The following alphabetical list represents papers published in 2018 with at least one Whitehead author (in red). Not all of this work was done at the Whitehead Institute. Some of these papers are collaborations with scientists elsewhere. The papers are gathered from PubMed and from Science Citation Index (also known as the Web of Science) Preceding the bibliography is an alphabetical list of the titles of the papers followed by the first author.

P.S. The journal links only work if you have a license to those respective online journals.

2018 Titles :

Agarwal , V., Subtelny, A.O., Thiru, P., Ulitsky, I., and Bartel, D.P. (2018). Predicting microRNA targeting efficacy in Drosophila. Genome biology 19(1) : 152. MicroRNAs (miRNAs) are short regulatory RNAs that derive from hairpin precursors. Important for understanding the functional roles of miRNAs is the ability to predict the messenger RNA (mRNA) targets most responsive to each miRNA. Progress towards developing quantitative models of miRNA targeting in Drosophila and other invertebrate species has lagged behind that of mammals due to the paucity of datasets measuring the effects of miRNAs on mRNA levels. RESULTS: We acquired datasets suitable for the quantitative study of miRNA targeting in Drosophila. Analyses of these data expanded the types of regulatory sites known to be effective in flies, expanded the mRNA regions with detectable targeting to include 5' untranslated regions, and identified features of site context that correlate with targeting efficacy in fly cells. Updated evolutionary analyses evaluated the probability of conserved targeting for each predicted site and indicated that more than a third of the Drosophila genes are preferentially conserved targets of miRNAs. Based on these results, a quantitative model was developed to predict targeting efficacy in insects. This model performed better than existing models, and it drives the most recent version, v7, of TargetScanFly. CONCLUSIONS: Our evolutionary and functional analyses expand the known scope of miRNA targeting in flies and other insects. The existence of a quantitative model that has been developed and trained using Drosophila data will provide a valuable resource for placing miRNAs into gene regulatory networks of this important experimental organism. Full Text

Ahn, W.S., Dong, W.T., Zhang, Z., Cantor, J.R., Sabatini, D.M., Iliopoulos, O., and Stephanopoulos, G. (2018). Glyceraldehyde 3-phosphate dehydrogenase modulates nonoxidative pentose phosphate pathway to provide anabolic precursors in hypoxic tumor cells. Aiche Journal 64, 4289-4296. Cancer cells exhibit enhanced lactate production to satisfy biosynthetic adenosine triphosphate requirements and also supply ribose 5-phosphate (R5P) and nicotinamide adenine dinucleotide phosphate via the pentose phosphate pathway (PPP). Yet, little is known about the mechanism by which glycolytic flux is diverted to PPP to fulfill the increased demand for anabolic precursors and reducing equivalents. Here we show, using a C-13-labeling methodology quantifying glycolysis and the PPP metabolism, that hypoxic cancer cells not only increase net glycolytic flux but also activate the exchange fluxes catalyzed by aldolase and transaldolase. The increased carbon exchange in the upward direction promotes the supplementation of R5P through the nonoxidative PPP and essentially controls the anaplerosis of upper glycolytic metabolites consumed for biosynthesis. This cascade of events is regulated by glyceraldehyde 3-phosphate dehydrogenase which plays a critical role in diverting metabolites for the synthesis of nucleotide precursors and thus acts as a limiting enzyme under hypoxia. Full Text

Alkan, H.F., Walter, K.E., Luengo, A., Madreiter-Sokolowski, C.T., Stryeck, S., Lau, A.N., Al-Zoughbi, W., Lewis, C.A. , Thomas, C.J., Hoefler, G., et al. (2018). Cytosolic Aspartate Availability Determines Cell Survival When Glutamine Is Limiting. Cell metabolism [Epub ahead of print] . Mitochondrial function is important for aspartate biosynthesis in proliferating cells. Here, we show that mitochondrial aspartate export via the aspartate-glutamate carrier 1 (AGC1) supports cell proliferation and cellular redox homeostasis. Insufficient cytosolic aspartate delivery leads to cell death when TCA cycle carbon is reduced following glutamine withdrawal and/or glutaminase inhibition. Moreover, loss of AGC1 reduces allograft tumor growth that is further compromised by treatment with the glutaminase inhibitor CB-839. Together, these findings argue that mitochondrial aspartate export sustains cell survival in low-glutamine environments and AGC1 inhibition can synergize with glutaminase inhibition to limit tumor growth. Full Text

Arranz-Solis, D., Regidor-Cerrillo, J., Lourido, S.., Ortega-Mora, L.M., and Saeij, J.P.J. (2018). Toxoplasma CRISPR/Cas9 constructs are functional for gene disruption in Neospora caninum. International journal for parasitology [Epub ahead of print]. Herein we describe, to our knowledge for the first time the use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 (CRISPR/Cas9) system for genome editing of Neospora caninum, an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide. By using plasmids containing the CRISPR/Cas9 components adapted to the closely related parasite Toxoplasma gondii, we successfully knocked out a green fluorescent protein (GFP) in an Nc-1 GFP-expressing strain, and efficiently disrupted the NcGRA7 gene in the Nc-Spain7 isolate by insertion of a pyrimethamine resistance cassette. The successful use of this technology in N. caninum lays the foundation for an efficient, targeted gene modification tool in this parasite. Full Text

Atabay, K.D., LoCascio, S.A., de Hoog, T., and Reddien, P.W. (2018). Self-organization and progenitor targeting generate stable patterns in planarian regeneration. Science[Epub ahead of print] . During animal regeneration cells must organize into discrete and functional systems. We show that self-organization, along with patterning cues, govern progenitor behavior in planarian regeneration. Surgical paradigms allowed manipulation of planarian eye regeneration in predictable locations and numbers, generating alternative stable neuroanatomical states for wild-type animals with multiple functional ectopic eyes. We utilized animals with multiple ectopic eyes and eye transplantation to demonstrate that broad progenitor specification, combined with self-organization, allows anatomy maintenance during regeneration. We propose a model for regenerative progenitors involving (i) migratory targeting cues, (ii) self-organization into existing or regenerating eyes, and (iii) a broad zone, associated with coarse progenitor specification, in which eyes can be targeted by progenitors. These three properties help explain how tissues can be organized during regeneration. Full Text

Aviles-Pagan , E.E., and Orr-Weaver, T.L. (2018). Activating Embryonic Development in Drosophila. Seminars in cell & developmental biology . [Epub ahead of print]. The transition from oocyte to embryo marks the onset of development. This process requires complex regulation to link developmental signals with profound changes in mRNA translation, cell cycle control, and metabolism. This control is beginning to be understood for most organisms, and research in the fruit fly Drosophila melanogaster has generated new insights. Recent findings have increased our understanding of the roles played by hormone and Ca(2+) signaling events as well as metabolic remodeling crucial for this transition. Specialized features of the structure and assembly of the meiotic spindle have been identified. The changes in protein levels, mRNA translation, and polyadenylation that occur as the oocyte becomes an embryo have been identified together with key aspects of their regulation. Here we highlight these important developments and the insights they provide on the intricate regulation of this dramatic transition.

Bakthavatchalu, V., Wert, K.J., Feng, Y., Mannion, A., Ge, Z., Garcia, A., Scott, K.E., Caron, T.J., Madden, C.M., Jacobsen, J.T., Victora G, Jaenisch R, and Fox JG. (2018). Cytotoxic Escherichia coli strains encoding colibactin isolated from immunocompromised mice with urosepsis and meningitis. PloS one 13(3):e0194443. Immune-compromised mouse models allow for testing the preclinical efficacy of human cell transplantations and gene therapy strategies before moving forward to clinical trials. However, CRISPR/Cas9 gene editing of the Wsh/Wsh mouse strain to create an immune-compromised model lacking function of Rag2 and Il2rgamma led to unexpected morbidity and mortality. This warranted an investigation to ascertain the cause and predisposing factors associated with the outbreak. Postmortem examination was performed on 15 moribund mice. The main lesions observed in these mice consisted of ascending urogenital tract infections, suppurative otitis media, pneumonia, myocarditis, and meningoencephalomyelitis. As Escherichia coli strains harboring polyketide synthase (pks) genomic island were recently isolated from laboratory mice, the tissue sections from the urogenital tract, heart, and middle ear were subjected to E. coli specific PNA-FISH assay that revealed discrete colonies of E. coli associated with the lesions. Microbiological examination and 16S rRNA sequencing confirmed E. coli-induced infection and septicemia in the affected mice. Further characterization by clb gene analysis and colibactin toxicity assays of the pks+ E. coli revealed colibactin-associated cytotoxicity. Rederivation of the transgenic mice using embryo transfer produced mice with an intestinal flora devoid of pks+ E. coli. Importantly, these barrier-maintained rederived mice have produced multiple litters without adverse health effects. This report is the first to describe acute morbidity and mortality associated with pks+ E. coli urosepsis and meningitis in immunocompromised mice, and highlights the importance of monitoring and exclusion of colibactin-producing pks+ E. coli. Full Text

Ban, Z., Qin, H., Mitchell, A.J., Liu, B., Zhang, F., Weng, J.K., Dixon, R.A., and Wang, G. (2018). Noncatalytic chalcone isomerase-fold proteins in Humulus lupulus are auxiliary components in prenylated flavonoid biosynthesis. PNAS 115(22): E5223-E5232. Xanthohumol (XN) and demethylxanthohumol (DMX) are specialized prenylated chalconoids with multiple pharmaceutical applications that accumulate to high levels in the glandular trichomes of hops (Humulus lupulus L.). Although all structural enzymes in the XN pathway have been functionally identified, biochemical mechanisms underlying highly efficient production of XN have not been fully resolved. In this study, we characterized two noncatalytic chalcone isomerase (CHI)-like proteins (designated as HlCHIL1 and HlCHIL2) using engineered yeast harboring all genes required for DMX production. HlCHIL2 increased DMX production by 2.3-fold, whereas HlCHIL1 significantly decreased DMX production by 30%. We show that CHIL2 is part of an active DMX biosynthetic metabolon in hop glandular trichomes that encompasses a chalcone synthase (CHS) and a membrane-bound prenyltransferase, and that type IV CHI-fold proteins of representative land plants contain conserved function to bind with CHS and enhance its activity. Binding assays and structural docking uncover a function of HlCHIL1 to bind DMX and naringenin chalcone to stabilize the ring-open configuration of these chalconoids. This study reveals the role of two HlCHILs in DMX biosynthesis in hops, and provides insight into their evolutionary development from the ancestral fatty acid-binding CHI-fold proteins to specialized auxiliary proteins supporting flavonoid biosynthesis in plants.Full Text

Bartel , D.P. (2018). Metazoan MicroRNAs. Cell 173, 20-51. MicroRNAs (miRNAs) are approximately 22 nt RNAs that direct posttranscriptional repression of mRNA targets in diverse eukaryotic lineages. In humans and other mammals, these small RNAs help sculpt the expression of most mRNAs. This article reviews advances in our understanding of the defining features of metazoan miRNAs and their biogenesis, genomics, and evolution. It then reviews how metazoan miRNAs are regulated, how they recognize and cause repression of their targets, and the biological functions of this repression, with a compilation of knockout phenotypes that shows that important biological functions have been identified for most of the broadly conserved miRNAs of mammals. Full Text

Bayraktar, E.C., Baudrier, L., Ozerdem, C., Lewis, C.A., Chan, S.H., Kunchok, T., Abu-Remaileh, M., Cangelosi, A.L., Sabatini, D.M., Birsoy, K., and Chen, W.W.. (2018). MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo. PNAS[Epub ahead of print]. Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo. Full Text

Bellott, D.W., Cho, T.J., Hughes, J.F., Skaletsky, H., and Page, D.C. (2018). Cost-effective high-throughput single-haplotype iterative mapping and sequencing for complex genomic structures. Nature protocols 13, 787-809. The reference sequences of structurally complex regions can be obtained only through highly accurate clone-based approaches. We and others have successfully used single-haplotype iterative mapping and sequencing (SHIMS) 1.0 to assemble structurally complex regions across the sex chromosomes of several vertebrate species and to allow for targeted improvements to the reference sequences of human autosomes. However, SHIMS 1.0 is expensive and time consuming, requiring resources that only a genome center can provide. Here we introduce SHIMS 2.0, an improved SHIMS protocol that allows even a small laboratory to generate high-quality reference sequence from complex genomic regions. Using a streamlined and parallelized library-preparation protocol, and taking advantage of inexpensive high-throughput short-read-sequencing technologies, a small laboratory with both molecular biology and bioinformatics experience can sequence and assemble 192 large-insert bacterial artificial chromosome (BAC) or fosmid clones in 1 week. In SHIMS 2.0, in contrast to other pooling strategies, each clone is sequenced with a unique barcode, thus enabling clones containing nearly identical sequences to be multiplexed in a single sequencing run and assembled separately. Relative to SHIMS 1.0, SHIMS 2.0 decreases the required cost and time by two orders of magnitude while preserving high sequencing accuracy. Full Text

Binnewies, M., Roberts, E.W., Kersten, K., Chan, V., Fearon, D.F., Merad, M., Coussens, L.M., Gabrilovich, D.I., Ostrand-Rosenberg, S., Hedrick, C.C., Weinberg, R.A., et al. (2018). Understanding the tumor immune microenvironment (TIME) for effective therapy. Nature medicine [Epub ahead of print].The clinical successes in immunotherapy have been both astounding and at the same time unsatisfactory. Countless patients with varied tumor types have seen pronounced clinical response with immunotherapeutic intervention; however, many more patients have experienced minimal or no clinical benefit when provided the same treatment. As technology has advanced, so has the understanding of the complexity and diversity of the immune context of the tumor microenvironment and its influence on response to therapy. It has been possible to identify different subclasses of immune environment that have an influence on tumor initiation and response and therapy; by parsing the unique classes and subclasses of tumor immune microenvironment (TIME) that exist within a patient's tumor, the ability to predict and guide immunotherapeutic responsiveness will improve, and new therapeutic targets will be revealed. Full Text

Boija , A., Klein, I.A., Sabari, B.R., Dall'Agnese, A., Coffey, E.L., Zamudio, A.V., Li, C.H., Manteiga, J.C., Hannett, N.M., Hannett NM, Abraham BJ, Afeyan LK, Guo YE , Schuijers J, Lee TI, and Young RA. (2018). Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains. Cell [Epub ahead of print]. Gene expression is controlled by transcription factors (TFs) that consist of DNA-binding domains (DBDs) and activation domains (ADs). The DBDs have been well characterized, but little is known about the mechanisms by which ADs effect gene activation. Here, we report that diverse ADs form phase-separated condensates with the Mediator coactivator. For the OCT4 and GCN4 TFs, we show that the ability to form phase-separated droplets with Mediator in vitro and the ability to activate genes in vivo are dependent on the same amino acid residues. For the estrogen receptor (ER), a ligand-dependent activator, we show that estrogen enhances phase separation with Mediator, again linking phase separation with gene activation. These results suggest that diverse TFs can interact with Mediator through the phase-separating capacity of their ADs and that formation of condensates with Mediator is involved in gene activation. Full Text

Bourne, P.E., Lewitter, F., Markel, S., and Papin, J.A. (2018). One thousand simple rules.PLoS computational biology 14(12):e1006670 . What began as a one-off in 2005 as Ten Simple Rules for Getting Published [1] has, in thirteen years, now multiplied a hundredfold to become One Thousand Simple Rules for many aspects of one’s professional development and led to Quick Tips in the journal’s Education section. Full Text

Bourque, G., Burns, K.H., Gehring, M., Gorbunova, V., Seluanov, A., Hammell, M., Imbeault, M., Izsvak, Z., Levin, H.L., Macfarlan, T.S., et al. (2018). Ten things you should know about transposable elements. Genome biology 19, 199. Transposable elements (TEs) are major components of eukaryotic genomes. However, the extent of their impact on genome evolution, function, and disease remain a matter of intense interrogation. The rise of genomics and large-scale functional assays has shed new light on the multi-faceted activities of TEs and implies that they should no longer be marginalized. Here, we introduce the fundamental properties of TEs and their complex interactions with their cellular environment, which are crucial to understanding their impact and manifold consequences for organismal biology. While we draw examples primarily from mammalian systems, the core concepts outlined here are relevant to a broad range of organisms.Full Text

Brabletz, T., Kalluri, R., Nieto, M.A., and Weinberg, R.A .. (2018). EMT in cancer. Nature reviews Cancer [Epub ahead of print] Similar to embryonic development, changes in cell phenotypes defined as an epithelial to mesenchymal transition (EMT) have been shown to play a role in the tumorigenic process. Although the first description of EMT in cancer was in cell cultures, evidence for its role in vivo is now widely reported but also actively debated. Moreover, current research has exemplified just how complex this phenomenon is in cancer, leaving many exciting, open questions for researchers to answer in the future. With these points in mind, we asked four scientists for their opinions on the role of EMT in cancer and the challenges faced by scientists working in this fast-moving field. Full Text

Brown, J.D., Feldman, Z.B., Doherty, S.P., Reyes, J.M., Rahl, P.B., Lin, C.Y., Sheng, Q., Duan, Q., Federation, A.J., Kung, A.L., Young RA, et al. (2018). BET bromodomain proteins regulate enhancer function during adipogenesis. PNAS [Epub ahead of print]. Developmental transitions are guided by master regulatory transcription factors. During adipogenesis, a transcriptional cascade culminates in the expression of PPARgamma and C/EBPalpha, which orchestrate activation of the adipocyte gene expression program. However, the coactivators controlling PPARgamma and C/EBPalpha expression are less well characterized. Here, we show the bromodomain-containing protein, BRD4, regulates transcription of PPARgamma and C/EBPalpha. Analysis of BRD4 chromatin occupancy reveals that induction of adipogenesis in 3T3L1 fibroblasts provokes dynamic redistribution of BRD4 to de novo super-enhancers proximal to genes controlling adipocyte differentiation. Inhibition of the bromodomain and extraterminal domain (BET) family of bromodomain-containing proteins impedes BRD4 occupancy at these de novo enhancers and disrupts transcription of Pparg and Cebpa, thereby blocking adipogenesis. Furthermore, silencing of these BRD4-occupied distal regulatory elements at the Pparg locus by CRISPRi demonstrates a critical role for these enhancers in the control of Pparg gene expression and adipogenesis in 3T3L1s. Together, these data establish BET bromodomain proteins as time- and context-dependent coactivators of the adipocyte cell state transition. Full Text

Castano, Z., San Juan, B.P., Spiegel, A., Pant, A., DeCristo, M.J., Laszewski, T., Ubellacker, J.M., Janssen, S.R., Dongre, A., Reinhardt, F., Gifford, A.M., Weinberg, R.A., et al. (2018). IL-1beta inflammatory response driven by primary breast cancer prevents metastasis-initiating cell colonization. Nature cell biology 20, 1084-1097Lack of insight into mechanisms governing breast cancer metastasis has precluded the development of curative therapies. Metastasis-initiating cancer cells (MICs) are uniquely equipped to establish metastases, causing recurrence and therapeutic resistance. Using various metastasis models, we discovered that certain primary tumours elicit a systemic inflammatory response involving interleukin-1beta (IL-1beta)-expressing innate immune cells that infiltrate distant MIC microenvironments. At the metastatic site, IL-1beta maintains MICs in a ZEB1-positive differentiation state, preventing MICs from generating highly proliferative E-cadherin-positive progeny. Thus, when the inherent plasticity of MICs is impeded, overt metastases cannot be established. Ablation of the pro-inflammatory response or inhibition of the IL-1 receptor relieves the differentiation block and results in metastatic colonization. Among patients with lymph node-positive breast cancer, high primary tumour IL-1beta expression is associated with better overall survival and distant metastasis-free survival. Our data reveal complex interactions that occur between primary tumours and disseminated MICs that could be exploited to improve patient survival. Full Text

