Genome-wide control of H4 K16 acetylation by the SAS-I complex in Saccharomyces cerevisiae

Die Histonacetyltransferase (HAT) Sas2 in Saccharomyces cerevisiae gehört zur Familie der MYST HATs und bildet mit den Untereinheiten Sas4 und Sas5 den SAS-I Komplex, der Histon H4 an Lysin 16 (H4 K16Ac) acetyliert. Diese Sas2-vermittelte H4 K16Ac verhindert eine Ausbreitung des telomerischen Heterochromatins in euchromatische Bereiche und ist weiterhin an der transkriptionellen Stilllegung der HM Loci und des rDNA Locus beteiligt. In der vorliegenden Arbeit wurde die Sas2-vermittelte H4 K16Ac auf genomweiter Ebene unter Anwendung von Chromatinimmunpräzipitation (ChIP) in Kombination mit hoch auflösenden genomischen Tiling Arrays untersucht. Da Sas2 mit den Chromatin-Assemblierungsfaktoren CAF-I und Asf1 interagiert, wurde weiterhin die Abhängigkeit der Sas2-vermittelten H4 K16Ac von diesen Faktoren untersucht. Dabei wurde ein partieller Einfluss von CAF-I und Asf1 auf die Sas2-vermittelte H4 K16Ac festgestellt. In Abwesenheit von Sas2 war die H4 K16Ac auf globaler Ebene reduziert. Interessanterweise verursachte der Verlust der H4 K16Ac in sas2∆ Zellen ein charakteristisches Muster außerhalb der telomerischen Region. H4 K16Ac war an der Mehrzahl der offenen Leseraster (ORFs) stark reduziert, während die H4 K16Ac in intergenischen Regionen kaum Veränderungen aufwies. Bezeichnenderweise korrelierte die Sas2-abhängige H4 K16Ac mit einem geringen Histon H3 Austausch und einem geringen Maß an H3 K56 Acetylierung. Dies deutete darauf hin, dass H4 K16Ac unabhängig von transkriptionsgekoppeltem Histonaustausch im Chromatin platziert wurde. Dementsprechend wurden in dieser Arbeit Hinweise gefunden, dass die Sas2 vermittelte H4 K16Ac im Zellzyklus gekoppelt an die S Phase eingeführt wird. In Übereinstimmung mit dem Effekt von Sas2 innerhalb von ORFs wurde weiterhin festgestellt, dass eine Deletion von SAS2 zur Resistenz gegenüber 6 Azauracil führte sowie die Konzentration der RNA Polymerase II (PolII) an den 3’ Regionen der Gene erhöhte. Diese Resultate ließen auf einen positiven Effekt der fehlenden H4 K16Ac auf die Elongationphase der Transkription schließen. Zusätzlich dazu wurde eine geringfügige Akkumulation von Transkripten an den 3’-Enden in der Mehrzahl der Gene in sas2∆ Zellen beobachtet. Zusammenfassend lassen die Resultate dieser Arbeit darauf schließen, dass die Sas2-abhängige H4 K16Ac global im Genom von S. cerevisiae positioniert wird, unabhängig von Transkription und Histonaustausch in das Chromatin eingebaut wird und daher vor allem in schwach transkribierten ORFs verbleibt. Es wurde gezeigt, dass die Acetylierung von H4 K16 gekoppelt an die DNA-Replikation erfolgt und dass die Sas2-vermittelte H4 K16Ac die Fähigkeit der PolII, ein Gen zu transkribieren, inhibiert.The MYST HAT Sas2 is part of the SAS-I complex, which includes the subunits Sas4 and Sas5 and acetylates histone H4 lysine 16 (H4 K16Ac). This Sas2-mediated H4 K16Ac blocks the propagation of heterochromatin at the telomeres of Saccharomyces cerevisiae and is further involved in silencing at the HM loci and the rDNA locus. In this study, we investigated Sas2 mediated H4 K16Ac on a genome-wide scale, by using chromatin immunoprecipitations combined with high resolution tiling arrays. Because Sas2 interacts with the chromatin assembly factors CAF-I and Asf1, we furthermore investigated the dependence of the Sas2-mediated H4 K16Ac on these factors and found a partial influence of CAF-I and Asf1 on H4 K16Ac. Globally, H4 K16Ac was reduced in the absence of Sas2. Interestingly, H4 K16Ac loss in sas2∆ cells outside of the telomeric regions showed a distinctive pattern in that there was a pronounced decrease of H4 K16Ac within the majority of open reading frames (ORFs), but little change in intergenic regions. Significantly, high Sas2-dependent H4 K16Ac correlated with low histone H3 exchange and low H3 K56 acetylation, indicating that this modification was placed on chromatin independently of histone exchange. Consistent with this notion we found evidence that Sas2 mediated H4 K16 acetylation coupled to the S-Phase of the cell cycle. In agreement with the effect of Sas2 within ORFs, sas2∆ caused resistance to 6-azauracil, and RNA polymerase II (PolII) occupancy in the 3’ region of genes was increased in sas2∆ cells, suggesting a positive effect on transcription elongation in the absence of H4 K16Ac. Additionally, we observed a slight accumulation of transcripts at the 3’ end of the majority of genes in sas2∆ cells. This effect for several reasons was distinct from short transcripts that are caused by cryptic transcription initiation. Nonetheless, this finding completed our picture of the positive impact of sas2∆ on PolII-dependent transcription. In summary, our data suggest that Sas2 dependent H4 K16Ac is distributed globally and deposited into chromatin independently of transcription and histone exchange but coupled to DNA replication, and that it has an inhibitory effect on the ability of PolII to travel through the body of the gene

