68 results on '"Hublitz P"'
Search Results
2. 1515 Gene edited iPSC-derived macrophages (iMACs) show increased phagocytosis in pre-clinical tumor models
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Daniel Sommermeyer, Monika Braun, Michael Epstein, Tanja Schneider, Kathrin Haake, Michela Mirenda, Quentin Bernard, Philip Hublitz, Lucie Gouxette, Martin Briscadieu, Philine Scheinpflug, Garima Singh, Alica Hinkelmann, Michael Esquerré, Audrey Holtzinger, Michael Paillasse, Andreas Scheel, Markus Dangl, and Nadja Wagner
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Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Published
- 2023
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3. Generation and characterization of an Advillin-Cre driver mouse line
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Moreira Pedro, Al Banchaabouchi Mumna, Hublitz Philip, Martínez Conception, Piszczek Agnieszka, Zurborg Sandra, Perlas Emerald, and Heppenstall Paul A
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Pathology ,RB1-214 - Abstract
Abstract Progress in the somatosensory field has been restricted by the limited number of genetic tools available to study gene function in peripheral sensory neurons. Here we generated a Cre-driver mouse line that expresses Cre-recombinase from the locus of the sensory neuron specific gene Advillin. These mice displayed almost exclusive Cre-mediated recombination in all peripheral sensory neurons. As such, the Advillin-Cre-driver line will be a powerful tool for targeting peripheral neurons in future investigations.
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- 2011
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4. SARS-CoV-2 mutations affect antigen processing by the proteasome to alter CD8+ T cell responses
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Dannielle Wellington, Zixi Yin, Zhanru Yu, Raphael Heilig, Simon Davis, Roman Fischer, Suet Ling Felce, Elie Antoun, Philip Hublitz, Ryan Beveridge, Danning Dong, Guihai Liu, Xuan Yao, Yanchun Peng, Benedikt M. Kessler, and Tao Dong
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Science (General) ,Q1-390 ,Social sciences (General) ,H1-99 - Abstract
Mutations within viral epitopes can result in escape from T cells, but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two SARS-CoV-2 nucleoprotein CD8+ epitopes, we investigated the contribution of these flanking mutations to proteasomal processing and T cell activation. We found decreased NP9-17-B*27:05 CD8+ T cell responses to the NP-Q7K mutation, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103 N/Y mutations flanking the NP9-17-B*27:05 and NP105-113-B*07:02 epitopes, respectively, increased CD8+ T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on proteasomal processing, either contributing to T cell escape or enhancement that may be exploited for future vaccine design.
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- 2023
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5. Glycosylated clusterin species facilitate Aβ toxicity in human neurons
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Evangeline M. Foster, Marco Fernandes, Adria Dangla-Valls, Philip Hublitz, Menelaos Pangalos, Simon Lovestone, Elena M. Ribe, and Noel J. Buckley
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Medicine ,Science - Abstract
Abstract Clusterin (CLU) is one of the most significant genetic risk factors for late onset Alzheimer’s disease (AD). However, the mechanisms by which CLU contributes to AD development and pathogenesis remain unclear. Studies have demonstrated that the trafficking and localisation of glycosylated CLU proteins is altered by CLU-AD mutations and amyloid-β (Aβ), which may contribute to AD pathogenesis. However, the roles of non-glycosylated and glycosylated CLU proteins in mediating Aβ toxicity have not been studied in human neurons. iPSCs with altered CLU trafficking were generated following the removal of CLU exon 2 by CRISPR/Cas9 gene editing. Neurons were generated from control (CTR) and exon 2 −/− edited iPSCs and were incubated with aggregated Aβ peptides. Aβ induced changes in cell death and neurite length were quantified to determine if altered CLU protein trafficking influenced neuronal sensitivity to Aβ. Finally, RNA-Seq analysis was performed to identify key transcriptomic differences between CLU exon 2 −/− and CTR neurons. The removal of CLU exon 2, and the endoplasmic reticulum (ER)-signal peptide located within, abolished the presence of glycosylated CLU and increased the abundance of intracellular, non-glycosylated CLU. While non-glycosylated CLU levels were unaltered by Aβ25–35 treatment, the trafficking of glycosylated CLU was altered in control but not exon 2 −/− neurons. The latter also displayed partial protection against Aβ-induced cell death and neurite retraction. Transcriptome analysis identified downregulation of multiple extracellular matrix (ECM) related genes in exon 2 −/− neurons, potentially contributing to their reduced sensitivity to Aβ toxicity. This study identifies a crucial role of glycosylated CLU in facilitating Aβ toxicity in human neurons. The loss of these proteins reduced both, cell death and neurite damage, two key consequences of Aβ toxicity identified in the AD brain. Strikingly, transcriptomic differences between exon 2 −/− and control neurons were small, but a significant and consistent downregulation of ECM genes and pathways was identified in exon 2 −/− neurons. This may contribute to the reduced sensitivity of these neurons to Aβ, providing new mechanistic insights into Aβ pathologies and therapeutic targets for AD.
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- 2022
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6. Evaluation of T cell responses to naturally processed variant SARS-CoV-2 spike antigens in individuals following infection or vaccination
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Zixi Yin, Ji-Li Chen, Yongxu Lu, Beibei Wang, Leila Godfrey, Alexander J. Mentzer, Xuan Yao, Guihai Liu, Dannielle Wellington, Yiqi Zhao, Peter A.C. Wing, Wanwisa Dejnirattisa, Piyada Supasa, Chang Liu, Philip Hublitz, Ryan Beveridge, Craig Waugh, Sally-Ann Clark, Kevin Clark, Paul Sopp, Timothy Rostron, Juthathip Mongkolsapaya, Gavin R. Screaton, Graham Ogg, Katie Ewer, Andrew J. Pollard, Sarah Gilbert, Julian C. Knight, Teresa Lambe, Geoffrey L. Smith, Tao Dong, and Yanchun Peng
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CP: Immunology ,Biology (General) ,QH301-705.5 - Abstract
Summary: Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, the rVACV system can be used to evaluate the cross-reactivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations.
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- 2023
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7. Methods and Protocols—Aims and Scope Update
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Fernando Albericio and Philip Hublitz
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n/a ,Biology (General) ,QH301-705.5 - Abstract
As our readers know, Methods and Protocols is a multidisciplinary peer-reviewed scientific journal that provides a forum to the publication of novel approaches in the fields of Life Sciences, Chemistry, and Biomedical Sciences and their intersection with other related scientific fields such as Physics, Earth Sciences, and Environmental Research [...]
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- 2023
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8. Dual RNA sequencing reveals dendritic cell reprogramming in response to typhoidal Salmonella invasion
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Aulicino, Anna, Antanaviciute, Agne, Frost, Joe, Sousa Geros, Ana, Mellado, Esther, Attar, Moustafa, Jagielowicz, Marta, Hublitz, Philip, Sinz, Julia, Preciado-Llanes, Lorena, Napolitani, Giorgio, Bowden, Rory, Koohy, Hashem, Drakesmith, Hal, and Simmons, Alison
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- 2022
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9. Glycosylated clusterin species facilitate Aβ toxicity in human neurons
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Foster, Evangeline M., Fernandes, Marco, Dangla-Valls, Adria, Hublitz, Philip, Pangalos, Menelaos, Lovestone, Simon, Ribe, Elena M., and Buckley, Noel J.
