-mutant analysis (TMA). We have used this approach to interrogate partially redundant pairs of genes in S. cerevisiae , including ASF1 and CAC1 , two histone chaperones. After subjecting asf1 Δ cac1 Δ to TMA, we found that the Swi/Snf Rdh54 protein compensates for the absence of Asf1 and Cac1. Rdh54 more strongly associates with the chromatin apparatus and the pericentromeric region in the double mutant. Moreover, Asf1 is responsible for the synthetic lethality observed in cac1 Δ strains lacking the HIRA-like proteins. A similar TMA was carried out after deleting both CLB5 and CLB6 , cyclins that regulate DNA replication, revealing a strong functional connection to chromosome segregation. This approach can reveal functional redundancies that cannot be uncovered through traditional double-mutant analyses. Graphical abstract

Description: Available online 21 March 2013 Publication year: 2013 Source: Cell Reports Effective defense responses involve the entire organism. To maintain body homeostasis after tissue damage, a systemic wound response is induced in which the response of each tissue is tightly orchestrated to avoid incomplete recovery or an excessive, damaging response. Here, we provide evidence that in the systemic response to wounding, an apoptotic caspase pathway is activated downstream of reactive oxygen species in the midgut enterocytes (ECs), cells distant from the wound site, in Drosophila . We show that a caspase-pathway mutant has defects in homeostatic gut cell renewal and that inhibiting caspase activity in fly ECs results in the production of systemic lethal factors after wounding. Our results indicate that wounding remotely controls caspase activity in ECs, which activates the tissue stem cell regeneration pathway in the gut to dampen the dangerous systemic wound reaction. Graphical abstract Highlights ► Caspase activity is required for gut cell turnover ► Caspase is specifically activated in ECs of midgut after wounding ► Caspase activity in ECs is required for fly survival after wounding ► EC turnover is required for dampening the production of lethal factors after wounding

Description: Available online 21 March 2013 Publication year: 2013 Source: Cell Reports The nitrate/nitrite transporters NarK and NarU play an important role in nitrogen homeostasis in bacteria and belong to the nitrate/nitrite porter family (NNP) of the major facilitator superfamily (MFS) fold. The structure and functional mechanism of NarK and NarU remain unknown. Here, we report the crystal structure of NarU at a resolution of 3.1 Å and systematic biochemical characterization. The two molecules of NarU in an asymmetric unit exhibit two distinct conformational states: occluded and partially inward-open. The substrate molecule nitrate appears to be coordinated by four highly conserved, charged, or polar amino acids. Structural and biochemical analyses allowed the identification of key amino acids that are involved in substrate gating and transport. The observed conformational differences of NarU, together with unique sequence features of the NNP family transporters, suggest a transport mechanism that might deviate from the canonical rocker-switch model. Graphical abstract Highlights ► Structure of the nitrate transporter NarU in substrate-free and -bound states ► Distinct conformations of NarU in substrate-free and -bound states ► Identification of a substrate binding site and transport path ► Transport mechanism may deviate from the rocker-switch model

Description: Available online 21 March 2013 Publication year: 2013 Source: Cell Reports X chromosome inactivation (XCI) is a dynamically regulated developmental process with inactivation and reactivation accompanying the loss and gain of pluripotency, respectively. A functional relationship between pluripotency and lack of XCI has been suggested, whereby pluripotency transcription factors repress the master regulator of XCI, the noncoding transcript Xist , by binding to its first intron (intron 1). To test this model, we have generated intron 1 mutant embryonic stem cells (ESCs) and two independent mouse models. We found that Xist ’s repression in ESCs, its transcriptional upregulation upon differentiation, and its silencing upon reprogramming to pluripotency are not dependent on intron 1. Although we observed subtle effects of intron 1 deletion on the randomness of XCI and in the absence of the antisense transcript Tsix in differentiating ESCs, these have little relevance in vivo because mutant mice do not deviate from Mendelian ratios of allele transmission. Altogether, our findings demonstrate that intron 1 is dispensable for the developmental dynamics of Xist expression. Video Abstract Graphical abstract Highlights ► Mice lacking intron 1 are not defective in dosage compensation ► Male and female ESCs lacking intron 1 maintain Xist repression ► Intron 1 is not required for silencing of Xist during reprogramming to iPSCs ► Xist intron 1 displays enhancer activity in differentiating ESCs

