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Als sich der großstädtische Verkehr um 1900 unter die Erde zurückzog, hatte die Moderne ihr neues Spielfeld gefunden. Die U-Bahn machte alle Umbrüche des letzten Jahrhunderts mit: vom Kaiserpavillon über das elektrifizierte Symbol des Wiederaufbaus bis zum postmodernen Stadtteilbahnhof. Das neue moderneREGIONAL-Sommer-Heft “Untergründig. Als die Moderne fahren lernte” (Redaktion: M. Bredenbeck/K. Berkemann) folgt dem neuen alten Verkehrsmittel auf seinem Weg durch die Jahrzehnte. In seinem Leitartikel blickt der Architekturkritiker Nikolaus Bernau auf die Geschichte der Metro. Für die Fachbeiträge waren die Autoren in ihren (Wahl-)Heimatstädten unterirdisch unterwegs: Martin Bredenbeck in Bonn, Verena Pfeiffer-Kloss in West-Berlin, Elisabeth und Lorenz Intichar in Wien, Julius Reinsberg in Moskau. Für das Porträt ging Sebastian Bank auf kermanische Spurensuche durch Essen und im Interview schwärmt die langjährige Kölner Dombaumeisterin Barabara Schock-Werner von den schönen Seiten des rheinischen Untergrunds. Die Rechte für die einzelnen Textbeiträge liegen bei den Autor:innen, die Bildrechte sind jeweils am Bild selbst angegeben. Die einzelnen Heftbeiträge sind (in der jeweils aktuellen Form) online zugänglich., ISSN (online): 2365-0370, HBZ-ID: HT018260134, ZDB-ID: 1050988183

SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing protein 1) has been implicated in X-chromosome inactivation, imprinting and DNA damage repair. Mutations inSMCHD1can also cause facioscapulohumoral muscular dystrophy. More recently, SMCHD1 has also been detected as component of telomeric chromatin. Here, we identify requirements of SMCHD1 for DNA damage signaling and non-homologous end joining (NHEJ) at unprotected telomeres. Co-depletion of SMCHD1 with TRF2 reduced the rate of 3’ overhang removal in time course experiments and the number of telomere end fusions. In SMCHD1 deficient cells, the formation of ATM pS1981, γH2AX and 53BP1 containing telomere dysfunction induced foci (TIFs) were diminished indicating defects in checkpoint signaling. Strikingly, removal of TPP1 and subsequent activation of ATR signaling rescued telomere fusion events in TRF2-depletedSMCHD1knockout cells. Together, these data indicate that SMCHD1 depletion reduces telomere fusions in TRF2-depleted cells due to defects in ATM-dependent DNA checkpoint signaling. SMCHD1 mediates DNA damage signaling activation upstream of ATM phosphorylation at uncapped telomeres.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance. Consistently, mice with null mutation of Ceacam1 (Cc1(-/-)) exhibit impaired insulin clearance with increased lipid production in liver and redistribution to white adipose tissue, leading to visceral obesity at 2 months of age. When the mutation is propagated on the C57/BL6J genetic background, total fat mass rises significantly with age, and glucose intolerance and systemic insulin resistance develop at 6 months of age. This study was carried out to determine the mechanisms underlying the marked increase in total fat mass in 6-month-old mutants. Indirect calorimetry analysis showed that Cc1(-/-) mice develop hyperphagia and a significant reduction in physical activity, in particular in the early hours of the dark cycle, during which energy expenditure is only slightly lower than in wild-type mice. They also exhibit increased triglyceride accumulation in skeletal muscle, due in part to incomplete fatty acid β-oxidation. Mechanistically, hypothalamic leptin signaling is reduced, as demonstrated by blunted STAT3 phosphorylation in coronal sections in response to an intracerebral ventricular injection of leptin. Hypothalamic fatty-acid synthase activity is also elevated in the mutants. Together, the data show that the increase in total fat mass in Cc1(-/-) mice is mainly attributed to hyperphagia and reduced spontaneous physical activity. Although the contribution of the loss of CEACAM1 from anorexigenic proopiomelanocortin neurons in the arcuate nucleus is unclear, leptin resistance and elevated hypothalamic fatty-acid synthase activity could underlie altered energy balance in these mice.

