The following alphabetical list represents papers published in 2019 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.

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2019 Titles :

Abe, T., Yabuuchi, A., Ezoe, K., Skaletsky, H., Fukuda, J., Ueno, S., Fan, Y., Goldsmith, S., Kobayashi, T., Silber, S., et al. (2019). Success rates in minimal stimulation cycle IVF with clomiphene citrate only. Journal of Assisted Reproduction and Genetics [Epub ahead of print]. PURPOSE: To determine age-adjusted overall success rates for patients undergoing clomiphene citrate only minimal stimulation cycle (mini) in vitro fertilization (IVF) without any gonadotropin administration. METHODS: Eight hundred thirty-nine women (mean age: 38.4 +/- 0.1 years; 2488 cycles) underwent clomiphene citrate only mini-IVF. Their first oocyte retrieval was between January 2009 and December 2009, with follow-up until December 2014. The cumulative live birth rate (CLBR) per oocyte retrieval cycle started and live birth rate per oocyte was retrospectively analyzed. The basic CLBR was calculated as the number of women who achieved a live birth divided by the total number of women who started oocyte retrieval. RESULTS: The mean number of oocytes retrieved was 1.5. The basic CLBRs for all ages after the first and third cycles were 22.6% and 39.2%, respectively. For /= 43 years, CLBRs after the first and third cycles were 42.5% and 70.1%, 32.9% and 49.1%, 20.0% and 38.6%, 12.6% and 25.2%, and 4.4% and 8.8%, respectively. These rates had a significant relationship with age (P < 0.01). The LBR per oocyte for all ages was 9.6%. CONCLUSION: Acceptable overall IVF success rates can be achieved in clomiphene citrate only mini-IVF, as well as acceptable LBR. The CLBRs and LBRs per oocyte are evidently influenced by women's age. Full Text

Abetov, D.A., Kiyan, V.S., Zhylkibayev, A.A., Sarbassova, D.A., Alybayev, S.D.,Spooner, E. , Song, M.S., Bersimbaev, R.I., and Sarbassov, D. (2019). Formation of mammalian preribosomes proceeds from intermediate to composed state during ribosome maturation. Journal of Biological Chemistry 294, 10746-10757 .In eukaryotes, ribosome assembly is a rate-limiting step in ribosomal biogenesis that takes place in a distinctive subnuclear organelle, the nucleolus. How ribosomes get assembled at the nucleolar site by forming initial preribosomal complexes remains poorly characterized. In this study, using several human and murine cell lines, we developed a method for isolation of native mammalian preribosomal complexes by lysing cell nuclei through mild sonication. A sucrose gradient fractionation of the nuclear lysate resolved several ribonucleoprotein (RNP) complexes containing rRNAs and ribosomal proteins. Characterization of the RNP complexes with MS-based protein identification and Northern blotting-based rRNA detection approaches identified two types of preribosomes we named here as intermediate preribosomes (IPRibs) and composed preribosome (CPRib). IPRib complexes comprised large preribosomes (105S to 125S in size) containing the rRNA modification factors and premature rRNAs. We further observed that a distinctive CPRib complex consists of an 85S preribosome assemled with mature rRNAs and a ribosomal biogenesis factor, Ly1 antibody-reactive (LYAR), that does not associate with premature rRNAs and rRNA modification factors. rRNA-labeling experiments uncovered that IPRib assembly precedes CPRib complex formation. We also found that formation of the preribosomal complexes is nutrient-dependent because the abundances of IPRib and CPRib decreased substantially when cells were either deprived of amino acids or exposed to an mTOR kinase inhibitor. These findings indicate that preribosomes form via dynamic and nutrient-dependent processing events and progress from an intermediate to a composed state during ribosome maturation. Full Text

Abu-Remaileh, M., Abu-Remaileh, M., Akkawi, R., Knani, I., Udi, S., Pacold, M.E., Tam, J., and Aqeilan, R.I. (2019). WWOX somatic ablation in skeletal muscles alters glucose metabolism. Molecular metabolism [Epub ahead of print]. WWOX, a well-established tumor suppressor, is frequently lost in cancer and plays important roles in DNA damage response and cellular metabolism. METHODS: We re-analyzed several genome-wide association studies (GWAS) using the Type 2 Diabetes Knowledge Portal website to uncover WWOX's association with metabolic syndrome (MetS). Using several engineered mouse models, we studied the effect of somatic WWOX loss on glucose homeostasis. RESULTS: Several WWOX variants were found to be strongly associated with MetS disorders. In mouse models, somatic ablation of Wwox in skeletal muscle (Wwox(DeltaSKM)) results in weight gain, glucose intolerance, and insulin resistance. Furthermore, Wwox(DeltaSKM) mice display reduced amounts of slow-twitch fibers, decreased mitochondrial quantity and activity, and lower glucose oxidation levels. Mechanistically, we found that WWOX physically interacts with the cellular energy sensor AMP-activated protein kinase (AMPK) and that its loss is associated with impaired activation of AMPK, and with significant accumulation of the hypoxia inducible factor 1 alpha (HIF1alpha) in SKM. CONCLUSIONS: Our studies uncover an unforeseen role of the tumor suppressor WWOX in whole-body glucose homeostasis and highlight the intimate relationship between cancer progression and metabolic disorders, particularly obesity and type-2 diabetes. SUBJECT AREAS: Genetics, Metabolic Syndrome, Diabetes. Full Text

Adelmann, C.H., Wang, T., Sabatini, D.M., and Lander, E.S. (2019). Genome-Wide CRISPR/Cas9 Screening for Identification of Cancer Genes in Cell Lines.Methods in Molecular Biology 1907 : 125-136. In this protocol, pooled sgRNA libraries targeting thousands of genes are computationally designed, generated using microarray-based synthesis techniques, and packaged into lentiviral particles. Target cells of interest are transduced with the lentiviral sgRNA pools to generate a collection of knockout mutants-via Cas9-mediated genomic cleavage-and screened for a phenotype of interest. The relative abundance of each mutant in the population can be monitored over time through high-throughput sequencing of the integrated sgRNA expression cassettes. Using this technique, we outline strategies for the identification of cancer driver genes and genes mediating drug response. Full Text

Anandapadamanaban, M., Masson, G.R., Perisic, O., Berndt, A., Kaufman, J., Johnson, C.M., Santhanam, B., Rogala, K.B., Sabatini, D.M., and Williams, R.L. (2019). Architecture of human Rag GTPase heterodimers and their complex with mTORC1. Science 366(6462):203-210. The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo-electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagAGTP/RagCGDP nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes. Full Text

Bevis , B.J. (2019). Editorial : In Memory of Susan Lindquist. FEMS Yeast Research 19(1). This special issue of FEMS Yeast Research is dedicated to the memory and legacy of Professor Susan Lindquist, a pioneer in the protein folding field and champion of using yeast as a ‘living test tube’ to study biological problems ranging from heat shock proteins to cancer and neurodegenerative disease. Full Text

Brown, E.M., Ke, X., Hitchcock, D., Jeanfavre, S., Avila-Pacheco, J., Nakata, T., Arthur, T.D., Fornelos, N., Heim, C., Franzosa, E.A., Nikki Watson, et al. (2019). Bacteroides-Derived Sphingolipids Are Critical for Maintaining Intestinal Homeostasis and Symbiosis. Cell host & microbe 25, 668-680.Sphingolipids are structural membrane components and important eukaryotic signaling molecules. Sphingolipids regulate inflammation and immunity and were recently identified as the most differentially abundant metabolite in stool from inflammatory bowel disease (IBD) patients. Commensal bacteria from the Bacteroidetes phylum also produce sphingolipids, but the impact of these metabolites on host pathways is largely uncharacterized. To determine whether bacterial sphingolipids modulate intestinal health, we colonized germ-free mice with a sphingolipid- deficient Bacteroides thetaiotaomicron strain. A lack of Bacteroides- derived sphingolipids resulted in intestinal inflammation and altered host ceramide pools in mice. Using lipidomic analysis, we described a sphingolipid biosynthesis pathway and revealed a variety of Bacteroides- derived sphingolipids including ceramide phosphoinositol and deoxy- sphingolipids. Annotating Bacteroides sphingolipids in an IBD metabolomic dataset revealed lower abundances in IBD and negative correlations with inflammation and host sphingolipid production. These data highlight the role of bacterial sphingolipids in maintaining homeostasis and symbiosis in the gut.Full Text

Browne, C.M., Jiang, B.S., Ficarro, S.B., Doctor, Z.M., Johnson, J.L., Card, J.D., Sivakumaren, S.C., Alexander, W.M., Yaron, T.M., Murphy, C.J., Kwiatkowski, N.P., et al. (2019). A Chemoproteomic Strategy for Direct and Proteome-Wide Covalent Inhibitor Target-Site Identification. Journal of the American Chemical Society 141 : 191-203. Despite recent clinical successes for irreversible drugs, potential toxicities mediated by unpredictable modification of off-target cysteines represents a major hurdle for expansion of covalent drug programs. Understanding the proteome-wide binding profile of covalent inhibitors can significantly accelerate their development; however, current mass spectrometry strategies typically do not provide a direct, amino acid level readout of covalent activity for complex, selective inhibitors. Here we report the development of CITe-Id, a novel chemoproteomic approach that employs covalent pharmacologic inhibitors as enrichment reagents in combination with an optimized proteomic platform to directly quantify dose-dependent binding at cysteine-thiols across the proteome. CITe-Id analysis of our irreversible CDK inhibitor THZ1 identified dose-dependent covalent modification of several unexpected kinases, including a previously unannotated cysteine (C840) on the understudied kinase PKN3. These data streamlined our development of JZ128 as a new selective covalent inhibitor of PKN3. Using JZ128 as a probe compound, we identified novel potential PKN3 substrates, thus offering an initial molecular view of PKN3 cellular activity. CITe-Id provides a powerful complement to current chemoproteomic platforms to characterize the selectivity of covalent inhibitors, identify new, pharmacologically addressable cysteine-thiols, and inform structure-based drug design programs.Full Text

Bushkin, G.G., Pincus, D., Morgan, J.T., Richardson, K., Lewis, C., Chan, S.H., Bartel, D.P., and Fink, G.R. (2019). modim(6)Afication of a 3' UTR site reduces RME1 mRNA levels to promote meiosis. Nature Communications 10, 3414.Despite the vast number of modification sites mapped within mRNAs, known examples of consequential mRNA modifications remain rare. Here, we provide multiple lines of evidence to show that Ime4p, an N6-methyladenosine (m(6)A) methyltransferase required for meiosis in yeast, acts by methylating a site in the 3' UTR of the mRNA encoding Rme1p, a transcriptional repressor of meiosis. Consistent with this mechanism, genetic analyses reveal that IME4 functions upstream of RME1. Transcriptome-wide, RME1 is the primary message that displays both increased methylation and reduced expression in an Ime4p-dependent manner. In yeast strains for which IME4 is dispensable for meiosis, a natural polymorphism in the RME1 promoter reduces RME1 transcription, obviating the requirement for methylation. Mutation of a single m(6)A site in the RME1 3' UTR increases Rme1p repressor production and reduces meiotic efficiency. These results reveal the molecular and physiological consequences of a modification in the 3' UTR of an mRNA.Full Text

Canzio, D., Nwakeze, C.L., Horta, A., Rajkumar, S.M., Coffey, E.L., Duffy, E.E., Duffie, R., Monahan, K., O'Keeffe, S., Simon, M.D., et al. (2019). Antisense lncRNA Transcription Mediates DNA Demethylation to Drive Stochastic Protocadherin alpha Promoter Choice. Cell [Epub ahead of print] . Stochastic activation of clustered Protocadherin (Pcdh) alpha, beta, and gamma genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdhalpha gene choice involves the activation of an antisense promoter located in the first exon of each Pcdhalpha alternate gene. Transcription of an antisense long noncoding RNA (lncRNA) from this antisense promoter extends through the sense promoter, leading to DNA demethylation of the CTCF binding sites proximal to each promoter. Demethylation-dependent CTCF binding to both promoters facilitates cohesin-mediated DNA looping with a distal enhancer (HS5-1), locking in the transcriptional state of the chosen Pcdhalpha gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdhalpha promoters, resulting in proximity-biased DNA looping of the HS5-1 enhancer. Thus, antisense transcription-mediated promoter demethylation functions as a mechanism for distance-independent enhancer/promoter DNA looping to ensure stochastic Pcdhalpha promoter choice. Full Text

Carter, V., LaCava, J., Taylor, M.S. , Liang, S.Y., Mustelin, C., Ukadike, K.C., Bengtsson, A., Lood, C., and Mustelin, T. (2019). High prevalence and disease correlation of autoantibodies against p40 encoded by long interspersed nuclear elements (LINE-1) in systemic lupus erythematosus.Arthritis & rheumatology [Epub ahead of print]. OBJECTIVE: The long interspersed nuclear element 1 (LINE-1) encodes two proteins, the RNA-binding p40 and the endonuclease and reverse transcriptase (ORF2p); both required for LINE-1 to retrotranspose. In cells expressing LINE-1, these proteins assemble with the LINE-1 RNA and additional RNA-binding proteins, some of which are well-known autoantigens in patients with systemic lupus erythematosus (SLE). We asked if SLE patients also make autoantibodies against the LINE-1 p40. METHODS: Highly purified p40 protein was used to quantitate IgG autoantibodies in the serum of 172 SLE patients, disease controls, and age-matched healthy subjects by immunoblotting and ELISA. Preparations of p40 that also contained associated proteins were analyzed by immunoblotting with patient sera. RESULTS: Antibodies reactive with p40 were detected in the majority of patients and many healthy controls: they were higher in patients with SLE, but not systemic sclerosis, compared to healthy subjects (p=0.01). The anti-p40 reactivity was higher in patients during a flare than in remission (p=0.03), correlated with SLEDAI (p=0.0002), type I interferon score (p=0.006), complement C3 decrease (p=0.0001), anti-DNA antibodies (p<0.0001), anti-C1q antibodies (p=0.004), current or past history of nephritis (p=0.02 and 0.003), and they correlated inversely with age (r=-0.49, p<0.0001). SLE patient sera also reacted with p40-associated proteins. CONCLUSIONS: Autoantibodies reacting with LINE-1 p40 characterize a population of SLE patients with severe and active disease. These autoantibodies may represent an early immune response against LINE-1 p40 that does not yet by itself imply clinically significant autoimmunity, but may represent an early, and still reversible, step towards SLE pathogenesis. Full Text

Chau , Y., Li, F.S., Levsh, O., and Weng, J.K. (2019). Exploration of icariin analog structure space reveals key features driving potent inhibition of human phosphodiesterase-5. PloS one 14: e0222803. The natural product icariin inhibits human phosphodiesterase-5 (PDE5) and represents a unique pharmacophore for treating erectile dysfunction, pulmonary hypertension, and other diseases. In this study, we explore the available icariin-derived chemical scaffolds through medicinal chemistry to develop novel icariin PDE5 inhibitors with improved potency and specificity. We synthesized six novel semi-synthetic icariin analogs as well as three naturally occurring icariin analogs, and characterized the structure-activity relationship in the context of human PDE5 inhibition using in vitro enzyme inhibition and kinetics assays and molecular modeling. Mammalian-cell-based assays and in vitro enzyme inhibition assays against human PDE6C further helped to identify the most potent and selective icariin analogs. Our results reveal the synergistic contribution of functional groups at the C3 and C7 positions of the icariin backbone towards PDE5 inhibition. Whereas a hydrophobic and flexible alkanol group at the C7 position is sufficient to enhance icariin analog potency, combining this group with a hydrophilic sugar group at the C3 position leads to further enhancement of potency and promotes specificity towards PDE5 versus PDE6C. In particular, compounds 3 and 7 exhibit Ki values of 0.036 +/- 0.005 muM and 0.036 +/- 0.007 muM towards PDE5 respectively, which are approaching those of commercial PDE5 inhibitors, and can effectively reduce GMP levels in cultured human BJ-hTERT cells. This study identifies novel icariin analogs as potent and selective PDE5 inhibitors poised to become lead compounds for further pharmaceutical development. Full Text

Cheng, J., Hu, G., Xu, Y., Torrens-Spence, M.P., Zhou, X., Wang, D., Weng, J.K., and Wang, Q. (2019). Production of nonnatural straight-chain amino acid 6-aminocaproate via an artificial iterative carbon-chain-extension cycle. Metabolic engineering 55, 23-32. Bioplastics produced from microbial source are promising green alternatives to traditional petrochemical-derived plastics. Nonnatural straight-chain amino acids, especially 5-aminovalerate, 6-aminocaproate and 7-aminoheptanoate are potential monomers for the synthesis of polymeric bioplastics as their primary amine and carboxylic acid are ideal functional groups for polymerization. Previous pathways for 5-aminovalerate and 6-aminocaproate biosynthesis in microorganisms are derived from L-lysine catabolism and the citric acid cycle, respectively. Here, we show the construction of an artificial iterative carbon-chain-extension cycle in Escherichia coli for simultaneous production of a series of nonnatural amino acids with varying chain length. Overexpression of L-lysine alpha-oxidase in E. coli yields 2-keto-6-aminocaproate (2K6AC) as a non-native substrate for the artificial iterative carbon-chain-extension cycle. The chain-extended alpha-ketoacid products are decarboxylated and oxidized by an alpha-ketoacid decarboxylase and an aldehyde dehydrogenase, respectively, to yield their corresponding nonnatural straight-chain amino acids. The engineered system demonstrated simultaneous in vitro production of 99.16mg/L of 5-aminovalerate, 46.96mg/L of 6-aminocaproate and 4.78mg/L of 7-aminoheptanoate after 8h of enzyme catalysis starting from 2K6AC as the substrate. Furthermore, simultaneous production of 2.15g/L of 5-aminovalerate, 24.12mg/L of 6-aminocaproate and 4.74mg/L of 7-aminoheptanoate was achieved in engineered E. coli. This work illustrates a promising metabolic-engineering strategy to access other medium-chain organic acids with -NH2, -SCH3, -SOCH3, -SH, -COOH, -COH, or -OH functional groups through carbon-chain-elongation chemistry. Full Text