Comparative functional analysis of the adhesion and invasion factor YadA of enteropathogenic Yersiniae

Das multifunktionelle, afimbrilliäre Adhäsin YadA ist ein wichtiger Adhäsionsfaktor der enteropathogenen Bakterien Yersinia pseudotuberculosis und Yersinia enterocolitica. Es trägt durch die Bindung an Proteine der extrazellulären Matrix (EZM) maßgeblich zur Etablierung einer Infektion bei. Obwohl die YadA-Proteine beider Yersinia spp. (YadApstb und YadAent) sehr homolog sind, vermittelt nur YadApstb effiziente Invasion in Epithelzellen. Es konnte gezeigt werden, dass eine in der N-terminalen Kopfregion des YadApstb-Proteins lokalisierte Region von 31 Aminosäuren, die dem YadAent-Protein fehlt, essentiell für die YadApstb-vermittelte Invasion ist. Die Deletion dieser Domäne in YadApstb führt zu einem Verlust der Invasionsfähigkeit, während die adhäsiven Eigenschaften des Moleküls erhalten bleiben. Die Invasionsdomäne ist darüber hinaus für die aggregativen Eigenschaften und die Bindekapazität von YadA für Proteine der EZM entscheidend. Ihr Fehlen bewirkt einen Verlust der Fähigkeit Auto- und Hämagglutination zu induzieren, vermittelt demgegenüber jedoch eine deutlich gesteigerte Bindung an Kollagen und Laminin. YadApstb hingegen bindet hauptsächlich an Fibronektin, und es konnte nachgewiesen werden, dass die YadApstb-vermittelte Invasion nur auf Grundlage der Bindung an Fibronektin-gebundene a5b1-Integrine erfolgen kann. Darüber hinaus konnte gezeigt werden, dass die invasiven und aggregativen Eigenschaften des YadApstb-Proteins für die effiziente Besiedlung tiefer gelegener Gewebe und Organe während der Infektion von Bedeutung sind. Die Identifikation der YadApstb- Invasionsdomäne ermöglicht es, den Invasions- und Adhäsionsprozess deutlich voneinander abzugrenzen, so dass die Unterschiede der zu Grunde liegenden eukaryotischen Signaltransduktion erstmalig untersucht werden können. Hierfür wurde ein in vitro System etabliert, das es erlaubt, die Aktivierung der Signalmoleküle in der Wirtszelle unbeeinflusst von anderen bakteriellen Faktoren zu analysieren.The multifunctional, afimbrial adhesin YadA is the major adhesion factor of the enteropathogenic bacteria Yersinia pseudotuberculosis and Yersinia enterocolitica. It contributes to the establishment of an infection by promoting tight adherence to extracellular matrix (ECM) proteins of the host cell. Although the YadA proteins of both Yersinia spp. (YadApstb and YadAent) are highly homologous, only YadApstb promotes efficient invasion into epithelial cells. Functional and structural analyses of YadApstb and YadAent revealed that an unique N-terminal region of 31 amino acids of YadApstb, which is absent in YadAent, is crucial for the YadApstb-mediated invasion. A deletion of this domain in YadApstb abrogates cell invasion, while the adhesion remains unaffected. The uptake domain also affects the aggregation behaviour and the specificity of ECM substrate binding of YadA. The loss of this motif causes YadA variants, which are deficient in auto- and hemagglutination but promote strong binding to collagen and laminin. YadApstb however exhibits a high binding efficiency for fibronectin and it was shown that the YadApstb-mediated invasion occurs only via fibronectin-bound a5b1-integrins. Furthermore, it was demonstrated that the invasive and aggregative properties of YadApstb contribute to the efficient colonization of deeper tissues and organs during infection. The identification of the uptake domain dissects invasion from adhesion and allows for the first time the analysis of the differences in the host cell signalling during these processes. For this purpose an in vitro system was developed, which facilitates an analysis unaffected by other bacterial factors