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- 2022
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10. Dual RNA sequencing reveals dendritic cell reprogramming in response to typhoidal Salmonella invasion
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Anna Aulicino, Agne Antanaviciute, Joe Frost, Ana Sousa Geros, Esther Mellado, Moustafa Attar, Marta Jagielowicz, Philip Hublitz, Julia Sinz, Lorena Preciado-Llanes, Giorgio Napolitani, Rory Bowden, Hashem Koohy, Hal Drakesmith, and Alison Simmons
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Biology (General) ,QH301-705.5 - Abstract
Aulicino, Antanaviciute et al investigate the transcriptional response to invasive Salmonella strains in dendritic cells (DCs). They show that S. Typhi mount a response against nitrosative stress pathways and propose a role of iron uptake and transport in preventing infection, which the pathogen can bypass. In parallel, they find that invasive Salmonella employs several mechanisms targeting more classic aspects of immunity to impair DC function.
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- 2022
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11. An immunodominant NP105–113-B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease
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Peng, Yanchun, Felce, Suet Ling, Dong, Danning, Penkava, Frank, Mentzer, Alexander J., Yao, Xuan, Liu, Guihai, Yin, Zixi, Chen, Ji-Li, Lu, Yongxu, Wellington, Dannielle, Wing, Peter A. C., Dominey-Foy, Delaney C. C., Jin, Chen, Wang, Wenbo, Hamid, Megat Abd, Fernandes, Ricardo A., Wang, Beibei, Fries, Anastasia, Zhuang, Xiaodong, Ashley, Neil, Rostron, Timothy, Waugh, Craig, Sopp, Paul, Hublitz, Philip, Beveridge, Ryan, Tan, Tiong Kit, Dold, Christina, Kwok, Andrew J., Rich-Griffin, Charlotte, Dejnirattisa, Wanwisa, Liu, Chang, Kurupati, Prathiba, Nassiri, Isar, Watson, Robert A., Tong, Orion, Taylor, Chelsea A., Kumar Sharma, Piyush, Sun, Bo, Curion, Fabiola, Revale, Santiago, Garner, Lucy C., Jansen, Kathrin, Ferreira, Ricardo C., Attar, Moustafa, Fry, Jeremy W., Russell, Rebecca A., Stauss, Hans J., James, William, Townsend, Alain, Ho, Ling-Pei, Klenerman, Paul, Mongkolsapaya, Juthathip, Screaton, Gavin R., Dendrou, Calliope, Sansom, Stephen N., Bashford-Rogers, Rachael, Chain, Benny, Smith, Geoffrey L., McKeating, Jane A., Fairfax, Benjamin P., Bowness, Paul, McMichael, Andrew J., Ogg, Graham, Knight, Julian C., and Dong, Tao
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- 2022
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12. Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5
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S. Galiani, K. Reglinski, P. Carravilla, A. Barbotin, I. Urbančič, J. Ott, J. Sehr, E. Sezgin, F. Schneider, D. Waithe, P. Hublitz, W. Schliebs, R. Erdmann, and C. Eggeling
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Physics ,QC1-999 ,Biology (General) ,QH301-705.5 - Abstract
Cellular functions rely on proper actions of organelles such as peroxisomes. These organelles rely on the import of proteins from the cytosol. The peroxisomal import receptor PEX5 takes up target proteins in the cytosol and transports them to the peroxisomal matrix. However, its cytosolic molecular interactions have so far not directly been disclosed. Here, we combined advanced optical microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy and stimulated emission depletion microscopy with biochemical tools to present a detailed characterization of the cytosolic diffusion and interaction dynamics of PEX5. Among other features, we highlight a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol as well as highlighting the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol by studying their diffusion dynamics.
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- 2022
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13. Mouse model of the human serotonin transporter-linked polymorphic region
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Piszczek, Lukasz, Memoli, Simone, Raggioli, Angelo, Viosca, José, Rientjes, Jeanette, Hublitz, Philip, Czaban, Weronika, Wyrzykowska, Anna, and Gross, Cornelius
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- 2019
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14. SCL/TAL1 cooperates with Polycomb RYBP-PRC1 to suppress alternative lineages in blood-fated cells
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Hedia Chagraoui, Maiken S. Kristiansen, Juan Pablo Ruiz, Ana Serra-Barros, Johanna Richter, Elisa Hall-Ponselé, Nicki Gray, Dominic Waithe, Kevin Clark, Philip Hublitz, Emmanouela Repapi, Georg Otto, Paul Sopp, Stephen Taylor, Supat Thongjuea, Paresh Vyas, and Catherine Porcher
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Science - Abstract
Mechanisms that operate during embryonic development to restrict cell fate are currently under investigation. Here the authors characterise the role of SCL/TAL1 at the onset of blood specification in embryonic development using mouse EB differentiation culture as a model system.
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- 2018
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15. Colonic epithelial cell diversity in health and inflammatory bowel disease
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Parikh, Kaushal, Antanaviciute, Agne, Fawkner-Corbett, David, Jagielowicz, Marta, Aulicino, Anna, Lagerholm, Christoffer, Davis, Simon, Kinchen, James, Chen, Hannah H., Alham, Nasullah Khalid, Ashley, Neil, Johnson, Errin, Hublitz, Philip, Bao, Leyuan, Lukomska, Joanna, Andev, Rajinder Singh, Björklund, Elisabet, Kessler, Benedikt M., Fischer, Roman, Goldin, Robert, Koohy, Hashem, and Simmons, Alison
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- 2019
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16. Rapid Emergence of Chronic Lymphocytic Leukemia During JAK2 Inhibitor Therapy in a Patient With Myelofibrosis
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Nikolaos Sousos, Gemma Buck, Alba Rodriguez-Meira, Ruggiero Norfo, Angela Hamblin, Francesco Pezzella, Jennifer Davies, Philip Hublitz, Bethan Psaila, and Adam J. Mead
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Published
- 2020
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17. Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia
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Sachith Mettananda, Chris A. Fisher, Deborah Hay, Mohsin Badat, Lynn Quek, Kevin Clark, Philip Hublitz, Damien Downes, Jon Kerry, Matthew Gosden, Jelena Telenius, Jackie A. Sloane-Stanley, Paula Faustino, Andreia Coelho, Jessica Doondeea, Batchimeg Usukhbayar, Paul Sopp, Jacqueline A. Sharpe, Jim R. Hughes, Paresh Vyas, Richard J. Gibbons, and Douglas R. Higgs
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Science - Abstract
β-thalassemia is characterised by the presence of an excess of α-globin chains, which contribute to erythrocyte pathology. Here the authors use CRISP/Cas9 to reduce α-globin expression in hematopoietic precursors, and show effectiveness in xenograft assays in mice.
