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Björklund, Stefan
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Publications (10 of 37) Show all publications
Kumar, K. R., Blomberg, J. & Björklund, S. (2018). The MED7 subunit paralogs of Mediator function redundantly in development of etiolated seedlings in Arabidopsis. The Plant Journal, 96(3), 578-594
Open this publication in new window or tab >>The MED7 subunit paralogs of Mediator function redundantly in development of etiolated seedlings in Arabidopsis
2018 (English)In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 96, no 3, p. 578-594Article in journal (Refereed) Published
Abstract [en]

MED7 is a subunit of the Mediator middle module and is encoded by two paralogs in Arabidopsis. We generated MED7 silenced lines using RNAi to study its impact on Arabidopsis growth and development. Compared with wild type, etiolated seedlings of the MED7 silenced lines exhibited reduced hypocotyl length caused by reduced cell elongation when grown in the dark. The hypocotyl length phenotype was rescued by exogenously supplied brassinosteroid. In addition, MED7 silenced seedlings exhibited defective hook opening in the dark as well as defective cotyledon expansion in the presence of the brassinosteroid inhibitor brassinazole. Whole transcriptome analysis on etiolated seedlings using RNA sequencing revealed several genes known to be regulated by auxin and brassinosteroids, and a broad range of cell wall-related genes that were differentially expressed in the MED7 silenced lines. This was especially evident for genes involved in cell wall extension and remodeling, such as EXPANSINs and XTHs. Conditional complementation with each MED7 paralog individually restored the hypocotyl phenotype as well as the gene expression defects. Additionally, conditional expression of MED7 had no effects that were independent of the Mediator complex on the observed phenotypes. We concluded that the MED7 paralogs function redundantly in regulating genes required for the normal development of etiolated Arabidopsis seedlings.

Place, publisher, year, edition, pages
John Wiley & Sons, 2018
Keywords
Arabidopsis thaliana, MED7, Mediator complex, paralogs, skotomorphogenesis, transcriptional regulation
National Category
Forest Science
Identifiers
urn:nbn:se:umu:diva-153697 (URN)10.1111/tpj.14052 (DOI)000447881200008 ()30058106 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research CouncilKnut and Alice Wallenberg FoundationThe Kempe FoundationsCarl Tryggers foundation
Available from: 2018-12-05 Created: 2018-12-05 Last updated: 2018-12-05Bibliographically approved
Chereji, R. V., Bharatula, V., Elfving, N., Blomberg, J., Larsson, M., Morozov, A. V., . . . Björklund, S. (2017). Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly. Nucleic Acids Research, 45(15), 8806-8821
Open this publication in new window or tab >>Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly
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2017 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 15, p. 8806-8821Article in journal (Refereed) Published
Abstract [en]

Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Sac-charomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization.

Place, publisher, year, edition, pages
Oxford University Press, 2017
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-139789 (URN)10.1093/nar/gkx491 (DOI)000409380900020 ()
Available from: 2017-10-03 Created: 2017-10-03 Last updated: 2018-06-09Bibliographically approved
Fallath, T., Kidd, B. N., Stiller, J., Davoine, C., Björklund, S., Manners, J. M., . . . Schenk, P. M. (2017). MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana. PLoS ONE, 12(4), Article ID e0176022.
Open this publication in new window or tab >>MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana
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2017 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 4, article id e0176022Article in journal (Refereed) Published
Abstract [en]

The conserved protein complex known as Mediator conveys transcriptional signals by acting as an intermediary between transcription factors and RNA polymerase II. As a result, Mediator subunits play multiple roles in regulating developmental as well as abiotic and biotic stress pathways. In this report we identify the head domain subunits MEDIATOR18 and MEDIATOR20 as important susceptibility factors for Fusarium oxysporum infection in Arabidopsis thaliana. Mutants of MED18 and MED20 display down-regulation of genes associated with jasmonate signaling and biosynthesis while up-regulation of salicylic acid associated pathogenesis related genes and reactive oxygen producing and scavenging genes. We propose that MED18 and MED20 form a sub-domain within Mediator that controls the balance of salicylic acid and jasmonate associated defense pathways.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2017
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-136206 (URN)10.1371/journal.pone.0176022 (DOI)000400308800020 ()28441405 (PubMedID)
Available from: 2017-07-03 Created: 2017-07-03 Last updated: 2018-06-09Bibliographically approved
Shaikhali, J., Davoine, C., Björklund, S. & Wingsle, G. (2016). Redox regulation of the MED28 and MED32 mediator subunits is important for development and senescence. Protoplasma, 253(3), 957-963
Open this publication in new window or tab >>Redox regulation of the MED28 and MED32 mediator subunits is important for development and senescence
2016 (English)In: Protoplasma, ISSN 0033-183X, E-ISSN 1615-6102, Vol. 253, no 3, p. 957-963Article in journal (Refereed) Published
Abstract [en]

