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Ubiquitin orchestrates proteasome dynamics between proliferation and quiescence in yeast.

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Ubiquitin orchestrates proteasome dynamics between proliferation and quiescence in yeast.

Mol Biol Cell. 2017 Sep 15;28(19):2479-2491

Authors: Gu ZC, Wu E, Sailer C, Jando J, Styles E, Eisenkolb I, Kuschel M, Bitschar K, Wang X, Huang L, Vissa A, Yip CM, Yedidi RS, Friesen H, Enenkel C

Abstract
Proteasomes are essential for protein degradation in proliferating cells. Little is known about proteasome functions in quiescent cells. In nondividing yeast, a eukaryotic model of quiescence, proteasomes are depleted from the nucleus and accumulate in motile cytosolic granules termed proteasome storage granules (PSGs). PSGs enhance resistance to genotoxic stress and confer fitness during aging. Upon exit from quiescence PSGs dissolve, and proteasomes are rapidly delivered into the nucleus. To identify key players in PSG organization, we performed high-throughput imaging of green fluorescent protein (GFP)-labeled proteasomes in the yeast null-mutant collection. Mutants with reduced levels of ubiquitin are impaired in PSG formation. Colocalization studies of PSGs with proteins of the yeast GFP collection, mass spectrometry, and direct stochastic optical reconstitution microscopy of cross-linked PSGs revealed that PSGs are densely packed with proteasomes and contain ubiquitin but no polyubiquitin chains. Our results provide insight into proteasome dynamics between proliferating and quiescent yeast in response to cellular requirements for ubiquitin-dependent degradation.

PMID: 28768827 [PubMed - indexed for MEDLINE]



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RNAi screen identifies essential regulators of human brain metastasis-initiating cells.

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RNAi screen identifies essential regulators of human brain metastasis-initiating cells.

Acta Neuropathol. 2017 Dec;134(6):923-940

Authors: Singh M, Venugopal C, Tokar T, Brown KR, McFarlane N, Bakhshinyan D, Vijayakumar T, Manoranjan B, Mahendram S, Vora P, Qazi M, Dhillon M, Tong A, Durrer K, Murty N, Hallet R, Hassell JA, Kaplan DR, Cutz JC, Jurisica I, Moffat J, Singh SK

Abstract
Brain metastases (BM) are the most common brain tumor in adults and are a leading cause of cancer mortality. Metastatic lesions contain subclones derived from their primary lesion, yet their functional characterization is limited by a paucity of preclinical models accurately recapitulating the metastatic cascade, emphasizing the need for a novel approach to BM and their treatment. We identified a unique subset of stem-like cells from primary human patient brain metastases, termed brain metastasis-initiating cells (BMICs). We now establish a BMIC patient-derived xenotransplantation (PDXT) model as an investigative tool to comprehensively interrogate human BM. Using both in vitro and in vivo RNA interference screens of these BMIC models, we identified SPOCK1 and TWIST2 as essential BMIC regulators. SPOCK1 in particular is a novel regulator of BMIC self-renewal, modulating tumor initiation and metastasis from the lung to the brain. A prospective cohort of primary lung cancer specimens showed that SPOCK1 was overexpressed only in patients who ultimately developed BM. Protein-protein interaction network mapping between SPOCK1 and TWIST2 identified novel pathway interactors with significant prognostic value in lung cancer patients. Of these genes, INHBA, a TGF-β ligand found mutated in lung adenocarcinoma, showed reduced expression in BMICs with knockdown of SPOCK1. In conclusion, we have developed a useful preclinical model of BM, which has served to identify novel putative BMIC regulators, presenting potential therapeutic targets that block the metastatic process, and transform a uniformly fatal systemic disease into a locally controlled and eminently more treatable one.

PMID: 28766011 [PubMed - in process]



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Functional annotation of chemical libraries across diverse biological processes.

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Functional annotation of chemical libraries across diverse biological processes.

