Development of a novel high resolution melting assay for identification and differentiation of all known 19 serovars of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory infectious disease responsible for global economic losses in the pig industry. From a monitoring perspective as well as due to the different courses of disease associated with the various serovars, it is essential to distinguish them in different herds or countries. In this study, we developed a novel high resolution melting (HRM) assay based on reference strains for each of the 19 known serovars and additional 15 clinical A. pleuropneumoniae isolates. The novel HRM comprises the species-specific APP-HRM1 and two serovar-specific HRM assays (APP-HRM2 and APP-HRM3). APP-HRM1 allowed polymerase chain reaction (PCR) amplification of apxIV resulting in an A. pleuropneumoniae specific melting curve, while nadV specific primers differentiated biovar 2 from biovar 1 isolates. Using APP-HRM2 and APP-HRM3, 13 A. pleuropneumoniae serovars can be determined by inspecting the assigned melting temperature. In contrast, serovar 3 and 14, serovar 9 and 11, and serovar 5 and 15 have partly overlapping melting temperatures and thus represent a challenge to accurately distinguish them. Consequently, to unambiguously ensure the correct assignment of the serovar, it is recommended to perform the serotyping HRM assay using a positive control for each serovar. This rapid and user-friendly assay showed high sensitivity with 1.25 fg-125 pg of input DNA and a specificity of 100% to identify A. pleuropneumoniae. Characteristic melting patterns of amplicons might allow detecting new serovars. The novel HRM assay has the potential to be implemented in diagnostic laboratories for better surveillance of this pathogen

Establishment of a mass-spectrometry-based method for the identification of the in vivo whole blood and plasma ADP-Ribosylomes

Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modification states of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove these modifications after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we discovered that plasma phosphodiesterases and/or ADP-ribosylhydrolases rapidly demodify in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a mass-spectrometry-based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa domesticus), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions

Functional characterization of chromatin-associated protein ADP-ribosylation

Summary Protein ADP-ribosylation is a post-translational modification (PTM) that consists of the addition of ADP-ribose moieties to target proteins. In the nucleus, the modification is catalyzed by members of the diphtheria toxin-like ADP-ribosyl- transferases (ARTDs), of which ARTD1 is the nuclear most abundant and best studied. ADP-ribosylation has been implicated in a variety of cellular processes, ranging from maintenance of genome integrity and DNA repair and gene transcription. However, very little is known about the localization and molecular function of chromatin-associated ADP-ribosylation. In this thesis, we have investigated the mechanistic role of ARTD1-mediated chromatin poly-ADP-ribosylation (PARylation) for different cellular conditions. In the first paradigm, we investigated the role of ADP-ribosylation for the transcription of SRY (sex determining region Y)-box 2 (SOX2) target genes during the early phase of fibroblasts reprogramming to induced pluripotent stem cells (iPSC). We could show that ARTD1 and PARylation are necessary for iPS colony formation and that PAR formation keeps SOX2 associated to the DNA to efficiently transcribe SOX2 target genes, including fibroblast growth factor 4 (Fgf4). We found that exogenous FGF4 administration functionally rescues ARTD1 ablation or inhibition of PARylation. In addition, we have developed a novel chromatin affinity precipitation (ChAP) protocol to enrich ADP-ribosylated chromatin for elucidating where ADP- ribosylation is localized. We have applied this protocol to an oxidative stress paradigm and found that ADP-ribosylation induced by hydrogen peroxide (H2O2) preferentially localizes to ARTD1 and nucleosome-dense heterochromatic regions, as well as to repetitive elements within the genome. Chromatin ADP-ribosylation induced at these sites was correlated with a higher accessibility. Moreover during in vitro induced adipogenesis, ADP-ribosylation was tightly associated with peroxisome proliferator-activated receptor (PPARγ) at PPARγ target genes. Together, the thesis reveals that chromatin-associated ADP-ribosylation is a PTM whose induction and genomic distribution varies with the stimulus and the cell type. Thus, for all tested conditions targeted ADP-ribosylation to defined chromatin loci either functionally regulated chromatin structure (as for H2O2) or gene transcription (SOX2 and PPARγ). How the target specificity of ADP-ribosylation in the chromatin context is achieved needs further investigation. Zusammenfassung Protein ADP-Ribosylierung ist eine post-translatare Modifikation bei der ADP-ribose an Zielproteine angehängt wird. Die verantwortlichen nuklearen Enzyme werden ADP-ribosyl-transferases diphteria toxin like (ARTDs) genannt, wobei ARTD1 am besten beschrieben ist. Die zellulären Prozesse bei denen ADP-Ribosylierung involviert ist, reichen von der Erhaltung der Genomintegrität und der DNS Reparatur bis hin zur Gentranskription. Dennoch ist sehr wenig über die Lokalisation und die molekulare Funktion von Chromatin-assoziierter ADP-Ribosylierung bekannt. In dieser Arbeit haben wir die mechanistische Rolle von ARTD1 erzeugter poly- ADP-Ribosylierung (PARylierung) unter verschiedenen zellulären Konditionen untersucht. Im ersten Paradigma untersuchten wir die Rolle der ADP-Ribosylierung von SOX2 Zielgenen während der frühen Phase der Fibroblasten Reprogrammierung in induzierte pluripotente Stammzellen (iPSZ). Wir konnten zeigen, dass ARTD1 für die Formierung von iPS Zellkolonien nötig ist und dass PARylierung von SOX2 wichtig ist, damit Fgf4 effizient transkribiert wird. Die exogenen Zugabe von FGF4 kompensierte die Inhibierung von PARylierung oder die Ablation von ARTD1. Des weiteren haben wir ein neues Chromatin Affinität Präzipitations Protokoll (ChAP) entwickelt, womit ADP-Ribosyliertes Chromatin angereichert werden kann und die Lokalisation der ADP-Ribosylierung geklärt werden kann. Wir haben dieses Protokoll auf ein oxidatives Stress Paradigma angewandt und gefunden, dass Wasserstoffperoxid (H2O2) induzierte ADP-Ribosylierung vorzugsweise zu Nukleosom-dichten heterochromatischen Regionen sowie zu repetitiven Elementen im Genom lokalisiert. Die an diesen Loci induzierte Chromatin ADP-Ribosylierung korrelierte mit einer höheren Zugänglichkeit des Chromatins. Zusätzlich war während der in vitro induzierten Adipogenese, die ADP-Ribosylierung eng mit PPARγ bei dessen Zielgenen assoziiert. Zusammengenommen zeigt diese Arbeit, dass die Induktion und genomische Distribution von Chromatin-assoziierter ADP-Ribosylierung vom Stimulus und Zelltyp abhängt. Unter allen getesteten Konditionen hat die zielgerichtete ADP- Ribosylierung zu bestimmten Chromatin Loci entweder die funktionelle Chromatin Struktur (bei H2O2), oder die Gentranskription (bei SOX2 und PPARγ) reguliert. Wie die zielgerichtete Spezifität der ADP-Ribosylierung im Kontext von Chromatin erreicht wird bedarf weiterer Untersuchungen