Mediator is a conserved multi-protein complex that acts as a bridge between promoter-bound transcriptional regulators and RNA polymerase II. While redox signaling is important in adjusting plant metabolism and development, the involvement of Mediator in redox homeostasis and regulation only recently started to emerge. Our previous results show that the MED10a, MED28, and MED32 Mediator subunits form various types of covalent oligomers linked by intermolecular disulfide bonds in vitro. To link that with biological significance we have characterized Arabidopsis med32 and med28 mutants and found that they are affected in root development and senescence, phenotypes possibly associated to redox changes.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-121313 (URN)10.1007/s00709-015-0853-y (DOI)000373578500029 ()26195288 (PubMedID)
Note

Special Issue on Cell Biology in Agricultural and Food Science

Available from: 2016-05-31 Created: 2016-05-31 Last updated: 2018-06-07Bibliographically approved
Shaikhali, J., Davoine, C., Brännström, K., Rouhier, N., Bygdell, J., Björklund, S. & Wingsle, G. (2015). Biochemical and redox characterization of the mediator complex and its associated transcription factor GeBPL, a GLABROUS1 enhancer binding protein. Biochemical Journal, 468(3), 385-400
Open this publication in new window or tab >>Biochemical and redox characterization of the mediator complex and its associated transcription factor GeBPL, a GLABROUS1 enhancer binding protein
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2015 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 468, no 3, p. 385-400Article in journal (Refereed) Published
Abstract [en]

The eukaryotic mediator integrates regulatory signals from promoter-bound transcription factors (TFs) and transmits them to RNA polymerase II (Pol II) machinery. Although redox signalling is important in adjusting plant metabolism and development, nothing is known about a possible redox regulation of mediator. In the present study, using pull-down and yeast two-hybrid assays, we demonstrate the association of mediator (MED) subunits MED10a, MED28 and MED32 with the GLABROUS1 (GL1) enhancer-binding protein-like (GeBPL), a plant-specific TF that binds a promoter containing cryptochrome 1 response element 2 (CryR2) element. All the corresponding recombinant proteins form various types of covalent oligomers linked by intermolecular disulfide bonds that are reduced in vitro by the thioredoxin (TRX) and/or glutathione/glutaredoxin (GRX) systems. The presence of recombinant MED10a, MED28 and MED32 subunits or changes of its redox state affect the DNA-binding capacity of GeBPL suggesting that redox-driven conformational changes might modulate its activity. Overall, these results advance our understanding of how redox signalling affects transcription and identify mediator as a novel actor in redox signalling pathways, relaying or integrating redox changes in combinationwith specific TFs as GeBPL.

Keywords
Arabidopsis thaliana, CryR2, deoxyribonucleic acid (DNA)-binding, GLABROUS1 enhancer-binding otein (GeBP), mediator, redox
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-109821 (URN)10.1042/BJ20150132 (DOI)000360966500004 ()25877331 (PubMedID)
Available from: 2015-10-06 Created: 2015-10-06 Last updated: 2018-06-07Bibliographically approved
Broach, J. R., Bharatula, V., Chereji, R., Elfving, N., Björklund, S. & Morozov, A. (2015). The Msn2 mediated stress response: Survival based on "hedging your bet" and a dynamic interplay of transcription factor binding and nucleosome occupancy. Paper presented at Yeast 2015: 27th International Conference on Yeast Genetics and Molecular Biology, (ICYGMB), Levico Terme, Italy, September 6-12, 2015. Yeast, 32(Suppl. 1), S221-S222
Open this publication in new window or tab >>The Msn2 mediated stress response: Survival based on "hedging your bet" and a dynamic interplay of transcription factor binding and nucleosome occupancy
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2015 (English)In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 32, no Suppl. 1, p. S221-S222Article in journal, Meeting abstract (Other academic) Published
Abstract [en]