Rationale: Regeneration of lost cardiomyocytes is a fundamental unresolved problem leading to heart failure. Despite several strategies developed from intensive studies performed in the past decades, endogenous regeneration of heart tissue is still limited and presents a big challenge that needs to be overcome to serve as a successful therapeutic option for myocardial infarction. Objective: One of the essential prerequisites for cardiac regeneration is the identification of endogenous cardiomyocyte progenitors and their niche that can be targeted by new therapeutic approaches. In this context, we hypothesized that the vascular wall, which was shown to harbor different types of stem and progenitor cells, might serve as a source for cardiac progenitors. Methods and Results: We describe generation of spontaneously beating mouse aortic wall-derived cardiomyocytes without any genetic manipulation. Using aortic wall-derived cells (AoCs) of WT (wild type), αMHC (α-myosin heavy chain), and Flk1 (fetal liver kinase 1)-reporter mice and magnetic bead-associated cell sorting sorting of Flk1 + AoCs from GFP (green fluorescent protein) mice, we identified Flk1 + CD (cluster of differentiation) 34 + Sca-1 (stem cell antigen-1)-CD44 − AoCs as the population that gives rise to aortic wall-derived cardiomyocytes. This AoC subpopulation delivered also endothelial cells and macrophages with a particular accumulation within the aortic wall-derived cardiomyocyte containing colonies. In vivo, cardiomyocyte differentiation capacity was studied by implantation of fluorescently labeled AoCs into chick embryonic heart. These cells acquired cardiomyocyte-like phenotype as shown by αSRA (α-sarcomeric actinin) expression. Furthermore, coronary adventitial Flk1 + and CD34 + cells proliferated, migrated into the myocardium after mouse myocardial infarction, and expressed Isl-1 + (insulin gene enhancer protein-1) indicative of cardiovascular progenitor potential. Conclusions: Our data suggest Flk1 + CD34 + vascular adventitia-resident stem cells, including those of coronary adventitia, as a novel endogenous source for generating cardiomyocytes. This process is essentially supported by endothelial cells and macrophages. In summary, the therapeutic manipulation of coronary adventitia-resident cardiac stem and their supportive cells may open new avenues for promoting cardiac regeneration and repair after myocardial infarction and for preventing heart failure.

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is known to be crucial to vasculogenesis and angiogenesis. Recently, CEACAM1 deficiency was shown to result in the formation of aortic plaque-like lesions, indicating a role for CEACAM1 in adult vessels as well. The underlying mechanisms remained largely elusive. Therefore, we aimed to elucidate the role of CEACAM1 in endothelial homeostasis. Here, we show that CEACAM1 deficiency causes subcellular eNOS redistribution in endothelial cells ( i.e., by eNOS depalmitoylation) and alters endothelial glycocalyx that confers antiadhesive properties to the endothelium ( i.e., by repression of glycocalyx-degrading enzymes). Accordingly, our analysis revealed an increased leukocyte-endothelial interaction in CEACAM1-deficient endothelium. In addition, CEACAM1 age dependently modulated basal and TNF-α-mediated endothelial barrier (EB) leakiness. In younger mice, CEACAM1 was protective for EB, whereas in aged mice it promoted EB leakiness. EB function depends on interendothelial adherence junctions formed by β-catenin/vascular endothelial-cadherin complexes. We show here that CEACAM1 influenced basal and TNF-α-mediated phosphorylation of β-catenin and caveolin-1, which are essential players in EB modulation. Both increased adhesiveness to leukocytes and EB modulation due to CEACAM1 deficiency may facilitate inflammatory cell transmigration into the vascular wall and subsequent plaque formation. Collectively, these results identify a crucial role for CEACAM1 in endothelial homeostasis of adult blood vessels.-Ghavampour, S., Kleefeldt, F., Bommel, H., Volland, J., Paus, A., Horst, A., Pfeiffer, V., Hubner, S., Wagner, N., Rueckschloss, U., Ergun, S. Endothelial barrier function is differentially regulated by CEACAM1-mediated signaling.

Tumors are often greatly dependent on signaling cascades promoting cell growth or survival and may become hypersensitive to inactivation of key components within these signaling pathways. Ras and RAF mutations found in human cancer confer constitutive activity to these signaling molecules thereby converting them into an oncogenic state. RAF dimerization is required for normal Ras-dependent RAF activation and is required for the oncogenic potential of mutant RAFs. Here we describe a new mouse model for lung tumor development to investigate the role of B-RAF in oncogenic C-RAF-mediated adenoma initiation and growth. Conditional elimination of B-RAF in C-RAF BxB-expressing embryonic alveolar epithelial type II cells did not block adenoma formation. However, loss of B-RAF led to significantly reduced tumor growth. The diminished tumor growth upon B-RAF inactivation was due to reduced cell proliferation in absence of senescence and increased apoptosis. Furthermore, B-RAF elimination inhibited C-RAF BxB-mediated activation of the mitogenic cascade. In line with these data, mutation of Ser-621 in C-RAF BxB abrogated in vitro the dimerization with B-RAF and blocked the ability to activate the MAPK cascade. Taken together these data indicate that B-RAF is an important factor in oncogenic C-RAF-mediated tumorigenesis.

