91 results on '"Tilman Borggrefe"'
Search Results
2. Monitoring autochthonous lung tumors induced by somatic CRISPR gene editing in mice using a secreted luciferase
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Nastasja Merle, Sabrina Elmshäuser, Florian Strassheimer, Michael Wanzel, Alexander M. König, Julianne Funk, Michelle Neumann, Katharina Kochhan, Frederik Helmprobst, Axel Pagenstecher, Andrea Nist, Marco Mernberger, André Schneider, Thomas Braun, Tilman Borggrefe, Rajkumar Savai, Oleg Timofeev, and Thorsten Stiewe
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Autochthonous mouse tumor ,Lung cancer ,Orthotopic tumor ,Luciferase ,CRISPR ,Adenovirus ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Abstract Background In vivo gene editing of somatic cells with CRISPR nucleases has facilitated the generation of autochthonous mouse tumors, which are initiated by genetic alterations relevant to the human disease and progress along a natural timeline as in patients. However, the long and variable, orthotopic tumor growth in inner organs requires sophisticated, time-consuming and resource-intensive imaging for longitudinal disease monitoring and impedes the use of autochthonous tumor models for preclinical studies. Methods To facilitate a more widespread use, we have generated a reporter mouse that expresses a Cre-inducible luciferase from Gaussia princeps (GLuc), which is secreted by cells in an energy-consuming process and can be measured quantitatively in the blood as a marker for the viable tumor load. In addition, we have developed a flexible, complementary toolkit to rapidly assemble recombinant adenoviruses (AVs) for delivering Cre recombinase together with CRISPR nucleases targeting cancer driver genes. Results We demonstrate that intratracheal infection of GLuc reporter mice with CRISPR-AVs efficiently induces lung tumors driven by mutations in the targeted cancer genes and simultaneously activates the GLuc transgene, resulting in GLuc secretion into the blood by the growing tumor. GLuc blood levels are easily and robustly quantified in small-volume blood samples with inexpensive equipment, enable tumor detection already several months before the humane study endpoint and precisely mirror the kinetics of tumor development specified by the inducing gene combination. Conclusions Our study establishes blood-based GLuc monitoring as an inexpensive, rapid, high-throughput and animal-friendly method to longitudinally monitor autochthonous tumor growth in preclinical studies.
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- 2022
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3. Hydroxylation of the NOTCH1 intracellular domain regulates Notch signaling dynamics
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Francesca Ferrante, Benedetto Daniele Giaimo, Tobias Friedrich, Toshiya Sugino, Daniel Mertens, Sabrina Kugler, Bernd Martin Gahr, Steffen Just, Leiling Pan, Marek Bartkuhn, Michael Potente, Franz Oswald, and Tilman Borggrefe
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Cytology ,QH573-671 - Abstract
Abstract Notch signaling plays a pivotal role in the development and, when dysregulated, it contributes to tumorigenesis. The amplitude and duration of the Notch response depend on the posttranslational modifications (PTMs) of the activated NOTCH receptor – the NOTCH intracellular domain (NICD). In normoxic conditions, the hydroxylase FIH (factor inhibiting HIF) catalyzes the hydroxylation of two asparagine residues of the NICD. Here, we investigate how Notch-dependent gene transcription is regulated by hypoxia in progenitor T cells. We show that the majority of Notch target genes are downregulated upon hypoxia. Using a hydroxyl-specific NOTCH1 antibody we demonstrate that FIH-mediated NICD1 hydroxylation is reduced upon hypoxia or treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG). We find that a hydroxylation-resistant NICD1 mutant is functionally impaired and more ubiquitinated. Interestingly, we also observe that the NICD1-deubiquitinating enzyme USP10 is downregulated upon hypoxia. Moreover, the interaction between the hydroxylation-defective NICD1 mutant and USP10 is significantly reduced compared to the NICD1 wild-type counterpart. Together, our data suggest that FIH hydroxylates NICD1 in normoxic conditions, leading to the recruitment of USP10 and subsequent NICD1 deubiquitination and stabilization. In hypoxia, this regulatory loop is disrupted, causing a dampened Notch response.
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- 2022
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4. SPEN is required for Xist upregulation during initiation of X chromosome inactivation
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Teresa Robert-Finestra, Beatrice F. Tan, Hegias Mira-Bontenbal, Erika Timmers, Cristina Gontan, Sarra Merzouk, Benedetto Daniele Giaimo, François Dossin, Wilfred F. J. van IJcken, John W. M. Martens, Tilman Borggrefe, Edith Heard, and Joost Gribnau
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Science - Abstract
SPEN is a key player in initiation of X chromosome inactivation. Here, the authors show that SPEN is required for Xist-mediated silencing of its own antisense regulator Tsix.
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- 2021
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5. A Drosophila Su(H) model of Adams-Oliver Syndrome reveals cofactor titration as a mechanism underlying developmental defects.
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Ellen K Gagliani, Lisa M Gutzwiller, Yi Kuang, Yoshinobu Odaka, Phillipp Hoffmeister, Stefanie Hauff, Aleksandra Turkiewicz, Emily Harding-Theobald, Patrick J Dolph, Tilman Borggrefe, Franz Oswald, Brian Gebelein, and Rhett A Kovall
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Genetics ,QH426-470 - Abstract
Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that heterozygous Drosophila with the AOS-like Su(H) allele behave in an opposing manner to heterozygous flies with a Su(H) null allele, due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants have decreased DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby the AOS-associated RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.
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- 2022
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6. SUMOylated non-canonical polycomb PRC1.6 complex as a prerequisite for recruitment of transcription factor RBPJ
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Małgorzata Sotomska, Robert Liefke, Francesca Ferrante, Heiko Schwederski, Franz Oswald, and Tilman Borggrefe
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Notch signaling ,Polycomb repressive complex ,RBPJ ,Sumoylation ,Epigenetics ,Genetics ,QH426-470 - Abstract
Abstract Background Notch signaling controls cell fate decisions in many contexts during development and adult stem cell homeostasis and, when dysregulated, leads to carcinogenesis. The central transcription factor RBPJ assembles the Notch coactivator complex in the presence of Notch signaling, and represses Notch target gene expression in its absence. Results We identified L3MBTL2 and additional members of the non-canonical polycomb repressive PRC1.6 complex in DNA-bound RBPJ associated complexes and demonstrate that L3MBTL2 directly interacts with RBPJ. Depletion of RBPJ does not affect occupancy of PRC1.6 components at Notch target genes. Conversely, absence of L3MBTL2 reduces RBPJ occupancy at enhancers of Notch target genes. Since L3MBTL2 and additional members of the PRC1.6 are known to be SUMOylated, we investigated whether RBPJ uses SUMO-moieties as contact points. Indeed, we found that RBPJ binds to SUMO2/3 and that this interaction depends on a defined SUMO-interaction motif. Furthermore, we show that pharmacological inhibition of SUMOylation reduces RBPJ occupancy at Notch target genes. Conclusions We propose that the PRC1.6 complex and its conjugated SUMO-modifications provide a favorable environment for binding of RBPJ to Notch target genes.
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- 2021
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7. Enhancer-promoter communication: unraveling enhancer strength and positioning within a given topologically associating domain (TAD)
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Benedetto Daniele Giaimo and Tilman Borggrefe
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Medicine ,Biology (General) ,QH301-705.5 - Published
- 2022
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8. Loss of the transcription factor RBPJ induces disease-promoting properties in brain pericytes
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Rodrigo Diéguez-Hurtado, Katsuhiro Kato, Benedetto Daniele Giaimo, Melina Nieminen-Kelhä, Hendrik Arf, Francesca Ferrante, Marek Bartkuhn, Tobias Zimmermann, M. Gabriele Bixel, Hanna M. Eilken, Susanne Adams, Tilman Borggrefe, Peter Vajkoczy, and Ralf H. Adams
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Science - Abstract
Pericytes are perivascular cells essential for blood-brain barrier maintenance. Here Diéguez-Hurtado et al. show that depletion of the transcription factor RBPJ in pericytes affects their molecular identity and disturbs endothelial cell behaviour, inducing the formation of vascular lesions in the brain.
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- 2019
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9. The histone variant H2A.Z in gene regulation
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Benedetto Daniele Giaimo, Francesca Ferrante, Andreas Herchenröther, Sandra B. Hake, and Tilman Borggrefe
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H2A.Z ,H2Av ,Histone variant ,p400 ,Domino ,Tip60 ,Genetics ,QH426-470 - Abstract
Abstract The histone variant H2A.Z is involved in several processes such as transcriptional control, DNA repair, regulation of centromeric heterochromatin and, not surprisingly, is implicated in diseases such as cancer. Here, we review the recent developments on H2A.Z focusing on its role in transcriptional activation and repression. H2A.Z, as a replication-independent histone, has been studied in several model organisms and inducible mammalian model systems. Its loading machinery and several modifying enzymes have been recently identified, and some of the long-standing discrepancies in transcriptional activation and/or repression are about to be resolved. The buffering functions of H2A.Z, as supported by genome-wide localization and analyzed in several dynamic systems, are an excellent example of transcriptional control. Posttranslational modifications such as acetylation and ubiquitination of H2A.Z, as well as its specific binding partners, are in our view central players in the control of gene expression. Understanding the key-mechanisms in either turnover or stabilization of H2A.Z-containing nucleosomes as well as defining the H2A.Z interactome will pave the way for therapeutic applications in the future.
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- 2019
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10. Phospho-Site Mutations in Transcription Factor Suppressor of Hairless Impact Notch Signaling Activity During Hematopoiesis in Drosophila
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Lisa Frankenreiter, Bernd M. Gahr, Hannes Schmid, Mirjam Zimmermann, Sebastian Deichsel, Philipp Hoffmeister, Aleksandra Turkiewicz, Tilman Borggrefe, Franz Oswald, and Anja C. Nagel
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Notch signaling ,Suppressor of Hairless ,phosphorylation ,hematopoiesis ,Drosophila ,Biology (General) ,QH301-705.5 - Abstract
The highly conserved Notch signaling pathway controls a multitude of developmental processes including hematopoiesis. Here, we provide evidence for a novel mechanism of tissue-specific Notch regulation involving phosphorylation of CSL transcription factors within the DNA-binding domain. Earlier we found that a phospho-mimetic mutation of the Drosophila CSL ortholog Suppressor of Hairless [Su(H)] at Ser269 impedes DNA-binding. By genome-engineering, we now introduced phospho-specific Su(H) mutants at the endogenous Su(H) locus, encoding either a phospho-deficient [Su(H)S269A] or a phospho-mimetic [Su(H)S269D] isoform. Su(H)S269D mutants were defective of Notch activity in all analyzed tissues, consistent with impaired DNA-binding. In contrast, the phospho-deficient Su(H)S269A mutant did not generally augment Notch activity, but rather specifically in several aspects of blood cell development. Unexpectedly, this process was independent of the corepressor Hairless acting otherwise as a general Notch antagonist in Drosophila. This finding is in agreement with a novel mode of Notch regulation by posttranslational modification of Su(H) in the context of hematopoiesis. Importantly, our studies of the mammalian CSL ortholog (RBPJ/CBF1) emphasize a potential conservation of this regulatory mechanism: phospho-mimetic RBPJS221D was dysfunctional in both the fly as well as two human cell culture models, whereas phospho-deficient RBPJS221A rather gained activity during fly hematopoiesis. Thus, dynamic phosphorylation of CSL-proteins within the DNA-binding domain provides a novel means to fine-tune Notch signal transduction in a context-dependent manner.
