Publications by authors named "Stefan Björklund"

27 Publications

  • Page 1 of 1

Specific functions for Mediator complex subunits from different modules in the transcriptional response of Arabidopsis thaliana to abiotic stress.

Sci Rep 2020 03 19;10(1):5073. Epub 2020 Mar 19.

Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, 901 87, Sweden.

Adverse environmental conditions are detrimental to plant growth and development. Acclimation to abiotic stress conditions involves activation of signaling pathways which often results in changes in gene expression via networks of transcription factors (TFs). Mediator is a highly conserved co-regulator complex and an essential component of the transcriptional machinery in eukaryotes. Some Mediator subunits have been implicated in stress-responsive signaling pathways; however, much remains unknown regarding the role of plant Mediator in abiotic stress responses. Here, we use RNA-seq to analyze the transcriptional response of Arabidopsis thaliana to heat, cold and salt stress conditions. We identify a set of common abiotic stress regulons and describe the sequential and combinatorial nature of TFs involved in their transcriptional regulation. Furthermore, we identify stress-specific roles for the Mediator subunits MED9, MED16, MED18 and CDK8, and putative TFs connecting them to different stress signaling pathways. Our data also indicate different modes of action for subunits or modules of Mediator at the same gene loci, including a co-repressor function for MED16 prior to stress. These results illuminate a poorly understood but important player in the transcriptional response of plants to abiotic stress and identify target genes and mechanisms as a prelude to further biochemical characterization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-61758-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081235PMC
March 2020

Exploiting Suspension Plasma Spraying to Deposit Wear-Resistant Carbide Coatings.

Materials (Basel) 2019 Jul 24;12(15). Epub 2019 Jul 24.

Department of Engineering Science, University West, 46132 Trollhättan, Sweden.

Titanium- and chromium-based carbides are attractive coating materials to impart wear resistance. Suspension plasma spraying (SPS) is a relatively new thermal spray process which has shown a facile ability to use sub-micron and nano-sized feedstock to deposit high-performance coatings. The specific novelty of this work lies in the processing of fine-sized titanium and chromium carbides (TiC and CrC) in the form of aqueous suspensions to fabricate wear-resistant coatings by SPS. The resulting coatings were characterized by surface morphology, microstructure, phase constitution, and micro-hardness. The abrasive, erosive, and sliding wear performance of the SPS-processed TiC and CrC coatings was also evaluated. The results amply demonstrate that SPS is a promising route to manufacture superior wear-resistant carbide-based coatings with minimal in situ oxidation during their processing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ma12152344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6696411PMC
July 2019

Durability of Gadolinium Zirconate/YSZ Double-Layered Thermal Barrier Coatings under Different Thermal Cyclic Test Conditions.

Materials (Basel) 2019 Jul 11;12(14). Epub 2019 Jul 11.

Department of Engineering Science, University West, 46186 Trollhattan, Sweden.

Higher durability in thermal barrier coatings (TBCs) is constantly sought to enhance the service life of gas turbine engine components such as blades and vanes. In this study, three double layered gadolinium zirconate (GZ)-on-yttria stabilized zirconia (YSZ) TBC variants with varying individual layer thickness but identical total thickness produced by suspension plasma spray (SPS) process were evaluated. The objective was to investigate the role of YSZ layer thickness on the durability of GZ/YSZ double-layered TBCs under different thermal cyclic test conditions i.e., thermal cyclic fatigue (TCF) at 1100 °C and a burner rig test (BRT) at a surface temperature of 1400 °C, respectively. Microstructural characterization was performed using SEM (Scanning Electron Microscopy) and porosity content was measured using image analysis technique. Results reveal that the durability of double-layered TBCs decreased with YSZ thickness under both TCF and BRT test conditions. The TBCs were analyzed by SEM to investigate microstructural evolution as well as failure modes during TCF and BRT test conditions. It was observed that the failure modes varied with test conditions, with all the three double-layered TBC variants showing failure in the TGO (thermally grown oxide) during the TCF test and in the ceramic GZ top coat close to the GZ/YSZ interface during BRT. Furthermore, porosity analysis of the as-sprayed and TCF failed TBCs revealed differences in sintering behavior for GZ and YSZ. The findings from this work provide new insights into the mechanisms responsible for failure of SPS processed double-layered TBCs under different thermal cyclic test conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ma12142238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678775PMC
July 2019

Axial Suspension Plasma Spraying: An ultimate technique to tailor Ti6Al4V surface with HAp for orthopaedic applications.

