Toward a molecular understanding of yeast silent chromatin : roles for H4K16 acetylation and the Sir3 C-terminus

Discrete regions of the eukaryotic genome assume a heritable chromatin structure that is refractory to gene expression. In budding yeast, silent chromatin is characterized by the loading of the Silent Information Regulatory (Sir) proteins (Sir2, Sir3 and Sir4) onto unmodified nucleosomes. This requires the deacetylase activity of Sir2, extensive contacts between Sir3 and the nucleosome, as well as interactions between Sir proteins forming the Sir2-3-4 complex. During my PhD thesis I sought to advance our understanding of these phenomena from a molecular perspective. Previous studies of Sir-chromatin interactions made use of histone peptides and recombinant Sir protein fragments. This gave us an idea of possible interactions, but could not elucidate the role of histone modifications in the assembly of silent chromatin. This required that we examine nucleosomal arrays exposed to full length Sir proteins or the holo Sir complex. In Chapter 2, I made use of an in vitro reconstitution system, that allows the loading of Sir proteins (Sir3, Sir2-4 or Sir2-3-4) onto arrays of regularly spaced nucleosomes, to examine the impact of specific histone modifications (methylation of H3K79, acetylation of H3K56 and H4K16) on Sir protein binding and linker DNA accessibility. The “active” H4K16ac mark is thought to limit the loading of the Sir proteins to silent domain thus favoring the formation of silent regions indirectly by increasing Sir concentration locally. Strikingly, I found that the Sir2-4 subcomplex, unlike Sir3, has a slight higher affinity for H4K16ac-containing chromatin in vitro, consistent with H4K16ac being a substrate for Sir2. In addition the NAD-dependent deacetylation of H4K16ac promotes the binding of the holo Sir complex to chromatin beyond generating hypoacetylated histone tails. We conclude that the Sir2-dependent turnover of the “active” H4K16ac mark directly helps to seed repression. The tight association of the holo Sir complex within silent domains relies on the ability of Sir3 to bind unmodified nucleosomes. In addition, Sir3 dimerization is thought to reinforce and propagate silent domains. However, no Sir3 mutants that fail to dimerize were characterized to date. It was unclear which domain of Sir3 mediates dimerization in vivo. In Chapter 3, we present the X-ray crystal structure of the Sir3 extreme C-terminus (aa 840-978), which folds into a variant winged helix-turn-helix (Sir3 wH) and forms a stable homodimer through a large hydrophobic interface. Loss of wH homodimerization impairs holo Sir3 dimerization in vitro showing that the Sir3 wH module is key to Sir3-Sir3 interaction. Homodimerization mediated by the wH domain can be fully recapitulated by an unrelated bacterial homodimerization domain and is essential for stable association of the Sir2-3-4 complex with chromatin and the formation of silent chromatin in vivo

Auswirkungen erhöhter sozialer Unterstützung auf das Wohlbefinden kognitiv beeinträchtigter älterer Menschen

Zusammenfassung: Soziale Beziehungen stellen einen wichtigen Faktor für den Erhalt der Lebenszufriedenheit im Alter dar. Wenig bekannt ist, welchen Einfluss erhöhte soziale Unterstützung auf das Wohlbefinden kognitiv beeinträchtigter Menschen hat. In zwei vergleichenden Studien in der Schweiz und Österreich mit insgesamt 84 kognitiv beeinträchtigten Personen (65-98Jahre) in stationärer Betreuung wurde deshalb in einem Kontrollgruppendesign untersucht, ob sich die Erhöhung sozialer Unterstützung positiv auf das Wohlbefinden auswirkt. Dazu wurden Interventionen bei kognitiv beeinträchtigten Menschen in Form von emotionaler sozialer Unterstützung durch freiwillige Besucher durchgeführt. In beiden Studien konnte ein positiver Zusammenhang zwischen der Erhöhung sozialer Unterstützung durch freiwillige Helfer und dem Wohlbefinden der Probanden festgestellt werden. Die Untersuchungen zeigen auf, dass soziale Unterstützung und Zuwendung generell von kognitiv beeinträchtigten Menschen registriert und als wohltuend empfunden werde

Wie erleben Erfasserinnen den Umgang mit der Observed Emotional Rating Scale?