Chen , C., Whitney, I.P., Banerjee, A., Sacristan, C., Sekhri, P., Kern, D.M., Fontan, A., Kops, G., Tyson, J.J., Cheeseman, I.M.., et al. (2018). Ectopic Activation of the Spindle Assembly Checkpoint Signaling Cascade Reveals Its Biochemical Design. Current biology [Epub ahead of print]. CBSwitch-like activation of the spindle assembly checkpoint (SAC) is critical for accurate chromosome segregation and for cell division in a timely manner. To determine the mechanisms that achieve this, we engineered an ectopic, kinetochore-independent SAC activator: the "eSAC." The eSAC stimulates SAC signaling by artificially dimerizing Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the kinetochore signaling scaffold. By exploiting variable eSAC expression in a cell population, we defined the dependence of the eSAC-induced mitotic delay on eSAC concentration in a cell to reveal the dose-response behavior of the core signaling cascade of the SAC. These quantitative analyses and subsequent mathematical modeling of the dose-response data uncover two crucial properties of the core SAC signaling cascade: (1) a cellular limit on the maximum anaphase-inhibitory signal that the cascade can generate due to the limited supply of SAC proteins and (2) the ability of the KNL1 phosphodomain to produce the anaphase-inhibitory signal synergistically, when it recruits multiple SAC proteins simultaneously. We propose that these properties together achieve inverse, non-linear scaling between the signal output per kinetochore and the number of signaling kinetochores. When the number of kinetochores is low, synergistic signaling by KNL1 enables each kinetochore to produce a disproportionately strong signal output. However, when many kinetochores signal concurrently, they compete for a limited supply of SAC proteins. This frustrates synergistic signaling and lowers their signal output. Thus, the signaling activity of unattached kinetochores will adapt to the changing number of signaling kinetochores to enable the SAC to approximate switch-like behavior. Full Text

Chen , W.W., Freinkman, E., and Sabatini, D.M. (2017). Rapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites. Nature protocols 12, 2215-2231. Mitochondria carry out numerous metabolic reactions that are critical to cellular homeostasis. Here we present a protocol for interrogating mitochondrial metabolites and measuring their matrix concentrations. Our workflow uses high-affinity magnetic immunocapture to rapidly purify HA-tagged mitochondria from homogenized mammalian cells in approximately 12 min. These mitochondria are extracted with methanol and water. Liquid chromatography and mass spectrometry (LC/MS) is used to determine the identities and mole quantities of mitochondrial metabolites using authentic metabolite standards and isotopically labeled internal standards, whereas the corresponding mitochondrial matrix volume is determined via immunoblotting, confocal microscopy of intact cells, and volumetric analysis. Once all values have been obtained, the matrix volume is combined with the aforementioned mole quantities to calculate the matrix concentrations of mitochondrial metabolites. With shortened isolation times and improved mitochondrial purity when compared with alternative methods, this LC/MS-compatible workflow allows for robust profiling of mitochondrial metabolites and serves as a strategy generalizable to the study of other mammalian organelles. Once all the necessary reagents have been prepared, quantifying the matrix concentrations of mitochondrial metabolites can be accomplished within a week. Full Text

Chiang, Y.C., Levsh, O., Lam, C.K., Weng, J.K., and Wang, Y. (2018). Structural and dynamic basis of substrate permissiveness in hydroxycinnamoyltransferase (HCT). PLoS computational biology 14, e1006511. Substrate permissiveness has long been regarded as the raw materials for the evolution of new enzymatic functions. In land plants, hydroxycinnamoyltransferase (HCT) is an essential enzyme of the phenylpropanoid metabolism. Although essential enzymes are normally associated with high substrate specificity, HCT can utilize a variety of non-native substrates. To examine the structural and dynamic basis of substrate permissiveness in this enzyme, we report the crystal structure of HCT from Selaginella moellendorffii and molecular dynamics (MD) simulations performed on five orthologous HCTs from several major lineages of land plants. Through altogether 17-mus MD simulations, we demonstrate the prevalent swing motion of an arginine handle on a submicrosecond timescale across all five HCTs, which plays a key role in native substrate recognition by these intrinsically promiscuous enzymes. Our simulations further reveal how a non-native substrate of HCT engages a binding site different from that of the native substrate and diffuses to reach the catalytic center and its co-substrate. By numerically solving the Smoluchowski equation, we show that the presence of such an alternative binding site, even when it is distant from the catalytic center, always increases the reaction rate of a given substrate. However, this increase is only significant for enzyme-substrate reactions heavily influenced by diffusion. In these cases, binding non-native substrates 'off-center' provides an effective rationale to develop substrate permissiveness while maintaining the native functions of promiscuous enzymes. Full Text

Chiu, A.C., Suzuki, H.I., Wu, X., Mahat, D.B., Kriz, A.J., and Sharp, P.A. (2018). Transcriptional Pause Sites Delineate Stable Nucleosome-Associated Premature Polyadenylation Suppressed by U1 snRNP. Molecular cell [Epub ahead of print]. Regulation of RNA polymerase II (Pol II) elongation is a critical step in gene regulation. Here, we report that U1 snRNP recognition and transcription pausing at stable nucleosomes are linked through premature polyadenylation signal (PAS) termination. By generating RNA exosome conditional deletion mouse embryonic stem cells, we identified a large class of polyadenylated short transcripts in the sense direction destabilized by the RNA exosome. These PAS termination events are enriched at the first few stable nucleosomes flanking CpG islands and suppressed by U1 snRNP. Thus, promoter-proximal Pol II pausing consists of two processes: TSS-proximal and +1 stable nucleosome pausing, with PAS termination coinciding with the latter. While pausing factors NELF/DSIF only function in the former step, flavopiridol-sensitive mechanism(s) and Myc modulate both steps. We propose that premature PAS termination near the nucleosome-associated pause site represents a common transcriptional elongation checkpoint regulated by U1 snRNP recognition, nucleosome stability, and Myc activity. Full Text

Chmatal , L., Schultz, R.M., Black, B.E., and Lampson, M.A. (2017). Cell Biology of Cheating-Transmission of Centromeres and Other Selfish Elements Through Asymmetric Meiosis. In : Centromeres and Kinetochores: Discovering the Molecular Mechanisms Underlying Chromosome Inheritance, pp. 377-396. Mendel's First Law of Genetics states that a pair of alleles segregates randomly during meiosis so that one copy of each is represented equally in gametes. Whereas male meiosis produces four equal sperm, in female meiosis only one cell, the egg, survives, and the others degenerate. Meiotic drive is a process in which a selfish DNA element exploits female meiotic asymmetry and segregates preferentially to the egg in violation of Mendel's First Law, thereby increasing its transmission to the offspring and frequency in a population. In principle, the selfish element can consist either of a centromere that increases its transmission via an altered kinetochore connection to the meiotic spindle or a centromere-like element that somehow bypasses the kinetochore altogether in doing so. There are now examples from eukaryotic model systems for both types of meiotic drive. Although meiotic drive has profound evolutionary consequences across many species, relatively little is known about the underlying mechanisms. We discuss examples in various systems and open questions about the underlying cell biology, and propose a mechanism to explain biased segregation in mammalian female meiosis. Full Text

Cho, W.K., Spille, J.H., Hecht, M., Lee, C., Li, C., Grube, V., and Cisse, II (2018). Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science [Epub ahead of print]. Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. Here we used live cell super-resolution and light sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo. Full Text

Christ , B., Pluskal, T., Aubry, S., and Weng, J.K. (2018). Contribution of Untargeted Metabolomics for Future Assessment of Biotech Crops. Trends in plant science S1360-1385 (18) 30216-4. The nutritional value and safety of food crops are ultimately determined by their chemical composition. Recent developments in the field of metabolomics have made it possible to characterize the metabolic profile of crops in a comprehensive and high-throughput manner. Here, we propose that state-of-the-art untargeted metabolomics technology should be leveraged for safety assessment of new crop products. We suggest generally applicable experimental design principles that facilitate the efficient and rigorous identification of both intended and unintended metabolic alterations associated with a newly engineered trait. Our proposition could contribute to increased transparency of the safety assessment process for new biotech crops.

Cohen , M.A., Markoulaki, S., and Jaenisch, R. (2018). Matched Developmental Timing of Donor Cells with the Host Is Crucial for Chimera Formation. Stem cell reports [Epub ahead of print]. Chimeric mice have been generated by injecting pluripotent stem cells into morula-to-blastocyst stage mouse embryo or by introducing more mature cells into later stage embryos that correspond to the differentiation stage of the donor cells. It has not been rigorously tested, however, whether successful chimera formation requires the developmental stage of host embryo and donor cell to be matched. Here, we compared the success of chimera formation following injection of primary neural crest cells (NCCs) into blastocysts or of embryonic stem cells (ESCs) into E8.5 embryos (heterochronic injection) with that of injecting ESCs cells into the blastocyst or NCCs into the E8.5 embryos (isochronic injection). Chimera formation was efficient when donor and host were matched, but no functional chimeric contribution was found in heterochronic injections. This suggests that matching the developmental stage of donor cells with the host embryo is crucial for functional engraftment of donor cells into the developing embryo. Full Text

Creixell, P., Pandey, J.P.., Palmeri, A., Bhattacharyya, M., Creixell, M., Ranganathan, R., Pincus, D.., and Yaffe, M.B. (2018). Hierarchical Organization Endows the Kinase Domain with Regulatory Plasticity. Cell systems.[Epub ahead of print]. The functional diversity of kinases enables specificity in cellular signal transduction. Yet how more than 500 members of the human kinome specifically receive regulatory inputs and convey information to appropriate substrates-all while using the common signaling output of phosphorylation-remains enigmatic. Here, we perform statistical co-evolution analysis, mutational scanning, and quantitative live-cell assays to reveal a hierarchical organization of the kinase domain that facilitates the orthogonal evolution of regulatory inputs and substrate outputs while maintaining catalytic function. We find that three quasi-independent "sectors"-groups of evolutionarily coupled residues-represent functional units in the kinase domain that encode for catalytic activity, substrate specificity, and regulation. Sector positions impact both disease and pharmacology: the catalytic sector is significantly enriched for somatic cancer mutations, and residues in the regulatory sector interact with allosteric kinase inhibitors. We propose that this functional architecture endows the kinase domain with inherent regulatory plasticity. Full Text

Das, A., Christ, B., and Hortensteiner, S. (2018). Characterization of the pheophorbide a oxygenase/phyllobilin pathway of chlorophyll breakdown in grasses. Planta [Epub ahead of print]. Although the PAO/phyllobilin pathway of chlorophyll breakdown is active in grass leaf senescence, the abundance of phyllobilins is far below the amount of degraded chlorophyll. The yellowing of fully developed leaves is the most prominent visual symptom of plant senescence. Thereby, chlorophyll is degraded via the so-called pheophorbide a oxygenase (PAO)/phyllobilin pathway to a species-specific set of phyllobilins, linear tetrapyrrolic products of chlorophyll breakdown. Here, we investigated the diversity and abundance of phyllobilins in cereal and forage crops, i.e. barley, rice, ryegrass, sorghum and wheat, using liquid chromatography-mass spectrometry. A total of thirteen phyllobilins were identified, among them four novel, not yet described ones, pointing to a rather high diversity of phyllobilin-modifying activities present in the Gramineae. Along with these phyllobilins, barley orthologs of known Arabidopsis thaliana chlorophyll catabolic enzymes were demonstrated to localize in the chloroplast, and two of them, i.e. PAO and pheophytin pheophorbide hydrolase, complemented respective Arabidopsis mutants. These data confirm functionality of the PAO/phyllobilin pathway in grasses. Interestingly, when comparing phyllobilin abundance with amounts of degraded chlorophyll in senescent leaves, in most analyzed grass species only minor fractions of chlorophyll were recovered as phyllobilins, opposite to A. thaliana where phyllobilin quantities match degraded chlorophyll rather well. These data show that, despite the presence and activity of the PAO/phyllobilin pathway in barley (and other cereals), phyllobilins do not accumulate stoichiometrically, implying possible degradation of chlorophyll beyond the phyllobilin level. Full Text

Dongre , A., and Weinberg, R.A. (2018). New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nature Reviews Molecular Cell Biology [Epub ahead of print]. Epithelial-mesenchymal transition (EMT) is a cellular programme that is known to be crucial for embryogenesis, wound healing and malignant progression. During EMT, cell-cell and cell-extracellular matrix interactions are remodelled, which leads to the detachment of epithelial cells from each other and the underlying basement membrane, and a new transcriptional programme is activated to promote the mesenchymal fate. In the context of neoplasias, EMT confers on cancer cells increased tumour-initiating and metastatic potential and a greater resistance to elimination by several therapeutic regimens. In this Review, we discuss recent findings on the mechanisms and roles of EMT in normal and neoplastic tissues, and the cell-intrinsic signals that sustain expression of this programme. We also highlight how EMT gives rise to a variety of intermediate cell states between the epithelial and the mesenchymal state, which could function as cancer stem cells. In addition, we describe the contributions of the tumour microenvironment in inducing EMT and the effects of EMT on the immunobiology of carcinomas. Full Text

Durbin, A.D., Zimmerman, M.W., Dharia, N.V., Abraham, B.J., Iniguez, A.B., Weichert-Leahey, N., He, S., Krill-Burger, J.M., Root, D.E., Vazquez, F., Richard A. Young ,et al. (2018). Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry. Nature genetics[Epub ahead of print]. Childhood high-risk neuroblastomas with MYCN gene amplification are difficult to treat effectively(1). This has focused attention on tumor-specific gene dependencies that underlie tumorigenesis and thus provide valuable targets for the development of novel therapeutics. Using unbiased genome-scale CRISPR-Cas9 approaches to detect genes involved in tumor cell growth and survival(2-6), we identified 147 candidate gene dependencies selective for MYCN-amplified neuroblastoma cell lines, compared to over 300 other human cancer cell lines. We then used genome-wide chromatin-immunoprecipitation coupled to high-throughput sequencing analysis to demonstrate that a small number of essential transcription factors-MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2-are members of the transcriptional core regulatory circuitry (CRC) that maintains cell state in MYCN-amplified neuroblastoma. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which act synergistically, in vitro and in vivo, with rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma that are essential for cell state and survival in this tumor. Full Text

Eliades, P., Abraham, B.J., Ji, Z., Miller, D.M., Christensen, C.L., Kwiatkowski, N., Kumar, R., Njauw, C.N., Taylor, M., Miao, B., Richard A. Young, et al. (2018). High MITF Expression is Associated with Super-enhancers and Suppressed by CDK7 Inhibition in Melanoma. The Journal of investigative dermatology [Epub ahead of print]. Cutaneous melanoma is an aggressive tumor which accounts for most of the skin cancer deaths. Among the physiological barriers against therapeutic success is a strong survival program driven by genes that specify melanocyte identity such as MITF - a phenomenon known in melanoma biology as "lineage dependency." MITF overexpression is occasionally explained by gene amplification, but here we demonstrate that "super-enhancers" are also important determinants of MITF overexpression in some melanoma cell lines and tumors. While compounds that directly inhibit MITF are unavailable, a covalent CDK7 inhibitor (THZ1) has recently been shown to potently suppress the growth of various cancers through the depletion of master transcription-regulating oncogenes and the disruption of their attendant super-enhancers. We also show that melanoma cells are highly sensitive to CDK7 inhibition both in vitro and in vivo and that THZ1 can dismantle the super-enhancer apparatus at MITF and SOX10 in some cell lines thereby extinguishing their intracellular levels. Our results reveal a dimension to MITF regulation in melanoma cells and point to CDK7 inhibition as a potential strategy to deprive oncogenic transcription and suppress tumor growth in melanoma. Full Text

Fallon , T.R., Lower, S.E., Chang, C.H., Bessho-Uehara, M., Martin, G.J., Bewick, A.J., Behringer, M., Debat, H.J., Wong, I., Day, J.C., Weng. J.K. ,et al. (2018). Firefly genomes illuminate parallel origins of bioluminescence in beetles. eLife 7:e36495. Fireflies and their luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of bioluminescence within beetles remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North American Photinus pyralis and Japanese Aquatica lateralis. To compare bioluminescent origins, we also sequenced the genome of a related click beetle, the Caribbean Ignelater luminosus, with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing hypothesis of parallel gains of bioluminescence. Our analyses support independent gains of bioluminescence in fireflies and click beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle. Full Text

Fanning S, Haque A, Imberdis T, Baru V., Barrasa MI, Nuber S, Termine D., Ramalingam N, Ho GPH, Noble T, Sandoe J, Lou Y, Landgraf D, Freyzon Y, Newby G, Soldner F, Terry-Kantor E, Kim TE, Hofbauer HF, Becuwe M, Jaenisch R, Pincus D, Clish CB, Walther TC, Farese RV Jr, Srinivasan S, Welte MA, Kohlwein SD, Dettmer U, Lindquist S, Selkoe D (2018). Lipidomic Analysis of alpha-Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment. Molecular cell [Epub ahead of print]. In Parkinson's disease (PD), alpha-synuclein (alphaS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in alphaS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human alphaS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of alphaS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased alphaS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in alphaS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented alphaS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on alphaS homeostasis: in human neural cells, excess OA caused alphaS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for alphaS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.Full Text

Fincher , C.T., Wurtzel, O., de Hoog, T., Kravarik, K.M., and Reddien, P.W. (2018). Cell type transcriptome atlas for the planarian Schmidtea mediterranea. Science [Epub ahead of print] The transcriptome of a cell dictates its unique cell-type biology. We used single-cell RNA sequencing to determine the transcriptomes for essentially every cell type of a complete animal: the regenerative planarian Schmidtea mediterranea. Planarians contain a diverse array of cell types, possess lineage progenitors for differentiated cells (including pluripotent stem cells), and constitutively express positional information, making them ideal for this undertaking. We generated data for 66,783 cells, defining transcriptomes for known and many previously unknown planarian cell types and for putative transition states between stem and differentiated cells. We also uncovered regionally expressed genes in muscle, which harbors positional information. Identifying the transcriptomes for potentially all cell types for many organisms should be readily attainable and is a powerful new approach to metazoan biology. Full Text

Frose , J., Chen, M.B., Hebron, K.E., Reinhardt, F. , Hajal, C., Zijlstra, A., Kamm, R.D., and Weinberg, R.A. (2018). Epithelial-Mesenchymal Transition Induces Podocalyxin to Promote Extravasation via Ezrin Signaling. Cell reports 24, 962-972.The epithelial-mesenchymal transition (EMT) endows carcinoma cells with traits needed to complete many of the steps leading to metastasis formation, but its contributions specifically to the late step of extravasation remain understudied. We find that breast cancer cells that have undergone an EMT extravasate more efficiently from blood vessels both in vitro and in vivo. Analysis of gene expression changes associated with the EMT program led to the identification of an EMT-induced cell-surface protein, podocalyxin (PODXL), as a key mediator of extravasation in mesenchymal breast and pancreatic carcinoma cells. PODXL promotes extravasation through direct interaction of its intracellular domain with the cytoskeletal linker protein ezrin. Ezrin proceeds to establish dorsal cortical polarity, enabling the transition of cancer cells from a non-polarized, rounded cell morphology to an invasive extravasation-competent shape. Hence, the EMT program can directly enhance the efficiency of extravasation and subsequent metastasis formation through a PODXL-ezrin signaling axis. Full Text

Fulciniti, M., Lin, C.Y., Samur, M.K., Lopez, M.A., Singh, I., Lawlor, M.A., Szalat, R.E., Ott, C.J., Avet-Loiseau, H., Anderson, K.C., Young, R.A.,et al. (2018). Non-overlapping Control of Transcriptome by Promoter- and Super-Enhancer-Associated Dependencies in Multiple Myeloma. Cell reports 25(13):3693-3705. The relationship between promoter proximal transcription factor-associated gene expression and super-enhancer-driven transcriptional programs are not well defined. However, their distinct genomic occupancy suggests a mechanism for specific and separable gene control. We explored the transcriptional and functional interrelationship between E2F transcription factors and BET transcriptional co-activators in multiple myeloma. We found that the transcription factor E2F1 and its heterodimerization partner DP1 represent a dependency in multiple myeloma cells. Global chromatin analysis reveals distinct regulatory axes for E2F and BETs, with E2F predominantly localized to active gene promoters of growth and/or proliferation genes and BETs disproportionately at enhancer-regulated tissue-specific genes. These two separate gene regulatory axes can be simultaneously targeted to impair the myeloma proliferative program, providing an important molecular mechanism for combination therapy. This study therefore suggests a sequestered cellular functional control that may be perturbed in cancer with potential for development of a promising therapeutic strategy. Full Text

