Deciphering context-dependent amber suppression efficiency in mammalian cells with an expanded genetic code

The genetic code of organisms can be expanded by introducing orthogonal translation systems (OTSs). One of the most commonly applied OTSs in mammalian cells is the archaeal pyrrolysyl-tRNA synthetase/tRNA_Pyl_CUA (PylRS/PylT) pair from Methanosarcina species. Thereby, usually in-frame amber stop codons (UAG) are suppressed to site-specifically incorporate non-canonical amino acids (ncAAs) into target proteins. These ncAAs can harbor unique chemical moieties, allowing to probe or engineer protein structure and function with high precision. To date, applicability of an expanded genetic code has been particularly advanced in bacteria by optimizing OTS components, modifying host translation, and developing mutually orthogonal translation systems. In mammalian cells, development of genetic code expansion tools has been largely focused on intrinsic properties of the OTS itself, for instance by engineering OTS components or tuning their expression levels. However, several-fold differences in ncAA incorporation efficiency are frequently observed between different amber stop codon positions within a target protein. These unpredictable variations in incorporation efficiencies substantially hamper the theoretical advantage of ncAAs to modify any user-defined site within a target protein. Here, applying a proteomics-based approach and fluorescent reporter system, we compute and validate a linear regression model that predicts ncAA incorporation efficiency in mammalian cells based on the nucleotide context. Thereby, we demonstrate that the immediate context directly modulates the competition between ncAA incorporation and termination at UAG. Moreover, our data support a molecular model in which the identity of up- and downstream nucleotides influences translational efficiency independent of amino acid and tRNA identity. Instead, base stacking of neighboring nucleotides might uniquely affect codon-anticodon base pairing during decoding of UAG. Additionally, context-specific ribosomal pausing and speed could contribute to varying ncAA incorporation efficiency. Furthermore, treatment with aminoglycosides and inhibition of nonsense mediated decay are proposed to improve yields of ncAA-modified proteins in mammalian cells. Taken together, our strategy not only facilitates the applicability of an expanded genetic code in mammalian cells, but should also prove useful in further deciphering the molecular mechanisms that govern context effects in translational efficiency. A better general understanding of context effects in translation would in turn benefit synthetic expansion of the genetic code.Der genetische Code von Organismen kann durch die Einbringung orthogonaler Translationssysteme (OTSe) erweitert werden. Das Pyrrolysyl-tRNA Synthetase/tRNA_Pyl_CUA (PylRS/PylT) Paar der Spezies Methanosarcina ist eines der am häufigsten angewendeten OTSe in Säugerzellen. Üblicherweise wird damit das amber Stoppcodon (UAG) innerhalb eines Leserasters supprimiert, um an spezifischen Stellen eines Zielproteins nicht-kanonische Aminosäuren (nkASn) einzubauen. Diese nkASn können einzigartige chemische Motive enthalten, die es ermöglichen die Struktur und Funktion von Proteinen mit hoher Präzision zu untersuchen und zu manipulieren. Bisher wurde insbesondere in Bakterien die Anwendbarkeit eines erweiterten genetischen Codes verbessert, indem OTS Komponenten optimiert, die Translation in Wirtsorganismen modifiziert und wechselseitig orthogonale Translationssysteme entwickelt wurden. Die Weiterentwicklung von Methoden, um den genetischen Code in Säugerzellen zu erweitern, fokussierte sich überwiegend auf intrinsische Eigenschaften der OTSe selbst, zum Beispiel der Modifizierung von OTS Komponenten oder der Anpassung ihrer Expressionslevel. Häufig unterscheiden sich jedoch verschiedene UAG Positionen in ihrer Effizienz eine nkAS einzubauen in mehrfacher Höhe. Diese unvorhersehbaren Schwankungen in der Einbaueffizienz schränken den Vorteil von nkASn erheblich ein, theoretisch jede benutzerdefinierte Position innerhalb eines Zielproteins modifizieren zu können. In dieser Publikation berechnen und validieren wir mit Hilfe einer proteomischen Methode und eines fluoreszierenden Reportersystems ein lineares Regressionsmodell, das anhand des Nukleotidkontextes die Effizienz des nkAS Einbaus in Säugerzellen vorhersagt. Wir zeigen dadurch, dass der unmittelbare Kontext direkt das Verhältnis zwischen nkAS Einbau und Termination an UAG moduliert. Unsere Daten unterstützen zudem ein molekulares Modell, in dem die Identität der vorherigen und nachfolgenden Nukleotide die Effizienz der Translation unabhängig von der Identität der Aminosäure und tRNA beeinflusst. Hingegen könnte sich ein Basen-Stacking über benachbarte Nukleotide in einzigartiger Weise auf die Codon-Anticodon Basenpaarung während der Dekodierung von UAG auswirken. Zusätzlich könnten ein Pausieren sowie die Geschwindigkeit des Ribosoms in Abhängigkeit vom Kontext zu der uneinheitlichen Effizienz des nkAS Einbaus beitragen. Des Weiteren werden ein Behandlungsverfahren mit Aminoglycosiden und eine Inhibierung des Nonsense-mediated Decay vorgeschlagen, um die Ausbeute an nkAS-modifizierten Proteinen zu verbessern. Zusammenfassend vereinfacht unsere Strategie nicht nur die Anwendbarkeit eines erweiterten genetischen Codes in Säugerzellen, sondern sollte sich auch als nützlich erweisen, um die molekularen Mechanismen, über die der Kontext die Translationseffizienz beeinflusst, weiter zu entschlüsseln. Ein besseres allgemeines Verständnis der Kontexteffekte bei der Translation würde wiederum die synthetische Erweiterung des genetischen Codes fördern