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- 2017
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18. Amplification of autoimmune organ damage by NKp46-activated ILC1s
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Biniaris-Georgallis, Stylianos-Iason, Aschman, Tom, Stergioula, Katerina, Schreiber, Frauke, Jafari, Vajiheh, Taranko, Anna, Karmalkar, Tejal, Kasapi, Ana, Lenac Rovis, Tihana, Jelencic, Vedrana, Bejarano, David A., Fabry, Lea, Papacharalampous, Michail, Mattiola, Irene, Molgora, Martina, Hou, Jinchao, Hublitz, Karolin W., Heinrich, Frederik, Guerra, Gabriela Maria, Durek, Pawel, Patone, Giannino, Lindberg, Eric L., Maatz, Henrike, Hölsken, Oliver, Krönke, Gerhard, Mortha, Arthur, Voll, Reinhard E., Clarke, Alexander J., Hauser, Anja E., Colonna, Marco, Thurley, Kevin, Schlitzer, Andreas, Schneider, Christoph, Stamatiades, Efstathios G., Mashreghi, Mir-Farzin, Jonjic, Stipan, Hübner, Norbert, Diefenbach, Andreas, Kanda, Masatoshi, and Triantafyllopoulou, Antigoni
- Abstract
In systemic lupus erythematosus, loss of immune tolerance, autoantibody production and immune complex deposition are required but not sufficient for organ damage1. How inflammatory signals are initiated and amplified in the setting of autoimmunity remains elusive. Here we set out to dissect layers and hierarchies of autoimmune kidney inflammation to identify tissue-specific cellular hubs that amplify autoinflammatory responses. Using high-resolution single-cell profiling of kidney immune and parenchymal cells, in combination with antibody blockade and genetic deficiency, we show that tissue-resident NKp46+innate lymphoid cells (ILCs) are crucial signal amplifiers of disease-associated macrophage expansion and epithelial cell injury in lupus nephritis, downstream of autoantibody production. NKp46 signalling in a distinct subset of group 1 ILCs (ILC1s) instructed an unconventional immune-regulatory transcriptional program, which included the expression of the myeloid cell growth factor CSF2. CSF2 production by NKp46+ILCs promoted the population expansion of monocyte-derived macrophages. Blockade of the NKp46 receptor (using the antibody clone mNCR1.15; ref. 2) or genetic deficiency of NKp46 abrogated epithelial cell injury. The same cellular and molecular patterns were operative in human lupus nephritis. Our data provide support for the idea that NKp46+ILC1s promote parenchymal cell injury by granting monocyte-derived macrophages access to epithelial cell niches. NKp46 activation in ILC1s therefore constitutes a previously unrecognized, crucial tissue rheostat that amplifies organ damage in autoimmune hosts, with broad implications for inflammatory pathologies and therapies.
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- 2024
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19. SCL/TAL1 cooperates with Polycomb RYBP-PRC1 to suppress alternative lineages in blood-fated cells
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Chagraoui, Hedia, Kristiansen, Maiken S., Ruiz, Juan Pablo, Serra-Barros, Ana, Richter, Johanna, Hall-Ponselé, Elisa, Gray, Nicki, Waithe, Dominic, Clark, Kevin, Hublitz, Philip, Repapi, Emmanouela, Otto, Georg, Sopp, Paul, Taylor, Stephen, Thongjuea, Supat, Vyas, Paresh, and Porcher, Catherine
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- 2018
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20. Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions
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Joseph Blayney, Evangeline M. Foster, Marta Jagielowicz, Mira Kreuzer, Matteo Morotti, Katharina Reglinski, Julie Huiyuan Xiao, and Philip Hublitz
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CRISPR/Cas9 ,dual sgRNA ,genomic knock-out ,NHEJ ,PCR screen ,inverted re-insertion ,Biology (General) ,QH301-705.5 - Abstract
Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions.
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- 2020
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21. RIG-I Plays a Dominant Role in the Induction of Transcriptional Changes in Zika Virus-Infected Cells, which Protect from Virus-Induced Cell Death
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Mirjam Schilling, Anne Bridgeman, Nicki Gray, Jonny Hertzog, Philip Hublitz, Alain Kohl, and Jan Rehwinkel
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Zika virus ,IFN ,RIG-I ,MDA5 ,apoptosis ,NS5 ,Cytology ,QH573-671 - Abstract
The Zika virus (ZIKV) has received much attention due to an alarming increase in cases of neurological disorders including congenital Zika syndrome associated with infection. To date, there is no effective treatment available. An immediate response by the innate immune system is crucial for effective control of the virus. Using CRISPR/Cas9-mediated knockouts in A549 cells, we investigated the individual contributions of the RIG-I-like receptors MDA5 and RIG-I to ZIKV sensing and control of this virus by using a Brazilian ZIKV strain. We show that RIG-I is the main sensor for ZIKV in A549 cells. Surprisingly, we observed that loss of RIG-I and consecutive type I interferon (IFN) production led to virus-induced apoptosis. ZIKV non-structural protein NS5 was reported to interfere with type I IFN receptor signaling. Additionally, we show that ZIKV NS5 inhibits type I IFN induction. Overall, our study highlights the importance of RIG-I-dependent ZIKV sensing for the prevention of virus-induced cell death and shows that NS5 inhibits the production of type I IFN.
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- 2020
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22. Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5
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Galiani, S., primary, Reglinski, K., additional, Carravilla, P., additional, Barbotin, A., additional, Urbančič, I., additional, Ott, J., additional, Sehr, J., additional, Sezgin, E., additional, Schneider, F., additional, Waithe, D., additional, Hublitz, P., additional, Schliebs, W., additional, Erdmann, R., additional, and Eggeling, C., additional
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- 2022
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23. Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5
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Galiani, S, primary, Reglinski, K, additional, Carravilla, P, additional, Barbotin, A, additional, Urbančič, I, additional, Ott, J, additional, Sehr, J, additional, Sezgin, E, additional, Schneider, F, additional, Waithe, D, additional, Hublitz, P, additional, Schliebs, W, additional, Erdmann, R, additional, and Eggeling, C, additional
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- 2021
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24. Renal inflamm-aging provokes intra-graft inflammation following experimental kidney transplantation
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He, An, Sarwar, Attia, Thole, Linda Marie Laura, Siegle, Janine, Sattler, Arne, Ashraf, Muhammad Imtiaz, Proß, Vanessa, Stahl, Carolin, Dornieden, Theresa, Bergmann, Yasmin, Ritschl, Paul Viktor, Ebner, Susanne, Hublitz, Karolin Wiebke, Stamatiades, Efstathios Gregorios, Bülow, Roman David, Boor, Peter, and Kotsch, Katja
- Abstract
Donor age is a major risk factor for allograft outcome in kidney transplantation. The underlying cellular mechanisms and the recipient’s immune response within an aged allograft have yet not been analyzed. A comprehensive immunophenotyping of naïve and transplanted young versus aged kidneys revealed that naïve aged murine kidneys harbor significantly higher frequencies of effector/memory T cells, whereas regulatory T cells were reduced. Aged kidney-derived CD8+T cells produced more IFNγ than their young counterparts. Senescent renal CD8+T and NK cells upregulated the cytotoxicity receptor NKG2D and the enrichment of memory-like CD49a+CXCR6+NK cells was documented in aged naïve kidneys. In the C57BL/6 to BALB/c kidney transplantation model, recipient-derived T cells infiltrating an aged graft produced significantly more IFNγ, granzyme B and perforin on day 7 post-transplantation, indicating an enhanced inflammatory, cytotoxic response towards the graft. Pre-treatment of aged kidney donors with the senolytic drug ABT-263 changed the recipient-derived effector molecule profile to significantly reduced levels of IFNγ and IL-10 compared to controls. Graft function after ABT-263 pre-treatment was significantly improved 28 days post kidney transplantation. In conclusion, renal senescence also occurs at the immunological level (inflamm-aging) and aged organs provoke an altered recipient-dominated immune response in the graft.