Nat Chem Biol. 2017 Sep;13(9):982-993

Authors: Piotrowski JS, Li SC, Deshpande R, Simpkins SW, Nelson J, Yashiroda Y, Barber JM, Safizadeh H, Wilson E, Okada H, Gebre AA, Kubo K, Torres NP, LeBlanc MA, Andrusiak K, Okamoto R, Yoshimura M, DeRango-Adem E, van Leeuwen J, Shirahige K, Baryshnikova A, Brown GW, Hirano H, Costanzo M, Andrews B, Ohya Y, Osada H, Yoshida M, Myers CL, Boone C

Abstract
Chemical-genetic approaches offer the potential for unbiased functional annotation of chemical libraries. Mutations can alter the response of cells in the presence of a compound, revealing chemical-genetic interactions that can elucidate a compound's mode of action. We developed a highly parallel, unbiased yeast chemical-genetic screening system involving three key components. First, in a drug-sensitive genetic background, we constructed an optimized diagnostic mutant collection that is predictive for all major yeast biological processes. Second, we implemented a multiplexed (768-plex) barcode-sequencing protocol, enabling the assembly of thousands of chemical-genetic profiles. Finally, based on comparison of the chemical-genetic profiles with a compendium of genome-wide genetic interaction profiles, we predicted compound functionality. Applying this high-throughput approach, we screened seven different compound libraries and annotated their functional diversity. We further validated biological process predictions, prioritized a diverse set of compounds, and identified compounds that appear to have dual modes of action.

PMID: 28759014 [PubMed - indexed for MEDLINE]



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Quiescent Oct4(+) Neural Stem Cells (NSCs) Repopulate Ablated Glial Fibrillary Acidic Protein(+) NSCs in the Adult Mouse Brain.

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Quiescent Oct4(+) Neural Stem Cells (NSCs) Repopulate Ablated Glial Fibrillary Acidic Protein(+) NSCs in the Adult Mouse Brain.

Stem Cells. 2017 Sep;35(9):2071-2082

Authors: Reeve RL, Yammine SZ, Morshead CM, van der Kooy D

Abstract
Adult primitive neural stem cells (pNSCs) are a rare population of glial fibrillary acidic protein (GFAP)(-) Oct4(+) cells in the mouse forebrain subependymal zone bordering the lateral ventricles that give rise to clonal neurospheres in leukemia inhibitory factor in vitro. pNSC neurospheres can be passaged to self-renew or give rise to GFAP(+) NSCs that form neurospheres in epidermal growth factor and fibroblast growth factor 2, which we collectively refer to as definitive NSCs (dNSCs). Label retention experiments using doxycycline-inducible histone-2B (H2B)-green fluorescent protein (GFP) mice and several chase periods of up to 1 year quantified the adult pNSC cell cycle time as 3-5 months. We hypothesized that while pNSCs are not very proliferative at baseline, they may exist as a reserve pool of NSCs in case of injury. To test this function of pNSCs, we obtained conditional Oct4 knockout mice, Oct4(fl/fl) ;Sox1(Cre) (Oct4(CKO) ), which do not yield adult pNSC-derived neurospheres. When we ablated the progeny of pNSCs, namely all GFAP(+) dNSCs, in these Oct4(CKO) mice, we found that dNSCs did not recover as they do in wild-type mice, suggesting that pNSCs are necessary for dNSC repopulation. Returning to the H2B-GFP mice, we observed that the cytosine β-d-arabinofuranoside ablation of proliferating cells including dNSCs-induced quiescent pNSCs to proliferate and significantly dilute their H2B-GFP label. In conclusion, we demonstrate that pNSCs are the most quiescent stem cells in the adult brain reported to date and that their lineage position upstream of GFAP(+) dNSCs allows them to repopulate a depleted neural lineage. Stem Cells 2017;35:2071-2082.

PMID: 28733998 [PubMed - in process]



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Steric Hindrance Assay for Secreted Factors in Stem Cell Culture.

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Steric Hindrance Assay for Secreted Factors in Stem Cell Culture.

ACS Sens. 2017 Apr 28;2(4):495-500

Authors: Zhou W, Mahshid SS, Wang W, Vallée-Bélisle A, Zandstra PW, Sargent EH, Kelley SO

Abstract
The ex vivo expansion of hematopoietic stem cells is significantly inhibited by secreted proteins that induce negative feedback loops. The ability to effectively monitor these factors is critical for their real-time regulation and control and, by extension, enhancing stem cell expansion. Here, we describe a novel monitoring strategy for the detection of soluble signaling factors in stem cell cultures using a DNA-based sensing mechanism on a chip-based nanostructured microelectrode platform. We combine DNA hybridization engineering with antibody-capturing chemistry in an amplified steric hindrance hybridization assay. This method enables the quantification of important secreted proteins, showcased by the detection of 10 pg·mL(-1) level concentrations of three proteins in stem cell culture samples. This approach is the first universal nonsandwich technique that permits pg·mL(-1) level quantification of small proteins in stem cell culture media without signal loss.

