The role of Schizosaccharomyces pombe cyclophilin Rct1 in RNA polymerase II transcription

Die größte Untereinheit der RNA Polymerase II (RNAP II) wird in ihrer C-terminalen Domäne (CTD) aus multiplen heptameren Peptidsequenzwiederholungen (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) aufgebaut. Die CTD wird während der Transkription an Ser2 und Ser5 phosphoryliert, Unterschiede in diesem Phosphorylierungsmuster bewirken differentielle Regulierungen von Transkription, mRNA-Prozessierung und Histonmodifizierung. Rct1, ein nukleäres Cyclophilin der Spalthefe Schizosaccharomyces pombe, enthält eine Peptidyl-Prolyl cis-trans Isomerase (PPIase) Domäne, ein RNA Erkennungsmotif (RRM) und eine CTD mit Arg-Ser/Arg-Asp (RS/RD) Sequenzwiederholungen. Es wurde bereits nachgewiesen, dass Rct1 die Phosphorylierung der CTD von RNAP II negativ reguliert und mit transkriptionell aktivem Chromatin assoziiert ist. Die zugrunde liegenden Mechanismen wurden jedoch nicht erforscht. In dieser Arbeit wurden sowohl die Interaktionen zwischen Rct1 und der CTD von RNAP II als auch von Rct1 mit den beschriebenen RNAP II Kinasen und Phosphatasen durchgeführt. Mit in vitro Experimenten konnte nachgewiesen werden, dass die PPIase Domäne von Rct1 sowohl für die Interaktion mit der RNAP II CTD als auch für die Interaktionen mit den RNAP II CTD Kinasen Cdk9 und Lsk1 verantwortlich ist. Cdk9 und Lsk1, welche die Transkription durch Phosphorylierung des Ser2 der CTD regulieren, interagieren mit Rct1 über ihre nicht-katalytischen Domänen. Kinase-Aktivitätsmessungen ergaben, dass Rct1 über die PPIase Domäne die Aktivität von Cdk9 auf die RNAP II CTD negativ reguliert. Mit Hilfe von Chromatin-Immunopräzipitation (ChIP) konnte gezeigt werden, dass die Assoziation von RNAP II an Chromatin während Elongation und Termination der Transkription durch Rct1 beeinflusst wird. Eine ChIP-Analyse des Histons H3 konnte nachweisen, dass die Azetylierung der Lysinreste 9 und 12 sowohl bei Rct1-Überexpression als auch bei -Verminderung reduziert ist, wobei jedoch die Reduktion durch Rct1-Überexpression stärker betont ist. Nuclear run-on Experimente bestätigten weiters eine Reduktion der mRNA-Transkriptionsrate, welche sowohl unabhängig von Rct1-Überexpression als auch von Rct1-Reduktion war. Diese Ergebnisse zeigen, dass veränderte Rct1-Expression unterschiedliche Effekte auf die Bindung von RNAP II an Chromatin ausübt, die Transkription jedoch immer negativ reguliert wird.The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) consists of multiple tandem heptapeptide repeats (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7). The CTD undergoes dynamic phosphorylation on Ser2 and Ser5 residues during transcription. Changes in the CTD phosphorylation pattern orchestrate recruitment of different transcription, mRNA-processing and histone-modifying factors. Rct1, a nuclear multidomain cyclophilin from Schizosaccharomyces pombe, consists of a peptidyl-prolyl cis-trans isomerase (PPIase) domain, an RNA recognition motif (RRM) and a C-terminal domain enriched in arginine-serine/arginine-aspartic acid (RS/RD) repeats. Previous work has shown that Rct1 negatively regulates RNA polymerase II (RNAP II) C-terminal domain (CTD) phosphorylation and associates with transcriptionally active chromatin. However, the mechanism of this regulation remained elusive. Therefore, in this work Rct1 interactions with the RNAP II CTD and CTD kinases and phosphatases were checked. In vitro pull-downs indicate that the PPIase domain of Rct1 is responsible for binding to the RNAP II CTD as well as to CTD kinases Cdk9 and Lsk1. Cdk9 and Lsk1, which are known to regulate transcription by phosphorylating Ser2 of the CTD, bind Rct1 with the help of their non-kinase parts. The performed kinase assays have revealed that Rct1 negatively controls specific activity of Cdk9 towards the RNAP II CTD via its PPIase domain. Chromatin immunoprecipitation (ChIP) analysis of RNAP II occupancy along transcription units indicates that amount of RNAP II bound to chromatin during transcription elongation and termination steps is significantly increased in Rct1 overexpressing cells and decreased in Rct1 depleted cells. ChIP of histone H3 acetylated at lysines 9 and 12 (an active transcription mark) has revealed that the acetylation is reduced in both cases (Rct1 overexpression and depletion). However, the reduction is more pronounced when Rct1 is overexpressed. Moreover, the outcome of nuclear run-on experiment shows that under the conditions of either Rct1 over- or underexpression mRNA production is decreased. The evidence presented suggests that although over- and underexpression of Rct1 cause opposite effect on the amount of RNAP II bound to chromatin, both changes in Rct1 expression level negatively regulate transcription

Humanized Mouse Model Mimicking Pathology of Human Tuberculosis for in vivo Evaluation of Drug Regimens

Human immune system mice are highly valuable for in vivo dissection of human immune responses. Although they were employed for analyzing tuberculosis (TB) disease, there is little data on the spatial organization and cellular composition of human immune cells in TB granuloma pathology in this model. We demonstrate that human immune system mice, generated by transplanted human fetal liver derived hematopoietic stem cells develop a continuum of pulmonary lesions upon Mycobacterium tuberculosis aerosol infection. In particular, caseous necrotic granulomas, which contribute to prolonged TB treatment time, developed, and had cellular phenotypic spatial-organization similar to TB patients. By comparing two recommended drug regimens, we confirmed observations made in clinical settings: Adding Moxifloxacin to a classical chemotherapy regimen had no beneficial effects on bacterial eradication. We consider this model instrumental for deeper understanding of human specific features of TB pathogenesis and of particular value for the pre-clinical drug development pipeline

Nucleic Acids Research / Identification of RNA targets for the nuclear multidomain cyclophilin atCyp59 and their effect on PPIase activity

AtCyp59 is a multidomain cyclophilin containing a peptidyl-prolyl cis/trans isomerase (PPIase) domain and an evolutionarily highly conserved RRM domain. Deregulation of this class of cyclophilins has been shown to affect transcription and to influence phosphorylation of the C-terminal repeat domain of the largest subunit of the RNA polymerase II. We used a genomic SELEX method for identifying RNA targets of AtCyp59. Analysis of the selected RNAs revealed an RNA-binding motif (G[U/C]N[G/A]CC[A/G]) and we show that it is evolutionarily conserved. Binding to this motif was verified by gel shift assays in vitro and by RNA immunopreciptation assays of AtCyp59 in vivo. Most importantly, we show that binding also occurs on unprocessed transcripts in vivo and that binding of specific RNAs inhibits the PPIase activity of AtCyp59 in vitro. Surprisingly, genome-wide analysis showed that the RNA motif is present in about 70% of the annotated transcripts preferentially in exons. Taken together, the available data suggest that these cyclophilins might have an important function in transcription regulation.(VLID)458944

AhR sensing of bacterial pigments regulates antibacterial defence

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns