ALBERT

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 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: 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: 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 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 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.

Description: Publication date: Available online 12 September 2013 Source: Cell Reports Author(s): Brandon L. Taylor , Fen-Fen Liu , Maike Sander Recently, loss of beta-cell-specific traits has been proposed as an early cause of beta cell failure in diabetes. However, the molecular mechanisms that underlie the loss of beta cell features remain unclear. Here, we identify an Nkx6.1-controlled gene regulatory network as essential for maintaining the functional and molecular traits of mature beta cells. Conditional Nkx6.1 inactivation in adult mice caused rapid-onset diabetes and hypoinsulinemia. Genome-wide analysis of Nkx6.1-regulated genes and functional assays further revealed a critical role for Nkx6.1 in the control of insulin biosynthesis, insulin secretion, and beta cell proliferation. Over time, Nkx6.1-deficient beta cells acquired molecular characteristics of delta cells, revealing a molecular link between impaired beta cell functional properties and loss of cell identity. Given that Nkx6.1 levels are reduced in human type 2 diabetic beta cells, our study lends support to the concept that loss of beta cell features could contribute to the pathogenesis of diabetes. Graphical abstract Teaser Type 2 diabetes is caused by impaired function of pancreatic beta cells. Beta cell failure in diabetes has been shown to coincide with reduced expression of beta-cell-enriched transcription factors, including Nkx6.1. Here, Sander and colleagues show that loss of Nkx6.1 causes diabetes due to reduced insulin production and secretion. By transcriptionally regulating genes controlling glycolytic flux, Nkx6.1 also maintains the proliferative capacity of beta cells. These results identify Nkx6.1 as an essential regulator of the functional beta cell state.

Description: Publication date: Available online 12 September 2013 Source: Cell Reports Author(s): Kubilay Demir , Nadine Kirsch , Carlo A. Beretta , Gerrit Erdmann , Dierk Ingelfinger , Enrico Moro , Francesco Argenton , Matthias Carl , Christof Niehrs , Michael Boutros Wnt/β-catenin signaling plays an important role in embryonic development and adult tissue homeostasis. When Wnt ligands bind to the receptor complex, LRP5/6 coreceptors are activated by phosphorylation and concomitantly endocytosed. In vertebrates, Wnt ligands induce caveolin-dependent endocytosis of LRP6 to relay signal downstream, whereas antagonists such as Dickkopf promote clathrin-dependent endocytosis, leading to inhibition. However, little is known about how LRP6 is directed to different internalization mechanisms, and how caveolin-dependent endocytosis is mediated. In an RNAi screen, we identified the Rab GTPase RAB8B as being required for Wnt/β-catenin signaling. RAB8B depletion reduces LRP6 activity, β-catenin accumulation, and induction of Wnt target genes, whereas RAB8B overexpression promotes LRP6 activity and internalization and rescues inhibition of caveolar endocytosis. In Xenopus laevis and  Danio rerio , RAB8B morphants show lower Wnt activity during embryonic development. Our results implicate RAB8B as an essential evolutionary conserved component of Wnt/β-catenin signaling through regulation of LRP6 activity and endocytosis. Graphical abstract Teaser The regulation of receptor function is a critical step in many signal transduction pathways. In an RNAi screen, Boutros and colleagues identified RAB8B GTPase as a positive regulator of Wnt/β-catenin signaling. RAB8B regulates Wnt coreceptor LRP6 activity and its caveolar endocytosis. RAB8B’s activation and subcellular localization are modulated by upstream Wnt pathway components. Furthermore, the authors show that RAB8B’s function is required for the control of Wnt target genes in cultured cells and Wnt activity during vertebrate embryonic development.

Description: Publication date: Available online 19 September 2013 Source: Cell Reports Author(s): Miguel Casanova , Michał Pasternak , Fatima El Marjou , Patricia Le Baccon , Aline V. Probst , Geneviève Almouzni The equalization of pericentric heterochromatin from distinct parental origins following fertilization is essential for genome function and development. The recent implication of noncoding transcripts in this process raises questions regarding the connection between RNA and the nuclear organization of distinct chromatin environments. Our study addresses the interrelationship between replication and transcription of the two parental pericentric heterochromatin (PHC) domains and their reorganization during early embryonic development. We demonstrate that the replication of PHC is dispensable for its clustering at the late two-cell stage. In contrast, using parthenogenetic embryos, we show that pericentric transcripts are essential for this reorganization independent of the chromatin marks associated with the PHC domains. Finally, our discovery that only reverse pericentric transcripts are required for both the nuclear reorganization of PHC and development beyond the two-cell stage challenges current views on heterochromatin organization. Graphical abstract Teaser Following fertilization in mice, extensive chromatin and nuclear rearrangements of pericentric domains are required for proper developmental progression. Now, Almouzni and colleagues show that transcription of major satellites, but not their replication, is necessary for the nuclear reorganization observed at the two-cell stage. They also provide evidence that these transcripts are required for reorganization of pericentric heterochromatin irrespective of its parental origin. In addition, they show that only reverse major satellite transcripts are required for developmental progression.

Description: Publication date: Available online 19 September 2013 Source: Cell Reports Author(s): Shifeng Zhu , Rae-Dong Jeong , Gah-Hyun Lim , Keshun Yu , Caixia Wang , A.C. Chandra-Shekara , Duroy Navarre , Daniel F. Klessig , Aardra Kachroo , Pradeep Kachroo Plant viruses often encode suppressors of host RNA silencing machinery, which occasionally function as avirulence factors that are recognized by host resistance (R) proteins. For example, the Arabidopsis R protein, hypersensitive response to TCV (HRT), recognizes the turnip crinkle virus (TCV) coat protein (CP). HRT-mediated resistance requires the RNA-silencing component double-stranded RNA-binding protein 4 (DRB4) even though it neither is associated with the accumulation of TCV-specific small RNA nor requires the RNA silencing suppressor function of CP. HRT interacts with the cytosolic fraction of DRB4. Interestingly, TCV infection both increases the cytosolic DRB4 pool and inhibits the HRT-DRB4 interaction. The virulent R8A CP derivative, which induces a subset of HRT-derived responses, also disrupts this interaction. The differential localization of DRB4 in the presence of wild-type and R8A CP implies the importance of subcellular compartmentalization of DRB4. The requirement of DRB4 in resistance to bacterial infection suggests a universal role in R-mediated defense signaling. Graphical abstract Teaser RNA silencing pathways are highly conserved between plants and animals and confer basal resistance against viral pathogens by promoting degradation of viral RNA. Here, Kachroo and colleagues show that components of plant RNA silencing pathway participate in induced resistance by regulating levels of resistance (R) proteins and R-mediated signaling. Their results suggest a distinct and unique role for RNA silencing machinery in effector-triggered immunity that does not overlap with their function in basal immunity.

Description: Publication date: Available online 19 September 2013 Source: Cell Reports Author(s): Jessica Rios-Esteves , Marilyn D. Resh Wnt proteins contain palmitoleic acid, an unusual lipid modification. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on the attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here, we show that stearoyl desaturase (SCD) generates a monounsaturated fatty acid substrate that is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation. Graphical abstract Teaser Wnt signaling is dependent on the modification of Wnt proteins with palmitoleate, a relatively rare monounsaturated fatty acid. In this study, Rios-Esteves and Resh show that stearoyl CoA desaturase (SCD) generates the palmitoleate substrate for the Wnt acyltransferase Porcupine and that Porcupine transfers monounsaturated, but not saturated, fatty acids to Wnt proteins. Their finding that SCD inhibition blocks Wnt3a secretion and renders Wnt inactive suggests that SCD inhibitors could be used to prevent Wnt pathway activation in normal and disease states.

Description: Publication date: Available online 19 September 2013 Source: Cell Reports Author(s): Jessie Villanueva , Jeffrey R. Infante , Clemens Krepler , Patricia Reyes-Uribe , Minu Samanta , Hsin-Yi Chen , Bin Li , Rolf K. Swoboda , Melissa Wilson , Adina Vultur , Mizuho Fukunaba-Kalabis , Bradley Wubbenhorst , Thomas Y. Chen , Qin Liu , Katrin Sproesser , Douglas J. DeMarini , Tona M. Gilmer , Anne-Marie Martin , Ronen Marmorstein , David C. Schultz , David W. Speicher , Giorgos C. Karakousis , Wei Xu , Ravi K. Amaravadi , Xiaowei Xu , Lynn M. Schuchter , Meenhard Herlyn , Katherine L. Nathanson Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF -V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it. Graphical abstract Teaser The therapeutic efficacy of MEK and BRAF inhibitors is limited by the emergence of drug resistance. Hence, it is critical to understand all mechanisms of resistance and develop strategies to overcome them. Here, Villanueva, Herlyn, and colleagues demonstrate that concurrent MEK2 mutations and BRAF amplification confer resistance to BRAF and MEK inhibitors in melanoma. The authors demonstrate that resistant melanoma cells had sustained S6K phosphorylation; a triple combination of BRAF, MEK, and PI3K/mTOR inhibitors caused sustained growth inhibition of resistant tumors.

