| 1. |
- Carlsten, Jonas O P, et al.
(författare)
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Loss of the Mediator subunit Med20 affects transcription of tRNA and other non-coding RNA genes in fission yeast
- 2016
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Ingår i: Biochimica Et Biophysica Acta-Gene Regulatory Mechanisms. - : Elsevier BV. - 1874-9399. ; 1859:2, s. 339-347
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Tidskriftsartikel (refereegranskat)abstract
- Mediator is a co-regulator of RNA polymerase II (Pol II), transducing signals from regulatory elements and transcription factors to the general transcription machinery at the promoter. We here demonstrate that Med20 influences ribosomal protein expression in fission yeast. In addition, loss of Med20 leads to an accumulation of aberrant, readthrough tRNA transcripts. These transcripts are polyadenylated and targeted for degradation by the exosome. Similarly, other non-coding RNA molecules, such as snRNA, snoRNA and rRNA, are also enriched in the polyadenylate preparations in the absence of Med20. We suggest that fission yeast Mediator takes part in a regulatory pathway that affects Pol III-dependent transcripts.
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| 2. |
- Carlsten, Jonas O P
(författare)
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Mediator and its role in non-coding RNA and chromatin regualtion
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mediator is a multiprotein complex required for the regulation of RNA Polymerase II (Pol II) transcription. Mediator transmits regulatory signals from activators and repressors to the Pol II machinery at the promoter, but the complex has also many other functions related to control of gene transcription. This thesis aims to expand our knowledge of Mediator’s involvement in regulation of the specialized chromatin structures found at telomeres and centromeres as well as its role in regulation of non-coding RNA transcription. A fine-tuned balance between the histone deacetylase Sir2 and the histone acetyltransferase Sas2 determines the location of the boundary between active and inactive chromatin at budding yeast telomeres. In our work, we demonstrate that Mediator interacts with heterochromatin at telomeres and directs the position of this boundary. Mutations in Mediator subunits cause a depletion of the complex from heterochromatin, which changes the balance between Sir2 and Sas2, and ultimately results in desilencing of subtelomeric regions. Telomeres are important regulators of replicative life span, which is reduced as a consequence of mutations in the Mediator complex. The Schizosaccharomcyes pombe centromeres are also characterized by silent heterochromatin, which is assembled and maintained through a complex multifactorial system. In our work, we find that Mediator is involved in formation of these heterochromatin structures. Loss of the Mediator subunit Med20 causes disruption of heterochromatin and leads to increased transcriptional activity at the centromere. The med20∆ mutant also causes reduced levels of CENP-ACnp1, a centromere specific form of histone H3 found at centromeres, and chromosome instability during cell division. In our work, we find that inactivation of the RNA degrading complex the exosome can reverse the increased levels of pericentromeric transcription observed in med20∆ cells, but that it fails to alleviate the chromosome segregation defects. Furthermore, loss of Med20 leads to a changed pattern of siRNA products, which is not further affected in the med20∆/rrp6∆ strain. Our results therefore suggest that Mediator and the exosome act in partially independent pathways to influence centromere function. We also demonstrate that Mediator influences RNA polymerase III (Pol III) transcription. Deletion of med20+ results in increased transcription of ribosomal protein genes, but also affects Pol III transcription causing an accumulation of aberrant tRNA transcripts with evidence of incorrect transcription termination. The aberrant transcripts are polyadenylated and targeted for degradation by the exosome. The effects of Mediator on Pol III transcription are distinct from those involving Maf1, the classical repressor of Pol III activity. Based on our findings we suggest that fission yeast Mediator takes part in a pathway that coordinates expression of ribosomal protein genes with Pol III transcription. Work in this thesis demonstrates that Mediator regulates the chromatin structure of several regions characterized by silenced chromatin. Mediator mutations cause loss of heterochromatin at both telomeres and centromeres, which has implications for replicative aging and cell division. Our observation of chromosome segregation defects in med20∆ cells may also have more general implications. Chromosomal instability is a driving force in tumorigenesis and mutations in genes encoding Mediator subunits have been linked to the development of several forms of cancer. The thesis also introduces the unexpected finding that Mediator influences Pol III transcription. All together, our results support the view that Mediator does not only mediate signals from gene specific transcription factors to the Pol II transcription machinery. Instead Mediator is a multifaceted protein complex involved in many processes connected to transcription.