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- 2021
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11. ATP Purinergic Receptor P2X1-Dependent Suicidal NETosis Induced by Cryptosporidium parvum under Physioxia Conditions
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Seyed Sajjad Hasheminasab, Iván Conejeros, Zahady D. Velásquez, Tilman Borggrefe, Ulrich Gärtner, Faustin Kamena, Anja Taubert, and Carlos Hermosilla
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Cryptosporidium parvum ,NETosis ,purinergic receptor P2X1 ,Notch ,glycolysis ,PMN ,Biology (General) ,QH301-705.5 - Abstract
Cryptosporidiosis is a zoonotic intestinal disease that affects humans, wildlife, and neonatal cattle, caused by Cryptosporidium parvum. Neutrophil extracellular traps (NETs), also known as suicidal NETosis, are a powerful and ancient innate effector mechanism by which polymorphonuclear neutrophils (PMN) battle parasitic organisms like protozoa and helminths. Here, C. parvum oocysts and live sporozoites were utilized to examine suicidal NETosis in exposed bovine PMN under both 5% O2 (physiological conditions within small intestinal tract) and 21% O2 (normal hyperoxic conditions in research facilities). Both sporozoites and oocysts induced suicidal NETosis in exposed PMN under physioxia (5% O2) and hyperoxia (21% O2). Besides, C. parvum-induced suicidal NETosis was affirmed by total break of PMN, co-localization of extracellular DNA decorated with pan-histones (H1A, H2A/H2B, H3, H4) and neutrophil elastase (NE) by means of confocal- and immunofluorescence microscopy investigations. C. parvum-triggered NETs entrapped sporozoites and impeded sporozoite egress from oocysts covered by released NETs, according to scanning electron microscopy (SEM) examination. Live cell 3D-holotomographic microscopy analysis visualized early parasite-induced PMN morphological changes, such as the formation of membrane protrusions towards C. parvum while undergoing NETosis. Significant reduction of C. parvum-induced suicidal NETosis was measured after PMN treatments with purinergic receptor P2X1 inhibitor NF449, under both oxygen circumstances, this receptor was found to play a critical role in the induction of NETs, indicating its importance. Similarly, inhibition of PMN glycolysis via 2-deoxy glucose treatments resulted in a reduction of C. parvum-triggered suicidal NETosis but not significantly. Extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) were not increased in C. parvum-exposed cells, according to measurements of PMN energetic state. Treatments with inhibitors of plasma membrane monocarboxylate transporters (MCTs) of lactate failed to significantly reduce C. parvum-mediated NET extrusion. Concerning Notch signaling, no significant reduction was detected after PMN treatments with two specific Notch inhibitors, i.e., DAPT and compound E. Overall, we here describe for the first time the pivotal role of ATP purinergic receptor P2X1 in C. parvum-mediated suicidal NETosis under physioxia (5% O2) and its anti-cryptosporidial properties.
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- 2022
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12. JAZF1, A Novel p400/TIP60/NuA4 Complex Member, Regulates H2A.Z Acetylation at Regulatory Regions
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Tara Procida, Tobias Friedrich, Antonia P. M. Jack, Martina Peritore, Clemens Bönisch, H. Christian Eberl, Nadine Daus, Konstantin Kletenkov, Andrea Nist, Thorsten Stiewe, Tilman Borggrefe, Matthias Mann, Marek Bartkuhn, and Sandra B. Hake
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JAZF1 ,H2A.Z ,histone variants ,TIP60 ,acetylation ,enhancer ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Histone variants differ in amino acid sequence, expression timing and genomic localization sites from canonical histones and convey unique functions to eukaryotic cells. Their tightly controlled spatial and temporal deposition into specific chromatin regions is accomplished by dedicated chaperone and/or remodeling complexes. While quantitatively identifying the chaperone complexes of many human H2A variants by using mass spectrometry, we also found additional members of the known H2A.Z chaperone complexes p400/TIP60/NuA4 and SRCAP. We discovered JAZF1, a nuclear/nucleolar protein, as a member of a p400 sub-complex containing MBTD1 but excluding ANP32E. Depletion of JAZF1 results in transcriptome changes that affect, among other pathways, ribosome biogenesis. To identify the underlying molecular mechanism contributing to JAZF1’s function in gene regulation, we performed genome-wide ChIP-seq analyses. Interestingly, depletion of JAZF1 leads to reduced H2A.Z acetylation levels at > 1000 regulatory sites without affecting H2A.Z nucleosome positioning. Since JAZF1 associates with the histone acetyltransferase TIP60, whose depletion causes a correlated H2A.Z deacetylation of several JAZF1-targeted enhancer regions, we speculate that JAZF1 acts as chromatin modulator by recruiting TIP60’s enzymatic activity. Altogether, this study uncovers JAZF1 as a member of a TIP60-containing p400 chaperone complex orchestrating H2A.Z acetylation at regulatory regions controlling the expression of genes, many of which are involved in ribosome biogenesis.
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- 2021
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13. Setting the Stage for Notch: The Drosophila Su(H)-Hairless Repressor Complex.
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Tilman Borggrefe and Franz Oswald
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Biology (General) ,QH301-705.5 - Abstract
Notch signaling is iteratively used throughout development to maintain stem cell potential or in other instances allow differentiation. The central transcription factor in Notch signaling is CBF-1/RBP-J, Su(H), Lag-1 (CSL)-Su(H) in Drosophila-which functions as a molecular switch between transcriptional activation and repression. Su(H) represses transcription by forming a complex with the corepressor Hairless (H). The Su(H)-repressor complex not only competes with the Notch intracellular domain (NICD) but also configures the local chromatin landscape. In this issue, Yuan and colleagues determined the structure of the Su(H)/H complex, showing that a major conformational change within Su(H) explains why the binding of NICD and H is mutually exclusive.
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- 2016
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14. Data from Endothelial RBPJ Is Essential for the Education of Tumor-Associated Macrophages
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Juan Rodriguez-Vita, Andreas Fischer, Tilman Borggrefe, Adelheid Cerwenka, Benedetto Daniele Giaimo, Adrian Stögbauer, Francesca De Angelis Rigotti, Jacqueline Taylor, Eleni Zimmer, Tara Ziegelbauer, Lorea Jordana-Urriza, Lena Wiedmann, Sarah Böhn, Iris Moll, Ronja Mülfarth, and Elisenda Alsina-Sanchis
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Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic cancers worldwide. EOC cells educate tumor-associated macrophages (TAM) through CD44-mediated cholesterol depletion to generate an immunosuppressive tumor microenvironment (TME). In addition, tumor cells frequently activate Notch1 receptors on endothelial cells (EC) to facilitate metastasis. However, further work is required to establish whether the endothelium also influences the education of recruited monocytes. Here, we report that canonical Notch signaling through RBPJ in ECs is an important player in the education of TAMs and EOC progression. Deletion of Rbpj in the endothelium of adult mice reduced infiltration of monocyte-derived macrophages into the TME of EOC and prevented the acquisition of a typical TAM gene signature; this was associated with stronger cytotoxic activity of T cells and decreased tumor burden. Mechanistically, CXCL2 was identified as a novel Notch/RBPJ target gene that regulated the expression of CD44 on monocytes and subsequent cholesterol depletion of TAMs. Bioinformatic analysis of ovarian cancer patient data showed that increased CXCL2 expression is accompanied by higher expression of CD44 and TAM education. Together, these findings indicate that EOC cells induce the tumor endothelium to secrete CXCL2 to establish an immunosuppressive microenvironment.Significance:Endothelial Notch signaling favors immunosuppression by increasing CXCL2 secretion to stimulate CD44 expression in macrophages, facilitating their education by tumor cells.
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- 2023
15. Supplementary material from Endothelial RBPJ Is Essential for the Education of Tumor-Associated Macrophages
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Juan Rodriguez-Vita, Andreas Fischer, Tilman Borggrefe, Adelheid Cerwenka, Benedetto Daniele Giaimo, Adrian Stögbauer, Francesca De Angelis Rigotti, Jacqueline Taylor, Eleni Zimmer, Tara Ziegelbauer, Lorea Jordana-Urriza, Lena Wiedmann, Sarah Böhn, Iris Moll, Ronja Mülfarth, and Elisenda Alsina-Sanchis
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It includes extended material and methods, and supplementary figures
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- 2023
16. Notch-dependent and -independent functions of transcription factor RBPJ
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Tobias Friedrich, Francesca Ferrante, Léo Pioger, Andrea Nist, Thorsten Stiewe, Jean-Christophe Andrau, Marek Bartkuhn, Benedetto Daniele Giaimo, and Tilman Borggrefe
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Gene Expression Regulation ,Receptors, Notch ,Immunoglobulin J Recombination Signal Sequence-Binding Protein ,T-Lymphocytes ,Genetics ,Signal Transduction - Abstract
Signal transduction pathways often involve transcription factors that promote activation of defined target gene sets. The transcription factor RBPJ is the central player in Notch signaling and either forms an activator complex with the Notch intracellular domain (NICD) or a repressor complex with corepressors like KYOT2/FHL1. The balance between these two antagonizing RBPJ-complexes depends on the activation state of the Notch receptor regulated by cell-to-cell interaction, ligand binding and proteolytic cleavage events. Here, we depleted RBPJ in mature T-cells lacking active Notch signaling and performed RNA-Seq, ChIP-Seq and ATAC-seq analyses. RBPJ depletion leads to upregulation of many Notch target genes. Ectopic expression of NICD1 activates several Notch target genes and enhances RBPJ occupancy. Based on gene expression changes and RBPJ occupancy we define four different clusters, either RBPJ- and/or Notch-regulated genes. Importantly, we identify early (Hes1 and Hey1) and late Notch-responsive genes (IL2ra). Similarly, to RBPJ depletion, interfering with transcriptional repression by squelching with cofactor KYOT2/FHL1, leads to upregulation of Notch target genes. Taken together, RBPJ is not only an essential part of the Notch co-activator complex but also functions as a repressor in a Notch-independent manner.