Description: Publication date: Available online 26 September 2013 Source: Cell Reports Author(s): Stephen M. Hinshaw , Stephen C. Harrison Accurate segregation of genetic material in eukaryotes relies on the kinetochore, a multiprotein complex that connects centromeric DNA with microtubules. In yeast and humans, two proteins—Mif2/CENP-C and Chl4/CNEP-N—interact with specialized centromeric nucleosomes and establish distinct but cross-connecting axes of chromatin-microtubule linkage. Proteins recruited by Chl4/CENP-N include a subset that regulates chromosome transmission fidelity. We show that Chl4 and a conserved member of this subset, Iml3, both from Saccharomyces cerevisiae , form a stable protein complex that interacts with Mif2 and Sgo1. We have determined the structures of an Iml3 homodimer and an Iml3-Chl4 heterodimer, which suggest a mechanism for regulating the assembly of this functional axis of the kinetochore. We propose that at the core centromere, the Chl4-Iml3 complex participates in recruiting factors, such as Sgo1, that influence sister chromatid cohesion and encourage sister kinetochore biorientation. Graphical abstract Teaser Hinshaw and Harrison describe a crystal structure of heterodimerized yeast-kinetochore proteins, Chl4 and Iml3 (human orthologs: CENP-N and CENP-L, respectively). Mutations at the Chl4-Iml3 interface perturb plasmid segregation. An N-terminal region of CENP-N (and by inference, Chl4) is known to associate with centromeric nucleosomes. The work presented here shows that the Chl4-Iml3 heterodimer binds both Mif2/CENP-C and Sgo1. The authors propose that, at centromeres, the Chl4-Iml3 complex helps recruit factors such as Sgo1, which influence sister chromatid cohesion.

Description: Publication date: Available online 26 September 2013 Source: Cell Reports Author(s): Juliane Zantke , Tomoko Ishikawa-Fujiwara , Enrique Arboleda , Claudia Lohs , Katharina Schipany , Natalia Hallay , Andrew D. Straw , Takeshi Todo , Kristin Tessmar-Raible Life is controlled by multiple rhythms. Although the interaction of the daily (circadian) clock with environmental stimuli, such as light, is well documented, its relationship to endogenous clocks with other periods is little understood. We establish that the marine worm Platynereis dumerilii possesses endogenous circadian and circalunar (monthly) clocks and characterize their interactions. The RNAs of likely core circadian oscillator genes localize to a distinct nucleus of the worm’s forebrain. The worm’s forebrain also harbors a circalunar clock entrained by nocturnal light. This monthly clock regulates maturation and persists even when circadian clock oscillations are disrupted by the inhibition of casein kinase 1δ/ε. Both circadian and circalunar clocks converge on the regulation of transcript levels. Furthermore, the circalunar clock changes the period and power of circadian behavior, although the period length of the daily transcriptional oscillations remains unaltered. We conclude that a second endogenous noncircadian clock can influence circadian clock function. Graphical abstract Teaser Tessmar-Raible and colleagues investigate the circalunar and circadian rhythms of the marine worm Platynereis dumerilii . They identify the worm’s putative core circadian clock genes, describe their transcriptional oscillations, and show that locomotor activity is a circadian-clock-controlled behavior. A pharmacological blocker of casein kinase 1∂/ε abolishes circadian clock oscillations on the molecular and behavioral level, but not the monthly synchronization of maturation, suggesting an independent circalunar clock. This circalunar clock modulates circadian behavior and transcription.

Description: Publication date: Available online 26 September 2013 Source: Cell Reports Author(s): Yadi Wu , Yifan Wang , Xiuwei H. Yang , Tiebang Kang , Yongxiang Zhao , Chi Wang , B. Mark Evers , Binhua P. Zhou LSD1 is a critical chromatin modulator that controls cellular pluripotency and differentiation through the demethylation of H3K4me1/2. Overexpression of LSD1 has been observed in many types of tumors and is correlated with its oncogenic effects in tumorigenesis. However, the mechanism leading to LSD1 upregulation in tumors remains unclear. Using an unbiased siRNA screening against all the human deubiquitinases, we identified USP28 as a bona fide deubiquitinase of LSD1. USP28 interacted with and stabilized LSD1 via deubiquitination. USP28 overexpression correlated with LSD1 upregulation in multiple cancer cell lines and breast tumor samples. Knockdown of USP28 resulted in LSD1 destabilization, leading to the suppression of cancer stem cell (CSC)-like characteristics in vitro and inhibition of tumorigenicity in vivo, which can be rescued by ectopic LSD1 expression. Our study reveals a critical mechanism underlying the epigenetic regulation by USP28 and provides another treatment approach against breast cancer. Graphical abstract Teaser LSD1 protein stabilization has been observed in many aggressive tumors. In this study, Wu, Zhou, and colleagues identified USP28 as a bona fide deubiquitinase of LSD1. USP28 overexpression correlated with LSD1 upregulation in multiple cancer cell lines and breast tumor samples. Knockdown of USP28 resulted in LSD1 destabilization, leading to the suppression of cancer stem cell (CSC)-like characteristics and inhibition of tumorigenicity. This study unveils the critical role of USP28-LSD1 axis and offers a therapeutic option for treating breast cancer.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Changmeng Cai , Housheng Hansen He , Shuai Gao , Sen Chen , Ziyang Yu , Yanfei Gao , Shaoyong Chen , Mei Wei Chen , Jesse Zhang , Musaddeque Ahmed , Yang Wang , Eric Metzger , Roland Schüle , X. Shirley Liu , Myles Brown , Steven P. Balk Lysine-Specific Demethylase 1 (LSD1, KDM1A) functions as a transcriptional corepressor through demethylation of histone 3 lysine 4 (H3K4) but has a coactivator function on some genes through mechanisms that are unclear. We show that LSD1, interacting with CoREST, associates with and coactivates androgen receptor (AR) on a large fraction of androgen-stimulated genes. A subset of these AR/LSD1-associated enhancer sites have histone 3 threonine 6 phosphorylation (H3T6ph), and these sites are further enriched for androgen-stimulated genes. Significantly, despite its coactivator activity, LSD1 still mediates H3K4me2 demethylation at these androgen-stimulated enhancers. FOXA1 is also associated with LSD1 at AR-regulated enhancer sites, and a FOXA1 interaction with LSD1 enhances binding of both proteins at these sites. These findings show that LSD1 functions broadly as a regulator of AR function, that it maintains a transcriptional repression function at AR-regulated enhancers through H3K4 demethylation, and that it has a distinct AR-linked coactivator function mediated by demethylation of other substrates. Graphical abstract Teaser Cai et al. show that lysine-specific demethylase 1 (LSD1), although generally a transcriptional corepressor through H3K4 demethylation, functions broadly as a coactivator for androgen receptor and interacts with FOXA1 on androgen-stimulated genes. LSD1-mediated H3K4 demethylation persists at these sites, indicating a distinct coactivator function mediated by demethylation of other substrates.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Raajit Rampal , Altuna Alkalin , Jozef Madzo , Aparna Vasanthakumar , Elodie Pronier , Jay Patel , Yushan Li , Jihae Ahn , Omar Abdel-Wahab , Alan Shih , Chao Lu , Patrick S. Ward , Jennifer J. Tsai , Todd Hricik , Valeria Tosello , Jacob E. Tallman , Xinyang Zhao , Danette Daniels , Qing Dai , Luisa Ciminio , Iannis Aifantis , Chuan He , Francois Fuks , Martin S. Tallman , Adolfo Ferrando , Stephen Nimer , Elisabeth Paietta , Craig B. Thompson , Jonathan D. Licht , Christopher E. Mason , Lucy A. Godley , Ari Melnick , Maria E. Figueroa , Ross L. Levine Somatic mutations in IDH1/IDH2 and TET2 result in impaired TET2-mediated conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The observation that WT1 inactivating mutations anticorrelate with TET2/IDH1/IDH2 mutations in acute myeloid leukemia (AML) led us to hypothesize that WT1 mutations may impact TET2 function. WT1 mutant AML patients have reduced 5hmC levels similar to TET2/IDH1/IDH2 mutant AML. These mutations are characterized by convergent, site-specific alterations in DNA hydroxymethylation, which drive differential gene expression more than alterations in DNA promoter methylation. WT1 overexpression increases global levels of 5hmC, and WT1 silencing reduced 5hmC levels. WT1 physically interacts with TET2 and TET3, and WT1 loss of function results in a similar hematopoietic differentiation phenotype as observed with TET2 deficiency. These data provide a role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/IDH2 and WT1 mutations define an AML subtype defined by dysregulated DNA hydroxymethylation. Graphical abstract Teaser Mutational studies in patients with acute myeloid leukemia (AML) have identified recurrent mutations in TET2 and IDH1/IDH2 , and these mutations result in a reduction in 5-hydroxymethylcytosine (5hmC) levels. Rampal et al. demonstrate that WT1 mutations anticorrelate with TET2 and IDH1/IDH2 mutations, and WT1 mutant AMLs have decreased 5hmC levels, consistent with reduced TET2 function.