Gene dosage in mammals: characterization of haploid embryonic stem cells

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Ring1B is crucial for the regulation of developmental control genes and PRC1 proteins but not X inactivation in embryonic cells

The Polycomb group (PcG) gene Ring1B has been implicated in the repression of developmental control genes and X inactivation and is essential for embryogenesis. Ring1B protein contains a RING finger domain and functions as an E3 ubiquitin ligase that is crucial for the monoubiquitination of histone H2A (H2AK119ub1). Here, we study the function of Ring1B in mouse embryonic stem (ES) cells. The deletion of Ring1B causes the loss of several PcG proteins, showing an unanticipated function in the regulation of PcG protein levels. Derepression of lineage genes and an aberrant differentiation potential is observed in Ring1B-deficient ES cells. Despite a crucial function of Ring1B in establishing the chromosome-wide ubiquitination of histone H2A lysine 119 (H2AK119ub1) upon Xist expression in ES cells, the initiation of silencing by Xist is independent of Ring1B. Other chromatin marks associated with the initiation of X inactivation are not affected in Ring1B-deficient cells, suggesting compensation for the loss of Ring1B in X inactivation in contrast to the repression of lineage genes

Long-Distance Quantum Communication with Entangled Photons using Satellites

The use of satellites to distribute entangled photon pairs (and single photons) provides a unique solution for long-distance quantum communication networks. This overcomes the principle limitations of Earth-bound technology, i.e. the narrow range of some 100 km provided by optical fiber and terrestrial free-space links.Comment: 12 pages, 7 figures; submitted to IEEE Journal of Selected Topics in Quantum Electronics, special issue on "Quantum Internet Technologies

Four Quarks from Lattice to the Continuum

A continuum extrapolation of static four- and two-quark energies calculated in quenched SU(2) is done based on Sommer's method of setting the scale. A model for four-quark energies with explicit gluonic degrees of freedom removed is fitted to these energies and the behavior of the parameters of the model is investigated.Comment: 4 pages, 3 figures. Talk presented at LATTICE96(phenomenology

The role of Polycomb group proteins in X chromosome inactivation and embryonic stem cell biology