ADP-ribose-specific chromatin-affinity purification for investigating genome-wide or locus-specific chromatin ADP-ribosylation

Protein ADP-ribosylation is a structurally heterogeneous post-translational modification (PTM) that influences the physicochemical and biological properties of the modified protein. ADP-ribosylation of chromatin changes its structural properties, thereby regulating important nuclear functions. A lack of suitable antibodies for chromatin immunoprecipitation (ChIP) has so far prevented a comprehensive analysis of DNA-associated protein ADP-ribosylation. To analyze chromatin ADP-ribosylation, we recently developed a novel ADP-ribose-specific chromatin-affinity purification (ADPr-ChAP) methodology that uses the recently identified ADP-ribose-binding domains RNF146 WWE and Af1521. In this protocol, we describe how to use this robust and versatile method for genome-wide and loci-specific localization of chromatin ADP-ribosylation. ADPr-ChAP enables bioinformatic comparisons of ADP-ribosylation with other chromatin modifications and is useful for understanding how ADP-ribosylation regulates biologically important cellular processes. ADPr-ChAP takes 1 week and requires standard skills in molecular biology and biochemistry. Although not covered in detail here, this technique can also be combined with conventional ChIP or DNA analysis to define the histone marks specifically associated with the ADP-ribosylated chromatin fractions and dissect the molecular mechanism and functional role of chromatin ADP-ribosylation

Analysis of chromatin ADP-Ribosylation at the genome-wide level and at specific loci by ADPr-ChAP

Chromatin ADP-ribosylation regulates important cellular processes. However, the exact location and magnitude of chromatin ADP-ribosylation are largely unknown. A robust and versatile method for assessing chromatin ADP-ribosylation is therefore crucial for further understanding its function. Here, we present a chromatin affinity precipitation method based on the high specificity and avidity of two well-characterized ADP-ribose binding domains to map chromatin ADP-ribosylation at the genome-wide scale and at specific loci. Our ADPr-ChAP method revealed that in cells exposed to oxidative stress, ADP-ribosylation of chromatin scales with histone density, with highest levels at heterochromatic sites and depletion at active promoters. Furthermore, in growth factor-induced adipocyte differentiation, increased chromatin ADP-ribosylation was observed at PPARγ target genes, whose expression is ADP-ribosylation dependent. In combination with deep-sequencing and conventional chromatin immunoprecipitation, the established ADPr-ChAP provides a valuable resource for the bioinformatic comparison of ADP-ribosylation with other chromatin modifications and for addressing its role in other biologically important processes

Establishment of a Mass-Spectrometry-Based Method for the Identification of the In Vivo Whole Blood and Plasma ADP-Ribosylomes

Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modification states of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove these modifications after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we discovered that plasma phosphodiesterases and/or ADP-ribosylhydrolases rapidly demodify in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a mass-spectrometry-based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa domesticus), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions.ISSN:1535-3893ISSN:1535-390

Establishment of a Mass-Spectrometry-Based Method for the Identification of the In Vivo Whole Blood and Plasma ADP-Ribosylomes

Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modification states of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove these modifications after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we discovered that plasma phosphodiesterases and/or ADP-ribosylhydrolases rapidly demodify in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a mass-spectrometry-based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa domesticus), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions.ISSN:1535-3893ISSN:1535-390

Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure

The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1(-/-) compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function