Telomere maintenance by the conventional DNA replication machinery and telomerase is assisted by specialized DNA helicases, nucleases and telomere binding proteins. Here, we identify the THO components at telomeres and define critical roles of this complex in telomere stability. Deletion of the THO-subunit THP2 leads to telomere shortening. We discover that telomeres contain RNA: DNA hybrid structures or R-loops which involve the long-non-coding RNA TERRA and which accumulate in thp2-Delta cells. Telomere length is not restored by R-loop removal upon RNase H overexpression, but by deletion of Exonuclease 1 (Exo1). Replication stress further enhances the short telomere phenotype of THP2 mutants. Similar events occur upon induced transcription of TERRA and genetic analysis links Thp2 to TERRA function. Altogether, our data indicate that THO, through the interplay with TERRA, regulates chromosome end processing activities and prevents interference with semiconservative DNA replication of telomeric DNA.

Reference EPFL-CONF-219859View record in Web of Science Record created on 2016-07-19, modified on 2017-03-26

Telomeric repeat-containing RNA (TERRA) has been implicated in the control of heterochromatin and telomerase. We demonstrate that yeast TERRA is regulated by telomere-binding proteins in a chromosome-end-specific manner that is dependent on subtelomeric repetitive DNA elements. At telomeres that contain only X-elements, the Rap1 carboxy-terminal domain recruits the Sir2/3/4 and Rif1/2 complexes to repress transcription in addition to promoting Rat1-nuclease-dependent TERRA degradation. At telomeres that contain Y' elements, however, Rap1 represses TERRA through recruitment of Rif1 and Rif2. Our work emphasizes the importance of subtelomeric DNA in the control of telomeric protein composition and telomere transcription.

Bacterial small noncoding RNAs (sRNAs) generally recognize target mRNAs in the 5' region to prevent 30S ribosomes from initiating translation. It was thought that the mRNA coding sequence (CDS) was refractory to sRNA-mediated repression, because elongating 70S ribosomes have an efficient RNA helicase activity that prevents stable target pairing. We report that the Hfq-associated MicC sRNA silences Salmonella typhimurium ompD mRNA via aor=12-bp RNA duplex within the CDS (codons 23-26) that is essential and sufficient for repression. MicC does not inhibit translational initiation at this downstream position but instead acts by accelerating RNase E-dependent ompD mRNA decay. We propose an alternative gene-silencing pathway within bacterial CDS wherein sRNAs repress targets by endonucleolytic mRNA destabilization rather than by the prototypical inhibition of translational initiation. The discovery of CDS targeting markedly expands the sequence space for sRNA target predictions in bacteria.

The RNA chaperone, Hfq, plays a diverse role in bacterial physiology beyond its original role as a host factor required for replication of Qbeta RNA bacteriophage. In this study, we show that Hfq is involved in the expression and secretion of virulence factors in the facultative intracellular pathogen, Salmonella typhimurium. A Salmonella hfq deletion strain is highly attenuated in mice after both oral and intraperitoneal infection, and shows a severe defect in invasion of epithelial cells and a growth defect in both epithelial cells and macrophages in vitro. Surprisingly, we find that these phenotypes are largely independent of the previously reported requirement of Hfq for expression of the stationary phase sigma factor, RpoS. Our results implicate Hfq as a key regulator of multiple aspects of virulence including regulation of motility and outer membrane protein (OmpD) expression in addition to invasion and intracellular growth. These pleiotropic effects are suggested to involve a network of regulatory small non-coding RNAs, placing Hfq at the centre of post-transcriptional regulation of virulence gene expression in Salmonella. In addition, the hfq mutation appears to cause a chronic activation of the RpoE-mediated envelope stress response which is likely due to a misregulation of membrane protein expression.