Chowdhary, S., Kainth, A.S., Pincus, D., and Gross, D.S. (2019). Heat Shock Factor 1 Drives Intergenic Association of Its Target Gene Loci upon Heat Shock. Cell reports 26, 18-28.Transcriptional induction of heat shock protein (HSP) genes is accompanied by dynamic changes in their 3D structure and spatial organization, yet the molecular basis for these phenomena remains unknown. Using chromosome conformation capture and single-cell imaging, we show that genes transcriptionally activated by Hsf1 specifically interact across chromosomes and coalesce into diffraction-limited intranuclear foci. Genes activated by the alternative stress regulators Msn2/Msn4, in contrast, do not interact among themselves nor with Hsf1 targets. Likewise, constitutively expressed genes, even those interposed between HSP genes, show no detectable interaction. Hsf1 forms discrete subnuclear puncta when stress activated, and these puncta dissolve in concert with transcriptional attenuation, paralleling the kinetics of HSP gene coalescence and dissolution. Nuclear Hsf1 and RNA Pol II are both necessary for intergenic HSP gene interactions, while DNA-bound Hsf1 is necessary and sufficient to drive heterologous gene coalescence. Our findings demonstrate that Hsf1 can dynamically restructure the yeast genome. Full Text

Christ<="" a="" data_temp_dwid="2"> , B., Xu, C., Xu, M., Li, F.S.<="" a="" data_temp_dwid="3">, Wada, N., Mitchell, A.J., Han, X.L., Wen, M.L., Fujita, M., and Weng, J.K. (2019). Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants.Nature Communications 10(1):3206 . Diosgenin is a spiroketal steroidal natural product extracted from plants and used as the single most important precursor for the world steroid hormone industry. The sporadic occurrences of diosgenin in distantly related plants imply possible independent biosynthetic origins. The characteristic 5,6-spiroketal moiety in diosgenin is reminiscent of the spiroketal moiety present in anthelmintic avermectins isolated from actinomycete bacteria. How plants gained the ability to biosynthesize spiroketal natural products is unknown. Here, we report the diosgenin-biosynthetic pathways in himalayan paris (Paris polyphylla), a monocot medicinal plant with hemostatic and antibacterial properties, and fenugreek (Trigonella foenum-graecum), an eudicot culinary herb plant commonly used as a galactagogue. Both plants have independently recruited pairs of cytochromes P450 that catalyze oxidative 5,6-spiroketalization of cholesterol to produce diosgenin, with evolutionary progenitors traced to conserved phytohormone metabolism. This study paves the way for engineering the production of diosgenin and derived analogs in heterologous hosts. Full Text

Condon , K.J., and Sabatini, D.M. (2019). Nutrient regulation of mTORC1 at a glance. Journal of cell science 132(21). The mechanistic target of rapamycin (mTOR) signaling pathway coordinates environmental and intracellular cues to control eukaryotic cell growth. As a pivot point between anabolic and catabolic processes, mTOR complex 1 (mTORC1) signaling has established roles in regulating metabolism, translation and autophagy. Hyperactivity of the mTOR pathway is associated with numerous human diseases, including diabetes, cancer and epilepsy. Pharmacological inhibition of the mTOR pathway can extend lifespan in a variety of model organisms. Given its broad control of essential cellular processes and clear relevance to human health, there is extensive interest in elucidating how upstream inputs regulate mTORC1 activation. In this Cell Science at a Glance article and accompanying poster, we summarize our understanding of how extracellular and intracellular signals feed into the mTOR pathway, how the lysosome acts as an mTOR signaling hub, and how downstream signaling controls autophagy and lysosome biogenesis. Full Text

Cote , L.E., Simental, E., and Reddien, P.W. (2019). Muscle functions as a connective tissue and source of extracellular matrix in planarians. Nature communications 10(1):1592. Regeneration and tissue turnover require new cell production and positional information. Planarians are flatworms capable of regenerating all body parts using a population of stem cells called neoblasts. The positional information required for tissue patterning is primarily harbored by muscle cells, which also control body contraction. Here we produce an in silico planarian matrisome and use recent whole-animal single-cell-transcriptome data to determine that muscle is a major source of extracellular matrix (ECM). No other ECM-secreting, fibroblast-like cell type was detected. Instead, muscle cells express core ECM components, including all 19 collagen-encoding genes. Inhibition of muscle-expressed hemicentin-1 (hmcn-1), which encodes a highly conserved ECM glycoprotein, results in ectopic peripheral localization of cells, including neoblasts, outside of the muscle layer. ECM secretion and hmcn-1-dependent maintenance of tissue separation indicate that muscle functions as a planarian connective tissue, raising the possibility of broad roles for connective tissue in adult positional information. Full Text

Dall'Agnese , A., Caputo, L., Nicoletti, C., di Iulio, J., Schmitt, A., Gatto, S., Diao, Y., Ye, Z., Forcato, M., Perera, R., et al. (2019). Transcription Factor-Directed Re-wiring of Chromatin Architecture for Somatic Cell Nuclear Reprogramming toward trans-Differentiation. Molecular cell [Epub ahead of print]. MYOD-directed fibroblast trans-differentiation into skeletal muscle provides a unique model to investigate how one transcription factor (TF) reconfigures the three-dimensional chromatin architecture to control gene expression, which is otherwise achieved by the combinatorial activities of multiple TFs. Integrative analysis of genome-wide high-resolution chromatin interactions, MYOD and CTCF DNA-binding profile, and gene expression, revealed that MYOD directs extensive re-wiring of interactions involving cis-regulatory and structural genomic elements, including promoters, enhancers, and insulated neighborhoods (INs). Re-configured INs were hot-spots of differential interactions, whereby MYOD binding to highly constrained sequences at IN boundaries and/or inside INs led to alterations of promoter-enhancer interactions to repress cell-of-origin genes and to activate muscle-specific genes. Functional evidence shows that MYOD-directed re-configuration of chromatin interactions temporally preceded the effect on gene expression and was mediated by direct MYOD-DNA binding. These data illustrate a model whereby a single TF alters multi-loop hubs to drive somatic cell trans-differentiation. Full Text

D'Almeida, O., Mothar, O., Bondzie, E.A., Lieumo, Y., Tagne, L., Gupta, S., Volkert, T., Levine, S., and Tagne, J.B. (2019) Encapsulated miR-200c and Nkx2.1 in a nuclear/mitochondria transcriptional regulatory network of non-metastatic and metastatic lung cancer cells. BMC cancer 19(1): 136. MicroRNAs are noncoding RNA molecules of ~ 22 nucleotides with diagnostic and therapeutic action [Curr Drug Targets, 2015. 16(12): p. 1381-403], affecting the expression of mRNAs involved in invasion, migration, and development [Oncotarget, 2015. 6(9): p. 6472-98, Cancer Manag Res, 2014. 6: p. 205-16]. miR-200c is part of the miR-200c/141 cluster on chromosome 12p13. Its mechanism of action when encapsulated is critical in lung cancer when patients express changes in miRNAs. miR-200c be a potential biomarkers for various lung diseases. As a potential therapy, miR-200c can impacts lives as target lung cancer is a leading cause of death with about 234,000 cases annually, high heterogeneity, complex screening, and a 5-year survival rate of 16% [CA Cancer J Clin, 2016.66(1): p. 7-30]. Encapsulated miR-200c efficiently enhances bioavailability, pharmacokinetics of therapeutics and targeting to cells, improves efficacy and provides potential cure. METHODS: The functions of miR-200c were determined in non-metastatic KW-634 and metastatic 821-T4 and 821-LN mouse lung cancer cell lines after various Nano vehicle treatments. Viability and cytotoxicity were determined by cell cycle and quantitative real-time PCR analyses were used to quantify levels of miR-200c and its target genes. In situ hybridization was used to visualize patterns of expression of miR-200c and others in the lung and many organs. Next-generation sequencing accession number GSE125000, invasion and migration assays using transwell chambers, and ActivSignal were used to elucidate the activation and inhibition profiles and perform direct expression measurements and modification of cellular components. RESULTS: Due to their effectiveness as intracellular vesicles transporting miR-200c into, out, and between parts of the cells, miR-200c is encapsulated with cholesterol, an integral part of the biological membranes with very important physical properties of the vehicle. Nano miR-200c showed efficient cellular uptake in KW-634, 821-T4, and 821-LN cells with important changes in gene expression and new isoforms. In KW-634, when treated with encapsulated miR-200c and compare to the non-encapsulated control; miR-29b increased by 5261-fold, and in 821-T4/LN, miR-1247 increased by 150-fold. Conversely, miR-1247 and miR-675 decreased by 348 and 1029.5-fold, respectively. miR-189 decreased by 34-fold in treated 821-T4 cells. A reduction of growth was observed only after 48 h of treatment with Nano miR-200c. Moreover, labeling the vehicle with carboxy-fluorescein showed that the encapsulated particles enter the nucleus and mitochondria. Encapsulated miR-200c by entering the cells, the nucleus and mitochondria, trigger changes in cell cycle phases with 4 up to 12 fold percentage in G2 and S phase respectively compare to miR-200c. Endogenous expression of Nkx2.1, miR-200c, and their targets Myb, Nfib, Six4 and Six1 showed an inverse correlation, as observed in development. CONCLUSIONS: Little is known about miR-200c involvement in regulatory processes. Nano miR-200c affects invasion and migration mechanisms. The expression of encapsulated miR-200c contributes to the inhibition/activation of Kras, EMT, Hippo, regulatory pathways and blockers of metastasis. Delivery of miR-200c increases the expression of miR-29b, an EMY regulator, and miR-1247, an inhibitor of cancer genes, both tumor suppressors involved in lung metastasis. Encapsulated miR-200c act on different proteins that regulates cell cycle pathways. These findings represent a part of a regulatory network providing new insights towards improvement of therapy. Full Text

Debruyne D.N., Dries, R., Sengupta, S., Seruggia, D., Gao, Y., Sharma, B., Huang, H., Moreau, L., McLane, M., Day, D.S., Young, R.A. , et al. (2019). BORIS promotes chromatin regulatory interactions in treatment-resistant cancer cells. Nature [Epub ahead of print]. The CCCTC-binding factor (CTCF), which anchors DNA loops that organize the genome into structural domains, has a central role in gene control by facilitating or constraining interactions between genes and their regulatory elements(1,2). In cancer cells, the disruption of CTCF binding at specific loci by somatic mutation(3,4) or DNA hypermethylation(5) results in the loss of loop anchors and consequent activation of oncogenes. By contrast, the germ-cell-specific paralogue of CTCF, BORIS (brother of the regulator of imprinted sites, also known as CTCFL)(6), is overexpressed in several cancers(7-9), but its contributions to the malignant phenotype remain unclear. Here we show that aberrant upregulation of BORIS promotes chromatin interactions in ALK-mutated, MYCN-amplified neuroblastoma(10) cells that develop resistance to ALK inhibition. These cells are reprogrammed to a distinct phenotypic state during the acquisition of resistance, a process defined by the initial loss of MYCN expression followed by subsequent overexpression of BORIS and a concomitant switch in cellular dependence from MYCN to BORIS. The resultant BORIS-regulated alterations in chromatin looping lead to the formation of super-enhancers that drive the ectopic expression of a subset of proneural transcription factors that ultimately define the resistance phenotype. These results identify a previously unrecognized role of BORIS-to promote regulatory chromatin interactions that support specific cancer phenotypes. Full Text

Demircioglu, F.E., Zheng, W., McQuown, A.J., Maier, N.K., Watson, N., Cheeseman, I.M. , Denic, V., Egelman, E.H., and Schwartz, T.U. (2019). The AAA + ATPase TorsinA polymerizes into hollow helical tubes with 8.5 subunits per turn.Nature Communications 10(1):3262. TorsinA is an ER-resident AAA + ATPase, whose deletion of glutamate E303 results in the genetic neuromuscular disease primary dystonia. TorsinA is an unusual AAA + ATPase that needs an external activator. Also, it likely does not thread a peptide substrate through a narrow central channel, in contrast to its closest structural homologs. Here, we examined the oligomerization of TorsinA to get closer to a molecular understanding of its still enigmatic function. We observe TorsinA to form helical filaments, which we analyzed by cryo-electron microscopy using helical reconstruction. The 4.4 A structure reveals long hollow tubes with a helical periodicity of 8.5 subunits per turn, and an inner channel of ~ 4 nm diameter. We further show that the protein is able to induce tubulation of membranes in vitro, an observation that may reflect an entirely new characteristic of AAA + ATPases. We discuss the implications of these observations for TorsinA function.Full Text

Derynck, R., and Weinberg, R.A. (2019). EMT and Cancer: More Than Meets the Eye. Developmental Cell 49, 313-316. Epithelial cells acquire mesenchymal characteristics during development, wound healing and inflammation, and in cancer and fibrosis. With increasing appreciation of different roles of epithelial-mesenchymal transition (EMT), we address the question of how to define and recognize EMT processes and discuss their properties in cancer progression. Full Text

Diehl, F.F., Lewis, C.A.., Fiske, B.P., and Vander Heiden, M.G. (2019). Cellular redox state constrains serine synthesis and nucleotide production to impact cell proliferation. Nature Metabolism 1, 861-867. The de novo serine synthesis pathway is upregulated in many cancers. However, even cancer cells with increased serine synthesis take up large amounts of serine from the environment1 and we confirm that exogenous serine is needed for maximal proliferation of these cells. Here we show that even when enzymes in the serine synthesis pathway are genetically upregulated, the demand for oxidized NAD+ constrains serine synthesis, rendering serine-deprived cells sensitive to conditions that decrease the cellular NAD+/NADH ratio. Further, purine depletion is a major consequence of reduced intracellular serine availability, particularly when NAD+ regeneration is impaired. Thus, cells rely on exogenous serine consumption to maintain purine biosynthesis. In support of this explanation, providing exogenous purine nucleobases, or increasing NAD+ availability to facilitate de novo serine and purine synthesis, both rescue maximal proliferation even in the absence of extracellular serine. Together, these data indicate that NAD+ is an endogenous limitation for cancer cells to synthesize the serine needed for purine production to support rapid proliferation. Full Text

Do, M.H., Wang, X., Zhang, X., Chou, C., Nixon, B.G., Capistrano, K.J., Peng, M., Efeyan, A., Sabatini, D.M., and Li, M.O. (2019). Nutrient mTORC1 signaling underpins regulatory T cell control of immune tolerance. The Journal of experimental medicine. [Epub ahead of print]. Foxp3(+) regulatory T (T reg) cells are pivotal regulators of immune tolerance, with T cell receptor (TCR)-driven activated T reg (aT reg) cells playing a central role; yet how TCR signaling propagates to control aT reg cell responses remains poorly understood. Here we show that TCR signaling induces expression of amino acid transporters, and renders amino acid-induced activation of mTORC1 in aT reg cells. T reg cell-specific ablation of the Rag family small GTPases RagA and RagB impairs amino acid-induced mTORC1 signaling, causing defective amino acid anabolism, reduced T reg cell proliferation, and a rampant autoimmune disorder similar in severity to that triggered by T reg cell-specific TCR deficiency. Notably, T reg cells in peripheral tissues, including tumors, are more sensitive to Rag GTPase-dependent nutrient sensing. Ablation of RagA alone impairs T reg cell accumulation in the tumor, resulting in enhanced antitumor immunity. Thus, nutrient mTORC1 signaling is an essential component of TCR-initiated T reg cell reprogramming, and Rag GTPase activities may be titrated to break tumor immune tolerance. Full Text

Dokshin, G.A., Davis, G.M., Sawle, A.D., Eldridge, M.D., Nicholls, P.K., Gourley, T.E., Romer, K.A., Molesworth, L.W., Tatnell, H.R., Ozturk, A.R., de Rooij DG, Hannon GJ, Page DC, Mello CC,Carmell MA(2019). GCNA Interacts with Spartan and Topoisomerase II to Regulate Genome Stability. Developmental Cell [Epub ahead of print]. GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan (DVC-1) and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. Here, we show that GCNA mutants in mouse and C. elegans display defects in genome maintenance including DNA damage, aberrant chromosome condensation, and crossover defects in mouse spermatocytes and spontaneous genomic rearrangements in C. elegans. We show that GCNA and topoisomerase II (TOP2) physically interact in both mice and worms and colocalize on condensed chromosomes during mitosis in C. elegans embryos. Moreover, C. elegans gcna-1 mutants are hypersensitive to TOP2 poison. Together, our findings support a model in which GCNA provides genome maintenance functions in the germline and may do so, in part, by promoting the resolution of TOP2 DPCs. Full Text