Gallagher , M.D., and Chen-Plotkin, A.S. (2018). The Post-GWAS Era: From Association to Function. American journal of human genetics 102, 717-730.During the past 12 years, genome-wide association studies (GWASs) have uncovered thousands of genetic variants that influence risk for complex human traits and diseases. Yet functional studies aimed at delineating the causal genetic variants and biological mechanisms underlying the observed statistical associations with disease risk have lagged. In this review, we highlight key advances in the field of functional genomics that may facilitate the derivation of biological meaning post-GWAS. We highlight the evidence suggesting that causal variants underlying disease risk often function through regulatory effects on the expression of target genes and that these expression effects might be modest and cell-type specific. We moreover discuss specific studies as proof-of-principle examples for current statistical, bioinformatic, and empirical bench-based approaches to downstream elucidation of GWAS-identified disease risk loci. Full Text

Garcia-Bermudez, J., Baudrier, L., La, K., Zhu, X.G., Fidelin, J., Sviderskiy, V.O., Papagiannakopoulos, T., Molina, H., Snuderl, M., Lewis, C.A., et al. (2018). Aspartate is a limiting metabolite for cancer cell proliferation under hypoxia and in tumours. Nature Cell Biology 20(7):775-781. As oxygen is essential for many metabolic pathways, tumour hypoxia may impair cancer cell proliferation(1-4). However, the limiting metabolites for proliferation under hypoxia and in tumours are unknown. Here, we assessed proliferation of a collection of cancer cells following inhibition of the mitochondrial electron transport chain (ETC), a major metabolic pathway requiring molecular oxygen (5) . Sensitivity to ETC inhibition varied across cell lines, and subsequent metabolomic analysis uncovered aspartate availability as a major determinant of sensitivity. Cell lines least sensitive to ETC inhibition maintain aspartate levels by importing it through an aspartate/glutamate transporter, SLC1A3. Genetic or pharmacologic modulation of SLC1A3 activity markedly altered cancer cell sensitivity to ETC inhibitors. Interestingly, aspartate levels also decrease under low oxygen, and increasing aspartate import by SLC1A3 provides a competitive advantage to cancer cells at low oxygen levels and in tumour xenografts. Finally, aspartate levels in primary human tumours negatively correlate with the expression of hypoxia markers, suggesting that tumour hypoxia is sufficient to inhibit ETC and, consequently, aspartate synthesis in vivo. Therefore, aspartate may be a limiting metabolite for tumour growth, and aspartate availability could be targeted for cancer therapy. Full Text

Guenther, U.P., Weinberg, D.E., Zubradt, M.M., Tedeschi, F.A., Stawicki, B.N., Zagore, L.L., Brar, G.A., Licatalosi, D.D., Bartel, D.P., Weissman, J.S., et al. (2018). The helicase Ded1p controls use of near-cognate translation initiation codons in 5' UTRs. Nature 559 (7712),130–134 . The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation (1) . Mutations in DDX3 are linked to tumorigenesis(2-4) and intellectual disability (5) , and the enzyme is targeted by a range of viruses (6) . How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions. Full Text

Gupta , P.B., Pastushenko, I., Skibinski, A., Blanpain, C., and Kuperwasser, C. (2018). Phenotypic Plasticity: Driver of Cancer Initiation, Progression, and Therapy Resistance. Cell stem cell [Epub ahead of print]. Our traditional understanding of phenotypic plasticity in adult somatic cells comprises dedifferentiation and transdifferentiation in the context of tissue regeneration or wound healing. Although dedifferentiation is central to tissue repair and stemness, this process inherently carries the risk of cancer initiation. Consequently, recent research suggests phenotypic plasticity as a new paradigm for understanding cancer initiation, progression, and resistance to therapy. Here, we discuss how cells acquire plasticity and the role of plasticity in initiating cancer, cancer progression, and metastasis and in developing therapy resistance. We also highlight the epithelial-to-mesenchymal transition (EMT) and known molecular mechanisms underlying plasticity and we consider potential therapeutic avenues. Full Text

Gutzman , J.H., Graeden, E., Brachmann, I., Yamazoe, S., Chen, J.K., and Sive, H. (2018). Basal constriction during midbrain-hindbrain boundary morphogenesis is mediated by Wnt5b and focal adhesion kinase. Biology open bio.034520 [Epub ahead of print]. Basal constriction occurs at the zebrafish midbrain-hindbrain boundary constriction (MHBC) and is likely a widespread morphogenetic mechanism. 3D reconstruction demonstrates that MHBC cells are wedge-shaped, and initially constrict basally, with subsequent apical expansion. wnt5b is expressed in the MHB and is required for basal constriction. Consistent with a requirement for this pathway, expression of dominant negative Gsk3beta overcomes wnt5b knockdown. Immunostaining identifies focal adhesion kinase (Fak) as active in the MHB region, and knockdown demonstrates Fak is a regulator of basal constriction. Tissue specific knockdown further indicates that Fak functions cell autonomously within the MHBC. Fak acts downstream of wnt5b, suggesting that Wnt5b signals locally as an early step in basal constriction and acts together with more widespread Fak activation. This study delineates signaling pathways that regulate basal constriction during brain morphogenesis. Full Text

Hara , M., Lourido, S., Petrova, B., Lou, H.J., Von Stetina, J.R., Kashevsky, H., Turk, B.E., and Orr-Weaver, T.L. (2018). Identification of PNG kinase substrates uncovers interactions with the translational repressor TRAL in the oocyte-to-embryo transition. eLife 7:e33150. The Drosophila Pan Gu (PNG) kinase complex regulates hundreds of maternal mRNAs that become translationally repressed or activated as the oocyte transitions to an embryo. In a previous paper (Hara et al., 2017), we demonstrated PNG activity is under tight developmental control and restricted to this transition. Here, examination of PNG specificity showed it to be a Thr-kinase yet lacking a clear phosphorylation site consensus sequence. An unbiased biochemical screen for PNG substrates identified the conserved translational repressor Trailer Hitch (TRAL). Phosphomimetic mutation of the PNG phospho-sites in TRAL reduced its ability to inhibit translation in vitro. In vivo, mutation of tral dominantly suppressed png mutants and restored Cyclin B protein levels. The repressor Pumilio (PUM) has the same relationship with PNG, and we also show that PUM is a PNG substrate. Furthermore, PNG can phosphorylate BICC and ME31B, repressors that bind TRAL in cytoplasmic RNPs. Therefore, PNG likely promotes translation at the oocyte-to-embryo transition by phosphorylating and inactivating translational repressors.Full Text

Hill , M., and Weng, J.K. (2018). Piece de Self-Resistance: A New Paradigm for Natural-Product Herbicide Discovery. Molecular plant [Epub ahead of print].

Heo, J.M., Ordureau, A., Swarup, S., Paulo, J.A., Shen, K., Sabatini, D.M., and Harper, J.W. (2018). RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway.Sci Adv 4(11): eaav0443. Removal of damaged mitochondria is orchestrated by a pathway involving the PINK1 kinase and the PARKIN ubiquitin ligase. Ubiquitin chains assembled by PARKIN on the mitochondrial outer membrane recruit autophagy cargo receptors in complexes with TBK1 protein kinase. While TBK1 is known to phosphorylate cargo receptors to promote ubiquitin binding, it is unknown whether TBK1 phosphorylates other proteins to promote mitophagy. Using global quantitative proteomics, we identified S72 in RAB7A, a RAB previously linked with mitophagy, as a dynamic target of TBK1 upon mitochondrial depolarization. TBK1 directly phosphorylates RAB7A(S72), but not several other RABs known to be phosphorylated on the homologous residue by LRRK2, in vitro, and this modification requires PARKIN activity in vivo. Interaction proteomics using nonphosphorylatable and phosphomimetic RAB7A mutants revealed loss of association of RAB7A(S72E) with RAB GDP dissociation inhibitor and increased association with the DENN domain-containing heterodimer FLCN-FNIP1. FLCN-FNIP1 is recruited to damaged mitochondria, and this process is inhibited in cells expressing RAB7A(S72A). Moreover, nonphosphorylatable RAB7A failed to support efficient mitophagy, as well as recruitment of ATG9A-positive vesicles to damaged mitochondria. These data reveal a novel function for TBK1 in mitophagy, which parallels that of LRRK2-mediated phosphorylation of the homologous site in distinct RABs to control membrane trafficking. Full Text

Herschlag, D., Bonilla, S., and Bisaria, N. (2018). The Story of RNA Folding, as Told in Epochs. Cold Spring Harbor perspectives in biology 10(10) : a032433. The past decades have witnessed tremendous developments in our understanding of RNA biology. At the core of these advances have been studies aimed at discerning RNA structure and at understanding the forces that influence the RNA folding process. It is easy to take the present state of understanding for granted, but there is much to be learned by considering the path to our current understanding, which has been tortuous, with the birth and death of models, the adaptation of experimental tools originally developed for characterization of protein structure and catalysis, and the development of novel tools for probing RNA. In this review we tour the stages of RNA folding studies, considering them as "epochs" that can be generalized across scientific disciplines. These epochs span from the discovery of catalytic RNA, through biophysical insights into the putative primordial RNA World, to characterization of structured RNAs, the building and testing of models, and, finally, to the development of models with the potential to yield generalizable predictive and quantitative models for RNA conformational, thermodynamic, and kinetic behavior. We hope that this accounting will aid others as they navigate the many fascinating questions about RNA and its roles in biology, in the past, present, and future. Full Text

Ho, N., Xu, C., and Thibault, G. (2018). From the unfolded protein response to metabolic diseases - lipids under the spotlight. Journal of cell science - Review. The unfolded protein response (UPR) is classically viewed as a stress response pathway to maintain protein homeostasis at the endoplasmic reticulum (ER). However, it has recently emerged that the UPR can be directly activated by lipid perturbation, independently of misfolded proteins. Comprising primarily phospholipids, sphingolipids and sterols, individual membranes can contain hundreds of distinct lipids. Even with such complexity, lipid distribution in a cell is tightly regulated by mechanisms that remain incompletely understood. It is therefore unsurprising that lipid dysregulation can be a key factor in disease development. Recent advances in analysis of lipids and their regulators have revealed remarkable mechanisms and connections to other cellular pathways including the UPR. In this Review, we summarize the current understanding in UPR transducers functioning as lipid sensors and the interplay between lipid metabolism and ER homeostasis in the context of metabolic diseases. We attempt to provide a framework consisting of a few key principles to integrate the different lines of evidence and explain this rather complicated mechanism. Full Text

Hua, B.L., Bell, G.W., Kashevsky, H., Von Stetina, J.R., and Orr-Weaver, T.L. (2018). Dynamic changes in ORC localization and replication fork progression during tissue differentiation. BMC genomics 19(1):623. Genomic regions repressed for DNA replication, resulting in either delayed replication in S phase or underreplication in polyploid cells, are thought to be controlled by inhibition of replication origin activation. Studies in Drosophila polytene cells, however, raised the possibility that impeding replication fork progression also plays a major role. RESULTS: We exploited genomic regions underreplicated (URs) with tissue specificity in Drosophila polytene cells to analyze mechanisms of replication repression. By localizing the Origin Recognition Complex (ORC) in the genome of the larval fat body and comparing this to ORC binding in the salivary gland, we found that sites of ORC binding show extensive tissue specificity. In contrast, there are common domains nearly devoid of ORC in the salivary gland and fat body that also have reduced density of ORC binding sites in diploid cells. Strikingly, domains lacking ORC can still be replicated in some polytene tissues, showing absence of ORC and origins is insufficient to repress replication. Analysis of the width and location of the URs with respect to ORC position indicates that whether or not a genomic region lacking ORC is replicated is controlled by whether replication forks formed outside the region are inhibited. CONCLUSIONS: These studies demonstrate that inhibition of replication fork progression can block replication across genomic regions that constitutively lack ORC. Replication fork progression can be inhibited in both tissue-specific and genome region-specific ways. Consequently, when evaluating sources of genome instability it is important to consider altered control of replication forks in response to differentiation. Full Text

Huang , N.J., Lin, Y.C., Lin, C.Y., Pishesha, N., Lewis, C.A., Freinkman, E., Farquharson, C., Millan, J.L., and Lodish, H. (2018). Enhanced phosphocholine metabolism is essential for terminal erythropoiesis. Blood [Epub ahead of print]. Red cells contain a unique constellation of membrane lipids. While much is known about regulated protein expression, the regulation of lipid metabolism during erythropoiesis is poorly studied. Here we show that transcription of PHOSPHO1, a phosphoethanolamine and phosphocholine phosphatase that mediates the hydrolysis of phosphocholine to choline, is strongly upregulated during the terminal stages of erythropoiesis of both human and mouse erythropoiesis, concomitant with increased catabolism of phosphatidylcholine and phosphocholine as shown by global lipidomic analyses of mouse and human terminal erythropoiesis. Depletion of PHOSPHO1 impaired differentiation of fetal mouse and human erythroblasts, and in adult mice depletion impaired phenylhydrazine-induced stress erythropoiesis. Loss of PHOSPHO1 also impaired phosphocholine catabolism in mouse fetal liver progenitors and resulted in accumulation of several lipids; ATP production was reduced as a result of decreased oxidative phosphorylation. Glycolysis replaced oxidative phosphorylation in PHOSPHO1 knockout erythroblasts and the increased glycolysis was used for the production of serine or glycine. Our study elucidates the dynamic changes in lipid metabolism during terminal erythropoiesis and reveals the key roles of phosphatidylcholine and phosphocholine metabolism in energy balance and amino acid supply. Full Text

Huet , D., Rajendran, E., van Dooren, G.G., and Lourido, S. (2018). Identification of cryptic subunits from an apicomplexan ATP synthase. eLife 7 : e38097. The mitochondrial ATP synthase is a macromolecular motor that uses the proton gradient to generate ATP. Proper ATP synthase function requires a stator linking the catalytic and rotary portions of the complex. However, sequence-based searches fail to identify genes encoding stator subunits in apicomplexan parasites like Toxoplasma gondii or the related organisms that cause malaria. Here, we identify 11 previously unknown subunits from the Toxoplasma ATP synthase, which lack homologs outside the phylum. Modeling suggests that two of them, ICAP2 and ICAP18, are distantly related to mammalian stator subunits. Our analysis shows that both proteins form part of the ATP synthase complex. Depletion of ICAP2 leads to aberrant mitochondrial morphology, decreased oxygen consumption, and disassembly of the complex, consistent with its role as an essential component of the Toxoplasma ATP synthase. Our findings highlight divergent features of the central metabolic machinery in apicomplexans, which may reveal new therapeutic opportunities. Full Text

Iniguez, A.B., Stolte, B., Wang, E.J., Conway, A.S., Alexe, G., Dharia, N.V., Kwiatkowski N., Zhang, T., Abraham, B.J., Mora, J., Richard A. Young , et al. (2018). EWS/FLI Confers Tumor Cell Synthetic Lethality to CDK12 Inhibition in Ewing Sarcoma. Cancer cell [Epub ahead of print]Many cancer types are driven by oncogenic transcription factors that have been difficult to drug. Transcriptional inhibitors, however, may offer inroads into targeting these cancers. Through chemical genomics screening, we identified that Ewing sarcoma is a disease with preferential sensitivity to THZ1, a covalent small-molecule CDK7/12/13 inhibitor. The selective CDK12/13 inhibitor, THZ531, impairs DNA damage repair in an EWS/FLI-dependent manner, supporting a synthetic lethal relationship between response to THZ1/THZ531 and EWS/FLI expression. The combination of these molecules with PARP inhibitors showed striking synergy in cell viability and DNA damage assays in vitro and in multiple models of Ewing sarcoma, including a PDX, in vivo without hematopoietic toxicity. Full Text

Jacobsen, J.T., Mesin, L., Markoulaki, S., Schiepers, A., Cavazzoni, C.B., Bousbaine, D., Jaenisch, R., and Victora, G.D. (2018). One-step generation of monoclonal B cell receptor mice capable of isotype switching and somatic hypermutation. The Journal of experimental medicine 20172064. [Epub ahead of print]. We developed a method for rapid generation of B cell receptor (BCR) monoclonal mice expressing prerearranged Igh and Igk chains monoallelically from the Igh locus by CRISPR-Cas9 injection into fertilized oocytes. B cells from these mice undergo somatic hypermutation (SHM), class switch recombination (CSR), and affinity-based selection in germinal centers. This method combines the practicality of BCR transgenes with the ability to study Ig SHM, CSR, and affinity maturation. Full Text

Jaenisch , R., Dubois, N., Rasko, J.E.J., Deng, H.K., Alvarado, A.S., Fuchs, E., Vunjak-Novakovic, G., and Baldwin, K. (2018). Challenging Stem Cells (Leading Edge Views). Cell 173 (5): 1063-1065. Over the last years the stem cell field has matured. We now have a basic understanding of how somatic cells are reprogrammed and can define the epigenetic state of pluripotent cells (PSCs). However, in my opinion, two major issues need to be addressed before the full power of the iPSC system for investigating human diseases can be realized: differentiation of PSCs into mature functional cells and their use in modeling with a disease-relevant readout.Full Text

Janecka, J.E., Davis, B.W., Ghosh, S., Paria, N., Das, P.J., Orlando, L., Schubert, M., Nielsen, M.K., Stout, T.A.E., Brashear, W., Bellott, D.W. , et al. (2018). Horse Y chromosome assembly displays unique evolutionary features and putative stallion fertility genes. Nature communications 9, 2945. Dynamic evolutionary processes and complex structure make the Y chromosome among the most diverse and least understood regions in mammalian genomes. Here, we present an annotated assembly of the male specific region of the horse Y chromosome (eMSY), representing the first comprehensive Y assembly in odd-toed ungulates. The eMSY comprises single-copy, equine specific multi-copy, PAR transposed, and novel ampliconic sequence classes. The eMSY gene density approaches that of autosomes with the highest number of retained X-Y gametologs recorded in eutherians, in addition to novel Y-born and transposed genes. Horse, donkey and mule testis RNAseq reveals several candidate genes for stallion fertility. A novel testis-expressed XY ampliconic sequence class, ETSTY7, is shared with the parasite Parascaris genome, providing evidence for eukaryotic horizontal transfer and inter-chromosomal mobility. Our study highlights the dynamic nature of the Y and provides a reference sequence for improved understanding of equine male development and fertility. Full Text

Jiang, W., Wei, Y., Long, Y., Owen, A., Wang, B., Wu, X., Luo, S., Dang, Y., and Ma, D.K. (2018). A genetic program mediates cold-warming response and promotes stress-induced phenoptosis in C. elegans. eLife 7 : e35037. How multicellular organisms respond to and are impacted by severe hypothermic stress is largely unknown. From C. elegans screens for mutants abnormally responding to cold-warming stimuli, we identify a molecular genetic pathway comprising ISY-1, a conserved uncharacterized protein, and ZIP-10, a bZIP-type transcription factor. ISY-1 gatekeeps the ZIP-10 transcriptional program by regulating the microRNA mir-60. Downstream of ISY-1 and mir-60, zip-10 levels rapidly and specifically increase upon transient cold-warming exposure. Prolonged zip-10 up-regulation induces several protease-encoding genes and promotes stress-induced organismic death, or phenoptosis, of C. elegans. zip-10 deficiency confers enhanced resistance to prolonged cold-warming stress, more prominently in adults than larvae. We conclude that the ZIP-10 genetic program mediates cold-warming response and may have evolved to promote wild-population kin selection under resource-limiting and thermal stress conditions. Full Text

