The ribosome assembly gene network is controlled by the feedback regulation of transcription elongation

Ribosome assembly requires the concerted expression of hundreds of genes, which are transcribed by all three nuclear RNA polymerases. Transcription elongation involves dynamic interactions between RNA polymerases and chromatin. We performed a synthetic lethal screening in Saccharomyces cerevisiae with a conditional allele of SPT6, which encodes one of the factors that facilitates this process. Some of these synthetic mutants corresponded to factors that facilitate pre-rRNA processing and ribosome biogenesis. We found that the in vivo depletion of one of these factors, Arb1, activated transcription elongation in the set of genes involved directly in ribosome assembly. Under these depletion conditions, Spt6 was physically targeted to the upregulated genes, where it helped maintain their chromatin integrity and the synthesis of properly stable mRNAs. The mRNA profiles of a large set of ribosome biogenesismutants confirmed the existence of a feedback regulatory network among ribosome assembly genes. The transcriptional response in this network depended on both the specific malfunction and the role of the regulated gene. In accordance with our screening, Spt6 positively contributed to the optimal operation of this global network. On the whole, this work uncovers a feedback control of ribosome biogenesis by fine-tuning transcription elongation in ribosome assembly factor-coding genes.Ministerio de Economía y Competitividad BFU2013-48643-C3-1-P, BFU2016-77728-C3-1-P, BFU2013-48643-C3- 3-P, BFU2013-42958-PJunta de Andalucía P12-BIO1938MO, P08-CVI-03508Comunidad Valenciana 2015/00

Two distinct repressive mechanisms for histone 3 lysine 4 methylation through promoting 3'-end antisense transcription

International audienceHistone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3'-end, indicating that repression is coupled to 3'-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3'-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3'-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3'-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3'-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3'-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. As novel and improved immunotherapies may fill this need, we dissected the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 25 tumors (10 pre- and 15 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas were infiltrated by NK, T and B cells, and immunosuppressive myeloid populations. NK cells showed reduced cytotoxicity and T cells had a dysfunctional profile. Interaction analysis revealed a vast immunoregulatory network and identified NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduced neuroblastoma growth, with complete responses in vivo. Moreover, addition of TIGIT blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model significantly improved survival. Concluding, our integrative analysis of neuroblastoma’s vast immunoregulatory network provides novel targets and a rationale for immunotherapeutic combination strategies

Single cell derived mRNA signals across human kidney tumors.

Funder: Department of HealthTumor cells may share some patterns of gene expression with their cell of origin, providing clues into the differentiation state and origin of cancer. Here, we study the differentiation state and cellular origin of 1300 childhood and adult kidney tumors. Using single cell mRNA reference maps of normal tissues, we quantify reference "cellular signals" in each tumor. Quantifying global differentiation, we find that childhood tumors exhibit fetal cellular signals, replacing the presumption of "fetalness" with a quantitative measure of immaturity. By contrast, in adult cancers our assessment refutes the suggestion of dedifferentiation towards a fetal state in most cases. We find an intimate connection between developmental mesenchymal populations and childhood renal tumors. We demonstrate the diagnostic potential of our approach with a case study of a cryptic renal tumor. Our findings provide a cellular definition of human renal tumors through an approach that is broadly applicable to human cancer