Tunable light and drug induced depletion of target proteins

Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to investigate the role of CED-3 in apoptosis during Caenorhabditis elegans development. These independent tools can be combined for spatial and temporal depletion of different sets of proteins, can help to distinguish immediate cellular responses from long-term adaptation effects and can facilitate the exploration of complex networks

Cloning Standard for Mammalian BioBrick Parts and Devices

To introduce a common cloning standard for BioBrick parts that find application in mammalian cells

Complement inhibitor CSMD1 acts as tumor suppressor in human breast cancer

Human CUB and Sushi multiple domains 1 (CSMD1) is a membrane-bound complement inhibitor suggested to act as a putative tumor suppressor gene, since allelic loss of this region encompassing 8p23 including CSMD1 characterizes various malignancies. Here, we assessed the role of CSMD1 as a tumor suppressor gene in the development of breast cancer in vitro and in vivo. We found that human breast tumor tissues expressed CSMD1 at lower levels compared to that in normal mammary tissues. The decreased expression of CSMD1 was linked to a shorter overall survival of breast cancer patients. We also revealed that expression of CSMD1 in human breast cancer cells BT-20 and MDA-MB-231 significantly inhibited their malignant phenotypes, including migration, adhesion and invasion. Conversely, stable silencing of CSMD1 expression in T47D cells enhanced cancer cell migratory, adherent and clonogenic abilities. Moreover, expression of CSMD1 in the highly invasive MDA-MB-231 cells diminished their signaling potential as well as their stem cell-like properties as assessed by measurement of aldehyde dehydrogenase activity. In a xenograft model, expression of CSMD1 blocked the ability of cancer cells to metastasize to secondary sites in vivo, likely via inhibiting local invasion but not the extravasation into distant tissues. Taken together, these findings demonstrate the role of CSMD1 as a tumor suppressor gene in breast cancer

Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency

Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation

Correction: Complement inhibitor CSMD1 acts as tumor suppressor in human breast cancer