Kanarek , N., Keys, H.R., Cantor, J.R., Lewis, C.A., Chan, S.H., Kunchok, T., Abu-Remaileh, M., Freinkman, E., Schweitzer, L.D., and Sabatini, D.M. (2018). Histidine catabolism is a major determinant of methotrexate sensitivity.Nature [Epub ahead of print] . The chemotherapeutic drug methotrexate inhibits the enzyme dihydrofolate reductase(1), which generates tetrahydrofolate, an essential cofactor in nucleotide synthesis(2). Depletion of tetrahydrofolate causes cell death by suppressing DNA and RNA production(3). Although methotrexate is widely used as an anticancer agent and is the subject of over a thousand ongoing clinical trials(4), its high toxicity often leads to the premature termination of its use, which reduces its potential efficacy(5). To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR-Cas9-based screen(6,7). This screen yielded FTCD, which encodes an enzyme-formimidoyltransferase cyclodeaminase-that is required for the catabolism of the amino acid histidine(8), a process that has not previously been linked to methotrexate sensitivity. In cultured cancer cells, depletion of several genes in the histidine degradation pathway markedly decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of tetrahydrofolate, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukaemia xenografts to methotrexate. The histidine degradation pathway markedly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention. Full Text

Kang, I., Choi, Y., Jung, S., Lim, J.Y., Lee, D., Gupta, S.., Moon, W., and Shin, C. (2018). Identification of target genes regulated by the Drosophila histone methyltransferase Eggless reveals a role of Decapentaplegic in apoptotic signaling. Scientific reports 8(1) : 7123. Epigenetic gene regulation is essential for developmental processes. Eggless (Egg), the Drosophila orthologue of the mammalian histone methyltransferase, SETDB1, is known to be involved in the survival and differentiation of germline stem cells and piRNA cluster transcription during Drosophila oogenesis; however the detailed mechanisms remain to be determined. Here, using high-throughput RNA sequencing, we investigated target genes regulated by Egg in an unbiased manner. We show that Egg plays diverse roles in particular piRNA pathway gene expression, some long non-coding RNA expression, apoptosis-related gene regulation, and Decapentaplegic (Dpp) signaling during Drosophila oogenesis. Furthermore, using genetic and cell biological approaches, we demonstrate that ectopic upregulation of dpp caused by loss of Egg in the germarium can trigger apoptotic cell death through activation of two pro-apoptotic genes, reaper and head involution defective. We propose a model in which Egg regulates germ cell differentiation and apoptosis through canonical and noncanonical Dpp pathways in Drosophila oogenesis. Full Text

Kaplanis, J., Gordon, A., Shor, T., Weissbrod, O., Geiger, D., Wahl, M., Gershovits, M., Markus, B., Sheikh, M., Gymrek, M., and Erlich H (2018). Quantitative analysis of population-scale family trees with millions of relatives. Science [Epub ahead of print] Family trees have vast applications in multiple fields from genetics to anthropology and economics. However, the collection of extended family trees is tedious and usually relies on resources with limited geographical scope and complex data usage restrictions. Here, we collected 86 million profiles from publicly-available online data shared by genealogy enthusiasts. After extensive cleaning and validation, we obtained population-scale family trees, including a single pedigree of 13 million individuals. We leveraged the data to partition the genetic architecture of longevity by inspecting millions of relative pairs and to provide insights into the geographical dispersion of families. We also report a simple digital procedure to overlay other datasets with our resource in order to empower studies with population-scale genealogical data. Full Text

Kaur, M., Kumar, D., Butty, V., Singh, S., Esteban, A., Fink, G.R., Ploegh, H.L., and Sehrawat, S. (2018). Galectin-3 Regulates gamma-Herpesvirus Specific CD8 T Cell Immunity. iScience 9, 101-119. To gain insights into the molecular mechanisms and pathways involved in the activation of gamma-herpesvirus (MHV68)-specific T cell receptor transnuclear (TN) CD8(+) T cells, we performed a comprehensive transcriptomic analysis. Upon viral infection, we observed differential expression of several thousand transcripts encompassing various networks and pathways in activated TN cells compared with their naive counterparts. Activated cells highly upregulated galectin-3. We therefore explored the role of galectin-3 in influencing anti-MHV68 immunity. Galectin-3 was recruited at the immunological synapse during activation of CD8(+) T cells and helped constrain their activation. The localization of galectin-3 to immune synapse was evident during the activation of both naive and memory CD8(+) T cells. Galectin-3 knockout mice mounted a stronger MHV68-specific CD8(+) T cell response to the majority of viral epitopes and led to better viral control. Targeting intracellular galectin-3 in CD8(+) T cells may therefore serve to enhance response to efficiently control infections. Full Text

Kayatekin , C., Amasino, A., Gaglia, G., Flannick, J., Bonner, J.M., Fanning, S., Narayan, P., Barrasa, M.I., Pincus, D., Landgraf, D., Lindquist, S. et al. (2018). Translocon Declogger Ste24 Protects against IAPP Oligomer-Induced Proteotoxicity. Cell 173(1):62-73. Aggregates of human islet amyloid polypeptide (IAPP) in the pancreas of patients with type 2 diabetes (T2D) are thought to contribute to beta cell dysfunction and death. To understand how IAPP harms cells and how this might be overcome, we created a yeast model of IAPP toxicity. Ste24, an evolutionarily conserved protease that was recently reported to degrade peptides stuck within the translocon between the cytoplasm and the endoplasmic reticulum, was the strongest suppressor of IAPP toxicity. By testing variants of the human homolog, ZMPSTE24, with varying activity levels, the rescue of IAPP toxicity proved to be directly proportional to the declogging efficiency. Clinically relevant ZMPSTE24 variants identified in the largest database of exomes sequences derived from T2D patients were characterized using the yeast model, revealing 14 partial loss-of-function variants, which were enriched among diabetes patients over 2-fold. Thus, clogging of the translocon by IAPP oligomers may contribute to beta cell failure. Full Text

Kersten , R.D., and Weng, J.K. (2018). Gene-guided discovery and engineering of branched cyclic peptides in plants. PNAS [Epub ahead of print]. The plant kingdom contains vastly untapped natural product chemistry, which has been traditionally explored through the activity-guided approach. Here, we describe a gene-guided approach to discover and engineer a class of plant ribosomal peptides, the branched cyclic lyciumins. Initially isolated from the Chinese wolfberry Lycium barbarum, lyciumins are protease-inhibiting peptides featuring an N-terminal pyroglutamate and a macrocyclic bond between a tryptophan-indole nitrogen and a glycine alpha-carbon. We report the identification of a lyciumin precursor gene from L. barbarum, which encodes a BURP domain and repetitive lyciumin precursor peptide motifs. Genome mining enabled by this initial finding revealed rich lyciumin genotypes and chemotypes widespread in flowering plants. We establish a biosynthetic framework of lyciumins and demonstrate the feasibility of producing diverse natural and unnatural lyciumins in transgenic tobacco. With rapidly expanding plant genome resources, our approach will complement bioactivity-guided approaches to unlock and engineer hidden plant peptide chemistry for pharmaceutical and agrochemical applications. Full Text

Kleaveland , B., Shi, C.Y., Stefano, J., and Bartel, D.P. (2018). A Network of Noncoding Regulatory RNAs Acts in the Mammalian Brain. Cell [Epub ahead of print]. Noncoding RNAs (ncRNAs) play increasingly appreciated gene-regulatory roles. Here, we describe a regulatory network centered on four ncRNAs-a long ncRNA, a circular RNA, and two microRNAs-using gene editing in mice to probe the molecular consequences of disrupting key components of this network. The long ncRNA Cyrano uses an extensively paired site to miR-7 to trigger destruction of this microRNA. Cyrano-directed miR-7 degradation is much more effective than previously described examples of target-directed microRNA degradation, which come primarily from studies of artificial and viral RNAs. By reducing miR-7 levels, Cyrano prevents repression of miR-7-targeted mRNAs and enables accumulation of Cdr1as, a circular RNA known to regulate neuronal activity. Without Cyrano, excess miR-7 causes cytoplasmic destruction of Cdr1as in neurons, in part through enhanced slicing of Cdr1as by a second miRNA, miR-671. Thus, several types of ncRNAs can collaborate to establish a sophisticated regulatory network. Full Text

Kory , N., Wyant, G.A., Prakash, G., Uit de Bos, J., Pacold, M.E., Chan, S.H., Lewis, C.A., Wang, T., Keys, H.R., Guo YE, and Sabatini DM. (2018). SFXN1 is a mitochondrial serine transporter required for one-carbon metabolism.Science 362(6416) . One-carbon metabolism generates the one-carbon units required to synthesize many critical metabolites, including nucleotides. The pathway has cytosolic and mitochondrial branches, and a key step is the entry, through an unknown mechanism, of serine into mitochondria, where it is converted into glycine and formate. In a CRISPR-based genetic screen in human cells for genes of the mitochondrial pathway, we found sideroflexin 1 (SFXN1), a multipass inner mitochondrial membrane protein of unclear function. Like cells missing mitochondrial components of one-carbon metabolism, those null for SFXN1 are defective in glycine and purine synthesis. Cells lacking SFXN1 and one of its four homologs, SFXN3, have more severe defects, including being auxotrophic for glycine. Purified SFXN1 transports serine in vitro. Thus, SFXN1 functions as a mitochondrial serine transporter in one-carbon metabolism. Full Text

Krakowiak, J., Zheng, X., Patel, N., Feder, Z.A., Anandhakumar, J., Valerius, K.., Gross, D.S., Khalil, A.S., and Pincus, D. (2018). Hsf1 and Hsp70 constitute a two-component feedback loop that regulates the yeast heat shock response. eLife e31668 [Epub ahead of print]. Models for regulation of the eukaryotic heat shock response typically invoke a negative feedback loop consisting of the transcriptional activator Hsf1 and a molecular chaperone. Previously we identified Hsp70 as the chaperone responsible for Hsf1 repression and constructed a mathematical model that recapitulated the yeast heat shock response (Zheng et al., 2016). The model was based on two assumptions: dissociation of Hsp70 activates Hsf1, and transcriptional induction of Hsp70 deactivates Hsf1. Here we validate these assumptions. First, we severed the feedback loop by uncoupling Hsp70 expression from Hsf1 regulation. As predicted by the model, Hsf1 was unable to efficiently deactivate in the absence of Hsp70 transcriptional induction. Next, we mapped a discrete Hsp70 binding site on Hsf1 to a C-terminal segment known as conserved element 2 (CE2). In vitro, CE2 binds to Hsp70 with low affinity (9 microM), in agreement with model requirements. In cells, removal of CE2 resulted in increased basal Hsf1 activity and delayed deactivation during heat shock, while tandem repeats of CE2 sped up Hsf1 deactivation. Finally, we uncovered a role for the N-terminal domain of Hsf1 in negatively regulating DNA binding. These results reveal the quantitative control mechanisms underlying the heat shock response. Full Text

Krall , J.A., Reinhardt, F., Mercury, O.A., Pattabiraman, D.R., Brooks, M.W., Dougan, M., Lambert, A.W., Bierie, B., Ploegh, H.L., Dougan, S.K., and Weinberg RA. (2018). The systemic response to surgery triggers the outgrowth of distant immune-controlled tumors in mouse models of dormancy.Science translational medicine 10, 436, eaan346. 4The systemic response to surgery triggers the outgrowth of distant immune-controlled tumors in mouse models of dormancy.Patients undergoing surgical resection of primary breast tumors confront a risk for metastatic recurrence that peaks sharply 12 to 18 months after surgery. The cause of early metastatic relapse in breast cancer has long been debated, with many ascribing these relapses to the natural progression of the disease. Others have proposed that some aspect of surgical tumor resection triggers the outgrowth of otherwise-dormant metastases, leading to the synchronous pattern of relapse. Clinical data cannot distinguish between these hypotheses, and previous experimental approaches have not provided clear answers. Such uncertainty hinders the development and application of therapeutic approaches that could potentially reduce early metastatic relapse. We describe an experimental model system that definitively links surgery and the subsequent wound-healing response to the outgrowth of tumor cells at distant anatomical sites. Specifically, we find that the systemic inflammatory response induced after surgery promotes the emergence of tumors whose growth was otherwise restricted by a tumor-specific T cell response. Furthermore, we demonstrate that perioperative anti-inflammatory treatment markedly reduces tumor outgrowth in this model, suggesting that similar approaches might substantially reduce early metastatic recurrence in breast cancer patients. Full Text

Kritzer, J.A., Freyzon, Y., and Lindquist, S.. (2018). Yeast can accommodate phosphotyrosine: v-Src toxicity in yeast arises from a single disrupted pathway.FEMS Yeast Research [Epub ahead of print]. Tyrosine phosphorylation is a key biochemical signal that controls growth and differentiation in multicellular organisms. Saccharomyces cerevisiae and nearly all other unicellular eukaryotes lack intact phosphotyrosine signaling pathways. However, many of these organisms have primitive phosphotyrosine-binding proteins and tyrosine phosphatases, leading to the assumption that the major barrier for emergence of phosphotyrosine signaling was the negative consequences of promiscuous tyrosine kinase activity. In this work, we reveal that the classic oncogene v-Src, which phosphorylates many dozens of proteins in yeast, is toxic because it disrupts a specific spore wall remodeling pathway. Using genetic selections, we find that expression of a specific cyclic peptide, or overexpression of SMK1, a MAP kinase that controls spore wall assembly, both lead to robust growth despite a continuous high level of phosphotyrosine in the yeast proteome. Thus, minimal genetic manipulations allow yeast to tolerate high levels of phosphotyrosine. These results indicate that the introduction of tyrosine kinases within single-celled organisms may not have been a major obstacle to the evolution of phosphotyrosine signaling. Full Text

Li, C., Singh, B., Graves-Deal, R., Ma, H., Starchenko, A., Fry, W.H., Lu, Y., Wang, Y., Bogatcheva, G., Khan, M.P., et al. (2017). Three-dimensional culture system identifies a new mode of cetuximab resistance and disease-relevant genes in colorectal cancer. PNAS 114 (14) : E2852-e2861. We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors. Full Text

Li , F.S., Phyo, P., Jacobowitz, J.., Hong, M., and Weng, J.K. .(2018). The molecular structure of plant sporopollenin. Nature plants [Epub ahead of print]. Sporopollenin is a ubiquitous and extremely chemically inert biopolymer that constitutes the outer wall of all land-plant spores and pollen grains(1). Sporopollenin protects the vulnerable plant gametes against a wide range of environmental assaults, and is considered a prerequisite for the migration of early plants onto land(2). Despite its importance, the chemical structure of plant sporopollenin has remained elusive(1). Using a newly developed thioacidolysis degradative method together with state-of-the-art solid-state NMR techniques, we determined the detailed molecular structure of pine sporopollenin. We show that pine sporopollenin is primarily composed of aliphatic-polyketide-derived polyvinyl alcohol units and 7-O-p-coumaroylated C16 aliphatic units, crosslinked through a distinctive dioxane moiety featuring an acetal. Naringenin was also identified as a minor component of pine sporopollenin. This discovery answers the long-standing question about the chemical make-up of plant sporopollenin, laying the foundation for future investigations of sporopollenin biosynthesis and for the design of new biomimetic polymers with desirable inert properties. Full Text

Li, H., Lodish, H.F. , and Sieff, C.A. (2018). Critical Issues in Diamond-Blackfan Anemia and Prospects for Novel Treatment.Hematology/oncology Clinics of North America 32(4), 701-712. Diamond-Blackfan anemia (DBA) is a severe congenital hypoplastic anemia caused by mutation in a ribosomal protein gene. Major clinical issues concern the optimal management of patients resistant to steroids, the first-line therapy. Hematopoietic stem cell transplantation is indicated in young patients with an HLA-matched unaffected sibling donor, and recent results with matched unrelated donor transplants indicate that these patients also do well. When neither steroids nor a transplant is possible red cell transfusions are required, and iron loading is rapid in some DBA patients, so effective chelation is vital. Also discussed are novel treatments under investigation for DBA.

Li, J., Choi, P.S., Chaffer, C.L., Labella, K., Hwang, J.H., Giacomelli, A.O., Kim, J.W., Ilic, N., Doench, J.G., Ly, S.H., Weinberg R.A., et al. (2018). An alternative splicing switch in FLNB promotes the mesenchymal cell state in human breast cancer. eLife e37184.[Epub ahead of print]. Alternative splicing of mRNA precursors represents a key gene expression regulatory step and permits the generation of distinct protein products with diverse functions. In a genome-scale expression screen for inducers of the epithelial-to-mesenchymal transition (EMT), we found a striking enrichment of RNA-binding proteins. We validated that QKI and RBFOX1 were necessary and sufficient to induce an intermediate mesenchymal cell state and increased tumorigenicity. Using RNA-seq and eCLIP analysis, we found that QKI and RBFOX1 coordinately regulated the splicing and function of the actin-binding protein FLNB, which plays a causal role in the regulation of EMT. Specifically, the skipping of FLNB exon 30 induced EMT by releasing the FOXC1 transcription factor. Moreover, skipping of FLNB exon 30 is strongly associated with EMT gene signatures in basal-like breast cancer patient samples. These observations identify a specific dysregulation of splicing, which regulates tumor cell plasticity and is frequently observed in human cancer. Full Text

Liu, F.D., Tam, K., Pishesha, N., Poon, Z., and Van Vliet, K.J. (2018). Improving hematopoietic recovery through modeling and modulation of the mesenchymal stromal cell secretome. Stem cell research & therapy 9 (1) : 268. Efficient and sustained hematopoietic recovery after hematopoietic stem cell or bone marrow transplantation is supported by paracrine signaling from specific subpopulations of mesenchymal stromal cells (MSCs). Here, we considered whether in vitro mechanopriming of human MSCs could be administered to predictively and significantly improve in vivo hematopoietic recovery after irradiation injury. METHODS: First, we implemented regression modeling to identify eight MSC-secreted proteins that correlated strongly with improved rescue from radiation damage, including hematopoietic recovery, in a murine model of hematopoietic failure. Using these partial least squares regression (PLSR) model parameters, we then predicted recovery potential of MSC populations that were culture expanded on substrata of varying mechanical stiffness. Lastly, we experimentally validated these predictions using an in vitro co-culture model of hematopoiesis and using new in vivo experiments for the same irradiation injury model used to generate survival predictions. RESULTS: MSCs grown on the least stiff (elastic moduli ~ 1 kPa) of these polydimethylsiloxane (PDMS) substrata secreted high concentrations of key proteins identified in regression modeling, at concentrations comparable to those secreted by minor subpopulations of MSCs shown previously to be effective in supporting such radiation rescue. We confirmed that these MSCs expanded on PDMS could promote hematopoiesis in an in vitro co-culture model with hematopoietic stem and progenitor cells (HSPCs). Further, MSCs cultured on PDMS of highest stiffness (elastic moduli ~ 100 kPa) promoted expression of CD123(+) HSPCs, indicative of myeloid differentiation. Systemic administration of mechanoprimed MSCs resulted in improved mouse survival and weight recovery after bone marrow ablation, as compared with both standard MSC expansion on stiffer materials and with biophysically sorted MSC subpopulations. Additionally, we observed recovery of white blood cells, platelets, and red blood cells, indicative of complete recovery of all hematopoietic lineages. CONCLUSIONS: These results demonstrate that computational techniques to identify MSC biomarkers can be leveraged to predict and engineer therapeutically effective MSC phenotypes defined by mechanoprimed secreted factors, for translational applications including hematopoietic recovery. Full Text

Liu, X.S., Wu, H., Krzisch, M., Wu, X., Graef, J., Muffat, J., Hnisz, D., Li, C.H., Yuan, B., Xu, C.., Vershkov D, Cacace A, Young RA , and Jaenisch R. (2018). Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of the FMR1 Gene. Cell [Epub ahead of print] Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS. Full Text

Ma , H., Wert, K.J., Shvartsman, D., Melton, D.A., and Jaenisch, R. (2018). Establishment of human pluripotent stem cell-derived pancreatic beta-like cells in the mouse pancreas. PNAS [Epub ahead of print]. Type 1 diabetes is characterized by autoimmune destruction of beta cells located in pancreatic islets. However, tractable in vivo models of human pancreatic beta cells have been limited. Here, we generated xenogeneic human pancreatic beta-like cells in the mouse pancreas by orthotopic transplantation of stem cell-derived beta (SC-beta) cells into the pancreas of neonatal mice. The engrafted beta-like cells expressed beta cell transcription factors and markers associated with functional maturity. Engrafted human cells recruited mouse endothelial cells, suggesting functional integration. Human insulin was detected in the blood circulation of transplanted mice for months after transplantation and increased upon glucose stimulation. In addition to beta-like cells, human cells expressing markers for other endocrine pancreas cell types, acinar cells, and pancreatic ductal cells were identified in the pancreata of transplanted mice, indicating that this approach allows studying other human pancreatic cell types in the mouse pancreas. Our results demonstrate that orthotopic transplantation of human SC-beta cells into neonatal mice is an experimental platform that allows the generation of mice with human pancreatic beta-like cells in the endogenous niche. Full Text