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- 2022
17. The H2A.Z.1/PWWP2A/NuRD-associated protein HMG20A controls early head and heart developmental transcription programs
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Andreas Herchenröther, Stefanie Gossen, Tobias Friedrich, Alexander Reim, Nadine Daus, Felix Diegmüller, Jörg Leers, Hakimeh Moghaddas Sani, Sarah Gerstner, Leah Schwarz, Inga Stellmacher, Laura Victoria Szymkowiak, Andrea Nist, Thorsten Stiewe, Tilman Borggrefe, Matthias Mann, Joel P. Mackay, Marek Bartkuhn, Annette Borchers, Jie Lan, and Sandra B. Hake
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/SummarySpecialized chromatin-binding proteins are required for DNA-based processes during development. We recently established PWWP2A as direct histone variant H2A.Z interactor involved in mitosis and cranial-facial development. Here, we identify the H2A.Z/PWWP2A-associated protein HMG20A as part of several chromatin-modifying complexes including NuRD, and show that it localizes to genomic regulatory regions. Hmg20a depletion causes severe head and heart developmental defects in Xenopus laevis. Our data indicate that craniofacial malformations are caused by defects in neural crest cell (NCC) migration and cartilage formation. These developmental defects are pheno-copied in HMG20A-depleted mESCs, which show inefficient differentiation into NCCs and cardiomyocytes (CMs). Accordingly, loss of HMG20A caused striking deregulation of transcription programs involved in epithelial- mesenchymal transition (EMT) and cardiac differentiation, thereby providing insights into the regulatory circuits controlled by HMG20A. Collectively, our findings implicate HMG20A as part of the H2A.Z/PWWP2A/NuRD-axis and reveal it as a key modulator of the intricate developmental transcription programs that guide NCC and cardiomyocyte differentiation.
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- 2022
18. Correction to: Enhancers and Promoters
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Tilman, Borggrefe and Benedetto, Daniele Giaimo
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- 2022
19. The structure, binding, and function of a Notch transcription complex involving RBPJ and the epigenetic reader protein L3MBTL3
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Daniel Hall, Benedetto Daniele Giaimo, Sung-Soo Park, Wiebke Hemmer, Tobias Friedrich, Francesca Ferrante, Marek Bartkuhn, Zhenyu Yuan, Franz Oswald, Tilman Borggrefe, Jean-François Rual, and Rhett A Kovall
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Genetics - Abstract
The highly conserved Notch pathway transmits signals between neighboring cells to elicit distinct downstream transcriptional programs. In given contexts, Notch is a major regulator of cell fate specification, proliferation, and apoptosis, such that aberrant Notch signaling leads to a pleiotropy of human diseases, including developmental disorders and cancers. The canonical pathway signals through the transcription factor CSL (RBPJ in mammals), which forms a transcriptional activation complex with the intracellular domain of the Notch receptor and the coactivator Mastermind. CSL can also function as a transcriptional repressor by forming complexes with one of several different corepressor proteins, such as FHL1 or SHARP in mammals and Hairless in Drosophila. Recently, we identified the malignant brain tumor (MBT) family member L3MBTL3 as a bona fide RBPJ binding corepressor that recruits the repressive lysine demethylase LSD1/KDM1A to Notch target genes. Here we define the RBPJ-interacting domain (RBP-ID) of L3MBTL3 and report the 2.06 Å crystal structure of the complex formed between RBPJ, the RBP-ID of L3MBTL3 and DNA. The structure reveals the molecular interactions underlying L3MBTL3 complexation with RBPJ, which we comprehensively analyze with a series of L3MBTL3 and RBPJ mutations that span the binding interface. Compared to other RBPJ-binding proteins, we find that L3MBTL3 interacts with RBPJ via an unusual binding motif, which is sensitive to mutations throughout its RBPJ-interacting region. We also show that these disruptive mutations affect RBPJ and L3MBTL3 function in cells, providing further insights into Notch mediated transcriptional regulation.
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- 2022
20. HDAC3 functions as a positive regulator in Notch signal transduction
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Tilman Borggrefe, Marek Bartkuhn, Thorsten Stiewe, Patricia Klöble, Daniel Mertens, Francesca Ferrante, Andrea Nist, Steffen Just, Viola Close, Johanna Meier-Soelch, Benedetto Daniele Giaimo, Franz Oswald, Michael Kracht, and Tobias Zimmermann
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AcademicSubjects/SCI00010 ,Chronic lymphocytic leukemia ,Notch signaling pathway ,Regulator ,Biology ,Peptides, Cyclic ,Histone Deacetylases ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Downregulation and upregulation ,hemic and lymphatic diseases ,Cell Line, Tumor ,Genetics ,medicine ,Animals ,Humans ,Receptor, Notch1 ,030304 developmental biology ,0303 health sciences ,Leukemia ,Protein Stability ,Lysine ,Gene regulation, Chromatin and Epigenetics ,medicine.disease ,HDAC3 ,Cell biology ,Histone Deacetylase Inhibitors ,Histone ,chemistry ,030220 oncology & carcinogenesis ,Mutation ,biology.protein ,Signal transduction ,Apicidin ,Signal Transduction - Abstract
Aberrant Notch signaling plays a pivotal role in T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic leukemia (CLL). Amplitude and duration of the Notch response is controlled by ubiquitin-dependent proteasomal degradation of the Notch1 intracellular domain (NICD1), a hallmark of the leukemogenic process. Here, we show that HDAC3 controls NICD1 acetylation levels directly affecting NICD1 protein stability. Either genetic loss-of-function of HDAC3 or nanomolar concentrations of HDAC inhibitor apicidin lead to downregulation of Notch target genes accompanied by a local reduction of histone acetylation. Importantly, an HDAC3-insensitive NICD1 mutant is more stable but biologically less active. Collectively, these data show a new HDAC3- and acetylation-dependent mechanism that may be exploited to treat Notch1-dependent leukemias.
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- 2020
21. Endothelial RBPJ Is Essential for the Education of Tumor-Associated Macrophages
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Elisenda Alsina-Sanchis, Ronja Mülfarth, Iris Moll, Sarah Böhn, Lena Wiedmann, Lorea Jordana-Urriza, Tara Ziegelbauer, Eleni Zimmer, Jacqueline Taylor, Francesca De Angelis Rigotti, Adrian Stögbauer, Benedetto Daniele Giaimo, Adelheid Cerwenka, Tilman Borggrefe, Andreas Fischer, and Juan Rodriguez-Vita
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Ovarian Neoplasms ,Cancer Research ,Endothelial Cells ,Carcinoma, Ovarian Epithelial ,Mice ,Cholesterol ,Oncology ,Immunoglobulin J Recombination Signal Sequence-Binding Protein ,Tumor-Associated Macrophages ,Tumor Microenvironment ,Humans ,Animals ,Female ,Endothelium - Abstract
Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic cancers worldwide. EOC cells educate tumor-associated macrophages (TAM) through CD44-mediated cholesterol depletion to generate an immunosuppressive tumor microenvironment (TME). In addition, tumor cells frequently activate Notch1 receptors on endothelial cells (EC) to facilitate metastasis. However, further work is required to establish whether the endothelium also influences the education of recruited monocytes. Here, we report that canonical Notch signaling through RBPJ in ECs is an important player in the education of TAMs and EOC progression. Deletion of Rbpj in the endothelium of adult mice reduced infiltration of monocyte-derived macrophages into the TME of EOC and prevented the acquisition of a typical TAM gene signature; this was associated with stronger cytotoxic activity of T cells and decreased tumor burden. Mechanistically, CXCL2 was identified as a novel Notch/RBPJ target gene that regulated the expression of CD44 on monocytes and subsequent cholesterol depletion of TAMs. Bioinformatic analysis of ovarian cancer patient data showed that increased CXCL2 expression is accompanied by higher expression of CD44 and TAM education. Together, these findings indicate that EOC cells induce the tumor endothelium to secrete CXCL2 to establish an immunosuppressive microenvironment. Significance: Endothelial Notch signaling favors immunosuppression by increasing CXCL2 secretion to stimulate CD44 expression in macrophages, facilitating their education by tumor cells.
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- 2022
22. Endothelial Rbpj is essential for the education of tumour-associated macrophages
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Ronja Mülfarth, Elisenda Alsina-Sanchis, Iris Moll, Sarah Böhn, Lena Wiedmann, Lorea Jordana, Tara Ziegelbauer, Jacqueline Taylor, Francesca De Angelis Rigotti, Adrian Stögbauer, Benedetto Daniele Giaimo, Adelheid Cerwenka, Tilman Borggrefe, Andreas Fischer, and Juan Rodriguez-Vita
- Subjects
endocrine system diseases - Abstract
Epithelial ovarian cancer (EOC) is one of the most lethal gynaecological cancers worldwide. EOC cells educate tumour-associated macrophages (TAMs) through CD44-mediated cholesterol depletion to generate an immunosuppressive tumour microenvironment (TME). In addition, tumour cells frequently activate Notch1 receptors on endothelial cells (ECs) to facilitate metastasis. However, little is known whether the endothelium would also influence the education of recruited monocytes. Here, we report that canonical Notch signalling through RBPJ in ECs is an important player in the education of TAMs and EOC progression. Deletion of Rbpj in the endothelium of adult mice reduced infiltration of monocyte-derived macrophages into the TME of EOC and prevented the acquisition of a typical TAM gene signature. This was associated with stronger cytotoxic activity of T cells and decreased tumour burden. Mechanistically, we identified CXCL2 as a novel Notch/RBPJ target gene. This angiocrine factor regulates the expression of CD44 on monocytes and subsequent cholesterol depletion of TAMs. Bioinformatic analysis of ovarian cancer patient data showed that increased CXCL2 expression is accompanied by higher expression of CD44 and TAM education. As such, EOC cells employ the tumour endothelium to secrete CXCL2 in order to facilitate an immunosuppressive microenvironment.
