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Functional studies of Mediator in Arabidopsis thaliana and Saccharomyces cerevisiae
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Stefan Björklund)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mediator has been shown to be essential for regulation of RNA Polymerase II mediated transcription. Mediator functions as an interface between the general transcriptional machinery and a multitude of DNA binding transcriptional regulators, although the molecular mechanism for the process is elusive. Mediator is a large complex of over twenty subunits, most of which are conserved from yeast to plants to mammals. Many of these subunits are essential for viability in yeast, and mutations in the corresponding genes have global effects on transcription. Mediator was originally identified in Saccharomyces cerevisiae, but has since been described in most eukaryotes. However, until recently the Mediator complex was not identified in plants. This thesis describes the first successful identification and isolation of the Mediator complex from the plant Arabidopsis thaliana. By raising antibodies against candidate A. thaliana Mediator subunits, we were able to purify a multisubunit protein complex. Mass spectrometry and bioinformatics analysis allowed us to identify 21 of these subunits as conserved Mediator components and six as A. thaliana specific subunits. Some of the genes that encode the identified Mediator subunits had earlier been described as components of specific regulatory pathways controlling for example cell proliferation and flowering time. Subsequent genetic analysis confirmed that the A. thaliana Mediator complex is important for several plant signaling pathways, including flowering and stress pathways. This thesis also describes identification of regulators that interact with the A. thaliana Mediator subunit Med25, previously identified as PFT1 (Phytochrome and Flowering Time 1) and implicated in regulation of flowering time in response to light quality. Finally, we describe the function of Mediator in S. cerevisiae using genome-wide approaches. We have carried out a transcriptional switch where half of the genome changes expression and determined Mediator occupancy across the genome before and after such a switch, using ChIP-SEQ on tagged subunits from different Mediator domains. Unexpectedly, we find that Mediator occupancy is limited at most promoters. However, at the highly occupied promoters, we see different modes of changes in occupancy as a result of the transcriptional switch. These highly occupied promoters control genes involved in different stress response pathways. Thus, our results suggest that Mediator function and composition differ considerably between different promoters.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2013. , 55 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1541
Keyword [en]
Transcriptional regulation, chromatin, stress, Pol II, mediator, DREB2A, ZFHD1, MYB-like, Msn2
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-62905ISBN: 978-91-7459-540-6 (print)OAI: oai:DiVA.org:umu-62905DiVA: diva2:580034
Public defence
2013-01-18, BiA 201, Biologihuset, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2012-12-21 Created: 2012-12-20 Last updated: 2013-01-11Bibliographically approved
List of papers
1. Purification of a plant mediator from Arabidopsis thaliana identifies PFT1 as the Med25 subunit
Open this publication in new window or tab >>Purification of a plant mediator from Arabidopsis thaliana identifies PFT1 as the Med25 subunit
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2007 (English)In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 26, no 5, 717-729 p.Article in journal (Refereed) Published
Keyword
Amino Acid Sequence, Arabidopsis/genetics/*metabolism, Arabidopsis Proteins/chemistry/genetics/*isolation & purification, Genes; Plant, Models; Biological, Molecular Sequence Data, Multiprotein Complexes/chemistry/isolation & purification, Nuclear Proteins/chemistry/genetics/*isolation & purification, Protein Subunits, Sequence Homology; Amino Acid, Signal Transduction, Tandem Mass Spectrometry, Trans-Activators/chemistry/genetics/*isolation & purification
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-6333 (URN)10.1016/j.molcel.2007.05.007 (DOI)17560376 (PubMedID)
Available from: 2007-12-09 Created: 2007-12-09 Last updated: 2017-12-14Bibliographically approved
2. The Arabidopsis thaliana Med25 mediator subunit integrates environmental cues to control plant development
Open this publication in new window or tab >>The Arabidopsis thaliana Med25 mediator subunit integrates environmental cues to control plant development
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2011 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 20, 8245-8250 p.Article in journal (Refereed) Published
Abstract [en]

Development in plants is controlled by abiotic environmental cues such as day length, light quality, temperature, drought, and salinity. These signals are sensed by a variety of systems and transmitted by different signal transduction pathways. Ultimately, these pathways are integrated to control expression of specific target genes, which encode proteins that regulate development and differentiation. The molecular mechanisms for such integration have remained elusive. We here show that a linear 130-amino-acids-long sequence in the Med25 subunit of the Arabidopsis thaliana Mediator is a common target for the drought response element binding protein 2A, zinc finger homeodomain 1, and Myb-like transcription factors which are involved in different stress response pathways. In addition, our results show that Med25 together with drought response element binding protein 2A also function in repression of PhyB-mediated light signaling and thus integrate signals from different regulatory pathways.

Keyword
transcriptional regulation, phytochrome flowering time 1, RNA polymerase II
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-46758 (URN)10.1073/pnas.1002981108 (DOI)21536906 (PubMedID)
Available from: 2011-09-13 Created: 2011-09-13 Last updated: 2017-12-08Bibliographically approved
3. Mediator exists in multiple forms and is predominantly associated to promoters with low nuclesome density
Open this publication in new window or tab >>Mediator exists in multiple forms and is predominantly associated to promoters with low nuclesome density
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(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-62898 (URN)
Funder
Swedish Research CouncilSwedish Cancer Society
Available from: 2012-12-20 Created: 2012-12-20 Last updated: 2012-12-21Bibliographically approved

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