Mansour, M.R., He, S., Li, Z., Lobbardi, R., Abraham, B.J. , Hug, C., Rahman, S., Leon, T.E., Kuang, Y.Y., Zimmerman, M.W., Anders, L., Young, R.A. , et al. (2018). JDP2: An oncogenic bZIP transcription factor in T cell acute lymphoblastic leukemia. The Journal of experimental medicine 215(7):1929-1945. A substantial subset of patients with T cell acute lymphoblastic leukemia (T-ALL) develops resistance to steroids and succumbs to their disease. JDP2 encodes a bZIP protein that has been implicated as a T-ALL oncogene from insertional mutagenesis studies in mice, but its role in human T-ALL pathogenesis has remained obscure. Here we show that JDP2 is aberrantly expressed in a subset of T-ALL patients and is associated with poor survival. JDP2 is required for T-ALL cell survival, as its depletion by short hairpin RNA knockdown leads to apoptosis. Mechanistically, JDP2 regulates prosurvival signaling through direct transcriptional regulation of MCL1. Furthermore, JDP2 is one of few oncogenes capable of initiating T-ALL in transgenic zebrafish. Notably, thymocytes from rag2:jdp2 transgenic zebrafish express high levels of mcl1 and demonstrate resistance to steroids in vivo. These studies establish JDP2 as a novel oncogene in high-risk T-ALL and implicate overexpression of MCL1 as a mechanism of steroid resistance in JDP2-overexpressing cells. Full Text

McKenney, A.S., Lau, A.N., Somasundara, A.V.H., Spitzer, B., Intlekofer, A.M., Ahn, J., Shank, K., Rapaport, F.T., Patel, M.A., Papalexi, E., Freinkman, E., et al. (2018). JAK2/IDH-mutant-driven myeloproliferative neoplasm is sensitive to combined targeted inhibition. The Journal of clinical investigation [Epub ahead of print].Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617F and mutant IDH1R132H or Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617F Idh2R140Q-mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mut and IDH2mut mutations. Taken together, these data suggest that combined JAK and IDH inhibition may offer a therapeutic advantage in this high-risk MPN subtype. Full Text

Mellios, N., Feldman, D.A., Sheridan, S.D., Ip, J.P.K., Kwok, S., Amoah, S.K., Rosen, B., Rodriguez, B.A., Crawford, B., Swaminathan, R., Chou S, Li Y, Ziats M, Ernst C, Jaenisch R, Haggarty SJ, and Sur M.(2018). MeCP2-regulated miRNAs control early human neurogenesis through differential effects on ERK and AKT signaling. Molecular psychiatry 23, 1051-1065. Rett syndrome (RTT) is an X-linked, neurodevelopmental disorder caused primarily by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, which encodes a multifunctional epigenetic regulator with known links to a wide spectrum of neuropsychiatric disorders. Although postnatal functions of MeCP2 have been thoroughly investigated, its role in prenatal brain development remains poorly understood. Given the well-established importance of microRNAs (miRNAs) in neurogenesis, we employed isogenic human RTT patient-derived induced pluripotent stem cell (iPSC) and MeCP2 short hairpin RNA knockdown approaches to identify novel MeCP2-regulated miRNAs enriched during early human neuronal development. Focusing on the most dysregulated miRNAs, we found miR-199 and miR-214 to be increased during early brain development and to differentially regulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase and protein kinase B (PKB/AKT) signaling. In parallel, we characterized the effects on human neurogenesis and neuronal differentiation brought about by MeCP2 deficiency using both monolayer and three-dimensional (cerebral organoid) patient-derived and MeCP2-deficient neuronal culture models. Inhibiting miR-199 or miR-214 expression in iPSC-derived neural progenitors deficient in MeCP2 restored AKT and ERK activation, respectively, and ameliorated the observed alterations in neuronal differentiation. Moreover, overexpression of miR-199 or miR-214 in the wild-type mouse embryonic brains was sufficient to disturb neurogenesis and neuronal migration in a similar manner to Mecp2 knockdown. Taken together, our data support a novel miRNA-mediated pathway downstream of MeCP2 that influences neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders. Full Text

Mihaylova , M.M., Cheng, C.W., Cao, A.Q., Tripathi, S., Mana, M.D., Bauer-Rowe, K.E., Abu-Remaileh, M., Clavain, L., Erdemir, A., Lewis, C.A., Freinkman, E., Huang, Y., Bell, G.W., Sabatini, D.M., et al. (2018). Fasting Activates Fatty Acid Oxidation to Enhance Intestinal Stem Cell Function during Homeostasis and Aging. Cell stem cell 22, 769-778. Diet has a profound effect on tissue regeneration in diverse organisms, and low caloric states such as intermittent fasting have beneficial effects on organismal health and age-associated loss of tissue function. The role of adult stem and progenitor cells in responding to short-term fasting and whether such responses improve regeneration are not well studied. Here we show that a 24 hr fast augments intestinal stem cell (ISC) function in young and aged mice by inducing a fatty acid oxidation (FAO) program and that pharmacological activation of this program mimics many effects of fasting. Acute genetic disruption of Cpt1a, the rate-limiting enzyme in FAO, abrogates ISC-enhancing effects of fasting, but long-term Cpt1a deletion decreases ISC numbers and function, implicating a role for FAO in ISC maintenance. These findings highlight a role for FAO in mediating pro-regenerative effects of fasting in intestinal biology, and they may represent a viable strategy for enhancing intestinal regeneration. Full Text

Miller, D.H., Jin, D.X., Sokol, E.S., Cabrera, J.R., Superville, D.A., Gorelov, R.A.,., Kuperwasser, C., and .Gupta, P.B., (2018). BCL11B Drives Human Mammary Stem Cell Self-Renewal In Vitro by Inhibiting Basal Differentiation. Stem cell reports 10, 1131-1145. The epithelial compartment of the mammary gland contains basal and luminal cell lineages, as well as stem and progenitor cells that reside upstream in the differentiation hierarchy. Stem and progenitor cell differentiation is regulated to maintain adult tissue and mediate expansion during pregnancy and lactation. The genetic factors that regulate the transition of cells between differentiation states remain incompletely understood. Here, we present a genome-scale method to discover genes driving cell-state specification. Applying this method, we identify a transcription factor, BCL11B, which drives stem cell self-renewal in vitro, by inhibiting differentiation into the basal lineage. To validate BCL11B's functional role, we use two-dimensional colony-forming and three-dimensional tissue differentiation assays to assess the lineage differentiation potential and functional abilities of primary human mammary cells. These findings show that BCL11B regulates mammary cell differentiation and demonstrate the utility of our proposed genome-scale strategy for identifying lineage regulators in mammalian tissues. Full Text

Minton, D.R., Nam, M., McLaughlin, D.J., Shin, J., Bayraktar, E.C., Alvarez, S.W., Sviderskiy, V.O., Papagiannakopoulos, T., Sabatini, D.M., Birsoy, K., et al. (2018). Serine Catabolism by SHMT2 Is Required for Proper Mitochondrial Translation Initiation and Maintenance of Formylmethionyl-tRNAs.Molecular cell 69, 610-621.e615. Upon glucose restriction, eukaryotic cells upregulate oxidative metabolism to maintain homeostasis. Using genetic screens, we find that the mitochondrial serine hydroxymethyltransferase (SHMT2) is required for robust mitochondrial oxygen consumption and low glucose proliferation. SHMT2 catalyzes the first step in mitochondrial one-carbon metabolism, which, particularly in proliferating cells, produces tetrahydrofolate (THF)-conjugated one-carbon units used in cytoplasmic reactions despite the presence of a parallel cytoplasmic pathway. Impairing cytoplasmic one-carbon metabolism or blocking efflux of one-carbon units from mitochondria does not phenocopy SHMT2 loss, indicating that a mitochondrial THF cofactor is responsible for the observed phenotype. The enzyme MTFMT utilizes one such cofactor, 10-formyl THF, producing formylmethionyl-tRNAs, specialized initiator tRNAs necessary for proper translation of mitochondrially encoded proteins. Accordingly, SHMT2 null cells specifically fail to maintain formylmethionyl-tRNA pools and mitochondrially encoded proteins, phenotypes similar to those observed in MTFMT-deficient patients. These findings provide a rationale for maintaining a compartmentalized one-carbon pathway in mitochondria. Full Text

Monda , J.K., and Cheeseman, I.M. (2018). Dynamic regulation of dynein localization revealed by small molecule inhibitors of ubiquitination enzymes. Open Biology 8(9): 180095. Cytoplasmic dynein is a minus-end-directed microtubule-based motor that acts at diverse subcellular sites. During mitosis, dynein localizes simultaneously to the mitotic spindle, spindle poles, kinetochores and the cell cortex. However, it is unclear what controls the relative targeting of dynein to these locations. As dynein is heavily post-translationally modified, we sought to test a role for these modifications in regulating dynein localization. We find that dynein rapidly and strongly accumulates at mitotic spindle poles following treatment with NSC697923, a small molecule that inhibits the ubiquitin E2 enzyme, Ubc13, or treatment with PYR-41, a ubiquitin E1 inhibitor. Subsets of dynein regulators such as Lis1, ZW10 and Spindly accumulate at the spindle poles, whereas others do not, suggesting that NSC697923 differentially affects specific dynein populations. We additionally find that dynein relocalization induced by NSC697923 or PYR-41 can be suppressed by simultaneous treatment with the non-selective deubiquitinase inhibitor, PR-619. However, we did not observe altered dynein localization following treatment with the selective E1 inhibitor, TAK-243. Although it is possible that off-target effects of NSC697923 and PYR-41 are responsible for the observed changes in dynein localization, the rapid relocalization upon drug treatment highlights the highly dynamic nature of dynein regulation during mitosis. Full Text

Monda , J.K., and Cheeseman, I.M. (2018). The kinetochore-microtubule interface at a glance. Journal of cell science 131 : jcs214577. Accurate chromosome segregation critically depends on the formation of attachments between microtubule polymers and each sister chromatid. The kinetochore is the macromolecular complex that assembles at the centromere of each chromosome during mitosis and serves as the link between the DNA and the microtubules. In this Cell Science at a Glance article and accompanying poster, we discuss the activities and molecular players that are involved in generating kinetochore-microtubule attachments, including the initial stages of lateral kinetochore-microtubule interactions and maturation to stabilized end-on attachments. We additionally explore the features that contribute to the ability of the kinetochore to track with dynamic microtubules. Finally, we examine the contributions of microtubule-associated proteins to the organization and stabilization of the mitotic spindle and the control of microtubule dynamics. Full Text

Monda , J.K., and Cheeseman, I.M. (2018). Nde1 promotes diverse dynein functions through differential interactions and exhibits an isoform-specific proteasome association.Molecular biology of the cell [Epub ahead of print] . Nde1 is a key regulator of cytoplasmic dynein, binding directly to both dynein itself and the dynein adaptor, Lis1. Nde1 and Lis1 are thought to function together to promote dynein function, yet mutations in each result in distinct neurodevelopment phenotypes. To reconcile these phenotypic differences, we sought to dissect the contribution of Nde1 to dynein regulation and explore the cellular functions of Nde1. Here, we show that an Nde1-Lis1 interaction is required for spindle pole focusing and Golgi organization, but is largely dispensable for centrosome placement, despite Lis1 itself being required. Thus, diverse functions of dynein rely on distinct Nde1- and Lis1-mediated regulatory mechanisms. Additionally, we discovered a robust, isoform-specific interaction between human Nde1 and the 26S proteasome and identify precise mutations in Nde1 that disrupt the proteasome interaction. Together, our work suggests that Nde1 makes unique contributions to human neurodevelopment through its regulation of both dynein and proteasome function. Full Text

Muffat , J., Li, Y., Omer, A., Durbin, A., Bosch, I., Bakiasi, G., Richards, E., Meyer, A., Gehrke, L., and Jaenisch R.(2018). Human induced pluripotent stem cell-derived glial cells and neural progenitors display divergent responses to Zika and dengue infections. PNAS [Epub ahead of print]. Maternal Zika virus (ZIKV) infection during pregnancy is recognized as the cause of an epidemic of microcephaly and other neurological anomalies in human fetuses. It remains unclear how ZIKV accesses the highly vulnerable population of neural progenitors of the fetal central nervous system (CNS), and which cell types of the CNS may be viral reservoirs. In contrast, the related dengue virus (DENV) does not elicit teratogenicity. To model viral interaction with cells of the fetal CNS in vitro, we investigated the tropism of ZIKV and DENV for different induced pluripotent stem cell-derived human cells, with a particular focus on microglia-like cells. We show that ZIKV infected isogenic neural progenitors, astrocytes, and microglia-like cells (pMGLs), but was only cytotoxic to neural progenitors. Infected glial cells propagated ZIKV and maintained ZIKV load over time, leading to viral spread to susceptible cells. DENV triggered stronger immune responses and could be cleared by neural and glial cells more efficiently. pMGLs, when cocultured with neural spheroids, invaded the tissue and, when infected with ZIKV, initiated neural infection. Since microglia derive from primitive macrophages originating in proximity to the maternal vasculature, they may act as a viral reservoir for ZIKV and establish infection of the fetal brain. Infection of immature neural stem cells by invading microglia may occur in the early stages of pregnancy, before angiogenesis in the brain rudiments. Our data are also consistent with ZIKV and DENV affecting the integrity of the blood-brain barrier, thus allowing infection of the brain later in life. Full Text

Naqvi , S., Bellott, D.W., Lin, K.S., and Page, D.C. (2018). Conserved microRNA targeting reveals preexisting gene dosage sensitivities that shaped amniote sex chromosome evolution. Genome research [Epub ahead of print]. Mammalian X and Y Chromosomes evolved from an ordinary autosomal pair. Genetic decay of the Y led to X Chromosome inactivation (XCI) in females, but some Y-linked genes were retained during the course of sex chromosome evolution, and many X-linked genes did not become subject to XCI. We reconstructed gene-by-gene dosage sensitivities on the ancestral autosomes through phylogenetic analysis of microRNA (miRNA) target sites and compared these preexisting characteristics to the current status of Y-linked and X-linked genes in mammals. Preexisting heterogeneities in dosage sensitivity, manifesting as differences in the extent of miRNA-mediated repression, predicted either the retention of a Y homolog or the acquisition of XCI following Y gene decay. Analogous heterogeneities among avian Z-linked genes predicted either the retention of a W homolog or gene-specific dosage compensation following W gene decay. Genome-wide analyses of human copy number variation indicate that these heterogeneities consisted of sensitivity to both increases and decreases in dosage. We propose a model of XY/ZW evolution incorporating such preexisting dosage sensitivities in determining the evolutionary fates of individual genes. Our findings thus provide a more complete view of the role of dosage sensitivity in shaping the mammalian and avian sex chromosomes, and reveal an important role for post-transcriptional regulatory sequences (miRNA target sites) in sex chromosome evolution. Full Text

Neckers, L., Blagg, B., Haystead, T., Trepel, J.B., Whitesell, L., and Picard, D. (2018). Methods to validate Hsp90 inhibitor specificity, to identify off-target effects, and to rethink approaches for further clinical development.Cell stress & chaperones [Epub ahead of print] . The molecular chaperone Hsp90 is one component of a highly complex and interactive cellular proteostasis network (PN) that participates in protein folding, directs misfolded and damaged proteins for destruction, and participates in regulating cellular transcriptional responses to environmental stress, thus promoting cell and organismal survival. Over the last 20 years, it has become clear that various disease states, including cancer, neurodegeneration, metabolic disorders, and infection by diverse microbes, impact the PN. Among PN components, Hsp90 was among the first to be pharmacologically targeted with small molecules. While the number of Hsp90 inhibitors described in the literature has dramatically increased since the first such small molecule was described in 1994, it has become increasingly apparent that not all of these agents have been sufficiently validated for specificity, mechanism of action, and lack of off-target effects. Given the less than expected activity of Hsp90 inhibitors in cancer-related human clinical trials, a re-evaluation of potentially confounding off-target effects, as well as confidence in target specificity and mechanism of action, is warranted. In this commentary, we provide feasible approaches to achieve these goals and we discuss additional considerations to improve the clinical efficacy of Hsp90 inhibitors in treating cancer and other diseases. Full Text

Newby , G.A., and Kayatekin, C. (2018). Microbial specialization by prions. Prion [Epub ahead of print] Microbial prions facilitate a variety of phenotypic switches. Recently-developed tools that can directly interrogate, in the living cell, the aggregation state of a protein have enabled a wider range of experiments for prion-mediated behaviors. With such tools, the roles of the yeast prion [SWI(+)] in migration and mating were studied. Although [SWI(+)] cells were consistently less fit than their [swi-] counterparts under traditional laboratory conditions, in these new phenotypic paradigms [SWI(+)] cells demonstrated a distinct advantage. [SWI(+)] cells dispersed over a larger area under conditions resembling rainfall and outcrossed more frequently. We postulate that many behaviors in microorganisms may be modulated by stochastic prion switching. In diverse and changing natural environments, prion switching at low frequency may promote greater fitness of the population by specializing a small number of individuals with altered responses to their environments. Full Text

Ni, T.K., Elman, J.S., Jin, D.X., Gupta, P.B. , and Kuperwasser, C. (2018). Premature polyadenylation of MAGI3 is associated with diminished N(6)-methyladenosine in its large internal exon. Scientific reports 8, 1415. In cancer, tumor suppressor genes (TSGs) are frequently truncated, causing their encoded products to be non-functional or dominant-negative. We previously showed that premature polyadenylation (pPA) of MAGI3 truncates the gene, switching its functional role from a TSG to a dominant-negative oncogene. Here we report that MAGI3 undergoes pPA at the intron immediately downstream of its large internal exon, which is normally highly modified by N(6)-methyladenosine (m(6)A). In breast cancer cells that upregulate MAGI3 (pPA) , m(6)A levels in the large internal exon of MAGI3 are significantly reduced compared to cells that do not express MAGI3 (pPA) . We further find that MAGI3 (pPA) transcripts are significantly depleted of m(6)A modifications, in contrast to highly m(6)A-modified full-length MAGI3 mRNA. Finally, we analyze public expression data and find that other TSGs, including LATS1 and BRCA1, also undergo intronic pPA following large internal exons, and that m(6)A levels in these exons are reduced in pPA-activated breast cancer cells relative to untransformed mammary cells. Our study suggests that m(6)A may play a role in regulating intronic pPA of MAGI3 and possibly other TSGs, warranting further investigation. Full Text

Omer , Javed A., Li, Y., Muffat, J., Su, K.C., Cohen, M.A., Lungjangwa, T., Aubourg, P., Cheeseman, I.M., and Jaenisch, R. (2018). Microcephaly Modeling of Kinetochore Mutation Reveals a Brain-Specific Phenotype. Cell reports 25, 368-382.e365. Most genes mutated in microcephaly patients are expressed ubiquitously, and yet the brain is the only major organ compromised in most patients. Why the phenotype remains brain specific is poorly understood. In this study, we used in vitro differentiation of human embryonic stem cells to monitor the effect of a point mutation in kinetochore null protein 1 (KNL1; CASC5), identified in microcephaly patients, during in vitro brain development. We found that neural progenitors bearing a patient mutation showed reduced KNL1 levels, aneuploidy, and an abrogated spindle assembly checkpoint. By contrast, no reduction of KNL1 levels or abnormalities was observed in fibroblasts and neural crest cells. We established that the KNL1 patient mutation generates an exonic splicing silencer site, which mainly affects neural progenitors because of their higher levels of splicing proteins. Our results provide insight into the brain-specific phenomenon, consistent with microcephaly being the only major phenotype of patients bearing KNL1 mutation. Full Text

