Die Rolle des HP1β über die genomische Stabilität und zelluläre Seneszenz

Heterochromatin protein 1 (HP1) proteins are fundamental units of heterochromatin packaging that are enriched at the centromeres and telomeres of nearly all eukaryotic chromosomes. HP1 homologues are found in a variety of organisms and are involved in the establishment and maintenance of higher-order chromatin structures by specifically recognizing and binding to (tri- and di-) methylated lysine 9 on histone H3. In mammals, there are three HP1 homologues termed HP1α (Cbx5), HP1β (Cbx1), and HP1γ (Cbx3). Among these three isoforms HP1β is the best characterized. Murine HP1β is essential for organismal survival. Cbx1-/- knockout mice are perinatal lethal and exhibit aberrant cerebral cortex development, reduced proliferation of neuronal precursors, widespread cell death and edema. Cbx1-/- neurospheres cultured in vitro show a dramatic genomic instability. This study demonstrates that Cbx1+/- and Cbx1-/- mouse embryonic fibroblasts (MEFs) escape senescence crisis that is associated with gross chromosomal aberrations including aneuploidy, premature chromosome separations and telomere-telomere fusions. Telomeres of Cbx1-/- MEFs show reduced binding of the shelterin protein TRF1 with no change in the cellular localizations of POT1a, POT1b and TPP1 proteins compared to wild type (WT) cells. Telomere length analysis revealed that the telomeres of late passage Cbx1-/- MEFs are longer (~20 kb) than the WT controls. There was no change in the localization of cohesin protein SMC3, spindle assembly checkpoint protein BUB1 and SGO1. In an in vitro model of oncogene-induced senescence (OIS), introduction of Cbx1-/- mutation in cells expressing H-rasV12 oncogene did not result senescence bypass. In vivo experiments utilizing the inducible K-rasV12 oncogene expression in Cbx1+/- mice resulted in increased malignant adenocarcinomas in lungs, which were negative for the markers of senescence. Taken together these data indicate that HP1β acts as a tumour suppressor rather than mediating senescence in response to oncogenic stress. GST pull down experiments with WT HP1β, HP1β mutants and recombinant histone H3 showed that the binding of HP1β to histone H3 is resistant to 0.75 M NaCl concentrations and that HP1β chromoshadow domain is sufficient for this interaction. Isothermal calorimetry experiments confirmed that the binding affinity of HP1β for recombinant histone H3 was 4 times higher than its affinity for H3K9me3. V23M and F45E (“aromatic cage”) mutations in the HP1β chromo domain were also shown to inhibit binding to H3K9me3 while retaining binding to histone H3. In this study, the role of murine HP1β protein in the regulation of genome stability and senescence has been investigated, which provided insights into the role of HP1β in both processes. It has been shown that there is a high affinity binding of HP1β to the histone H3 histone-fold domain that is stronger than the affinity to H3K9me3. It is proposed that the loss of this high affinity interaction might result in the perinatal lethal phenotype seen in Cbx1-/- mice.Heterochromatin-Protein-1- (HP1-) Proteine sind grundlegende strukturelle Komponenten des Heterochromatins, die bevorzugt in den Centromeren und Telomeren fast aller eukaryotischer Chromosomen vorkommen. HP1-Homologe finden sich in zahlreichen Organismen und sind an der Bildung und Aufrechterhaltung von Chromatinstrukturen höherer Ordnung beteiligt. Dazu erkennen und binden sie spezifisch (tri- und di-) methylierte Lysin-9-Reste am Histon H3. In Säugern gibt es drei HP1-Homologe, nämlich HP1α (Cbx5), HP1β (Cbx1) und HP1γ (Cbx3), von denen HP1β das am besten charakterisierte darstellt. Das murine HP1β ist unabdingbar für das Überleben des Organismus. Cbx1-/- -Nullmutanten sind perinatal letal und zeigen eine anomale Entwicklung des cerebralen Cortex, eine verringerte Proliferation von neuronalen Vorläuferzellen, ausgedehnten Zelltod und Ödeme. In vitro kultivierte Cbx1-/- -Neurosphären weisen eine signifikante genomische Instabilität auf. Diese Arbeit zeigt, dass murine embryonale Fibroblasten (MEF) der Genotypen Cbx1+/- und Cbx1-/- der Seneszenzkrise entkommen, die mit erheblichen Chromosomenanomalien einschließlich Aneuploidie, vorzeitiger Chromosomenseperation und Telomer-Telomer-Fusionen einhergeht. Telomere von Cbx1-/- -MEF zeigen eine verringerte Bindung des Shelterin-Proteins TRF1, aber keine Veränderung der zellulären Lokalisation der Proteine POT1a, POT1b und TPP1 im Vergleich zu Wildtyp- (WT-) Zellen. Eine Analyse der Telomerlängen ergab, dass die Telomere von späten Cbx1-/- -MEF-Passagen ~20 kb länger waren als WT-Kontrollen. Es konnten dabei keine Änderungen der Lokalisationen des Kohesinproteins SMC3, des Spindelkontrollpunkt-Proteins BUB1 sowie des SGO1-Proteins festgestellt werden. In einem In-vitro-Modell der Onkogen-induzierten Seneszenz (OIS) resultierte die Einführung der Cbx1-/- -Mutation in H-rasV12-Onkogen exprimierende Zellen nicht in einer Umgehung der Seneszenz. In-vivo-Experimente mit Hilfe einer induzierbaren K-rasV12-Onkogenexpression in Cbx1+/- -Mäusen führten zum vermehrten Auftreten maligner Adenokarzinome der Lunge, wobei diese keine Seneszenzmarker aufweisen. Zusammengenommen weisen diese Daten darauf hin, dass HP1β keine Induktion der Seneszenz als Antwort auf onkogenen Stress bewirkt, sondern vielmehr als Tumorsuppressor fungiert. GST-pull-down-Experimente mit WT-HP1β oder HP1β-Mutanten und rekombinantem Histon H3 zeigten, dass die Bindung von HP1β an Histon H3 resistent gegenüber einer Konzentration von 0.75 M NaCl ist und dass die Chromoshadow-Domäne ausreichend für diese Interaktion ist. Isothermale Kalorimetrie-Experimente zeigten, dass die Bindungsaffinität von HP1β zu rekombinantem Histon H3 viermal höher liegt als die Affinität zu H3K9me3. V23M- und F45E- (“aromatischer Käfig-”) Mutationen in der HP1β-Chromo-Domäne zeigten weiterhin eine Inhibition der H3K9me3-Bindung während die Bindung zu Histon H3 erhalten bleibt. In dieser Arbeit wurde die Rolle des murinen HP1β-Proteins bei der Regulation der Genomstabilität und Seneszenz untersucht, wobei sich Einblicke in die Involvierung von HP1β in beide Prozesse ergaben. Es wurde gezeigt, dass HP1β eine hohe Affinität zur histone-fold-Domäne des Histons H3 besitzt, welche über der Affinität zu H3K9me3 liegt. Hieraus ergibt sich, dass der Verlust dieser hochaffinen Interaktion den perinatal letalen Phänotyp von Cbx1-/- Mäusen bedingen könnte

HP1γ function is required for male germ cell survival and spermatogenesis

<p>Abstract</p> <p>Background</p> <p>HP1 proteins are conserved components of eukaryotic constitutive heterochromatin. In mammals, there are three genes that encode HP1-like proteins, termed HP1α, HP1β and HP1γ, which have a high degree of homology This paper describes for the first time, to our knowledge, the physiological function of HP1γ using a gene-targeted mouse.</p> <p>Results</p> <p>While targeting the <it>Cbx3 </it>gene (encoding the HP1γ protein) with a conditional targeting vector, we generated a hypomorphic allele (<it>Cbx3</it>^{<it>hypo</it>}), which resulted in much reduced (barely detectable) levels of HP1γ protein. Homozygotes for the hypomorphic allele (<it>Cbx3</it>^{<it>hypo</it>/<it>hypo</it>}) are rare, with only 1% of <it>Cbx3</it>^{<it>hypo</it>/<it>hypo </it>}animals reaching adulthood. Adult males exhibit a severe hypogonadism that is associated with a loss of germ cells, with some seminiferous tubules retaining only the supporting Sertoli cells (Sertoli cell-only phenotype). The percentage of seminiferous tubules that are positive for L1 ORF1 protein (ORF1p) in <it>Cbx3</it>^{<it>hypo</it>/<it>hypo </it>}testes is greater than that for wild-type testes, indicating that L1 retrotransposon silencing is reversed, leading to ectopic expression of ORF1p in <it>Cbx3</it>^{<it>hypo</it>/<it>hypo </it>}germ cells.</p> <p>Conclusions</p> <p>The <it>Cbx3 </it>gene product (the HP1γ protein) has a non-redundant function during spermatogenesis that cannot be compensated for by the other two HP1 isotypes. The <it>Cbx3</it>^{<it>hypo</it>/<it>hypo </it>}spermatogenesis defect is similar to that found in <it>Miwi2 </it>and <it>Dnmt3L </it>mutants. The <it>Cbx3 </it>gene-targeted mice generated in this study provide an appropriate model for the study of HP1γ in transposon silencing and parental imprinting.</p

