TY - JOUR A1 - Gierlich, Philipp A1 - Lex, Veronika A1 - Technau, Antje A1 - Keupp, Anne A1 - Morper, Lorenz A1 - Glunz, Amelie A1 - Sennholz, Hanno A1 - Rachor, Johannes A1 - Sauer, Sascha A1 - Marcu, Ana A1 - Grigoleit, Götz Ulrich A1 - Wölfl, Matthias A1 - Schlegel, Paul G. A1 - Eyrich, Matthias T1 - Prostaglandin E\(_2\) in a TLR3‑ and 7/8‑agonist‑based DC maturation cocktail generates mature, cytokine‑producing, migratory DCs but impairs antigen cross‑presentation to CD8\(^+\) T cells JF - Cancer Immunology, Immunotherapy N2 - Mature dendritic cells (DCs) represent cellular adjuvants for optimal antigen presentation in cancer vaccines. Recently, a combination of prostaglandin E\(_2\) (PGE\(_2\)) with Toll-like receptor agonists (TLR-P) was proposed as a new standard to generate superior cytokine-producing DCs with high migratory capacity. Here, we compare TLR-P DCs with conventional DCs matured only with the proinflammatory cytokines TNFα and IL-1ß (CDCs), focussing on the interaction of resulting DCs with CD8\(^+\) T-cells. TLR-P matured DCs showed elevated expression of activation markers such as CD80 and CD83 compared to CDCs, together with a significantly higher migration capacity. Secretion of IL-6, IL-8, IL-10, and IL-12 was highest after 16 h in TLR-P DCs, and only TLR-P DCs secreted active IL-12p70. TLR-P DCs as well as CDCs successfully primed multifunctional CD8\(^+\) T-cells from naïve precursors specific for the peptide antigens Melan-A, NLGN4X, and PTP with comparable priming efficacy and T-cell receptor avidity. CD8\(^+\) T-cells primed by TLR-P DCs showed significantly elevated expression of the integrin VLA-4 and a trend for higher T-cell numbers after expansion. In contrast, TLR-P DCs displayed a substantially reduced capability to cross-present CMVpp65 protein antigen to pp65-specific T cells, an effect that was dose-dependent on PGE2 during DC maturation and reproducible with several responder T-cell lines. In conclu-sion, TLR-P matured DCs might be optimal presenters of antigens not requiring processing such as short peptides. However, PGE\(_2\) seems less favorable for maturation of DCs intended to process and cross-present more complex vaccine antigens such as lysates, proteins or long peptides. KW - dendritic cells KW - cancer vaccines KW - prostaglandin E2 KW - TLR agonists KW - tumor-specific CD8+ T cells Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232311 SN - 0340-7004 VL - 69 ER - TY - JOUR A1 - Van Haute, Lindsey A1 - Dietmann, Sabine A1 - Kremer, Laura A1 - Hussain, Shobbir A1 - Pearce, Sarah F. A1 - Powell, Christopher A. A1 - Rorbach, Joanna A1 - Lantaff, Rebecca A1 - Blanco, Sandra A1 - Sauer, Sascha A1 - Kotzaeridou, Urania A1 - Hoffmann, Georg F. A1 - Memari, Yasin A1 - Kolb-Kokocinski, Anja A1 - Durbin, Richard A1 - Mayr, Johannes A. A1 - Frye, Michaela A1 - Prokisch, Holger A1 - Minczuk, Michal T1 - Deficient methylation and formylation of mt-tRNA(Met) wobble cytosine in a patient carrying mutations in NSUN3 JF - Nature Communications N2 - Epitranscriptome modifications are required for structure and function of RNA and defects in these pathways have been associated with human disease. Here we identify the RNA target for the previously uncharacterized 5-methylcytosine (m5C) methyltransferase NSun3 and link m5C RNA modifications with energy metabolism. Using whole-exome sequencing, we identified loss-of-function mutations in NSUN3 in a patient presenting with combined mitochondrial respiratory chain complex deficiency. Patient-derived fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of NSun3. We show that NSun3 is required for deposition of m5C at the anticodon loop in the mitochondrially encoded transfer RNA methionine (mt-tRNAMet). Further, we demonstrate that m5C deficiency in mt-tRNAMet results in the lack of 5-formylcytosine (f5C) at the same tRNA position. Our findings demonstrate that NSUN3 is necessary for efficient mitochondrial translation and reveal that f5C in human mitochondrial RNA is generated by oxidative processing of m5C. KW - Methylation KW - RNA KW - Transferases Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165998 VL - 7 ER - TY - JOUR A1 - Plauth, Annabell A1 - Geikowski, Anne A1 - Cichon, Susanne A1 - Wowro, Sylvia J. A1 - Liedgens, Linda