DNA microarrays for monitoring and analysis of gene expession

The aim of the work described in this thesis is the development of microarray technology using new methods and tools, on the one hand and, on the other, its employment to address basic scientific questions. The starting point is the development of three methodologies for microarray image analysis which increase the reproducibility of data, as well as increase the dynamic range of the measurements. Then, we employ microarray analysis to focus on the in vivo requirement of gene expression for the general transcription complex TFIID in hepatocytes in different developmental stages. The results of this work showed that TFIID is needed during the initial steps of the assembly of the pre-initiation complex, while it is dispensable for the following steps. It also revealed for the first time the involvement of the complex in silencing of previously expressed genes. After that, we shift the focus of the study from global to gene-specific transcription factors. We constructed a boutique microarray to analyze the differential recruitment of Gcn4, a model acidic transcription factor in S. cerevisiae, on its target genes and the differences in the transcription efficiency between the activation domains of Gcn4 and Gal4. We show differential requirements of the domains for components of SAGA complex and propose possible explanations. Transcription regulation can be more complex than simply employing transcription factors and the transcription machinery. Later on, we analyze the effect of Rad9, a DNA damage-dependent checkpoint protein, on transcription. It was revealed to affect specific pathways under different conditions. The physical presence of the protein in the promoters and bodies of regulated genes implies that the DNA damage response and gene transcription are intimately connected. Finally, we constructed a DNA microarray using a Cistus creticus cDNA library. We used it to identify trichome-overexpressed and trichome-specific genes in an effort to identify genes responsible for the generation of trichomes’ metabolites of important pharmaceutical properties. Of the 16 proposed trichome-specific genes the only one related to secondary metabolism was isolated and characterized as a trichome-specific germacrene B synthase, an important member of the biosynthesis of the active metabolites. In the individual chapters an introduction to the individual studies and goals, is followed by the results and the conclusions.Ο σκοπός της εργασίας που περιγράφεται σε αυτή τη διατριβή απέβλεπαν στην ανάπτυξη της τεχνολογίας των μικροσυστοιχιών και εφαρμογή νέων μεθόδων και εργαλείων αφενός και αφεταίρου στην εφαρμογή της στην απάντηση βασικών βιολογικών ερωτημάτων. Αρχικά, παρουσιάζεται η ανάπτυξη τριών μεθοδολογιών για την ανάλυση εικόνας από μικροσυστοιχίες που αυξάνουν την επαναληψιμότητα των παραγόμενων αποτελεσμάτων, καθώς και την αύξηση της δυναμικής διακύμανσής τους. Στην επόμενη ενότητα, με τη χρήση μικροσυστοιχιών αναλύεται η in vivo συμμετοχή του βασικού μεταγραφικού συμπλόκου TFIID στη μεταγραφή ηπατοκυττάρων σε διαφορετικά αναπτυξιακά στάδια. Τα αποτελέσματα αυτής της ανάλυσης έδειξαν ότι το TFIID είναι απαραίτητο μόνο κατά τη μεταγραφική έναρξη και όχι στα επόμενα στάδια της μεταγραφής. Επίσης αποκάλυψε για πρώτη φορά τη συμμετοχή του συμπλόκου στη μεταγραφική σίγηση γονιδίων που εκφράζονταν σε προηγούμενα στάδια. Κατόπιν, η εστίαση των ερευνών αλλάζει από τη βασική μεταγραφική μηχανή στους μεταγραφικούς παράγοντες και τις απαιτήσεις τους. Με την κατασκευή μικροσυστοιχίας που περιέχει μικρό αριθμό επιλεγμένων γονιδίων εξετάσαμε τη διαφορική πρόσδεση του μεταγραφικού παράγοντα Gcn4 του σακχαρομύκητα στα γονίδια-στόχους του και τις διαφορές στις απαιτήσεις για μεταγραφική ενεργοποίηση των περιοχών ενεργοποίησης του Gal4 και του Gcn4 μεταγραφικού παράγοντα. Η ανάλυση έδειξε διαφορετικές απαιτήσεις των περιοχών ενεργοποίησης για υπομονάδες του συμπλόκου SAGA και προτάθηκαν πιθανές εξηγήσεις. Η μεταγραφική ρύθμιση είναι πιο σύνθετη από την απλή αλληλεπίδραση μεταγραφικών παραγόντων με τη μεταγραφική μηχανή. Μετά αναλύθηκε η επίδραση της Rad9, μιας πρωτεΐνης που παίζει ρόλο στην απάντηση σε καταστροφή του DNA, στη μεταγραφική ρύθμιση συγκεκριμένων μονοπατιών στο σακχαρομύκητα. Η φυσική παρουσία της πρωτεΐνης σε ρυθμιζόμενα γονίδια υπονοεί ότι η μεταγραφική ρύθμιση είναι άρρηκτα συνδεδεμένη με την απόκριση σε καταστροφή του DNA. Τέλος, κατασκευάστηκε μια μικροσυστοιχία από μια cDNA βιβλιοθήκη του φυτού Cistus creticus. Αυτή χρησιμοποιήθηκε για την ανίχνευση γονιδίων που υπερεκφράζονται ή εκφράζονται αποκλειστικά στα τριχώματα, σε μια προσπάθεια να αναγνωριστούν γονίδια που είναι υπεύθυνα για την βιοσύνθεση μεταβολιτών που παράγονται στα τριχώματα με σημαντικές φαρμακευτικές ιδιότητες. Από τα πιθανά ιστο-ειδικά εκφραζόμενα γονίδια, το γονίδιο που σχετίζεται με δευτερογενή μεταβολισμό απομονώθηκε και χαρακτηρίστηκε ως μια συνθάση Β γερμακρενίου, ενός σημαντικού ενζύμου για τη βιοσύνθεση των ενεργών μεταβολιτών. Στα επιμέρους κεφάλαια, πριν τα αποτελέσματα, περιλαμβάνεται μια σύντομη εισαγωγή για τα αντίστοιχα ερευνητικά ερωτήματα