Pedley, A.M., Karras, G.I., Zhang, X., Lindquist, S., and Benkovic, S.J. (2018). Role of HSP90 in the Regulation of de Novo Purine Biosynthesis. Biochemistry 57, 3217-3221. Despite purines making up one of the largest classes of metabolites in a cell, little is known about the regulatory mechanisms that facilitate efficient purine production. Under conditions resulting in high purine demand, enzymes within the de novo purine biosynthetic pathway cluster into multienzyme assemblies called purinosomes. Purinosome formation has been linked to molecular chaperones HSP70 and HSP90; however, the involvement of these molecular chaperones in purinosome formation remains largely unknown. Here, we present a new-found biochemical mechanism for the regulation of de novo purine biosynthetic enzymes mediated through HSP90. HSP90-client protein interaction assays were employed to identify two enzymes within the de novo purine biosynthetic pathway, PPAT and FGAMS, as client proteins of HSP90. Inhibition of HSP90 by STA9090 abrogated these interactions and resulted in a decrease in the level of available soluble client protein while having no significant effect on their interactions with HSP70. These findings provide a mechanism to explain the dependence of purinosome assembly on HSP90 activity. The combined efforts of molecular chaperones in the maturation of PPAT and FGAMS result in purinosome formation and are likely essential for enhancing the rate of purine production to meet intracellular purine demand. Full Text

Pelka, K., Bertheloot, D., Reimer, E., Phulphagar, K., Schmidt, S.V., Christ, A., Stahl, R., Watson, N., Miyake, K., Hacohen, N., et al. (2018). The Chaperone UNC93B1 Regulates Toll-like Receptor Stability Independently of Endosomal TLR Transport. Immunity 48, 911-922.e917.Unc-93 homolog B1 (UNC93B1) is a key regulator of nucleic acid (NA)-sensing Toll-like receptors (TLRs). Loss of NA-sensing TLR responses in UNC93B1-deficient patients facilitates Herpes simplex virus type 1 (HSV-1) encephalitis. UNC93B1 is thought to guide NA-sensing TLRs from the endoplasmic reticulum (ER) to their respective endosomal signaling compartments and to guide the flagellin receptor TLR5 to the cell surface, raising the question of how UNC93B1 mediates differential TLR trafficking. Here, we report that UNC93B1 regulates a step upstream of the differential TLR trafficking process. We discovered that UNC93B1 deficiency resulted in near-complete loss of TLR3 and TLR7 proteins in primary splenic mouse dendritic cells and macrophages, showing that UNC93B1 is critical for maintaining TLR expression. Notably, expression of an ER-retained UNC93B1 version was sufficient to stabilize TLRs and largely restore endosomal TLR trafficking and activity. These data are critical for an understanding of how UNC93B1 can regulate the function of a broad subset of TLRs. Full Text

Petkau, G., Kawano, Y., Wolf, I., Knoll, M.,, and Melchers, F. (2018). MiR221 promotes precursor B-cell retention in the bone marrow by amplifying the PI3K-signaling pathway in mice. European journal of immunology[Epub ahead of print]. Hematopoietic stem cells and lineage-uncommitted progenitors are able to home to the bone marrow upon transplantation and reconstitute the host with hematopoietic progeny. Expression of miR221 in B-lineage committed preBI-cells induces their capacity to home to the bone marrow. However, the molecular mechanisms underlying miR221-controlled bone marrow homing and retention remain poorly understood. Here, we demonstrate, that miR221 regulates bone marrow retention of such B-cell precursors by targeting PTEN, thus enhancing PI3K signaling in response to the chemokine CXCL12. MiR221-enhanced PI3K signaling leads to increased expression of the anti-apoptotic protein Bcl2 and VLA4 integrin-mediated adhesion to VCAM1 in response to CXCL12 in vitro. Ablation of elevated PI3K activity abolishes the retention of miR221 expressing preBI-cells in the bone marrow. These results suggest that amplification of PI3K signaling by miR221 could be a general mechanism for bone marrow residence, shared by miR221-expressing hematopoietic cells. Full Text

Petrova , B., Liu, K., Tian, C., Kitaoka, M., Freinkman, E. , Yang, J., and Orr-Weaver, T.L. (2018). Dynamic redox balance directs the oocyte-to-embryo transition via developmentally controlled reactive cysteine changes. PNAS [Epub ahead of print].The metabolic and redox state changes during the transition from an arrested oocyte to a totipotent embryo remain uncharacterized. Here, we applied state-of-the-art, integrated methodologies to dissect these changes in Drosophila We demonstrate that early embryos have a more oxidized state than mature oocytes. We identified specific alterations in reactive cysteines at a proteome-wide scale as a result of this metabolic and developmental transition. Consistent with a requirement for redox change, we demonstrate a role for the ovary-specific thioredoxin Deadhead (DHD). dhd-mutant oocytes are prematurely oxidized and exhibit meiotic defects. Epistatic analyses with redox regulators link dhd function to the distinctive redox-state balance set at the oocyte-to-embryo transition. Crucially, global thiol-redox profiling identified proteins whose cysteines became differentially modified in the absence of DHD. We validated these potential DHD substrates by recovering DHD-interaction partners using multiple approaches. One such target, NO66, is a conserved protein that genetically interacts with DHD, revealing parallel functions. As redox changes also have been observed in mammalian oocytes, we hypothesize a link between developmental control of this cell-cycle transition and regulation by metabolic cues. This link likely operates both by general redox state and by changes in the redox state of specific proteins. The redox proteome defined here is a valuable resource for future investigation of the mechanisms of redox-modulated control at the oocyte-to-embryo transition. Full Text

Pignatta , D., Novitzky, K., Satyaki, P.R.V., and Gehring, M. (2018). A variably imprinted epiallele impacts seed development. PLoS genetics 14, e1007469. The contribution of epigenetic variation to phenotypic variation is unclear. Imprinted genes, because of their strong association with epigenetic modifications, represent an opportunity for the discovery of such phenomena. In mammals and flowering plants, a subset of genes are expressed from only one parental allele in a process called gene imprinting. Imprinting is associated with differential DNA methylation and chromatin modifications between parental alleles. In flowering plants imprinting occurs in a seed tissue-endosperm. Proper endosperm development is essential for the production of viable seeds. We previously showed that in Arabidopsis thaliana intraspecific imprinting variation is correlated with naturally occurring DNA methylation polymorphisms. Here, we investigated the mechanisms and function of allele-specific imprinting of the class IV homeodomain-Leucine zipper (HD-ZIP) transcription factor HDG3. In imprinted strains, HDG3 is expressed primarily from the methylated paternally inherited allele. We manipulated the methylation state of endogenous HDG3 in a non-imprinted strain and demonstrated that methylation of a proximal transposable element is sufficient to promote HDG3 expression and imprinting. Gain of HDG3 imprinting was associated with earlier endosperm cellularization and changes in seed weight. These results indicate that epigenetic variation alone is sufficient to explain imprinting variation and demonstrate that epialleles can underlie variation in seed development phenotypes. Full Text

Pincus , D., Pandey, J.P., Feder, Z.A., Creixell, P., Resnekov, O., and Reynolds, K.A. (2018). Engineering allosteric regulation in protein kinases. Science signaling Vol. 11, Issue 555, eaar3250. Phosphoregulation, in which the addition of a negatively charged phosphate group modulates protein activity, enables dynamic cellular responses. To understand how new phosphoregulation might be acquired, we mutationally scanned the surface of a prototypical yeast kinase (Kss1) to identify potential regulatory sites. The data revealed a set of spatially distributed "hotspots" that might have coevolved with the active site and preferentially modulated kinase activity. By engineering simple consensus phosphorylation sites at these hotspots, we rewired cell signaling in yeast. Using the same approach with a homolog yeast mitogen-activated protein kinase, Hog1, we introduced new phosphoregulation that modified its localization and signaling dynamics. Beyond revealing potential use in synthetic biology, our findings suggest that the identified hotspots contribute to the diversity of natural allosteric regulatory mechanisms in the eukaryotic kinome and, given that some are mutated in cancers, understanding these hotspots may have clinical relevance to human disease.Full Text

Pincus , D., Anandhakumar, J., Thiru, P. , Guertin, M.J., Erkine, A.M., and Gross, D.S. (2018). Genetic and epigenetic determinants establish a continuum of Hsf1 occupancy and activity across the yeast genome. Molecular biology of the cell, mbc E18060353 [Epub ahead of print]. Heat Shock Factor 1 is the master transcriptional regulator of molecular chaperones and binds to the same cis-acting Heat Shock Element (HSE) across the eukaryotic lineage. In budding yeast, Hsf1 drives the transcription of approximately 20 genes essential to maintain proteostasis under basal conditions, yet its specific targets and extent of inducible binding during heat shock remain unclear. Here, we combine Hsf1 ChIP-seq, nascent RNA-seq and Hsf1 nuclear depletion to quantify Hsf1 binding and transcription across the yeast genome. We find that Hsf1 binds 74 loci during acute heat shock, and these are linked to 46 genes with strong Hsf1-dependent expression. Notably, Hsf1's induced DNA binding leads to a disproportionate ( approximately 7.5-fold) increase in nascent transcription. Promoters with high basal Hsf1 occupancy have nucleosome-depleted regions due to the presence of 'pioneer factors'. These accessible sites are likely critical for Hsf1 occupancy as the activator is incapable of binding HSEs within a stably positioned, reconstituted nucleosome. In response to heat shock, however, Hsf1 accesses nucleosomal sites and promotes chromatin disassembly in concert with the RSC remodeling complex. Our data suggest that the interplay between nucleosome positioning, HSE strength and active Hsf1 levels allows cells to precisely tune expression of the proteostasis network. Full Text

Rajbhandari, P., Lopez, G., Capdevila, C., Salvatori, B., Yu, J.Y., Rodriguez-Barrueco, R., Martinez, D., Yarmarkovich, M., Weichert-Leahey, N., Abraham, B.J., Young, R.A., et al. (2018). Cross-Cohort Analysis Identifies a TEAD4-MYCN Positive Feedback Loop as the Core Regulatory Element of High-Risk Neuroblastoma. Cancer Discovery 8, 582-599. High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module-centered around a TEAD4-MYCN positive feedback loop-emerged as the regulatory driver of the high-risk subtype associated with MYCN amplification. Silencing of either gene collapsed MYCN-amplified (MYCNAmp) neuroblastoma transcriptional hallmarks and abrogated viability in vitro and in vivo. Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas. SIGNIFICANCE: Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4-MYCN positive feedback loop in MYCNAmp neuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention. Full Text

Reddien , P.W. (2018). The Cellular and Molecular Basis for Planarian Regeneration.Cell 175, 327-345. Regeneration is one of the great mysteries of biology. Planarians are flatworms capable of dramatic feats of regeneration, which have been studied for over 2 centuries. Recent findings identify key cellular and molecular principles underlying these feats. A stem cell population (neoblasts) generates new cells and is comprised of pluripotent stem cells (cNeoblasts) and fate-specified cells (specialized neoblasts). Positional information is constitutively active and harbored primarily in muscle, where it acts to guide stem cell-mediated tissue turnover and regeneration. I describe here a model in which positional information and stem cells combine to enable regeneration. Full Text

Renault, H., Werck-Reichhart, D., and Weng, J.K. (2018). Harnessing lignin evolution for biotechnological applications. Current opinion in biotechnology 56, 105-111 Lignin evolved concomitantly with the rise of vascular plants on planet earth approximately 450 million years ago. Several iterations of exploiting ancestral phenylpropanoid metabolism for biopolymers occurred prior to lignin that facilitated early plants' adaptation to terrestrial environments. The first true lignin was constructed via oxidative coupling of a number of simple phenylpropanoid alcohols to form a sturdy polymer that supports long-distance water transport. This invention has directly contributed to the dominance of vascular plants in the Earth's flora, and has had a profound impact on the establishment of the rich terrestrial ecosystems as we know them today. Within vascular plants, new lignin traits continued to emerge with expanded biological functions pertinent to host fitness under complex environmental niches. Understanding the chemical and biochemical basis for lignin's evolution in diverse plants therefore offers new opportunities and tools for engineering desirable lignin traits in crops with economic significance. Full Text

Rodriguez-Rodriguez, J.A., Lewis, C., McKinley, K.L., Sikirzhytski, V., Corona, J., Maciejowski, J., Khodjakov, A., Cheeseman, I.M.., and Jallepalli, P.V. (2018). Distinct Roles of RZZ and Bub1-KNL1 in Mitotic Checkpoint Signaling and Kinetochore Expansion. Current biology : CB 28(21):3422-3429.The Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls M phase progression through a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1, 2]. During interphase, Mad1-Mad2 generates MCC at nuclear pores [3]. After nuclear envelope breakdown (NEBD), kinetochore-associated Mad1-Mad2 catalyzes MCC assembly until all chromosomes achieve bipolar attachment [1, 2]. Mad1-Mad2 and other factors are also incorporated into the fibrous corona, a phospho-dependent expansion of the outer kinetochore that precedes microtubule attachment [4-6]. The factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes remain controversial [7-12], and the specific phosphorylation event(s) that trigger corona formation remain elusive [5, 13]. We used genome editing to eliminate Bub1, KNL1, and the Rod-Zw10-Zwilch (RZZ) complex in human cells. We show that RZZ's sole role in SAC activation is to tether Mad1-Mad2 to kinetochores. Separately, Mps1 kinase triggers fibrous corona formation by phosphorylating two N-terminal sites on Rod. In contrast, Bub1 and KNL1 activate kinetochore-bound Mad1-Mad2 to produce a "wait anaphase" signal but are not required for corona formation. We also show that clonal lines isolated after BUB1 disruption recover Bub1 expression and SAC function through nonsense-associated alternative splicing (NAS). Our study reveals a fundamental division of labor in the mammalian SAC and highlights a transcriptional response to nonsense mutations that can reduce or eliminate penetrance in genome editing experiments. Full Text

Roessler , R., Goldmann, J., Shivalila, C., and Jaenisch, R. (2018). JIP2 haploinsufficiency contributes to neurodevelopmental abnormalities in human pluripotent stem cell-derived neural progenitors and cortical neurons.Life Science Alliance 1(4):e201800094. Phelan-McDermid syndrome (also known as 22q13.3 deletion syndrome) is a syndromic form of autism spectrum disorder and currently thought to be caused by heterozygous loss of SHANK3. However, patients most frequently present with large chromosomal deletions affecting several additional genes. We used human pluripotent stem cell technology and genome editing to further dissect molecular and cellular mechanisms. We found that loss of JIP2 (MAPK8IP2) may contribute to a distinct neurodevelopmental phenotype in neural progenitor cells (NPCs) affecting neuronal maturation. This is most likely due to a simultaneous down-regulation of c-Jun N-terminal kinase (JNK) proteins, leading to impaired generation of mature neurons. Furthermore, semaphorin signaling appears to be impaired in patient NPCs and neurons. Pharmacological activation of neuropilin receptor 1 (NRP1) rescued impaired semaphorin pathway activity and JNK expression in patient neurons. Our results suggest a novel disease-specific mechanism involving the JIP/JNK complex and identify NRP1 as a potential new therapeutic target. Full Text

Rohde, J.M., Brimacombe, K.R., Liu, L., Pacold, M.E., Yasgar, A., Cheff, D.M., Lee, T.D., Rai, G., Baljinnyam, B., Li, Z., Simeonov A, Hall MD, Shen M, Sabatini DM, and Boxer MB. (2018). Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors. Bioorganic & medicinal chemistry [Epub ahead of print]. Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a marked improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling in vitro assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells. This manuscript reports the assay development and medicinal chemistry leading to the development of NCT-502 and -503 reported in Pacold et al. Full Text

Romer, K.A., de Rooij, D.G., Kojima, M.L., and Page, D.C. (2018). Isolating mitotic and meiotic germ cells from male mice by developmental synchronization, staging, and sorting. Developmental biology [Epub ahead of print].Isolating discrete populations of germ cells from the mouse testis is challenging, because the adult testis contains germ cells at every step of spermatogenesis, in addition to somatic cells. We present a novel method for isolating precise, high-purity populations of male germ cells. We first synchronize germ cell development in vivo by manipulating retinoic acid metabolism, and perform histological staging to verify synchronization. We use fluorescence-activated cell sorting to separate the synchronized differentiating germ cells from contaminating somatic cells and undifferentiated spermatogonia. We achieve ~90% purity at each step of development from undifferentiated spermatogonia through late meiotic prophase. Utilizing this "3S" method (synchronize, stage, and sort), we can separate germ cell types that were previously challenging or impossible to distinguish, with sufficient yield for epigenetic and biochemical studies. 3S expands the toolkit of germ cell sorting methods, and should facilitate detailed characterization of molecular and biochemical changes that occur during the mitotic and meiotic phases of spermatogenesis. Full Text

Sabari , B.R., Dall'Agnese, A., Boija, A., Klein, I.A., Coffey, E.L., Shrinivas, K., Abraham, B.J., Hannett, N.M., Zamudio, A.V., Manteiga, J.C., Li CH, Guo YE, Day DS, Schuijers J, Hnisz D, Lee TI, Young RA, et al. (2018). Coactivator condensation at super-enhancers links phase separation and gene control. Science [Epub ahead of print]. Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here, we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets and MED1-IDR droplets can compartmentalize and concentrate transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in control of key cell identity genes. Full Text

Saha, K., Hurlbut, J.B., Jasanoff, S., Ahmed, A., Appiah, A., Bartholet, E., Baylis, F., Bennett, G., Church, G., Cohen, I.G., Jaenisch R., et al. (2018). Building Capacity for a Global Genome Editing Observatory : Institutional Design. Trends in biotechnology [Epub ahead of print] A new infrastructure is urgently needed at the global level to facilitate exchange on key issues concerning genome editing. We advocate the establishment of a global observatory to serve as a center for international, interdisciplinary, and cosmopolitan reflection. Full Text

Sajiki, K., Tahara, Y., Villar-Briones, A., Pluskal, T., Teruya, T., Mori, A., Hatanaka, M., Ebe, M., Nakamura, T., Aoki, K., et al. (2018). Genetic defects in SAPK signalling, chromatin regulation, vesicle transport and CoA-related lipid metabolism are rescued by rapamycin in fission yeast.Open Biology 8(3): 170261. Rapamycin inhibits TOR (target of rapamycin) kinase, and is being used clinically to treat various diseases ranging from cancers to fibrodysplasia ossificans progressiva. To understand rapamycin mechanisms of action more comprehensively, 1014 temperature-sensitive (ts) fission yeast (Schizosaccharomyces pombe) mutantswere screened in order to isolate strains in which the ts phenotype was rescued by rapamycin. Rapamycin-rescued 45 strains, among which 12 genes responsible for temperature sensitivity were identified. These genes are involved in stress-activated protein kinase (SAPK) signalling, chromatin regulation, vesicle transport, and CoA- and mevalonate-related lipid metabolism. Subsequent metabolome analyses revealed that rapamycin upregulated stress-responsive metabolites, while it downregulated purine biosynthesis intermediates and nucleotide derivatives. Rapamycin alleviated abnormalities in cell growth and cell division caused by sty1 mutants (Delta sty1) of SAPK. Notably, in Delta sty1, rapamycin reduced greater than 75% of overproduced metabolites (greater than 2 x WT), like purine biosynthesis intermediates and nucleotide derivatives, to WT levels. This suggests that these compounds may be the points at which the SAPK/TOR balance regulates continuous cell proliferation. Rapamycin might be therapeutically useful for specific defects of these gene functions. Full Text