Eisen T.J., Eichhorn, S.W., Subtelny, A.O., and Bartel, D.P. (2019). MicroRNAs Cause Accelerated Decay of Short-Tailed Target mRNAs. Molecular Cell [Epub ahead of print]. MicroRNAs (miRNAs) specify the recruitment of deadenylases to mRNA targets. Despite this recruitment, we find that miRNAs have almost no effect on steady-state poly(A)-tail lengths of their targets in mouse fibroblasts, which motivates the acquisition of pre-steady-state measurements of the effects of miRNAs on tail lengths, mRNA levels, and translational efficiencies. Effects on translational efficiency are minimal compared to effects on mRNA levels, even for newly transcribed target mRNAs. Effects on target mRNA levels accumulate as the mRNA population approaches steady state, whereas effects on tail lengths peak for recently transcribed target mRNAs and then subside. Computational modeling of this phenomenon reveals that miRNAs cause not only accelerated deadenylation of their targets but also accelerated decay of short-tailed target molecules. This unanticipated effect of miRNAs largely prevents short-tailed target mRNAs from accumulating despite accelerated target deadenylation. The net result is a nearly imperceptible change to the steady-state tail-length distribution of targeted mRNAs. Full Text

Eisen , T.J., Eichhorn, S.W., Subtelny, A.O., Lin, K.S., McGeary, S.E., Gupta, S., and Bartel, D.P. (2019). The Dynamics of Cytoplasmic mRNA Metabolism. Molecular Cell [Epub ahead of print]. For all but a few mRNAs, the dynamics of metabolism are unknown. Here, we developed an experimental and analytical framework for examining these dynamics for mRNAs from thousands of genes. mRNAs of mouse fibroblasts exit the nucleus with diverse intragenic and intergenic poly(A)-tail lengths. Once in the cytoplasm, they have a broad (1000-fold) range of deadenylation rate constants, which correspond to cytoplasmic lifetimes. Indeed, with few exceptions, degradation appears to occur primarily through deadenylation-linked mechanisms, with little contribution from either endonucleolytic cleavage or deadenylation-independent decapping. Most mRNA molecules degrade only after their tail lengths fall below 25 nt. Decay rate constants of short-tailed mRNAs vary broadly (1000-fold) and are larger for short-tailed mRNAs that have previously undergone more rapid deadenylation. This coupling helps clear rapidly deadenylated mRNAs, enabling the large range in deadenylation rate constants to impart a similarly large range in stabilities. Full Text

Elman, J.S., Ni, T.K., Mengwasser, K.E., Jin, D. , Wronski, A., Elledge, S.J., and Kuperwasser, C. (2019). Identification of FUBP1 as a Long Tail Cancer Driver and Widespread Regulator of Tumor Suppressor and Oncogene Alternative Splicing. Cell Reports 28, 3435-3449.Comprehensive sequencing approaches have allowed for the identification of the most frequent contributors to cancer, known as drivers. They have also revealed a class of mutations in understudied, infrequently altered genes, referred to as "long tail" (LT) drivers. A key challenge has been to find clinically relevant LT drivers and to understand how they cooperate to drive disease. Here, we identified far upstream binding protein 1 (FUBP1) as an LT driver using an in vivo CRISPR screen. FUBP1 cooperates with other tumor suppressor genes to transform mammary epithelial cells by disrupting cellular differentiation and tissue architecture. Mechanistically, FUBP1 participates in regulating N(6)-methyladenosine (m(6)A) RNA methylation, and its loss leads to global changes in RNA splicing and widespread expression of aberrant driver isoforms. These findings suggest that somatic alteration of a single gene involved in RNA splicing and m(6)A methylation can produce the necessary panoply of contributors for neoplastic transformation. Full Text

Endo, T., Mikedis, M.M., Nicholls, P.K., Page, D.C., and de Rooij, D.G. (2019). Retinoic Acid and Germ Cell Development in the Ovary and Testis. Biomolecules 9 (12) : 10.3390/biom9120775 Retinoic acid (RA), a derivative of vitamin A, is critical for the production of oocytes and sperm in mammals. These gametes derive from primordial germ cells, which colonize the nascent gonad, and later undertake sexual differentiation to produce oocytes or sperm. During fetal development, germ cells in the ovary initiate meiosis in response to RA, whereas those in the testis do not yet initiate meiosis, as they are insulated from RA, and undergo cell cycle arrest. After birth, male germ cells resume proliferation and undergo a transition to spermatogonia, which are destined to develop into haploid spermatozoa via spermatogenesis. Recent findings indicate that RA levels change periodically in adult testes to direct not only meiotic initiation, but also other key developmental transitions to ensure that spermatogenesis is precisely organized for the prodigious output of sperm. This review focuses on how female and male germ cells develop in the ovary and testis, respectively, and the role of RA in this process. Full Text

Garcia-Bermudez, J., Baudrier, L., Bayraktar, E.C., Shen, Y., La, K., Guarecuco, R., Yucel, B., Fiore, D., Tavora, B., Freinkman, E., Chan SH, Lewis C, Sabatini DM., et al. (2019). Squalene accumulation in cholesterol auxotrophic lymphomas prevents oxidative cell death.Nature [Epub ahead of print] . Cholesterol is essential for cells to grow and proliferate. Normal mammalian cells meet their need for cholesterol through its uptake or de novo synthesis(1), but the extent to which cancer cells rely on each of these pathways remains poorly understood. Here, using a competitive proliferation assay on a pooled collection of DNA-barcoded cell lines, we identify a subset of cancer cells that is auxotrophic for cholesterol and thus highly dependent on its uptake. Through metabolic gene expression analysis, we pinpoint the loss of squalene monooxygenase expression as a cause of cholesterol auxotrophy, particularly in ALK(+) anaplastic large cell lymphoma (ALCL) cell lines and primary tumours. Squalene monooxygenase catalyses the oxidation of squalene to 2,3-oxidosqualene in the cholesterol synthesis pathway and its loss results in accumulation of the upstream metabolite squalene, which is normally undetectable. In ALK(+) ALCLs, squalene alters the cellular lipid profile and protects cancer cells from ferroptotic cell death, providing a growth advantage under conditions of oxidative stress and in tumour xenografts. Finally, a CRISPR-based genetic screen identified cholesterol uptake by the low-density lipoprotein receptor as essential for the growth of ALCL cells in culture and as patient-derived xenografts. This work reveals that the cholesterol auxotrophy of ALCLs is a targetable liability and, more broadly, that systematic approaches can be used to identify nutrient dependencies unique to individual cancer types. Full Text

Gehring , M. (2019). Epigenetic dynamics during flowering plant reproduction: evidence for reprogramming? The New phytologist [Epub ahead of print]. Over the last ten years there have been major advances in documenting and understanding dynamic changes to DNA methylation, small RNAs, chromatin modifications, and chromatin structure that accompany reproductive development in flowering plants from germline specification to seed maturation. Here I highlight recent advances in the field, mostly made possible by microscopic analysis of epigenetic states or by the ability to isolate specific cell types or tissues and apply omics approaches. I consider in which contexts there is potentially reprogramming vs maintenance or reinforcement of epigenetic states. Full Text

Gehrke, A.R., Neverett, E., Luo, Y.J., Brandt, A., Ricci, L., Hulett, R.E., Gompers, A., Ruby, J.G., Rokhsar, D.S., Reddien, P.W., et al. (2019). Acoel genome reveals the regulatory landscape of whole-body regeneration. Science 363 (6432). Whole-body regeneration is accompanied by complex transcriptomic changes, yet the chromatin regulatory landscapes that mediate this dynamic response remain unexplored. To decipher the regulatory logic that orchestrates regeneration, we sequenced the genome of the acoel worm Hofstenia miamia, a highly regenerative member of the sister lineage of other bilaterians. Epigenomic profiling revealed thousands of regeneration-responsive chromatin regions and identified dynamically bound transcription factor motifs, with the early growth response (EGR) binding site as the most variably accessible during Hofstenia regeneration. Combining egr inhibition with chromatin profiling suggests that Egr functions as a pioneer factor to directly regulate early wound-induced genes. The genetic connections inferred by this approach allowed the construction of a gene regulatory network for whole-body regeneration, enabling genomics-based comparisons of regeneration across species. Full Text

Goldman, A., Khiste, S., Freinkman, E.., Dhawan, A., Majumder, B., Mondal, J., Pinkerton, A.B., Eton, E., Medhi, R., Chandrasekar, V., et al. (2019). Targeting tumor phenotypic plasticity and metabolic remodeling in adaptive cross-drug tolerance. Science signaling 12(595). Metastable phenotypic state transitions in cancer cells can lead to the development of transient adaptive resistance or tolerance to chemotherapy. Here, we report that the acquisition of a phenotype marked by increased abundance of CD44 (CD44(Hi)) by breast cancer cells as a tolerance response to routinely used cytotoxic drugs, such as taxanes, activated a metabolic switch that conferred tolerance against unrelated standard-of-care chemotherapeutic agents, such as anthracyclines. We characterized the sequence of molecular events that connected the induced CD44(Hi) phenotype to increased activity of both the glycolytic and oxidative pathways and glucose flux through the pentose phosphate pathway (PPP). When given in a specific order, a combination of taxanes, anthracyclines, and inhibitors of glucose-6-phosphate dehydrogenase (G6PD), an enzyme involved in glucose metabolism, improved survival in mouse models of breast cancer. The same sequence of the three-drug combination reduced the viability of patient breast tumor samples in an explant system. Our findings highlight a convergence between phenotypic and metabolic state transitions that confers a survival advantage to cancer cells against clinically used drug combinations. Pharmacologically targeting this convergence could overcome cross-drug tolerance and could emerge as a new paradigm in the treatment of cancer. Full Text

Grabek, K.R., Cooke, T.F., Epperson, L.E., Spees, K.K., Cabral, G.F., Sutton, S.C., Merriman, D.K., Martin, S.L., and Bustamante, C.D. (2019). Genetic variation drives seasonal onset of hibernation in the 13-lined ground squirrel. Communications Biology 2(1):478. Hibernation in sciurid rodents is a dynamic phenotype timed by a circannual clock. When housed in an animal facility, 13-lined ground squirrels exhibit variation in seasonal onset of hibernation, which is not explained by environmental or biological factors. We hypothesized that genetic factors instead drive variation in timing. After increasing genome contiguity, here, we employ a genotype-by-sequencing approach to characterize genetic variation in 153 ground squirrels. Combined with datalogger records (n = 72), we estimate high heritability (61-100%) for hibernation onset. Applying a genome-wide scan with 46,996 variants, we identify 2 loci significantly (p < 7.14 x 10(-6)), and 12 loci suggestively (p < 2.13 x 10(-4)), associated with onset. At the most significant locus, whole-genome resequencing reveals a putative causal variant in the promoter of FAM204A. Expression quantitative trait loci (eQTL) analyses further reveal gene associations for 8/14 loci. Our results highlight the power of applying genetic mapping to hibernation and present new insight into genetics driving its onset. Full Text

Graef, J.D., Wu, H., Ng, C., Sun, C., Villegas, V., Qadir, D., Jesseman, K., Warren, S.T., Jaenisch, R., Cacace, A., et al. (2019). Partial FMRP expression is sufficient to normalize neuronal hyperactivity in Fragile X neurons. The European Journal of Neuroscience [Epub ahead of print]. Fragile X Syndrome (FXS) is the most common genetic form of intellectual disability caused by a CGG repeat expansion in the 5'-UTR of the Fragile X mental retardation gene FMR1, triggering epigenetic silencing and the subsequent absence of the protein, FMRP. Reactivation of FMR1 represents an attractive therapeutic strategy targeting the genetic root cause of FXS. However, largely missing in the FXS field is an understanding of how much FMR1 reactivation is required to rescue FMRP-dependent mutant phenotypes. Here, we utilize FXS patient derived excitatory neurons to model FXS in vitro and confirm that the absence of FMRP leads to neuronal hyperactivity. We further determined the levels of FMRP and the percentage of FMRP positive cells necessary to correct this phenotype utilizing a mixed and mosaic neuronal culture system and a combination of CRISPR, antisense and expression technologies to titrate FMRP in FXS and WT neurons. Our data demonstrate that restoration of greater than 5% of overall FMRP expression levels or greater than 20% FMRP expressing neurons in a mosaic pattern is sufficient to normalize a FMRP-dependent, hyperactive phenotype in FXS iPSC-derived neurons. Full Text

Graindorge, A., Pinheiro, I., Nawrocka, A., Mallory, A.C., Tsvetkov, P., Gil, N., Carolis, C., Buchholz, F., Ulitsky, I., Heard, E., et al. (2019). In-cell identification and measurement of RNA-protein interactions. Nature Communications 10(1) : 5317. Regulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long noncoding RNAs (lncRNAs), we identify RBPs specifically interacting with the lncRNA Firre and three functionally distinct regions of the lncRNA Xist. incPRINT confirms previously known lncRNA-protein interactions and identifies additional interactions that had evaded detection with other approaches. Importantly, the majority of the incPRINT-defined interactions are specific to individual functional regions of the large Xist transcript. Thus, we present an RNA-centric method that enables reliable identification of RNA-region-specific RBPs and is applicable to any RNA of interest. Full Text

Gross, K.M., Zhou, W., Breindel, J.L., Ouyang, J., Jin, D.X., Sokol, E.S., Gupta, P.B. , Huber, K., Zou, L., and Kuperwasser, C. (2019). Loss of Slug Compromises DNA Damage Repair and Accelerates Stem Cell Aging in Mammary Epithelium. Cell Reports 28(2):394-407. DNA damage activates checkpoints that limit the replicative potential of stem cells, including differentiation. These checkpoints protect against cancer development but also promote tissue aging. Because mice lacking Slug/Snai2 exhibit limited stem cell activity, including luminobasal differentiation, and are protected from mammary cancer, we reasoned that Slug might regulate DNA damage checkpoints in mammary epithelial cells. Here, we show that Slug facilitates efficient execution of RPA32-mediated DNA damage response (DDR) signaling. Slug deficiency leads to delayed phosphorylation of ataxia telangiectasia mutated and Rad3-related protein (ATR) and its effectors RPA32 and CHK1. This leads to impaired RAD51 recruitment to DNA damage sites and persistence of unresolved DNA damage. In vivo, Slug/Snai2 loss leads to increased DNA damage and premature aging of mammary epithelium. Collectively, our work demonstrates that the mammary stem cell regulator Slug controls DDR checkpoints by dually inhibiting differentiation and facilitating DDR repair, and its loss causes unresolved DNA damage and accelerated aging.Full Text

Guida, M.C., Birse, R.T., Dall'Agnese, A., Toto, P.C., Diop, S.B., Mai, A., Adams, P.D., Puri, P.L., and Bodmer, R. (2019). Intergenerational inheritance of high fat diet-induced cardiac lipotoxicity in Drosophila. Nature communications 10(1):193. Obesity is strongly correlated with lipotoxic cardiomyopathy, heart failure and thus mortality. The incidence of obesity has reached alarming proportions worldwide, and increasing evidence suggests that the parents' nutritional status may predispose their offspring to lipotoxic cardiomyopathy. However, to date, mechanisms underlying intergenerational heart disease risks have yet to be elucidated. Here we report that cardiac dysfunction induced by high-fat-diet (HFD) persists for two subsequent generations in Drosophila and is associated with reduced expression of two key metabolic regulators, adipose triglyceride lipase (ATGL/bmm) and transcriptional cofactor PGC-1. We provide evidence that targeted expression of ATGL/bmm in the offspring of HFD-fed parents protects them, and the subsequent generation, from cardio-lipotoxicity. Furthermore, we find that intergenerational inheritance of lipotoxic cardiomyopathy correlates with elevated systemic H3K27 trimethylation. Lowering H3K27 trimethylation genetically or pharmacologically in the offspring of HFD-fed parents prevents cardiac pathology. This suggests that metabolic homeostasis is epigenetically regulated across generations. Full Text

Guo , Y.E., Manteiga, J.C., Henninger, J.E., Sabari, B.R., Dall'Agnese, A., Hannett, N.M., Spille, J.H., Afeyan, L.K., Zamudio, A.V., Shrinivas, K., Abraham BJ, Boija A, Lee TI, and Young RA., et al. (2019). Pol II phosphorylation regulates a switch between transcriptional and splicing condensates.Nature [Epub ahead of print] . The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex(1-4). The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus(5,6). Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain(7-12). Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers(7,8), and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites(9-12). Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference. Full Text