- Published
- 2021
23. SPEN is required for Xist upregulation during initiation of X chromosome inactivation
- Author
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Hegias Mira-Bontenbal, François Dossin, John W.M. Martens, Sarra Merzouk, Tilman Borggrefe, Edith Heard, Beatrice F. Tan, Teresa Robert-Finestra, Wilfred F. J. van IJcken, Joost Gribnau, Benedetto Daniele Giaimo, Erika Timmers, Cristina Gontan, Developmental Biology, Cell biology, and Medical Oncology
- Subjects
Male ,Transcriptional Activation ,Molecular biology ,Science ,General Physics and Astronomy ,Stem cells ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,X-inactivation ,Mice ,Downregulation and upregulation ,Genes, X-Linked ,X Chromosome Inactivation ,Developmental biology ,Animals ,Gene silencing ,Promoter Regions, Genetic ,Psychological repression ,Mice, Knockout ,Multidisciplinary ,Gene Expression Regulation, Developmental ,RNA-Binding Proteins ,Cell Differentiation ,Mouse Embryonic Stem Cells ,General Chemistry ,Chromatin Assembly and Disassembly ,Up-Regulation ,Chromatin ,Antisense RNA ,Cell biology ,DNA-Binding Proteins ,Mice, Inbred C57BL ,Female ,RNA, Long Noncoding ,XIST ,Tsix ,Transcriptome ,human activities - Abstract
At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen−/− ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in initiation of the XCI process., SPEN is a key player in initiation of X chromosome inactivation. Here, the authors show that SPEN is required for Xist-mediated silencing of its own antisense regulator Tsix.
- Published
- 2021
24. A Drosophila Su(H) model of Adams-Oliver Syndrome reveals cofactor titration as a mechanism underlying developmental defects
- Author
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Ellen K. Gagliani, Lisa M. Gutzwiller, Yi Kuang, Yoshinobu Odaka, Phillipp Hoffmeister, Stefanie Hauff, Aleksandra Turkiewicz, Emily Harding-Theobald, Patrick J. Dolph, Tilman Borggrefe, Franz Oswald, Brian Gebelein, and Rhett A. Kovall
- Subjects
Mammals ,Cancer Research ,Scalp ,Receptors, Notch ,Skull ,Limb Deformities, Congenital ,DNA ,Scalp Dermatoses ,Ectodermal Dysplasia ,Genetics ,Animals ,Drosophila Proteins ,Humans ,Drosophila ,Molecular Biology ,Co-Repressor Proteins ,Genetics (clinical) ,Ecology, Evolution, Behavior and Systematics - Abstract
Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that heterozygous Drosophila with the AOS-like Su(H) allele behave in an opposing manner to heterozygous flies with a Su(H) null allele, due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants have decreased DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby the AOS-associated RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.
- Published
- 2021
25. A Drosophila Su(H) Model of Adams-Oliver Syndrome Reveals Notch Cofactor Titration as a Mechanism Underlying Developmental Defects
- Author
-
Ellen K. Gagliani, Brian Gebelein, Tilman Borggrefe, Yi Kuang, Patrick J. Dolph, Rhett A. Kovall, Phillipp Hoffmeister, Aleksandra Turkiewicz, Emily Harding-Theobald, Yoshinobu Odaka, Franz Oswald, Stefanie Hauff, and Lisa M. Gutzwiller
- Subjects
Cofactor binding ,RBPJ ,Chemistry ,Notch signaling pathway ,Wild type ,Allele ,Transcription factor ,Null allele ,Cell biology ,Hairless - Abstract
Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that Drosophila with a single copy of the AOS-like Su(H) allele behave in an opposing manner as flies with a Su(H) null allele due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants decrease DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby a single RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.
- Published
- 2021
26. Loss of the transcription factor RBPJ induces disease-promoting properties in brain pericytes
- Author
-
Tilman Borggrefe, Marek Bartkuhn, M. Gabriele Bixel, Hanna M. Eilken, Melina Nieminen-Kelhä, Ralf H. Adams, Hendrik Arf, Rodrigo Diéguez-Hurtado, Susanne Adams, Peter Vajkoczy, Benedetto Daniele Giaimo, Katsuhiro Kato, Tobias Zimmermann, and Francesca Ferrante
- Subjects
Male ,0301 basic medicine ,Hemangioma, Cavernous, Central Nervous System ,Science ,General Physics and Astronomy ,Vascular permeability ,02 engineering and technology ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,medicine ,Animals ,Humans ,lcsh:Science ,Transcription factor ,Pathological ,Stroke ,Mice, Knockout ,Multidisciplinary ,RBPJ ,Neurodegeneration ,Mesenchymal stem cell ,Brain ,General Chemistry ,021001 nanoscience & nanotechnology ,medicine.disease ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Blood-Brain Barrier ,Immunoglobulin J Recombination Signal Sequence-Binding Protein ,Disease Progression ,Female ,lcsh:Q ,Angiogenesis ,Pericyte ,Pericytes ,0210 nano-technology - Abstract
Sufficient vascular supply is indispensable for brain development and function, whereas dysfunctional blood vessels are associated with human diseases such as vascular malformations, stroke or neurodegeneration. Pericytes are capillary-associated mesenchymal cells that limit vascular permeability and protect the brain by preserving blood-brain barrier integrity. Loss of pericytes has been linked to neurodegenerative changes in genetically modified mice. Here, we report that postnatal inactivation of the Rbpj gene, encoding the transcription factor RBPJ, leads to alteration of cell identity markers in brain pericytes, increases local TGFβ signalling, and triggers profound changes in endothelial behaviour. These changes, which are not mimicked by pericyte ablation, imperil vascular stability and induce the acquisition of pathological landmarks associated with cerebral cavernous malformations. In adult mice, loss of Rbpj results in bigger stroke lesions upon ischemic insult. We propose that brain pericytes can acquire deleterious properties that actively enhance vascular lesion formation and promote pathogenic processes., Pericytes are perivascular cells essential for blood-brain barrier maintenance. Here Diéguez-Hurtado et al. show that depletion of the transcription factor RBPJ in pericytes affects their molecular identity and disturbs endothelial cell behaviour, inducing the formation of vascular lesions in the brain.
- Published
- 2019
27. Phospho-site mutations in transcription factor suppressor of hairless impact notch signaling activity during hematopoiesis in drosophila
- Author
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Aleksandra Turkiewicz, Hannes Schmid, Sebastian Deichsel, Anja C. Nagel, Tilman Borggrefe, Mirjam Zimmermann, Bernd M. Gahr, Franz Oswald, Lisa Frankenreiter, and Philipp Hoffmeister
- Subjects
Phosphorylierung ,Notch signaling pathway ,Context (language use) ,Suppressor of Hairless ,Cell and Developmental Biology ,ddc:150 ,ddc:570 ,Phosphorylation ,Transcription factor ,lcsh:QH301-705.5 ,Notch signaling ,Original Research ,Chemistry ,RBPJ ,phosphorylation ,Hämatopoese ,Cell Biology ,hematopoiesis ,Hairless ,Cell biology ,Hematopoiesis ,lcsh:Biology (General) ,Drosophila ,Signal transduction ,Taufliege ,Corepressor ,Developmental Biology - Abstract
The highly conserved Notch signaling pathway controls a multitude of developmental processes including hematopoiesis. Here, we provide evidence for a novel mechanism of tissue-specific Notch regulation involving phosphorylation of CSL transcription factors within the DNA-binding domain. Earlier we found that a phospho-mimetic mutation of theDrosophilaCSL ortholog Suppressor of Hairless [Su(H)] at Ser269impedes DNA-binding. By genome-engineering, we now introduced phospho-specificSu(H)mutants at the endogenousSu(H)locus, encoding either a phospho-deficient [Su(H)S269A] or a phospho-mimetic [Su(H)S269D] isoform.Su(H)S269Dmutants were defective of Notch activity in all analyzed tissues, consistent with impaired DNA-binding. In contrast, the phospho-deficientSu(H)S269Amutant did not generally augment Notch activity, but rather specifically in several aspects of blood cell development. Unexpectedly, this process was independent of the corepressorHairlessacting otherwise as a general Notch antagonist inDrosophila. This finding is in agreement with a novel mode of Notch regulation by posttranslational modification of Su(H) in the context of hematopoiesis. Importantly, our studies of the mammalian CSL ortholog (RBPJ/CBF1) emphasize a potential conservation of this regulatory mechanism: phospho-mimetic RBPJS221Dwas dysfunctional in both the fly as well as two human cell culture models, whereas phospho-deficient RBPJS221Arather gained activity during fly hematopoiesis. Thus, dynamic phosphorylation of CSL-proteins within the DNA-binding domain provides a novel means to fine-tune Notch signal transduction in a context-dependent manner.
- Published
- 2021
28. Transcription Factors in Blood Cell Development
- Author
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Tilman Borggrefe, Benedetto Daniele Giaimo, Tilman Borggrefe, and Benedetto Daniele Giaimo
- Subjects
- Genetic transcription, Cytology, Post-translational modification, Hematopoietic system, Developmental biology
- Abstract
This book describes transcription factors in the context of hematopoietic development and reveal mechanistic insights how they orchestrate specific gene expression patterns. Several examples from lymphoid and myeloid lineages are given as well as particular examples of leukemia fusion proteins that contain a part of a transcription factor. In the given chapters covering each one particular example, the function of transcription factors is characterized in normal as well as leukemic contexts. New technologies such as ChIP-Seq or RNA-Seq, that make use of genome-wide sequencing, have shown that such master regulators of blood cell development often work in combinatorial fashion and, when dysregulated, results in leukemogenesis. This book is not only intended for experts but it should also be a useful resource for younger scientists or scientists from other disciplines, who may use this book as a stimulating starting point for further discoveries and/ or translational endeavors.
- Published
- 2024
29. A Comprehensive Toolbox to Analyze Enhancer–Promoter Functions
- Author
-
Tobias Friedrich, Benedetto Daniele Giaimo, and Tilman Borggrefe
- Subjects
Transcription (biology) ,biology.protein ,Promoter ,Context (language use) ,RNA polymerase II ,Computational biology ,Biology ,Enhancer ,Transcription factor ,Chromatin immunoprecipitation ,Chromatin - Abstract
Knowledge in gene transcription and chromatin regulation has been intensely studied for decades, but thanks to next-generation sequencing (NGS) techniques there has been a major leap forward in the last few years. Historically, identification of specific enhancer elements has led to the identification of master transcription factors (TFs) in the 1990s. Genetic and biochemical experiments have identified the key regulators controlling RNA polymerase II (RNAPII) transcription and structurally analyses have elucidated detailed mechanisms. NGS and the development of chromatin immunoprecipitation (ChIP) have accelerated the gain of knowledge in the recent years. By now, we have a dazzling wealth of techniques that are currently used to put gene expression into a genome-wide context. This book is an attempt to assemble useful protocols for many researchers within and nearby research areas. In general, these innovative techniques focus on enhancer and promoter studies. The techniques should also be of interest for related fields such as DNA repair and replication.