Description: Available online 21 March 2013 Publication year: 2013 Source: Cell Reports The specification of neuronal subtypes in the cerebral cortex proceeds in a temporal manner; however, the regulation of the transitions between the sequentially generated subtypes is poorly understood. Here, we report that the forkhead box transcription factor Foxg1 coordinates the production of neocortical projection neurons through the global repression of a default gene program. The delayed activation of Foxg1 was necessary and sufficient to induce deep-layer neurogenesis, followed by a sequential wave of upper-layer neurogenesis. A genome-wide analysis revealed that Foxg1 binds to mammalian-specific noncoding sequences to repress over 12 transcription factors expressed in early progenitors, including Ebf2/3, Dmrt3, Dmrta1, and Eya2. These findings reveal an unexpected prolonged competence of progenitors to initiate corticogenesis at a progressed stage during development and identify Foxg1 as a critical initiator of neocorticogenesis through spatiotemporal repression, a system that balances the production of nonradially and radially migrating glutamatergic subtypes during mammalian cortical expansion. Graphical abstract Highlights ► Foxg1 directs projection neuron production onset in the neocortex ► Foxg1 acts cell autonomously to switch mammalian cortical subtypes ► Progenitors retain a prolonged competence to initiate corticogenesis in vivo ► Ebf2/3 , Dmrta1 , and Eya2 are mammalian-specific repression targets of Foxg1

Description: Available online 21 March 2013 Publication year: 2013 Source: Cell Reports During an infection the antigen-nonspecific memory CD8 T cell compartment is not simply an inert pool of cells, but becomes activated and cytotoxic. It is unknown how these cells contribute to the clearance of an infection. We measured the strength of T cell receptor (TCR) signals that bystander-activated, cytotoxic CD8 T cells (BA-CTLs) receive in vivo and found evidence of limited TCR signaling. Given this marginal contribution of the TCR, we asked how BA-CTLs identify infected target cells. We show that target cells express NKG2D ligands following bacterial infection and demonstrate that BA-CTLs directly eliminate these target cells in an innate-like, NKG2D-dependent manner. Selective inhibition of BA-CTL-mediated killing led to a significant defect in pathogen clearance. Together, these data suggest an innate role for memory CD8 T cells in the early immune response before the onset of a de novo generated, antigen-specific CD8 T cell response. Graphical abstract Highlights ► Memory CD8 T cells become cytotoxic when activated by inflammation (BA-CTLs) ► Bystander activation of memory CD8 T cells occurs with minimal TCR signaling ► BA-CTLs eliminate target cells in an innate-like, NKG2D-dependent manner ► BA-CTLs are necessary to limit pathogen replication early after an infection

Description: Available online 21 March 2013 Publication year: 2013 Source: Cell Reports Many organisms, including plants, use the circadian clock to measure the duration of day and night. Daily rhythms in the plant circadian system are generated by multiple interlocked transcriptional/translational loops and also by spatial regulations such as nuclear translocation. GIGANTEA (GI), one of the key clock components in Arabidopsis , makes distinctive nuclear bodies like other nuclear-localized circadian regulators. However, little is known about the dynamics or roles of GI subnuclear localization. Here, we characterize GI subnuclear compartmentalization and identify unexpected dynamic changes under diurnal conditions. We further identify EARLY FLOWERING 4 (ELF4) as a regulator of GI nuclear distribution through a physical interaction. ELF4 sequesters GI from the nucleoplasm, where GI binds the promoter of CONSTANS ( CO ), to discrete nuclear bodies. We suggest that the subnuclear compartmentalization of GI by ELF4 contributes to the regulation of photoperiodic flowering. Graphical abstract Highlights ► GI forms dynamic subnuclear structures ► GI and ELF4 physically interact at nuclear bodies ► ELF4 regulates subnuclear localization of GI ► ELF4 sequesters GI to nuclear bodies from the CO promoter