Colloids Surf B Biointerfaces 2019 Jan 26;173:806-815. Epub 2018 Oct 26.

Centre for Biomaterials, Cellular & Molecular Theranostics (CBCMT), Vellore Institute of Technology, Vellore, 632014, India. Electronic address:

Dissolution of atmospheric plasma sprayed (APS) hydroxyapatite (HAp) coatings on Ti-6Al-4 V medical implants have always been a challenge to overcome in the field of biomedical industry. In the present work, an attempt has been made to develop a HAp coating using a novel thermal spray process called axial suspension plasma spraying (SPS), which leads to thin adherent coatings. Two HAp coatings fabricated by APS (P1 and P2) and four SPS HAp coatings (S1, S2, S3 and S4) produced with varying spraying parameters were characterized in terms of (1) microstructure, porosity, hardness, adhesion strength, contact angle and phase purity; (2) corrosion resistance in 10% Fetal bovine serum (FBS); (3) in-vitro cell adherence and cell viability using human umbilical cord blood-derived mesenchymal stem cells (hMSCs). Amongst different APS and SPS coatings, P1 and S3 exhibited superior properties. S3 coating developed using SPS exhibited 1.3 times higher adhesion strength when compared to APS coating (P1) and 9.5 times higher corrosion resistance than P1. In addition, both S3 and P1 exhibited comparatively higher biocompatibility as evidenced by the presence of more than 92% viable hMSCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.colsurfb.2018.10.071DOI Listing
January 2019

The MED7 subunit paralogs of Mediator function redundantly in development of etiolated seedlings in Arabidopsis.

Plant J 2018 11 15;96(3):578-594. Epub 2018 Sep 15.

Department of Medical Biochemistry and Biophysics, Umeå Plant Science Centre, Umeå University, Umeå, S-90187, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tpj.14052DOI Listing
November 2018

Functional metabolomics as a tool to analyze Mediator function and structure in plants.

PLoS One 2017 22;12(6):e0179640. Epub 2017 Jun 22.

Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

Mediator is a multiprotein transcriptional co-regulator complex composed of four modules; Head, Middle, Tail, and Kinase. It conveys signals from promoter-bound transcriptional regulators to RNA polymerase II and thus plays an essential role in eukaryotic gene regulation. We describe subunit localization and activities of Mediator in Arabidopsis through metabolome and transcriptome analyses from a set of Mediator mutants. Functional metabolomic analysis based on the metabolite profiles of Mediator mutants using multivariate statistical analysis and heat-map visualization shows that different subunit mutants display distinct metabolite profiles, which cluster according to the reported localization of the corresponding subunits in yeast. Based on these results, we suggest localization of previously unassigned plant Mediator subunits to specific modules. We also describe novel roles for individual subunits in development, and demonstrate changes in gene expression patterns and specific metabolite levels in med18 and med25, which can explain their phenotypes. We find that med18 displays levels of phytoalexins normally found in wild type plants only after exposure to pathogens. Our results indicate that different Mediator subunits are involved in specific signaling pathways that control developmental processes and tolerance to pathogen infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179640PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5480960PMC
September 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 Res 2017 Sep;45(15):8806-8821

Department of Medical Biochemistry and Biophysics Umeå University, 901 87 Umeå, Sweden.

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 Saccharomyces 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkx491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587782PMC
September 2017

MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana.

PLoS One 2017 25;12(4):e0176022. Epub 2017 Apr 25.

Plant-Microbe Interactions Laboratory, School of Agriculture and Food Sciences, The University of Queensland, St Lucia, Australia.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176022PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5404846PMC
September 2017

Redox regulation of the MED28 and MED32 mediator subunits is important for development and senescence.

Protoplasma 2016 May 21;253(3):957-963. Epub 2015 Jul 21.

Department of Forest Genetics and Plant Physiology, Umeå Plant Science Center, Swedish University of Agricultural Sciences, 901 83, Umeå, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00709-015-0853-yDOI Listing
May 2016

Biochemical and redox characterization of the mediator complex and its associated transcription factor GeBPL, a GLABROUS1 enhancer binding protein.

Biochem J 2015 Jun 16;468(3):385-400. Epub 2015 Apr 16.

Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences SLU, 901 83 Umeå, Sweden

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 combination with specific TFs as GeBPL.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20150132DOI Listing
June 2015

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 2014 29;9(5):e98575. Epub 2014 May 29.

Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

Mediator is an evolutionary conserved multi-protein complex present in all eukaryotes. It functions as a transcriptional co-regulator 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098575PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038590PMC
January 2015

A dynamic interplay of nucleosome and Msn2 binding regulates kinetics of gene activation and repression following stress.

Nucleic Acids Res 2014 May 5;42(9):5468-82. Epub 2014 Mar 5.

Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, PA 17033, USA

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gku176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027177PMC
May 2014

Functional studies of the yeast med5, med15 and med16 mediator tail subunits.

PLoS One 2013 22;8(8):e73137. Epub 2013 Aug 22.

Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0073137PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3750046PMC
June 2014

Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes.

Nucleic Acids Res 2012 Jul 24;40(13):5938-50. Epub 2012 Mar 24.

Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gks265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401450PMC
July 2012

The Arabidopsis thaliana Med25 mediator subunit integrates environmental cues to control plant development.

Proc Natl Acad Sci U S A 2011 May 2;108(20):8245-50. Epub 2011 May 2.

Department of Medical Biochemistry and Biophysics, Umeå Plant Science Center, Umeå University, SE-901 87 Umeå, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1002981108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3100966PMC
May 2011

Gene expression profiling and chromatin immunoprecipitation identify DBN1, SETMAR and HIG2 as direct targets of SOX11 in mantle cell lymphoma.

PLoS One 2010 Nov 22;5(11):e14085. Epub 2010 Nov 22.

Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet and Karolinska University Hospital Huddinge, Stockholm, Sweden.

The SRY (sex determining region Y)-box 11 (SOX11) gene, located on chromosome 2p25, encodes for a transcription factor that is involved in tissue remodeling during embryogenesis and is crucial for neurogenesis. The role for SOX11 in hematopoiesis has not yet been defined. Two genes under direct control of SOX11 are the class- III β-tubulin gene (TUBB3) in neural cells and the transcription factor TEA domain family member 2 (TEAD2) in neural and mesenchymal progenitor cells. Normal, mature lymphocytes lack SOX11 but express SOX4, another member of the same group of SOX transcription factors. We and others recently identified SOX11 as aberrantly expressed in mantle cell lymphoma (MCL). Since SOX11 is variably expressed in MCL it may not be essential for tumorigenesis, but may carry prognostic information. Currently, no specific functional effects have been linked to SOX11 expression in MCL and it is not known which genes are under influence of SOX11 in lymphoma. In this study we found variable expression of SOX11, SOX4 and SOX12 mRNA in mantle cell lymphoma cell lines. Downregulation of SOX11 expression by siRNA verified that SOX11 controlled the expression of the gene TUBB3 in the MCL cell line Granta 519. Furthermore we identified, by global gene expression analysis, 26 new target genes influenced by siRNA SOX11 downmodulation. Among these genes, DBN1, SETMAR and HIG2 were found to be significantly correlated to SOX11 expression in two cohorts of primary mantle cell lymphomas. Chromatin immunoprecipitation (ChIP) analysis showed that these genes are direct targets of the SOX11 protein. In spite of almost complete downregulation of the SOX11 protein no significant effects on Granta 519 cell proliferation or survival in short term in vitro experiments was found. In summary we have identified a number of genes influenced by SOX11 expression in MCL cell lines and primary MCL. Among these genes, DBN1, SETMAR and HIG2 are direct transcriptional targets of the SOX11 protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0014085PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2989913PMC
November 2010

MAP1B binds to the NMDA receptor subunit NR3A and affects NR3A protein concentrations.

Neurosci Lett 2010 May 19;475(1):33-7. Epub 2010 Mar 19.

Division of Neurodegeneration, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Novum, 141 86 Stockholm, Sweden.

Incorporation of the N-methyl-d-aspartate receptor (NMDAR) subunit NR3A into functional NMDARs results in reduced channel conductance and Ca(2+) permeability. To further investigate the function of NR3A, we have set out to characterize its intracellular binding partners. Here, we report a novel protein interaction between NR3A and microtubule associated-protein (MAP) 1B, which both are localized to dendritic shafts and filopodia. NR3A protein levels were increased in MAP1B deficient (-/-) mice, with a corresponding decrease in NR1 levels, but the fraction of filopodia immunoreactive for NR3A was equal in cells from -/- and wild type (WT) mice. NR3A has previously been shown to interact with another member of the MAP1 family, MAP1S. We showed that MAP1S binds to microtubules in a similar manner as MAP1B, and suggest that MAP1S and MAP1B both are involved in regulating trafficking of NR3A-containing NMDAR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neulet.2010.03.039DOI Listing
May 2010

Med8, Med18, and Med20 subunits of the Mediator head domain are interdependent upon each other for folding and complex formation.