Polycomb Proteine haben eine wichtige Bedeutung für die Regulation von zellulären Differenzierungsprozessen, Tumorigenese und Imprinting in Säugetieren. Sie sind evolutionär konserviert und dafür verantwortlich die Transkription von Genen stabil über mehrere Zellteilungen epigenetisch zu hemmen. In genomweiten Studien wurde nachgewiesen, dass Polycomb Proteine in embryonalen Stammzellen an mehr als 2500 Promotoren binden. Gene, die von Polycomb Proteinen gebunden werden, haben eine zentrale Steuerfunktion in der embryonalen Entwicklung. Es wurde die Hypothese aufgestellt, dass in frühen embryonalen Zellen durch Polycomb Proteine epigenetische Muster gesetzt werden, die für Differenzierungsprozesse notwendige dynamische Änderungen im Transkriptionsprofil ermöglichen. Zwei unterschiedliche Polycomb Komplexe wurden biochemisch nachgewiesen. Beide besitzen enzymatische Aktivität, welche gegen Histone gerichtet ist. Der „Polycomb repressive complex 1“ (PRC1) vermittelt die Mono-Ubiquitinierung von Histon H2A, wogegen PRC2 für die Histon H3 Tri-Methylierung an Lysin 27 verantwortlich ist. Beide Histon Modifikationen sind charakteristisch für transkriptionell inaktives Chromatin. In Knockout Studien wurde gezeigt dass beide Komplexe essentiell für die embryonale Entwicklung sind und dass das Fehlen eines der beiden Komplexe zum Entwicklungsstopp in der Gastrulation führt. In meiner Dissertation beschreibe ich zuerst die Funktion des PRC1 Komplexes in embryonalen Stammzellen bezüglich der Regulation von Differenzierungs-Kontrollgenen sowie der X Chromosom Inaktivierung. Meine Resultate belegen, dass der Verlust eines funktionellen PRC1 Komplexes zur Expression von Differenzierungs-Kontrollgenen führt, welche in Wiltdtyp ES Zellen inaktiv sind. Im Gegensatz dazu bleibt die transkriptionelle Repression des inaktiven X Chromosoms aufrecht. Meine Resultate und Ergebnisse aus früheren Studien werfen die Frage auf, in welchem Ausmaß die PRC1 and PRC2 Komplexe überlappende Funktionen im Bezug auf die Regulation von Polycomb gebundenen Genen sowie in der frühen Differenzierung haben. Um eine mögliche Redundanz zwischen den beiden Polycomb Komplexen zu analysieren wurden die zentralen Proteine beider Komplexe durch homologe Rekombination zerstört. In diesen doppelt defizienten (dKO) ES Zellen wurden Gene identifiziert, die von PRC1 und PRC2 redundant inhibiert werden. Im Gegensatz zu Eed-/- oder Ring1B-/- ES Zellen, resultiert der Verlust von beiden PcG Komplexen darin, dass keine Teratome gebildet werden können, was darauf hindeutet, dass beide PcG Komplexe eine redundante Funktion in der Differenzierung ausüben. Meine Resultate weisen darauf hin, dass eine Anzahl von PcG gebundenen Genen von beiden Polycomb Komplexen redundant reguliert wird. Die transkriptionelle Regulation durch PcG Proteine ist jedoch nicht auf Gene beschränkt. Meine Ergebnisse zeigen, dass endogene Retroviren von Polycomb Proteinen gebunden und reguliert werden. Zusammenfassend zeigen meine Resultate eine unerwartete Redundanz zwischen PRC1 und PRC2 in der Regulation der Genexpression und in Differenzierungsprozessen, sowie eine essentielle Funktion von Polycomb Proteinen im Schutz der genomischen Integrität durch die Repression von parasitären DNA Sequenzen.Polycomb complexes establish epigenetic patterns for maintaining gene repression by modifying histone tails. Polycomb complex mediated epigenetic marks are thought to be important for establishing and maintaining cellular identity. In mammals, PcG proteins regulate tumorigenesis, imprinting and dosage compensation. A role in the regulation of self renewal and pluripotency of embryonic stem (ES) cells has been proposed. In order to delineate the function of PcG complexes their genomic binding sites have been determined. In mouse ES cells approximately 2500 PcG target genes have been identified, many of which have central functions in embryonic development. PcG proteins have been proposed to maintain pluripotency by inhibiting the transcription of lineage specifying genes. Developmental plasticity is conferred by the lineage specific activation of PcG target genes in the course of differentiation. Two catalytically active PcG complexes have been biochemically characterized. Polycomb repressive complex 1 (PRC1) contains the ubiquitin E3 ligase Ring1B and catalyses mono-ubiquitination of Lysine 119 on histone H2A (ubH2A). PRC2 contains the PcG proteins Eed, Suz12 and Ezh2, which catalyses tri-methylation of Lysine 27 on histone H3 (H3K27me3). The catalytic functions of PRC1 and PRC2 are essential for development and mutations in either Eed or Ring1B lead to developmental arrest soon after implantation. In my thesis I investigated the function of the PcG system in embryonic stem cell biology and X chromosome inactivation. For this, I first generated Ring1B deficient ES cells by homologous recombination. I found that the deletion of Ring1B disrupts the PRC1 complex, and several PRC1 member proteins as well as ubH2A are absent in Ring1B-/- cells. Furthermore, the deletion of PRC1 function results in derepression of lineage control genes which are normally not expressed in ES cells. Despite the strong perturbance of epigenetic gene repression and the destabilization of the ES cell state, X chromosome inactivation was unaffected by the loss of PRC1 activity. The activity of PRC2 remained unaffected and H3K27me3 was recruited to the inactive X chromosome. Vice versa, PRC2 deficiency leads to the absence of H3K27me3 genome wide and on the Xi, whereas PRC1 recruitment to the Xi and genomic ubH2A levels remain largely unaffected. This indicates that parallel modes of PRC1 and PRC2 recruitment exist and that these marks are, to a large extent, independent of each other. To address the redundancy of PRC1 and PRC2 and the role of the PcG system in maintaining pluripotency, I have established an ES cell line deficient for PRC1 and PRC2 by concomitant deletion of Ring1B and Eed. My data provide genetic evidence for an unexpected redundancy between PRC1 and PRC2 and show that the PcG system is essential for the maintenance of differentiated cell types. Furthermore, endogenous retroelements are identified as novel targets for PcG regulation and are activated in the absence of PcG repression. This indicates that apart from its function in gene regulation, the PcG system plays a role in maintaining genome integrity by the repression of parasitic DNA sequences

Proof-of-Concept Experiments for Quantum Physics in Space

Quantum physics experiments in space using entangled photons and satellites are within reach of current technology. We propose a series of fundamental quantum physics experiments that make advantageous use of the space infrastructure with specific emphasis on the satellite-based distribution of entangled photon pairs. The experiments are feasible already today and will eventually lead to a Bell-experiment over thousands of kilometers, thus demonstrating quantum correlations over distances which cannot be achieved by purely earth-bound experiments.Comment: 15 pages, 10 figures, to appear in: SPIE Proceedings on Quantum Communications and Quantum Imaging (2003