Carcinoembryonic antigen-related cell adhesion molecule 2 (CEACAM2) regulates food intake as demonstrated by hyperphagia in mice with the Ceacam2 null mutation (Cc2(-/-)). This study investigated whether CEACAM2 also regulates insulin secretion. Ceacam2 deletion caused an increase in β-cell secretory function, as assessed by hyperglycemic clamp analysis, without affecting insulin response. Although CEACAM2 is expressed in pancreatic islets predominantly in non-β-cells, basal plasma levels of insulin, glucagon and somatostatin, islet areas, and glucose-induced insulin secretion in pooled Cc2(-/-) islets were all normal. Consistent with immunofluorescence analysis showing CEACAM2 expression in distal intestinal villi, Cc2(-/-) mice exhibited a higher release of oral glucose-mediated GLP-1, an incretin that potentiates insulin secretion in response to glucose. Compared with wild type, Cc2(-/-) mice also showed a higher insulin excursion during the oral glucose tolerance test. Pretreating with exendin(9-39), a GLP-1 receptor antagonist, suppressed the effect of Ceacam2 deletion on glucose-induced insulin secretion. Moreover, GLP-1 release into the medium of GLUTag enteroendocrine cells was increased with siRNA-mediated Ceacam2 down-regulation in parallel to an increase in Ca(2+) entry through L-type voltage-dependent Ca(2+) channels. Thus, CEACAM2 regulates insulin secretion, at least in part, by a GLP-1-mediated mechanism, independent of confounding metabolic factors.

The bacterial envelope stress response (ESR) is triggered by the accumulation of misfolded outer membrane proteins (OMPs) upon envelope damage or excessive OMP synthesis, and is mediated by the alternative sigma factor, sigmaE. Activation of the GE pathway causes a rapid downregulation of major omp mRNAs, which prevents further build-up of unassembled OMPs and liberates the translocation and folding apparatus under conditions that require envelope remodelling. The factors that facilitate the rapid removal of the unusually stable omp mRNAs in the ESR were previously unknown. We report that in Salmonella the ESR relies upon two highly conserved, sigmaE-controlled small non-coding RNAs, RybB and MicA. By using a transcriptomic approach and kinetic analyses of target mRNA decay in vivo, RybB was identified as the factor that selectively accelerates the decay of multiple major omp mRNAs upon induction of the ESR, while MicA is proposed to facilitate rapid decay of the single ompA mRNA. In unstressed bacterial cells, the two oE-dependent small RNAs function within a surveillance loop to maintain envelope homeostasis and to achieve autoregulation of oE.

We have previously shown that mice lacking the protein kinase B-RAF have defects in both neural and endothelial cell lineages and die around embryonic day 12 (E12). To delineate the function of B-RAF in the brain, B-RAF KIN/KIN mice lacking B-RAF and expressing A-RAF under the control of the B-RAF locus were created. B-RAF KIN/KIN embryos displayed no vascular defects, no endothelial and neuronal apoptosis, or gross developmental abnormalities, and a significant proportion of these animals survived for up to 8 weeks. Cell proliferation in the neocortex was reduced from E14.5 onwards. Newborn cortical neurons were impaired in their migration toward the cortical plate, causing a depletion of Brn-2-expressing pyramidal neurons in layers II, III, and V of the postnatal cortex. Our data reveal that B-RAF is an important mediator of neuronal survival, migration, and dendrite formation and that A-RAF cannot fully compensate for these functions.

Numerous small non-coding RNAs (sRNAs) in bacteria modulate rates of translation initiation and degradation of target mRNAs, which they recognize through base-pairing facilitated by the RNA chaperone Hfq. Recent evidence indicates that the ternary complex of Hfq, sRNA and mRNA guides endoribonuclease RNase E to initiate turnover of both the RNAs. We show that a sRNA not only guides RNase E to a defined site in a target RNA, but also allosterically activates the enzyme by presenting a monophosphate group at the 5'-end of the cognate-pairing "seed." Moreover, in the absence of the target the 5'-monophosphate makes the sRNA seed region vulnerable to an attack by RNase E against which Hfq confers no protection. These results suggest that the chemical signature and pairing status of the sRNA seed region may help to both 'proofread' recognition and activate mRNA cleavage, as part of a dynamic process involving cooperation of RNA, Hfq and RNase E.

Post-transcriptional repression of porin synthesis has emerged as a major function of Hfq-dependent, small non-coding RNAs (sRNAs). Many enterobacteria express OmpX-like porins, a family of outer membrane proteins whose physiological roles and structural properties have been studied intensively. While regulatory sRNAs have been identified for most major and many minor porins of Salmonella and Escherichia coli, a post-transcriptional regulator of OmpX levels has never been found. Here, we have taken a 'reverse target search' approach by systematic inactivation of Salmonella sRNA genes, and screening 35 sRNA deletion strains for effects on OmpX synthesis. We have identified the Hfq-dependent CyaR (formerly RyeE) sRNA as an ompX repressor. Global transcriptomic profiling following induction of CyaR expression suggests that ompX mRNA is the primary target of this sRNA under standard growth conditions. The results of phylogenetic and mutational analyses suggest that a conserved RNA hairpin of CyaR, featuring a C-rich apical loop, acts to sequester the Shine-Dalgarno sequence of ompX mRNA and to inhibit translational initiation. We have also discovered that cyaR expression is tightly controlled by the cyclic AMP receptor protein, CRP. This represents a new link between porin repression and nutrient availability that is likely to be widely conserved among enterobacteria.