Yeast cell subjected to many different stresses elicit an acute transcriptional stress response mediated by the Msn2 transcription factor, which alters expression of both a stress specific-cohort of genes as well as a common cohort of genes that changes expression in a stereotypic fashion upon exposure to any of a wide variety of stresses. We have shown by dynamic single cell analysis that stresses regulate Msn2 activity through cytoplasm to nuclear relocalization but do so in an unusual way: stresses induce increased frequency of bursts of short-lived, recurrent periods of Msn2 nuclear localization with different stresses eliciting different patterns of bursts. Moreover, genetically identical cells subject to an identical stress can behave quite differently, with some cells mounting a robust nuclear occupancy of Msn2 while others show no nuclear localization at all. We have proposed that this idiosyncratic behavior allows populations of cells to “hedge their bet” as to what will be the optimum strategy for surviving the ensuing stress. We have used computational modeling and single cell analysis to determine that bursting is a consequence of noise in the stress signaling pathways amplified by the small number of Msn2 molecules in the cell. Moreover, we have applied genome wide chromatin immunoprecipitation and nucleosome profiling to address how different stresses determine where Msn2 binds under a particular stressful conditions, and thus what genes are regulated by that stress, and how that binding affects, and is affected by, nucleosome positioning and other transcription factor binding. These results provide in vivo validation of Widon's model of indirect cooperativity of transcription factor binding, mediated by partial unwinding of nucleosomes by one transcription factor to allow access for a second transcription factor to a previously occluded binding site. Finally, we have addressed the “bet hedging” hypothesis by showing that persistence of the Msn2-mediated stress response yields cell growth arrest and have identified the targets responsible for that growth arrest. We have applied experimental evolution paradigms to address the relative fitness of cells exhibiting stochastic stress responses versus those with a uniform response. In short, our results indicate that the stress response is complex and that complexity is critical for cell survival.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2015
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-109915 (URN)000361466200377 ()
Conference
Yeast 2015: 27th International Conference on Yeast Genetics and Molecular Biology, (ICYGMB), Levico Terme, Italy, September 6-12, 2015
Available from: 2015-10-16 Created: 2015-10-09 Last updated: 2018-06-07Bibliographically approved
Elfving, N., Chereji, R. V., Bharatula, V., Björklund, S., Morozov, A. V. & Broach, J. R. (2014). A dynamic interplay of nucleosome and Msn2 binding regulates kinetics of gene activation and repression following stress. Nucleic Acids Research, 42(9), 5468-5482
Open this publication in new window or tab >>A dynamic interplay of nucleosome and Msn2 binding regulates kinetics of gene activation and repression following stress
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2014 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 9, p. 5468-5482Article in journal (Refereed) Published
Abstract [en]

The transcription factor Msn2 mediates a significant proportion of the environmental stress response, in which a common cohort of genes changes expression in a stereotypic fashion upon exposure to any of a wide variety of stresses. We have applied genome-wide chromatin immunoprecipitation and nucleosome profiling to determine where Msn2 binds under stressful conditions and how that binding affects, and is affected by, nucleosome positioning. We concurrently determined the effect of Msn2 activity on gene expression following stress and demonstrated that Msn2 stimulates both activation and repression. We found that some genes responded to both intermittent and continuous Msn2 nuclear occupancy while others responded only to continuous occupancy. Finally, these studies document a dynamic interplay between nucleosomes and Msn2 such that nucleosomes can restrict access of Msn2 to its canonical binding sites while Msn2 can promote reposition, expulsion and recruitment of nucleosomes to alter gene expression. This interplay may allow the cell to discriminate between different types of stress signaling.

National Category
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-90879 (URN)10.1093/nar/gku176 (DOI)000336495400015 ()
Available from: 2014-07-01 Created: 2014-07-01 Last updated: 2018-06-07Bibliographically approved
Aguilar, X., Blomberg, J., Brännström, K., Olofsson, A., Schleucher, J. & Björklund, S. (2014). Interaction Studies of the Human and Arabidopsis thaliana Med25-ACID Proteins with the Herpes Simplex Virus VP16-and Plant-Specific Dreb2a Transcription Factors. PLoS ONE, 9(5), e98575
Open this publication in new window or tab >>Interaction Studies of the Human and Arabidopsis thaliana Med25-ACID Proteins with the Herpes Simplex Virus VP16-and Plant-Specific Dreb2a Transcription Factors
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 5, p. e98575-Article in journal (Refereed) Published
Abstract [en]