Harding , C.R., Gow, M., Kang, J.H., Shortt, E., Manalis, S.R., Meissner, M., and Lourido, S. (2019). Alveolar proteins stabilize cortical microtubules in Toxoplasma gondii. Nature communications 10(1):401.Single-celled protists use elaborate cytoskeletal structures, including arrays of microtubules at the cell periphery, to maintain polarity and rigidity. The obligate intracellular parasite Toxoplasma gondii has unusually stable cortical microtubules beneath the alveoli, a network of flattened membrane vesicles that subtends the plasmalemma. However, anchoring of microtubules along alveolar membranes is not understood. Here, we show that GAPM1a, an integral membrane protein of the alveoli, plays a role in maintaining microtubule stability. Degradation of GAPM1a causes cortical microtubule disorganisation and subsequent depolymerisation. These changes in the cytoskeleton lead to parasites becoming shorter and rounder, which is accompanied by a decrease in cellular volume. Extended GAPM1a depletion leads to severe defects in division, reminiscent of the effect of disrupting other alveolar proteins. We suggest that GAPM proteins link the cortical microtubules to the alveoli and are required to maintain the shape and rigidity of apicomplexan zoites.Full Text

Hou, J., Tan, G., Fink, G.R., Andrews, B.J., and Boone, C. (2019). Complex modifier landscape underlying genetic background effects. PNAS [Epub ahead of print]. The phenotypic consequence of a given mutation can be influenced by the genetic background. For example, conditional gene essentiality occurs when the loss of function of a gene causes lethality in one genetic background but not another. Between two individual Saccharomyces cerevisiae strains, S288c and Sigma1278b, approximately 1% of yeast genes were previously identified as "conditional essential." Here, in addition to confirming that some conditional essential genes are modified by a nonchromosomal element, we show that most cases involve a complex set of genomic modifiers. From tetrad analysis of S288C/Sigma1278b hybrid strains and whole-genome sequencing of viable hybrid spore progeny, we identified complex sets of multiple genomic regions underlying conditional essentiality. For a smaller subset of genes, including CYS3 and CYS4, each of which encodes components of the cysteine biosynthesis pathway, we observed a segregation pattern consistent with a single modifier associated with conditional essentiality. In natural yeast isolates, we found that the CYS3/CYS4 conditional essentiality can be caused by variation in two independent modifiers, MET1 and OPT1, each with roles associated with cellular cysteine physiology. Interestingly, the OPT1 allelic variation appears to have arisen independently from separate lineages, with rare allele frequencies below 0.5%. Thus, while conditional gene essentiality is usually driven by genetic interactions associated with complex modifier architectures, our analysis also highlights the role of functionally related, genetically independent, and rare variants. Full Text

Jacobowitz , J.R., Doyle, W.C., and Weng, J.K. (2019). PRX9 and PRX40 are extensin peroxidases essential for maintaining tapetum and microspore cell wall integrity during Arabidopsis anther development. The Plant cell [Epub ahead of print]. Pollen and microspore development are essential steps in the life cycle of all land plants that generate male gametes. Within flowering plants, pollen development occurs inside of the anther. Here, we report the identification of two class III peroxidase-encoding genes, PEROXIDASE9 and PEROXIDASE40, that are genetically redundant and essential for proper anther and pollen development in Arabidopsis thaliana. Arabidopsis double mutants devoid of functional PRX9 and PRX40 are male-sterile. The mutant anthers display swollen, hypertrophic tapetal cells and pollen grains, suggesting disrupted cell wall integrity. These phenotypes lead to nearly 100%-penetrant pollen degeneration upon anther maturation. Using immunochemical and biochemical approaches, we show that PRX9 and PRX40 likely cross-link extensins to contribute to tapetal cell wall integrity during anther development. This work suggests that PRX9 and PRX40 encode Arabidopsis extensin peroxidases and highlights the importance of extensin cross-linking during pollen development. Full Text

Johnstone D.L., Al-Shekaili, H.H., Tarailo-Graovac, M., Wolf, N.I., Ivy, A.S., Demarest, S., Roussel, Y., Ciapaite, J., van Roermund, C.W.T., Kernohan, K.D., Pena, I.A., et al. (2019). PLPHP deficiency: clinical, genetic, biochemical, and mechanistic insights. Brain [Epub ahead of print]. Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery. Full Text

Kikani, C.K., Wu, X., Fogarty, S., Kang, S.A.W. , Dephoure, N., Gygi, S.P., Sabatini, D.M. , and Rutter, J. (2019). Activation of PASK by mTORC1 is required for the onset of the terminal differentiation program. PNAS 116, 10382-10391. During skeletal muscle regeneration, muscle stem cells (MuSCs) respond to multiple signaling inputs that converge onto mammalian target of rapamycin complex 1 (mTORC1) signaling pathways. mTOR function is essential for establishment of the differentiation-committed progenitors (early stage of differentiation, marked by the induction of myogenin expression), myotube fusion, and, ultimately, hypertrophy (later stage of differentiation). While a major mTORC1 substrate, p70S6K, is required for myotube fusion and hypertrophy, an mTORC1 effector for the induction of myogenin expression remains unclear. Here, we identified Per-Arnt-Sim domain kinase (PASK) as a downstream phosphorylation target of mTORC1 in MuSCs during differentiation. We have recently shown that the PASK phosphorylates Wdr5 to stimulate MuSC differentiation by epigenetically activating the myogenin promoter. We show that phosphorylation of PASK by mTORC1 is required for the activation of myogenin transcription, exit from self-renewal, and induction of the myogenesis program. Our studies reveal that mTORC1-PASK signaling is required for the rise of myogenin- positive committed myoblasts (early stage of myogenesis), whereas mTORC1-S6K signaling is required for myoblast fusion (later stage of myogenesis). Thus, our discoveries allow molecular dissection of mTOR functions during different stages of the myogenesis program driven by two different substrates. Full Text

Kojima , M.L., de Rooij, D.G., and Page, D.C. (2019). Amplification of a broad transcriptional program by a common factor triggers the meiotic cell cycle in mice. eLife 8:e43738. The germ line provides the cellular link between generations of multicellular organisms, its cells entering the meiotic cell cycle only once each generation. However, the mechanisms governing this initiation of meiosis remain poorly understood. Here, we examined cells undergoing meiotic initiation in mice, and we found that initiation involves the dramatic upregulation of a transcriptional network of thousands of genes whose expression is not limited to meiosis. This broad gene expression program is directly upregulated by STRA8, encoded by a germ cell-specific gene required for meiotic initiation. STRA8 binds its own promoter and those of thousands of other genes, including meiotic prophase genes, factors mediating DNA replication and the G1-S cell-cycle transition, and genes that promote the lengthy prophase unique to meiosis I. We conclude that, in mice, the robust amplification of this extraordinarily broad transcription program by a common factor triggers initiation of meiosis. Full Text

Krajewska, M., Dries, R., Grassetti, A.V., Dust, S., Gao, Y., Huang, H., Sharma, B., Day, D.S., Kwiatkowski, N., Pomaville, M., Young, R.A., et al. (2019). CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation. Nature communications 10(1):1757. Cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and selectively affects the expression of genes involved in the DNA damage response (DDR) and mRNA processing. Yet, the mechanisms underlying such selectivity remain unclear. Here we show that CDK12 inhibition in cancer cells lacking CDK12 mutations results in gene length-dependent elongation defects, inducing premature cleavage and polyadenylation (PCPA) and loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlates with an increased number of intronic polyadenylation sites, a feature especially prominent among DDR genes. Phosphoproteomic analysis indicated that CDK12 directly phosphorylates pre-mRNA processing factors, including those regulating PCPA. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition primarily due to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites. Full Text

Kroger , C., Afeyan, A., Mraz, J., Eaton, E.N., Reinhardt, F., Khodor, Y.L., Thiru, P., Bierie, B., Ye, X., Burge, C.B., and Robert A. Weinberg.. (2019). Acquisition of a hybrid E/M state is essential for tumorigenicity of basal breast cancer cells. PNAS [Epub ahead of print]. Carcinoma cells residing in an intermediate phenotypic state along the epithelial-mesenchymal (E-M) spectrum are associated with malignant phenotypes, such as invasiveness, tumor-initiating ability, and metastatic dissemination. Using the recently described CD104(+)/CD44(hi) antigen marker combination, we isolated highly tumorigenic breast cancer cells residing stably-both in vitro and in vivo-in an intermediate phenotypic state and coexpressing both epithelial (E) and mesenchymal (M) markers. We demonstrate that tumorigenicity depends on individual cells residing in this E/M hybrid state and cannot be phenocopied by mixing two cell populations that reside stably at the two ends of the spectrum, i.e., in the E and in the M state. Hence, residence in a specific intermediate state along the E-M spectrum rather than phenotypic plasticity appears critical to the expression of tumor-initiating capacity. Acquisition of this E/M hybrid state is facilitated by the differential expression of EMT-inducing transcription factors (EMT-TFs) and is accompanied by the expression of adult stem cell programs, notably, active canonical Wnt signaling. Furthermore, transition from the highly tumorigenic E/M state to a fully mesenchymal phenotype, achieved by constitutive ectopic expression of Zeb1, is sufficient to drive cells out of the E/M hybrid state into a highly mesenchymal state, which is accompanied by a substantial loss of tumorigenicity and a switch from canonical to noncanonical Wnt signaling. Identifying the gatekeepers of the various phenotypic states arrayed along the E-M spectrum is likely to prove useful in developing therapeutic approaches that operate by shifting cancer cells between distinct states along this spectrum. Full Text

Kruszka, P., Buscetta, A., Acosta, M.T., Banks, N., Addissie, Y.A., Toro, C., Luby, M., Latour, L., Vezina, G., Page, D.C., et al. (2019). Circle of Willis anomalies in Turner syndrome: Absent A1 segment of the anterior cerebral artery. Birth Defects Research [Epub ahead of print]. Turner syndrome (TS) is the most common sex chromosome disorder in women and is associated with a higher than expected death rate secondary to cerebrovascular disease, including stroke. This study evaluates the cerebral vascular anatomy of individuals with TS. METHODS: Twenty-one women with TS had brain magnetic resonance angiography (MRA). These MRAs were evaluated in a blinded manner with a control group of 25 men and 25 women who had MRA imaging for multiple indications including migraine headaches, psychiatric disorders, and seizures. RESULTS: Twenty-nine percent of women with TS were missing an A1 segment of the anterior cerebral artery (ACA) compared to 0% in the control group (p < .001). There were no other significant differences in the circle of Willis (COW) in women with TS compared with the control group. A complete COW was found in 3 of 21 (14%) of women with TS and 12 of 47 (26%) controls (p = .36). CONCLUSION: Women with TS have a significantly different intracranial vascular anatomy, specifically the absence of the A1 segment of the ACA when compared to male and female controls. More research in brain imaging in women with TS and stroke and other cerebrovascular diseases is needed to determine the clinical significance of this anomaly.

Kuroda, K., Hammer, S.K., Watanabe, Y., Montano Lopez, J., Fink, G.R., Stephanopoulos, G., Ueda, M., and Avalos, J.L. (2019). Critical Roles of the Pentose Phosphate Pathway and GLN3 in Isobutanol-Specific Tolerance in Yeast. Cell Systems [Epub ahead of print]. Branched-chain alcohols are attractive advanced biofuels; however, their cellular toxicity is an obstacle to engineering microbes to produce them at high titers. We performed genome-wide screens on the Saccharomyces cerevisiae gene deletion library to identify cell systems involved in isobutanol-specific tolerance. Deletion of pentose phosphate pathway genes GND1 or ZWF1 causes hypersensitivity to isobutanol but not to ethanol. By contrast, deletion of GLN3 increases yeast tolerance specifically to branched-chain alcohols. Transcriptomic analyses revealed that isobutanol induces a nitrogen starvation response via GLN3 and GCN4, upregulating amino acid biosynthesis and nitrogen scavenging while downregulating glycolysis, cell wall biogenesis, and membrane lipid biosynthesis. Disruption of this response by deleting GLN3 is enough to enhance tolerance and boost isobutanol production 4.9-fold in engineered strains. This study illustrates how adaptive mechanisms to tolerate stress can lead to toxicity in microbial fermentations for chemical production and how genetic interventions can boost production by evading such mechanisms.Full Text

Kwasnieski , J.C., Orr-Weaver, T.L., and Bartel, D.P. (2019). Early genome activation in Drosophila is extensive with an initial tendency for aborted transcripts and retained introns. Genome Research 29, 1188-1197. Control of metazoan embryogenesis shifts from maternal to zygotic gene products as the zygotic genome becomes transcriptionally activated. In Drosophila, zygotic genome activation (ZGA) has been thought to occur in two phases, starting with a minor wave, in which a small number of genes become expressed, and progressing to the major wave, in which many more genes are activated. However, technical challenges have hampered the identification of early transcripts or obscured the onset of their transcription. Here, we develop an approach to isolate transcribed mRNAs and apply it over the course of Drosophila early genome activation. Our results increase by 10-fold the genes reported to be activated during what has been thought of as the minor wave and show that early genome activation is continuous and gradual. Transposable-element mRNAs are also produced, but discontinuously. Genes transcribed in the early and middle part of ZGA are short with few if any introns, and their transcripts are frequently aborted and tend to have retained introns, suggesting that inefficient splicing as well as rapid cell divisions constrain the lengths of early transcripts. Full Text

Lesch , B.J., Tothova, Z., Morgan, E.A., Liao, Z., Bronson, R.T., Ebert, B.L., and Page, D.C. (2019). Intergenerational epigenetic inheritance of cancer susceptibility in mammals. eLife 2019 8 : e39380. Susceptibility to cancer is heritable, but much of this heritability remains unexplained. Some 'missing' heritability may be mediated by epigenetic changes in the parental germ line that do not involve transmission of genetic variants from parent to offspring. We report that deletion of the chromatin regulator Kdm6a (Utx) in the paternal germ line results in elevated tumor incidence in genetically wild type mice. This effect increases following passage through two successive generations of Kdm6a male germline deletion, but is lost following passage through a wild type germ line. The H3K27me3 mark is redistributed in sperm of Kdm6a mutants, and we define approximately 200 H3K27me3-marked regions that exhibit increased DNA methylation, both in sperm of Kdm6a mutants and in somatic tissue of progeny. Hypermethylated regions in enhancers may alter regulation of genes involved in cancer initiation or progression. Epigenetic changes in male gametes may therefore impact cancer susceptibility in adult offspring. Full Text

Li , D.J., McMann, C.L., and Reddien, P.W. (2019). Nuclear receptor NR4A is required for patterning at the ends of the planarian anterior-posterior axis.eLife 8 : e42015. Positional information is fundamental to animal regeneration and tissue turnover. In planarians, muscle cells express signaling molecules to promote positional identity. At the ends of the anterior-posterior (AP) axis, positional identity is determined by anterior and posterior poles, which are putative organizers. We identified a gene, nr4A, that is required for anterior- and posterior-pole localization to axis extremes. nr4A encodes a nuclear receptor expressed predominantly in planarian muscle, including strongly at AP-axis ends and the poles. nr4A RNAi causes patterning gene expression domains to retract from head and tail tips, and ectopic anterior and posterior anatomy (e.g., eyes) to iteratively appear more internally. Our study reveals a novel patterning phenotype, in which pattern-organizing cells (poles) shift from their normal locations (axis extremes), triggering abnormal tissue pattern that fails to reach equilibrium. We propose that nr4A promotes pattern at planarian AP axis ends through restriction of patterning gene expression domains. Full Text

Li, H., Ericsson, M., Rabasha, B., Budnik, B., Chan, S.H., Freinkman, E., Lewis, C.A., Doench, J.G., Wagner, B.K., Garraway, L.A., et al. (2019). 6-Phosphogluconate Dehydrogenase Links Cytosolic Carbohydrate Metabolism to Protein Secretion via Modulation of Glutathione Levels. Cell chemical biology [Epub ahead of print]. The proteinaceous extracellular matrix (ECM) is vital for the survival, proliferation, migration, and differentiation of many types of cancer. However, little is known regarding metabolic pathways required for ECM secretion. By using an unbiased computational approach, we searched for enzymes whose suppression may lead to disruptions in protein secretion. Here, we show that 6-phosphogluconate dehydrogenase (PGD), a cytosolic enzyme involved in carbohydrate metabolism, is required for ER structural integrity and protein secretion. Chemical inhibition or genetic suppression of PGD activity led to cell stress accompanied by significantly expanded ER volume and was rescued by compensating endogenous glutathione supplies. Our results also suggest that this characteristic ER-dilation phenotype may be a general marker indicating increased ECM protein congestion inside cells and decreased secretion. Thus, PGD serves as a link between cytosolic carbohydrate metabolism and protein secretion. Full Text

Li , H., Natarajan, A., Ezike, J., Barrasa, M.I., Le, Y., Feder, Z.A., Yang, H., Ma, C., Markoulaki, S., and Lodish, H.F. (2019). Rate of Progression through a Continuum of Transit-Amplifying Progenitor Cell States Regulates Blood Cell Production. Developmental cell [Epub ahead of print]. The nature of cell-state transitions during the transit-amplifying phases of many developmental processes-hematopoiesis in particular-is unclear. Here, we use single-cell RNA sequencing to demonstrate a continuum of transcriptomic states in committed transit-amplifying erythropoietic progenitors, which correlates with a continuum of proliferative potentials in these cells. We show that glucocorticoids enhance erythrocyte production by slowing the rate of progression through this developmental continuum of transit-amplifying progenitors, permitting more cell divisions prior to terminal erythroid differentiation. Mechanistically, glucocorticoids prolong expression of genes that antagonize and slow induction of genes that drive terminal erythroid differentiation. Erythroid progenitor daughter cell pairs have similar transcriptomes with or without glucocorticoid stimulation, indicating largely symmetric cell division. Thus, the rate of progression along a developmental continuum dictates the absolute number of erythroid cells generated from each transit-amplifying progenitor, suggesting a paradigm for regulating the total output of differentiated cells in numerous other developmental processes. Full Text