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- 2022
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25. Generation of immune checkpoint knock-out human antigen-specific T cells via CRISPR/Cas9-mediated genetic engineering
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Zhang, C., primary, Peng, Y., additional, Hublitz, P., additional, and Dong, T., additional
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- 2016
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26. 3O - Generation of immune checkpoint knock-out human antigen-specific T cells via CRISPR/Cas9-mediated genetic engineering
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Zhang, C., Peng, Y., Hublitz, P., and Dong, T.
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- 2016
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27. Evaluation of T cell responses to naturally processed variant SARS-CoV-2 spike antigens in individuals following infection or vaccination
- Author
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Yin, Zixi, Chen, Ji-Li, Lu, Yongxu, Wang, Beibei, Godfrey, Leila, Mentzer, Alexander J., Yao, Xuan, Liu, Guihai, Wellington, Dannielle, Zhao, Yiqi, Wing, Peter A.C., Dejnirattisa, Wanwisa, Supasa, Piyada, Liu, Chang, Hublitz, Philip, Beveridge, Ryan, Waugh, Craig, Clark, Sally-Ann, Clark, Kevin, Sopp, Paul, Rostron, Timothy, Mongkolsapaya, Juthathip, Screaton, Gavin R., Ogg, Graham, Ewer, Katie, Pollard, Andrew J., Gilbert, Sarah, Knight, Julian C., Lambe, Teresa, Smith, Geoffrey L., Dong, Tao, and Peng, Yanchun
- Abstract
Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, the rVACV system can be used to evaluate the cross-reactivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations.
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- 2023
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28. Mechanisms of transcriptional repression by histone lysine methylation.
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HUBLITZ, PHILIP, ALBERT, MAREIKE, and PETERS, ANTOINE H. F. M.
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GENETIC transcription regulation ,CELLULAR control mechanisms ,METHYLATION ,HISTONES ,LYSINE ,METHYLTRANSFERASES - Abstract
During development, covalent modification of both, histones and DNA contribute to the specification and maintenance of cell identity. Repressive modifications are thought to stabilize cell type specific gene expression patterns, reducing the likelihood of reactivation of lineage-unrelated genes. In this report, we review the recent literature to deduce mechanisms underlying Polycomb and H3K9 methylation mediated repression, and describe the functional interplay with activating H3K4 methylation. We summarize recent data that indicate a close relationship between GC density of promoter sequences, transcription factor binding and the antagonizing activities of distinct epigenetic regulators such as histone methyltransferases (HMTs) and histone demethylases (HDMs). Subsequently, we compare chromatin signatures associated with different types of transcriptional outcomes from stable repression to highly dynamic regulated genes, strongly suggesting that the interplay of different epigenetic pathways is essential in defining specific types of heritable chromatin and associated transcriptional states. [ABSTRACT FROM AUTHOR]
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- 2009
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29. CRISPR/Cas9 genome editing of CLU to examine clusterin's contribution to neurodegeneration and Alzheimer's disease: Molecular and cell biology/neurodegeneration and neuroprotection.
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Foster, Evangeline M., Ribe, Elena M., Robbins, Jacqueline, Hublitz, Philip, Pangalos, Menelas, Buckley, Noel, Lovestone, Simon, Neuroscience, Translational, and Research, Dementia
- Abstract
Background: Traditionally, clusterin is referred to as a secreted protein however, more recent evidence indicates clusterin may exist intracellularly where it may have a pro‐apoptotic role. Previously, our lab observed altered clusterin trafficking in rodent neurons in response to amyloid‐β suggesting it may be a key factor in neurodegeneration. We aim to use CRISPR/Cas9 in human iPSCs to explore the contribution that clusterin plays in cellular vulnerability to amyloid‐β. Method: We aim to remove exon 2 from CLU since this exon contains the ER‐signal peptide thought to be required for secretion of clusterin protein. So far, we have generated heterozygous exon 2 knockout iPSCs (exon 2+/‐) which have been differentiated into cortical neurons and astrocytes. Clusterin gene and protein expression has been characterised in these iPSCs compared to control iPSCs. Response of control and exon 2+/‐ neurons and astrocytes to amyloid‐β treatment has also been explored. Result: Surprisingly, exon 2+/‐ iPSCs express significantly less intracellular clusterin than control iPSCs. Secreted clusterin expression is unaltered by the edit achieved but glycosylation state is altered. We build on previous findings in rodent neurons, showing that clusterin trafficking is also altered by amyloid‐β in both control iPSC‐neurons and iPSC‐astrocytes. This response is observed in exon 2+/‐ neurons but is absent in exon 2+/‐ astrocytes. Divergence in gene expression changes of DKK1‐signature genes is observed. Here, we describe differences in both gene and protein expression changes in response to amyloid‐β treatment in control and exon 2+/‐ cells. Conclusion: Currently, we are exploring whether differences observed in neuronal and astrocytic responses of exon 2+/‐ cells convey protection or vulnerability to amyloid‐β induced toxicity. To do so, we are using imaging techniques to quantify changes in cell death and neurite projections for both genotypes in response to treatment. [ABSTRACT FROM AUTHOR]
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- 2020
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30. Characterization of the DNA-Binding and Dimerization Properties of the Nuclear Orphan Receptor Germ Cell Nuclear Factor
- Author
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Greschik, Holger, Wurtz, Jean-Marie, Hublitz, Philip, Köhler, Fabian, Moras, Dino, and Schüle, Roland
- Abstract
The orphan receptor germ cell nuclear factor (GCNF) is a member of the superfamily of nuclear receptors. During development, GCNF exhibits a restricted brain-specific expression pattern, whereas GCNF expression in the adult is germ cell specific. Therefore, the receptor may participate in the regulation of neurogenesis and reproductive functions. No natural GCNF target gene has yet been identified, but recent data demonstrate specific and high-affinity binding of GCNF either to the direct repeat DNA element AGGTCAAGGTCA (DR0) or to extended half-sites, such as TCAAGGTCA. In this study, we show that murine GCNF (mGCNF) can bind as a homodimer to extended half-sites, thus describing a novel property within the nuclear receptor superfamily. Homodimeric binding to extended half-sites requires the presence of a dimerization function within the mGCNF DNA-binding domain (DBD) and a novel dimerization surface encompassing the putative helix 3 and the helix 12 region of the mGCNF ligand-binding domain (LBD). In addition, the mGCNF LBD has the potential to adopt different conformations with distinct dimerization properties. The helix 12 region of the mGCNF LBD not only regulates the switch between these dimerization conformations but also dictates the DNA-binding behavior and transcriptional properties of the different dimerization conformations. In summary, our findings describe unique DNA-binding and dimerization properties of a nuclear receptor and suggest a novel mechanism that allows mGCNF to modulate target gene activity.