Description: Available online 21 March 2013 Publication year: 2013 Source: Cell Reports Mammalian iron metabolism is regulated systemically by the hormone hepcidin and cellularly by iron regulatory proteins (IRPs) that orchestrate a posttranscriptional regulatory network. Through ligand-inducible genetic ablation of both IRPs in the gut epithelium of adult mice, we demonstrate that IRP deficiency impairs iron absorption and promotes mucosal iron retention via a ferritin-mediated “mucosal block.” We show that IRP deficiency does not interfere with intestinal sensing of body iron loading and erythropoietic iron need, but rather alters the basal expression of the iron-absorption machinery. IRPs thus secure sufficient iron transport across absorptive enterocytes by restricting the ferritin “mucosal block” and define a basal set point for iron absorption upon which IRP-independent systemic regulatory inputs are overlaid. Graphical abstract Highlights ► Disruption of intestinal IRP function constrains iron absorption in adult mice ► IRPs must limit mucosal ferritin for efficient iron absorption ► IRPs control ferroportin directly and DMT1 directly or through HIF2α ► IRPs define a set point for hepcidin-mediated regulation of iron absorption

Description: Available online 21 March 2013 Publication year: 2013 Source: Cell Reports The control of memory retention is important for proper responses to constantly changing environments, but the regulatory mechanisms underlying forgetting have not been fully elucidated. Our genetic analyses in C. elegans revealed that mutants of the TIR-1/JNK-1 pathway exhibited prolonged retention of olfactory adaptation and salt chemotaxis learning. In olfactory adaptation, conditioning induces attenuation of odor-evoked Ca 2+ responses in olfactory neurons, and this attenuation is prolonged in the TIR-1/JNK-1-pathway mutant animals. We also found that a pair of neurons in which the pathway functions is required for the acceleration of forgetting, but not for sensation or adaptation, in wild-type animals. In addition, the neurosecretion from these cells is important for the acceleration of forgetting. Therefore, we propose that these neurons accelerate forgetting through the TIR-1/JNK-1 pathway by sending signals that directly or indirectly stimulate forgetting. Graphical abstract Highlights ► Food signals actively regulate forgetting of olfactory adaptation ► TIR-1/JNK-1 pathway accelerates forgetting of adaptation and of associative learning ► Response to diacetyl in AWA neurons is diminished after exposure to diacetyl ► Neurosecretion from AWC neurons is important for the acceleration of forgetting

Description: Available online 21 March 2013 Publication year: 2013 Source: Cell Reports The targeting of type III secretion (TTS) proteins at the injectisome is an important process in bacterial virulence. Nevertheless, how the injectisome specifically recognizes TTS substrates among all bacterial proteins is unknown. A TTS peripheral membrane ATPase protein located at the base of the injectisome has been implicated in the targeting process. We have investigated the targeting of the EspA filament protein and its cognate chaperone, CesAB, to the EscN ATPase of the enteropathogenic E. coli (EPEC). We show that EscN selectively engages the EspA-loaded CesAB but not the unliganded CesAB. Structure analysis revealed that the targeting signal is encoded in a disorder-order structural transition in CesAB that is elicited only upon the binding of its physiological substrate, EspA. Abrogation of the interaction between the CesAB-EspA complex and EscN resulted in severe secretion and infection defects. Additionally, we show that the targeting and secretion signals are distinct and that the two processes are likely regulated by different mechanisms. Graphical abstract Highlights ► The targeting signal of TTS substrates to the ATPase was identified ► The ATPase engages the chaperone-substrate complex but not the free chaperone ► The ATPase recognizes a conformational change induced by the substrate to the chaperone ► Abrogation of the ternary complex results in secretion and infection defects