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Shong Lau , Daniella Rylander Ottosson , Johan Jakobsson , Malin Parmar Recent findings show that human fibroblasts can be directly programmed into functional neurons without passing via a proliferative stem cell intermediate. These findings open up the possibility of generating subtype-specific neurons of human origin for therapeutic use from fetal cell, from patients themselves, or from matched donors. In this study, we present an improved system for direct neural conversion of human fibroblasts. The neural reprogramming genes are regulated by the neuron-specific microRNA, miR-124, such that each cell turns off expression of the reprogramming genes once the cell has reached a stable neuronal fate. The regulated system can be combined with integrase-deficient vectors, providing a nonintegrative and self-regulated conversion system that rids problems associated with the integration of viral transgenes into the host genome. These modifications make the system suitable for clinical use and therefore represent a major step forward in the development of induced neurons for cell therapy. Graphical abstract Teaser Lau et al. now use miRNA targeting to build a self-regulating neural conversion system. Combined with nonintegrating vectors, this system can efficiently drive conversion of human fibroblasts into functional induced neurons (iNs) suitable for clinical applications.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Lucas Leclère , Fabian Rentzsch Patterning of the metazoan dorsoventral axis is mediated by a complex interplay of BMP signaling regulators. Repulsive guidance molecule (RGM) is a conserved BMP coreceptor that has not been implicated in axis specification. We show that NvRGM is a key positive regulator of BMP signaling during secondary axis establishment in the cnidarian Nematostella vectensis . NvRGM regulates first the generation and later the shape of a BMP-dependent Smad1/5/8 gradient with peak activity on the side opposite the  NvBMP/NvRGM/NvChordin expression domain. Full knockdown of Smad1/5/8 signaling blocks the formation of endodermal structures, the mesenteries, and the establishment of bilateral symmetry, while altering the gradient through partial NvRGM or NvBMP knockdown shifts the boundaries of asymmetric gene expression and the positioning of the mesenteries along the secondary axis. These findings provide insight into the diversification of axis specification mechanisms and identify a previously unrecognized role for RGM in BMP-mediated axial patterning. Graphical abstract Teaser Leclère and Rentzsch identify repulsive guidance molecule (RGM) as an essential regulator of the BMP morphogen gradient that controls the formation and patterning of the secondary body axis in the sea anemone Nematostella . The evolutionary conservation of RGM-BMP interactions indicates that this function might also be important for bilaterian embryogenesis.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Grace E. Kim , Jack Kronengold , Giulia Barcia , Imran H. Quraishi , Hilary C. Martin , Edward Blair , Jenny C. Taylor , Olivier Dulac , Laurence Colleaux , Rima Nabbout , Leonard K. Kaczmarek Disease-causing mutations in ion channels generally alter intrinsic gating properties such as activation, inactivation, and voltage dependence. We examined nine different mutations of the KCNT1 (Slack) Na + -activated K + channel that give rise to three distinct forms of epilepsy. All produced many-fold increases in current amplitude compared to the wild-type channel. This could not be accounted for by increases in the intrinsic open probability of individual channels. Rather, greatly increased opening was a consequence of cooperative interactions between multiple channels in a patch. The degree of cooperative gating was much greater for all of the mutant channels than for the wild-type channel, and could explain increases in current even in a mutant with reduced unitary conductance. We also found that the same mutation gave rise to different forms of epilepsy in different individuals. Our findings indicate that a major consequence of these mutations is to alter channel-channel interactions. Graphical abstract Teaser Slack KCNT1 channels regulate how neurons respond to sustained stimulation. Kim et al. characterized nine KCNT1 mutations found in epilepsy patients with severe intellectual disabilities and showed that, in isolation, channel behavior is unaltered. However, in groups, mutant channels interact with each other abnormally, increasing current that flows through the channels.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Juanjuan Ou , Hongming Miao , Yinyan Ma , Feng Guo , Jia Deng , Xing Wei , Jie Zhou , Ganfeng Xie , Hang Shi , Bingzhong Xue , Houjie Liang , Liqing Yu How cancer cells shift metabolism to aerobic glycolysis is largely unknown. Here, we show that deficiency of α/β-hydrolase domain-containing 5 (Abhd5), an intracellular lipolytic activator that is also known as comparative gene identification 58 (CGI-58), promotes this metabolic shift and enhances malignancies of colorectal carcinomas (CRCs). Silencing of Abhd5 in normal fibroblasts induces malignant transformation. Intestine-specific knockout of Abhd5 in Apc Min/+ mice robustly increases tumorigenesis and malignant transformation of adenomatous polyps. In colon cancer cells, Abhd5 deficiency induces epithelial-mesenchymal transition by suppressing the AMPKα-p53 pathway, which is attributable to increased aerobic glycolysis. In human CRCs, Abhd5 expression falls substantially and correlates negatively with malignant features. Our findings link Abhd5 to CRC pathogenesis and suggest that cancer cells develop aerobic glycolysis by suppressing Abhd5-mediated intracellular lipolysis. Graphical abstract Teaser Cancer cells shift their metabolism to aerobic glycolysis (i.e., fermentation of glucose as energy in the presence of ample oxygen), but the underlying mechanisms remain elusive. Ou et al. identify Abhd5, an activator of intracellular fat breakdown, as a suppressor of this metabolic shift and associated malignancies in colon cancer.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Emmanuel Eggermann , Yves Kremer , Sylvain Crochet , Carl C.H. Petersen Internal brain states affect sensory perception, cognition, and learning. Many neocortical areas exhibit changes in the pattern and synchrony of neuronal activity during quiet versus active behaviors. Active behaviors are typically associated with desynchronized cortical dynamics. Increased thalamic firing contributes importantly to desynchronize mouse barrel cortex during active whisker sensing. However, a whisking-related cortical state change persists after thalamic inactivation, which is mediated at least in part by acetylcholine, as we show here by using whole-cell recordings, local pharmacology, axonal calcium imaging, and optogenetic stimulation. During whisking, we find prominent cholinergic signals in the barrel cortex, which suppress spontaneous cortical activity. The desynchronized state of barrel cortex during whisking is therefore driven by at least two distinct signals with opposing functions: increased thalamic activity driving glutamatergic excitation of the cortex and increased cholinergic input suppressing spontaneous cortical activity. Graphical abstract Teaser Eggermann et al. now find that the desynchronized state of the barrel cortex during active whisker sensing is accompanied by increased cholinergic input, which suppresses slow spontaneous cortical activity in excitatory layer 2/3 barrel cortex neurons.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Nancy A. Chow , Luke D. Jasenosky , Anne E. Goldfeld Interferon γ (IFN-γ) priming sensitizes monocytes and macrophages to lipopolysaccharide (LPS) stimulation, resulting in augmented expression of a set of genes including TNF . Here, we demonstrate that IFN-γ priming of LPS-stimulated TNF transcription requires a distal TNF/LT locus element 8 kb upstream of the TNF transcription start site (hHS-8). IFN-γ stimulation leads to increased DNase I accessibility of hHS-8 and its recruitment of interferon regulatory factor 1 (IRF1), and subsequent LPS stimulation enhances H3K27 acetylation and induces enhancer RNA synthesis at hHS-8. Ablation of IRF1 or targeting the hHS-8 IRF1 binding site in vivo with Cas9 linked to the KRAB repressive domain abolishes IFN-γ priming, but does not affect LPS induction of the gene. Thus, IFN-γ poises a distal enhancer in the TNF/LT locus by chromatin remodeling and IRF1 recruitment, which then drives enhanced TNF gene expression in response to a secondary toll-like receptor (TLR) stimulus. Graphical abstract Teaser Interferon γ (IFN-γ) priming is a critical immune event that enhances the monocyte and macrophage response, particularly expression of the TNF gene, to toll-like receptor (TLR) signaling. Chow et al. demonstrate that IFN-γ priming requires a distal enhancer element within the TNF/LT locus, thereby expanding the role of distal regulatory elements in the innate immune response.