Schuijers , J., Manteiga, J.C., Weintraub, A.S., Day, D.S., Zamudio, A.V., Hnisz, D., Lee, T.I., and Young, R.A. (2018). Transcriptional Dysregulation of MYC Reveals Common Enhancer-Docking Mechanism. Cell reports 23, 349-360.Transcriptional dysregulation of the MYC oncogene is among the most frequent events in aggressive tumor cells, and this is generally accomplished by acquisition of a super-enhancer somewhere within the 2.8 Mb TAD where MYC resides. We find that these diverse cancer-specific super-enhancers, differing in size and location, interact with the MYC gene through a common and conserved CTCF binding site located 2 kb upstream of the MYC promoter. Genetic perturbation of this enhancer-docking site in tumor cells reduces CTCF binding, super-enhancer interaction, MYC gene expression, and cell proliferation. CTCF binding is highly sensitive to DNA methylation, and this enhancer-docking site, which is hypomethylated in diverse cancers, can be inactivated through epigenetic editing with dCas9-DNMT. Similar enhancer-docking sites occur at other genes, including genes with prominent roles in multiple cancers, suggesting a mechanism by which tumor cell oncogenes can generally hijack enhancers. These results provide insights into mechanisms that allow a single target gene to be regulated by diverse enhancer elements in different cell types. Full Text

Scimone , M.L., Wurtzel, O., Malecek, K., Fincher, C.T., Oderberg, I.M., Kravarik, K.M., and Reddien, P.W. (2018). foxF-1 Controls Specification of Non-body Wall Muscle and Phagocytic Cells in Planarians. Current biology : CB.[Epub ahead of print].Planarians are flatworms capable of regenerating any missing body part in a process requiring stem cells and positional information. Muscle is a major source of planarian positional information and consists of several types of fibers with distinct regulatory roles in regeneration. The transcriptional regulatory programs used to specify different muscle fibers are poorly characterized. Using single-cell RNA sequencing, we define the transcriptomes of planarian dorsal-ventral muscle (DVM), intestinal muscle (IM), and pharynx muscle. This analysis identifies foxF-1, which encodes a broadly conserved Fox-family transcription factor, as a master transcriptional regulator of all non-body wall muscle. The transcription factors encoded by nk4 and gata4/5/6-2 specify two different subsets of DVM, lateral and medial, respectively, whereas gata4/5/6-3 specifies IM. These muscle types all express planarian patterning genes. Both lateral and medial DVM are required for medial-lateral patterning in regeneration, whereas medial DVM and IM have a role in maintaining and regenerating intestine morphology. In addition to the role in muscle, foxF-1 is required for the specification of multiple cell types with transcriptome similarities, including high expression levels of cathepsin genes. These cells include pigment cells, glia, and several other cells with unknown function. cathepsin(+) cells phagocytose E. coli, suggesting these are phagocytic cells. In conclusion, we describe a regulatory program for planarian muscle cell subsets and phagocytic cells, both driven by foxF-1. FoxF proteins specify different mesoderm-derived tissues in other organisms, suggesting that FoxF regulates formation of an ancient and broadly conserved subset of mesoderm derivatives in the Bilateria. Full Text

Shen , K., and Sabatini, D.M. (2018). Ragulator and SLC38A9 activate the Rag GTPases through noncanonical GEF mechanisms. PNAS 115(38):9545-9550. The mechanistic target of rapamycin complex 1 (mTORC1) growth pathway detects nutrients through a variety of sensors and regulators that converge on the Rag GTPases, which form heterodimers consisting of RagA or RagB tightly bound to RagC or RagD and control the subcellular localization of mTORC1. The Rag heterodimer uses a unique "locking" mechanism to stabilize its active ((GTP)RagA-RagC(GDP)) or inactive ((GDP)RagA-RagC(GTP)) nucleotide states. The Ragulator complex tethers the Rag heterodimer to the lysosomal surface, and the SLC38A9 transmembrane protein is a lysosomal arginine sensor that upon activation stimulates mTORC1 activity through the Rag GTPases. How Ragulator and SLC38A9 control the nucleotide loading state of the Rag GTPases remains incompletely understood. Here we find that Ragulator and SLC38A9 are each unique guanine exchange factors (GEFs) that collectively push the Rag GTPases toward the active state. Ragulator triggers GTP release from RagC, thus resolving the locked inactivated state of the Rag GTPases. Upon arginine binding, SLC38A9 converts RagA from the GDP- to the GTP-loaded state, and therefore activates the Rag GTPase heterodimer. Altogether, Ragulator and SLC38A9 act on the Rag GTPases to activate the mTORC1 pathway in response to nutrient sufficiency. Full Text

Shen , K., Huang, R.K., Brignole, E.J., Condon, K.J., Valenstein, M.L., Chantranupong, L., Bomaliyamu, A., Choe, A., Hong, C., Yu, Z., and Sabatini DM. (2018). Architecture of the human GATOR1 and GATOR1-Rag GTPases complexes. Nature [Epub ahead of print]. Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex-comprising DEPDC5, NPRL2 and NPRL3-regulates the Rag GTPases as a GTPase-activating protein (GAP) for RAGA; loss of GATOR1 desensitizes mTORC1 signalling to nutrient starvation. GATOR1 components have no sequence homology to other proteins, so the function of GATOR1 at the molecular level is currently unknown. Here we used cryo-electron microscopy to solve structures of GATOR1 and GATOR1-Rag GTPases complexes. GATOR1 adopts an extended architecture with a cavity in the middle; NPRL2 links DEPDC5 and NPRL3, and DEPDC5 contacts the Rag GTPase heterodimer. Biochemical analyses reveal that our GATOR1-Rag GTPases structure is inhibitory, and that at least two binding modes must exist between the Rag GTPases and GATOR1. Direct interaction of DEPDC5 with RAGA inhibits GATOR1-mediated stimulation of GTP hydrolysis by RAGA, whereas weaker interactions between the NPRL2-NPRL3 heterodimer and RAGA execute GAP activity. These data reveal the structure of a component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase. Full Text

Sidik, S.M., Huet, D., and Lourido, S.(2018). CRISPR-Cas9-based genome-wide screening of Toxoplasma gondii. Nature protocols 13, 307-323. Apicomplexan parasites, such as Toxoplasma gondii, cause extensive morbidity and mortality in humans and livestock, highlighting the need for a deeper understanding of their molecular biology. Although techniques for the generation of targeted gene disruptions have long been available for apicomplexans, such methods are not readily scalable to the entire genome. We recently used CRISPR-Cas9 to disrupt all nuclear protein-coding genes in T. gondii using a pooled format. The method relies on transfection of a guide RNA library into parasites constitutively expressing Cas9. Here, we present the complete workflow of such a screen, including preparation of the guide RNA library, growth and testing of the recipient strain, generation of the mutant population, culture conditions for the screen, preparation of genomic DNA libraries, next-generation sequencing of the guide RNA loci, and analysis to detect fitness-conferring genes. This method can be deployed to study how culture conditions affect the repertoire of genes needed by parasites, which will enable studies of their metabolic needs, host specificity, and drug-resistance mechanisms. In addition, by manipulating the background in which the screen is performed, researchers will be able to investigate genetic interactions, which may help uncover redundancy or epistasis in the parasite genome. Using this method, a genome-wide screen and its analysis can be completed in 3 weeks, after approximately 1 month of preparation to generate the library and grow the cells needed, making it a powerful tool for uncovering functionally important genes in apicomplexan parasites. Full Text

Silber, S.J., DeRosa, M., Goldsmith, S., Fan, Y., Castleman, L., and Melnick, J. (2018). Cryopreservation and transplantation of ovarian tissue: results from one center in the USA. Journal of assisted reproduction and genetics [Epub ahead of print]. Cryopreservation and transplantation of ovarian tissue: results from one center in the USA. PURPOSE: To report the results of cryopreserved ovary tissue transplantation for leukemia and other cancers, in a single US center. METHODS: One hundred eight females between age 6 and (median age 24) 35 were referred for possible ovary tissue cryopreservation over a 20-year period, with either slow freeze or vitrification. Thus far 13 patients returned up to 18 years later to have their tissue transplanted back. RESULTS: All 13 patients had return of ovarian function 5 months post transplant with regular menstrual cycling. AMH rose to very high levels as the FSH declined to normal. Four months later, the AMH again declined to very low levels. Nonetheless, the grafts remained functional for up to 5 years or longer. Ten of the 13 (77%) became spontaneously pregnant at least once, resulting in 13 healthy babies. A total of 24 healthy babies have been born 11 from fresh transplanted ovarian tissue and 13 from cryopreserved transplanted ovarian tissue. CONCLUSIONS: (1) Ovary tissue cryopreservation is a robust method for preserving a woman's fertility. (2) Cortical tissue pressure may be a key regulator of primordial follicle arrest, recruitment, and ovarian longevity. (3) This is the only such series yet reported in the USA. Full Text

Soldner , F., and Jaenisch, R. (2018). Stem Cells, Genome Editing, and the Path to Translational Medicine. Cell 175, 615-632.The derivation of human embryonic stem cells (hESCs) and the stunning discovery that somatic cells can be reprogrammed into human induced pluripotent stem cells (hiPSCs) holds the promise to revolutionize biomedical research and regenerative medicine. In this Review, we focus on disorders of the central nervous system and explore how advances in human pluripotent stem cells (hPSCs) coincide with evolutions in genome engineering and genomic technologies to provide realistic opportunities to tackle some of the most devastating complex disorders. Full Text

Su , K.C., Tsang, M.J., Emans, N., and Cheeseman, I.M. (2018). CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses. Molecular biology of the cell [Epub ahead of print]. A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. For the past 25 years, the predominant strategy utilized in human tissue culture cells has been RNAi-mediated protein depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible protein depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells. However, this approach is still under-utilized for conducting visual phenotypic analyses, particularly for essential genes that require conditional strategies to eliminate their gene products. Optimizing this strategy requires effective and streamlined approaches to introduce the Cas9 guide RNA into target cells. Here, we assess the efficacy of synthetic guide RNA transfection to eliminate gene products for cell biological studies. Based on three representative gene targets (KIF11, CENPN, and RELA), we demonstrate that transfection of synthetic sgRNA and crRNA guides works comparably for protein depletion as cell lines stably expressing lentiviral-delivered RNA guides. We additionally demonstrate that synthetic sgRNAs can be introduced by reverse transfection on an array. Together, these strategies provide a robust, flexible, and scalable approach for conducting functional studies in human cells. Movie S1 Movie S1 Time-lapse movie of HeLa Cas9 cells on sgRNA array. From Figure 4. Cells were stained for DNA. Left - Control, Right - sgKIF11. Initial image displays area of printed fluorescence dye-conjugated RNA, which is highlighted by white circle. Time post seeding is indicated. Full Text

Sullivan, L.B., Luengo, A., Danai, L.V., Bush, L.N., Diehl, F.F., Hosios, A.M., Lau, A.N., Elmiligy, S., Malstrom, S., Lewis, C.A. , et al. (2018). Aspartate is an endogenous metabolic limitation for tumour growth. Nature cell biology 20(7):782-788. Defining the metabolic limitations of tumour growth will help to develop cancer therapies (1) . Cancer cells proliferate slower in tumours than in standard culture conditions, indicating that a metabolic limitation may restrict cell proliferation in vivo. Aspartate synthesis can limit cancer cell proliferation when respiration is impaired(2-4); however, whether acquiring aspartate is endogenously limiting for tumour growth is unknown. We confirm that aspartate has poor cell permeability, which prevents environmental acquisition, whereas the related amino acid asparagine is available to cells in tumours, but cancer cells lack asparaginase activity to convert asparagine to aspartate. Heterologous expression of guinea pig asparaginase 1 (gpASNase1), an enzyme that produces aspartate from asparagine (5) , confers the ability to use asparagine to supply intracellular aspartate to cancer cells in vivo. Tumours expressing gpASNase1 grow at a faster rate, indicating that aspartate acquisition is an endogenous metabolic limitation for the growth of some tumours. Tumours expressing gpASNase1 are also refractory to the growth suppressive effects of metformin, suggesting that metformin inhibits tumour growth by depleting aspartate. These findings suggest that therapeutic aspartate suppression could be effective to treat cancer. Full Text

Teitz , L.S., Pyntikova, T., Skaletsky, H., and Page, D.C. (2018). Selection Has Countered High Mutability to Preserve the Ancestral Copy Number of Y Chromosome Amplicons in Diverse Human Lineages. American Journal of Human Genetics 103(2):261-275. Amplicons-large, highly identical segmental duplications-are a prominent feature of mammalian Y chromosomes. Although they encode genes essential for fertility, these amplicons differ vastly between species, and little is known about the selective constraints acting on them. Here, we develop computational tools to detect amplicon copy number with unprecedented accuracy from high-throughput sequencing data. We find that one-sixth (16.9%) of 1,216 males from the 1000 Genomes Project have at least one deleted or duplicated amplicon. However, each amplicon's reference copy number is scrupulously maintained among divergent branches of the Y chromosome phylogeny, including the ancient branch A00, indicating that the reference copy number is ancestral to all modern human Y chromosomes. Using phylogenetic analyses and simulations, we demonstrate that this pattern of variation is incompatible with neutral evolution and instead displays hallmarks of mutation-selection balance. We also observe cases of amplicon rescue, in which deleted amplicons are restored through subsequent duplications. These results indicate that, contrary to the lack of constraint suggested by the differences between species, natural selection has suppressed amplicon copy number variation in diverse human lineages.Full Text

Tewari , A.G., Stern, S.R., Oderberg, I.M., and Reddien, P.W. (2018). Cellular and Molecular Responses Unique to Major Injury Are Dispensable for Planarian Regeneration.Cell Reports 25 (9) : 2577-2590. The fundamental requirements for regeneration are poorly understood. Planarians can robustly regenerate all tissues after injury, involving stem cells, positional information, and a set of cellular and molecular responses collectively called the "missing tissue" or "regenerative" response. follistatin, which encodes an extracellular Activin inhibitor, is required for the missing tissue response after head amputation and for subsequent regeneration. We found that follistatin is required for the missing tissue response regardless of the wound context, but causes regeneration failure only after head amputation. This head regeneration failure involves follistatin-mediated regulation of Wnt signaling at wounds and is not a consequence of a diminished missing tissue response. All tested contexts of regeneration, including head regeneration, could occur with a defective missing tissue response, but at a slower pace. Our findings suggest that major cellular and molecular programs induced specifically by large injuries function to accelerate regeneration but are dispensable for regeneration itself. Full Text

Title, A.C., Hong, S.J., Pires, N.D., Hasenohrl, L., Godbersen, S., Stokar-Regenscheit, N., Bartel, D.P., and Stoffel, M. (2018). Genetic dissection of the miR-200-Zeb1 axis reveals its importance in tumor differentiation and invasion. Nature communications 9(1):4671. The epithelial-to-mesenchymal transition (EMT) is an important mechanism for cancer progression and metastasis. Numerous in vitro and tumor-profiling studies point to the miR-200-Zeb1 axis as crucial in regulating this process, yet in vivo studies involving its regulation within a physiological context are lacking. Here, we show that miR-200 ablation in the Rip-Tag2 insulinoma mouse model induces beta-cell dedifferentiation, initiates an EMT expression program, and promotes tumor invasion. Strikingly, disrupting the miR-200 sites of the endogenous Zeb1 locus causes a similar phenotype. Reexpressing members of the miR-200 superfamily in vitro reveals that the miR-200c family and not the co-expressed and closely related miR-141 family is responsible for regulation of Zeb1 and EMT. Our results thus show that disrupting the in vivo regulation of Zeb1 by miR-200c is sufficient to drive EMT, thus highlighting the importance of this axis in tumor progression and invasion and its potential as a therapeutic target. Full Text

Truttmann, M.C., Pincus, D., and Ploegh, H.L. (2018). Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides. PNAS [Epub ahead of print]. Proteostasis is critical to maintain organismal viability, a process counteracted by aging-dependent protein aggregation. Chaperones of the heat shock protein (HSP) family help control proteostasis by reducing the burden of unfolded proteins. They also oversee the formation of protein aggregates. Here, we explore how AMPylation, a posttranslational protein modification that has emerged as a powerful modulator of HSP70 activity, influences the dynamics of protein aggregation. We find that adjustments of cellular AMPylation levels in Caenorhabditis elegans directly affect aggregation properties and associated toxicity of amyloid-beta (Abeta), of a polyglutamine (polyQ)-extended polypeptide, and of alpha-synuclein (alpha-syn). Expression of a constitutively active C. elegans AMPylase FIC-1(E274G) under its own promoter expedites aggregation of Abeta and alpha-syn, and drastically reduces their toxicity. A deficiency in AMPylation decreases the cellular tolerance for aggregation-prone polyQ proteins and alters their aggregation behavior. Overexpression of FIC-1(E274G) interferes with cell survival and larval development, underscoring the need for tight control of AMPylase activity in vivo. We thus define a link between HSP70 AMPylation and the dynamics of protein aggregation in neurodegenerative disease models. Our results are consistent with a cytoprotective, rather than a cytotoxic, role for such protein aggregates. Full Text

Tycko, J., Barrera, L.A., Huston, N.C., Friedland, A.E., Wu, X. , Gootenberg, J.S., Abudayyeh, O.O., Myer, V.E., Wilson, C.J., and Hsu, P.D. (2018). Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells.Nature Communications 9(1):2962. Therapeutic genome editing with Staphylococcus aureus Cas9 (SaCas9) requires a rigorous understanding of its potential off-target activity in the human genome. Here we report a high-throughput screening approach to measure SaCas9 genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporate randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with 21-mer or 22-mer spacer sequences are generally more active, although high efficiency 20-mer spacers are markedly less tolerant of mismatches. Using this dataset, we developed an SaCas9 specificity model that performs robustly in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions. Full Text

Uranukul, B., Woolston, B.M., Fink, G.R., and Stephanopoulos, G. (2018). Biosynthesis of monoethylene glycol in Saccharomyces cerevisiae utilizing native glycolytic enzymes. Metabolic engineering [Epub ahead of print]. Monoethylene glycol (MEG) is an important commodity chemical with applications in numerous industrial processes, primarily in the manufacture of polyethylene terephthalate (PET) polyester used in packaging applications. In the drive towards a sustainable chemical industry, bio-based production of MEG from renewable biomass has attracted growing interest. Recent attempts for bio-based MEG production have investigated metabolic network modifications in Escherichia coli, specifically rewiring the xylose assimilation pathways for the synthesis of MEG. In the present study, we examined the suitability of Saccharomyces cerevisiae, a preferred organism for industrial applications, as platform for MEG biosynthesis. Based on combined genetic, biochemical and fermentation studies, we report evidence for the existence of an endogenous biosynthetic route for MEG production from D-xylose in S. cerevisiae which consists of phosphofructokinase and fructose-bisphosphate aldolase, the two key enzymes in the glycolytic pathway. Further metabolic engineering and process optimization yielded a strain capable of producing up to 4.0g/L MEG, which is the highest titer reported in yeast to-date. Full Text

van Dijk, D., Sharma, R., Nainys, J., Yim, K., Kathail, P., Carr, A.J., Burdziak, C., Moon, K.R., Chaffer, C.L., Pattabiraman, D., Bierie, B. , et al. (2018). Recovering Gene Interactions from Single-Cell Data Using Data Diffusion.Cell [Epub ahead of print]. Single-cell RNA sequencing technologies suffer from many sources of technical noise, including under-sampling of mRNA molecules, often termed "dropout," which can severely obscure important gene-gene relationships. To address this, we developed MAGIC (Markov affinity-based graph imputation of cells), a method that shares information across similar cells, via data diffusion, to denoise the cell count matrix and fill in missing transcripts. We validate MAGIC on several biological systems and find it effective at recovering gene-gene relationships and additional structures. Applied to the epithilial to mesenchymal transition, MAGIC reveals a phenotypic continuum, with the majority of cells residing in intermediate states that display stem-like signatures, and infers known and previously uncharacterized regulatory interactions, demonstrating that our approach can successfully uncover regulatory relations without perturbations. Full Text

Veri, A.O., Miao, Z., Shapiro, R.S., Tebbji, F., O'Meara, T.R., Kim, S.H., Colazo, J., Tan, K., Vyas, V.K., Whiteway, M., et al. (2018). Tuning Hsf1 levels drives distinct fungal morphogenetic programs with depletion impairing Hsp90 function and overexpression expanding the target space. PLoS genetics 14, e1007270. The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen Candida albicans is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of C. albicans HSF1 induces morphogenesis in the absence of external cues. HSF1 depletion compromises Hsp90 function, thereby driving filamentation. HSF1 overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition. Full Text

