| License: Publishing license for publications excluding print on demand (7MB) |
- URN to cite this document:
- urn:nbn:de:bvb:355-epub-404135
| Item type: | Thesis of the University of Regensburg (PhD) |
|---|---|
| Publisher: | University of Regensburg |
| Place of Publication: | Regensburg |
| Number of Pages: | 223 |
| Date: | 24 June 2020 |
| Referee: | Prof. Dr. Klaus Grasser and apl. Prof. Dr Joachim Griesenbeck |
| Date of exam: | 24 June 2019 |
| Institutions: | Biology, Preclinical Medicine > Institut für Pflanzenwissenschaften > Lehrstuhl für Zellbiologie und Pflanzenphysiologie (Prof. Dr. Klaus Grasser) |
| Keywords: | transcription, TFIIS, polymerase, chromatin, arabidopsis |
| Dewey Decimal Classification: | 500 Science > 570 Life sciences |
| Status: | Published |
| Refereed: | Yes, this version has been refereed |
| Created at the University of Regensburg: | Yes |
| Item ID: | 40413 |
Abstract (English)
The regulation of gene expression is a fundamental process in eukaryotes encompassing many molecular mechanisms. Transcript elongation has emerged over the last decades as a highly regulated and very dynamic step in determining the transcriptional outcome. Accordingly, precisely controlled gene expression may be attributed to the properties of transcriptionally engaged RNAPII properties as well ...
Abstract (English)
The regulation of gene expression is a fundamental process in eukaryotes encompassing many molecular mechanisms. Transcript elongation has emerged over the last decades as a
highly regulated and very dynamic step in determining the transcriptional outcome.
Accordingly, precisely controlled gene expression may be attributed to the properties of transcriptionally engaged RNAPII properties as well as the coordination with ongoing
co-transcriptional processes, both largely influenced by the action of so-called transcript elongation factors (TEFs). However, these regulatory processes are still poorly understood in plants.
In this study the molecular and functional characterisation of TFIIS has been performed by using its dominant negative version (TFIISmut), revealing TFIIS importance in regulating
transcript elongation in vivo. Additionally, the mutual impact of various TEFs on gene expression and consequently plant development has been addressed.
TFIIS is a well characterised TEF which directly influences RNAPII properties and its molecular function has been extensively studied in vitro. Accordingly, TFIIS has been demonstrated to stimulate intrinsic RNAPII endonucleolytic cleavage activity, allowing the control of RNAPII
pausing and read-through. Still, the details regarding TFIIS molecular role in vivo as well as its biological importance remain largely unknown and those aspects have been addressed in this study by using the inducibly expressed TFIISmut.
The -estradiol inducible system was successfully adapted in this study to allow temporally controlled expression of TFIISmut in plants lacking functional TFIIS, leading to severe growth defects of created mutants. Observed morphological defects could be subsequently linked to generally compromised transcription triggered by TFIISmut expression, supporting the biological functionality of the created inducible system. Accordingly, the replacement of invariant Asp309 and Glu310 residues renders Arabidopsis TFIIS as a negative dominant form which presumably inhibits intrinsic RNAPII cleavage activity in vivo. TFIISmut was additionally
demonstrated to associate with actively transcribing RNAPII in the chromatin context as a component of the Arabidopsis TEC.
In this study several previously unknown characteristic of plant-specific transcript elongation were demonstrated in the context of active RNAPII occupancy. Mutation of TFIIS was
subsequently shown to significantly influence genome-wide distribution of active RNAPII leading to its clear enrichment in the promoter-proximal region. Observed RNAPII accumulation has been linked with RNAPII arrest which consequently lowered TEC mobility, impeded cell cycle progression and resulted in the proteasomal degradation of NRPB1. Spatial
determination of RNAPII arrest in the presence of mutated TFIIS revealed strong overlap with the position of the +1 nucleosome. Thus, the molecular role of TFIIS in vivo may primarily encompass nucleosome traversal by RNAPII but also other pausing-related mechanisms, such as promoter-proximal pausing, transcription fidelity and transcript elongation rate. Thus, TFIIS emerges from this study as a general regulator of many processes related to RNAPII pausing, supporting the previously suggested role of RNAPII backtracking in the regulation of many cellular processes. Biologically, TFIIS was suggested in this study as important for the adjustment of transcriptomic programs and metabolic adaptation when facing the external stimuli or transition through various developmental stages. Thus, the usage of a negative dominant version of TFIIS framed into an inducible system consists a valuable molecular tool for studding the role of TFIIS in vivo as well as the properties of conditionally controlled transcript elongation.
In the second part of the thesis the composition of Arabidopsis TEC has been studied in detail in order to better understand the mutual regulation of transcript elongation by different TEFs.