- Published
- 2021
30. Correction to: Enhancers and Promoters
- Author
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Tilman Borggrefe and Benedetto Daniele Giaimo
- Published
- 2021
31. SPEN is Required forXistUpregulation during Initiation of X Chromosome Inactivation
- Author
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Hegias Mira-Bontenbal, François Dossin, Beatrice F. Tan, Wilfred F. J. van IJcken, Edith Heard, Benedetto Daniele Giaimo, Cristina Gontan-Pardo, Tilman Borggrefe, John W.M. Martens, Joost Gribnau, Teresa Robert-Finestra, Erika Timmers, and Sarra Merzouk
- Subjects
Downregulation and upregulation ,Gene silencing ,XIST ,Tsix ,Biology ,Psychological repression ,X-inactivation ,Antisense RNA ,Chromatin ,Cell biology - Abstract
At initiation of X chromosome inactivation (XCI),Xistis monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcriptTsix.Xistrecruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes incisand establishment of the Xi. Here, we show that SPEN plays an important role in the initiation of XCI.Spennull female mouse embryonic stem cells (ESCs) are defective inXistupregulation upon differentiation. We find thatXist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of theTsixpromoter is required forXistupregulation. Accordingly, failedXistupregulation inSpen−/−ESCs can be rescued by concomitant removal ofTsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in the initiation of the XCI process.
- Published
- 2020
32. Transcription Factor RBPJ as a Molecular Switch in Regulating the Notch Response
- Author
-
Benedetto Daniele, Giaimo, Ellen K, Gagliani, Rhett A, Kovall, and Tilman, Borggrefe
- Subjects
Gene Expression Regulation ,Receptors, Notch ,Immunoglobulin J Recombination Signal Sequence-Binding Protein ,Humans ,Chromatin ,Signal Transduction - Abstract
The Notch signal transduction cascade requires cell-to-cell contact and results in the proteolytic processing of the Notch receptor and subsequent assembly of a transcriptional coactivator complex containing the Notch intracellular domain (NICD) and transcription factor RBPJ. In the absence of a Notch signal, RBPJ remains at Notch target genes and dampens transcriptional output. Like in other signaling pathways, RBPJ is able to switch from activation to repression by associating with corepressor complexes containing several chromatin-modifying enzymes. Here, we focus on the recent advances concerning RBPJ-corepressor functions, especially in regard to chromatin regulation. We put this into the context of one of the best-studied model systems for Notch, blood cell development. Alterations in the RBPJ-corepressor functions can contribute to the development of leukemia, especially in the case of acute myeloid leukemia (AML). The versatile role of transcription factor RBPJ in regulating pivotal target genes like c-MYC and HES1 may contribute to the better understanding of the development of leukemia.
- Published
- 2020
33. Transcription Factor RBPJ as a Molecular Switch in Regulating the Notch Response
- Author
-
Tilman Borggrefe, Ellen K. Gagliani, Rhett A. Kovall, and Benedetto Daniele Giaimo
- Subjects
03 medical and health sciences ,0302 clinical medicine ,RBPJ ,Notch signaling pathway ,Context (language use) ,030212 general & internal medicine ,Signal transduction ,HES1 ,Biology ,Corepressor ,Transcription factor ,Cell biology ,Chromatin - Abstract
The Notch signal transduction cascade requires cell-to-cell contact and results in the proteolytic processing of the Notch receptor and subsequent assembly of a transcriptional coactivator complex containing the Notch intracellular domain (NICD) and transcription factor RBPJ. In the absence of a Notch signal, RBPJ remains at Notch target genes and dampens transcriptional output. Like in other signaling pathways, RBPJ is able to switch from activation to repression by associating with corepressor complexes containing several chromatin-modifying enzymes. Here, we focus on the recent advances concerning RBPJ-corepressor functions, especially in regard to chromatin regulation. We put this into the context of one of the best-studied model systems for Notch, blood cell development. Alterations in the RBPJ-corepressor functions can contribute to the development of leukemia, especially in the case of acute myeloid leukemia (AML). The versatile role of transcription factor RBPJ in regulating pivotal target genes like c-MYC and HES1 may contribute to the better understanding of the development of leukemia.
- Published
- 2020
34. <scp>RBPJ</scp> / <scp>CBF</scp> 1 interacts with L3 <scp>MBTL</scp> 3/ <scp>MBT</scp> 1 to promote repression of Notch signaling via histone demethylase <scp>KDM</scp> 1A/ <scp>LSD</scp> 1
- Author
-
Yali Dou, Rhett A. Kovall, Kevin P. Conlon, Honglai Zhang, Yang Zhang, Kimberly Ha, Kazuya Hori, Tilman Borggrefe, Marek Bartkuhn, Venkatesha Basrur, Kojo S.J. Elenitoba-Johnson, Lucas Anhezini, Julián Cerón, Jean François Rual, Sung Soo Park, Yuqing Sun, Tao Xu, Cheng Yu Lee, Eléna Milon, Benedetto Daniele Giaimo, Daniel Hall, Alexey I. Nesvizhskii, Rork Kuick, Diana M. Ho, Brandon Govindarajoo, Iris Ertl, and Francesca Ferrante
- Subjects
0301 basic medicine ,Genetics ,General Immunology and Microbiology ,biology ,RBPJ ,General Neuroscience ,Notch signaling pathway ,KDM1A ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Histone ,Notch proteins ,biology.protein ,Demethylase ,Enhancer ,Molecular Biology ,Transcription factor ,030217 neurology & neurosurgery - Abstract
Notch signaling is an evolutionarily conserved signal transduction pathway that is essential for metazoan development. Upon ligand binding, the Notch intracellular domain (NOTCH ICD) translocates into the nucleus and forms a complex with the transcription factor RBPJ (also known as CBF1 or CSL) to activate expression of Notch target genes. In the absence of a Notch signal, RBPJ acts as a transcriptional repressor. Using a proteomic approach, we identified L3MBTL3 (also known as MBT1) as a novel RBPJ interactor. L3MBTL3 competes with NOTCH ICD for binding to RBPJ. In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (also known as LSD1) to the enhancers of Notch target genes, leading to H3K4me2 demethylation and to transcriptional repression. Importantly, in vivo analyses of the homologs of RBPJ and L3MBTL3 in Drosophila melanogaster and Caenorhabditis elegans demonstrate that the functional link between RBPJ and L3MBTL3 is evolutionarily conserved, thus identifying L3MBTL3 as a universal modulator of Notch signaling in metazoans.
- Published
- 2017
35. Heterodimerization of AML1/ETO with CBFβ is required for leukemogenesis but not for myeloproliferation
- Author
-
Peggy Schwarz, Virág Vas, Karin Soller, Hartmut Geiger, Tilman Borggrefe, Marek Bartkuhn, Verena N. Thiel, Franz Oswald, Benedetto Daniele Giaimo, Lars Bullinger, Konstanze Döhner, and Tamara J. Blätte
- Subjects
0301 basic medicine ,Cancer Research ,Oncogene Proteins, Fusion ,Biology ,Core Binding Factor beta Subunit ,Cell Line ,Fusion gene ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,RUNX1 Translocation Partner 1 Protein ,0302 clinical medicine ,hemic and lymphatic diseases ,Myeloproliferation ,Animals ,Humans ,neoplasms ,Transcription factor ,Leukemia ,Myeloid leukemia ,Cell Differentiation ,Hematology ,Fusion protein ,Mice, Inbred C57BL ,030104 developmental biology ,Oncology ,RUNX1 ,chemistry ,030220 oncology & carcinogenesis ,Core Binding Factor Alpha 2 Subunit ,Cancer research ,Original Article ,Dimerization ,Corepressor - Abstract
The AML1/Runx1 transcription factor and its heterodimerization partner CBFβ are essential regulators of myeloid differentiation. The chromosomal translocation t(8;21), fusing the DNA binding domain of AML1 to the corepressor eight-twenty-one (ETO), is frequently associated with acute myeloid leukemia and generates the AML1/ETO (AE) fusion protein. AE represses target genes usually activated by AML1 and also affects the endogenous repressive function of ETO at Notch target genes. In order to analyze the contribution of CBFβ in AE-mediated leukemogenesis and deregulation of Notch target genes, we introduced two point mutations in a leukemia-initiating version of AE in mice, called AE9a, that disrupt the AML1/CBFβ interaction (AE9aNT). We report that the AE9a/CBFβ interaction is not required for the AE9a-mediated aberrant expression of AML1 target genes, while upregulation/derepression of Notch target genes does require the interaction with CBFβ. Using retroviral transduction to express AE9a in murine adult bone marrow-derived hematopoietic progenitors, we observed that both AE9a and AE9aNT lead to increased myeloproliferation in vivo. However, both development of leukemia and long-term replating capacity are only observed with AE9a but not with AE9aNT. Thus, deregulation of both AML1 and Notch target genes is required for the development of AE9a-driven leukemia.
- Published
- 2017
36. Dynamic chromatin regulation at Notch target genes
- Author
-
Benedetto Daniele Giaimo, Franz Oswald, and Tilman Borggrefe
- Subjects
SHARP ,0301 basic medicine ,Notch ,Transcription, Genetic ,casein kinase 2 ,RBPJ ,Methylation ,environment and public health ,Biochemistry ,Histones ,03 medical and health sciences ,0302 clinical medicine ,NICD ,transcriptional repression ,Histone methylation ,Histone H2A ,Genetics ,Animals ,Humans ,transcriptional activation ,Histone code ,Gene Regulatory Networks ,Point-of-View ,Phosphorylation ,Transcription factor ,Receptors, Notch ,biology ,Chemistry ,KMT2D ,Acetylation ,Chromatin ,030104 developmental biology ,Histone ,Gene Expression Regulation ,Immunoglobulin J Recombination Signal Sequence-Binding Protein ,Multiprotein Complexes ,Histone methyltransferase ,NCoR ,biology.protein ,Cancer research ,transcription ,030217 neurology & neurosurgery ,Protein Binding ,Biotechnology - Abstract
RBPJ is the central transcription factor that controls the Notch-dependent transcriptional response by coordinating repressing histone H3K27 deacetylation and activating histone H3K4 methylation. Here, we discuss the molecular mechanisms how RBPJ interacts with opposing NCoR/HDAC-corepressing or KMT2D/UTX-coactivating complexes and how this is controlled by phosphorylation of chromatin modifiers.