A1 - Rousseau, Morten A1 - Weidner, Christopher A1 - Fuhr, Luise A1 - Kliem, Magdalena A1 - Jenkins, Gail A1 - Lotito, Silvina A1 - Wainwright, Linda J. A1 - Sauer, Sascha T1 - Hormetic shifting of redox environment by pro-oxidative resveratrol protects cells against stress JF - Free Radical Biology and Medicine N2 - Resveratrol has gained tremendous interest owing to multiple reported health-beneficial effects. However, the underlying key mechanism of action of this natural product remained largely controversial. Here, we demonstrate that under physiologically relevant conditions major biological effects of resveratrol can be attributed to its generation of oxidation products such as reactive oxygen species (ROS). At low nontoxic concentrations (in general < 50 mu M), treatment with resveratrol increased viability in a set of representative cell models, whereas application of quenchers of ROS completely truncated these beneficial effects. Notably, resveratrol treatment led to mild, Nrf2-specific gene expression reprogramming. For example, in primary epidermal keratinocytes derived from human skin this coordinated process resulted in a 1.3-fold increase of endogenously generated glutathione (GSH) and subsequently in a quantitative reduction of the cellular redox environment by 2.61 mV mmol GSH per g protein. After induction of oxidative stress by using 0.78% (v/v) ethanol, endogenous generation of ROS was consequently reduced by 24% in resveratrol pre-treated cells. In contrast to the common perception that resveratrol acts mainly as a chemical antioxidant or as a target protein-specific ligand, we propose that the cellular response to resveratrol treatment is essentially based on oxidative triggering. In physiological microenvironments this molecular training can lead to hormetic shifting of cellular defense towards a more reductive state to improve physiological resilience to oxidative stress. KW - Trans-reservatrol KW - Hydrogen-peroxide KW - In-vitro KW - Hormesis KW - Ethanol KW - Oxygen KW - Nrf2 KW - Glutathione KW - Metabolism KW - Polyphenols KW - ROS KW - Oxidative stress KW - Redox environment KW - Skin KW - Epidermis Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187186 VL - 99 ER - TY - JOUR A1 - Volckmar, Anna-Lena A1 - Han, Chung Ting A1 - Pütter, Carolin A1 - Haas, Stefan A1 - Vogel, Carla I. G. A1 - Knoll, Nadja A1 - Struve, Christoph A1 - Göbel, Maria A1 - Haas, Katharina A1 - Herrfurth, Nikolas A1 - Jarick, Ivonne A1 - Grallert, Harald A1 - Schürmann, Annette A1 - Al-Hasani, Hadi A1 - Hebebrand, Johannes A1 - Sauer, Sascha A1 - Hinney, Anke T1 - Analysis of Genes Involved in Body Weight Regulation by Targeted Re-Sequencing JF - PLoS ONE N2 - Introduction Genes involved in body weight regulation that were previously investigated in genome-wide association studies (GWAS) and in animal models were target-enriched followed by massive parallel next generation sequencing. Methods We enriched and re-sequenced continuous genomic regions comprising FTO, MC4R, TMEM18, SDCCAG8, TKNS, MSRA and TBC1D1 in a screening sample of 196 extremely obese children and adolescents with age and sex specific body mass index (BMI) ≥ 99th percentile and 176 lean adults (BMI ≤ 15th percentile). 22 variants were confirmed by Sanger sequencing. Genotyping was performed in up to 705 independent obesity trios (extremely obese child and both parents), 243 extremely obese cases and 261 lean adults. Results and Conclusion We detected 20 different non-synonymous variants, one frame shift and one nonsense mutation in the 7 continuous genomic regions in study groups of different weight extremes. For SNP Arg695Cys (rs58983546) in TBC1D1 we detected nominal association with obesity (pTDT = 0.03 in 705 trios). Eleven of the variants were rare, thus were only detected heterozygously in up to ten individual(s) of the complete screening sample of 372 individuals. Two of them (in FTO and MSRA) were found in lean individuals, nine in extremely obese. In silico analyses of the 11 variants did not reveal functional implications for the mutations. Concordant with our hypothesis we detected a rare variant that potentially leads to loss of FTO function in a lean individual. For TBC1D1, in contrary to our hypothesis, the loss of function