Stefan Björklund | Umeå University (original) (raw)

Papers by Stefan Björklund

Scientific Reports, 2020

Adverse environmental conditions are detrimental to plant growth and development. Acclimation to ... more 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 differ...

The Plant Journal, 2018

MED7 is a subunit of the Mediator middle module and is encoded by two paralogs in Arabidopsis. We... more 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.

PLOS ONE, 2017

Mediator is a multiprotein transcriptional co-regulator complex composed of four modules; Head, M... more 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.

Protoplasma, 2015

Mediator is a conserved multi-protein complex that acts as a bridge between promoter-bound transc... more 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.

The Biochemical journal, Jan 16, 2015

The eukaryotic Mediator integrates regulatory signals from promoter-bound transcription factors a... more The eukaryotic Mediator integrates regulatory signals from promoter-bound transcription factors and transmits them to RNA polymerase II machinery. Although redox signaling is important in adjusting plant metabolism and development, nothing is known about a possible redox regulation of Mediator. Here, using pull-down and yeast two-hybrid assays, we demonstrate the association of MED10a, MED28 and MED32 Mediator subunits with the GLABROUS1 enhancer binding protein-like (GeBPL), a plant-specific transcription factor that binds a promoter containing 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 and/or glutathione/glutaredoxin systems. The presence of 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 res...

European Journal of Biochemistry, 2001

Unregulated transcription of protein-encoding genes in vitro is dependent on 12-subunit core RNA ... more Unregulated transcription of protein-encoding genes in vitro is dependent on 12-subunit core RNA polymerase II and five general transcription factors; TATA binding protein (TBP), transcription factor (TF)IIB, TFIIE, TFIIF, and TFIIH. Here we describe cloning of the mouse cDNAs encoding TFIIB and the small and large TFIIE and TFIIF subunits. The cDNAs have been used to express the corresponding proteins in recombinant form in Escherichia coli and in Sf21 insect cells, and all proteins have been purified to > 90% homogeneity. We have also purified a recombinant His6-tagged mouse TBP to near homogeneity and show that it is active in both a reconstituted mouse in vitro transcription system and a TBP-dependent in vitro transcription system from Saccharomyces cerevisiae. The more complex general transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse cells. When combined, the purified factors were sufficient to initiate transcription from different promoters in vitro. Functional studies of the S-phase-specific mouse ribonucleotide reductase R2 promoter using both the highly purified system described here (a mouse cell nuclear extract in vitro transcription system) and in vivo R2-promoter reporter gene assays together identify an NF-Y interacting promoter proximal CCAAT-box as being essential for high-level expression from the R2 promoter.

Advances in Protein Chemistry, 2004

ABSTRACT This chapter describes the understanding of the structure and possible models for the fu... more ABSTRACT This chapter describes the understanding of the structure and possible models for the function of mediator in yeast and metazoan cells. Mediator complexes were recently identified also in metazoans, confirming a role for mediator in transcription regulation in higher eukaryotes as well. In spite of its general significance for transcription control, the exact mechanisms of mediator function remain unclear. The discovery of mediator has changed the view of transcriptional regulation. This multi-protein complex is now established as the main transducer of regulator information from enhancers and other control elements to the promoter. The mediator complex acts as a bridge, conveying regulatory information from enhancers and other control elements to the general transcription machinery. The mediator was originally identified in saccharomyces cerevisiae and is required for the basal and regulated expression of nearly all RNA polymerase II (Pol II)-dependent genes. Mediator seems to form an interface between gene-specific regulatory proteins and the highly conserved basal transcription machinery. A conserved core of only eight proteins found in all eukaryotic cells is responsible for contacts formed with RNA Pol II and transcription factor IIH. Other, species-specific subunits are mainly responsible for direct interactions with regulatory proteins. The subunit composition of mediator may, therefore, vary among different eukaryotic cell types, but the mechanisms of mediator-dependent transcriptional regulation are highly conserved.

Journal of Sound and Vibration, 2015

ABSTRACT A state-dependent method to model contact nonlinearities in rolling contacts is proposed... more ABSTRACT A state-dependent method to model contact nonlinearities in rolling contacts is proposed. By pre-calculation of contact stiffness and contact filters as functions of vertical relative displacement, a computationally efficient modelling approach based on a moving point force description is developed. Simulations using the state-dependent model have been analysed by comparison with measurements. Results from the investigated case – consisting of a steel ball rolling over a steel beam having two different degrees of roughness – show good agreement between nonlinear simulations and measured beam vibrations. The promising results obtained with the proposed method are potentially applicable to wheel–rail interaction and rolling element bearings.

Proceedings of the National Academy of Sciences of the United States of America, 2011

PloS one, 2014

Mediator is an evolutionary conserved multi-protein complex present in all eukaryotes. It functio... more 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 D...

Trends in Biochemical Sciences, 2005

The Mediator complex acts as a bridge, conveying regulatory information from enhancers and other ... more 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. Mediator is a conserved co-regulator of gene transcription The Saccharomyces cerevisiae Mediator complex was originally identified as a requirement for activatordependent stimulation of RNA polymerase II (pol II) transcription [1,2]. The S. cerevisiae Mediator complex comprises 21 subunits and it is found both in free form and as a holoenzyme in a complex with pol II [3,4]. Mediator structure and function seems to be conserved in mammalian cells and a unifying nomenclature was recently proposed [5]. Here, we describe recent advances in our understanding of yeast Mediator and its regulation by post-translational modification.

European Journal of Biochemistry, 2003

Ribonucleotide reductase is essential for the synthesis of all four dNTPs required for DNA replic... more 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&amp;amp;amp;amp;amp;amp;amp;#39;-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.

Proceedings of the National Academy of Sciences, 1995

Sin4 and Rgr1 proteins, previously shown by genetic studies to play both positive and negative ro... more Sin4 and Rgr1 proteins, previously shown by genetic studies to play both positive and negative roles in the transcriptional regulation of many genes, are identified here as components of mediator and RNA polymerase II holoenzyme complexes. Results with Sin4 deletion and Rgr1 truncation strains indicate the association of these proteins in a subcomplex comprising Sin4, Rgr1, Gal11, and a 50-kDa polypeptide. Taken together with the previous genetic evidence, our findings point to a role of the mediator in repression as well as in transcriptional activation.

Proceedings of the National Academy of Sciences, 1993

Mammalian ribonucleotide reductase (EC 1.17.4.1) is composed of two nonidentical subunits, protei... more Mammalian ribonucleotide reductase (EC 1.17.4.1) is composed of two nonidentical subunits, proteins R1 and R2, both required for enzyme activity. The structure of the genomic mouse ribonucleotide reductase R1 gene was compiled from a number of overlapping lambda clones isolated from a Charon 4A mouse sperm genomic library. The R1-encoding gene covers 26 kb and consists of 19 exons. All exon-intron boundaries were located by dideoxynucleotide sequencing, showing that intron 7 starts with the variant GC instead of GT. About 3.5 kb of DNA from the 5'-flanking region of the R1-encoding gene were cloned and sequenced, and the transcriptional start site was determined by nuclease S1 mapping of RNA. DNase I footprinting assays on the R1 promoter identified two nearly identical 23-bp-long protein-binding regions. Three protein complexes binding to one of the 23-mer regions were resolved and partially identified by using gel-retardation mobility-shift assays and UV crosslinking. One comp...

Nucleic Acids Research, 2014

Nucleic Acids Research, 2012

Molecular Genetics and Genomics, 2006

Med21 (Srb7) is a small essential subunit of the middle domain of the Mediator, which is conserve... more 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.