- Published
- 2016
37. Functional and clinical characterization of the alternatively spliced isoform AML1-ETO9a in adult patients with translocation t(8;21)(q22;q22.1) acute myeloid leukemia (AML)
- Author
-
Mohammed Wattad, Daniela Weber, Arnold Ganser, Felicitas Thol, Nikolaus Jahn, Frank G. Rücker, Ivan Bedzhov, Mridul Agrawal, Elisabeth Koller, Franz Oswald, Thomas Schroeder, Peggy Schwarz, Peter Paschka, Hartmut Döhner, Thomas Kindler, Katharina Götze, Mark Ringhoffer, Tilman Borggrefe, Michael Heuser, Konstanze Döhner, Verena I. Gaidzik, Hans Salwender, Benedetto Daniele Giaimo, Elisabeth Lange, Hartmut Geiger, Andrea Corbacioglu, Michael Lübbert, and Lars Bullinger
- Subjects
Gene isoform ,Adult ,Male ,Cancer Research ,Myeloid ,Letter ,Oncogene Proteins ,Adolescent ,Oncogene Proteins, Fusion ,Chromosomes, Human, Pair 21 ,Chromosomal translocation ,Translocation, Genetic ,Young Adult ,RUNX1 Translocation Partner 1 Protein ,medicine ,Humans ,Protein Isoforms ,Young adult ,Oncogenesis ,Aged ,business.industry ,Myeloid leukemia ,Hematology ,Oncogenes ,Middle Aged ,medicine.disease ,ddc ,Leukemia ,Alternative Splicing ,Leukemia, Myeloid, Acute ,medicine.anatomical_structure ,Oncology ,Core Binding Factor Alpha 2 Subunit ,Cancer research ,Female ,T(8 ,21)(q22 ,q22) ,business ,Chromosomes, Human, Pair 8 - Published
- 2019
38. Nucleo-cytoplasmic shuttling of Drosophila Hairless/Su(H) heterodimer as a means of regulating Notch dependent transcription
- Author
-
Anette Preiss, Ludmilla Kober, Dieter Maier, Tilman Borggrefe, Mirjam Zimmermann, Thomas K. Smylla, Philipp Hoffmeister, Jan Reichmuth, Anja C. Nagel, Dorina B. Wolf, Franz Oswald, and Aleksandra Turkiewicz
- Subjects
Cytoplasm ,animal structures ,Nuclear Localization Signals ,Repressor ,03 medical and health sciences ,Transcription (biology) ,Animals ,Drosophila Proteins ,Wings, Animal ,Nuclear export signal ,Molecular Biology ,Transcription factor ,030304 developmental biology ,Nuclear Export Signals ,0303 health sciences ,integumentary system ,Receptors, Notch ,Chemistry ,RBPJ ,030302 biochemistry & molecular biology ,Wild type ,Cell Biology ,Hairless ,Cell biology ,Drosophila melanogaster ,Phenotype ,Female ,Nuclear localization sequence ,Transcription Factors - Abstract
Activation and repression of Notch target genes is mediated by transcription factor CSL, known as Suppressor of Hairless (Su(H)) in Drosophila and CBF1 or RBPJ in human. CSL associates either with co-activator Notch or with co-repressors such as Drosophila Hairless. The nuclear translocation of transcription factor CSL relies on co-factor association, both in mammals and in Drosophila. The Drosophila CSL orthologue Su(H) requires Hairless for repressor complex formation. Based on its role in transcriptional silencing, H protein would be expected to be strictly nuclear. However, H protein is also cytosolic, which may relate to its role in the stabilization and nuclear translocation of Su(H) protein. Here, we investigate the function of the predicted nuclear localization signals (NLS 1–3) and single nuclear export signal (NES) of co-repressor Hairless using GFP-fusion proteins, reporter assays and in vivo analyses using Hairless wild type and shuttling-defective Hairless mutants. We identify NLS3 and NES to be critical for Hairless function. In fact, H⁎NLS3 mutant flies match H null mutants, whereas H⁎NLS3⁎NES double mutants display weaker phenotypes in agreement with a crucial role for NES in H export. As expected for a transcriptional repressor, Notch target genes are deregulated in H⁎NLS3 mutant cells, demonstrating nuclear requirement for its activity. Importantly, we reveal that Su(H) protein strictly follows Hairless protein localization. Together, we propose that shuttling between the nucleo-cytoplasmic compartments provides the possibility to fine tune the regulation of Notch target gene expression by balancing of Su(H) protein availability for Notch activation.
- Published
- 2019
39. A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes
- Author
-
Melanie Rothe, Michael Kühl, Gerhard Mittler, Patrick Rodriguez, Nassif Tabaja, Rhett A. Kovall, Franz Oswald, Kelly J. Collins, Tilman Borggrefe, Laura Mira, Kerstin Hein, Verena N. Thiel, Susanne J. Kühl, Benedetto Daniele Giaimo, Maria Dominguez, Wiebke Cizelsky, Zeus A. Antonello, Francesca Ferrante, German Research Foundation, University of Freiburg, Federal Ministry of Education and Research (Germany), Fundación Botín, Ministerio de Economía y Competitividad (España), Leukemia & Lymphoma Society (US), National Institutes of Health (US), and American Heart Association
- Subjects
0301 basic medicine ,Transcription, Genetic ,Cell Line ,Mice ,Xenopus laevis ,03 medical and health sciences ,Histone H3 ,Cell Line, Tumor ,Coactivator ,Genetics ,Animals ,Drosophila Proteins ,Humans ,Histone code ,Protein Interaction Domains and Motifs ,Phosphorylation ,Casein Kinase II ,ChIA-PET ,Homeodomain Proteins ,Receptors, Notch ,biology ,Gene regulation, Chromatin and Epigenetics ,Nuclear Proteins ,RNA-Binding Proteins ,Histone-Lysine N-Methyltransferase ,Chromatin ,Cell biology ,DNA-Binding Proteins ,Histone Code ,030104 developmental biology ,Histone ,Gene Expression Regulation ,Histone methyltransferase ,biology.protein ,Cancer research ,Co-Repressor Proteins ,Corepressor ,Myeloid-Lymphoid Leukemia Protein - Abstract
The transcriptional shift from repression to activation of target genes is crucial for the fidelity of Notch responses through incompletely understood mechanisms that likely involve chromatin-based control. To activate silenced genes, repressive chromatin marks are removed and active marks must be acquired. Histone H3 lysine-4 (H3K4) demethylases are key chromatin modifiers that establish the repressive chromatin state at Notch target genes. However, the counteracting histone methyltransferase required for the active chromatin state remained elusive. Here, we show that the RBP-J interacting factor SHARP is not only able to interact with the NCoR corepressor complex, but also with the H3K4 methyltransferase KMT2D coactivator complex. KMT2D and NCoR compete for the C-terminal SPOC-domain of SHARP. We reveal that the SPOC-domain exclusively binds to phosphorylated NCoR. The balance between NCoR and KMT2D binding is shifted upon mutating the phosphorylation sites of NCoR or upon inhibition of the NCoR kinase CK2β. Furthermore, we show that the homologs of SHARP and KMT2D in Drosophila also physically interact and control Notch-mediated functions in vivo. Together, our findings reveal how signaling can fine-tune a committed chromatin state by phosphorylation of a pivotal chromatin-modifier., Collaborative research [TRR81]; Heisenberg program [BO 1639/5-1] by the Deutsche Forschungsgemeinschaft (DFG) and the Max-Planck society, regarding [to T.B.]; Excellence Initiative of the German Federal and State Governments [GSC4, Spemann Graduate School Freiburg, to B.D.G.]; DFG [collaborative research grant SFB 1074/A3 to F.O.]; BMBF (research nucleus SyStAR), regarding [to F.O. and M.K.]; International Graduate School in Molecular Medicine, Ulm [GSC270 to W.C. and M.R.]; National Institutes of Health Grant [CA178974], regarding [to R.A.K]; Leukemia and Lymphoma Society [to R.A.K.]; Spanish Grants [SAF2012-35181, SEV-2013-0317, PROMETEO II/2013/001]; Botin Foundation [to M.D.]; American Heart Association [to K.J.C.]., Funding for open access charge: DFG collaborative research [TRR81].
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- 2016
40. The Notch intracellular domain integrates signals from Wnt, Hedgehog, TGFβ/BMP and hypoxia pathways
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Danny Huylebroeck, Benedetto Daniele Giaimo, Franz Oswald, Tilman Borggrefe, Matthias Lauth, An Zwijsen, and Cell biology
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0301 basic medicine ,Notch signaling pathway ,Biology ,Notch signal transduction ,Methylation ,Models, Biological ,03 medical and health sciences ,Transforming Growth Factor beta ,Animals ,Humans ,Hedgehog Proteins ,Hypoxia ,Molecular Biology ,Transcription factor ,Receptors, Notch ,Wnt signaling pathway ,Cell Biology ,Cell biology ,Wnt Proteins ,Crosstalk (biology) ,030104 developmental biology ,Notch proteins ,Hes3 signaling axis ,Bone Morphogenetic Proteins ,Cyclin-dependent kinase 8 ,Signal transduction ,Signal Transduction - Abstract
Notch signaling is a highly conserved signal transduction pathway that regulates stem cell maintenance and differentiation in several organ systems. Upon activation, the Notch receptor is proteolytically processed, its intracellular domain (NICD) translocates into the nucleus and activates expression of target genes. Output, strength and duration of the signal are tightly regulated by post-translational modifications. Here we review the intracellular post-translational regulation of Notch that fine-tunes the outcome of the Notch response. We also describe how crosstalk with other conserved signaling pathways like the Wnt, Hedgehog, hypoxia and TGFβ/BMP pathways can affect Notch signaling output. This regulation can happen by regulation of ligand, receptor or transcription factor expression, regulation of protein stability of intracellular key components, usage of the same cofactors or coregulation of the same key target genes. Since carcinogenesis is often dependent on at least two of these pathways, a better understanding of their molecular crosstalk is pivotal. publisher: Elsevier articletitle: The Notch intracellular domain integrates signals from Wnt, Hedgehog, TGFβ/BMP and hypoxia pathways journaltitle: Biochimica et Biophysica Acta (BBA) - Molecular Cell Research articlelink: http://dx.doi.org/10.1016/j.bbamcr.2015.11.020 content_type: article copyright: Copyright © 2015 The Authors. Published by Elsevier B.V. ispartof: Biochimica et Biophysica Acta vol:1863 issue:2 pages:303-13 ispartof: location:Netherlands status: published
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- 2016
41. Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination
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Bernardo Reina-San-Martin, Pedro P. Rocha, Anne-Sophie Thomas-Claudepierre, Jane A. Skok, Isabelle Robert, Vincent M. Luo, Tilman Borggrefe, Ebe Schiavo, Janardan K. Reddy, Richard Bonneau, Vincent Heyer, and Ramya Raviram
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0301 basic medicine ,Transcriptional Activation ,Transcription, Genetic ,Immunology ,Mediator Complex Subunit 1 ,chemical and pharmacologic phenomena ,Biology ,MED1 ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Mediator ,Transcription (biology) ,Immunology and Allergy ,Animals ,Enhancer ,Research Articles ,Cells, Cultured ,Genetics ,Recombination, Genetic ,B-Lymphocytes ,Mediator Complex ,Brief Definitive Report ,Promoter ,Cytidine deaminase ,Immunoglobulin Class Switching ,030104 developmental biology ,Immunoglobulin class switching ,Genetic Loci ,Gene Knockdown Techniques ,Immunoglobulin Heavy Chains ,030215 immunology ,Protein Binding - Abstract
Thomas-Claudepierre et al. report that mediator facilitates the long-range contacts between acceptor switch regions and the IgH locus enhancers during class switch recombination and their transcriptional activation., Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation.