The bovine dilated cardiomyopathy locus maps to a 1.0-Mb interval on chromosome 18

Cardiomyopathies are myocardial diseases that lead to cardiac dysfunction, heart failure, arrhythmia, and sudden death. In human medicine, cardiomyopathies frequently warrant heart transplantation in children and adults. Bovine dilated cardiomyopathy (BDCMP) is a heart muscle disorder that has been observed during the last 30years in cattle of Holstein-Friesian origin. In Switzerland BDCMP affects Swiss Fleckvieh and Red Holstein breeds. BDCMP is characterized by a cardiac enlargement with ventricular remodeling and chamber dilatation. The common symptoms in affected animals are subacute subcutaneous edema, congestion of the jugular veins, and tachycardia with gallop rhythm. A cardiomegaly with dilatation and hypertrophy of all heart chambers, myocardial degeneration, and fibrosis are typical postmortem findings. It was shown that all BDCMP cases reported worldwide traced back to a red factor-carrying Holstein-Friesian bull, ABC Reflection Sovereign. An autosomal recessive mode of inheritance was proposed for BDCMP. Recently, the disease locus was mapped to a 6.7-Mb interval MSBDCMP06-BMS2785 on bovine Chr 18 (BTA18). In the present study the BDCMP locus was fine mapped by using a combined strategy of homozygosity mapping and association study. A BAC contig of 2.9Mb encompassing the crucial interval was constructed to establish the correct marker order on BTA18. We show that the disease locus is located in a gene-rich interval of 1.0Mb and is flanked by the microsatellite markers DIK3006 and MSBDCMP5

Mo.Hub – Co-Developing Cooperative Mobility Hubs in Vienna

Cities face the challenge that urban public spaces are often dominated by moving and parked motorised vehicles. In particular in existing neighbourhoods, the question arises as to where space for active mobility, greening and recreation can be taken from and how public space can be redesigned to meet users’ needs. The availability of diverse mobility solutions - as concentrated at mobility hubs - promotes inter-modal and multi-modal, seamless mobility and helps to reduce motorisation (Villareal 2018; Pais 2019; Claasen 2020). Thus, mobility hubs are used in some European cities, to concentrate different mobility options and functions of the public urban space spatially and digitally concentrate (Villareal 2018; Pais 2019; Claasen 2020). The implementation of such mobility hubs has often taken place in new neighbourhoods and is organised in a top-down manner by transport companies, city administrations or developers. The research project Mo.Hub (https://mohub.at) aims to tackle the challenge of implementing mobility hubs in existing neighbourhoods by co-developing and implementing three mobility hubs with a cooperative and co-creative approach for a pilot phase of six months in Vienna. The mobility hubs combine demand-oriented (shared) mobility offers in close distance to public transport and a jointly designed recreational area, in the form of a parklet. Thereby public space regained by transforming the behaviour from private car use to multi-modal mobility behaviour is made visible. A higher user acceptance is expected by involvement not only in the implementation process but also by testing new operating models that build upon active participation and self-organization

Bovine Cardiac Troponin I Gene (\u3cem\u3eTNNI3\u3c/em\u3e) as a Candidate Gene for Bovine Dilated Cardiomyopathy

The cardiac troponin complex, which is an important component of the contractile apparatus, is composed of the three subunits troponin I (TnI), troponin C (TnC) and troponin T (TnT). Troponin I is the inhibitory subunit and consists of three isoforms encoded by TNNI1, TNNI2 and TNNI3 genes, respectively. Due to the different types of cardiomyopathies caused by mutations in the TNNI3 gene and its fluorescence in situ hybridization (FISH) mapping on bovine chromosome 18q26, which was shown to be linked to the recessively inherited bovine dilated cardiomyopathy (BDCMP), bovine TNNI3 was considered as candidate gene for BDCMP. Real-time polymerase chain reaction (PCR) TNNI3 expression analysis resulted in a significant difference between BDCMP affected and unaffected animals when normalized to ACTB gene expression, but there was no significant difference in expression when normalized to GAPDH. Northen blotting experiment was in agreement with the expression analysis and did not reveal a significant difference between the group of BDCMP affected and unaffected animals. Sequencing of the bovine TNNI3 gene revealed a single nucleotide polymorphism in intron 6 (c.378+315G\u3eA), but this single nucleotide polymorphism (SNP)was present regardless of the BDCMP status. In summary our data provide evidence to exclude the bovine TNNI3 gene as a candidate for BDCMP