Genome-wide H4 K16 acetylation by SAS-I is deposited independently of transcription and histone exchange

The MYST HAT Sas2 is part of the SAS-I complex that acetylates histone H4 lysine 16 (H4 K16Ac) and blocks the propagation of heterochromatin at the telomeres of Saccharomyces cerevisiae. In this study, we investigated Sas2-mediated H4 K16Ac on a genome-wide scale. Interestingly, H4 K16Ac loss in sas2Δ cells outside of the telomeric regions showed a distinctive pattern in that there was a pronounced decrease of H4 K16Ac within the majority of open reading frames (ORFs), but little change in intergenic regions. Furthermore, regions of low histone H3 exchange and low H3 K56 acetylation showed the most pronounced loss of H4 K16Ac in sas2Δ, indicating that Sas2 deposited this modification on chromatin independently of histone exchange. In agreement with the effect of Sas2 within ORFs, sas2Δ caused resistance to 6-azauracil, indicating a positive effect on transcription elongation in the absence of H4 K16Ac. In summary, our data suggest that Sas2-dependent H4 K16Ac is deposited into chromatin independently of transcription and histone exchange, and that it has an inhibitory effect on the ability of PolII to travel through the body of the gene

The Effect of Micrococcal Nuclease Digestion on Nucleosome Positioning Data

Eukaryotic genomes are packed into chromatin, whose basic repeating unit is the nucleosome. Nucleosome positioning is a widely researched area. A common experimental procedure to determine nucleosome positions involves the use of micrococcal nuclease (MNase). Here, we show that the cutting preference of MNase in combination with size selection generates a sequence-dependent bias in the resulting fragments. This strongly affects nucleosome positioning data and especially sequence-dependent models for nucleosome positioning. As a consequence we see a need to re-evaluate whether the DNA sequence is a major determinant of nucleosome positioning in vivo. More generally, our results show that data generated after MNase digestion of chromatin requires a matched control experiment in order to determine nucleosome positions

Structural drivers and social protection:Mechanisms of HIV risk and HIV prevention for South African adolescents