PMID: 28723184 [PubMed]



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C. elegans SUP-46, an HNRNPM family RNA-binding protein that prevents paternally-mediated epigenetic sterility.

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C. elegans SUP-46, an HNRNPM family RNA-binding protein that prevents paternally-mediated epigenetic sterility.

BMC Biol. 2017 Jul 17;15(1):61

Authors: Johnston WL, Krizus A, Ramani AK, Dunham W, Youn JY, Fraser AG, Gingras AC, Dennis JW

Abstract
BACKGROUND: In addition to DNA, gametes contribute epigenetic information in the form of histones and non-coding RNA. Epigenetic programs often respond to stressful environmental conditions and provide a heritable history of ancestral stress that allows for adaptation and propagation of the species. In the nematode C. elegans, defective epigenetic transmission often manifests as progressive germline mortality. We previously isolated sup-46 in a screen for suppressors of the hexosamine pathway gene mutant, gna-2(qa705). In this study, we examine the role of SUP-46 in stress resistance and progressive germline mortality.
RESULTS: We identified SUP-46 as an HNRNPM family RNA-binding protein, and uncovered a highly novel role for SUP-46 in preventing paternally-mediated progressive germline mortality following mating. Proximity biotinylation profiling of human homologs (HNRNPM, MYEF2) identified proteins of ribonucleoprotein complexes previously shown to contain non-coding RNA. Like HNRNPM and MYEF2, SUP-46 was associated with multiple RNA granules, including stress granules, and also formed granules on active chromatin. SUP-46 depletion disrupted germ RNA granules and caused ectopic sperm, increased sperm transcripts, and chronic heat stress sensitivity. SUP-46 was also required for resistance to acute heat stress, and a conserved "MYEF2" motif was identified that was needed for stress resistance.
CONCLUSIONS: In mammals, non-coding RNA from the sperm of stressed males has been shown to recapitulate paternal stress phenotypes in the offspring. Our results suggest that HNRNPM family proteins enable stress resistance and paternally-mediated epigenetic transmission that may be conserved across species.

PMID: 28716093 [PubMed - in process]



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Correction of a splicing defect in a mouse model of congenital muscular dystrophy type 1A using a homology-directed-repair-independent mechanism.

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Correction of a splicing defect in a mouse model of congenital muscular dystrophy type 1A using a homology-directed-repair-independent mechanism.

Nat Med. 2017 Aug;23(8):984-989

Authors: Kemaladewi DU, Maino E, Hyatt E, Hou H, Ding M, Place KM, Zhu X, Bassi P, Baghestani Z, Deshwar AG, Merico D, Xiong HY, Frey BJ, Wilson MD, Ivakine EA, Cohn RD

Abstract
Splice-site defects account for about 10% of pathogenic mutations that cause Mendelian diseases. Prevalence is higher in neuromuscular disorders (NMDs), owing to the unusually large size and multi-exonic nature of genes encoding muscle structural proteins. Therapeutic genome editing to correct disease-causing splice-site mutations has been accomplished only through the homology-directed repair pathway, which is extremely inefficient in postmitotic tissues such as skeletal muscle. Here we describe a strategy using nonhomologous end-joining (NHEJ) to correct a pathogenic splice-site mutation. As a proof of principle, we focus on congenital muscular dystrophy type 1A (MDC1A), which is characterized by severe muscle wasting and paralysis. Specifically, we correct a splice-site mutation that causes the exclusion of exon 2 from Lama2 mRNA and the truncation of Lama2 protein in the dy(2J)/dy(2J) mouse model of MDC1A. Through systemic delivery of adeno-associated virus (AAV) carrying clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing components, we simultaneously excise an intronic region containing the mutation and create a functional donor splice site through NHEJ. This strategy leads to the inclusion of exon 2 in the Lama2 transcript and restoration of full-length Lama2 protein. Treated dy(2J)/dy(2J) mice display substantial improvement in muscle histopathology and function without signs of paralysis.

PMID: 28714989 [PubMed - indexed for MEDLINE]



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ASCL1 Reorganizes Chromatin to Direct Neuronal Fate and Suppress Tumorigenicity of Glioblastoma Stem Cells.

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ASCL1 Reorganizes Chromatin to Direct Neuronal Fate and Suppress Tumorigenicity of Glioblastoma Stem Cells.