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Daniel W. Hagey , Jonas Muhr Organ formation and maintenance depends on slowly self-renewing stem cells that supply an intermediate population of rapidly dividing progenitors, but how this proliferative hierarchy is regulated is unknown. By performing genome-wide single-cell and functional analyses in the cortex, we demonstrate that reduced Sox2 expression is a key regulatory signature of the transition between stem cells and rapidly dividing progenitors. In stem cells, Sox2 is expressed at high levels, which enables its repression of proproliferative genes, of which Cyclin D1 is the most potent target. Sox2 confers this function through binding to low-affinity motifs, which facilitate the recruitment of Gro/Tle corepressors in synergy with Tcf/Lef proteins. Upon differentiation, proneural factors reduce Sox2 expression, which derepresses Cyclin D1 and promotes proliferation. Our results show how concentration-dependent Sox2 occupancy of DNA motifs of varying affinities translates into recruitment of repressive complexes, which regulate the proliferative dynamics of neural stem and progenitor cells. Graphical abstract Teaser Hagey and Muhr show that high levels of Sox2 maintain stem cells of the developing cortex in a slowly self-renewing state by directly repressing cell-cycle genes. They further demonstrate that proneural protein-induced commitment to differentiation induces a rapidly dividing state via the reduction of Sox2 expression levels.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Jakub O. Westholm , Pedro Miura , Sara Olson , Sol Shenker , Brian Joseph , Piero Sanfilippo , Susan E. Celniker , Brenton R. Graveley , Eric C. Lai Circularization was recently recognized to broadly expand transcriptome complexity. Here, we exploit massive Drosophila total RNA-sequencing data, >5 billion paired-end reads from >100 libraries covering diverse developmental stages, tissues, and cultured cells, to rigorously annotate >2,500 fruit fly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and the circularization of hundreds of genes is conserved across multiple Drosophila species. We elucidate structural and sequence properties of Drosophila circular RNAs, which exhibit commonalities and distinctions from mammalian circles. Notably, Drosophila circular RNAs harbor >1,000 well-conserved canonical miRNA seed matches, especially within coding regions, and coding conserved miRNA sites reside preferentially within circularized exons. Finally, we analyze the developmental and tissue specificity of circular RNAs and note their preferred derivation from neural genes and enhanced accumulation in neural tissues. Interestingly, circular isoforms increase substantially relative to linear isoforms during CNS aging and constitute an aging biomarker. Graphical abstract Teaser Westholm et al. annotate Drosophila circular RNAs from a massive collection of total RNA-seq data, providing insights into their biogenesis and function. In particular, circularizing exons are predominantly associated with long flanking introns, are preferred locations of conserved coding miRNA sites, and accumulate to highest levels in the aging CNS.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Bo-Kuan Wu , Charles Brenner Hypermethylation-mediated tumor suppressor gene (TSG) silencing is a central epigenetic alteration in RAS-dependent tumorigenesis. Ten-eleven translocation (TET) enzymes can depress DNA methylation by hydroxylation of 5-methylcytosine (5mC) bases to 5-hydroxymethylcytosine (5hmC). Here, we report that suppression of TET1 is required for KRAS-induced DNA hypermethylation and cellular transformation. In distinct nonmalignant cell lines, oncogenic KRAS promotes transformation by inhibiting TET1 expression via the ERK-signaling pathway. This reduces chromatin occupancy of TET1 at TSG promoters, lowers levels of 5hmC, and increases levels of 5mC and 5mC-dependent transcriptional silencing. Restoration of TET1 expression by ERK pathway inhibition or ectopic TET1 reintroduction in KRAS-transformed cells reactivates TSGs and inhibits colony formation. KRAS knockdown increases TET1 expression and diminishes colony-forming ability, whereas KRAS/TET1 double knockdown bypasses the KRAS dependence of KRAS-addicted cancer cells. Thus, suppression of TET1-dependent DNA demethylation is critical for KRAS-mediated transformation. Graphical abstract Teaser Wu and Brenner now show that KRAS drives ERK-dependent mRNA suppression of TET1 and that this is necessary to achieve tumor suppressor gene hypermethylation and malignant transformation.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Zekiye Buket Yılmaz , Bente Kofahl , Patrick Beaudette , Katharina Baum , Inbal Ipenberg , Falk Weih , Jana Wolf , Gunnar Dittmar , Claus Scheidereit The mechanisms that govern proteolytic maturation or complete destruction of the precursor proteins p100 and p105 are fundamental to homeostasis and activation of NF-κB; however, they remain poorly understood. Using mass-spectrometry-based quantitative analysis of noncanonical LTβR-induced signaling, we demonstrate that stimulation induces simultaneous processing of both p100 and p105. The precursors not only form hetero-oligomers but also interact with the ATPase VCP/p97, and their induced proteolysis strictly depends on the signal response domain (SRD) of p100, suggesting that the SRD-targeting proteolytic machinery acts in cis and in trans . Separation of cellular pools by isotope labeling revealed synchronous dynamics of p105 and p100 proteolysis. The generation of p50 and p52 from their precursors depends on functional VCP/p97. We have developed quantitative mathematical models that describe the dynamics of the system and predict that p100-p105 complexes are signal responsive. Graphical abstract Teaser Proteolytic precursor processing is a hallmark of the NF-κB system. Yilmaz et al. show that in lymphotoxin-stimulated cells p100 acts upstream of p105, resulting in concurrent production of p52 and p50. Both precursors form complexes and bind to segregase (p97/VCP), which promotes proteasomal processing. The findings are supported by mass spectrometry and incorporated in quantitative mathematical models.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Lisa L. Korn , Harper G. Hubbeling , Paige M. Porrett , Qi Yang , Lisa G. Barnett , Terri M. Laufer Regulatory T cells (Tregs) are CD4 + T cells that maintain immune homeostasis and prevent autoimmunity. Like all CD4 + T cells, Tregs require antigen-specific signals via T cell receptor-major histocompatibility complex class II (TCR-MHCII) interactions for their development. However, the requirement for MHCII in Treg homeostasis in tissues such as intestinal lamina propria (LP) is unknown. We examined LP Treg homeostasis in a transgenic mouse model that lacks peripheral TCR-MHCII interactions and generation of extrathymic Tregs (iTregs). Thymically generated Tregs entered the LP of weanlings and proliferated independently of MHCII to fill the compartment. The adult LP was a closed niche; new thymic Tregs were excluded, and Tregs in parabiotic pairs were LP resident. The isolated LP niche was interleukin-2 (IL-2) independent but dependent on commensal bacteria. Thus, an LP Treg niche can be filled, isolated, and maintained independently of antigen signals and iTregs. This niche may represent a tissue-specific mechanism for maintaining immune tolerance. Graphical abstract Teaser Regulatory T cells (Tregs) maintain immune homeostasis and prevent autoimmunity. Korn et al. describe a unique Treg niche in the intestinal lamina propria that does not require T cell receptor signals for development or maintenance and is physiologically isolated from the Tregs that circulate through lymphoid organs. Maintenance of this niche is dependent upon local commensal bacteria. The authors propose that this isolated niche may represent a tissue-specific mechanism for maintaining immune tolerance.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Romain Christiano , Nagarjuna Nagaraj , Florian Fröhlich , Tobias C. Walther How cells maintain specific levels of each protein and whether that control is evolutionarily conserved are key questions. Here, we report proteome-wide steady-state protein turnover rate measurements for the evolutionarily distant but ecologically similar yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe . We find that the half-life of most proteins is much longer than currently thought and determined to a large degree by protein synthesis and dilution due to cell division. However, we detect a significant subset of proteins (∼15%) in both yeasts that are turned over rapidly. In addition, the relative abundances of orthologous proteins between the two yeasts are highly conserved across the 400 million years of evolution. In contrast, their respective turnover rates differ considerably. Our data provide a high-confidence resource for studying protein degradation in common yeast model systems. Graphical abstract Teaser Christiano et al. report protein turnover rates for S. cerevisiae and S. pombe . Overall, protein half-life is far less conserved than protein abundance. Protein turnover profiling of hrd1 Δ, an endoplasmic-reticulum-associated degradation (ERAD)-defective mutant, identifies candidate substrates for this pathway in budding yeast.