| 1. |
- Abdel-Fattah, Wael R., et al.
(författare)
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Growth-regulated co-occupancy of Mediator and Lsm3 at intronic ribosomal protein genes
- 2024
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 52:11, s. 6220-6233
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Tidskriftsartikel (refereegranskat)abstract
- Mediator is a well-known transcriptional co-regulator and serves as an adaptor between gene-specific regulatory proteins and RNA polymerase II. Studies on the chromatin-bound form of Mediator revealed interactions with additional protein complexes involved in various transcription-related processes, such as the Lsm2–8 complex that is part of the spliceosomal U6 small nuclear ribonucleoprotein complex. Here, we employ Chromatin Immunoprecipitation sequencing (ChIP-seq) of chromatin associated with the Lsm3 protein and the Med1 or Med15 Mediator subunits. We identify 86 genes co-occupied by both Lsm3 and Mediator, of which 73 were intron-containing ribosomal protein genes. In logarithmically growing cells, Mediator primarily binds to their promoter regions but also shows a second, less pronounced occupancy at their 3́-exons. During the late exponential phase, we observe a near-complete transition of Mediator from these promoters to a position in their 3́-ends, overlapping the Lsm3 binding sites ∼250 bp downstream of their last intron–exon boundaries. Using an unbiased RNA sequencing approach, we show that transition of Mediator from promoters to the last exon of these genes correlates to reduction of both their messenger RNA levels and splicing ratios, indicating that the Mediator and Lsm complexes cooperate to control growth-regulated expression of intron-containing ribosomal protein genes at the levels of transcription and splicing.
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| 2. |
- Balciunas, Darius, et al.
(författare)
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Functional interactions within yeast mediator and evidence of differential subunit modifications
- 2003
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:6, s. 3831-3839
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Tidskriftsartikel (refereegranskat)abstract
- It is possible to recruit RNA polymerase II to a target promoter and, thus, activate transcription by fusing Mediator subunits to a DNA binding domain. To investigate functional interactions within Mediator, we have tested such fusions of the lexA DNA binding domain to Med1, Med2, Gal11, Srb7, and Srb10 in wild type, med1, med2, gal11, sin4, srb8, srb10, and srb11 strains. We found that lexA-Med2 and lexA-Gal11 are strong activators that are independent of all Mediator subunits tested. lexA-Srb10 is a weak activator that depends on Srb8 and Srb11. lexA-Med1 and lexA-Srb7 are both cryptic activators that become active in the absence of Srb8, Srb10, Srb11, or Sin4. An unexpected finding was that lexA-VP16 differs from Gal4-VP16 in that it is independent of the activator binding Mediator module. Both lexA-Med1 and lexA-Srb7 are stably associated with Med4 and Med8, which suggests that they are incorporated into Mediator. Med4 and Med8 exist in two mobility forms that differ in their association with lexA-Med1 and lexA-Srb7. Within purified Mediator, Med4 is present as a phosphorylated lower mobility form. Taken together, these results suggest that assembly of Mediator is a multistep process that involves conversion of both Med4 and Med8 to their low mobility forms.
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| 3. |
- Balciunas, Darius, et al.
(författare)
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The Med1 subunit of the yeast mediator complex is involved in both transcriptional activation and repression
- 1999
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 96:2, s. 376-381
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Tidskriftsartikel (refereegranskat)abstract
- The mediator complex is essential for regulated transcription in vitro. In the yeast Saccharomyces cerevisiae, mediator comprises >15 subunits and interacts with the C-terminal domain of the largest subunit of RNA polymerase II, thus forming an RNA polymerase II holoenzyme. Here we describe the molecular cloning of the MED1 cDNA encoding the 70-kDa subunit of the mediator complex. Yeast cells lacking the MED1 gene are viable but show a complex phenotype including partial defects in both repression and induction of the GAL genes. Together with results on other mediator subunits, this implies that the mediator is involved in both transcriptional activation and repression. Similar to mutations in the SRB10 and SRB11 genes encoding cyclin C and the cyclin C-dependent kinase, a disruption of the MED1 gene can partially suppress loss of the Snf1 protein kinase. We further found that a lexA-Med1 fusion protein is a strong activator in srb11 cells, which suggests a functional link between Med1 and the Srb10/11 complex. Finally, we show that the Med2 protein is lost from the mediator on purification from Med1-deficient cells, indicating a physical interaction between Med1 and Med2.
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| 4. |
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| 5. |
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| 6. |
- Carlsson, Mattias, et al.