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| 3. |
- Carlsten, Jonas O P, et al.
(författare)
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Mediator Promotes CENP-A Incorporation at Fission Yeast Centromeres
- 2012
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Ingår i: Molecular and Cellular Biology. - : Informa UK Limited. - 0270-7306 .- 1098-5549. ; 32:19, s. 4035-4043
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Tidskriftsartikel (refereegranskat)abstract
- At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A(Cnp1), which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation through not only the RNA interference (RNAi) machinery but also RNAi-independent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here demonstrate that Mediator directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator colocalizes with Pol II at centromeres, and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired via both the RNAi-dependent and -independent pathways, resulting in loss of CENP-A(Cnp1) from the core centromere, a defect in kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20 Delta cells appears to directly block CENP-A(Cnp1) incorporation since inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function.
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| 4. |
- Carlsten, Jonas O P, et al.
(författare)
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The multitalented Mediator complex
- 2013
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Ingår i: Trends in Biochemical Sciences. - : Elsevier BV. - 0968-0004. ; 38:11, s. 531-537
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Tidskriftsartikel (refereegranskat)abstract
- The Mediator complex is needed for regulated transcription of RNA polymerase II (Pol II)-dependent genes. Initially, Mediator was only seen as a protein bridge that conveyed regulatory information from enhancers to the promoter. Later studies have added many other functions to the Mediator repertoire. Indeed, recent findings show that Mediator influences nearly all stages of transcription and coordinates these events with concomitant changes in chromatin organization. We review the multitude of activities associated with Mediator and discuss how this complex coordinates transcription with other cellular events. We also discuss the inherent difficulties associated with in vivo characterization of a coactivator complex that can indirectly affect diverse cellular processes via changes in gene transcription.
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| 5. |
- Zhu, Xuefeng, et al.
(författare)
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Mediator influences telomeric silencing and cellular life span.
- 2011
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Ingår i: Molecular and cellular biology. - 1098-5549. ; 31:12, s. 2413-21
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Tidskriftsartikel (refereegranskat)abstract
- The Mediator complex is required for the regulated transcription of nearly all RNA polymerase II-dependent genes. Here we demonstrate a new role for Mediator which appears to be separate from its function as a transcriptional coactivator. Mediator associates directly with heterochromatin at telomeres and influences the exact boundary between active and inactive chromatin. Loss of the Mediator Med5 subunit or mutations in Med7 cause a depletion of the complex from regions located near subtelomeric X elements, which leads to a change in the balance between the Sir2 and Sas2 proteins. These changes in turn result in increased levels of H4K16 acetylation near telomeres and in desilencing of subtelomeric genes. Increases in H4K16 acetylation have been observed at telomeres in aging cells. In agreement with this observation, we found that the loss of MED5 leads to shortening of the Saccharomyces cerevisiae (budding yeast) replicative life span.
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| 6. |
- Zhu, Xuefeng, et al.
(författare)
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Mediator tail subunits can form amyloid-like aggregates in vivo and affect stress response in yeast
- 2015
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 43:15, s. 7306-7314
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Tidskriftsartikel (refereegranskat)abstract
- The Med2, Med3 and Med15 proteins form a heterotrimeric subdomain in the budding yeast Mediator complex. This Med15 module is an important target for many gene specific transcription activators. A previous proteome wide screen in yeast identified Med3 as a protein with priogenic potential. In the present work, we have extended this observation and demonstrate that both Med3 and Med15 form amyloid-like protein aggregates under H2O2 stress conditions. Amyloid formation can also be stimulated by overexpression of Med3 or of a glutamine-rich domain present in Med15, which in turn leads to loss of the entire Med15 module from Mediator and a change in stress response. In combination with genome wide transcription analysis, our data demonstrate that amyloid formation can change the subunit composition of Mediator and thereby influence transcriptional output in budding yeast.
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