Proc Natl Acad Sci U S A 2009 Dec 23;106(49):20728-33. Epub 2009 Nov 23.

Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.

We have studied folding and complex formation of the yeast Mediator head-module protein subunits Med8, Med18, and Med20. Using a combination of immunoprecipitation, far-UV circular dichroism, and fluorescence measurements on recombinantly expressed and denatured proteins that were allowed to renature separately or in different combinations, we found that Med8, Med18, and Med20 can fold in different ways to form both soluble monomeric proteins and different distinct subcomplexes. However, the concurrent presence of all three protein subunits during the renaturation process is required for proper folding and trimer complex formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0907645106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781058PMC
December 2009

Purification of a plant mediator from Arabidopsis thaliana identifies PFT1 as the Med25 subunit.

Mol Cell 2007 Jun;26(5):717-29

Department of Medical Biochemistry and Biophysics, KBC, Umeå University, SE-901 87 Umeå, Sweden.

Mediator, a central coregulator of transcription, has been identified as a large protein complex in eukaryotes ranging from yeast to man. It is therefore remarkable that Mediator has not yet been identified within the plant kingdom. Here we identify Mediator in a plant, Arabidopsis thaliana. The plant Mediator subunits typically show very low homology to other species, but our biochemical purification identifies 21 conserved and six A. thaliana-specific Mediator subunits. Most notably, we identify the A. thaliana proteins STRUWWELPETER (SWP) and PHYTOCHROME AND FLOWERING TIME 1 (PFT1) as the Med14 and Med25 subunits, respectively. These findings show that specific plant Mediator subunits are linked to the regulation of specialized processes such as the control of cell proliferation and the regulation of flowering time in response to light quality. The identification of the plant Mediator will provide new tools and insights into the regulation of transcription in plants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2007.05.007DOI Listing
June 2007

Functional and physical interactions within the middle domain of the yeast mediator.

Mol Genet Genomics 2006 Aug 7;276(2):197-210. Epub 2006 Jun 7.

Department of Medical Biochemistry and Biophysics, Umeå University, 0187, Umeå, Sweden.

Med21 (Srb7) is a small essential subunit of the middle domain of the Mediator, which is conserved in all eukaryotes. It is thought to play an important role in both transcriptional activation and repression. In the yeast Saccharomyces cerevisiae, Med21 is known to interact both with the Mediator subunit Med6 and the global co-repressor Tup1. We have made a temperature-sensitive med21-ts mutant, which we used in a high copy number suppressor screen. We found ten yeast genes that can suppress the med21-ts mutation in high copy number. The three strongest suppressors were MED7 and MED10 (NUT2), which encode other Mediator subunits, and ASH1, which encodes a repressor of the HO gene. 2-Hybrid experiments confirmed multiple interactions between Med21, Med10, Med7 and Med4, and also revealed a Med21 self-interaction. The interactions of Med21 with Med7 and Med10 were verified by co-immunoprecipitation of tagged proteins produced in insect cells and E. coli, where both interactions were found to depend strongly on the amino acid residues 2-8 of Med21. These interactions, and the interactions of Med21 with Med6 and Tup1, suggest that Med21 may serve as a molecular switchboard that integrates different signals before they reach the core polymerase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00438-006-0135-7DOI Listing
August 2006

The yeast Mediator complex and its regulation.

Trends Biochem Sci 2005 May;30(5):240-4

Department of Medical Biochemistry, Umeå University, S-901 87 Umeå, Sweden.

The Mediator complex acts as a bridge, conveying regulatory information from enhancers and other control elements to the basal RNA polymerase II transcription machinery. Mediator is required for the regulated transcription of nearly all RNA polymerase II-dependent genes in Saccharomyces cerevisiae, and post-translational modifications of specific Mediator subunits can affect global patterns of gene transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tibs.2005.03.008DOI Listing
May 2005

Site-specific Srb10-dependent phosphorylation of the yeast Mediator subunit Med2 regulates gene expression from the 2-microm plasmid.