Mediator is an evolutionary conserved multi-protein complex present in all eukaryotes. It functions as a transcriptional coregulator by conveying signals from activators and repressors to the RNA polymerase II transcription machinery. The Arabidopsis thaliana Med25 (aMed25) ACtivation Interaction Domain (ACID) interacts with the Dreb2a activator which is involved in plant stress response pathways, while Human Med25-ACID (hMed25) interacts with the herpes simplex virus VP16 activator. Despite low sequence similarity, hMed25-ACID also interacts with the plant-specific Dreb2a transcriptional activator protein. We have used GST pull-down-, surface plasmon resonance-, isothermal titration calorimetry and NMR chemical shift experiments to characterize interactions between Dreb2a and VP16, with the hMed25 and aMed25-ACIDs. We found that VP16 interacts with aMed25-ACID with similar affinity as with hMed25-ACID and that the binding surface on aMed25-ACID overlaps with the binding site for Dreb2a. We also show that the Dreb2a interaction region in hMed25-ACID overlaps with the earlier reported VP16 binding site. In addition, we show that hMed25-ACID/Dreb2a and aMed25-ACID/Dreb2a display similar binding affinities but different binding energetics. Our results therefore indicate that interaction between transcriptional regulators and their target proteins in Mediator are less dependent on the primary sequences in the interaction domains but that these domains fold into similar structures upon interaction.

Keywords
Mediator, Med25, Dreb2a, VP16, conformational changes, NMR, ITC
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-91061 (URN)10.1371/journal.pone.0098575 (DOI)000336790800049 ()
Note

Originally included in thesis in manuscript form.

Available from: 2014-07-11 Created: 2014-07-10 Last updated: 2018-06-07Bibliographically approved
Larsson, M., Uvell, H., Sandström, J., Rydén, P., Selth, L. A. & Björklund, S. (2013). Functional Studies of the Yeast Med5, Med15 and Med16 Mediator Tail Subunits. PLoS ONE, 8(8), e73137
Open this publication in new window or tab >>Functional Studies of the Yeast Med5, Med15 and Med16 Mediator Tail Subunits
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 8, p. e73137-Article in journal (Refereed) Published
Abstract [en]

The yeast Mediator complex can be divided into three modules, designated Head, Middle and Tail. Tail comprises the Med2, Med3, Med5, Med15 and Med16 protein subunits, which are all encoded by genes that are individually non-essential for viability. In cells lacking Med16, Tail is displaced from Head and Middle. However, inactivation of MED5/MED15 and MED15/MED16 are synthetically lethal, indicating that Tail performs essential functions as a separate complex even when it is not bound to Middle and Head. We have used the N-Degron method to create temperature-sensitive (ts) mutants in the Mediator tail subunits Med5, Med15 and Med16 to study the immediate effects on global gene expression when each subunit is individually inactivated, and when Med5/15 or Med15/16 are inactivated together. We identify 25 genes in each double mutant that show a significant change in expression when compared to the corresponding single mutants and to the wild type strain. Importantly, 13 of the 25 identified genes are common for both double mutants. We also find that all strains in which MED15 is inactivated show down-regulation of genes that have been identified as targets for the Ace2 transcriptional activator protein, which is important for progression through the G1 phase of the cell cycle. Supporting this observation, we demonstrate that loss of Med15 leads to a G1 arrest phenotype. Collectively, these findings provide insight into the function of the Mediator Tail module.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-81837 (URN)10.1371/journal.pone.0073137 (DOI)000324470700057 ()
Available from: 2013-10-23 Created: 2013-10-22 Last updated: 2018-06-08Bibliographically approved
Blomberg, J., Aguilar, X., Brännström, K., Rautio, L., Olofsson, A., Wittung-Stafshede, P. & Björklund, S. (2012). Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes. Nucleic Acids Research, 40(13), 5938-5950
Open this publication in new window or tab >>Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes
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2012 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 13, p. 5938-5950Article in journal (Refereed) Published
Abstract [en]

Mediator is a multiprotein coregulatory complex that conveys signals from DNA-bound transcriptional regulators to the RNA polymerase II transcription machinery in eukaryotes. The molecular mechanisms for how these signals are transmitted are still elusive. By using purified transcription factor Dreb2a, mediator subunit Med25 from Arabidopsis thaliana, and a combination of biochemical and biophysical methods, we show that binding of Dreb2a to its canonical DNA sequence leads to an increase in secondary structure of the transcription factor. Similarly, interaction between the Dreb2a and Med25 in the absence of DNA results in conformational changes. However, the presence of the canonical Dreb2a DNA-binding site reduces the affinity between Dreb2a and Med25. We conclude that transcription regulation is facilitated by small but distinct changes in energetic and structural parameters of the involved proteins.

Place, publisher, year, edition, pages
Oxford: Oxford University Press, 2012
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-54407 (URN)10.1093/nar/gks265 (DOI)000306970700019 ()22447446 (PubMedID)
Available from: 2012-04-26 Created: 2012-04-25 Last updated: 2018-06-08Bibliographically approved
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