Li, L., Ng, S.R., Colon, C.I., Drapkin, B.J., Hsu, P.P., Li, Z., Nabel, C.S., Lewis, C.A.., Romero, R., Mercer, K.L., et al. (2019). Identification of DHODH as a therapeutic target in small cell lung cancer. Science Translational Medicine 11(517). Small cell lung cancer (SCLC) is an aggressive lung cancer subtype with extremely poor prognosis. No targetable genetic driver events have been identified, and the treatment landscape for this disease has remained nearly unchanged for over 30 years. Here, we have taken a CRISPR-based screening approach to identify genetic vulnerabilities in SCLC that may serve as potential therapeutic targets. We used a single-guide RNA (sgRNA) library targeting ~5000 genes deemed to encode "druggable" proteins to perform loss-of-function genetic screens in a panel of cell lines derived from autochthonous genetically engineered mouse models (GEMMs) of SCLC, lung adenocarcinoma (LUAD), and pancreatic ductal adenocarcinoma (PDAC). Cross-cancer analyses allowed us to identify SCLC-selective vulnerabilities. In particular, we observed enhanced sensitivity of SCLC cells toward disruption of the pyrimidine biosynthesis pathway. Pharmacological inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in this pathway, reduced the viability of SCLC cells in vitro and strongly suppressed SCLC tumor growth in human patient-derived xenograft (PDX) models and in an autochthonous mouse model. These results indicate that DHODH inhibition may be an approach to treat SCLC. Full Text

Li , P., and Elowitz, M.B. (2019). Communication codes in developmental signaling pathways.Development 146(12). A handful of core intercellular signaling pathways play pivotal roles in a broad variety of developmental processes. It has remained puzzling how so few pathways can provide the precision and specificity of cell-cell communication required for multicellular development. Solving this requires us to quantitatively understand how developmentally relevant signaling information is actively sensed, transformed and spatially distributed by signaling pathways. Recently, single cell analysis and cell-based reconstitution, among other approaches, have begun to reveal the 'communication codes' through which information is represented in the identities, concentrations, combinations and dynamics of extracellular ligands. They have also revealed how signaling pathways decipher these features and control the spatial distribution of signaling in multicellular contexts. Here, we review recent work reporting the discovery and analysis of communication codes and discuss their implications for diverse developmental processes. Full Text

Li , Y., Muffat, J., Omer Javed, A., Keys, H.R., Lungjangwa, T., Bosch, I., Khan, M., Virgilio, M.C., Gehrke, L., Sabatini, D.M., and Jaenisch, R.(2019). Genome-wide CRISPR screen for Zika virus resistance in human neural cells. PNAS [Epub ahead of print]. Zika virus (ZIKV) is a neurotropic and neurovirulent arbovirus that has severe detrimental impact on the developing human fetal brain. To date, little is known about the factors required for ZIKV infection of human neural cells. We identified ZIKV host genes in human pluripotent stem cell (hPSC)-derived neural progenitors (NPs) using a genome-wide CRISPR-Cas9 knockout screen. Mutations of host factors involved in heparan sulfation, endocytosis, endoplasmic reticulum processing, Golgi function, and interferon activity conferred resistance to infection with the Uganda strain of ZIKV and a more recent North American isolate. Host genes essential for ZIKV replication identified in human NPs also provided a low level of protection against ZIKV in isogenic human astrocytes. Our findings provide insights into host-dependent mechanisms for ZIKV infection in the highly vulnerable human NP cells and identify molecular targets for potential therapeutic intervention. Full Text

Liu, X.S., and Jaenisch, R. (2019). Editing the Epigenome to Tackle Brain Disorders. Trends in Neurosciences 42(12):861-870. Genetic studies of epigenetic modifiers such as DNA methyltransferases and histone acetyltransferases have revealed a critical role for epigenetic regulation during brain development and function. Alteration of epigenetic modifications have been documented in a variety of brain disorders, including neurodevelopmental, psychiatric, and neurodegenerative diseases. Development of epigenome editing tools enables a functional dissection of the link between altered epigenetic changes and disease outcomes. Here, we review the development of epigenome editing tools, summarize proof of concept applications focusing on brain disease-associated genes, and discuss the promising application and challenges of epigenome editing to tackle brain disorders. Full Text

Lourido , S. (2019). Toxoplasma gondii. Trends in Parasitology S1471-4922(19)30166-7. Toxoplasma gondii is an obligate intracellular eukaryotic parasite from the phylum Apicomplexa that infects up to one-third of the global population. Extensive genetic tools make T. gondii a tractable model to dissect the biology of the phylum. Full Text

Luengo, A., Abbott, K.L., Davidson, S.M., Hosios, A.M., Faubert, B., Chan, S.H., Freinkman, E., Zacharias, L.G., Mathews, T.P., Clish, C.B., DeBerardinis RJ, Lewis CA., Vander Heiden MG. (2019). Reactive metabolite production is a targetable liability of glycolytic metabolism in lung cancer. Nature Communications 10(1):5604. Increased glucose uptake and metabolism is a prominent phenotype of most cancers, but efforts to clinically target this metabolic alteration have been challenging. Here, we present evidence that lactoylglutathione (LGSH), a byproduct of methylglyoxal detoxification, is elevated in both human and murine non-small cell lung cancers (NSCLC). Methylglyoxal is a reactive metabolite byproduct of glycolysis that reacts non-enzymatically with nucleophiles in cells, including basic amino acids, and reduces cellular fitness. Detoxification of methylglyoxal requires reduced glutathione (GSH), which accumulates to high levels in NSCLC relative to normal lung. Ablation of the methylglyoxal detoxification enzyme glyoxalase I (Glo1) potentiates methylglyoxal sensitivity and reduces tumor growth in mice, arguing that targeting pathways involved in detoxification of reactive metabolites is an approach to exploit the consequences of increased glucose metabolism in cancer. Full Text

Ly, P., Brunner, S.F., Shoshani, O., Kim, D.H., Lan, W., Pyntikova, T., Flanagan, A.M., Behjati, S., Page, D.C., Campbell, P.J., et al. (2019). Chromosome segregation errors generate a diverse spectrum of simple and complex genomic rearrangements.Nature genetics [Epub ahead of print] . Cancer genomes are frequently characterized by numerical and structural chromosomal abnormalities. Here we integrated a centromere-specific inactivation approach with selection for a conditionally essential gene, a strategy termed CEN-SELECT, to systematically interrogate the structural landscape of mis-segregated chromosomes. We show that single-chromosome mis-segregation into a micronucleus can directly trigger a broad spectrum of genomic rearrangement types. Cytogenetic profiling revealed that mis-segregated chromosomes exhibit 120-fold-higher susceptibility to developing seven major categories of structural aberrations, including translocations, insertions, deletions, and complex reassembly through chromothripsis coupled to classical non-homologous end joining. Whole-genome sequencing of clonally propagated rearrangements identified random patterns of clustered breakpoints with copy-number alterations resulting in interspersed gene deletions and extrachromosomal DNA amplification events. We conclude that individual chromosome segregation errors during mitotic cell division are sufficient to drive extensive structural variations that recapitulate genomic features commonly associated with human disease.Full Text

Markus B.M., Bell, G.W., Lorenzi, H.A., and Lourido, S. (2019). Optimizing Systems for Cas9 Expression in Toxoplasma gondii. mSphere 4(3). CRISPR-Cas9 technologies have enabled genome engineering in an unprecedented array of species, accelerating biological studies in both model and nonmodel systems. However, Cas9 can be inherently toxic, which has limited its use in some organisms. We previously described the serendipitous discovery of a single guide RNA (sgRNA) that helped overcome Cas9 toxicity in the apicomplexan parasite Toxoplasma gondii, enabling the first genome-wide loss-of-function screens in any apicomplexan. Even in the presence of the buffering sgRNA, low-level Cas9 toxicity persists and results in frequent loss of Cas9 expression, which can affect the outcome of these screens. Similar Cas9-mediated toxicity has also been described in other organisms. We therefore sought to define the requirements for stable Cas9 expression, comparing different expression constructs and characterizing the role of the buffering sgRNA to understand the basis of Cas9 toxicity. We find that viral 2A peptides can substantially improve the selection and stability of Cas9 expression. We also demonstrate that the sgRNA has two functions: primarily facilitating integration of the Cas9-expression construct following initial genome targeting and secondarily improving long-term parasite fitness by alleviating Cas9 toxicity. We define a set of guidelines for the expression of Cas9 with improved stability and selection stringency, which are directly applicable to a variety of genetic approaches in diverse organisms. Our work also emphasizes the need for further characterizing the effects of Cas9 expression.IMPORTANCE Toxoplasma gondii is an intracellular parasite that causes life-threatening disease in immunocompromised patients and affects the developing fetus when contracted during pregnancy. Closely related species cause malaria and severe diarrhea, thereby constituting leading causes for childhood mortality. Despite their importance to global health, this family of parasites has remained enigmatic. Given its remarkable experimental tractability, T. gondii has emerged as a model also for the study of related parasites. Genetic approaches are important tools for studying the biology of organisms, including T. gondii As such, the recent developments of CRISPR-Cas9-based techniques for genome editing have vastly expanded our ability to study the biology of numerous species. In some organisms, however, CRISPR-Cas9 has been difficult to implement due to its inherent toxicity. Our research characterizes the basis of the observed toxicity, using T. gondii as a model, allowing us to develop approaches to aid the use of CRISPR-Cas9 in diverse species. Full Text

McGeary , S.E., Lin, K.S., Shi, C.Y., Pham, T., Bisaria, N., Kelley, G.M., and Bartel, D.P. (2019). The biochemical basis of microRNA targeting efficacy. Science [Epub ahead of print]. MicroRNAs (miRNAs) act within Argonaute proteins to guide repression of mRNA targets. Although various approaches have provided insight into target recognition, the sparsity of miRNA-target affinity measurements has limited understanding and prediction of targeting efficacy. Here, we adapted RNA bind-n-seq to enable measurement of relative binding affinities between Argonaute-miRNA complexes and all 12 or less nucleotide sequences. This approach revealed noncanonical target sites unique to each miRNA, miRNA-specific differences in canonical target-site affinities, and a 100-fold impact of dinucleotides flanking each site. These data enabled construction of a biochemical model of miRNA-mediated repression, which was extended to all miRNA sequences using a convolutional neural network. This model substantially improved prediction of cellular repression, thereby providing a biochemical basis for quantitatively integrating miRNAs into gene-regulatory networks. Full Text

Mertens, S., Gallone, B., Steensels, J., Herrera-Malaver, B., Cortebeek, J., Nolmans, R., Saels, V., Vyas, V.K., and Verstrepen, K.J. (2019). Reducing phenolic off-flavors through CRISPR-based gene editing of the FDC1 gene in Saccharomyces cerevisiae x Saccharomyces eubayanus hybrid lager beer yeasts. PloS One 14(1):e0209124. Today's beer market is challenged by a decreasing consumption of traditional beer styles and an increasing consumption of specialty beers. In particular, lager-type beers (pilsner), characterized by their refreshing and unique aroma and taste, yet very uniform, struggle with their sales. The development of novel variants of the common lager yeast, the interspecific hybrid Saccharomyces pastorianus, has been proposed as a possible solution to address the need of product diversification in lager beers. Previous efforts to generate new lager yeasts through hybridization of the ancestral parental species (S. cerevisiae and S. eubayanus) yielded strains with an aromatic profile distinct from the natural biodiversity. Unfortunately, next to the desired properties, these novel yeasts also inherited unwanted characteristics. Most notably is their phenolic off-flavor (POF) production, which hampers their direct application in the industrial production processes. Here, we describe a CRISPR-based gene editing strategy that allows the systematic and meticulous introduction of a natural occurring mutation in the FDC1 gene of genetically complex industrial S. cerevisiae strains, S. eubayanus yeasts and interspecific hybrids. The resulting cisgenic POF- variants show great potential for industrial application and diversifying the current lager beer portfolio.Full Text

Mitchell , A.J., and Weng, J.K. (2019). Unleashing the synthetic power of plant oxygenases: from mechanism to application. Plant physiology [Epub ahead of print]. Here we review families of plant oxygenases and their chemistry and suggest potential applications.Full Text

Morgan , J.T., Fink, G.R., and Bartel, D.P. (2019). Excised linear introns regulate growth in yeast. Nature [Epub ahead of print]. Spliceosomal introns are ubiquitous non-coding RNAs that are typically destined for rapid debranching and degradation. Here we describe 34 excised introns in Saccharomyces cerevisiae that-despite being rapidly degraded in log-phase growth-accumulate as linear RNAs under either saturated-growth conditions or other stresses that cause prolonged inhibition of TORC1, which is a key integrator of growth signalling. Introns that become stabilized remain associated with components of the spliceosome and differ from other spliceosomal introns in having a short distance between their lariat branch point and 3' splice site, which is necessary and sufficient for their stabilization. Deletion of these unusual introns is disadvantageous in saturated conditions and causes aberrantly high growth rates in yeast that are chronically challenged with the TORC1 inhibitor rapamycin. The reintroduction of native or engineered stable introns suppresses this aberrant rapamycin response. Thus, excised introns function within the TOR growth-signalling network of S. cerevisiae and, more generally, excised spliceosomal introns can have biological functions.Full Text

Naqvi, S., Godfrey, A.K., Hughes, J.F., Goodheart, M.L., Mitchell, R.N., and Page, D.C.(2019). Conservation, acquisition, and functional impact of sex-biased gene expression in mammals.Science 365(6450) . Sex differences abound in human health and disease, as they do in other mammals used as models. The extent to which sex differences are conserved at the molecular level across species and tissues is unknown. We surveyed sex differences in gene expression in human, macaque, mouse, rat, and dog, across 12 tissues. In each tissue, we identified hundreds of genes with conserved sex-biased expression-findings that, combined with genomic analyses of human height, explain ~12% of the difference in height between females and males. We surmise that conserved sex biases in expression of genes otherwise operating equivalently in females and males contribute to sex differences in traits. However, most sex-biased expression arose during the mammalian radiation, which suggests that careful attention to interspecies divergence is needed when modeling human sex differences. Full Text

Nicholls , P.K., Schorle, H., Naqvi, S., Hu, Y.C., Fan, Y., Carmell, M.A., Dobrinski, I., Watson, A.L., Carlson, D.F., Fahrenkrug, S.C.,and Page, D.C.(2019). Mammalian germ cells are determined after PGC colonization of the nascent gonad. PNAS [Epub ahead of print]. Mammalian primordial germ cells (PGCs) are induced in the embryonic epiblast, before migrating to the nascent gonads. In fish, frogs, and birds, the germline segregates even earlier, through the action of maternally inherited germ plasm. Across vertebrates, migrating PGCs retain a broad developmental potential, regardless of whether they were induced or maternally segregated. In mammals, this potential is indicated by expression of pluripotency factors, and the ability to generate teratomas and pluripotent cell lines. How the germline loses this developmental potential remains unknown. Our genome-wide analyses of embryonic human and mouse germlines reveal a conserved transcriptional program, initiated in PGCs after gonadal colonization, that differentiates germ cells from their germline precursors and from somatic lineages. Through genetic studies in mice and pigs, we demonstrate that one such gonad-induced factor, the RNA-binding protein DAZL, is necessary in vivo to restrict the developmental potential of the germline; DAZL's absence prolongs expression of a Nanog pluripotency reporter, facilitates derivation of pluripotent cell lines, and causes spontaneous gonadal teratomas. Based on these observations in humans, mice, and pigs, we propose that germ cells are determined after gonadal colonization in mammals. We suggest that germ cell determination was induced late in embryogenesis-after organogenesis has begun-in the common ancestor of all vertebrates, as in modern mammals, where this transition is induced by somatic cells of the gonad. We suggest that failure of this process of germ cell determination likely accounts for the origin of human testis cancer. Full Text

Nicholls P.K., Bellott, D.W., Cho, T.J., Pyntikova, T., and Page, D.C. ,(2019). Locating and Characterizing a Transgene Integration Site by Nanopore Sequencing. G3-Genes Genomes Genetics 9, 1481-1486. The introduction of foreign DNA into cells and organisms has facilitated much of modern biological research, and it promises to become equally important in clinical practice. Locating sites of foreign DNA incorporation in mammalian genomes has proven burdensome, so the genomic location of most transgenes remains unknown. To address this challenge, we applied nanopore sequencing in search of the site of integration of Tg(Pou5f1-EGFP)(2Mnn) (also known as Oct4:EGFP), a widely used fluorescent reporter in mouse germ line research. Using this nanopore-based approach, we identified the site of Oct4:EGFP transgene integration near the telomere of Chromosome 9. This methodology simultaneously yielded an estimate of transgene copy number, provided direct evidence of transgene inversions, revealed contaminating E. coli genomic DNA within the transgene array, validated the integrity of neighboring genes, and enabled definitive genotyping. We suggest that such an approach provides a rapid, cost-effective method for identifying and analyzing transgene integration sites. Full Text