- Published
- 1999
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31. Lactic Dehydrogenase in L1210 Cells
- Author
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KISLIUK, R. L., ZEL, G., WALTER, H. A., NORBERT, G. F., HUBLITZ, U. F., and D'AMICO, R. P.
- Abstract
PROMPTED by the striking difference in lactic dehydrogenase activity observed by Woodliff1in sensitive L1210 leukæmic cells compared with those resistant to amethopterin and 6-mercaptopurine we have examined lactic dehydrogenase-levels in sensitive L1210Z and a strain (L1210C59) resistant to both amethopterin and 6-mercaptopurine and partially resistant to cytoxan.
- Published
- 1963
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32. Methods and Protocols -Aims and Scope Update.
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Albericio F and Hublitz P
- Abstract
As our readers know, Methods and Protocols is a multidisciplinary peer-reviewed scientific journal that provides a forum to the publication of novel approaches in the fields of Life Sciences, Chemistry, and Biomedical Sciences and their intersection with other related scientific fields such as Physics, Earth Sciences, and Environmental Research [...].
- Published
- 2023
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33. GTP Cyclohydrolase Drives Breast Cancer Development and Promotes EMT in an Enzyme-Independent Manner.
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Wang Z, Zhang N, Zhang M, Jiang Y, Ng AS, Bridges E, Zhang W, Zeng X, Luo Q, Liang J, Győrffy B, Hublitz P, Liang Z, Fischer R, Kerr D, Harris AL, and Cai S
- Subjects
- Animals, Female, Humans, Mice, Biopterins analogs & derivatives, Biopterins metabolism, Cell Line, Tumor, Cell Proliferation, HSP90 Heat-Shock Proteins metabolism, HSP90 Heat-Shock Proteins genetics, Mice, Nude, Receptor, ErbB-2 metabolism, Receptor, ErbB-2 genetics, Triple Negative Breast Neoplasms pathology, Triple Negative Breast Neoplasms metabolism, Triple Negative Breast Neoplasms genetics, Vimentin metabolism, Breast Neoplasms pathology, Breast Neoplasms metabolism, Breast Neoplasms genetics, Epithelial-Mesenchymal Transition, GTP Cyclohydrolase metabolism, GTP Cyclohydrolase genetics
- Abstract
GTP cyclohydrolase (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis. The catalysis of BH4 biosynthesis is tightly regulated for physiological neurotransmission, inflammation, and vascular tone. Paradoxically, BH4 has emerged as an oncometabolite regulating tumor growth, but the effects on tumor development remain controversial. Here, we found that GCH1 potentiated the growth of triple-negative breast cancer (TNBC) and HER2+ breast cancer and transformed nontumor breast epithelial cells. Independent of BH4 production, GCH1 protein induced epithelial-to-mesenchymal transition by binding to vimentin (Vim), which was mediated by HSP90. Conversely, GCH1 ablation impaired tumor growth, suppressed Vim in TNBC, and inhibited EGFR/ERK signaling while activating the p53 pathway in estrogen receptor-positive tumor cells. GCH1 deficiency increases tumor cell sensitivity to HSP90 inhibition and endocrine treatments. In addition, high GCH1 correlated with poor breast cancer survival. Together, this study reveals an enzyme-independent oncogenic role of GCH1, presenting it as a potential target for therapeutic development., Significance: GTP cyclohydrolase functions as an oncogene in breast cancer and binds vimentin to induce epithelial-to-mesenchymal transition independently of its enzyme activity, which confers targetable vulnerabilities for developing breast cancer treatment strategies., (©2023 American Association for Cancer Research.)
- Published
- 2023
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34. SARS-CoV-2 mutations affect antigen processing by the proteasome to alter CD8 + T cell responses.
- Author
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Wellington D, Yin Z, Yu Z, Heilig R, Davis S, Fischer R, Felce SL, Antoun E, Hublitz P, Beveridge R, Dong D, Liu G, Yao X, Peng Y, Kessler BM, and Dong T
- Abstract
Mutations within viral epitopes can result in escape from T cells, but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two SARS-CoV-2 nucleoprotein CD8
+ epitopes, we investigated the contribution of these flanking mutations to proteasomal processing and T cell activation. We found decreased NP9-17 -B*27:05 CD8+ T cell responses to the NP-Q7K mutation, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103 N/Y mutations flanking the NP9-17 -B*27:05 and NP105-113 -B*07:02 epitopes, respectively, increased CD8+ T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on proteasomal processing, either contributing to T cell escape or enhancement that may be exploited for future vaccine design., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Published by Elsevier Ltd.)- Published
- 2023
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35. FOXN1 forms higher-order nuclear condensates displaced by mutations causing immunodeficiency.
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Rota IA, Handel AE, Maio S, Klein F, Dhalla F, Deadman ME, Cheuk S, Newman JA, Michaels YS, Zuklys S, Prevot N, Hublitz P, Charles PD, Gkazi AS, Adamopoulou E, Qasim W, Davies EG, Hanson I, Pagnamenta AT, Camps C, Dreau HM, White A, James K, Fischer R, Gileadi O, Taylor JC, Fulga T, Lagerholm BC, Anderson G, Sezgin E, and Holländer GA
- Abstract
The transcription factor FOXN1 is a master regulator of thymic epithelial cell (TEC) development and function. Here, we demonstrate that FOXN1 expression is differentially regulated during organogenesis and participates in multimolecular nuclear condensates essential for the factor’s transcriptional activity. FOXN1’s C-terminal sequence regulates the diffusion velocity within these aggregates and modulates the binding to proximal gene regulatory regions. These dynamics are altered in a patient with a mutant FOXN1 that is modified in its C-terminal sequence. This mutant is transcriptionally inactive and acts as a dominant negative factor displacing wild-type FOXN1 from condensates and causing athymia and severe lymphopenia in heterozygotes. Expression of the mutated mouse ortholog selectively impairs mouse TEC differentiation, revealing a gene dose dependency for individual TEC subtypes. We have therefore identified the cause for a primary immunodeficiency disease and determined the mechanism by which this FOXN1 gain-of-function mutant mediates its dominant negative effect.
- Published
- 2021
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36. Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions.
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Blayney J, Foster EM, Jagielowicz M, Kreuzer M, Morotti M, Reglinski K, Xiao JH, and Hublitz P
- Abstract
Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions.
- Published
- 2020
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37. RIG-I Plays a Dominant Role in the Induction of Transcriptional Changes in Zika Virus-Infected Cells, which Protect from Virus-Induced Cell Death.