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- 2016
42. Introduction to Molecular Mechanisms in Notch Signal Transduction and Disease Pathogenesis
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Benedetto Daniele, Giaimo and Tilman, Borggrefe
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Leukemia ,Receptors, Notch ,Animals ,Humans ,Neoplasm Proteins ,Signal Transduction - Abstract
The Notch signaling pathway plays a pivotal role in development, physiology and diseases such as cancer. In this chapter, we first give an overview of the different molecular mechanisms that regulate Notch signaling. Each subject is covered in more depth in the subsequent chapters of this book. Next, we will use the inflammatory system as an example to discuss the physiological function of Notch signaling. This is followed by a discussion of recent advances in the different pathophysiological roles of Notch signaling in leukemia as well as a wide range of solid cancers. Finally, we discuss how information about pathogenic mutations in Notch pathway components, combined with structural biological data, are beginning to provide important biological and mechanistic insights about the pathway.
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- 2018
43. Structural and functional studies of the RBPJ-SHARP complex reveal conserved corepressor binding site
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Kerstin Hein, Benedetto Daniele Giaimo, Franz Oswald, Tilman Borggrefe, Bradley D. VanderWielen, Rhett A. Kovall, Aleksandra Turkiewicz, Zhenyu Yuan, Leiling Pan, and Courtney E. Collins
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RBPJ ,Transcription (biology) ,Chemistry ,Notch signaling pathway ,Repressor ,Binding site ,Corepressor ,Transcription factor ,Cell biology ,Hairless - Abstract
The Notch pathway is a conserved signaling mechanism that is essential for cell fate decisions during pre and postnatal development. Dysregulated signaling underlies the pathophysiology of numerous human diseases, most notably T-cell acute lymphoblastic leukemia. Receptor-ligand interactions result in changes in gene expression, which are regulated by the transcription factor CSL. CSL forms a complex with the intracellular domain of the Notch receptor and the transcriptional coactivator Mastermind, which is required to activate transcription of all Notch target genes. CSL can also function as repressor by interacting with corepressor proteins, e.g. SHARP in mammals and Hairless in Drosophila melanogaster; however, its role as a transcriptional repressor is not well understood. Here we determine the high-resolution structure of RBPJ, the mouse CSL ortholog, bound to the corepressor SHARP and DNA, which reveals a new mode of corepressor binding to CSL and an interesting example for how ligand binding sites evolve in proteins. Based on the structure, we designed and tested a number of mutants in biophysical, biochemical, and cellular assays to characterize the role of RBPJ as a repressor of Notch target genes. Our cellular studies clearly demonstrate that RBPJ mutants that are deficient for binding SHARP are incapable of repressing transcription from genes responsive to Notch signaling. Altogether, our structure-function studies of the RBPJ-SHARP corepressor complex bound to DNA provide significant insights into the repressor function of RBPJ and identify a new binding pocket on RBPJ that could be targeted for therapeutic benefit.
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- 2018
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44. Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response
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Benedetto Daniele Giaimo, Andrea Nist, Thorsten Stiewe, Gerhard Mittler, Peggy Schwarz, Irene Gutierrez-Perez, Franz Oswald, Diana M. Vallejo, Tilman Borggrefe, Marek Bartkuhn, Kerstin Hein, Susanne Herold, Tobias Zimmermann, Maria Dominguez, Francesca Ferrante, German Research Foundation, Federal Ministry of Education and Research (Germany), Max Planck Society, European Commission, Ministerio de Economía y Competitividad (España), and Fundación Científica Asociación Española Contra el Cáncer
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0301 basic medicine ,Notch signaling pathway ,Lysine Acetyltransferase 5 ,Cell Line ,Histones ,03 medical and health sciences ,Cell Line, Tumor ,Genetics ,Animals ,Humans ,Enhancer ,Transcription factor ,Adaptor Proteins, Signal Transducing ,Regulation of gene expression ,Adenosine Triphosphatases ,Mice, Knockout ,biology ,Receptors, Notch ,Nuclear Proteins ,Acetylation ,Cell biology ,Chromatin ,030104 developmental biology ,Histone ,HEK293 Cells ,Gene Expression Regulation ,Immunoglobulin J Recombination Signal Sequence-Binding Protein ,biology.protein ,Signal transduction ,HeLa Cells ,Signal Transduction - Abstract
A fundamental as yet incompletely understood feature of Notch signal transduction is a transcriptional shift from repression to activation that depends on chromatin regulation mediated by transcription factor RBP-J and associated cofactors. Incorporation of histone variants alter the functional properties of chromatin and are implicated in the regulation of gene expression. Here, we show that depletion of histone variant H2A.Z leads to upregulation of canonical Notch target genes and that the H2A.Z-chaperone TRRAP/p400/Tip60 complex physically associates with RBP-J at Notch-dependent enhancers. When targeted to RBP-J-bound enhancers, the acetyltransferase Tip60 acetylates H2A.Z and upregulates Notch target gene expression. Importantly, the Drosophila homologs of Tip60, p400 and H2A.Z modulate Notch signaling response and growth in vivo. Together, our data reveal that loading and acetylation of H2A.Z are required to assure tight control of canonical Notch activation., This work was supported by the collaborative research grant TRR81 and the Heisenberg program (BO 1639/5–1) by the DFG (German Research Foundation), the Max Planck Society and the EXC 294 in Freiburg and the Excellence Cluster for Cardio Pulmonary System (ECCPS) in Giessen to T.B and S.H. This work was further supported by the DFG through collaborative research grants (SFB 1074/A3 and KFO309 service project Z1) and by the BMBF (Federal Ministry of Education and Research, research nucleus SyStAR) to F.O. Spanish Grants [BFU2015–64239-R co-financed by the European Regional Development Fund (ERDF), SEV-2013–0317, PROMETEO/2017/146]; Botin Foundation and AECC Foundation (CICP16001DOMI) to M.D. Funding for open access charge: DFG collaborative research [TRR81]. This study was supported in part by the Excellence Initiative of the German Federal and State Governments [GSC-4, Spemann Graduate School].
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- 2018
45. Protein arginine methyltransferase 5 mediates enolase-1 cell surface trafficking in human lung adenocarcinoma cells
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Klaus T. Preissner, Benedetto Daniele Giaimo, Liliana Schaefer, Dariusz Zakrzewicz, Tilman Borggrefe, Marcus Krüger, Andreas C. Hocke, Malgorzata Wygrecka, Anna Zakrzewicz, Maren Mieth, and Miroslava Didiasova
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0301 basic medicine ,Lipopolysaccharides ,Protein-Arginine N-Methyltransferases ,Methyltransferase ,Tudor domain ,Lung Neoplasms ,Arginine ,Cell ,Caveolin 1 ,Adenocarcinoma of Lung ,Adenocarcinoma ,03 medical and health sciences ,0302 clinical medicine ,Extracellular ,medicine ,Biomarkers, Tumor ,Humans ,Molecular Biology ,Chemistry ,Protein arginine methyltransferase 5 ,Tumor Suppressor Proteins ,Methylation ,Cell biology ,Neoplasm Proteins ,DNA-Binding Proteins ,Protein Transport ,030104 developmental biology ,medicine.anatomical_structure ,A549 Cells ,030220 oncology & carcinogenesis ,Phosphopyruvate Hydratase ,Cancer cell ,Molecular Medicine - Abstract
Objectives Enolase-1-dependent cell surface proteolysis plays an important role in cell invasion. Although enolase-1 (Eno-1), a glycolytic enzyme, has been found on the surface of various cells, the mechanism responsible for its exteriorization remains elusive. Here, we investigated the involvement of post-translational modifications (PTMs) of Eno-1 in its lipopolysaccharide (LPS)-triggered trafficking to the cell surface. Results We found that stimulation of human lung adenocarcinoma cells with LPS triggered the monomethylation of arginine 50 (R50me) within Eno-1. The Eno-1R50me was confirmed by its interaction with the tudor domain (TD) from TD-containing 3 (TDRD3) protein recognizing methylarginines. Substitution of R50 with lysine (R50K) reduced Eno-1 association with epithelial caveolar domains, thereby diminishing its exteriorization. Similar effects were observed when pharmacological inhibitors of arginine methyltransferases were applied. Protein arginine methyltransferase 5 (PRMT5) was identified to be responsible for Eno-1 methylation. Overexpression of PRMT5 and caveolin-1 enhanced levels of membrane-bound extracellular Eno-1 and, conversely, pharmacological inhibition of PRMT5 attenuated Eno-1 cell-surface localization. Importantly, Eno-1R50me was essential for cancer cell motility since the replacement of Eno-1 R50 by lysine or the suppression of PRMT 5 activity diminished Eno-1-triggered cell invasion. Conclusions LPS-triggered Eno-1R50me enhances Eno-1 cell surface levels and thus potentiates the invasive properties of cancer cells. Strategies to target Eno-1R50me may offer novel therapeutic approaches to attenuate tumor metastasis in cancer patients.