HP1-β is required for development of the cerebral neocortex and neuromuscular junctions

HP1 proteins are thought to be modulators of chromatin organization in all mammals, yet their exact physiological function remains unknown. In a first attempt to elucidate the function of these proteins in vivo, we disrupted the murine Cbx1 gene, which encodes the HP1-β isotype, and show that the Cbx1−/−-null mutation leads to perinatal lethality. The newborn mice succumbed to acute respiratory failure, whose likely cause is the defective development of neuromuscular junctions within the endplate of the diaphragm. We also observe aberrant cerebral cortex development in Cbx1−/− mutant brains, which have reduced proliferation of neuronal precursors, widespread cell death, and edema. In vitro cultures of neurospheres from Cbx1−/− mutant brains reveal a dramatic genomic instability. Our results demonstrate that HP1 proteins are not functionally redundant and that they are likely to regulate lineage-specific changes in heterochromatin organization

A MSFD complementary approach for the assessment of pressures, knowledge and data gaps in Southern European Seas : the PERSEUS experience

PERSEUS project aims to identify the most relevant pressures exerted on the ecosystems of the Southern European Seas (SES), highlighting knowledge and data gaps that endanger the achievement of SES Good Environmental Status (GES) as mandated by the Marine Strategy Framework Directive (MSFD). A complementary approach has been adopted, by a meta-analysis of existing literature on pressure/impact/knowledge gaps summarized in tables related to the MSFD descriptors, discriminating open waters from coastal areas. A comparative assessment of the Initial Assessments (IAs) for five SES countries has been also independently performed. The comparison between meta-analysis results and IAs shows similarities for coastal areas only. Major knowledge gaps have been detected for the biodiversity, marine food web, marine litter and underwater noise descriptors. The meta-analysis also allowed the identification of additional research themes targeting research topics that are requested to the achievement of GES. 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license.peer-reviewe

Reliability of caspase activity as a biomarker of hepatic apoptosis in nonalcoholic fatty liver disease

A letter in response to: Yilmaz Y, Kurt R, Kalayci C. Apoptosis in nonalcoholic steatohepatitis with normal aminotransferase values: zooming in on cytokeratin 18 fragments. Biomarkers Med. 4(5), 743–745 (2010). </jats:p

Analysis of the anomalous electromagnetic moments of the tau lepton in γ p collisions at the LHC

In this study, we investigate the potential of the process pp?p??p?p??¯?q?X at the LHC to examine the anomalous electromagnetic moments of the tau lepton. We obtain 95% confidence level bounds on the anomalous coupling parameters with various values of the integrated luminosity and center-of-mass energy. The improved bounds have been obtained on the anomalous coupling parameters of electric and magnetic moments of the tau lepton a? and |d?| compared to the current experimental sensitivity bounds. The ?p mode of photon reactions at the LHC have shown that it has great potential for the electromagnetic dipole moments studies of the tau lepton. © 2018 The Authors115F136This work has been supported by the Scientific and Technological Research Council of Turkey (TUBITAK) in the framework of Project No. 115F136

Search for the anomalous electromagnetic moments of tau lepton through electron-photon scattering at CLIC

WOS:000413405200022We have examined the anomalous electromagnetic moments of the tau lepton in the processes e(-)gamma -> nu(e)tau(nu) over bar tau (gamma is the Compton backscattering photon) and e(-)e(+)-> e(-)gamma*e(+) -> e(-)gamma -> nu(e)tau(nu) over bar tau (gamma* is the Weizsacker-Williams photon) with unpolarized and polarized electron beams at the CLIC. We have obtained 95% confidence level bounds on the anomalous magnetic and electric dipole moments for various values of the integrated luminosity and center-of-mass energy. Improved constraints of the anomalous magnetic and electric dipole moments have been obtained compared to the LEP sensitivity. (C) 2017 The Authors. Published by Elsevier B.V.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [115F136]This work has been supported by the Scientific and Technological Research Council of Turkey (TUBITAK) in the framework of Project No. 115F136

Apoptosis and Disease Severity is Associated with Insulin Resistance in Nonalcoholic Fatty Liver Disease

Background & Aims : Non-alcoholic fatty liver disease (NAFLD) is associated with insulin resistance (IR). We evaluated whether IR contributes to hepatocyte apoptosis, inflammation, and fibrosis in NAFLD