Introduction: Social protection is high on the HIV-prevention agenda for youth in sub-Saharan Africa. However, questions remain: How do unconditional cash transfers work? What is the effect of augmenting cash provision with social care? And can “cash plus care” social protection reduce risks for adolescents most vulnerable to infection? This study tackles these questions by first identifying mediated pathways to adolescent HIV risks and then examining potential main and moderating effects of social protection in South Africa. Methods: This study was a prospective observational study of 3515 10-to-17-year-olds (56.7% female; 96.8% one-year retention). Within randomly selected census areas in four rural and urban districts in two South African provinces, all homes with a resident adolescent were sampled between 2009/2010 and 2011/2012. Measures included 1) potential structural drivers of HIV infection such as poverty and community violence; 2) HIV risk behaviours; 3) hypothesized psychosocial mediating factors; and 4) types of social protection involving cash and care. Using gender-disaggregated analyses, longitudinal mediation models were tested for potential main and moderating effects of social protection. Results: Structural drivers were associated with increased onset of adolescent HIV risk behaviour (p<0.001, B=0.06, SE=0.01), fully mediated by increased psychosocial problems. Both cash and care aspects of social protection were associated with reductions in HIV risk behaviour and psychosocial deprivations. In addition, cash social protection moderated risk pathways: for adolescent girls and boys experiencing more acute structural deprivation, social protection had the greatest associations with HIV risk prevention (e.g. moderation effects for girls: B=−0.08, p<0.002 between structural deprivation and psychosocial problems, and B=−0.07, p<0.001 between psychosocial problems and HIV risk behaviour). Conclusions: Adolescents with the greatest structural deprivation are at higher risk of HIV, but social protection has the greatest prevention effects for the most vulnerable. Social protection comprising unconditional cash plus care was associated with reduced risk pathways through moderation and main effects, respectively. Our findings suggest the importance of social protection within a combination package of HIV-prevention approaches

Faster Removal of 2-Phosphoglycolate through Photorespiration Improves Abiotic Stress Tolerance of Arabidopsis

Photorespiration metabolizes 2-phosphoglyolate (2-PG) to avoid inhibition of carbon assimilation and allocation. In addition to 2-PG removal, photorespiration has been shown to play a role in stress protection. Here, we studied the impact of faster 2-PG degradation through overexpression of 2-PG phosphatase (PGLP) on the abiotic stress-response of Arabidopsis thaliana (Arabidopsis). Two transgenic lines and the wild type were subjected to short-time high light and elevated temperature stress during gas exchange measurements. Furthermore, the same lines were exposed to long-term water shortage and elevated temperature stresses. Faster 2-PG degradation allowed maintenance of photosynthesis at combined light and temperatures stress and under water-limiting conditions. The PGLP-overexpressing lines also showed higher photosynthesis compared to the wild type if grown in high temperatures, which also led to increased starch accumulation and shifts in soluble sugar contents. However, only minor effects were detected on amino and organic acid levels. The wild type responded to elevated temperatures with elevated mRNA and protein levels of photorespiratory enzymes, while the transgenic lines displayed only minor changes. Collectively, these results strengthen our previous hypothesis that a faster photorespiratory metabolism improves tolerance against unfavorable environmental conditions, such as high light intensity and temperature as well as drought. In case of PGLP, the likely mechanism is alleviation of inhibitory feedback of 2-PG onto the Calvin&ndash;Benson cycle, facilitating carbon assimilation and accumulation of transitory starch

Chapter 10. Isotopically Enriched Systems

Most solid-state NMR measurements employ rare-spin nuclei, such as 13C or 15N, for detection. However, the low natural abundance of those spins limits the possibility of obtaining multidimensional homo- or hetero-nuclear solid-state NMR-spectra, which rely on internuclear correlations between those rare spins, unless signal enhancement or isotopic labelling is applied. In this chapter, we first give an overview of different techniques for selective and uniform isotope labelling of biomolecules. In the following sections, we describe different homo- and hetero-nuclear recoupling techniques and their use in multidimensional NMR spectroscopy. In particular, we emphasize the difference between zeroth-order recoupling techniques, which are well-suited for dipolar transfers between spins close in space, and second- and higher-order recoupling schemes, which allow the detection of long-range correlations. We also provide some examples how these techniques are applied towards structure elucidation of biomolecules. Finally, we briefly outline the technique of signal enhancement by dynamic nuclear polarization, a method that may in part help to overcome the need for isotopic enrichment