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Brian C. Shonesy , Rebecca J. Bluett , Teniel S. Ramikie , Rita Báldi , Daniel J. Hermanson , Philip J. Kingsley , Lawrence J. Marnett , Danny G. Winder , Roger J. Colbran , Sachin Patel Endocannabinoid (eCB) signaling has been heavily implicated in the modulation of anxiety and depressive behaviors and emotional learning. However, the role of the most-abundant endocannabinoid 2-arachidonoylglycerol (2-AG) in the physiological regulation of affective behaviors is not well understood. Here, we show that genetic deletion of the 2-AG synthetic enzyme diacylglycerol lipase α (DAGLα) in mice reduces brain, but not circulating, 2-AG levels. DAGLα deletion also results in anxiety-like and sex-specific anhedonic phenotypes associated with impaired activity-dependent eCB retrograde signaling at amygdala glutamatergic synapses. Importantly, acute pharmacological normalization of 2-AG levels reverses both phenotypes of DAGLα-deficient mice. These data suggest 2-AG deficiency could contribute to the pathogenesis of affective disorders and that pharmacological normalization of 2-AG signaling could represent an approach for the treatment of mood and anxiety disorders. Graphical abstract Teaser The role of the primary endogenous cannabinoid 2-AG in mood and anxiety regulation is not well understood. Shonesy et al. show that deletion of a primary 2-AG synthetic enzyme, DAGLα, results in anxiety and sex-specific depressive phenotypes, which can be rapidly reversed by pharmacological normalization of endocannabinoid levels.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Kiran Batta , Magdalena Florkowska , Valerie Kouskoff , Georges Lacaud Recent reports have shown that somatic cells, under appropriate culture conditions, could be directly reprogrammed to cardiac, hepatic, or neuronal phenotype by lineage-specific transcription factors. In this study, we demonstrate that both embryonic and adult somatic fibroblasts can be efficiently reprogrammed to clonal multilineage hematopoietic progenitors by the ectopic expression of the transcription factors ERG, GATA2, LMO2, RUNX1c, and SCL. These reprogrammed cells were stably expanded on stromal cells and possessed short-term reconstitution ability in vivo. Loss of p53 function facilitated reprogramming to blood, and p53 −/− reprogrammed cells efficiently generated erythroid, megakaryocytic, myeloid, and lymphoid lineages. Genome-wide analyses revealed that generation of hematopoietic progenitors was preceded by the appearance of hemogenic endothelial cells expressing endothelial and hematopoietic genes. Altogether, our findings suggest that direct reprogramming could represent a valid alternative approach to the differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) for disease modeling and autologous blood cell therapies. Graphical abstract Teaser Batta et al. demonstrate that murine fibroblasts are reprogrammed to hematopoietic progenitors, with erythroid, megakaryocyte, and myeloid potential, by ectopic expression of hematopoietic transcription factors. Reprogramming efficiency is increased by loss of p53 function, and generation of blood cells is preceded by the appearance of hemogenic endothelium.

Description: Publication date: Available online 4 December 2014 Source: Cell Reports Author(s): Nancy F. Ramia , Michael Spilman , Li Tang , Yaming Shao , Joshua Elmore , Caryn Hale , Alexis Cocozaki , Nilakshee Bhattacharya , Rebecca M. Terns , Michael P. Terns , Hong Li , Scott M. Stagg The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA) and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes. Graphical abstract Teaser Ramia et al. show that the helical core of the type III-B Cmr CRISPR-Cas effector complex, made up of multiple Cmr4 subunits, forms the platform for a corresponding number of cleavages of the target RNA. Comparison with the type I-E Cascade structure reveals strikingly similar mechanisms of crRNA and target binding.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Christos G. Gkogkas , Arkady Khoutorsky , Ruifeng Cao , Seyed Mehdi Jafarnejad , Masha Prager-Khoutorsky , Nikolaos Giannakas , Archontia Kaminari , Apostolia Fragkouli , Karim Nader , Theodore J. Price , Bruce W. Konicek , Jeremy R. Graff , Athina K. Tzinia , Jean-Claude Lacaille , Nahum Sonenberg Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS phenotypes. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1 −/y ), we show that phosphorylation of the mRNA 5′ cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1 −/y mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS. Graphical abstract Teaser Fragile X syndrome (FXS) is caused by dysregulation of translation in the brain. Gkogkas et al. show that phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) is increased in FXS postmortem brains and Fmr1 −/y mice. Downregulation of eIF4E phosphorylation in Fmr1 −/y mice rescues defects in dendritic spine morphology, synaptic plasticity, and social interaction via normalization of MMP-9 expression.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Wei-Qun Fang , Wei-Wei Chen , Liwen Jiang , Kai Liu , Wing-Ho Yung , Amy K.Y. Fu , Nancy Y. Ip The functional integrity of the neocortex depends upon proper numbers of excitatory and inhibitory neurons; however, the consequences of dysregulated neuronal production during the development of the neocortex are unclear. As excess cortical neurons are linked to the neurodevelopmental disorder autism, we investigated whether the overproduction of neurons leads to neocortical malformation and malfunction in mice. We experimentally increased the number of pyramidal neurons in the upper neocortical layers by using the small molecule XAV939 to expand the intermediate progenitor population. The resultant overpopulation of neurons perturbs development of dendrites and spines of excitatory neurons and alters the laminar distribution of interneurons. Furthermore, these phenotypic changes are accompanied by dysregulated excitatory and inhibitory synaptic connection and balance. Importantly, these mice exhibit behavioral abnormalities resembling those of human autism. Thus, our findings collectively suggest a causal relationship between neuronal overproduction and autism-like features, providing developmental insights into the etiology of autism. Graphical abstract Teaser Fang et al. generated a mouse model with excessive excitatory neurons in the neocortex by manipulating embryonic neurogenesis. Overproduction of neurons results in autism-like anatomical and behavioral features. These findings suggest a causal relationship between overproduction of neurons and cortical malfunction and provide developmental insights into the etiology of autism.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Lauren J. Manderfield , Kurt A. Engleka , Haig Aghajanian , Mudit Gupta , Steven Yang , Li Li , Julie E. Baggs , John B. Hogenesch , Eric N. Olson , Jonathan A. Epstein Loss of Pax3, a developmentally regulated transcription factor expressed in premigratory neural crest, results in severe developmental defects and embryonic lethality. Although Pax3 mutations produce profound phenotypes, the intrinsic transcriptional activation exhibited by Pax3 is surprisingly modest. We postulated the existence of transcriptional coactivators that function with Pax3 to mediate developmental functions. A high-throughput screen identified the Hippo effector proteins Taz and Yap65 as Pax3 coactivators. Synergistic coactivation of target genes by Pax3-Taz/Yap65 requires DNA binding by Pax3, is Tead independent, and is regulated by Hippo kinases Mst1 and Lats2. In vivo, Pax3 and Yap65 colocalize in the nucleus of neural crest progenitors in the dorsal neural tube. Neural crest deletion of Taz and Yap65 results in embryo-lethal neural crest defects and decreased expression of the Pax3 target gene, Mitf . These results suggest that Pax3 activity is regulated by the Hippo pathway and that Pax factors are Hippo effectors. Graphical abstract Teaser Hippo signaling is a conserved kinase cascade that mediates transcription through a Yap65/Taz-Tead complex and governs organ size. Manderfield et al. have identified Yap65 and Taz as coactivators of Pax factors and established neural crest as a novel site of Hippo signaling. The Pax3-Yap65/Taz complex is regulated by upstream Hippo kinases and is Tead independent. Deletion of Yap65 and Taz in neural crest generated differentiation defects in specific neural crest derivatives.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Chris M. Woodard , Brian A. Campos , Sheng-Han Kuo , Melissa J. Nirenberg , Michael W. Nestor , Matthew Zimmer , Eugene V. Mosharov , David Sulzer , Hongyan Zhou , Daniel Paull , Lorraine Clark , Eric E. Schadt , Sergio Pablo Sardi , Lee Rubin , Kevin Eggan , Mathew Brock , Scott Lipnick , Mahendra Rao , Stephen Chang , Aiqun Li , Scott A. Noggle Parkinson’s disease (PD) has been attributed to a combination of genetic and nongenetic factors. We studied a set of monozygotic twins harboring the heterozygous glucocerebrosidase mutation ( GBA N370S) but clinically discordant for PD. We applied induced pluripotent stem cell (iPSC) technology for PD disease modeling using the twins’ fibroblasts to evaluate and dissect the genetic and nongenetic contributions. Utilizing fluorescence-activated cell sorting, we obtained a homogenous population of “footprint-free” iPSC-derived midbrain dopaminergic (mDA) neurons. The mDA neurons from both twins had ∼50% GBA enzymatic activity, ∼3-fold elevated α-synuclein protein levels, and a reduced capacity to synthesize and release dopamine. Interestingly, the affected twin’s neurons showed an even lower dopamine level, increased monoamine oxidase B (MAO-B) expression, and impaired intrinsic network activity. Overexpression of wild-type GBA and treatment with MAO-B inhibitors normalized α-synuclein and dopamine levels, suggesting a combination therapy for the affected twin. Graphical abstract Teaser Woodard et al. studied a set of identical twins harboring GBA N370S mutation but clinically discordant for Parkinson's disease (PD). Using iPSC technology, they found that enzymes GBA and MAO-B could be therapeutic targets in PD. These findings shed light into future studies using iPSCs to model idiopathic PD cases.