Von Stetina , J.R., Frawley, L.E., Unhavaithaya, Y., and Orr-Weaver, T.L. (2018). Variant cell cycles regulated by Notch signaling control cell size and ensure a functional blood-brain barrier. Development 145: dev157115 Regulation of cell size is crucial in development. In plants and animals two cell cycle variants are employed to generate large cells by increased ploidy: the endocycle and endomitosis. The rationale behind the choice of which of these cycles is implemented is unknown. We show that in the Drosophila nervous system the subperineurial glia (SPG) are unique in using both the endocycle and endomitosis to grow. In the brain, the majority of SPG initially endocycle, then switch to endomitosis during larval development. The Notch signaling pathway and the String Cdc25 phosphatase are crucial for the endocycle versus endomitosis choice, providing the means experimentally to change cells from one to the other. This revealed fundamental insights into the control of cell size and the properties of endomitotic cells. Endomitotic cells attain a higher ploidy and larger size than endocycling cells, and endomitotic SPG are necessary for the blood-brain barrier. Decreased Notch signaling promotes endomitosis even in the ventral nerve cord SPG that normally are mononucleate, but not in the endocycling salivary gland cells, revealing tissue-specific cell cycle responses. Full Text

Vyas , V.K., Bushkin, G.G., Bernstein, D.A., Getz, M.A., Sewastianik, M., Barrasa, M.I., Bartel, D.P., and Fink, G.R. (2018). New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi. mSphere 3(2) : e00154-18. We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms.IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata, as well as in the related yeasts S. cerevisiae and N. castellii The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species. Full Text

Wada, N., Kersten, R., Iwai, T., Lee, S., Sakurai, F., Kikuchi, T., Fujita, D., Fujita, M., and Weng, J.K.. (2018). Crystalline Sponge-based Structural Analysis of Crude Natural Product Extracts. Angewandte Chemie Int. Ed. [Epub ahead of print]. The characterization of complex natural product mixtures to the absolute structural level of their components often requires significant amounts of starting materials, lengthy purification process, followed by arduous structure elucidation efforts. The crystalline sponge (CS) method has demonstrated its utility in absolute structure elucidation of isolated organic compounds at miniscule quantity compared to conventional methodology. Here we develop a new CS-based workflow that greatly expedites in-depth structural analysis of crude natural product extracts. Using a crude extract of the red alga Laurencia pacifica, we showed that CS affinity screening prior to compound isolation enables prioritization of analytes present in the extract, and subsequently resolved the molecular structures of six sesquiterpenes with stereochemical clarity from ~10 mg crude extract. This study demonstrates a new chemotyping workflow that can greatly accelerate natural product discovery from complex samples. Full Text

Wang , I.E., Wagner, D.E., and Reddien, P.W. (2018). Clonal Analysis of Planarian Stem Cells by Subtotal Irradiation and Single-Cell Transplantation. Methods in molecular biology 1774, 479-495. Stem cells, which both self-renew and produce differentiated progeny, represent fundamental biological units for the development, growth, maintenance, and regeneration of adult tissues. Characterization of stem cell lineage potential can be accomplished with clonal assays that interrogate stem cell output at the single-cell level. Here we present two methods for clonal analysis of individual proliferative cells (neoblasts) in the planarian Schmidtea mediterranea. The first method utilizes "subtotal" gamma irradiation to study rare surviving neoblasts and their clonal descendants in their native environment. The second method utilizes a fluorescent-activated cell sorting (FACS) strategy to obtain neoblast-enriched cell fractions, followed by single-cell transplantation into lethally irradiated hosts. Together, these methods provide a framework for generation and analysis of stem cell-derived clones in planarians. Full Text

Wedel, J., Bruneau, S., Liu, K., Kong, S.W., Sage, P.T., Sabatini, D.M., Laplante, M., and Briscoe, D.M. (2018). Deptor modulates activation responses in CD4(+) T cells and enhances immunoregulation following transplantation. American journal of transplantation[Epub ahead of print]. DEPTOR is an evolutionarily conserved cell-intrinsic binding partner of mTOR that functions as a negative regulator of signaling responses. In this study, we show that DEPTOR is expressed within CD4(+) T cells, and we observed that its relative level of expression modulates differentiation as well as glucose utilization within CD4(+) T effectors in vitro. Using knockin mice, we also find that induced expression of DEPTOR within CD4(+) T regulatory cells stabilizes Foxp3 expression, shifts metabolism towards oxidative phosphorylation and increases survival and suppressive function. In vivo, fully MHC mismatched cardiac allograft survival is significantly prolonged in knockin recipients and sustained recipient expression of DEPTOR in combination with costimulatory blockade induces long-term graft survival. Furthermore, we show that the induced expression of DEPTOR in CD4(+) T effectors fails to inhibit acute allograft rejection. Rather, prolonged survival is dominantly mediated via induced expression and function of DEPTOR within recipient CD4(+) T regulatory cells. These collective findings identify DEPTOR as a novel protein that functions in CD4(+) T cells to augment immunoregulation in vitro and in vivo. Full Text

Weng, Y., Yu, X., Li, J., Dong, Q., Li, F., Cheng, F., Zhang, Y., Yao, C., Zou, Z., Zhou, W., et al. (2018).Abietane diterpenoids from Lycopodium complanatum. Fitoterapia [Epub ahead of print]. Five new abietane diterpenoids (complanatins A-E, 1-5) have been isolated from the club moss Lycopodium complanatum, along with two known abietane diterpenoids (xanthoperol and sugiol). Their structures were determined by comprehensive analysis of 1D, 2D NMR, CD and HRESIMS data. The cytotoxic effects of five compounds (1-4, 7) were evaluated in three human lung cancer cell lines (MSTO-211H, NCI-H2052 and NCI-H226). Compounds 3 and 4 exhibited cytotoxic activities against the three cell lines. In addition, a plausible biogenetic pathway of compounds 1-7 was proposed.

Wijeratne, E.M.K., Oliveira, M.C.F., Mafezoli, J., Xu, Y.M., Minguzzi, S., Batista, P.H.J., Pessoa, O.D.L., Whitesell, L., and Gunatilaka, A.A.L. (2018). Withaferin A and Withanolide D Analogues with Dual Heat-Shock-Inducing and Cytotoxic Activities: Semisynthesis and Biological Evaluation. Journal of Natural Products [Epub ahead of print]. Withanolides constitute a valuable class of bioactive natural products because some members of the class are known to exhibit cytotoxic activity and also induce a cytoprotective heat-shock response. In order to understand the relationship between their structures and these dual bioactivities of the withanolide scaffold, we obtained 25 analogues of withaferin A (WA) and withanolide D (WD) including 17 new compounds by semisynthesis involving chemical and microbial transformations. Hitherto unknown 16beta-hydroxy analogues of WA and WD were prepared by their reaction with triphenylphosphine/iodine, providing unexpected 5beta-hydroxy-6alpha-iodo analogues (iodohydrins) followed by microbial biotransformation with Cunninghamella echinulata and base-catalyzed cyclization of the resulting 16beta-hydroxy iodohydrins. Evaluation of these 25 withanolide analogues for their cytotoxicity and heat-shock-inducing activity (HSA) confirmed the known structure-activity relationships for WA-type withanolides and revealed that WD analogues were less active in both assays compared to their corresponding WA analogues. The 5beta,6beta-epoxide moiety of withanolides contributed to their cytotoxicity but not HSA. Introduction of a 16beta-OAc group to 4,27-di- O-acetyl-WA enhanced cytotoxicity and decreased HSA, whereas introduction of the same group to 4- O-acetyl-WD decreased both activities. Full Text

Wong, M.Y., Doan, N.D., DiChiara, A.S., Papa, L.J., Cheah, J.H., Soule, C.K., Watson, N., Hulleman, J.D., and Shoulders, M.D. (2018). A High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production. Biochemistry 57, 2814-2827. Collagen overproduction is a feature of fibrosis and cancer, while insufficient deposition of functional collagen molecules and/or the secretion of malformed collagen is common in genetic disorders like osteogenesis imperfecta. Collagen secretion is an appealing therapeutic target in these and other diseases, as secretion directly connects intracellular biosynthesis to collagen deposition and biological function in the extracellular matrix. However, small molecule and biological methods to tune collagen secretion are severely lacking. Their discovery could prove useful not only in the treatment of disease, but also in providing tools for better elucidating mechanisms of collagen biosynthesis. We developed a cell-based, high-throughput luminescent assay of collagen type I secretion and used it to screen for small molecules that selectively enhance or inhibit that process. Among several validated hits, the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) robustly decreases the secretion of collagen-I by our model cell line and by human primary cells. In these systems, 17-AAG and other pan-isoform Hsp90 inhibitors reduce collagen-I secretion post-translationally and are not global inhibitors of protein secretion. Surprisingly, the consequences of Hsp90 inhibitors cannot be attributed to inhibition of the endoplasmic reticulum's Hsp90 isoform, Grp94. Instead, collagen-I secretion likely depends on the activity of cytosolic Hsp90 chaperones, even though such chaperones cannot directly engage nascent collagen molecules. Our results highlight the value of a cell-based high-throughput screen for selective modulators of collagen secretion and suggest an unanticipated role for cytosolic Hsp90 in collagen secretion. Full Text

Woodham, A.W., Cheloha, R.W., Ling, J., Rashidian, M., Kolifrath, S.C., Mesyngier, M., Duarte, J.N., Bader, J.M., Skeate, J.G., Da Silva, D.M., et al. (2018). Nanobody-antigen conjugates elicit HPV-specific anti-tumor immune responses. Cancer immunology research [Epub ahead of print]. High-risk human papillomavirus-associated cancers express viral oncoproteins (e.g., E6 and E7) that induce and maintain the malignant phenotype. The viral origin of these proteins makes them attractive targets for development of a therapeutic vaccine. Camelid-derived single-domain antibody fragments (nanobodies or VHHs) that recognize cell surface proteins on antigen-presenting cells (APCs) can serve as targeted delivery vehicles for antigens attached to them. Such VHHs were shown to induce CD4+ and CD8+ T-cell responses against model antigens conjugated to them via sortase, but antitumor responses had not yet been investigated. Here, we tested the ability of an anti-CD11b VHH (VHHCD11b) to target APCs and serve as the basis for a therapeutic vaccine to induce CD8+ T cell responses against HPV+ tumors. Mice immunized with VHHCD11b conjugated to an H-2Db-restricted immunodominant E7 epitope (E749-57) had more E7-specific CD8+ T cells compared to those immunized with E749-57 peptide alone. These CD8+ T cells acted prophylactically and conferred protection against a subsequent challenge with HPV E7-expressing tumor cells. In a therapeutic setting, VHHCD11b-E749-57 vaccination resulted in greater numbers of CD8+ tumor-infiltrating lymphocytes compared to mice receiving E749-57 peptide alone in HPV+ tumor-bearing mice, as measured by in vivo noninvasive VHH-based immune-positron emission tomography (immunoPET), which correlated with tumor regression and survival outcome. Together, these results demonstrate that VHHs can serve as a therapeutic cancer vaccine platform for HPV-induced cancers. Full Text

Wu, Y., Shan, B., Dai, J., Xia, Z., Cai, J., Chen, T., Lv, S., Feng, Y., Zheng, L., Wang, Y., et al. (2018). Dual Role for Inositol-requiring Enzyme 1alpha in Promoting the Development of Hepatocellular Carcinoma during Diet-induced Obesity. Hepatology[Epub ahead of print] Obesity is associated with both endoplasmic reticulum (ER) stress and chronic metabolic inflammation. ER stress activates the unfolded protein response (UPR) and has been implicated in a variety of cancers, including hepatocellular carcinoma (HCC). It is unclear whether individual UPR pathways are mechanistically linked to HCC development, however. Here we report a dual role for inositol-requiring enzyme 1alpha (IRE1alpha), the ER-localized UPR signal transducer, in obesity-promoted HCC development. We found that genetic ablation of IRE1alpha in hepatocytes not only markedly reduced the occurrence of diethylnitrosamine (DEN)-induced HCC in LKO mice when fed a normal chow (NC) diet, but also protected against the acceleration of HCC progression during high-fat diet (HFD) feeding. Irrespective of their adiposity states, LKO mice showed decreased hepatocyte proliferation and STAT3 activation, even in the face of increased hepatic apoptosis. Furthermore, IRE1alpha abrogation blunted obesity-associated activation of hepatic IKKbeta-NF-kappaB pathway, leading to reduced production of the tumor-promoting inflammatory cytokines TNF and IL-6. Importantly, higher IRE1alpha expression along with elevated STAT3 phosphorylation was also observed in the tumor tissues from human HCC patients, correlating with their poorer survival rate. CONCLUSION: These results demonstrate that IRE1alpha acts in a feed-forward loop during obesity-induced metabolic inflammation to promote HCC development through STAT3-mediated hepatocyte proliferation. This article is protected by copyright. All rights reserved. Full Text

Wyant , G.A., Abu-Remaileh, M., Frenkel, E.M., Laqtom, N.N., Dharamdasani, V., Lewis, C.A., Chan, S.H., Heinze, I., Ori, A., and Sabatini, D.M. (2018). NUFIP1 is a ribosome receptor for starvation-induced ribophagy. Science [Epub ahead of print]. The lysosome degrades and recycles macromolecules, signals to the master growth regulator mTORC1, and is associated with human disease. Here, we performed quantitative proteomic analyses of lysosomes rapidly isolated using the LysoIP method and find that nutrient levels and mTOR dynamically modulate the lysosomal proteome. We focus on NUFIP1, a protein that upon mTORC1 inhibition redistributes from the nucleus to autophagosomes and lysosomes. Upon these conditions, NUFIP1 interacts with ribosomes and delivers them to autophagosomes by directly binding to LC3B. The starvation-induced degradation of ribosomes via autophagy (ribophagy) depends on the capacity of NUFIP1 to bind LC3B and promotes cell survival. We propose that NUFIP1 is a receptor for the selective autophagy of ribosomes. Full Text

Xu, H., Lee, M.S., Tsai, P.Y., Adler, A.S., Curry, N.L., Challa, S., Freinkman, E.., Hitchcock, D.S., Copps, K.D., White, M.F., et al. (2018). Ablation of insulin receptor substrates 1 and 2 suppresses Kras-driven lung tumorigenesis.PNAS 115, 4228-4233. Non-small-cell lung cancer (NSCLC) is a leading cause of cancer death worldwide, with 25% of cases harboring oncogenic Kirsten rat sarcoma (KRAS). Although KRAS direct binding to and activation of PI3K is required for KRAS-driven lung tumorigenesis, the contribution of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) in the context of mutant KRAS remains controversial. Here, we provide genetic evidence that lung-specific dual ablation of insulin receptor substrates 1/2 (Irs1/Irs2), which mediate insulin and IGF1 signaling, strongly suppresses tumor initiation and dramatically extends the survival of a mouse model of lung cancer with Kras activation and p53 loss. Mice with Irs1/Irs2 loss eventually succumb to tumor burden, with tumor cells displaying suppressed Akt activation and strikingly diminished intracellular levels of essential amino acids. Acute loss of IRS1/IRS2 or inhibition of IR/IGF1R in KRAS-mutant human NSCLC cells decreases the uptake and lowers the intracellular levels of amino acids, while enhancing basal autophagy and sensitivity to autophagy and proteasome inhibitors. These findings demonstrate that insulin/IGF1 signaling is required for KRAS-mutant lung cancer initiation, and identify decreased amino acid levels as a metabolic vulnerability in tumor cells with IR/IGF1R inhibition. Consequently, combinatorial targeting of IR/IGF1R with autophagy or proteasome inhibitors may represent an effective therapeutic strategy in KRAS-mutant NSCLC. Full Text

Yang, Y.S., Moynihan, K.D., Bekdemir, A., Dichwalkar, T.M., Noh, M.M., Watson, N., Melo, M., Ingram, J., Suh, H., Ploegh, H., et al. (2018). Targeting small molecule drugs to T cells with antibody-directed cell-penetrating gold nanoparticles.Biomaterials Science [Epub ahead of print]. We sought to develop a nanoparticle vehicle that could efficiently deliver small molecule drugs to target lymphocyte populations. The synthesized amphiphilic organic ligand-protected gold nanoparticles (amph-NPs) were capable of sequestering large payloads of small molecule drugs within hydrophobic pockets of their ligand shells. These particles exhibit membrane-penetrating activity in mammalian cells, and thus enhanced uptake of a small molecule TGF-beta inhibitor in T cells in cell culture. By conjugating amph-NPs with targeting antibodies or camelid-derived nanobodies, the particle' cell-penetrating properties could be temporarily suppressed, allowing targeted uptake in specific lymphocyte subpopulations. Degradation of the protein targeting moieties following particle endocytosis allowed the NPs to recover their cell-penetrating activity in situ to enter the cytoplasm of T cells. In vivo, targeted amph-NPs showed 40-fold enhanced uptake in CD8+ T cells relative to untargeted particles, and delivery of TGF-beta inhibitor-loaded particles to T cells enhanced their cytokine polyfunctionality in a cancer vaccine model. Thus, this system provides a facile approach to concentrate small molecule compounds in target lymphocyte populations of interest for immunotherapy in cancer and other diseases. Full Text

Yu, Z., Surface, L.E., Park, C.Y., Horlbeck, M.A., Wyant, G.A., Abu-Remaileh, M. Peterson, T.R., Sabatini, D.M., Weissman, J.S., and O'Shea, E.K. (2018).Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing-bisphosphonates. eLife 7 : e36620. Nitrogen-containing-bisphosphonates (N-BPs) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance. Full Text

Zhang, C., Konermann, S., Brideau, N.J., Lotfy, P., Wu, X., Novick, S.J., Strutzenberg, T., Griffin, P.R., Hsu, P.D., and Lyumkis, D. (2018). Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d. Cell 175, 212-223. CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 A resolution, respectively. Furthermore, a 6.5 A reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies. Full Text

Zhang , Y., and Weinberg, R.A. (2018). Epithelial-to-mesenchymal transition in cancer: complexity and opportunities.Frontiers of Medicine [Epub ahead of print]. The cell-biological program termed the epithelial-to-mesenchymal transition (EMT) plays an important role in both development and cancer progression. Depending on the contextual signals and intracellular gene circuits of a particular cell, this program can drive fully epithelial cells to enter into a series of phenotypic states arrayed along the epithelial-mesenchymal phenotypic axis. These cell states display distinctive cellular characteristics, including stemness, invasiveness, drug-resistance and the ability to form metastases at distant organs, and thereby contribute to cancer metastasis and relapse. Currently we still lack a coherent overview of the molecular and biochemical mechanisms inducing cells to enter various states along the epithelial-mesenchymal phenotypic spectrum. An improved understanding of the dynamic and plastic nature of the EMT program has the potential to yield novel therapies targeting this cellular program that may aid in the management of high-grade malignancies. Full Text

Zheng , X., Beyzavi, A., Krakowiak, J., Patel, N., Khalil, A.S., and Pincus, D. (2018). Hsf1 Phosphorylation Generates Cell-to-Cell Variation in Hsp90 Levels and Promotes Phenotypic Plasticity.Cell reports 22, 3099-3106. Clonal populations of cells exhibit cell-to-cell variation in the transcription of individual genes. In addition to this noise in gene expression, heterogeneity in the proteome and the proteostasis network expands the phenotypic diversity of a population. Heat shock factor 1 (Hsf1) regulates chaperone gene expression, thereby coupling transcriptional noise to proteostasis. Here we show that cell-to-cell variation in Hsf1 activity is an important determinant of phenotypic plasticity. Budding yeast cells with high Hsf1 activity were enriched for the ability to acquire resistance to an antifungal drug, and this enrichment depended on Hsp90, a known phenotypic capacitor and canonical Hsf1 target. We show that Hsf1 phosphorylation promotes cell-to-cell variation, and this variation, rather than absolute Hsf1 activity, promotes antifungal resistance. We propose that Hsf1 phosphorylation enables differential tuning of the proteostasis network in individual cells, allowing populations to access a range of phenotypic states. Full Text