Μελέτη του ρόλου των επιγενετικών μηχανισμών και των μηχανισμών μεταγωγής σήματος στη ρύθμιση της γονιδιακής έκφρασης: η λειτουργία των CS πρωτεϊνών

Η μελέτη μας αρχικά επικεντρώθηκε στον τρόπο με τον οποίο τα φυσιολογικά κύτταρα αποκρίνονται στις βλάβες που προκαλεί η υπεριώδης ακτινοβολία στο γονιδίωμα και οι οποίες διακόπτουν την πορεία την μεταγραφή. Βρέθηκε ότι αμέσως μετά την επαγωγή βλαβών ξεκινά η ταυτόχρονη απελευθέρωση RNA πολυμερασών II επιμήκυνσης από τις θέσεις παύσης της μεταγραφής πλησίον των υποκινητών (Promoter proximal pausing sites) προς το 3’ άκρο όλων των ενεργά μεταγραφόμενων γονιδίων. Με τον τρόπο αυτόν οι RNA πολυμεράσες, οι οποίες κινούνται κατά μήκος των ενεργών γονιδίων, μεταγράφοντάς τα, μεγιστοποιούν τις πιθανότητες να συναντήσουν κάποια βλάβη και να ενεργοποιήσουν άμεσα το υπεύθυνο μονοπάτι επιδιόρθωσης, συντελώντας με τον τρόπο αυτό στην επιτάχυνση της απομάκρυνσής τους. Μια σημαντική συνέπεια του μεταγραφικού αυτού μηχανισμού άμυνας είναι η ομογενής μείωση των μεταλλάξεων σε όλα σχεδόν τα εκφρασμένα γονίδια, όπως βρέθηκε μετά από αλληλούχιση γονιδιωμάτων που είχαν εκτεθεί σε τοξικούς παράγοντες όπως το μελάνωμα και το αδενοκαρκίνωμα του πνεύμονα. Τα αποτελέσματα της ενότητας αυτής έχουν δημοσιευθεί πρόσφατα (Lavigne et al., Nature Communications 2017). Στη συνέχεια μελετήθηκε η απόκριση των κυττάρων που φέρουν μεταλλαγή στο ERCC6 γονίδιο (κωδικοποιεί για την CSB πρωτεΐνη), ώστε να αποσαφηνιστεί ο ρόλος της πρωτεΐνης στη ρύθμιση της μεταγραφής πριν και μετά από την επίδραση γενοτοξικών παραγόντων. Χρησιμοποιώντας αλληλούχιση νεοσυντιθέμενων τμημάτων RNA βρήκαμε, σε αντίθεση με ότι πιστεύονταν, ότι και στα CS-B κύτταρα ξεκινά ένα νέο κύμα μεταγραφής στα αρχικά στάδια έπειτα από επίδραση υπεριώδους ακτινοβολίας, το οποίο όμως είναι πολύ πιο αργό από αυτό που παρατηρείται στα φυσιολογικά κύτταρα. Η μειωμένη ταχύτητα μεταγραφής που παρατηρείται απουσία λειτουργικής CSB πρωτεΐνης οφείλεται πιθανά στην εκτεταμένη παρακώλυση των RNA πολυμερασών στα σημεία DNA βλαβών λόγω της ελαττωματικής επιδιόρθωσης τους σε αυτά τα κύτταρα. Ενώ, η έναρξη της μεταγραφής φαίνεται ότι δεν επηρεάζεται, οι πολυμεράσες επιμήκυνσης παραμένουν μόνιμα παγιδευμένες στα αρχικά τμήματα των γονιδίων και δεν επιτρέπουν την επανάκαμψη της επιμήκυνσης της μεταγραφής, με αποτέλεσμα την απώλεια ζωτικών μεταγράφων και την ενεργοποίηση μηχανισμών απόπτωσης των CS-B κυττάρων. Τα αποτελέσματα της 2ης ενότητας έχουν συμπεριληφθεί σε μια δημοσίευση η οποία είναι στο στάδιο υποβολής σε διεθνές επιστημονικό περιοδικό (Ntakou-Zamplara et al.).In this study we focused on complex molecular responses that preserve gene expression accuracy and genome integrity in the face of UV irradiation. We revealed a new mechanism in response to UV , in which RNA polymerase II (RNAPII) molecules are dynamically and synchronously released from promoter-proximal pausing sites (PPP) into elongation to promote uniform and accelerated surveillance of the whole transcribed genome. The maximised influx of de novo released RNAPII correlates with increased damage-sensing. In turn, this transcription elongation ‘safe’ mode guarantees efficient DNA repair regardless of damage location, gene size and transcription level. Accordingly, we detect low and homogenous rates of mutational signatures associated with UV exposure or cigarette smoke across all active genes. Our results were recently published (Lavigne et al., Nature Communications 2017). Moreover, aiming to shed light on the role of Cockayne Syndrome protein B (CSB) protein in transcription reorganization after damage, we studied the response of cells with defective CSB to UV irradiation. Using nascent RNA sequencing, we revealed a new wave of transcription released from PPP as an early response to UV in contrast to previous studies. This de novo wave is characterized by a slower rate in comparison to normal cells possibly due to the blockage of RNAPII molecules at damaged sites. Whereas the initiation of transcription seems unaffected, the elongating polymerases remain trapped in areas close to the 5’ prime end of long genes, preventing the recovery of transcription. As a result, Cockayne Syndrome cells lacking vital transcripts eventually activate apoptotic pathways. The results of the second part of this study are included in a manuscript which is under submission for publication (Ntakou-Zamplara et al.)