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Hyokjoon Kwon , Sarnia Laurent , Yan Tang , Haihong Zong , Pratibha Vemulapalli , Jeffrey E. Pessin Adipose tissue inflammation is one pathway shown to mediate insulin resistance in obese humans and rodents. Obesity induces dynamic cellular changes in adipose tissue to increase proinflammatory cytokines and diminish anti-inflammatory cytokines. However, we have found that anti-inflammatory interleukin-13 (IL-13) is unexpectedly induced in adipose tissue of obese humans and high-fat diet (HFD)-fed mice, and the source of IL-13 is primarily the adipocyte. Moreover, HFD-induced proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and IL-1β mediate IL-13 production in adipocytes in an IKKβ-dependent manner. In contrast, adipocyte-specific IKKβ-deficient mice show diminished IL-13 expression and enhanced inflammation after HFD feeding, resulting in a worsening of the insulin-resistant state. Together these data demonstrate that although IKKβ activates the expression of proinflammatory mediators, in adipocytes, IKKβ signaling also induces the expression of the anti-inflammatory cytokine IL-13, which plays a unique protective role by limiting adipose tissue inflammation and insulin resistance. Graphical abstract Teaser IKKβ is known to be a proinflammatory mediator. However, IKKβ in adipocytes also mediates IL-13 expression to suppress high-fat-diet-induced inflammation in adipose tissue. This feedback mechanism may be the molecular basis of diet-induced chronic low-grade inflammation, resulting in systemic insulin resistance.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Morgane Belle , David Godefroy , Chloé Dominici , Céline Heitz-Marchaland , Pavol Zelina , Farida Hellal , Frank Bradke , Alain Chédotal Clearing techniques have been developed to transparentize mouse brains, thereby preserving 3D structure, but their complexity has limited their use. Here, we show that immunolabeling of axonal tracts followed by optical clearing with solvents (3DISCO) and light-sheet microscopy reveals brain connectivity in mouse embryos and postnatal brains. We show that the Robo3 receptor is selectively expressed by medial habenula axons forming the fasciculus retroflexus (FR) and analyzed the development of this commissural tract in mutants of the Slit/Robo and DCC/Netrin pathways. Netrin-1 and DCC are required to attract FR axons to the midline, but the two mutants exhibit specific and heterogeneous axon guidance defects. Moreover, floor-plate-specific deletion of Slit ligands with a conditional Slit2 allele perturbs not only midline crossing by FR axons but also their anteroposterior distribution. In conclusion, this method represents a unique and powerful imaging tool to study axonal connectivity in mutant mice. Graphical abstract Teaser Clearing techniques have recently been developed to look at mouse brains, but they are complex and expensive. Belle et al. now describe a simple procedure that combines immunolabeling, solvent-based clearing, and light-sheet fluorescence microscopy. This technique allows large-scale screening of axon guidance defects and other developmental disorders in mutant mice.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Emmanuelle Godefroy , Anne Gallois , Juliana Idoyaga , Miriam Merad , Navpreet Tung , Ngozi Monu , Yvonne Saenger , Yichun Fu , Rajesh Ravindran , Bali Pulendran , Francine Jotereau , Sergio Trombetta , Nina Bhardwaj Matrix metalloproteinase-2 (MMP-2) is involved in several physiological mechanisms, including wound healing and tumor progression. We show that MMP-2 directly stimulates dendritic cells (DCs) to both upregulate OX40L on the cell surface and secrete inflammatory cytokines. The mechanism underlying DC activation includes physical association with Toll-like receptor-2 (TLR2), leading to NF-κB activation, OX40L upregulation on DCs, and ensuing T H 2 differentiation. Significantly, MMP-2 polarizes T cells toward type 2 responses in vivo, in a TLR2-dependent manner. MMP-2-dependent type 2 polarization may represent a key immune regulatory mechanism for protection against a broad array of disorders, such as inflammatory, infectious, and autoimmune diseases, which can be hijacked by tumors to evade immunity. Graphical abstract Teaser Godefroy et al. now demonstrate that matrix metalloproteinase-2 (MMP-2) directly interacts with and activates dendritic cells (DCs) via Toll-like receptor-2. MMP-2-exposed DCs upregulate OX40L, promoting type 2 polarization both in vitro and in vivo. This may represent a key immune regulatory mechanism involved in a variety of inflammatory disorders.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Catalin Chimerel , Edward Emery , David K. Summers , Ulrich Keyser , Fiona M. Gribble , Frank Reimann It has long been speculated that metabolites, produced by gut microbiota, influence host metabolism in health and diseases. Here, we reveal that indole, a metabolite produced from the dissimilation of tryptophan, is able to modulate the secretion of glucagon-like peptide-1 (GLP-1) from immortalized and primary mouse colonic L cells. Indole increased GLP-1 release during short exposures, but it reduced secretion over longer periods. These effects were attributed to the ability of indole to affect two key molecular mechanisms in L cells. On the one hand, indole inhibited voltage-gated K + channels, increased the temporal width of action potentials fired by L cells, and led to enhanced Ca 2+ entry, thereby acutely stimulating GLP-1 secretion. On the other hand, indole slowed ATP production by blocking NADH dehydrogenase, thus leading to a prolonged reduction of GLP-1 secretion. Our results identify indole as a signaling molecule by which gut microbiota communicate with L cells and influence host metabolism. Graphical abstract Teaser Indole is the main metabolite produced by gut bacteria from tryptophan. Chimerel et al. demonstrate that indole modulates the hormone secretion of enteroendocrine L cells and reveal the molecular mechanism behind this modulation. These findings suggest that the production of indole by bacteria could have a major impact on host metabolism.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Qing-ming Gao , Keshun Yu , Ye Xia , M.B. Shine , Caixia Wang , DuRoy Navarre , Aardra Kachroo , Pradeep Kachroo The plant galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) have been linked to the anti-inflammatory and cancer benefits of a green leafy vegetable diet in humans due to their ability to regulate the levels of free radicals like nitric oxide (NO). Here, we show that DGDG contributes to plant NO as well as salicylic acid biosynthesis and is required for the induction of systemic acquired resistance (SAR). In contrast, MGDG regulates the biosynthesis of the SAR signals azelaic acid (AzA) and glycerol-3-phosphate (G3P) that function downstream of NO. Interestingly, DGDG is also required for AzA-induced SAR, but MGDG is not. Notably, transgenic expression of a bacterial glucosyltransferase is unable to restore SAR in dgd1 plants even though it does rescue their morphological and fatty acid phenotypes. These results suggest that MGDG and DGDG are required at distinct steps and function exclusively in their individual roles during the induction of SAR. Graphical abstract Teaser The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) constitute ∼80% of total membrane lipids in plants. Gao et al. now show that these galactolipids function nonredundantly to regulate systemic acquired resistance (SAR). Furthermore, they show that the terminal galactose on the α-galactose-β-galactose head group of DGDG is critical for SAR.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Rafael B. Blasco , Elif Karaca , Chiara Ambrogio , Taek-Chin Cheong , Emre Karayol , Valerio G. Minero , Claudia Voena , Roberto Chiarle Generation of genetically engineered mouse models (GEMMs) for chromosomal translocations in the endogenous loci by a knockin strategy is lengthy and costly. The CRISPR/Cas9 system provides an innovative and flexible approach for genome engineering of genomic loci in vitro and in vivo. Here, we report the use of the CRISPR/Cas9 system for engineering a specific chromosomal translocation in adult mice in vivo. We designed CRISPR/Cas9 lentiviral vectors to induce cleavage of the murine endogenous Eml4 and Alk loci in order to generate the Eml4-Alk gene rearrangement recurrently found in non-small-cell lung cancers (NSCLCs). Intratracheal or intrapulmonary inoculation of lentiviruses induced Eml4-Alk gene rearrangement in lung cells in vivo. Genomic and mRNA sequencing confirmed the genome editing and the production of the Eml4-Alk fusion transcript. All mice developed Eml4-Alk -rearranged lung tumors 2 months after the inoculation, demonstrating that the CRISPR/Cas9 system is a feasible and simple method for the generation of chromosomal rearrangements in vivo. Graphical abstract Teaser Blasco et al. demonstrate that CRISPR/Cas9 technology can be exploited to generate targeted chromosomal rearrangements in vivo in mice in a time- and cost-effective approach.