Proc Natl Acad Sci U S A 2004 Mar 26;101(10):3370-5. Epub 2004 Feb 26.

Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.

The yeast Mediator complex is required for transcriptional regulation both in vivo and in vitro, and its function is conserved in all eukaryotes. Mediator interacts with both transcriptional activators and RNA polymerase II, but little is known about the mechanisms by which it operates at the molecular level. Here, we show that the cyclin-dependent kinase Srb10 interacts with, and phosphorylates, the Med2 subunit of Mediator both in vivo and in vitro. A point mutation of the single phosphorylation site in Med2 results in a strongly reduced expression of the REP1, REP2, FLP1, and RAF1 genes, which are all located on the endogenous 2-microm plasmid. Combined with previous studies on the effects of SRB10/SRB11 deletions, our data suggest that posttranslational modifications of Mediator subunits are important for regulation of gene expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0400221101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373468PMC
March 2004

The mediator complex.

Adv Protein Chem 2004 ;67:43-65

Department of Medical Biochemistry, Umeå University, S-901 87 Umeå, Sweden.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S0065-3233(04)67002-1DOI Listing
April 2004

S Phase-specific transcription of the mouse ribonucleotide reductase R2 gene requires both a proximal repressive E2F-binding site and an upstream promoter activating region.

J Biol Chem 2004 Mar 19;279(11):10796-807. Epub 2003 Dec 19.

Department of Medical Biochemistry and Biophysics, Umeå University, Sweden.

Ribonucleotide reductase is essential for supplying a balanced pool of the four deoxyribonucleotides required for DNA synthesis and repair. The active enzyme consists of two non-identical subunits called proteins R1 and R2. There are multiple levels of regulation of ribonucleotide reductase activity, which is highest during the S and G(2) phases of an unperturbed cell cycle in mammalian cells. Previous reports in the literature have indicated that the S phase-specific transcription of the mammalian R2 gene is regulated by a transcriptional block, is dependent on the transcription factor E2F1, or is simply regulated by proteins that bind to promoter CCAAT boxes plus the TATA box. Here, we demonstrate that the S phase-specific transcription of the mouse R2 gene is dependent on an upstream promoter activating region (located at nucleotides (nt) -672 to -527 from the transcription start site) and one proximal promoter repressive element (located at nt -112 to -107) that binds E2F4. Binding to the E2F site is modulated by binding of nuclear factor-Y to an adjacent CCAAT element (nt -79 to -75). The upstream activating region is crucial for overall R2 promoter activity. Mutation of the E2F-binding site leads to premature promoter activation in G(1) and increases overall promoter activity but only when the upstream activating region is present and intact. Therefore, E2F-dependent repression is essential for cell cycle-specific R2 transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M312482200DOI Listing
March 2004

Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription.

Eur J Biochem 2003 Apr;270(8):1791-801

Department of Medical Biochemistry and Biophysics, Umeå University, Sweden.

Ribonucleotide reductase is essential for the synthesis of all four dNTPs required for DNA replication. The enzyme is composed of two proteins, R1 and R2, which are both needed for activity. Expression of the R1 and R2 mRNAs is restricted to the S-phase of the cell cycle, but the R1 and R2 promoters show no obvious sequence homologies that could indicate coordination of transcription. Here we study initiation of transcription at the natural mouse R2 promoter, which contains an atypical TATA-box with the sequence TTTAAA, using a combination of in vivo reporter gene assays and in vitro transcription. Our results indicate that in constructs where sequences from the R2 5'-UTR are present, the mouse R2 TATA-box is dispensable both for unregulated, basal transcription from the R2 promoter and for S-phase specific activity. Instead, initiation of R2 transcription is directed by sequences downstream from the transcription start. We report that this region contains a conserved palindrome sequence that interacts with TAFIIs. This interaction down-regulates basal transcription from the R2 promoter, both in the absence and in the presence of the TATA-box.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1046/j.1432-1033.2003.03541.xDOI Listing
April 2003

Functional interactions within yeast mediator and evidence of differential subunit modifications.

J Biol Chem 2003 Feb 4;278(6):3831-9. Epub 2002 Dec 4.

Department of Plant Biology, Swedish University of Agricultural Sciences, Uppsala Genetic Center, Box 7080, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M206946200DOI Listing
February 2003