Da im deutschsprachigen Raum bisher kaum empirische Daten zur Anwendung der Observed Emotion Rating Scale (OERS) bei demenzerkrankten Personen vorliegen, sollte im Rahmen des Pilotprojekts «Agitation» untersucht werden, wie Erfasserinnen den Einsatz des Instruments OERS erleben. Zu diesem Zweck konnten 12 Probandinnen gewonnen werden, die in drei Pflegezentren im Kanton Zürich Beobachtungen mit dem Instrument OERS durchführten. Der überwiegende Teil der Probandinnen verfügte über pflegerische Qualifikationen bzw. Erfahrung im Umgang mit demenzerkrankten Personen. Die Datensammlung erfolgte anhand von drei Fokusgruppeninterviews, die mittels einer qualitativen Inhaltsanalyse ausgewertet worden waren. Die vorläufigen Ergebnisse aus den Fokusgruppeninterviews wurden von denselben Probandinnen anhand eines Fragebogens validiert. Die Kategorien «fehlende Eindeutigkeit», «fehlende Kodiermöglichkeit», «Demenzschwere», «Einschätzen von Emotionen», «Einflussfaktoren» und «Anwenderschulung» zeigten, dass das Wissen um die Demenzerkrankung genauso bedeutsam ist wie eine geübte Handhabung der OERS. As barely any empirical data on the application of the Observed Emotion Rating Scale (OERS) on sufferers of dementia is available for German-speaking Europe, the pilot project titled “Agitation” aimed to see how those persons entering the data get along with the OERS instrument. For this purpose, 12 test persons were persuaded to carry out observations with the OERS instrument in three care centres located in the Swiss Canton of Zurich. The test persons were predominantly holders of nursing qualifications and/or experienced in caring for patients suffering from dementia. Data collection was undertaken on the basis of three focus group interviews which were evaluated via qualitative content analysis. The preliminary results gained in the focus group interviews were validated by the same people using a questionnaire. The categories were titled “Missing Clearness”, “Missing Possibility to Encode”, “Level of Dementia”, “Estimation of Emotions”, “Influencing Factors” and “User Training”. These indicated that know­ledge of dementia is just as important as skilled use of OERS

Welche Informationen benötigen Erfasserinnen für den Einsatz der Observed Emotion Rating Scale?

As there have so far not been any empirical results in German-speaking Europe with regard to training people in the use of the Observed Emotion Rating Scale (OERS) instrument, the investigation needs to establish what information is required for those involved in OERS data entry. The explorative analysis was carried out with 12 probands using a methodological approach based on the Mayring (2003) qualitative content analysis procedure. Based on the results, it became clear that the use of OERS needs to be well prepared in advance, as the estimation¨of emotions with sufferers of dementia requires background knowledge of the illness, as well as the adjustment of those under observation to the surroundings being required. Furthermore, it became apparent that the use of OERS is suited to reflecting the surroundings of those suffering from dementia, as the rarity of emotions being expressed can itself be a sign of a maladjusted environment. Da im deutschsprachigen Raum bisher keine empirischen Ergebnisse im Bedarf einer Schulung mit dem Instrument Observed Emotion Rating Scale (OERS) vorliegen, sollte mit der Untersuchung herausgefunden werden, welche Informationen Erfasserinnen für den Einsatz der OERS benötigen. Die Untersuchung wurde explorativ mit dem methodischen Ansatz der qualitativen Inhaltsanalyse nach Mayring (2003) bei 12 Probandinnen durchgeführt.Anhand der Ergebnisse wurde ersichtlich, dass der Einsatz der OERS gut vorbereitet werden muss, weil die Einschätzung von Emotionen bei demenzerkrankten Menschen Hintergrundwissen zur Erkrankung benötigt und eine Anpassung der Beobachtenden an die Umgebung Voraussetzung ist. Ferner stellte sich heraus, dass sich der Einsatz der OERS dazu eignet, die Umgebung von Demenzerkrankten zu reflektieren, weil der Umstand, dass nur selten Emotionen geäußert werden, auch ein Zeichen für eine unangepasste Umgebung sein kann