Oulhen, N., Swartz, S.Z., Wang, L., Wikramanayake, A., and Wessel, G.M. (2019). Distinct transcriptional regulation of Nanos2 in the germ line and soma by the Wnt and delta/notch pathways. Developmental biology [Epub ahead of print]. Specification of the primordial germ cells (PGCs) is essential for sexually reproducing animals. Although the mechanisms of PGC specification are diverse between organisms, the RNA binding protein Nanos is consistently required in the germ line in all species tested. How Nanos is selectively expressed in the germ line, however, remains largely elusive. We report that in sea urchin embryos, the early expression of Nanos2 in the PGCs requires the maternal Wnt pathway. During gastrulation, however, Nanos2 expression expands into adjacent somatic mesodermal cells and this secondary Nanos expression instead requires Delta/Notch signaling through the forkhead family member FoxY. Each of these transcriptional regulators were tested by chromatin immunoprecipitation analysis and found to directly interact with a DNA locus upstream of Nanos2. Given the conserved importance of Nanos in germ line specification, and the derived character of the micromeres and small micromeres in the sea urchin, we propose that the ancestral mechanism of Nanos2 expression in echinoderms was by induction in mesodermal cells during gastrulation. Full Text

Paredes-Santos, T., Wang, Y., Waldman, B., Lourido, S., and Saeij, J.P. (2019). The GRA17 Parasitophorous Vacuole Membrane Permeability Pore Contributes to Bradyzoite Viability. Frontiers in Cellular and Infection Microbiology 9:321.The Toxoplasma gondii parasitophorous vacuole membrane (PVM) offers protection from the host immune system but is also a barrier for uptake of nutrients from the host. Previously, we showed that GRA17 mediates the tachyzoite PVM permeability to small molecules. During the conversion from tachyzoites to encysted bradyzoites, the PVM become the cyst membrane that is the outer layer of the cyst wall. Little is known about how small molecules, such as nutrients, enter cysts. To characterize GRA17's role in cysts, we deleted GRA17 in the type II ME49 cyst-forming strain. ME49Deltagra17 parasites have reduced growth and formed grossly enlarged "bubble vacuoles," which have reduced PVM small molecule permeability. ME49Deltagra17 parasites formed cysts in vitro at rates comparable to the wild-type, but the viability of the bradyzoites inside these cysts was significantly reduced compared to wild-type bradyzoites. Genetic complementation of ME49Deltagra17 with GRA17 expressed from the endogenous or tachyzoite-specific SAG1 promoter recovered the viability of bradyzoites. Complementation with the bradyzoite-specific SRS9 promoter drastically increased the viability of bradyzoites, demonstrating the importance of GRA17 in regulating bradyzoite viability inside cysts. Mice infected with a high dose of ME49Deltagra17 parasites did not contain parasites in their brain nor did mice infected with ME49Deltagra17 complemented with GRA17 expressed from a bradyzoite-specific promoter. Our results suggest that the ME49Deltagra17 strain is avirulent and is cleared before it can reach the brain and that GRA17 not only plays an important role during acute infections but is also needed for viability of bradyzoites inside cysts. Full Text

Pentinmikko, N., Iqbal, S., Mana, M., Andersson, S., Cognetta, A.B., 3rd, Suciu, R.M., Roper, J., Luopajarvi, K., Markelin, E., Gopalakrishnan, S., Sabatini, D.M., et al. (2019). Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium. Nature [Epub ahead of print]. A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by actively cycling intestinal stem cells that are regulated by the Paneth cell niche(1,2). Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor alpha (PPAR-alpha)(3), and lowered PPAR-alpha activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues. Full Text

Pluskal , T., Torrens-Spence, M.P., Fallon, T.R., De Abreu, A., Shi, C.H., and Weng, J.K. (2019). The biosynthetic origin of psychoactive kavalactones in kava. Nature plants [Epub ahead of print]. Kava (Piper methysticum) is an ethnomedicinal shrub native to the Polynesian islands with well-established anxiolytic and analgesic properties. Its main psychoactive principles, kavalactones, form a unique class of polyketides that interact with the human central nervous system through mechanisms distinct from those of conventional psychiatric drugs. However, an unknown biosynthetic machinery and difficulty in chemical synthesis hinder the therapeutic use of kavalactones. In addition, kava also produces flavokavains, which are chalconoids with anticancer properties structurally related to kavalactones. Here, we report de novo elucidation of the key enzymes of the kavalactone and flavokavain biosynthetic network. We present the structural basis for the evolutionary development of a pair of paralogous styrylpyrone synthases that establish the kavalactone scaffold and the catalytic mechanism of a regio- and stereo-specific kavalactone reductase that produces a subset of chiral kavalactones. We further demonstrate the feasibility of engineering styrylpyrone production in heterologous hosts, thus opening a way to develop kavalactone-based non-addictive psychiatric therapeutics through synthetic biology. Full Text

Pontis, J., Planet, E., Offner, S., Turelli, P., Duc, J., Coudray, A., Theunissen, T.W., Jaenisch, R., and Trono, D. (2019). Hominoid-Specific Transposable Elements and KZFPs Facilitate Human Embryonic Genome Activation and Control Transcription in Naive Human ESCs. Cell stem cell[Epub ahead of print]. Expansion of transposable elements (TEs) coincides with evolutionary shifts in gene expression. TEs frequently harbor binding sites for transcriptional regulators, thus enabling coordinated genome-wide activation of species- and context-specific gene expression programs, but such regulation must be balanced against their genotoxic potential. Here, we show that Kruppel-associated box (KRAB)-containing zinc finger proteins (KZFPs) control the timely and pleiotropic activation of TE-derived transcriptional cis regulators during early embryogenesis. Evolutionarily recent SVA, HERVK, and HERVH TE subgroups contribute significantly to chromatin opening during human embryonic genome activation and are KLF-stimulated enhancers in naive human embryonic stem cells (hESCs). KZFPs of corresponding evolutionary ages are simultaneously induced and repress the transcriptional activity of these TEs. Finally, the same KZFP-controlled TE-based enhancers later serve as developmental and tissue-specific enhancers. Thus, by controlling the transcriptional impact of TEs during embryogenesis, KZFPs facilitate their genome-wide incorporation into transcriptional networks, thereby contributing to human genome regulation. Full Text

Prakash, S.K., San Roman, A.K., Crenshaw, M., Flink, B., Earle, K., Los, E., Bonnard, A., and Lin, A.E. (2019). "Donating our bodies to science": A discussion about autopsy and organ donation in Turner syndrome. American journal of medical genetics Part C, Seminars in medical genetics [Epub ahead of print]. At the Third Turner Resource Network Symposium, a working group presented the results of collaborative discussions about the importance of autopsy in Turner syndrome (TS). Considerable gaps in understanding the causes of death in TS can only be closed by more frequent death investigations and autopsies. The presentation included an overview of autopsy methods, strategies for utilizing autopsy, and biobanking to address research questions about TS, and the role of palliative care in the context of autopsy. This review highlights strategies to promote autopsy and tissue donation, culminating with an action plan to increase autopsy rates in the TS community.Full Text

Priyam, A., Woodcroft, B.J., Rai, V., Moghul, I., Mungala, A., Ter, F., Chowdhary, H., Pieniak, I.L., Gibbins, M.A., Moon, H., Pluskal T., et al. (2019). Sequenceserver: a modern graphical user interface for custom BLAST databases. Molecular biology and evolution [Epub ahead of print]. Comparing newly obtained and previously known nucleotide and amino-acid sequences underpins modern biological research. BLAST is a well-established tool for such comparisons but is challenging to use on new datasets. We combined a user-centric design philosophy with sustainable software development approaches to create Sequenceserver, a tool for running BLAST and visually inspecting BLAST results for biological interpretation. Sequenceserver uses simple algorithms to prevent potential analysis errors, and provides flexible text-based and visual outputs to support researcher productivity. Our software can be rapidly installed for use by individuals or on shared servers.Full Text

Rasheed, N., Lima, T.B., Mercaldi, G.F., Nascimento, A.F.Z., Silva, A.L.S., Righetto, G.L., Bar-Peled, L., Shen, K., Sabatini, D.M., Gozzo, F.C., et al. (2019). C7orf59/Lamtor4 phosphorylation and structural flexibility modulate Ragulator assembly. FEBS open bio. Ragulator is a pentamer composed of p18, MP1, p14, C7orf59 and HBXIP (LAMTOR 1 - 5) which acts as a lysosomal scaffold of the Rag GTPases in the amino acid sensitive branch of TORC1 signaling. Here we present the crystal structure of human HBXIP-C7orf59 dimer (LAMTOR 4/5) at 2.9A and identify a phosphorylation site on C7orf59 which modulates its interaction with p18. Additionally, we demonstrate the requirement of HBXIP-C7orf59 to stabilize p18 and allow further binding of MP1-p14. The structure of the dimer revealed an unfolded N-terminus in C7orf59 (residues 1-15) which was shown to be essential for p18 binding. Full length p18 does not interact stably with MP1-p14 in the absence of HBXIP-C7orf59, but deletion of p18 residues 108-161 rescues MP1-p14 binding. C7orf59 was phosphorylated by PKA in vitro and mutation of the conserved Ser67 residue to aspartate prevented phosphorylation and negatively affected the C7orf59 interaction with p18 both in cell culture and in vitro. C7orf59 Ser67 was phosphorylated in HEK293T cells. PKA activation with forskolin induced dissociation of p18 from C7orf59, which was prevented by the PKA inhibitor H-89. Our results highlight the essential role of HBXIP-C7orf59 dimer as a nucleator of pentameric Ragulator and support a sequential model of Ragulator assembly in which HBXIP-C7orf59 binds and stabilizes p18 which allows subsequent binding of MP1-p14. Full Text

Rashidian , M., LaFleur, M.W., Verschoor, V.L., Dongre, A., Zhang, Y. , Nguyen, T.H., Kolifrath, S., Aref, A.R., Lau, C.J., Paweletz, C.P., Bu X, Freeman GJ, Barrasa MI, Weinberg RA ,Sharpe AH, and Ploegh HL. (2019). Immuno-PET identifies the myeloid compartment as a key contributor to the outcome of the antitumor response under PD-1 blockade. PNAS [Epub ahead of print]. Immunotherapy using checkpoint-blocking antibodies against PD-1 has produced impressive results in a wide range of cancers. However, the response remains heterogeneous among patients. We used noninvasive immuno-positron emission tomography (PET), using (89)Zr-labeled PEGylated single-domain antibody fragments (nanobodies or VHHs), to explore the dynamics and distribution of intratumoral CD8(+) T cells and CD11b(+) myeloid cells in response to anti-PD-1 treatment in the MC38 colorectal mouse adenocarcinoma model. Responding and nonresponding tumors showed consistent differences in the distribution of CD8(+) and CD11b(+) cells. Anti-PD-1 treatment mobilized CD8(+) T cells from the tumor periphery to a more central location. Only those tumors fully infiltrated by CD8(+) T cells went on to complete resolution. All tumors contained CD11b(+) myeloid cells from the outset of treatment, with later recruitment of additional CD11b(+) cells. As tumors grew, the distribution of intratumoral CD11b(+) cells became more heterogeneous. Shrinkage of tumors in responders correlated with an increase in the CD11b(+) population in the center of the tumors. The changes in distribution of CD8(+) and CD11b(+) cells, as assessed by PET, served as biomarkers to gauge the efficacy of anti-PD-1 treatment. Single-cell RNA sequencing of RNA from intratumoral CD45(+) cells showed that CD11b(+) cells in responders and nonresponders were markedly different. The responders exhibited a dominant population of macrophages with an M1-like signature, while the CD45(+) population in the nonresponders displayed an M2-like transcriptional signature. Thus, by using immuno-PET and single-cell RNA sequencing, we show that anti-PD-1 treatment not only affects interactions of CD8(+) T cells with the tumor but also impacts the intratumoral myeloid compartment. Full Text

Reddien , P.W. (2019). The cells of regeneration.Science 365,314-316. The ability to regenerate missing body parts is a prominent feature of many animals. Investigation into the cellular and molecular basis of regeneration using highly regenerative model organisms should identify principles that explain how regeneration can occur and might clarify why such regenerative capacity is limited in humans. Full Text

Renault, H., Werck-Reichhart, D., and Weng, J.K. (2019). 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 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

Rogala , K.B., Gu, X., Kedir, J.F., Abu-Remaileh, M., Bianchi, L.F., Bottino, A.M.S., Dueholm, R., Niehaus, A., Overwijn, D., Fils, A.P.,Zhou, L.X.Leary D, Laqtom NN, Brignole EJ and Sabatini, D.M. (2019). Structural basis for the docking of mTORC1 on the lysosomal surface. Science [Epub ahead of print]. The mTORC1 protein kinase regulates growth in response to nutrients and growth factors. Nutrients promote its translocation to the lysosomal surface, where its Raptor subunit interacts with the Rag GTPase-Ragulator complex. Nutrients switch the heterodimeric Rag GTPases between four different nucleotide binding states, only one of which (RagA/B*GTP-RagC/D*GDP) permits mTORC1 association. We determined the structure of the supercomplex of Raptor with Rag-Ragulator to 3.2 A resolution by cryo-electron microscopy. The Raptor alpha-solenoid directly detects the nucleotide state of RagA, while the Raptor "claw" threads between the GTPase domains to detect that of RagC. Mutations that disrupt Rag-Raptor binding inhibit mTORC1 lysosomal localization and signaling. By comparison with a structure of mTORC1 bound to its activator Rheb, we develop a model of active mTORC1 docked on the lysosome. Full Text

San Roman, A.K., and Page, D.C. (2019). A strategic research alliance: Turner syndrome and sex differences. American journal of medical genetics Part C, Seminars in medical genetics [Epub ahead of print]. Sex chromosome constitution varies in the human population, both between the sexes (46,XX females and 46,XY males), and within the sexes (e.g., 45,X and 46,XX females, and 47,XXY and 46,XY males). Coincident with this genetic variation are numerous phenotypic differences between males and females, and individuals with sex chromosome aneuploidy. However, the molecular mechanisms by which sex chromosome constitution impacts phenotypes at the cellular, tissue, and organismal levels remain largely unexplored. Thus, emerges a fundamental question connecting the study of sex differences and sex chromosome aneuploidy syndromes: How does sex chromosome constitution influence phenotype? Here, we focus on Turner syndrome (TS), associated with the 45,X karyotype, and its synergies with the study of sex differences. We review findings from evolutionary studies of the sex chromosomes, which identified genes that are most likely to contribute to phenotypes as a result of variation in sex chromosome constitution. We then explore strategies for investigating the direct effects of the sex chromosomes, and the evidence for specific sex chromosome genes impacting phenotypes. In sum, we argue that integrating the study of TS with sex differences offers a mutually beneficial alliance to identify contributions of the sex chromosomes to human development, health, and disease. Full Text

Sangare, L.O., Olafsson, E.B., Wang, Y., Yang, N., Julien, L., Camejo, A., Pesavento, P., Sidik, S.M., Lourido, S., Barragan, A., et al. (2019). In Vivo CRISPR Screen Identifies TgWIP as a Toxoplasma Modulator of Dendritic Cell Migration. Cell Host & Microbe 26, 478-492. Toxoplasma can reach distant organs, especially the brain, leading to a lifelong chronic phase. However, genes involved in related in vivo processes are currently unknown. Here, we use focused CRISPR libraries to identify Toxoplasma genes that affect in vivo fitness. We focus on TgWIP, whose deletion affects Toxoplasma dissemination to distant organs. We show that TgWIP is secreted into the host cell upon invasion and interacts with the host WAVE regulatory complex and SHP2 phosphatase, both of which regulate actin dynamics. TgWIP affects the morphology of dendritic cells and mediates the dissolution of podosomes, which dendritic cells use to adhere to extracellular matrix. TgWIP enhances the motility and transmigration of parasitized dendritic cells, likely explaining its effect on in vivo fitness. Our results provide a framework for systemic identification of Toxoplasma genes with in vivo effects at the site of infection or on dissemination to distant organs, including the brain. Full Text

Satyaki , P.R.V., and Gehring, M. (2019). Paternally acting canonical RNA-directed DNA methylation pathway genes sensitize Arabidopsis endosperm to paternal genome dosage.The Plant cell [Epub ahead of print]. Paternally acting canonical RNA-directed DNA methylation pathway genes sensitize Arabidopsis endosperm to paternal genome dosage. Seed development is sensitive to parental dosage, with excess maternal or paternal genomes creating reciprocal phenotypes. Paternal genomic excess frequently results in extensive endosperm proliferation without cellularization and seed abortion. We previously showed that loss of the RNA Pol IV gene nrpd1 in tetraploid fathers represses seed abortion in paternal excess crosses. Here we show genetically that RNA-directed DNA methylation (RdDM) pathway activity in the paternal parent is sufficient to determine the viability of paternal excess seeds. We compared transcriptomes, DNA methylation, and small RNAs from endosperm of balanced crosses (diploid x diploid) and lethal (diploid x tetraploid) and viable paternal excess (diploid x tetraploid nrpd1). Endosperm from both lethal and viable paternal excess seeds share widespread transcriptional and DNA methylation changes at genes and TEs. Interploidy seed abortion is thus unlikely to be caused by either transposable element or imprinted gene mis-regulation, and its repression by loss of paternal RdDM is associated with only modest gene expression changes. Finally, using allele-specific transcription data, we present evidence for a transcriptional buffering system that increases expression of maternal alleles and represses paternal alleles in response to excess paternal genomic dosage. These findings prompt reconsideration of models for dosage sensitivity in endosperm. Full Text

Schiebinger, G., Shu, J., Tabaka, M., Cleary, B., Subramanian, V., Solomon, A., Gould, J., Liu, S., Lin, S., Berube, P., Jaenisch, R., et al. (2019). Optimal-Transport Analysis of Single-Cell Gene Expression Identifies Developmental Trajectories in Reprogramming. Cell 176(4):928-943. Understanding the molecular programs that guide differentiation during development is a major challenge. Here, we introduce Waddington-OT, an approach for studying developmental time courses to infer ancestor-descendant fates and model the regulatory programs that underlie them. We apply the method to reconstruct the landscape of reprogramming from 315,000 single-cell RNA sequencing (scRNA-seq) profiles, collected at half-day intervals across 18 days. The results reveal a wider range of developmental programs than previously characterized. Cells gradually adopt either a terminal stromal state or a mesenchymal-to-epithelial transition state. The latter gives rise to populations related to pluripotent, extra-embryonic, and neural cells, with each harboring multiple finer subpopulations. The analysis predicts transcription factors and paracrine signals that affect fates and experiments validate that the TF Obox6 and the cytokine GDF9 enhance reprogramming efficiency. Our approach sheds light on the process and outcome of reprogramming and provides a framework applicable to diverse temporal processes in biology. Full Text

Shen , K., Rogala, K.B., Chou, H.T., Huang, R.K., Yu, Z., and Sabatini, D.M. (2019). Cryo-EM Structure of the Human FLCN-FNIP2-Rag-Ragulator Complex. Cell 179(6):1319-1329. mTORC1 controls anabolic and catabolic processes in response to nutrients through the Rag GTPase heterodimer, which is regulated by multiple upstream protein complexes. One such regulator, FLCN-FNIP2, is a GTPase activating protein (GAP) for RagC/D, but despite its important role, how it activates the Rag GTPase heterodimer remains unknown. We used cryo-EM to determine the structure of FLCN-FNIP2 in a complex with the Rag GTPases and Ragulator. FLCN-FNIP2 adopts an extended conformation with two pairs of heterodimerized domains. The Longin domains heterodimerize and contact both nucleotide binding domains of the Rag heterodimer, while the DENN domains interact at the distal end of the structure. Biochemical analyses reveal a conserved arginine on FLCN as the catalytic arginine finger and lead us to interpret our structure as an on-pathway intermediate. These data reveal features of a GAP-GTPase interaction and the structure of a critical component of the nutrient-sensing mTORC1 pathway. Full Text

Shen K., Valenstein, M.L., Gu, X., and Sabatini, D.M. (2019). Arg-78 of Nprl2 catalyzes GATOR1-stimulated GTP hydrolysis by the Rag GTPases. Journal of Biological Chemistry 294, 2970-2975. mTOR complex 1 (mTORC1) is a major regulator of cell growth and proliferation that coordinates nutrient inputs with anabolic and catabolic processes. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which directly recruit mTORC1 onto the lysosomal surface, its site of activation. The Rag GTPase heterodimer has a unique architecture that consists of two GTPase subunits, RagA or RagB bound to RagC or RagD. Their nucleotide-loading states are strictly controlled by several lysosomal or cytosolic protein complexes that directly detect and transmit the amino acid signals. GATOR1 (GTPase-activating protein (GAP) activity toward Rags-1), a negative regulator of the cytosolic branch of the nutrient-sensing pathway, comprises three subunits, Depdc5 (DEP domain-containing protein 5), Nprl2 (NPR2-like GATOR1 complex subunit), and Nprl3 (NPR3-like GATOR1 complex subunit), and is a GAP for RagA. GATOR1 binds the Rag GTPases via two modes: an inhibitory mode that holds the Rag GTPase heterodimer and has previously been captured by structural determination, and a GAP mode that stimulates GTP hydrolysis by RagA but remains structurally elusive. Here, using site-directed mutagenesis, GTP hydrolysis assays, coimmunoprecipitation experiments, and structural analysis, we probed the GAP mode and found that a critical residue on Nprl2, Arg-78, is the arginine finger that carries out GATOR1's GAP function. Substitutions of this arginine residue rendered mTORC1 signaling insensitive to amino acid starvation and are found frequently in cancers such as glioblastoma. Our results reveal the biochemical bases of mTORC1 inactivation through the GATOR1 complex. Full Text

Shrinivas, K., Sabari, B.R., Coffey, E.L., Klein, I.A., Boija, A., Zamudio, A.V., Schuijers, J., Hannett, N.M.., Sharp, P.A., Young, R.A., et al. (2019). Enhancer Features that Drive Formation of Transcriptional Condensates.Molecular Cell 75, 549-561. Enhancers are DNA elements that are bound by transcription factors (TFs), which recruit coactivators and the transcriptional machinery to genes. Phase-separated condensates of TFs and coactivators have been implicated in assembling the transcription machinery at particular enhancers, yet the role of DNA sequence in this process has not been explored. We show that DNA sequences encoding TF binding site number, density, and affinity above sharply defined thresholds drive condensation of TFs and coactivators. A combination of specific structured (TF-DNA) and weak multivalent (TF-coactivator) interactions allows for condensates to form at particular genomic loci determined by the DNA sequence and the complement of expressed TFs. DNA features found to drive condensation promote enhancer activity and transcription in cells. Our study provides a framework to understand how the genome can scaffold transcriptional condensates at specific loci and how the universal phenomenon of phase separation might regulate this process. Full Text

Song , Y., van den Berg, P.R., Markoulaki, S., Soldner, F., Dall'Agnese, A., Henninger, J.E., Drotar, J., Rosenau, N., Cohen, M.A., Young, R.A., Jaenisch, R. et al. (2019).Dynamic Enhancer DNA Methylation as Basis for Transcriptional and Cellular Heterogeneity of ESCs. Molecular Cell [Epub ahead of print]. Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs in vivo. Full Text

Sullivan, M.R., Danai, L.V., Lewis, C.A., Chan, S.H.., Gui, D.Y., Kunchok, T.., Dennstedt, E.A., Vander Heiden, M.G., and Muir, A. (2019). Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability. eLife 8. pii: e44235. Cancer cell metabolism is heavily influenced by microenvironmental factors, including nutrient availability. Therefore, knowledge of microenvironmental nutrient levels is essential to understand tumor metabolism. To measure the extracellular nutrient levels available to tumors, we utilized quantitative metabolomics methods to measure the absolute concentrations of >118 metabolites in plasma and tumor interstitial fluid, the extracellular fluid that perfuses tumors. Comparison of nutrient levels in tumor interstitial fluid and plasma revealed that the nutrients available to tumors differ from those present in circulation. Further, by comparing interstitial fluid nutrient levels between autochthonous and transplant models of murine pancreatic and lung adenocarcinoma, we found that tumor type, anatomical location and animal diet affect local nutrient availability. These data provide a comprehensive characterization of the nutrients present in the tumor microenvironment of widely used models of lung and pancreatic cancer and identify factors that influence metabolite levels in tumors. Full Text

Sullivan, M.R., Mattaini, K.R., Dennstedt, E.A., Nguyen, A.A., Sivanand, S., Reilly, M.F., Meeth, K., Muir, A., Darnell, A.M., Bosenberg, M.W., Lewis, C.A., et al. (2019). Increased Serine Synthesis Provides an Advantage for Tumors Arising in Tissues Where Serine Levels Are Limiting. Cell metabolism [Epub ahead of print]. Tumors exhibit altered metabolism compared to normal tissues. Many cancers upregulate expression of serine synthesis pathway enzymes, and some tumors exhibit copy-number gain of the gene encoding the first enzyme in the pathway, phosphoglycerate dehydrogenase (PHGDH). However, whether increased serine synthesis promotes tumor growth and how serine synthesis benefits tumors is controversial. Here, we demonstrate that increased PHGDH expression promotes tumor progression in mouse models of melanoma and breast cancer, human tumor types that exhibit PHGDH copy-number gain. We measure circulating serine levels and find that PHGDH expression is necessary to support cell proliferation at lower physiological serine concentrations. Increased dietary serine or high PHGDH expression is sufficient to increase intracellular serine levels and support faster tumor growth. Together, these data suggest that physiological serine availability restrains tumor growth and argue that tumors arising in serine-limited environments acquire a fitness advantage by upregulating serine synthesis pathway enzymes. Full Text

Svoboda , D.S., Barrasa, M.I., Shu, J., Rietjens, R., Zhang, S., Mitalipova, M., Berube, P., Fu, D, Shultz, L.D., Bell, G.W., and Jaenisch, R. (2019).Human iPSC-derived microglia assume a primary microglia-like state after transplantation into the neonatal mouse brain. PNAS [Epub ahead of print]. Microglia are essential for maintenance of normal brain function, with dysregulation contributing to numerous neurological diseases. Protocols have been developed to derive microglia-like cells from human induced pluripotent stem cells (hiPSCs). However, primary microglia display major differences in morphology and gene expression when grown in culture, including down-regulation of signature microglial genes. Thus, in vitro differentiated microglia may not accurately represent resting primary microglia. To address this issue, we transplanted microglial precursors derived in vitro from hiPSCs into neonatal mouse brains and found that the cells acquired characteristic microglial morphology and gene expression signatures that closely resembled primary human microglia. Single-cell RNA-sequencing analysis of transplanted microglia showed similar cellular heterogeneity as primary human cells. Thus, hiPSCs-derived microglia transplanted into the neonatal mouse brain assume a phenotype and gene expression signature resembling that of resting microglia residing in the human brain, making chimeras a superior tool to study microglia in human disease. Full Text

Swartz , S.Z., McKay, L.S., Su, K.C., Bury, L., Padeganeh, A., Maddox, P.S., Knouse, K.A., and Cheeseman, I.M. (2019). Quiescent Cells Actively Replenish CENP-A Nucleosomes to Maintain Centromere Identity and Proliferative Potential. Developmental cell (In Press, Corrected Proof) . Centromeres provide a robust model for epigenetic inheritance as they are specified by sequence-independent mechanisms involving the histone H3-variant centromere protein A (CENP-A). Prevailing models indicate that the high intrinsic stability of CENP-A nucleosomes maintains centromere identity indefinitely. Here, we demonstrate that CENP-A is not stable at centromeres but is instead gradually and continuously incorporated in quiescent cells including G0-arrested tissue culture cells and prophase I-arrested oocytes. Quiescent CENP-A incorporation involves the canonical CENP-A deposition machinery but displays distinct requirements from cell cycle-dependent deposition. We demonstrate that Plk1 is required specifically for G1 CENP-A deposition, whereas transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence results in significantly reduced CENP-A levels and perturbs chromosome segregation following the resumption of cell division. In contrast to quiescent cells, terminally differentiated cells fail to maintain CENP-A levels. Our work reveals that quiescent cells actively maintain centromere identity providing an indicator of proliferative potential. Full Text

Tang X., Drotar, J.,, Li, K., Clairmont, C.D. , Brumm, A.S., Sullins, A.J., Wu, H., Liu, X.S. , Wang, J., Gray, N.S., Sur, M., and Jaenisch, R. (2019). Pharmacological enhancement of KCC2 gene expression exerts therapeutic effects on human Rett syndrome neurons and Mecp2 mutant mice. Science Translational Medicine 11(503). Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. There are currently no approved treatments for RTT. The expression of K(+)/Cl(-) cotransporter 2 (KCC2), a neuron-specific protein, has been found to be reduced in human RTT neurons and in RTT mouse models, suggesting that KCC2 might play a role in the pathophysiology of RTT. To develop neuron-based high-throughput screening (HTS) assays to identify chemical compounds that enhance the expression of the KCC2 gene, we report the generation of a robust high-throughput drug screening platform that allows for the rapid assessment of KCC2 gene expression in genome-edited human reporter neurons. From an unbiased screen of more than 900 small-molecule chemicals, we have identified a group of compounds that enhance KCC2 expression termed KCC2 expression-enhancing compounds (KEECs). The identified KEECs include U.S. Food and Drug Administration-approved drugs that are inhibitors of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3beta (GSK3beta) pathways and activators of the sirtuin 1 (SIRT1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment with hit compounds increased KCC2 expression in human wild-type (WT) and isogenic MECP2 mutant RTT neurons, and rescued electrophysiological and morphological abnormalities of RTT neurons. Injection of KEEC KW-2449 or piperine in Mecp2 mutant mice ameliorated disease-associated respiratory and locomotion phenotypes. The small-molecule compounds described in our study may have therapeutic effects not only in RTT but also in other neurological disorders involving dysregulation of KCC2. Full Text

Tewari , A.G., Owen, J.H., Petersen, C.P., Wagner, D.E., and Reddien, P.W. (2019). A small set of conserved genes, including sp5 and Hox, are activated by Wnt signaling in the posterior of planarians and acoels. PLoS Genetics 15:e1008401. Wnt signaling regulates primary body axis formation across the Metazoa, with high Wnt signaling specifying posterior identity. Whether a common Wnt-driven transcriptional program accomplishes this broad role is poorly understood. We identified genes acutely affected after Wnt signaling inhibition in the posterior of two regenerative species, the planarian Schmidtea mediterranea and the acoel Hofstenia miamia, which are separated by >550 million years of evolution. Wnt signaling was found to maintain positional information in muscle and regional gene expression in multiple differentiated cell types. sp5, Hox genes, and Wnt pathway components are down-regulated rapidly after beta-catenin RNAi in both species. Brachyury, a vertebrate Wnt target, also displays Wnt-dependent expression in Hofstenia. sp5 inhibits trunk gene expression in the tail of planarians and acoels, promoting separate tail-trunk body domains. A planarian posterior Hox gene, Post-2d, promotes normal tail regeneration. We propose that common regulation of a small gene set-Hox, sp5, and Brachyury-might underlie the widespread utilization of Wnt signaling in primary axis patterning across the Bilateria. Full Text

Tian, T.V., Di Stefano, B., Stik, G., Vila-Casadesus, M., Sardina, J.L., Vidal, E., Dasti, A., Segura-Morales, C., De Andres-Aguayo, L., Gomez, A., . Goldmann, J., Jaenisch, R., and Graf, T,. (2019) Whsc1 links pluripotency exit with mesendoderm specification. Nature Cell Biology 21, 824-834. How pluripotent stem cells differentiate into the main germ layers is a key question of developmental biology. Here, we show that the chromatin-related factor Whsc1 (also known as Nsd2 and MMSET) has a dual role in pluripotency exit and germ layer specification of embryonic stem cells. On induction of differentiation, a proportion of Whsc1-depleted embryonic stem cells remain entrapped in a pluripotent state and fail to form mesendoderm, although they are still capable of generating neuroectoderm. These functions of Whsc1 are independent of its methyltransferase activity. Whsc1 binds to enhancers of the mesendodermal regulators Gata4, T (Brachyury), Gata6 and Foxa2, together with Brd4, and activates the expression of these genes. Depleting each of these regulators also delays pluripotency exit, suggesting that they mediate the effects observed with Whsc1. Our data indicate that Whsc1 links silencing of the pluripotency regulatory network with activation of mesendoderm lineages. Full Text

Tomasello , D.L. (2019). Addressing Isolation in the Scientific Community. Trends in Molecular Medicine [Epub ahead of print]. In STEM, and particularly in science, many early career researchers find themselves isolated and lacking guidance. There is an enormous need to connect early career scientists with experienced professionals outside their immediate work environment. A new initiative aims to create a supportive community to foster communication between scientists through all stages of their careers. Full Text

Torrens-Spence , M.P., Bobokalonova, A., Carballo, V., Glinkerman, C.M., Pluskal, T., Shen, A., and Weng, J.K. (2019). PBS3 and EPS1 complete salicylic acid biosynthesis from isochorismate in Arabidopsis. Molecular Plant[Epub ahead of print]. Salicylic acid (SA) is an important phytohormone mediating both local and systemic defense responses in plants. Despite over half a century of research, how plants biosynthesize SA remains unresolved. In Arabidopsis, a major part of SA is derived from isochorismate, a key intermediate produced by the isochorismate synthase (ICS), which is reminiscent of SA biosynthesis in bacteria. Whereas bacteria employ an isochorismate pyruvate lyase (IPL) that catalyzes the turnover of isochorismate to pyruvate and SA, plants do not contain an IPL ortholog and generate SA from isochorismate through an unknown mechanism. Combining genetic and biochemical approaches, we delineated the SA biosynthetic pathway downstream of isochorismate in Arabidopsis. We show that PBS3, a GH3 acyl adenylase-family enzyme important for SA accumulation, catalyzes ATP- and Mg(2+)-dependent conjugation of L-glutamate primarily to the 8-carboxyl of isochorismate and yields the key SA biosynthetic intermediate isochorismoyl-glutamate A. Moreover, EPS1, a BAHD acyltransferase-family protein with previously implicated role in SA accumulation upon pathogen attack, harbors a noncanonical active site and an unprecedented isochorismoyl-glutamate A pyruvoyl-glutamate lyase (IPGL) activity that produces SA from the isochorismoyl-glutamate A substrate. Together, PBS3 and EPS1 form a two-step metabolic pathway to produce SA from isochorismate in Arabidopsis, which is distinct from how SA is biosynthesized in bacteria. This study closes a major knowledge gap in plant SA metabolism and would help develop new strategies for engineering disease resistance in crop plants. Full Text