- Author
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Schilling M, Bridgeman A, Gray N, Hertzog J, Hublitz P, Kohl A, and Rehwinkel J
- Subjects
- Animals, Chlorocebus aethiops virology, Humans, Immunity, Innate immunology, Signal Transduction immunology, Vero Cells virology, Viral Nonstructural Proteins metabolism, Zika Virus immunology, Zika Virus metabolism, Zika Virus Infection immunology, Cell Death physiology, DEAD Box Protein 58 metabolism, Receptors, Immunologic metabolism, Zika Virus Infection virology
- Abstract
The Zika virus (ZIKV) has received much attention due to an alarming increase in cases of neurological disorders including congenital Zika syndrome associated with infection. To date, there is no effective treatment available. An immediate response by the innate immune system is crucial for effective control of the virus. Using CRISPR/Cas9-mediated knockouts in A549 cells, we investigated the individual contributions of the RIG-I-like receptors MDA5 and RIG-I to ZIKV sensing and control of this virus by using a Brazilian ZIKV strain. We show that RIG-I is the main sensor for ZIKV in A549 cells. Surprisingly, we observed that loss of RIG-I and consecutive type I interferon (IFN) production led to virus-induced apoptosis. ZIKV non-structural protein NS5 was reported to interfere with type I IFN receptor signaling. Additionally, we show that ZIKV NS5 inhibits type I IFN induction. Overall, our study highlights the importance of RIG-I-dependent ZIKV sensing for the prevention of virus-induced cell death and shows that NS5 inhibits the production of type I IFN.
- Published
- 2020
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38. Rapid Emergence of Chronic Lymphocytic Leukemia During JAK2 Inhibitor Therapy in a Patient With Myelofibrosis.
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Sousos N, Buck G, Rodriguez-Meira A, Norfo R, Hamblin A, Pezzella F, Davies J, Hublitz P, Psaila B, and Mead AJ
- Published
- 2020
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- View/download PDF
39. Polyamines Control eIF5A Hypusination, TFEB Translation, and Autophagy to Reverse B Cell Senescence.
- Author
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Zhang H, Alsaleh G, Feltham J, Sun Y, Napolitano G, Riffelmacher T, Charles P, Frau L, Hublitz P, Yu Z, Mohammed S, Ballabio A, Balabanov S, Mellor J, and Simon AK
- Subjects
- Adaptive Immunity drug effects, Age Factors, Aging, Animals, B-Lymphocytes immunology, B-Lymphocytes metabolism, B-Lymphocytes pathology, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors deficiency, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors genetics, HEK293 Cells, Humans, Immunologic Memory drug effects, Jurkat Cells, Mice, Mice, Inbred C57BL, Mice, Knockout, NIH 3T3 Cells, Peptide Initiation Factors genetics, RNA-Binding Proteins genetics, Signal Transduction, Eukaryotic Translation Initiation Factor 5A, Autophagy drug effects, B-Lymphocytes drug effects, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors metabolism, Cellular Senescence drug effects, Immunosenescence drug effects, Peptide Initiation Factors metabolism, Protein Processing, Post-Translational drug effects, RNA-Binding Proteins metabolism, Spermidine pharmacology
- Abstract
Failure to make adaptive immune responses is a hallmark of aging. Reduced B cell function leads to poor vaccination efficacy and a high prevalence of infections in the elderly. Here we show that reduced autophagy is a central molecular mechanism underlying immune senescence. Autophagy levels are specifically reduced in mature lymphocytes, leading to compromised memory B cell responses in old individuals. Spermidine, an endogenous polyamine metabolite, induces autophagy in vivo and rejuvenates memory B cell responses. Mechanistically, spermidine post-translationally modifies the translation factor eIF5A, which is essential for the synthesis of the autophagy transcription factor TFEB. Spermidine is depleted in the elderly, leading to reduced TFEB expression and autophagy. Spermidine supplementation restored this pathway and improved the responses of old human B cells. Taken together, our results reveal an unexpected autophagy regulatory mechanism mediated by eIF5A at the translational level, which can be harnessed to reverse immune senescence in humans., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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40. Microhomologies are prevalent at Cas9-induced larger deletions.
- Author
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Owens DDG, Caulder A, Frontera V, Harman JR, Allan AJ, Bucakci A, Greder L, Codner GF, Hublitz P, McHugh PJ, Teboul L, and de Bruijn MFTR
- Subjects
- Animals, Cell Line, Chromosome Breakpoints, Chromosomes, Mammalian metabolism, DNA End-Joining Repair, Deoxyribonuclease I genetics, Deoxyribonuclease I metabolism, Endonucleases genetics, Endonucleases metabolism, Mice, Models, Genetic, RNA, Guide, CRISPR-Cas Systems genetics, RNA, Guide, CRISPR-Cas Systems metabolism, CRISPR-Cas Systems, Chromosome Deletion, Chromosomes, Mammalian genetics, Gene Editing methods, Sequence Deletion
- Abstract
The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2019
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41. Infection with a Brazilian isolate of Zika virus generates RIG-I stimulatory RNA and the viral NS5 protein blocks type I IFN induction and signaling.
- Author
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Hertzog J, Dias Junior AG, Rigby RE, Donald CL, Mayer A, Sezgin E, Song C, Jin B, Hublitz P, Eggeling C, Kohl A, and Rehwinkel J
- Subjects
- Active Transport, Cell Nucleus, Brazil, DEAD Box Protein 58 genetics, Down-Regulation, HEK293 Cells, Humans, Interferon Type I genetics, Phosphorylation, Receptors, Immunologic, Signal Transduction, Virus Replication, Zika Virus, Zika Virus Infection, DEAD Box Protein 58 immunology, Interferon Type I metabolism, RNA immunology, STAT1 Transcription Factor metabolism, STAT2 Transcription Factor metabolism, Viral Nonstructural Proteins metabolism
- Abstract
Zika virus (ZIKV) is a major public health concern in the Americas. We report that ZIKV infection and RNA extracted from ZIKV infected cells potently activated the induction of type I interferons (IFNs). This effect was fully dependent on the mitochondrial antiviral signaling protein (MAVS), implicating RIG-I-like receptors (RLRs) as upstream sensors of viral RNA. Indeed, RIG-I and the related RNA sensor MDA5 contributed to type I IFN induction in response to RNA from infected cells. We found that ZIKV NS5 from a recent Brazilian isolate blocked type I IFN induction downstream of RLRs and also inhibited type I IFN receptor (IFNAR) signaling. We defined the ZIKV NS5 nuclear localization signal and report that NS5 nuclear localization was not required for inhibition of signaling downstream of IFNAR. Mechanistically, NS5 blocked IFNAR signaling by both leading to reduced levels of STAT2 and by blocking phosphorylation of STAT1, two transcription factors activated by type I IFNs. Taken together, our observations suggest that ZIKV infection induces a type I IFN response via RLRs and that ZIKV interferes with this response by blocking signaling downstream of RLRs and IFNAR., (© 2018 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2018
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42. Genetic abrogation of immune checkpoints in antigen-specific cytotoxic T-lymphocyte as a potential alternative to blockade immunotherapy.