The Human Plasma Membrane Peripherome: Visualization and Analysis of Interactions

A major part of membrane function is conducted by proteins, both integral and peripheral. Peripheral membrane proteins temporarily adhere to biological membranes, either to the lipid bilayer or to integral membrane proteins with non-covalent interactions. The aim of this study was to construct and analyze the interactions of the human plasma membrane peripheral proteins (peripherome hereinafter). For this purpose, we collected a dataset of peripheral proteins of the human plasma membrane. We also collected a dataset of experimentally verified interactions for these proteins. The interaction network created from this dataset has been visualized using Cytoscape. We grouped the proteins based on their subcellular location and clustered them using the MCL algorithm in order to detect functional modules. Moreover, functional and graph theory based analyses have been performed to assess biological features of the network. Interaction data with drug molecules show that ~10% of peripheral membrane proteins are targets for approved drugs, suggesting their potential implications in disease. In conclusion, we reveal novel features and properties regarding the protein-protein interaction network created by peripheral proteins of the human plasma membrane.Comment: 39 pages, 5 figures, 3 supplement figures, under review in BMR

Expression of phosphorylated ribosomal protein S6 in mesothelioma patients - correlation with clinico-pathological characteristics and outcome: results from the European Thoracic Oncology Platform (ETOP) Mesoscape project

Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Although histology and pathologic stage are important prognostic factors, better prognostic biomarkers are needed. The ribosomal protein S6 is a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway involved in protein synthesis and cell proliferation. In previous studies, low phosphorylated S6 (pS6) immunoreactivity was significantly correlated with longer progression-free survival (PFS) and overall survival (OS) in PM patients. We aimed to correlate pS6 expression to clinical data in a large multi-centre PM cohort as part of the European Thoracic Oncology Platform (ETOP) Mesoscape project. Tissue Micro Arrays (TMAs) of PM were constructed and expression of pS6 was evaluated by a semiquantitatively aggregate H-score. Expression results were correlated to patient characteristics as well as OS/PFS. pS6 IHC results of 364 patients from 9 centres, diagnosed between 1999 and 2017 were available. The primary histology of included tumours was epithelioid (70.3%), followed by biphasic (24.2%) and sarcomatoid (5.5%). TMAs included both treatment-naive and tumour tissue taken after induction chemotherapy. High pS6 expression (181 patients with H-score>1.41) was significantly associated with less complete resection. In the overall cohort, OS/PFS were not significantly different between pS6-low and pS6-high patients. In a subgroup analysis nonepithelioid (biphasic and sarcomatoid) patients with high pS6 expression showed a significantly shorter OS (p< 0.001, 10.7 versus 16.9 months) and PFS (p < 0.001, 6.2 versus 10.8 months). In subgroup analysis, in non-epithelioid PM patients high pS6 expression was associated with significantly shorter OS and PFS. These exploratory findings suggest a clinically relevant PI3K pathway activation in non-epithelioid PM which might lay the foundation for future targeted treatment strategies

Measurement of the top quark forward-backward production asymmetry and the anomalous chromoelectric and chromomagnetic moments in pp collisions at √s = 13 TeV

Abstract The parton-level top quark (t) forward-backward asymmetry and the anomalous chromoelectric (d̂ t) and chromomagnetic (μ̂ t) moments have been measured using LHC pp collisions at a center-of-mass energy of 13 TeV, collected in the CMS detector in a data sample corresponding to an integrated luminosity of 35.9 fb−1. The linearized variable AFB(1) is used to approximate the asymmetry. Candidate t t ¯ events decaying to a muon or electron and jets in final states with low and high Lorentz boosts are selected and reconstructed using a fit of the kinematic distributions of the decay products to those expected for t t ¯ final states. The values found for the parameters are AFB(1)=0.048−0.087+0.095(stat)−0.029+0.020(syst),μ̂t=−0.024−0.009+0.013(stat)−0.011+0.016(syst), and a limit is placed on the magnitude of | d̂ t| &lt; 0.03 at 95% confidence level. [Figure not available: see fulltext.

Measurement of t(t)over-bar normalised multi-differential cross sections in pp collisions at root s=13 TeV, and simultaneous determination of the strong coupling strength, top quark pole mass, and parton distribution functions

Peer reviewe

An embedding technique to determine ττ backgrounds in proton-proton collision data

An embedding technique is presented to estimate standard model tau tau backgrounds from data with minimal simulation input. In the data, the muons are removed from reconstructed mu mu events and replaced with simulated tau leptons with the same kinematic properties. In this way, a set of hybrid events is obtained that does not rely on simulation except for the decay of the tau leptons. The challenges in describing the underlying event or the production of associated jets in the simulation are avoided. The technique described in this paper was developed for CMS. Its validation and the inherent uncertainties are also discussed. The demonstration of the performance of the technique is based on a sample of proton-proton collisions collected by CMS in 2017 at root s = 13 TeV corresponding to an integrated luminosity of 41.5 fb(-1).Peer reviewe

MUSiC : a model-unspecific search for new physics in proton-proton collisions at root s=13TeV

Results of the Model Unspecific Search in CMS (MUSiC), using proton-proton collision data recorded at the LHC at a centre-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 35.9 fb(-1), are presented. The MUSiC analysis searches for anomalies that could be signatures of physics beyond the standard model. The analysis is based on the comparison of observed data with the standard model prediction, as determined from simulation, in several hundred final states and multiple kinematic distributions. Events containing at least one electron or muon are classified based on their final state topology, and an automated search algorithm surveys the observed data for deviations from the prediction. The sensitivity of the search is validated using multiple methods. No significant deviations from the predictions have been observed. For a wide range of final state topologies, agreement is found between the data and the standard model simulation. This analysis complements dedicated search analyses by significantly expanding the range of final states covered using a model independent approach with the largest data set to date to probe phase space regions beyond the reach of previous general searches.Peer reviewe