Torrens-Spence , M.P., Liu, C.T., and Weng, J.K. (2019). Engineering New Branches of the Kynurenine Pathway To Produce Oxo-(2-aminophenyl) and Quinoline Scaffolds in Yeast. ACS synthetic biology [Epub ahead of print]. The kynurenine pathway, named after its nonproteinogenic amino acid precursor l-kynurenine, is responsible for the de novo biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) in eukaryotes. Oxo-(2-aminophenyl) and quinoline molecules downstream from l-kynurenine also serve as antagonists of several receptors of the central nervous system in mammals. In this study, we engineered new biosynthetic routes in yeast Saccharomyces cerevisiae to produce a suite of l-kynurenine-derived natural products. Overexpression of Homo sapiens l-tryptophan 2,3-dioxygenase (HsTDO2) in S. cerevisiae led to a marked increase in the production of l-kynurenine and downstream metabolites. Using this background, new branch points to the kynurenine pathway were added through the incorporation of a Psilocybe cubensis noncanonical L-aromatic amino acid decarboxylase (PcncAAAD) capable of catalyzing both decarboxylation and decarboxylation-dependent oxidative-deamination reactions of l-kynurenine and 3-hydroxy-l-kynurenine to yield their corresponding monoamines, aldehydes, and downstream nonenzymatically cyclized quinolines. The PcncAAAD-catalyzed decarboxylation products, kynuramine and 3-hydroxykynuramine, could further be converted to quinoline scaffolds through the addition of H. sapiens monoamine oxidase A (HsMAO-A). Finally, by incorporating upstream regiospecific l-tryptophan halogenases into the engineering scheme, we produced a number of halogenated oxo-(2-aminophenyl) and quinoline compounds. This work illustrates a synthetic biology approach to expand primary metabolic pathways in the production of novel natural-product-like scaffolds amenable for downstream functionalization. Full Text

Tsvetkov, P., Detappe, A., Cai, K., Keys, H.R., Brune, Z., Ying, W., Thiru, P., Reidy, M., Kugener, G., Rossen, J., Kory N., Whitesell L., Lindquist S., et al. (2019). Mitochondrial metabolism promotes adaptation to proteotoxic stress. Nature chemical biology [Epub ahead of print]. The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but are key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome-inhibitor resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small molecule compound elesclomol. Genome-wide CRISPR-Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and nuclear-magnetic-resonance-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies explain a fundamental mechanism by which cells adapt to proteotoxic stress and suggest strategies to mitigate proteasome inhibitor resistance. Full Text

Tye, B.W., Commins, N., Ryazanova, L.V., Wuhr, M., Springer, M., Pincus, D., and Churchman, L.S. (2019). Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness. eLife 8: e43002 [Epub ahead of print]. To achieve maximal growth, cells must manage a massive economy of ribosomal proteins (r-proteins) and RNAs (rRNAs) to produce thousands of ribosomes every minute. Although ribosomes are essential in all cells, natural disruptions to ribosome biogenesis lead to heterogeneous phenotypes. Here, we model these perturbations in Saccharomyces cerevisiae and show that challenges to ribosome biogenesis result in acute loss of proteostasis. Imbalances in the synthesis of r-proteins and rRNAs lead to the rapid aggregation of newly synthesized orphan r-proteins and compromise essential cellular processes, which cells alleviate by activating proteostasis genes. Exogenously bolstering the proteostasis network increases cellular fitness in the face of challenges to ribosome assembly, demonstrating the direct contribution of orphan r-proteins to cellular phenotypes. We propose that ribosome assembly is a key vulnerability of proteostasis maintenance in proliferating cells that may be compromised by diverse genetic, environmental, and xenobiotic perturbations that generate orphan r-proteins. Full Text

Valantine, H., Travis, E., El-Adhami, W., Vernos, I., Mosqueda, L., Wayne, E., Kearns-Zimmerman, F., Bonefont, L., Visweswariah, S.S., Akande-Sholabi, W., & Jessics Polka .(2019). A giant leap for womankind. Nature Medicine 25(5):704-707. We asked 11 thought leaders on how they would advise the research community to make real progress in the next 25 years to address gender inequality in medical research. They offer concrete ideas for change. Full Text

Vyas V.K., and Bernstein, D.A. (2019). An Introduction to CRISPR-Mediated Genome Editing in Fungi.Journal of Microbiology & Biology Education 20(3) . Central dogma, transformation, and genome editing are key biological concepts for which junior scientists must gain experience during training. Here we present an exercise that introduces these concepts in a single practical laboratory exercise. Our exercise utilizes CRISPR/Cas9 genome editing to introduce a stop codon into Saccharomyces cerevisiae ADE2. This edit leads to the buildup of an adenine precursor that dyes the edited cells red. As the repair template, guide RNA, and Cas9 are all encoded in our vector, transformation can be performed in 2 hours. Furthermore, since all components of the Cas9/CRISPR system are encoded by the vector, specialized equipment and reagents, such as a PCR machine or oligonucleotides, are not required to perform the experiments as designed. As such, these exercises provide an efficient cost-effective introduction to a wide variety of key molecular biology concepts and lay the foundation for more rigorous investigations in upper-level classes and independent research projects. Full Text

Wang, H., Xiang, D., Liu, B., He, A., Randle, H.J., Zhang, K.X., Dongre, A., Sachs, N., Clark, A.P., Tao, L., et al. (2019). Inadequate DNA Damage Repair Promotes Mammary Transdifferentiation, Leading to BRCA1 Breast Cancer. Cell 178, 135-151. Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to a mesenchymal state of cultured, human basal-like MECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB, or HES1 or chemically induced ICL damage in primary murine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. In vivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation. Full Text

Wang, L., Tan, T.K., Durbin, A.D., Zimmerman, M.W., Abraham, B.J., Tan, S.H., Ngoc, P.C.T., Weichert-Leahey, N., Akahane, K., Lawton, L.N., Rokita JL, Maris JM, Young RA, Look AT, and Sanda T. (2019). ASCL1 is a MYCN- and LMO1-dependent member of the adrenergic neuroblastoma core regulatory circuitry.Nature Communications 10(1):5622. A heritable polymorphism within regulatory sequences of the LMO1 gene is associated with its elevated expression and increased susceptibility to develop neuroblastoma, but the oncogenic pathways downstream of the LMO1 transcriptional co-regulatory protein are unknown. Our ChIP-seq and RNA-seq analyses reveal that a key gene directly regulated by LMO1 and MYCN is ASCL1, which encodes a basic helix-loop-helix transcription factor. Regulatory elements controlling ASCL1 expression are bound by LMO1, MYCN and the transcription factors GATA3, HAND2, PHOX2B, TBX2 and ISL1-all members of the adrenergic (ADRN) neuroblastoma core regulatory circuitry (CRC). ASCL1 is required for neuroblastoma cell growth and arrest of differentiation. ASCL1 and LMO1 directly regulate the expression of CRC genes, indicating that ASCL1 is a member and LMO1 is a coregulator of the ADRN neuroblastoma CRC. Full Text

Weinberg , R.A. (2019). How TP53 (almost) became an oncogene.Journal of Molecular Cell Biology [Epub ahead of print] .Like the histories of many other genes, the discovery and characterization of p53 are associated with some false leads, dead ends, and major detours. The chronicle of p53's early years is no exception. As is now well known, the protein was actually discovered independently by three groups –– those of Arnie Levine, David Lane, and Lloyd Old. Full Text

Wesselhoeft, R.A., Kowalski, P.S., Parker-Hale, F.C., Huang, Y., Bisaria, N., and Anderson, D.G. (2019). RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo. Molecular cell [Epub ahead of print]. Circular RNAs (circRNAs) are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Here, we show that unmodified exogenous circRNA is able to bypass cellular RNA sensors and thereby avoid provoking an immune response in RIG-I and Toll-like receptor (TLR) competent cells and in mice. The immunogenicity and protein expression stability of circRNA preparations are found to be dependent on purity, with small amounts of contaminating linear RNA leading to robust cellular immune responses. Unmodified circRNA is less immunogenic than unmodified linear mRNA in vitro, in part due to the evasion of TLR sensing. Finally, we provide the first demonstration to our knowledge of exogenous circRNA delivery and translation in vivo, and we show that circRNA translation is extended in adipose tissue in comparison to unmodified and uridine-modified linear mRNAs.Full Text

Whitesell , L., Robbins, N., Huang, D.S., McLellan, C.A., Shekhar-Guturja, T., LeBlanc, E.V., Nation, C.S., Hui, R., Hutchinson, A., Collins, C., Lindquist, S. et al. (2019). Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus. Nature communications 10, 402. New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy. Full Text

Yang, Y.S.S., Moynihan, K.D., Bekdemir, A., Dichwalkar, T.M., Noh, M.M., Watson, N., Melo, M., Ingram, J., Suh, H., Ploegh, H., et al. (2019). Targeting small molecule drugs to T cells with antibody-directed cell-penetrating gold nanoparticles. Biomaterials Science 7, 113-124. 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- inhibitor in T cells in cell culture. By conjugating amph-NPs with targeting antibodies or camelid-derived nanobodies, the particles' 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- 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

Zamudio , A.V., Dall'Agnese, A., Henninger, J.E., Manteiga, J.C., Afeyan, L.K., Hannett, N.M., Coffey, E.L., Li, C.H., Oksuz, O., Sabari, B.R., Boija A, Klein IA, Abraham BJ, Lee TI Schuijers J, Young RA, et al (2019). Mediator Condensates Localize Signaling Factors to Key Cell Identity Genes. Molecular Cell [Epub ahead of print]. The gene expression programs that define the identity of each cell are controlled by master transcription factors (TFs) that bind cell-type-specific enhancers, as well as signaling factors, which bring extracellular stimuli to these enhancers. Recent studies have revealed that master TFs form phase-separated condensates with the Mediator coactivator at super-enhancers. Here, we present evidence that signaling factors for the WNT, TGF-beta, and JAK/STAT pathways use their intrinsically disordered regions (IDRs) to enter and concentrate in Mediator condensates at super-enhancers. We show that the WNT coactivator beta-catenin interacts both with components of condensates and DNA-binding factors to selectively occupy super-enhancer-associated genes. We propose that the cell-type specificity of the response to signaling is mediated in part by the IDRs of the signaling factors, which cause these factors to partition into condensates established by the master TFs and Mediator at genes with prominent roles in cell identity. Full Text

Zhao, Q., Yang, J., Cui, M.Y., Liu, J., Fang, Y., Yan, M., Qiu, W., Shang, H., Xu, Z., Yidiresi, R., Pluskal T., Weng, J.K., et al. (2019). The Reference Genome Sequence of Scutellaria baicalensis Provides Insights into the Evolution of Wogonin Biosynthesis.Molecular plant [Epub ahead of print] .Scutellaria baicalensis Georgi is important in Chinese Traditional Medicine where preparations of dried roots, 'Huang Qin', are used for liver and lung complaints including complementary cancer treatments. We report a high-quality reference genome sequence for S. baicalensis where 93% of the 408.14 Mb genome has been assembled into 9 pseudochromosomes with a super-N50 of 33.2 Mb. Comparison of this sequence to those of closely related species in the order Lamiales, Sesamum indicum and Salvia splendens, revealed how the specialised metabolic pathway for the synthesis of 4'deoxyflavone bioactives evolved in the genus, Scutellaria. We found that the gene encoding a specific cinnamate CoA ligase likely obtained its new function following recent mutations, and four genes encoding enzymes in the 4'deoxyflavone pathway are present as tandem repeats in the genome of S. baicalensis. Further analyses revealed that gene duplications, segmental duplication, gene amplification and point mutations coupled to gene neo- and sub-functionalizations were involved in the evolution of 4'deoxyflavone synthesis in the genus, Scutellaria. The reference genome of S. baicalensis will facilitate the development of improved assemblies of genome sequences for other members of the mint family and offers an important foundation for decoding the synthetic pathways of bioactive compounds in medicinal plants. Our study not only provides significant insight into the evolution of specific flavone biosynthetic pathways in members of the mint family, Lamiaceae, but also would facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding in plants. Full Text

Zheng , X.Y., and Gehring, M. (2019). Low-input chromatin profiling in Arabidopsis endosperm using CUT&RUN. Plant reproduction [Epub ahead of print]. Application of a low-input chromatin profiling method, CUT&RUN, to FACS-purified Arabidopsis endosperm nuclei generates parental-specific genome-wide H3K27me3 landscapes with high sensitivity, specificity and reproducibility. Endosperm is an essential seed tissue with a unique epigenetic landscape. During endosperm development, differential epigenetic regulation of the maternal and paternal genomes plays important roles in regulating gene expression, especially at imprinted genes. In Arabidopsis, profiling the epigenetic landscape of endosperm on a genome-wide scale is challenging due to its small size, mode of development and close association with maternal tissue. Here, we applied a low-input chromatin profiling method, CUT&RUN (cleavage under targets and release using nuclease), to profile parental-specific chromatin modifications using limited numbers of Arabidopsis endosperm nuclei. We demonstrate that CUT&RUN generates genome-wide H3K27me3 landscapes with high sensitivity, specificity and reproducibility using around 20,000 endosperm nuclei purified by flow cytometry and fluorescence-activated cell sorting. H3K27me3 peaks identified by CUT&RUN and previous ChIP (chromatin immunoprecipitation) approaches were largely overlapping, with some distinctions in heterochromatin. The versatility and simplicity of CUT&RUN make it a viable alternative to ChIP, which requires greater amounts of starting material, and will enable further study of tissue- or cell-type-specific epigenomes in Arabidopsis and other plant species. Full Text

Zoppo, M., Di Luca, M., Franco, M., Rizzato, C., Lupetti, A., Stringaro, A., De Bernardis, F., Schaudinn, C., Barrasa, M.I., Vyas V.K., Bottai D., et al. (2019). CpALS4770 and CpALS4780 contribution to the virulence of Candida parapsilosis. Microbiological Research 231, 126351 [Epub ahead of print].The ability of yeast to adhere to biotic and abiotic surfaces represents an essential trait during the early stages of infection. Agglutinin-like sequence (Als) cell-wall proteins play a key role in adhesion of Candida species. Candida parapsilosis genome encompasses 5 ALS members, of which only the role of CPAR2_404800 has been elucidated. The present project was aimed at investigating the contribution of C. parapsilosis Als proteins by generating edited strains lacking functional Als proteins. CPAR2_404770 and CPAR2_404780, further indicated as CpALS4770 and CpALS4780, were selected for the generation of single and double edited strains using an episomal CRISPR/Cas9 technology. Phenotypic characterization of mutant strains revealed that editing of both genes had no impact on the in vitro growth of C. parapsilosis or on morphogenesis. Notably, CpALS4770-edited strain showed a reduction of biofilm formation and adhesive properties to human buccal cells (HBECs). Conversely, single CpALS4780-edited strain did not show any difference compared to the wild-type strain in all the assays performed, while the double CpALS4770-CpALS4780 mutant revealed an increased ability to produce biofilm, a hyper-adhesive phenotype to HBECs, and a marked tendency to form cellular aggregates. Murine vaginal infection experiments indicated a significant reduction in CFUs recovered from BALC/c mice infected with single and double edited strains, compared to those infected with the wild-type strain. These finding clearly indicate that CpAls4770 plays a role in adhesion to biotic and abiotic surfaces, while both CpALS4770 and CpALS4780 genes are required for C. parapsilosis ability to colonize and persist in the vaginal mucosa.

Zoppo, M., Luca, M.D., Villarreal, S.N., Poma, N., Barrasa, M.I., Vyas, V.K., Bottai, D., and Tavanti, A. (2019). A CRISPR/Cas9-based strategy to simultaneously inactivate the entire ALS gene family in Candida orthopsilosis. Future microbiology. [Epub ahead of print]. Aim: In this study, the CRISPR gene-editing approach was used to simultaneously inactivate all three members of the ALS gene family in the opportunistic pathogen Candida orthopsilosis. Materials & methods: Using a single gRNA and repair template, CRISPR-edited clones were successfully generated in a one-step process in both C. orthopsilosis reference and clinical strains. Results: The phenotypic characterization of the ALS triple-edited strains revealed no impact on growth in liquid or solid media. However, pseudohyphal formation and the ability to adhere to human buccal epithelial cells were significantly decreased in triple-edited clones. Conclusion: Our CRISPR/Cas9 system is a powerful tool for simultaneous editing of fungal gene families, which greatly accelerates the generation of multiple gene-edited Candida strains. Data deposition: Nucleotide sequence data are available in the GenBank databases under the accession numbers MK875971, MK875972, MK875973, MK875974, MK875975, MK875976, MK875977.