04 Fakultät Energie-, Verfahrens- und Biotechnik

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Institute: search.filters.UBSInstitut.Institut für Biomedizinische GenetikPublication Type: search.filters.UBSTyp.Zeitschriftenartikel

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    A timed off-switch for dynamic control of gene expression in Corynebacterium glutamicum
    (2021) Siebert, Daniel; Altenbuchner, Josef; Blombach, Bastian
    Dynamic control of gene expression mainly relies on inducible systems, which require supplementation of (costly) inducer molecules. In contrast, synthetic regulatory circuits, which allow the timed shutdown of gene expression, are rarely available and therefore represent highly attractive tools for metabolic engineering. To achieve this, we utilized the VanR/PvanABK* regulatory system of Corynebacterium glutamicum, which consists of the transcriptional repressor VanR and a modified promoter of the vanABK operon (PvanABK*). VanR activity is modulated by one of the phenolic compounds ferulic acid, vanillin or vanillic acid, which are co-metabolized with d-glucose. Thus, gene expression in the presence of d-glucose is turned off if one of the effector molecules is depleted from the medium. To dynamically control the expression of the aceE gene, encoding the E1 subunit of the pyruvate dehydrogenase complex that is essential for growth on d-glucose, we replaced the native promoter by vanR/PvanABK* yielding C. glutamicum ΔPaceE::vanR-PvanABK*. The biomass yield of this strain increased linearly with the supplemented amount of effector. After consumption of the phenolic compounds growth ceased, however, C. glutamicumΔPaceE::vanR-PvanABK* continued to utilize the residual d-glucose to produce significant amounts of pyruvate, l-alanine, and l-valine. Interestingly, equimolar concentrations of the three phenolic compounds resulted in different biomass yields; and with increasing effector concentration, the product spectrum shifted from pyruvate over l-alanine to l-valine. To further test the suitability of the VanR/PvanABK* system, we overexpressed the l-valine biosynthesis genes ilvBNCE in C. glutamicum ΔPaceE::vanR-PvanABK*, which resulted in efficient l-valine production with a yield of about 0.36 mol l-valine per mol d-glucose. These results demonstrate that the VanR/PvanABK* system is a valuable tool to control gene expression in C. glutamicum in a timed manner by the cheap and abundant phenolic compounds ferulic acid, vanillin, and vanillic acid.
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    Genetic and molecular interactions between HΔCT, a novel allele of the notch antagonist Hairless, and the histone chaperone Asf1 in Drosophila melanogaster
    (2023) Maier, Dieter; Bauer, Milena; Boger, Mike; Sanchez Jimenez, Anna; Yuan, Zhenyu; Fechner, Johannes; Scharpf, Janika; Kovall, Rhett A.; Preiss, Anette; Nagel, Anja C.
    Cellular differentiation relies on the highly conserved Notch signaling pathway. Notch activity induces gene expression changes that are highly sensitive to chromatin landscape. We address Notch gene regulation using Drosophila as a model, focusing on the genetic and molecular interactions between the Notch antagonist Hairless and the histone chaperone Asf1. Earlier work implied that Asf1 promotes the silencing of Notch target genes via Hairless (H). Here, we generate a novel HΔCT allele by genome engineering. Phenotypically, HΔCT behaves as a Hairless gain of function allele in several developmental contexts, indicating that the conserved CT domain of H has an attenuator role under native biological contexts. Using several independent methods to assay protein-protein interactions, we define the sequences of the CT domain that are involved in Hairless-Asf1 binding. Based on previous models, where Asf1 promotes Notch repression via Hairless, a loss of Asf1 binding should reduce Hairless repressive activity. However, tissue-specific Asf1 overexpression phenotypes are increased, not rescued, in the HΔCT background. Counterintuitively, Hairless protein binding mitigates the repressive activity of Asf1 in the context of eye development. These findings highlight the complex connections of Notch repressors and chromatin modulators during Notch target-gene regulation and open the avenue for further investigations.
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    Enzymatic depolymerization of alginate by two novel thermostable alginate lyases from Rhodothermus marinus
    (2022) Dobruchowska, Justyna M.; Bjornsdottir, Bryndis; Fridjonsson, Olafur H.; Altenbuchner, Josef; Watzlawick, Hildegard; Gerwig, Gerrit J.; Dijkhuizen, Lubbert; Kamerling, Johannis P.; Hreggvidsson, Gudmundur O.
    Alginate (alginic acid) is a linear polysaccharide, wherein (1->4)-linked β-D-mannuronic acid and its C5 epimer, α-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1->4) glycosidic linkages between the monomers by a β-elimination mechanism, to yield unsaturated 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid (Δ) at the non-reducing end of resulting oligosaccharides (α-L-erythro configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers. In this study, two alginate lyase genes, designated alyRm3 and alyRm4, from the marine thermophilic bacterium Rhodothermus marinus (strain MAT378), were cloned and expressed in Escherichia coli. The recombinant enzymes were characterized, and their substrate specificity and product structures determined. AlyRm3 (PL39) and AlyRm4 (PL17) are among the most thermophilic and thermostable alginate lyases described to date with temperature optimum of activity at ∼75 and 81°C, respectively. The pH optimum of activity of AlyRm3 is ∼5.5 and AlyRm4 at pH 6.5. Detailed NMR analysis of the incubation products demonstrated that AlyRm3 is an endolytic lyase, while AlyRm4 is an exolytic lyase, cleaving monomers from the non-reducing end of oligo/poly-alginates.