Description: Publication date: Available online 26 November 2014 Source: Cell Reports Author(s): Andrea Galmozzi , Si B. Sonne , Svetlana Altshuler-Keylin , Yutaka Hasegawa , Kosaku Shinoda , Ineke H.N. Luijten , Jae Won Chang , Louis Z. Sharp , Benjamin F. Cravatt , Enrique Saez , Shingo Kajimura Obesity develops when energy intake chronically exceeds energy expenditure. Because brown adipose tissue (BAT) dissipates energy in the form of heat, increasing energy expenditure by augmenting BAT-mediated thermogenesis may represent an approach to counter obesity and its complications. The ability of BAT to dissipate energy is dependent on expression of mitochondrial uncoupling protein 1 (UCP1). To facilitate the identification of pharmacological modulators of BAT UCP1 levels, which may have potential as antiobesity medications, we developed a transgenic model in which luciferase activity faithfully mimics endogenous UCP1 expression and its response to physiologic stimuli. Phenotypic screening of a library using cells derived from this model yielded a small molecule that increases UCP1 expression in brown fat cells and mice. Upon adrenergic stimulation, compound-treated mice showed increased energy expenditure. These tools offer an opportunity to identify pharmacologic modulators of UCP1 expression and uncover regulatory pathways that impact BAT-mediated thermogenesis. Graphical abstract Teaser Pharmacological activation of brown adipose tissue (BAT) thermogenesis and energy dissipation, a process mediated by UCP1, may be useful to counter the energy imbalance that engenders obesity. Galmozzi et al. have developed an in vivo model to monitor UCP1 expression in real time and identified a small molecule that increases UCP1 levels. Mice treated with this molecule show greater energy expenditure upon adrenergic stimulation. Discovery of compounds with this ability is an important stride toward enhancing BAT function in obese individuals.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Yanmeng Guo , Yuping Wang , Qingxiu Wang , Zuoren Wang In Drosophila larvae, the class IV dendritic arborization (da) neurons are polymodal nociceptors. Here, we show that ppk26 (CG8546) plays an important role in mechanical nociception in class IV da neurons. Our immunohistochemical and functional results demonstrate that ppk26 is specifically expressed in class IV da neurons. Larvae with mutant ppk26 showed severe behavioral defects in a mechanical nociception behavioral test but responded to noxious heat stimuli comparably to wild-type larvae. In addition, functional studies suggest that ppk26 and ppk (also called ppk1 ) function in the same pathway, whereas piezo functions in a parallel pathway. Consistent with these functional results, we found that PPK and PPK26 are interdependent on each other for their cell surface localization. Our work indicates that PPK26 and PPK might form heteromeric DEG/ENaC channels that are essential for mechanotransduction in class IV da neurons. Graphical abstract Teaser Sensing painful stimuli is of vital importance for animal survival. Guo et al. now find that PPK26 is selectively expressed in class IV dendritic arborization neurons and contributes to mechanical nociception but not thermal nociception in Drosophila larvae.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Amol Ugale , Gudmundur L. Norddahl , Martin Wahlestedt , Petter Säwén , Pekka Jaako , Cornelis Jan Pronk , Shamit Soneji , Jörg Cammenga , David Bryder Studies of developmental pathways of hematopoietic stem cells (HSCs) have defined lineage relationships throughout the blood system. This is relevant to acute myeloid leukemia (AML), where aggressiveness and therapeutic responsiveness can be influenced by the initial stage of transformation. To address this, we generated a mouse model in which the mixed-lineage leukemia/eleven-nineteen-leukemia (MLL-ENL) transcription factor can be conditionally activated in any cell type. We show that AML can originate from multiple hematopoietic progenitor subsets with granulocytic and monocytic potential, and that the normal developmental position of leukemia-initiating cells influences leukemic development. However, disease failed to arise from HSCs. Although it maintained or upregulated the expression of target genes associated with leukemic development, MLL-ENL dysregulated the proliferative and repopulating capacity of HSCs. Therefore, the permissiveness for development of AML may be associated with a narrower window of differentiation than was previously appreciated, and hijacking the self-renewal capacity of HSCs by a potent oncogene is insufficient for leukemic development. Graphical abstract Teaser The cellular origin of leukemia driven by MLL fusions has been suggested to underlie heterogeneity in aggressiveness and prognosis. Ugale et al. now use an inducible MLL-ENL mouse model to study leukemia initiation and competence throughout the hematopoietic hierarchy. Although AML development could originate from multiple progenitor subsets, the most primitive stem cells were unexpectedly unable to initiate disease.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Nila Roy Choudhury , Jakub S. Nowak , Juan Zuo , Juri Rappsilber , Steven H. Spoel , Gracjan Michlewski RNA binding proteins have thousands of cellular RNA targets and often exhibit opposite or passive molecular functions. Lin28a is a conserved RNA binding protein involved in pluripotency and tumorigenesis that was previously shown to trigger TuT4-mediated pre-let-7 uridylation, inhibiting its processing and targeting it for degradation. Surprisingly, despite binding to other pre-microRNAs (pre-miRNAs), only pre-let-7 is efficiently uridylated by TuT4. Thus, we hypothesized the existence of substrate-specific cofactors that stimulate Lin28a-mediated pre-let-7 uridylation or restrict its functionality on non-let-7 pre-miRNAs. Through RNA pull-downs coupled with quantitative mass spectrometry, we identified the E3 ligase Trim25 as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. We show that Trim25 binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing for more efficient Lin28a-mediated uridylation. These findings reveal that protein-modifying enzymes, only recently shown to bind RNA, can guide the function of canonical ribonucleoprotein (RNP) complexes in cis , thereby providing an additional level of specificity. Graphical abstract Teaser Lin28a triggers TuT4-mediated pre-let-7 uridylation. Despite binding to other pre-microRNAs, only pre-let-7 is efficiently uridylated by TuT4. Choudhury et al. show that Trim25 is an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. These findings reveal that Trim25 can guide the function of canonical RNP complexes in cis , thereby providing an additional level of specificity.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Anita Parmigiani , Aida Nourbakhsh , Boxiao Ding , Wei Wang , Young Chul Kim , Konstantin Akopiants , Kun-Liang Guan , Michael Karin , Andrei V. Budanov The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a sensor of different environmental conditions and regulator of cell growth, metabolism, and autophagy. mTORC1 is activated by Rag GTPases, working as RagA:RagB and RagC:RagD heterodimers. Rags control mTORC1 activity by tethering mTORC1 to the lysosomes where it is activated by Rheb GTPase. RagA:RagB, active in its GTP-bound form, is inhibited by GATOR1 complex, a GTPase-activating protein, and GATOR1 is in turn negatively regulated by GATOR2 complex. Sestrins are stress-responsive proteins that inhibit mTORC1 via activation of AMP-activated protein kinase (AMPK) and tuberous sclerosis complex. Here we report an AMPK-independent mechanism of mTORC1 inhibition by Sestrins mediated by their interaction with GATOR2. As a result of this interaction, the Sestrins suppress mTOR lysosomal localization in a Rag-dependent manner. This mechanism is potentially involved in mTORC1 regulation by amino acids, rotenone, and tunicamycin, connecting stress response with mTORC1 inhibition. Graphical abstract Teaser The mTORC1 kinase integrates various environmental signals to regulate cell growth and metabolism. Parmigiani et al. identified a mechanism of mTORC1 regulation by Sestrins via interaction with GATOR2 and suppression of mTOR lysosomal localization.