Cell Stem Cell. 2017 Aug 03;21(2):209-224.e7

Authors: Park NI, Guilhamon P, Desai K, McAdam RF, Langille E, O'Connor M, Lan X, Whetstone H, Coutinho FJ, Vanner RJ, Ling E, Prinos P, Lee L, Selvadurai H, Atwal G, Kushida M, Clarke ID, Voisin V, Cusimano MD, Bernstein M, Das S, Bader G, Arrowsmith CH, Angers S, Huang X, Lupien M, Dirks PB

Abstract
Glioblastomas exhibit a hierarchical cellular organization, suggesting that they are driven by neoplastic stem cells that retain partial yet abnormal differentiation potential. Here, we show that a large subset of patient-derived glioblastoma stem cells (GSCs) express high levels of Achaete-scute homolog 1 (ASCL1), a proneural transcription factor involved in normal neurogenesis. ASCL1(hi) GSCs exhibit a latent capacity for terminal neuronal differentiation in response to inhibition of Notch signaling, whereas ASCL1(lo) GSCs do not. Increasing ASCL1 levels in ASCL1(lo) GSCs restores neuronal lineage potential, promotes terminal differentiation, and attenuates tumorigenicity. ASCL1 mediates these effects by functioning as a pioneer factor at closed chromatin, opening new sites to activate a neurogenic gene expression program. Directing GSCs toward terminal differentiation may provide therapeutic applications for a subset of GBM patients and strongly supports efforts to restore differentiation potential in GBM and other cancers.

PMID: 28712938 [PubMed - in process]



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Regulatory Expansion in Mammals of Multivalent hnRNP Assemblies that Globally Control Alternative Splicing.

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Regulatory Expansion in Mammals of Multivalent hnRNP Assemblies that Globally Control Alternative Splicing.

Cell. 2017 Jul 13;170(2):324-339.e23

Authors: Gueroussov S, Weatheritt RJ, O'Hanlon D, Lin ZY, Narula A, Gingras AC, Blencowe BJ

Abstract
Alternative splicing (AS) patterns have diverged rapidly during vertebrate evolution, yet the functions of most species- and lineage-specific splicing events are not known. We observe that mammalian-specific AS events are enriched in transcript sequences encoding intrinsically disordered regions (IDRs) of proteins, in particular those containing glycine/tyrosine repeats that mediate formation of higher-order protein assemblies implicated in gene regulation and human disease. These evolutionary changes impact nearly all members of the hnRNP A and D families of RNA binding proteins. Regulation of these events requires formation of unusual, long-range mammalian-specific RNA duplexes. Differential inclusion of the alternative exons controls the formation of tyrosine-dependent multivalent hnRNP assemblies that, in turn, function to globally regulate splicing. Together, our results demonstrate that AS control of IDR-mediated interactions between hnRNPs represents an important and recurring mechanism underlying splicing regulation. Furthermore, this mechanism has expanded the regulatory capacity of mammalian cells.

PMID: 28709000 [PubMed - indexed for MEDLINE]



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Quantitative analysis of protein interaction network dynamics in yeast.

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Quantitative analysis of protein interaction network dynamics in yeast.

Mol Syst Biol. 2017 Jul 13;13(7):934

Authors: Celaj A, Schlecht U, Smith JD, Xu W, Suresh S, Miranda M, Aparicio AM, Proctor M, Davis RW, Roth FP, St Onge RP

Abstract
Many cellular functions are mediated by protein-protein interaction networks, which are environment dependent. However, systematic measurement of interactions in diverse environments is required to better understand the relative importance of different mechanisms underlying network dynamics. To investigate environment-dependent protein complex dynamics, we used a DNA-barcode-based multiplexed protein interaction assay in Saccharomyces cerevisiae to measure in vivo abundance of 1,379 binary protein complexes under 14 environments. Many binary complexes (55%) were environment dependent, especially those involving transmembrane transporters. We observed many concerted changes around highly connected proteins, and overall network dynamics suggested that "concerted" protein-centered changes are prevalent. Under a diauxic shift in carbon source from glucose to ethanol, a mass-action-based model using relative mRNA levels explained an estimated 47% of the observed variance in binary complex abundance and predicted the direction of concerted binary complex changes with 88% accuracy. Thus, we provide a resource of yeast protein interaction measurements across diverse environments and illustrate the value of this resource in revealing mechanisms of network dynamics.

PMID: 28705884 [PubMed - in process]



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