The composition of the Arabidopsis TEC was determined with the contribution of other lab members. The aspects related to mutual regulation of transcript elongation by TFIIS and
PAF1-C were further elaborated in this study. Accordingly, the genetic interaction between TFIIS and ELF7 was demonstrated and could be subsequently associated with the reduced
PAF1-C level within the TEC in the absence of N-terminal TFIIS. Arabidopsis ELF7 was additionally demonstrated to play a role in the response to replication stress and presumably
in the resolution of transcription-replication conflicts (TRCs), similarly to observations in yeast. The potential influence of TRCs accumulation on observed genetic interaction between
TFIIS and ELF7 was evaluated in this study. Additionally, mutual TFIIS and ELF7 contributions into other molecular processes have been proposed, including the regulation of nucleosome traversal and RNAPII and promoter-proximal pausing. Consequently, the data in this study suggest that the identified genetic interaction may be a combination of perturbations in PAF1-C level within the TEC as well as other molecular processes synergistically affected by the mutual absence of TFIIS and ELF7. Those findings imply many levels of interaction between various TEFs during transcript elongation.
Taken together, this study encompasses the optimisation and successful implementation of novel molecular tools and approaches in combination with conventional molecular assays and reverse genetic approaches. As a result, TFIIS importance in regulating transcript elongation in vivo as well as the mutual contribution of various TEFs into correctly regulated gene expression and plant development could be extensively demonstrated. Thus, findings presented in this study present a valuable insight into the regulation of transcript elongation in plants and additionally elucidate the general principles of gene expression in higher
eukaryotes.
Translation of the abstract (German)
Die Regulation der Genexpression ist ein grundlegender Prozess bei Eukaryoten, der vielemolekulare Mechanismen umfasst. Diese regulatorischen Prozesse sind in Pflanzen jedoch nochwenig verstanden. In dieser Studie wurde die molekulare und funktionelle Charakterisierung vonTFIIS unter Verwendung seiner dominant negativen Version (TFIISmut) durchgeführt, wodurch dieBedeutung von TFIIS für die ...
Translation of the abstract (German)
Die Regulation der Genexpression ist ein grundlegender Prozess bei Eukaryoten, der vielemolekulare Mechanismen umfasst. Diese regulatorischen Prozesse sind in Pflanzen jedoch nochwenig verstanden. In dieser Studie wurde die molekulare und funktionelle Charakterisierung vonTFIIS unter Verwendung seiner dominant negativen Version (TFIISmut) durchgeführt, wodurch dieBedeutung von TFIIS für die Regulation der Transkriptverlängerung in vivo deutlich wird. Darüberhinaus wurde der gegenseitige Einfluss verschiedener TEFs auf die Genexpression und damit auf die Pflanzenentwicklung untersucht. Es wurde gezeigt, dass TFIIS die intrinsische endonukleolytischeRNAPII-Spaltungsaktivität stimuliert und die Kontrolle der RNAPII-Pause und des Durchlesensermöglicht. Die Details bezüglich der molekularen Rolle von TFIIS in vivo sowie seiner biologischenBedeutung sind jedoch weitgehend unbekannt, und diese Aspekte wurden in dieser Studie unterVerwendung des induzierbar exprimierten TFIISmut angesprochen. Das durch Östradiol induzierbareSystem wurde in dieser Studie erfolgreich angepasst, um eine zeitlich kontrollierte Expression von FIISmut in Pflanzen ohne funktionelles TFIIS zu ermöglichen, was zu schweren Wachstumsdefektender erzeugten Mutanten führte. Der Ersatz invarianter Asp309- und Glu310-Reste macht ArabidopsisTFIIS zu einer negativ dominanten Form, die vermutlich die intrinsische RNAPII-Spaltungsaktivität invivo hemmt. Anschließend wurde gezeigt, dass die Mutation von TFIIS die genomweite Verteilungvon aktivem RNAPII signifikant beeinflusst, was zu seiner deutlichen Anreicherung in der Promotor-proximalen Region führt. Die beobachtete Akkumulation von RNAPII wurde mit einem RNAPII-Stillstand in Verbindung gebracht, der folglich die TEC-Mobilität verringerte, das Fortschreiten desZellzyklus behinderte und zum proteasomalen Abbau von NRPB1 führte. Die räumliche Bestimmungdes RNAPII-Arrests in Gegenwart von mutiertem TFIIS ergab eine starke Überlappung mit derPosition des +1-Nukleosoms. Daher kann die molekulare Rolle von TFIIS in vivo in erster Linie dieDurchquerung von Nucleosomen durch RNAPII umfassen, aber auch andere pausenbezogene Mechanismen, wie Promotor-proximale Pause, Transkriptionstreue undTranskriptionsverlängerungsrate. Daher zeight sich TFIIS aus dieser Studie als allgemeiner Regulatorvieler Prozesse hervor, die mit RNAPII-Pausen zusammenhängen.
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