variant (Arg443Stop) was found in an obese individual. Functional in vitro studies are warranted. KW - body weight regulation KW - genes KW - targeted re-sequencing Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167274 VL - 11 IS - 2 ER - TY - JOUR A1 - Weidner, Christopher A1 - Rousseau, Morten A1 - Plauth, Annabell A1 - Wowro, Sylvia J. A1 - Fischer, Cornelius A1 - Abdel-Aziz, Heba A1 - Sauer, Sascha T1 - Iberis amara Extract Induces Intracellular Formation of Reactive Oxygen Species and Inhibits Colon Cancer JF - PLoS ONE N2 - Massively increasing global incidences of colorectal cancer require efficient treatment and prevention strategies. Here, we report unexpected anticancerogenic effects of hydroethanolic Iberis amara extract (IAE), which is known as a widely used phytomedical product for treating gastrointestinal complaints. IAE significantly inhibited the proliferation of HT-29 and T84 colon carcinoma cells with an inhibitory concentration (IC\(_{50}\)) of 6 and 9 μg/ml, respectively, and further generated inhibitory effects in PC-3 prostate and MCF7 breast cancer cells. Inhibition of proliferation in HT-29 cells was associated with a G2/M phase cell cycle arrest including reduced expression of various regulatory marker proteins. Notably, in HT-29 cells IAE further induced apoptosis by intracellular formation of reactive oxygen species (ROS). Consistent with predictions derived from our in vitro experiments, bidaily oral gavage of 50 mg/kg of IAE over 4 weeks resulted in significant inhibition of tumor growth in a mouse HT-29 tumor xenograft model. Taken together, Iberis amara extracts could become useful alternatives for preventing and treating the progression of colon cancer. KW - iberis amara extract KW - colorectal cancer KW - treatment KW - prevention Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167044 VL - 11 IS - 4 ER - TY - JOUR A1 - Vučićević, Dubravka A1 - Gehre, Maja A1 - Dhamija, Sonam A1 - Friis-Hansen, Lennart A1 - Meierhofer, David A1 - Sauer, Sascha A1 - Ørom, Ulf Andersson T1 - The long non-coding RNA PARROT is an upstream regulator of c-Myc and affects proliferation and translation JF - Oncotarget N2 - Long non-coding RNAs are important regulators of gene expression and signaling pathways. The expression of long ncRNAs is dysregulated in cancer and other diseases. The identification and characterization of long ncRNAs is often challenging due to their low expression level and localization to chromatin. Here, we identify a functional long ncRNA, PARROT (Proliferation Associated RNA and Regulator Of Translation) transcribed by RNA polymerase II and expressed at a relatively high level in a number of cell lines. The PARROT long ncRNA is associated with proliferation in both transformed and normal cell lines. We characterize the long ncRNA PARROT as an upstream regulator of c-Myc affecting cellular proliferation and translation using RNA sequencing and mass spectrometry following depletion of the long ncRNA. PARROT is repressed during senescence of human mammary epithelial cells and overexpressed in some cancers, suggesting an important association with proliferation through regulation of c-Myc. With this study, we add to the knowledge of cytoplasmic functional long ncRNAs and extent the long ncRNA-Myc regulatory network in transformed and normal cells. KW - PARROT KW - c-Myc KW - long ncRNA KW - upstream regulator Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166519 VL - 7 IS - 23 ER - TY - JOUR A1 - Cui, Huanhuan A1 - Schlesinger, Jenny A1 - Schoenhals, Sophia A1 - Tonjes, Martje A1 - Dunkel, Ilona A1 - Meierhofer, David A1 - Cano, Elena A1 - Schulz, Kerstin A1 - Berger, Michael F. A1 - Haack, Timm A1 - Abdelilah-Seyfried, Salim A1 - Bulyk, Martha L. A1 - Sauer, Sascha A1 - Sperling, Silke R. T1 - Phosphorylation of the chromatin remodeling factor DPF3a induces cardiac hypertrophy through releasing HEY repressors from DNA JF - Nucleic Acids Research N2 - DPF3 (BAF45c) is a member of the BAF chromatin remodeling complex. Two isoforms have been described, namely DPF3a and DPF3b. The latter binds to acetylated and methylated lysine residues of histones. Here, we elaborate on the role of DPF3a and describe a novel pathway of cardiac gene transcription leading to pathological