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    Fate mapping of hematopoietic stem cells reveals two pathways of native thrombopoiesis
    (2022) Morcos, Mina N. F.; Li, Congxin; Munz, Clara M.; Greco, Alessandro; Dressel, Nicole; Reinhardt, Susanne; Sameith, Katrin; Dahl, Andreas; Becker, Nils B.; Roers, Axel; Höfer, Thomas; Gerbaulet, Alexander
    Hematopoietic stem cells (HSCs) produce highly diverse cell lineages. Here, we chart native lineage pathways emanating from HSCs and define their physiological regulation by computationally integrating experimental approaches for fate mapping, mitotic tracking, and single-cell RNA sequencing. We find that lineages begin to split when cells leave the tip HSC population, marked by high Sca-1 and CD201 expression. Downstream, HSCs either retain high Sca-1 expression and the ability to generate lymphocytes, or irreversibly reduce Sca-1 level and enter into erythro-myelopoiesis or thrombopoiesis. Thrombopoiesis is the sum of two pathways that make comparable contributions in steady state, a long route via multipotent progenitors and CD48 hi megakaryocyte progenitors (MkPs), and a short route from HSCs to developmentally distinct CD48 -/lo MkPs. Enhanced thrombopoietin signaling differentially accelerates the short pathway, enabling a rapid response to increasing demand. In sum, we provide a blueprint for mapping physiological differentiation fluxes from HSCs and decipher two functionally distinct pathways of native thrombopoiesis.
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    High-throughput mutagenesis identifies mutations and RNA-binding proteins controlling CD19 splicing and CART-19 therapy resistance
    (2022) Cortés-López, Mariela; Schulz, Laura; Enculescu, Mihaela; Paret, Claudia; Spiekermann, Bea; Quesnel-Vallières, Mathieu; Torres-Diz, Manuel; Unic, Sebastian; Busch, Anke; Orekhova, Anna; Kuban, Monika; Mesitov, Mikhail; Mulorz, Miriam M.; Shraim, Rawan; Kielisch, Fridolin; Faber, Jörg; Barash, Yoseph; Thomas-Tikhonenko, Andrei; Zarnack, Kathi; Legewie, Stefan; König, Julian
    Following CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to developing CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis-regulatory elements and trans-acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to pervasive CD19 mis-splicing. Our dataset represents a comprehensive resource for identifying predictive biomarkers for CART-19 therapy.
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    Zellen sind Individualisten : Entscheidungsprozesse auf Einzelzellebene
    (2024) Lenhardt, Sonja; Hartmann, Laura; Legewie, Stefan; Loewer, Alexander
    Decision-making is a fundamental aspect of life. However, our understanding of how cells encode and decode information to enable reliable fate decisions remains limited. Employing live cell imaging and automated analysis, our research unveils substantial heterogeneity in the cellular response to TGFβ and sheds light on the intricate link between the dynamics of SMAD signaling, the state of individual cells and their fate decisions.
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    LUBAC enables tumor-promoting LTβ receptor signaling by activating canonical NF-κB
    (2024) Chen, Yu-Guang; Rieser, Eva; Bhamra, Amandeep; Surinova, Silvia; Kreuzaler, Peter; Ho, Meng-Hsing; Tsai, Wen-Chiuan; Peltzer, Nieves; de Miguel, Diego; Walczak, Henning
    Lymphotoxin β receptor (LTβR), a member of the TNF receptor superfamily (TNFR-SF), is essential for development and maturation of lymphoid organs. In addition, LTβR activation promotes carcinogenesis by inducing a proinflammatory secretome. Yet, we currently lack a detailed understanding of LTβR signaling. In this study we discovered the linear ubiquitin chain assembly complex (LUBAC) as a previously unrecognized and functionally crucial component of the native LTβR signaling complex (LTβR-SC). Mechanistically, LUBAC-generated linear ubiquitin chains enable recruitment of NEMO, OPTN and A20 to the LTβR-SC, where they act coordinately to regulate the balance between canonical and non-canonical NF-κB pathways. Thus, different from death receptor signaling, where LUBAC prevents inflammation through inhibition of cell death, in LTβR signaling LUBAC is required for inflammatory signaling by enabling canonical and interfering with non-canonical NF-κB activation. This results in a LUBAC-dependent LTβR-driven inflammatory, protumorigenic secretome. Intriguingly, in liver cancer patients with high LTβR expression, high expression of LUBAC correlates with poor prognosis, providing clinical relevance for LUBAC-mediated inflammatory LTβR signaling.