This article has been corrected: In Figure 4B, the image of MDA-MB-231 cells expressing CSMD1 is an accidental duplicate of the image showing invaded BT-20 cells expressing CSMD1 in Figure 4A. The correct Figure 4, produced using the original data, is shown below. The authors declare that these corrections do not change the results or conclusions of this paper. Original article: Oncotarget. 2016; 7:76920–76933. https://doi.org/10.18632/oncotarget.1272

mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy

Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications

Exploring functional subsets of cancer-associated fibroblasts

The tumor microenvironment consists of several interacting cell types. Cancer research focssed mainly on the malignant cell in the past. The importance of the tumor microenvironment is increasingly appreciated, as endothelial cells and immune cells were identified as targets for anti-tumor therapy. Targeted therapy against cancer-associated fibroblasts (CAFs) are not in clinical use for the treatment of carcinomas, even though CAFs are involved in many tumor-supporting processes. CAFs are mesenchymal stromal cells and generate and modulate the extracellular matrix (ECM), which provides physical stability to the growing tumor. CAFs can alter cell-to-cell communication within the tumor microenvironment and thereby influence the immune reaction to cancer cells, the response to cancer therapy and the tumor metabolism.Breast cancer is the most common malignant disease and second most common reason for cancer-related death in women. Despite advancements in the treatment of breast cancer, some aggressive forms remain hard to treat.In the first paper we investigated the effect of complement oligomeric matrix protein (COMP) on breast cancer. Epithelial COMP expression is associated with reduced survival in breast cancer patients.We showed that COMP resolves endoplasmic reticulum stress and deregulates the cell metabolism, causing increased growth and metastasis in vivo. We propose COMP expression as a potential prognostic marker in breast cancer.In the second part of the thesis we analyzed the importance of platelet-derived growth factor (PDGF) signaling in solid tumors in general, and the effect of PDGF-CC signaling in breast cancer in particular. We showed that PDGF-CC signaling to CAFs and the subsequent release of CAF-derived stanniocalcin 1, hepatocyte growth factor, and insulin growth factor binding protein 3 maintain a basal-like phenotype in breast cancer. Genetic and pharmacologic disruption of this commuication loop resulted in conversion of a hormone receptor-negative into a hormone receptor-positive state, causing enhanced sensitivity to endocrine therapy in previously resistant tumors. We conclude that the breast cancer subtype is in part under the control of the tumor microenvironment.CAFs have many different functions in the tumor microenvironment and different origins for CAFs have been suggested. In the last paper we used single-cell RNA-sequencing of 786 mesenchymal cells derived from tumors of the MMTV-PyMT mouse model of breast cancer, to identify subclasses of CAFs in an unbiased approach. We detected and confirmed the existence of four subclasses that potentially derive from three different origins. Based on differential gene expression analysis we assigned functional properties to each CAF subgroup. Gene profiles of the main CAF subgroups held independent prognostic capability in large clinical cohorts. We showed that an in depth investigation of cellular constituents of the tumor microenvironment with increased resolution, can reveal a higher order of cellular organization in malignant disease

PDGF family function and prognostic value in tumor biology

The development and progression of a tumor depends on the close interaction of malignant cells and the supportive and suppressive tumor microenvironment. Paracrine signaling enables tumor cells to shape the surrounding tissue in order to decrease recognition by the immune system, attract blood vessels to fuel growth, change metabolic programs, and induce wound healing programs. In this study, we investigate the role of the platelet-derived growth factor (PDGF) family members PDGFA, PDGFB, PDGFC and PDGFD and their cognate tyrosine kinase receptors PDGFRA and PDGFRB, using publicly available data from The Cancer Genome Atlas and the Human Protein Atlas. Large scale analysis of expression correlation in RNA sequencing data from 7616 samples derived from 16 tumor types, revealed conserved functional programs in PDGF signaling in the majority of solid tumor types. Besides the well-known effects of PDGF signaling in mesenchymal cells, our analyses revealed a potential role of PDGF signaling in the composition of the immune microenvironment. We furthermore derived gene signatures with increased prognostic value for each PDGF family member. This study emphasizes the potential to impinge on specific paracrine signaling events to interfere with the crosstalk between malignant cells and their microenvironment