Description: Available online 4 April 2013 Publication year: 2013 Source: Cell Reports Extensive axonal pruning and neuronal cell death are critical events for the development of the nervous system. Like neuronal cell death, axonal elimination occurs in discrete steps; however, the regulators of these processes remain mostly elusive. Here, we identify the kinesin superfamily protein 2A (KIF2A) as a key executor of microtubule disassembly and axonal breakdown during axonal pruning. Knockdown of Kif2a , but not other microtubule depolymerization or severing proteins, protects axonal microtubules from disassembly upon trophic deprivation. We further confirmed and extended this result to demonstrate that the entire degeneration process is delayed in neurons from the Kif2a knockout mice. Finally, we show that the Kif2a -null mice exhibit normal sensory axon patterning early during development, but abnormal target hyperinnervation later on, as they compete for limited skin-derived trophic support. Overall, these findings reveal a central regulatory mechanism of axonal pruning during development. Graphical abstract Highlights ► During axonal pruning, microtubule disassembly is regulated by KIF2A ► Degradation of Tau precedes microtubule disassembly during axonal pruning ► Kif2a ablation causes skin hyperinnervation by sensory axons during development

Description: Publication date: Available online 12 September 2013 Source: Cell Reports Author(s): Ling Cai , Mark A. McCormick , Brian K. Kennedy , Benjamin P. Tu Ribosome biogenesis requires an enormous commitment of energy and resources in growing cells. In budding yeast, the transcriptional coactivator Ifh1p is an essential regulator of ribosomal protein (RP) gene transcription. Here, we report that Ifh1p is dynamically acetylated and phosphorylated as a function of the growth state of cells. Ifh1p is acetylated at numerous sites in its N-terminal region by Gcn5p and deacetylated by NAD + -dependent deacetylases of the sirtuin family. Acetylation of Ifh1p is responsive to intracellular acetyl-CoA levels and serves to regulate the stability of Ifh1p. The phosphorylation of Ifh1p is mediated by protein kinase A and is dependent on TORC1 signaling. Thus, multiple nutrient-sensing mechanisms converge on Ifh1p. However, instead of modulating overall rates of RP gene transcription or cell growth, the nutrient-responsive phosphorylation of Ifh1p plays a more prominent role in the regulation of cellular replicative lifespan. Graphical abstract Teaser In this study, Tu and colleagues show that an essential transcriptional coactivator required for ribosome biogenesis is subject to multiple nutrient-responsive posttranslational modifications. Ifh1p is dynamically acetylated and phosphorylated as a function of the growth state of yeast cells and regulates additional targets aside from ribosomal subunit genes. Ifh1p is a significant nonhistone substrate of sirtuins, and its phosphorylation unexpectedly regulates cellular replicative lifespan as opposed to overall rates of growth or ribosomal gene transcription.

Description: Publication date: Available online 12 September 2013 Source: Cell Reports Author(s): Robert M. Martin , José Rino , Célia Carvalho , Tomas Kirchhausen , Maria Carmo-Fonseca Removal of introns from pre-messenger RNAs (pre-mRNAs) via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20–30 s. Furthermore, we show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin μ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min −1 and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context. Graphical abstract Teaser Carmo-Fonseca, Kirchhausen, and colleagues have developed a system that makes it experimentally possible to observe in real time the transcription and turnover of fluorescently labeled introns in living cells with single-molecule resolution. They show that different types of introns have distinct splicing kinetics, depending on their relative position in the transcript, size, and splice-site strength. The approach outlined here will be useful for addressing fundamental questions concerning the dynamic control of pre-mRNA splicing in living cells.