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Nicholas J. Bessman , Atrish Bagchi , Kathryn M. Ferguson , Mark A. Lemmon The epidermal growth factor receptor (EGFR) plays pivotal roles in development and is mutated or overexpressed in several cancers. Despite recent advances, the complex allosteric regulation of EGFR remains incompletely understood. Through efforts to understand why the negative cooperativity observed for intact EGFR is lost in studies of its isolated extracellular region (ECR), we uncovered unexpected relationships between ligand binding and receptor dimerization. The two processes appear to compete. Surprisingly, dimerization does not enhance ligand binding (although ligand binding promotes dimerization). We further show that simply forcing EGFR ECRs into preformed dimers without ligand yields ill-defined, heterogeneous structures. Finally, we demonstrate that extracellular EGFR-activating mutations in glioblastoma enhance ligand-binding affinity without directly promoting EGFR dimerization, suggesting that these oncogenic mutations alter the allosteric linkage between dimerization and ligand binding. Our findings have important implications for understanding how EGFR and its relatives are activated by specific ligands and pathological mutations. Graphical abstract Teaser Although the epidermal growth factor receptor (EGFR) was the first growth factor receptor for which ligand-induced dimerization was established, the relationship between growth factor binding and EGFR dimerization remains unclear. Bessman et al. use a range of biophysical methods to study growth factor binding to EGFR variants, including those found in glioblastoma, to shed light on ligand-specific allosteric control of EGFR.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Kristen L. Karlin , Gourish Mondal , Jessica K. Hartman , Siddhartha Tyagi , Sarah J. Kurley , Chris S. Bland , Tiffany Y.T. Hsu , Alexander Renwick , Justin E. Fang , Ilenia Migliaccio , Celetta Callaway , Amritha Nair , Rocio Dominguez-Vidana , Don X. Nguyen , C. Kent Osborne , Rachel Schiff , Li-Yuan Yu-Lee , Sung Y. Jung , Dean P. Edwards , Susan G. Hilsenbeck , Jeffrey M. Rosen , Xiang H.-F. Zhang , Chad A. Shaw , Fergus J. Couch , Thomas F. Westbrook Defining the molecular networks that drive breast cancer has led to therapeutic interventions and improved patient survival. However, the aggressive triple-negative breast cancer subtype (TNBC) remains recalcitrant to targeted therapies because its molecular etiology is poorly defined. In this study, we used a forward genetic screen to discover an oncogenic network driving human TNBC. S CYL1, T EX14, and P LK1 (“STP axis”) cooperatively trigger degradation of the REST tumor suppressor protein, a frequent event in human TNBC. The STP axis induces REST degradation by phosphorylating a conserved REST phospho-degron and bridging REST interaction with the ubiquitin-ligase βTRCP. Inhibition of the STP axis leads to increased REST protein levels and impairs TNBC transformation, tumor progression, and metastasis. Expression of the STP axis correlates with low REST protein levels in human TNBCs and poor clinical outcome for TNBC patients. Our findings demonstrate that the STP-REST axis is a molecular driver of human TNBC. Graphical abstract Teaser Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype for which the molecular drivers are poorly understood. Karlin et al. now demonstrate that S CYL1, T EX14, and P LK1 (“STP axis”) cooperatively trigger degradation of the REST tumor suppressor protein, a frequent event that may provide a therapeutic entry point for human TNBC.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Christina L. Zheng , Nicholas J. Wang , Jongsuk Chung , Homayoun Moslehi , J. Zachary Sanborn , Joseph S. Hur , Eric A. Collisson , Swapna S. Vemula , Agne Naujokas , Kami E. Chiotti , Jeffrey B. Cheng , Hiva Fassihi , Andrew J. Blumberg , Celeste V. Bailey , Gary M. Fudem , Frederick G. Mihm , Bari B. Cunningham , Isaac M. Neuhaus , Wilson Liao , Dennis H. Oh , James E. Cleaver , Philip E. LeBoit , Joseph F. Costello , Alan R. Lehmann , Joe W. Gray , Paul T. Spellman , Sarah T. Arron , Nam Huh , Elizabeth Purdom , Raymond J. Cho Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs) arising in an XPC −/− background. XPC −/− cells lack global genome nucleotide excision repair (GG-NER), thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk. Graphical abstract Teaser Zheng et al. report that variable mutation densities within cancer genomes result from differential access of DNA repair machinery, imposed by chromatin state. By showing that transcription restores DNA repair to tightly packaged DNA, their study reveals natural differences in expression level as a potentially important modulator of oncogene mutation rate.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Ana Freije , Rut Molinuevo , Laura Ceballos , Marta Cagigas , Pilar Alonso-Lecue , René Rodriguez , Pablo Menendez , Daniel Aberdam , Ernesto De Diego , Alberto Gandarillas Tumor suppressor p53 is a major cellular guardian of genome integrity, and its inactivation is the most frequent genetic alteration in cancer, rising up to 80% in squamous cell carcinoma (SCC). By adapting the small hairpin RNA (shRNA) technology, we inactivated endogenous p53 in primary epithelial cells from the epidermis of human skin. We show that either loss of endogenous p53 or overexpression of a temperature-sensitive dominant-negative conformation triggers a self-protective differentiation response, resulting in cell stratification and expulsion. These effects follow DNA damage and exit from mitosis without cell division. p53 preserves the proliferative potential of the stem cell compartment and limits the power of proto-oncogene MYC to drive cell cycle stress and differentiation. The results provide insight into the role of p53 in self-renewal homeostasis and help explain why p53 mutations do not initiate skin cancer but increase the likelihood that cancer cells will appear. Graphical abstract Teaser The p53 tumor suppressor is frequently inactivated in squamous cell carcinoma, yet loss of p53 does not initiate nonmelanoma skin cancer, suggesting that epithelial skin cells have self-protective mechanisms. Freije et al. show that p53 enhances proliferation and inhibits differentiation in keratinocytes by preventing endoreplication. p53 loss leads to squamous differentiation and expulsion of mutant cells, which may confer the epidermis with a molecular protective response.

Description: Publication date: 20 November 2014 Source: Cell Reports, Volume 9, Issue 4 Author(s): Andreas Gewies , Oliver Gorka , Hanna Bergmann , Konstanze Pechloff , Franziska Petermann , Katharina M. Jeltsch , Martina Rudelius , Mark Kriegsmann , Wilko Weichert , Marion Horsch , Johannes Beckers , Wolfgang Wurst , Mathias Heikenwalder , Thomas Korn , Vigo Heissmeyer , Jürgen Ruland The paracaspase Malt1 is a central regulator of antigen receptor signaling that is frequently mutated in human lymphoma. As a scaffold, it assembles protein complexes for NF-κB activation, and its proteolytic domain cleaves negative NF-κB regulators for signal enforcement. Still, the physiological functions of Malt1-protease are unknown. We demonstrate that targeted Malt1-paracaspase inactivation induces a lethal inflammatory syndrome with lymphocyte-dependent neurodegeneration in vivo. Paracaspase activity is essential for regulatory T cell (Treg) and innate-like B cell development, but it is largely dispensable for overcoming Malt1-dependent thresholds for lymphocyte activation. In addition to NF-κB inhibitors, Malt1 cleaves an entire set of mRNA stability regulators, including Roquin-1, Roquin-2, and Regnase-1, and paracaspase inactivation results in excessive interferon gamma (IFNγ) production by effector lymphocytes that drive pathology. Together, our results reveal distinct threshold and modulatory functions of Malt1 that differentially control lymphocyte differentiation and activation pathways and demonstrate that selective paracaspase blockage skews systemic immunity toward destructive autoinflammation. Graphical abstract Teaser The paracaspase Malt1 is a key regulator of antigen receptor signaling and frequently mutated in human lymphoma. Gewies et al. demonstrate that the proteolytic function of Malt1 is largely dispensable for lymphocyte activation but critical for regulatory T cell (Treg) and innate-like B cell development and protection from IFNγ-mediated autoinflammation in vivo.