- Author
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Zhang C, Peng Y, Hublitz P, Zhang H, and Dong T
- Subjects
- CRISPR-Cas Systems, Humans, Lymphocyte Activation, Neoplasms immunology, Programmed Cell Death 1 Receptor genetics, Programmed Cell Death 1 Receptor immunology, Antigens, Neoplasm immunology, Gene Editing, Immunotherapy, Neoplasms drug therapy, Programmed Cell Death 1 Receptor antagonists & inhibitors, T-Lymphocytes, Cytotoxic immunology
- Abstract
T cell function can be compromised during chronic infections or through continuous exposure to tumor antigens by the action of immune checkpoint receptors, such as programmed cell death protein 1 (PD-1). Systemic administration of blocking antibodies against the PD-1 pathway can restore T cell function, and has been approved for the treatment of several malignancies, although there is a risk of adverse immune-related side-effects. We have developed a method for generating gene knockouts in human antigen (Ag)-specific cytotoxic T-Lymphocyte (CTLs) using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing. Using this method, we generated several transduced CD4
+ or CD8+ antigen-specific polyclonal CTL lines and clones, and validated gene modifications of the PD-1 gene. We compared these T-cell lines and clones with control groups in the presence of programmed death-ligand 1 (PD-L1) and observed improved effector functions in the PD1-disrupted cell group. Overall, we have developed a versatile tool for functional genomics in human antigen-specific CTL studies. Furthermore, we provide an alternative strategy for current cell-based immunotherapy that will minimize the side effects caused by antibody blockade therapy.- Published
- 2018
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43. Lack of Truncated IFITM3 Transcripts in Cells Homozygous for the rs12252-C Variant That is Associated With Severe Influenza Infection.
- Author
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Makvandi-Nejad S, Laurenson-Schafer H, Wang L, Wellington D, Zhao Y, Jin B, Qin L, Kite K, Moghadam HK, Song C, Clark K, Hublitz P, Townsend AR, Wu H, McMichael AJ, Zhang Y, and Dong T
- Subjects
- Dendritic Cells immunology, Humans, Influenza A Virus, H1N1 Subtype isolation & purification, Influenza, Human virology, Leukocytes, Mononuclear, Sequence Analysis, RNA, United Kingdom, Influenza, Human genetics, Influenza, Human pathology, Membrane Proteins genetics, Polymorphism, Single Nucleotide, RNA, Messenger genetics, RNA-Binding Proteins genetics
- Abstract
Interferon-induced transmembrane 3 (IFITM3) is known to restrict the entry of a range of enveloped viruses. The single nucleotide polymorphism rs12252-C within IFITM3 has been shown to be associated with severe influenza A virus infection. It has been suggested that rs12252-C results in expression of a truncated IFITM3 protein lacking the first 21 amino acids. By performing high-throughput RNA sequencing on primary dendritic cells and peripheral blood mononuclear cells isolated from pandemic H1N1 influenza and human immunodeficiency virus-1 (HIV-1) infected patients we show that full-length IFITM3 mRNA is dominantly expressed (>99%) across all rs12252 genotypes. Full-length IFITM3 protein can be detected in all genotypes., (© The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)
- Published
- 2018
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44. Autophagy-Dependent Generation of Free Fatty Acids Is Critical for Normal Neutrophil Differentiation.
- Author
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Riffelmacher T, Clarke A, Richter FC, Stranks A, Pandey S, Danielli S, Hublitz P, Yu Z, Johnson E, Schwerd T, McCullagh J, Uhlig H, Jacobsen SEW, and Simon AK
- Subjects
- Adaptation, Biological, Animals, Cluster Analysis, Energy Metabolism, Gene Expression Profiling, Gene Knockout Techniques, Glucose metabolism, Lipid Metabolism, Lipolysis, Myelopoiesis, Neutrophils ultrastructure, Oxidation-Reduction, Pyruvic Acid metabolism, Autophagy, Cell Differentiation, Fatty Acids, Nonesterified metabolism, Neutrophils cytology, Neutrophils metabolism
- Abstract
Neutrophils are critical and short-lived mediators of innate immunity that require constant replenishment. Their differentiation in the bone marrow requires extensive cytoplasmic and nuclear remodeling, but the processes governing these energy-consuming changes are unknown. While previous studies show that autophagy is required for differentiation of other blood cell lineages, its function during granulopoiesis has remained elusive. Here, we have shown that metabolism and autophagy are developmentally programmed and essential for neutrophil differentiation in vivo. Atg7-deficient neutrophil precursors had increased glycolytic activity but impaired mitochondrial respiration, decreased ATP production, and accumulated lipid droplets. Inhibiting autophagy-mediated lipid degradation or fatty acid oxidation alone was sufficient to cause defective differentiation, while administration of fatty acids or pyruvate for mitochondrial respiration rescued differentiation in autophagy-deficient neutrophil precursors. Together, we show that autophagy-mediated lipolysis provides free fatty acids to support a mitochondrial respiration pathway essential to neutrophil differentiation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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45. Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia.
- Author
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Mettananda S, Fisher CA, Hay D, Badat M, Quek L, Clark K, Hublitz P, Downes D, Kerry J, Gosden M, Telenius J, Sloane-Stanley JA, Faustino P, Coelho A, Doondeea J, Usukhbayar B, Sopp P, Sharpe JA, Hughes JR, Vyas P, Gibbons RJ, and Higgs DR
- Subjects
- Animals, Antigens, CD34 metabolism, Base Sequence, CRISPR-Cas Systems, Cells, Cultured, Female, Gene Knockdown Techniques, Genome, Human, Heterografts, Humans, Mice, Sequence Deletion genetics, Single-Cell Analysis, Enhancer Elements, Genetic genetics, Gene Editing, Hematopoietic Stem Cells metabolism, alpha-Globins genetics, beta-Thalassemia genetics, beta-Thalassemia therapy
- Abstract
β-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and β-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with β-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with β-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for β-thalassemia.β-thalassemia is characterised by the presence of an excess of α-globin chains, which contribute to erythrocyte pathology. Here the authors use CRISP/Cas9 to reduce α-globin expression in hematopoietic precursors, and show effectiveness in xenograft assays in mice.
- Published
- 2017
- Full Text
- View/download PDF
46. The K(+) channel GIRK2 is both necessary and sufficient for peripheral opioid-mediated analgesia.
- Author
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Nockemann D, Rouault M, Labuz D, Hublitz P, McKnelly K, Reis FC, Stein C, and Heppenstall PA
- Subjects
- Aged, Animals, Electrophysiology, Humans, Inflammation, Mice, Mice, Transgenic, Neurons metabolism, Peripheral Nervous System metabolism, Potassium Channels metabolism, Rats, Signal Transduction, Skin metabolism, Analgesia methods, Analgesics, Opioid therapeutic use, G Protein-Coupled Inwardly-Rectifying Potassium Channels physiology, Receptors, Opioid metabolism
- Abstract
The use of opioid agonists acting outside the central nervous system (CNS) is a promising therapeutic strategy for pain control that avoids deleterious central side effects such as apnea and addiction. In human clinical trials and rat models of inflammatory pain, peripherally restricted opioids have repeatedly shown powerful analgesic effects; in some mouse models however, their actions remain unclear. Here, we investigated opioid receptor coupling to K(+) channels as a mechanism to explain such discrepancies. We found that GIRK channels, major effectors for opioid signalling in the CNS, are absent from mouse peripheral sensory neurons but present in human and rat. In vivo transgenic expression of GIRK channels in mouse nociceptors established peripheral opioid signalling and local analgesia. We further identified a regulatory element in the rat GIRK2 gene that accounts for differential expression in rodents. Thus, GIRK channels are indispensable for peripheral opioid analgesia, and their absence in mice has profound consequences for GPCR signalling in peripheral sensory neurons., (© 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)
- Published
- 2013
- Full Text
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47. Tubulin acetyltransferase αTAT1 destabilizes microtubules independently of its acetylation activity.