(författare)
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A Ham1p-Dependent Mechanism and Modulation of the Pyrimidine Biosynthetic Pathway can both Confer Resistance to 5-Fluorouracil in Yeast
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:10, s. e52094-
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Tidskriftsartikel (refereegranskat)abstract
- 5-Fluorouracil (5-FU) is an anticancer drug and pyrimidine analogue. A problem in 5-FU therapy is acquired resistance to the drug. To find out more about the mechanisms of resistance, we screened a plasmid library in yeast for genes that confer 5-FU resistance when overexpressed. We cloned five genes: CPA1, CPA2, HMS1, YAE1 and YJL055W. CPA1 and CPA2 encode a carbamoyl phosphate synthase involved in arginine biosynthesis and HMS1 a helix-loop-helix transcription factor. Our results suggest that CPA1, CPA2, and HMS1 confer 5-FU resistance by stimulating pyrimidine biosynthesis. Thus, they are unable to confer 5-FU resistance in a ura2 mutant, and inhibit the uptake and incorporation into RNA of both uracil and 5-FU. In contrast, YAE1 and YJL055W confer 5-FU resistance in a ura2 mutant, and selectively inhibit incorporation into RNA of 5-FU but not uracil. YAE1 is the strongest resistance gene, but it partially depends on YJL055W for its function. This suggests that YAE1 and YJL055W function together in a novel mechanism for detoxification of 5-FU and other pyrimidine analogs. Yae1p belongs to a small protein family with only two members, which are conserved in all eukaryotes examined. One of the human homologs, TAOS1, is overexpressed in oral carcinomas.
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| 7. |
- Carlsson, Mattias, et al.
(författare)
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Gene dosage effects in yeast support broader roles for the LOG1, HAM1 and DUT1 genes in detoxification of nucleotide analogues
- 2018
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Purine and pyrimidine analogues have important uses in chemotherapies against cancer, and a better understanding of the mechanisms that cause resistance to these drugs is therefore of importance in cancer treatment. In the yeast Saccharomyces cerevisiae, overexpression of the HAM1 gene encoding inosine triphosphate pyrophosphatase confers resistance to both the purine analogue 6-N-hydroxylaminopurine (HAP) and the pyrimidine analogue 5-fluorouracil (5-FU) (Carlsson et al., 2013, PLoS One 8, e52094). To find out more about the mechanisms of resistance to nucleotide analogues, and possible interdependencies between purine and pyrimidine analogue resistance mechanisms, we screened a plasmid library in yeast for genes that confer HAP resistance when overexpressed. We cloned four such genes: ADE4, DUT1, APT2, and ATR1. We further looked for genetic interactions between these genes and genes previously found to confer resistance to 5-FU. We found that HMS1, LOG1 (YJL055W), HAM1, and ATR1 confer resistance to both 5-FU and HAP, whereas ADE4, DUT1 and APT2 are specific for HAP resistance, and CPA1 and CPA2 specific for 5-FU resistance. Possible mechanisms for 5-FU and HAP detoxification are discussed based on the observed genetic interactions. Based on the effect of LOG1 against both 5-FU and HAP toxicity, we propose that the original function of the LOG (LONELY GUY) family of proteins likely was to degrade non-canonical nucleotides, and that their role in cytokinin production is a later development in some organisms.
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| 8. |
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| 9. |
- Eklund, D. Magnus, et al.
(författare)
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Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens
- 2010
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Ingår i: Development. - : The Company of Biologists. - 0950-1991 .- 1477-9129. ; 137:8, s. 1275-1284
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Tidskriftsartikel (refereegranskat)abstract
- The plant hormone auxin plays fundamental roles in vascular plants. Although exogenous auxin also stimulates developmental transitions and growth in non-vascular plants, the effects of manipulating endogenous auxin levels have thus far not been reported. Here, we have altered the levels and sites of auxin production and accumulation in the moss Physcomitrella patens by changing the expression level of homologues of the Arabidopsis SHI/STY family proteins, which are positive regulators of auxin biosynthesis genes. Constitutive expression of PpSHI1 resulted in elevated auxin levels, increased and ectopic expression of the auxin response reporter GmGH3pro:GUS, and in an increased caulonema/chloronema ratio, an effect also induced by exogenous auxin application. In addition, we observed premature ageing and necrosis in cells ectopically expressing PpSHI1. Knockout of either of the two PpSHI genes resulted in reduced auxin levels and auxin biosynthesis rates in leafy shoots, reduced internode elongation, delayed ageing, a decreased caulonema/chloronema ratio and an increased number of axillary hairs, which constitute potential auxin biosynthesis sites. Some of the identified auxin functions appear to be analogous in vascular and non-vascular plants. Furthermore, the spatiotemporal expression of the PpSHI genes and GmGH3pro:GUS strongly overlap, suggesting that local auxin biosynthesis is important for the regulation of auxin peak formation in non-vascular plants.
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| 10. |
- Elfving, Nils, et al.
(författare)
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The Arabidopsis thaliana Med25 mediator subunit integrates environmental cues to control plant development
- 2011
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:20, s. 8245-8250
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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