The Salmonella pathogenicity island (SPI-1) encodes approximately 35 proteins involved in assembly of a type III secretion system (T3SS) which endows Salmonella with the ability to invade eukaryotic cells. We have discovered a novel SPI-1 gene, invR, which expresses an abundant small non-coding RNA (sRNA). The invR gene, which we identified in a global search for new Salmonella sRNA genes, is activated by the major SPI-1 transcription factor, HilD, under conditions that favour host cell invasion. The RNA chaperone, Hfq, is essential for the in vivo stability of the approximately 80 nt InvR RNA. Hfq binds InvR with high affinity in vitro, and InvR co-immunoprecipitates with FLAG epitope-tagged Hfq in Salmonella extracts. Surprisingly, deletion/overexpression of invR revealed no phenotype in SPI-1 regulation. In contrast, we find that InvR represses the synthesis of the abundant OmpD porin encoded by the Salmonella core genome. As invR is conserved in the early branching Salmonella bongori, we speculate that porin repression by InvR may have aided successful establishment of the SPI-1 T3SS after horizontal acquisition in the Salmonella lineage. This study identifies the first regulatory RNA of an enterobacterial pathogenicity island, and new roles for Hfq and HilD in SPI-1 gene expression.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License., The family of RAF kinases transduces extracellular information to the nucleus, and their activation is crucial for cellular regulation on many levels, ranging from embryonic development to carcinogenesis. B-RAF and C-RAF modulate neurogenesis and neuritogenesis during chicken inner ear development. C-RAF deficiency in humans is associated with deafness in the rare genetic insulin-like growth factor 1 (IGF-1), Noonan and Leopard syndromes. In this study, we show that RAF kinases are expressed in the developing inner ear and in adult mouse cochlea. A homozygous C-Raf deletion in mice caused profound deafness with no evident cellular aberrations except for a remarkable reduction of the K+ channel Kir4.1 expression, a trait that suffices as a cause of deafness. To explore the role of C-Raf in cellular protection and repair, heterozygous C-Raf +/− mice were exposed to noise. A reduced C-RAF level negatively affected hearing preservation in response to noise through mechanisms involving the activation of JNK and an exacerbated apoptotic response. Taken together, these results strongly support a role for C-RAF in hearing protection., This work was supported by Spanish grants from the Ministerio de Economia y Competitividad (SAF2011-24391 and SAF2014-53979-R) and European FP7-INNOVA2-AFHELO and FP7-PEOPLE-IAPP-TARGEAR to IVN. RdI hold a CSIC contract associated to SAF2011-24391.

Telomeres are the physical ends of eukaryotic chromosomes. They protect chromosome ends from DNA degradation, recombination, and DNA end fusions, and they are important for nuclear architecture. Telomeres provide a mechanism for their replication by semiconservative DNA replication and length maintenance by telomerase. Through telomerase repression and induced telomere shortening, telomeres provide the means to regulate cellular life span. In this review, we introduce the current knowledge on telomere composition and structure. We then discuss in depth the current understanding of how telomere components mediate their function during semiconservative DNA replication and how telomerase is regulated at the end of the chromosome. We focus our discussion on the telomeres from mammals and the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Telomeres are specialized nucleoprotein structures that protect chromosome ends from DNA damage response (DDR) and DNA rearrangements. The telomeric shelterin protein TRF2 suppresses the DDR, and this function has been attributed to its abilities to trigger t-loop formation or prevent massive decompaction and loss of density of telomeric chromatin. Here, we applied stochastic optical reconstruction microscopy (STORM) to measure the sizes and shapes of functional human telomeres of different lengths and dysfunctional telomeres that elicit a DDR. Telomeres have an ovoid appearance with considerable plasticity in shape. Examination of many telomeres demonstrated that depletion of TRF2, TRF1, or both affected the sizes of only a small subset of telomeres. Costaining of telomeres with DDR markers further revealed that the majority of DDR signaling telomeres retained a normal size. Thus, DDR signaling at telomeres does not require decompaction. We propose that telomeres are monitored by the DDR machinery in the absence of telomere expansion and that the DDR is triggered by changes at the molecular level in structure and protein composition.