Begleitumstände von Agitation bei demenzerkrankten Heimbewohnern

Die vorliegende Studie hatte das Ziel, die Begleitumstände von Agitation im Pflegeheimkontext zu erfassen sowie Rückschlüsse auf deren Ursachen zu ziehen. Dazu wurden in zwei Pflegezentren und in einem demenzspezialisierten Krankenheim während eines Zeitraums von elf Wochen 359 Tagebucheinträge zu agitationsbedingtem Verhalten erfasst. Die Ergebnisse zeigen, dass Agitation in Situationen auftrat, in denen die Bewohner mit Demenz in Interaktion mit anderen standen, aber dass sie auch in Situationen stattgefunden hat, in denen die Pflegepersonen keine direkten Einflüsse/Reize aus der Umgebung wahrnehmen konnten. Im Weiteren konnte ein gewisses Muster zwischen der Tageszeit und der Häufigkeit des Auftretens von Agitation festgestellt werden. Ergänzend konnten personenspezifische Verhaltensmuster nachgewiesen werden. Die Ergebnisse der Studie verstärken die Forderung nach individualisierten und dem Kontext sowie der aktuellen Situation Rechnung tragenden Interventionen des Pflegepersonals. The present study examined the circumstances of agitation in nursing homes and drew conclusions about the causes and context of agitated behaviour. It took place in two nursing homes as well as in one dementia-specialised home health care. The study utilized recordings created during a period of eleven weeks and 359 diary entries relating to the pre-agitation conditions. The results showed that agitation occurred during situations in which the person interacted with others, but also in situations in which no direct influences or stimuli from the environment were visible. Secondly, a certain pattern could be identified in the relationship between the time and the frequency of the occurrence of agitation. Person-specific patterns could also be detected. The results of this study reinforce the call for the individualised and situation-oriented behaviour intervention of nurses

Regulating repression : roles for the Sir4 N-terminus in linker DNA protection and stabilization of epigenetic states

The Gasser laboratory is supported by the Novartis Research Foundation and the EU training network Nucleosome 4D. SK was supported by an EMBO long-term fellowship, a Schrodinger fellowship from the FWF, and the Swiss SystemsX.ch initiative/C-CINA; HCF by an EMBO long-term fellowship.Silent information regulator proteins Sir2, Sir3, and Sir4 form a heterotrimeric complex that represses transcription at subtelomeric regions and homothallic mating type (HM) loci in budding yeast. We have performed a detailed biochemical and genetic analysis of the largest Sir protein, Sir4. The N-terminal half of Sir4 is dispensable for SIR-mediated repression of HM loci in vivo, except in strains that lack Yku70 or have weak silencer elements. For HM silencing in these cells, the C-terminal domain (Sir4C, residues 747-1,358) must be complemented with an N-terminal domain (Sir4N; residues 1-270), expressed either independently or as a fusion with Sir4C. Nonetheless, recombinant Sir4C can form a complex with Sir2 and Sir3 in vitro, is catalytically active, and has sedimentation properties similar to a full-length Sir4-containing SIR complex. Sir4C-containing SIR complexes bind nucleosomal arrays and protect linker DNA from nucleolytic digestion, but less effectively than wild-type SIR complexes. Consistently, full-length Sir4 is required for the complete repression of subtelomeric genes. Supporting the notion that the Sir4 N-terminus is a regulatory domain, we find it extensively phosphorylated on cyclin-dependent kinase consensus sites, some being hyperphosphorylated during mitosis. Mutation of two major phosphoacceptor sites (S63 and S84) derepresses natural subtelomeric genes when combined with a serendipitous mutation (P2A), which alone can enhance the stability of either the repressed or active state. The triple mutation confers resistance to rapamycin-induced stress and a loss of subtelomeric repression. We conclude that the Sir4 N-terminus plays two roles in SIR-mediated silencing: it contributes to epigenetic repression by stabilizing the SIR-mediated protection of linker DNA; and, as a target of phosphorylation, it can destabilize silencing in a regulated manner.Publisher PDFPeer reviewe

Characterization of rock slopes through slope mass rating using 3D point clouds

Rock mass classification systems are widely used tools for assessing the stability of rock slopes. Their calculation requires the prior quantification of several parameters during conventional fieldwork campaigns, such as the orientation of the discontinuity sets, the main properties of the existing discontinuities and the geo-mechanical characterization of the intact rock mass, which can be time-consuming and an often risky task. Conversely, the use of relatively new remote sensing data for modelling the rock mass surface by means of 3D point clouds is changing the current investigation strategies in different rock slope engineering applications. In this paper, the main practical issues affecting the application of Slope Mass Rating (SMR) for the characterization of rock slopes from 3D point clouds are reviewed, using three case studies from an end-user point of view. To this end, the SMR adjustment factors, which were calculated from different sources of information and processes, using the different softwares, are compared with those calculated using conventional fieldwork data. In the presented analysis, special attention is paid to the differences between the SMR indexes derived from the 3D point cloud and conventional field work approaches, the main factors that determine the quality of the data and some recognized practical issues. Finally, the reliability of Slope Mass Rating for the characterization of rocky slopes is highlighted.This work was partially funded by the University of Alicante (vigrob-157 Project and GRE1404 Project), the Generalitat Valenciana (Projects GV/2011/044 and ACOMP/2014/136), the Spanish Ministry of Economy and Competitiveness (MINECO) and EU FEDER under Projects TEC2011-28201-C02-02 and TIN2014-55413-C2-2-P