cardiac hypertrophy. Upon hypertrophic stimuli, casein kinase 2 phosphorylates DPF3a at serine 348. This initiates the interaction of DPF3a with the transcriptional repressors HEY, followed by the release of HEY from the DNA. Moreover, BRG1 is bound by DPF3a, and is thus recruited to HEY genomic targets upon interaction of the two components. Consequently, the transcription of downstream targets such as NPPA and GATA4 is initiated and pathological cardiac hypertrophy is established. In human, DPF3a is significantly up-regulated in hypertrophic hearts of patients with hypertrophic cardiomyopathy or aortic stenosis. Taken together, we show that activation of DPF3a upon hypertrophic stimuli switches cardiac fetal gene expression from being silenced by HEY to being activated by BRG1. Thus, we present a novel pathway for pathological cardiac hypertrophy, whose inhibition is a long-term therapeutic goal for the treatment of the course of heart failure. KW - phosphorylation KW - DPF3a KW - HEY repressors KW - DNA KW - cardiac hypertrophy Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166391 VL - 44 IS - 6 ER - TY - JOUR A1 - Telorac, Jonas A1 - Prykhozhij, Sergey V. A1 - Schöne, Stefanie A1 - Meierhofer, David A1 - Sauer, Sascha A1 - Thomas-Chollier, Morgane A1 - Meijsing, Sebastiaan H. T1 - Identification and characterization of DNA sequences that prevent glucocorticoid receptor binding to nearby response elements JF - Nucleic Acids Research N2 - Out of the myriad of potential DNA binding sites of the glucocorticoid receptor (GR) found in the human genome, only a cell-type specific minority is actually bound, indicating that the presence of a recognition sequence alone is insufficient to specify where GR binds. Cooperative interactions with other transcription factors (TFs) are known to contribute to binding specificity. Here, we reasoned that sequence signals preventing GR recruitment to certain loci provide an alternative means to confer specificity. Motif analyses uncovered candidate Negative Regulatory Sequences (NRSs) that interfere with genomic GR binding. Subsequent functional analyses demonstrated that NRSs indeed prevent GR binding to nearby response elements. We show that NRS activity is conserved across species, found in most tissues and that they also interfere with the genomic binding of other TFs. Interestingly, the effects of NRSs appear not to be a simple consequence of changes in chromatin accessibility. Instead, we find that NRSs interact with proteins found at sub-nuclear structures called paraspeckles and that these proteins might mediate the repressive effects of NRSs. Together, our studies suggest that the joint influence of positive and negative sequence signals partition the genome into regions where GR can bind and those where it cannot. KW - DNA sequencing KW - glucocorticoid receptor KW - DNA binding KW - transcription factors Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166330 VL - 44 IS - 13 ER - TY - JOUR A1 - Conrad, Thomas A1 - Albrecht, Anne-Susann A1 - Rodrigues de Melo Costa, Veronica A1 - Sauer, Sascha A1 - Meierhofer, David A1 - Andersson Ørom, Ulf T1 - Serial interactome capture of the human cell nucleus JF - Nature Communications N2 - Novel RNA-guided cellular functions are paralleled by an increasing number of RNA-binding proteins (RBPs). Here we present ‘serial RNA interactome capture’ (serIC), a multiple purification procedure of ultraviolet-crosslinked poly(A)–RNA–protein complexes that enables global RBP detection with high specificity. We apply serIC to the nuclei of proliferating K562 cells to obtain the first human nuclear RNA interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including few factors without known RNA-binding domains that are in good agreement with computationally predicted RNA binding. serIC extends the number of DNA–RNA-binding proteins (DRBPs), and reveals a network of RBPs involved in p53 signalling and double-strand break repair. serIC is an effective tool to couple global RBP capture with additional selection or labelling steps for specific detection of highly purified RBPs. KW - human cell nucleus KW - serial RNA interactome capture KW - RNA-binding proteins Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166172 VL - 7 IS - 11212 ER -