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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 Jul 17;:

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 - as supplied by publisher]



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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 Jul 11;:

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 - as supplied by publisher]



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

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 - in process]



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Quantitative analysis of protein interaction network dynamics in yeast.

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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Syp1 regulates the clathrin-mediated and clathrin-independent endocytosis of multiple cargo proteins through a novel sorting motif.

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Syp1 regulates the clathrin-mediated and clathrin-independent endocytosis of multiple cargo proteins through a novel sorting motif.

Mol Biol Cell. 2017 Jul 12;:

Authors: Apel AR, Hoban K, Chuartzman S, Tonikian R, Sidhu S, Schuldiner M, Wendland B, Prosser D

Abstract
Internalization of proteins from the plasma membrane (PM) allows for cell-surface composition regulation, signaling network modulation, and nutrient uptake. Clathrin-mediated endocytosis (CME) is a major internalization route for PM proteins. During CME, endocytic adaptor proteins bind cargos at the cell surface and link them to the PM and clathrin coat. Muniscins are a conserved family of endocytic adaptors, including Syp1 in budding yeast and its mammalian ortholog, FCHo1. These adaptors bind cargo via a C-terminal ì-homology domain (ìHD); however, few cargos exhibiting muniscin-dependent endocytosis have been identified, and the sorting sequence recognized by the μHD is unknown. To reveal Syp1 cargo-sorting motifs, we performed a phage display screen and used biochemical methods to demonstrate that the Syp1 μHD binds DxY motifs in the previously identified Syp1 cargo, Mid2, and the v-SNARE, Snc1. We also executed an unbiased visual screen, which identified the peptide transporter, Ptr2, and the ammonium permease, Mep3, as Syp1 cargos containing DxY motifs. Finally, we determined that, in addition to regulating cargo entry through CME, Syp1 can promote internalization of Ptr2 through a recently identified clathrin-independent endocytic pathway that requires the Rho1 GTPase. These findings elucidate the mechanism of Syp1 cargo recognition and its role in trafficking.

PMID: 28701344 [PubMed - as supplied by publisher]



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Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane.

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Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane.

J Biol Chem. 2017 Jul 11;:

Authors: Hirama T, Das R, Yang Y, Ferguson C, Won A, Yip CM, Kay JG, Grinstein S, Parton RG, Fairn GD

Abstract
Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (CAVs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled CAV1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveolae formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable CAV1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of CAV and cavin1 diminished, and cavin1 degradation increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns4,5P2 has minimal impact on caveola assembly, but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper CAV clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability.

PMID: 28698382 [PubMed - as supplied by publisher]



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Oncogenic Activation of the RNA Binding Protein NELFE and MYC Signaling in Hepatocellular Carcinoma.

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Oncogenic Activation of the RNA Binding Protein NELFE and MYC Signaling in Hepatocellular Carcinoma.

Cancer Cell. 2017 Jul 10;32(1):101-114.e8

Authors: Dang H, Takai A, Forgues M, Pomyen Y, Mou H, Xue W, Ray D, Ha KCH, Morris QD, Hughes TR, Wang XW

Abstract
Global transcriptomic imbalance is a ubiquitous feature associated with cancer, including hepatocellular carcinoma (HCC). Analyses of 1,225 clinical HCC samples revealed that a large numbers of RNA binding proteins (RBPs) are dysregulated and that RBP dysregulation is associated with poor prognosis. We further identified that oncogenic activation of a top candidate RBP, negative elongation factor E (NELFE), via somatic copy-number alterations enhanced MYC signaling and promoted HCC progression. Interestingly, NELFE induces a unique tumor transcriptome by selectively regulating MYC-associated genes. Thus, our results revealed NELFE as an oncogenic protein that may contribute to transcriptome imbalance in HCC through the regulation of MYC signaling.

PMID: 28697339 [PubMed - in process]



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Tracing the origins of relapse in acute myeloid leukaemia to stem cells.

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Tracing the origins of relapse in acute myeloid leukaemia to stem cells.

Nature. 2017 Jun 28;:

Authors: Shlush LI, Mitchell A, Heisler L, Abelson S, Ng SWK, Trotman-Grant A, Medeiros JJF, Rao-Bhatia A, Jaciw-Zurakowsky I, Marke R, McLeod JL, Doedens M, Bader G, Voisin V, Xu C, McPherson JD, Hudson TJ, Wang JCY, Minden MD, Dick JE

Abstract
In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving remission. Historically, the failure of therapy has been thought to be due to mutations that produce drug resistance, possibly arising as a consequence of the mutagenic properties of chemotherapy drugs. However, other lines of evidence have pointed to the pre-existence of drug-resistant cells. For example, deep sequencing of paired diagnosis and relapse acute myeloid leukaemia samples has provided direct evidence that relapse in some cases is generated from minor genetic subclones present at diagnosis that survive chemotherapy, suggesting that resistant cells are generated by evolutionary processes before treatment and are selected by therapy. Nevertheless, the mechanisms of therapy failure and capacity for leukaemic regeneration remain obscure, as sequence analysis alone does not provide insight into the cell types that are fated to drive relapse. Although leukaemia stem cells have been linked to relapse owing to their dormancy and self-renewal properties, and leukaemia stem cell gene expression signatures are highly predictive of therapy failure, experimental studies have been primarily correlative and a role for leukaemia stem cells in acute myeloid leukaemia relapse has not been directly proved. Here, through combined genetic and functional analysis of purified subpopulations and xenografts from paired diagnosis/relapse samples, we identify therapy-resistant cells already present at diagnosis and two major patterns of relapse. In some cases, relapse originated from rare leukaemia stem cells with a haematopoietic stem/progenitor cell phenotype, while in other instances relapse developed from larger subclones of immunophenotypically committed leukaemia cells that retained strong stemness transcriptional signatures. The identification of distinct patterns of relapse should lead to improved methods for disease management and monitoring in acute myeloid leukaemia. Moreover, the shared functional and transcriptional stemness properties that underlie both cellular origins of relapse emphasize the importance of developing new therapeutic approaches that target stemness to prevent relapse.

PMID: 28658204 [PubMed - as supplied by publisher]



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