- Author
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Kalebic N, Martinez C, Perlas E, Hublitz P, Bilbao-Cortes D, Fiedorczuk K, Andolfo A, and Heppenstall PA
- Subjects
- Acetylation, Animals, CHO Cells, Cell Line, Cricetinae, Lysine metabolism, Mice, Mice, Inbred C57BL, Microtubules enzymology, NIH 3T3 Cells, Acetyltransferases metabolism, Microtubules metabolism, Tubulin metabolism
- Abstract
Acetylation of α-tubulin at lysine 40 (K40) is a well-conserved posttranslational modification that marks long-lived microtubules but has poorly understood functional significance. Recently, αTAT1, a member of the Gcn5-related N-acetyltransferase superfamily, has been identified as an α-tubulin acetyltransferase in ciliated organisms. Here, we explored the function of αTAT1 with the aim of understanding the consequences of αTAT1-mediated microtubule acetylation. We demonstrate that α-tubulin is the major target of αTAT1 but that αTAT1 also acetylates itself in a regulatory mechanism that is required for effective modification of tubulin. We further show that in mammalian cells, αTAT1 promotes microtubule destabilization and accelerates microtubule dynamics. Intriguingly, this effect persists in an αTAT1 mutant with no acetyltransferase activity, suggesting that interaction of αTAT1 with microtubules, rather than acetylation per se, is the critical factor regulating microtubule stability. Our data demonstrate that αTAT1 has cellular functions that extend beyond its classical enzymatic activity as an α-tubulin acetyltransferase.
- Published
- 2013
- Full Text
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48. Generation and characterization of an Advillin-Cre driver mouse line.
- Author
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Zurborg S, Piszczek A, Martínez C, Hublitz P, Al Banchaabouchi M, Moreira P, Perlas E, and Heppenstall PA
- Subjects
- Aging metabolism, Animals, Behavior, Animal, Embryo, Mammalian metabolism, Gene Dosage genetics, Hyperalgesia pathology, Hyperalgesia physiopathology, Mice, Mice, Inbred C57BL, Mice, Transgenic, Nociception physiology, Sensory Receptor Cells metabolism, Staining and Labeling, beta-Galactosidase metabolism, Genetic Techniques, Integrases metabolism, Microfilament Proteins metabolism
- Abstract
Progress in the somatosensory field has been restricted by the limited number of genetic tools available to study gene function in peripheral sensory neurons. Here we generated a Cre-driver mouse line that expresses Cre-recombinase from the locus of the sensory neuron specific gene Advillin. These mice displayed almost exclusive Cre-mediated recombination in all peripheral sensory neurons. As such, the Advillin-Cre-driver line will be a powerful tool for targeting peripheral neurons in future investigations.
- Published
- 2011
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49. NIR is a novel INHAT repressor that modulates the transcriptional activity of p53.
- Author
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Hublitz P, Kunowska N, Mayer UP, Müller JM, Heyne K, Yin N, Fritzsche C, Poli C, Miguet L, Schupp IW, van Grunsven LA, Potiers N, van Dorsselaer A, Metzger E, Roemer K, and Schüle R
- Subjects
- Animals, Apoptosis, Gene Expression Regulation, Humans, Mice, Mice, Knockout, Promoter Regions, Genetic, Protein Interaction Mapping, RNA Interference, Repressor Proteins metabolism, Tumor Suppressor Protein p53 metabolism, Histone Acetyltransferases antagonists & inhibitors, Repressor Proteins physiology, Transcription, Genetic, Tumor Suppressor Protein p53 genetics
- Abstract
Most transcriptional repression pathways depend on the targeted deacetylation of histone tails. In this report, we characterize NIR, a novel transcriptional corepressor with inhibitor of histone acetyltransferase (INHAT) activity. NIR (Novel INHAT Repressor) is ubiquitously expressed throughout embryonic development and adulthood. NIR is a potent transcriptional corepressor that is not blocked by histone deacetylase inhibitors and is capable of silencing both basal and activator-driven transcription. NIR directly binds to nucleosomes and core histones and prevents acetylation by histone acetyltransferases, thus acting as a bona fide INHAT. Using a tandem affinity purification approach, we identified the tumor suppressor p53 as a NIR-interacting partner. Association of p53 and NIR was verified in vitro and in vivo. Upon recruitment by p53, NIR represses transcription of both p53-dependent reporters and endogenous target genes. Knock-down of NIR by RNA interference significantly enhances histone acetylation at p53-regulated promoters. Moreover, p53-dependent apoptosis is robustly increased upon depletion of NIR. In summary, our findings describe NIR as a novel INHAT that plays an important role in the control of p53 function.
- Published
- 2005
- Full Text
- View/download PDF
50. The LIM-only coactivator FHL2 modulates WT1 transcriptional activity during gonadal differentiation.
- Author
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Du X, Hublitz P, Günther T, Wilhelm D, Englert C, and Schüle R
- Subjects
- Animals, Anti-Mullerian Hormone, Cell Differentiation, Cell Line, DAX-1 Orphan Nuclear Receptor, DNA-Binding Proteins genetics, Female, Growth Inhibitors biosynthesis, Growth Inhibitors genetics, Homeodomain Proteins genetics, LIM-Homeodomain Proteins, Male, Mice, Models, Genetic, Ovary embryology, RNA, Messenger biosynthesis, Receptors, Retinoic Acid genetics, Sex Determination Processes, Testicular Hormones biosynthesis, Testicular Hormones genetics, Testis embryology, Transcription Factors genetics, Transcriptional Activation, Two-Hybrid System Techniques, Glycoproteins, Homeodomain Proteins physiology, Muscle Proteins, Ovary metabolism, Repressor Proteins, Testis metabolism, Transcription Factors physiology, WT1 Proteins physiology
- Abstract
An essential step during sex determination is the maintenance of the Müllerian duct in females and its regression in males caused by the expression of Müllerian inhibiting substance (MIS). In testes, the Wilms' tumor suppressor and the orphan nuclear receptor SF1 cooperatively bind to the promoter and activate transcription of MIS. In the ovaries, on the other hand, the orphan nuclear receptor DAX1 binds to SF1, inhibits transactivation by WT1/SF1 and thereby suppresses the induction of MIS expression. In addition, WT1 itself is responsible for the upregulation of DAX1 transcription. So far, little is known on which protein-protein interactions or cofactors elicit the spatiotemporal control of WT1-mediated transcription. Here we demonstrate coexpression of the LIM-only coactivator FHL2 and WT1. FHL2 and WT1 functionally interact both in vitro and in vivo. The importance of this interaction is revealed by the ability of FHL2 to potentiate the synergistic induction of MIS gene expression by WT1/SF1. Moreover, FHL2 coactivates transactivation of the DAX1 promoter by WT1. Hence, we present FHL2 as a novel transcriptional coactivator of WT1. The ability to modulate both DAX1 and MIS expression might allow FHL2 to act in the molecular fine tuning of WT1-dependent control mechanisms in the reproductive organs.
- Published
- 2002
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