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- 2017
46. Molecular Mechanisms of Notch Signaling
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Tilman Borggrefe, Benedetto Daniele Giaimo, Tilman Borggrefe, and Benedetto Daniele Giaimo
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- Cellular signal transduction, Notch proteins
- Abstract
This book describes the Notch signaling pathway with a focus on molecular mechanisms. The Notch signaling pathway is a seemingly simple pathway that does not involve any second messenger. Upon ligand binding two consecutive proteolytic cleavages of the NOTCH receptor release the Notch intracellular domain from the membrane. The Notch intracellular domain migrates into the nucleus and activates gene expression. Recently, new technologies allowed us to better understand this pivotal signaling cascade and revealed new regulatory mechanisms. The different chapters cover many aspects of the Notch signaling focusing on the mechanisms governing the receptor/ligand interaction as well as on the downstream intracellular signaling events. Aspects of both canonical and non-canonical signaling are discussed and the function of Notch signaling in physiological and pathological contexts are elucidated. This book is not only intended for experts but it should also be a usefulresource for young, sprouting scientists or interested scientists from other research areas, who may use this book as a stimulating starting point for further discoveries and developments.
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- 2018
47. Dll4 and Notch signalling couples sprouting angiogenesis and artery formation
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Urs H. Langen, Susana F. Rocha, Manuel Ehling, Daniel Biljes, Frank Berkenfeld, Mara E. Pitulescu, Tilman Borggrefe, Francesca Ferrante, Hongryeol Park, Napoleone Ferrara, Ralf H. Adams, Tobiah Antoine, Inga Schmidt, Benedetto Daniele Giaimo, Martin Stehling, and Takashi Nagasawa
- Subjects
0301 basic medicine ,Vascular Endothelial Growth Factor A ,Male ,Time Factors ,Angiogenesis ,Cellular differentiation ,Cell Communication ,Cardiovascular ,Inbred C57BL ,Medical and Health Sciences ,Transgenic ,Neovascularization ,Chemokine receptor ,Mice ,Cell Movement ,Receptors ,Receptor, Notch1 ,Cells, Cultured ,Cultured ,Intracellular Signaling Peptides and Proteins ,Adaptor Proteins ,Cell Differentiation ,Biological Sciences ,Cell biology ,Phenotype ,cardiovascular system ,Female ,medicine.symptom ,Signal Transduction ,Receptor ,Cell signaling ,Receptors, CXCR4 ,Genotype ,Retinal Artery ,1.1 Normal biological development and functioning ,Cells ,Notch signaling pathway ,Neovascularization, Physiologic ,Mice, Transgenic ,Biology ,03 medical and health sciences ,Vasculogenesis ,Underpinning research ,medicine ,Animals ,Cell Lineage ,Physiologic ,Adaptor Proteins, Signal Transducing ,Cell Proliferation ,Sprouting angiogenesis ,CXCR4 ,Notch1 ,Calcium-Binding Proteins ,Signal Transducing ,Endothelial Cells ,Membrane Proteins ,Cell Biology ,Mice, Inbred C57BL ,030104 developmental biology ,Gene Expression Regulation ,Jagged-1 Protein ,Developmental Biology - Abstract
Endothelial sprouting and proliferation are tightly coordinated processes mediating the formation of new blood vessels during physiological and pathological angiogenesis. Endothelial tip cells lead sprouts and are thought to suppress tip-like behaviour in adjacent stalk endothelial cells by activating Notch. Here, we show with genetic experiments in postnatal mice that the level of active Notch signalling is more important than the direct Dll4-mediated cell-cell communication between endothelial cells. We identify endothelial expression of VEGF-A and of the chemokine receptor CXCR4 as key processes controlling Notch-dependent vessel growth. Surprisingly, genetic experiments targeting endothelial tip cells in vivo reveal that they retain their function without Dll4 and are also not replaced by adjacent, Dll4-positive cells. Instead, activation of Notch directs tip-derived endothelial cells into developing arteries and thereby establishes that Dll4-Notch signalling couples sprouting angiogenesis and artery formation.
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- 2017
48. Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines
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Tilman Borggrefe, Francesca Ferrante, and Benedetto Daniele Giaimo
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0301 basic medicine ,sonication ,Chromatin Immunoprecipitation ,General Chemical Engineering ,T-Lymphocytes ,ChIP ,Cell Culture Techniques ,Biology ,Regulatory Sequences, Nucleic Acid ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Histones ,03 medical and health sciences ,Mice ,Genetics ,Nucleosome ,Animals ,Humans ,Enhancer ,Transcription factor ,General Immunology and Microbiology ,histone marks ,General Neuroscience ,H3K4me1 ,Reproducibility of Results ,regulation ,H3K4me3 ,DNA ,ChIP-on-chip ,H3K27ac ,shearing ,Molecular biology ,Chromatin ,Cell biology ,Nucleosomes ,DNA-Binding Proteins ,H3 ,030104 developmental biology ,Histone ,Issue 124 ,biology.protein ,Chromatin immunoprecipitation ,Protein Processing, Post-Translational ,transcription ,Protein Binding ,Transcription Factors - Abstract
Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.
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- 2017
49. RBPJ/CBF1 interacts with L3MBTL3/MBT1 to promote repression of Notch signaling via histone demethylase KDM1A/LSD1
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Tao, Xu, Sung-Soo, Park, Benedetto Daniele, Giaimo, Daniel, Hall, Francesca, Ferrante, Diana M, Ho, Kazuya, Hori, Lucas, Anhezini, Iris, Ertl, Marek, Bartkuhn, Honglai, Zhang, Eléna, Milon, Kimberly, Ha, Kevin P, Conlon, Rork, Kuick, Brandon, Govindarajoo, Yang, Zhang, Yuqing, Sun, Yali, Dou, Venkatesha, Basrur, Kojo Sj, Elenitoba-Johnson, Alexey I, Nesvizhskii, Julian, Ceron, Cheng-Yu, Lee, Tilman, Borggrefe, Rhett A, Kovall, and Jean-François, Rual
- Subjects
Transcription, Genetic ,RBPJ ,Article ,Histones ,Protein Domains ,Cell Line, Tumor ,Two-Hybrid System Techniques ,Animals ,Drosophila Proteins ,Humans ,L3MBTL3 ,Caenorhabditis elegans ,Conserved Sequence ,Notch signaling ,Histone Demethylases ,Receptors, Notch ,Articles ,Biological Evolution ,DNA-Binding Proteins ,Drosophila melanogaster ,Gene Expression Regulation ,KDM1A ,Immunoglobulin J Recombination Signal Sequence-Binding Protein ,Neuroglia ,Transcription ,Protein Binding ,Signal Transduction - Abstract
Notch signaling is an evolutionarily conserved signal transduction pathway that is essential for metazoan development. Upon ligand binding, the Notch intracellular domain (NOTCH ICD) translocates into the nucleus and forms a complex with the transcription factor RBPJ (also known as CBF1 or CSL) to activate expression of Notch target genes. In the absence of a Notch signal, RBPJ acts as a transcriptional repressor. Using a proteomic approach, we identified L3MBTL3 (also known as MBT1) as a novel RBPJ interactor. L3MBTL3 competes with NOTCH ICD for binding to RBPJ. In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (also known as LSD1) to the enhancers of Notch target genes, leading to H3K4me2 demethylation and to transcriptional repression. Importantly, in vivo analyses of the homologs of RBPJ and L3MBTL3 in Drosophila melanogaster and Caenorhabditis elegans demonstrate that the functional link between RBPJ and L3MBTL3 is evolutionarily conserved, thus identifying L3MBTL3 as a universal modulator of Notch signaling in metazoans.
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- 2017
50. Host-derived extracellular RNA promotes adhesion of Streptococcus pneumoniae to endothelial and epithelial cells
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Andreas C. Hocke, Maren Mieth, Sven Hammerschmidt, Malgorzata Wygrecka, Klaus T. Preissner, Guenter Lochnit, Dariusz Zakrzewicz, Benedetto Daniele Giaimo, Liliana Schaefer, Miroslava Didiasova, Tilman Borggrefe, Simone Bergmann, and Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
- Subjects
0301 basic medicine ,media_common.quotation_subject ,Amino Acid Motifs ,Glycine ,Biology ,600 Technik, Medizin, angewandte Wissenschaften::610 Medizin und Gesundheit ,medicine.disease_cause ,Article ,Bacterial Adhesion ,Pneumococcal Infections ,Microbiology ,Cell wall ,Extracellular matrix ,03 medical and health sciences ,Streptococcus pneumoniae ,medicine ,Extracellular ,Animals ,Humans ,ddc:610 ,Internalization ,Receptor ,Lung ,media_common ,A549 cell ,Binding Sites ,Multidisciplinary ,030102 biochemistry & molecular biology ,Nucleotides ,Lysine ,Endothelial Cells ,Epithelial Cells ,DNA ,Ribonuclease, Pancreatic ,030104 developmental biology ,A549 Cells ,Phosphopyruvate Hydratase ,Mutation ,RNA ,Cattle ,Extracellular Space ,Extracellular RNA - Abstract
Streptococcus pneumoniae is the most frequent cause of community-acquired pneumonia. The infection process involves bacterial cell surface receptors, which interact with host extracellular matrix components to facilitate colonization and dissemination of bacteria. Here, we investigated the role of host-derived extracellular RNA (eRNA) in the process of pneumococcal alveolar epithelial cell infection. Our study demonstrates that eRNA dose-dependently increased S. pneumoniae invasion of alveolar epithelial cells. Extracellular enolase (Eno), a plasminogen (Plg) receptor, was identified as a novel eRNA-binding protein on S. pneumoniae surface, and six Eno eRNA-binding sites including a C-terminal 15 amino acid motif containing lysine residue 434 were characterized. Although the substitution of lysine 434 for glycine (K434G) markedly diminished the binding of eRNA to Eno, the adherence to and internalization into alveolar epithelial cells of S. pneumoniae strain carrying the C-terminal lysine deletion and the mutation of internal Plg-binding motif were only marginally impaired. Accordingly, using a mass spectrometric approach, we identified seven novel eRNA-binding proteins in pneumococcal cell wall. Given the high number of eRNA-interacting proteins on pneumococci, treatment with RNase1 completely inhibited eRNA-mediated pneumococcal alveolar epithelial cell infection. Our data support further efforts to employ RNAse1 as an antimicrobial agent to combat pneumococcal infectious diseases.
- Published
- 2016
- Full Text
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